15.1B: Metabolism - Biology

15.1B: Metabolism - Biology

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All living things must have an unceasing supply of energy and matter. The transformation of this energy and matter within the body is called metabolism. Catabolism is destructive metabolism. Typically, in catabolism, larger organic molecules are broken down into smaller constituents. This usually occurs with the release of energy (usually as ATP). Anabolism is constructive metabolism. Typically, in anabolism, small precursor molecules are assembled into larger organic molecules. This always requires the input of energy (often as ATP).

Autotrophic and Heterotrophic Nutrition

Green plants, algae, and some bacteria are autotrophs ("self-feeders"). Most of them use the energy of sunlight to assemble inorganic precursors, chiefly carbon dioxide and water, into the array of organic macromolecules of which they are made. The process is photosynthesis. Photosynthesis makes the ATP needed for the anabolic reactions in the cell.

All other organisms, including ourselves, are heterotrophs. We secure all our energy from organic molecules taken in from our surroundings ("food"). Although heterotrophs may feed partially (as most of us do) or exclusively on other heterotrophs, all the food molecules come ultimately from autotrophs. We may eat beef but the steer ate grass. Heterotrophs degrade some of the organic molecules they take in (catabolism) to make the ATP that they need to synthesize the others into the macromolecules of which they are made (anabolism).

How humans (and other animals) do it

Humans are heterotrophs. We are totally dependent on ingested preformed organic molecules to meet all our energy needs. We are also dependent on preformed organic molecules as the building blocks to meet our anabolic needs.

  • Ingestion: taking food within the body (although as the figure shows, it is still topologically in the external world, not the internal).
  • Digestion.
    The enzyme-catalyzed hydrolysis of
    • polysaccharides (e.g., starch) to sugars
    • proteins to amino acids
    • fats to fatty acids and glycerol
    • nucleic acids to nucleotides
  • Absorption into the body and transport to the cells.
  • Absorption into cells

Within cells, these molecules are further degraded into still simpler molecules containing two to four carbon atoms. These fragments (acetyl-CoA for example) face one of two alternatives:

  • They may proceed up various metabolic pathways and serve as the building blocks of, for example, sugars and fatty acids. From these will be assembled the macromolecules of the cell:
    • polysaccharides
    • fats
    • proteins
    • nucleic acids
  • Or the molecules in this pool of two- to four-carbon fragments may be still further degraded — ultimately to simple inorganic molecules such as carbon dioxide (CO2), H2O, and ammonia (NH3). This phase of catabolism releases large amounts of energy (in the form of ATP). One use to which this energy is put is to run the anabolic activities of the cell.

21st European Symposium on Computer Aided Process Engineering

David Yeo , . Athanasios Mantalaris , in Computer Aided Chemical Engineering , 2011

3.2 Growth kinetics

Batch cultures underwent a lag phase for the first 72 hours of the culture whereas perfusion feeding experienced exponential growth almost immediately. Perfusion feeding enabled the expansion of high cellular density whereas batch cultures were unable to, resulting in a decline phase. Modeling results were converted from cell concentration (55 ml per vessel) to cell/bead (500 beads). The model predicts the expansion of a differentiated cell phase at day 6.5 in batch whereas perfusion feeding kept the initial population undifferentiated (X TOTAL MODEL = XU XD = 0).

Unit 15 - unit 15

15.1 Regulation of Gene Expression in Prokaryotes 15.1a The Operon is the Unit of Transcription ➔ When bacteria environments change, certain metabolic genes are turned off while others are turned on ➔ Operon Model: 1961 Jacob and Monod control of the expression of genes for lactose metabolism in E. coli ◆ 3 genes encode proteins for the metabolism of lactose by E. coli ◆ In the absence of lactose, the three genes are not expressed ◆ In the presence of lactose, the genes are expressed ➔ Operon: cluster of prokaryotic genes and the DNA sequences involved in their regulation ◆ Transcribed from the promoter into a transcription unit ◆ Operator: short segment to which a regulatory protein binds ● Protein is encoded by a gene separate from operon ● Repressor: a regulatory protein which, when active, prevents the genes of the operon from being expressed ● Activator: a regulatory protein which, when active, stimulates the expression of genes 15.1b The lac Operon for Lactose Metabolism is Transcribed When an Inducer Inactivates a Repressor ➔ Lac Operon: ◆ Inducible operon: an inducer molecule increases its expression ◆ The promoter for the transcription unit is upstream of lacZ ◆ The operator is a short DNA sequence between the promoter and the lacZ gene ◆ LacZ: encodes the enzyme β-galactosidase, which catalyzes the conversion of the disaccharide sugar, lactose, into the monosaccharide sugars, glucose and galactose ◆ LacY: encodes a permease enzyme that transports lactose actively into the cell ◆ LacA: encodes a transacetylase enzyme, the function of which is unknown ➔ Lac repressor: encoded by regulatory gene lacI nearby but separate from lac operon ◆ When lactose is absent, active Lac repressor binds to operator, blocking the RNA polymerase from binding to promoter ● Transcription cannot occur ◆ When lactose is present, β-galactosidase already present convert some of it to allolactose, an isomer ● This acts as an inducer: turns on the three genes in the operon by binding to the Lac repressor, inactivating it by altering its shape so that it can no longer bind to the operator ● RNA polymerase can then make mRNA (which lasts about 3 minutes)

15.1c Transcription of the trp Operon Genes for Tryptophan Biosynthesis is Repressed When Tryptophan Activates a Repressor ➔ If tryptophan is absent, E. Coli must make it so it can be used in protein synthesis ◆ Trp operon is expressed default state ➔ If tryptophan is present, E. Coli will use it instead of making its own ◆ Trp operon is shut off by tryptophan entering the cell, binding to the Trp repressor and activating it ◆ Active Trp repressor then binds to the operator of the trp operon and blocks RNA polymerase from binding to the promoter

➔ Trp Operon: ◆ Repressible Operon: presence of tryptophan represses the expression of the tryptophan biosynthesis genes ◆ 5 genes: TrpA-TrpE encode the enzymes for the steps in the tryptophan biosynthesis pathway ◆ Upstream of the trpE gene are the operon’s promoter and operator sequences ➔ Trp repressor: regulatory protein encoded by the trpR gene, which is located elsewhere in the genome ◆ Synthesized in the inactive form ◆ Corepressor: regulatory molecule that combines with a repressor to activate it and thus shut off the operon ● Ex. tryptophan ➔ Negative Gene Regulation: regulated by a repressor that turns off gene expression

15.2a In Eukaryotes, Regulation of Gene Expression Occurs at Several Levels 15.2b Chromatin Structure Plays an Important Role in Whether a Gene is Active or Inactive ➔ Genes in regions of the DNA that are tightly wound around histones in chromatin are inactive because their promoters are not accessible to the proteins that initiate transcription ➔ Chromatin Remodelling: changing the state of chromatin so proteins that initiate transcription can bind to the promoters ◆ 1st. An activator binds to a regulatory sequence upstream of the gene’s promoter and recruits a remodelling complex ● A protein complex that displaces a nucleosome from the chromatin ● This exposes the promoter ◆ 2nd. An activator binds to a regulatory sequence upstream of the gene’s promoter and recruits an enzyme that acetylates (adds acetyl groups: CH3CO —) to histones in the nucleosome where the promoter is located ● Histones loosen association with DNA promoter becomes accessible ● Reversed by deacetylation enzymes that remove the acetyl groups from the histones 15.2c Regulation of Transcription Initiation Involves the Effects of Proteins Binding to a Gene’s Promoter and Regulatory Sites ➔ Adjacent to the promoter, further upstream, is the promoter proximal region ◆ Contains regulatory sequences called promoter proximal elements ● Increase rate of transcription ◆ More distant from the beginning of the gene is the enhancer ● Determines whether the gene is transcribed at its maximum possible rate

➔ General transcription factors bind to the promoter in the area of the TATA box to initiate transcription ◆ Recruits RNA Polymerase II to create transcription initiation complex ● Brings about low rate of transcription and few mRNA strands ➔ Activators: bind to the promoter proximal elements to increase the rate of transcription ➔ A coactivator, a large multiprotein complex, forms a bridge between the activators at the enhancer and the proteins at the promoter and promoter proximal region ◆ Causes the DNA to loop around on itself for max transcription rate ➔ Some repressors bind to the same regulatory sequence to which activators bind (often in the enhancer), thereby preventing activators from binding to that site ◆ Others bind to their own specific site in the DNA near where the activator binds and interact with the activator so that it cannot interact with the coactivator ◆ Others recruit histone deacetylase to make promoter inaccessible ➔ Combinatorial Gene Regulation: By combining a few regulatory proteins in particular ways, the transcription of an array of genes can be controlled, and a large number of cell types can be specified ➔ A hormone is a molecule produced by one tissue and transported via the bloodstream to another specific tissue to alter its physiological activity ◆ A steroid is a type of lipid derived from cholesterol ◆ A steroid hormone acts on specific target tissues in the body because only cells in those tissues have steroid hormone receptors in their cytoplasm that recognize and bind the hormone ◆ The hormone–receptor complex enters the nucleus and binds to specific regulatory sequences adjacent to the genes whose expression is controlled ◆ Activates rapid transcription ◆ Steroid Hormone Response Element: All genes regulated by a specific steroid hormone have the same DNA sequence to which the hormone– receptor complex binds 15.2d Methylation of DNA Can Control Gene Transcription ➔ DNA methylation: a methyl group (—CH3) is added enzymatically to cytosine ◆ Methylation of cytosines in the promoter inhibits transcriptions and turns the genes off in silencing ◆ Genomic imprinting: methylation permanently silences transcription of either the inherited maternal or paternal allele of a particular gene 15.3 Posttranscriptional, Translational, and Posttranslational Regulation 15.3a Posttranscriptional Regulation Controls mRNA Availability ➔ Variations in pre-mRNA processing can regulate which proteins are made in cells ◆ I.e alternative splicing produces different mRNAs from the same pre-mRNA by removing different combinations of exons and introns ◆ Regulatory proteins specific to the type of cell control which exons are removed by binding to regulatory sequences within those molecules ➔ “Masking” proteins bind to mRNAs and make them unavailable for protein synthesis ➔ A regulatory molecule, such as a steroid hormone, directly or indirectly affects the mRNA breakdown steps, either slowing or increasing the rate of those steps

an active form under regulatory control ➔ The rate of degradation of proteins is also under regulatory control ◆ Short-lived proteins are marked for breakdown by enzymes that attach a “doom tag” consisting of a small protein called ubiquitin ● Recognized and attacked by a proteasome, a large cytoplasmic complex of a number of different proteins ○ Unfolds the protein, and protein-digesting enzymes within the core digest the protein into small peptides ○ Cytosolic enzymes further digest the peptides into individual amino acids, which are recycled for use in protein synthesis or oxidized as an energy source 15.4 The Loss of Regulatory Control in Cancer ➔ Mutations of genes regulating the cell cycle can cause problems with growth and division ➔ This can give rise to tumours ◆ Cells lose their normal regulatory controls and revert partially or completely to an embryonic developmental state, in a process called dedifferentiation ◆ Benign: if the cells stay together in a single mass not life-threatening ◆ Malignant: this is a cancer cell invade and disrupt surrounding tissues ● Metastasis: parts break off and move through blood or lymph to form tumours in new locations 15.4a Most Cancers are Caused by Genes That Have Lost Their Normal Controls ➔ Proto-oncogenes: genes in normal cells that encode various kinds of proteins that stimulate cell division ◆ Become oncogenes: genes that stimulate cell to progress to the cancerous state ◆ Caused by: ● Mutations in a gene’s promoter or other control sequences ● Mutations in the coding segment of the gene may produce an altered form of the encoded protein ● Translocation may move a gene that controls cell division to a new location near the promoter or enhancer sequence of a highly active gene ● Infecting viruses may introduce genes to regions in the chromosomes where the expression of the genes disrupts cell cycle control or alters regulatory proteins to turn genes on ➔ Tumour-Suppressor Genes: in normal cells, encode proteins that inhibit cell division ◆ Both alleles must be inactivated for inhibitory activity to be lost in cancer cells ◆ Ex. inactive TP53 which is supposed to code for p ● p53 stops cell division by combining with and inhibiting cyclin-dependent protein kinases that trigger entry into critical stages of DNA replication and mitosis 15.4b Cancer Develops Gradually By Multiple Steps ➔ Successive alterations in several to many genes gradually accumulate to change normal cells into cancer cells ➔ Offers some hope to those who quit smoking, for stopping the exposure to the carcinogenic smoke may halt multistep progression before it reaches its deadly

Quick Notes on Genetic Code | Cell Biology

Living things depend on proteins for exis­tence, the latter produce enzymes necessary for all chemical reactions. Structural infor­mation required to specify the synthesis of any given protein resides in the molecule of DNA which has the spatial configuration of a double helix proposed by Watson and Crick (1953).

The linear sequence of bases in DNA consti­tutes alphabet (hereditary lettering of 4 bases – A, T, C, C) which ‘codes’ for another linear structure, a protein, written in another alphabet of 20 amino acids.

The actual transfer of infor­mation is, however, indirect. DNA is a ‘tem­plate’ for the formation of RNAs, which are incorporated into ribosomes and in turn act as templates for protein synthesis.

All properties of protein, including its secondary and tertiary structure, are ultimately determined by chro­mosomal DNA, and all biological properties are in turn determined by the amino acid sequence of the proteins within an organism, through protein structure and enzyme activity.

The term ‘coding’ implies the relationship between DNA and protein. By coding, the hereditary lettering carried in the four alphabet of DNA is ultimately converted into the protein language composed of twenty letter alphabet of amino acids.

Co-linearity of Gene and Polypep­tide:

In 1958, Crick proposed the hypothesis that DNA determines the sequence of amino acids in a polypeptide. Fundamental to this relationship is that they are both linear in structures, in one case a sequence of nucleotides, in the other case a sequence of amino acids.

By comparing the nucleotide sequence of a gene with the amino acid sequence of a protein, we can determine directly whether the gene and the protein are co-linear or not. A gene of 3N base pairs is required to code for a protein of N amino acids.

The co-linearity of gene and protein was ori­ginally investigated in the tryptophan synthetase gene of E. coli by Yanofsky and his co-workers by utilizing a polypeptide chain A of tryptophan synthetase enzyme. It has been observed that different mutations in the DNA sequence were present in the same order as is observed in the alterations noticed in corresponding amino acid sequence in polypeptide chain A.

The recom­bination distances are relatively similar to the actual distances in the protein, so in this case there is much similarity between the recombina­tion map and the physical map.

For eukaryotic split gene having introns where all base sequences are not translated into amino acid in proteins demonstrates that co-linearity between base sequence of gene and amino acid sequence in protein may be interrupted but not violated.

Properties of Genetic Code:

Code is Triplet:

Researches have been carried out by Ochoa, Kornberg, Nirenberg, Brenner, Crick and others to detect the coding ratio, i.e., the number of units in one system required to specify one unit in the other system. Certainly no one-to-one correspondence can be observed between nucleotides and amino acids.

If each kind of nucleotide specified a single amino acid, only proteins consisting of four amino acids could be constructed. Similarly, the correspondence of an amino acid to two nucleotides would give a larger number of possibilities but still not enough, only = 16.

If a three digit code is employed, however, a total of = 64 kinds of units or codons are established (Fig. 15.1), more than enough to encode twenty amino acids. The surplus forty four triplets were initially thought to be nonsense codons and the remaining twenty as sense codons.

However, later studies have shown that several triplets can code for one amino acid. As such the number of nonsense triplets is very few. Some of the nonsense triplets might also be used as ‘punctuations’, designating the end of a chemical message.

Critical information on the nature of coding units (i.e., the code is in triplets) was gathered from studies of the muta­genic effect on polynucleotide chain (DNA).

Application of mutagen leads to the deletion or duplication of one nucleotide pair or several adjacent pairs. Addition or deletion of one or two bases respectively often causes a drastic effect and the organisms ultimately dies.

The addition or deletion of three bases together, on the other hand, though causing changes in the behaviour of the organism, yet may not necessarily induce a lethal effect and organism may survive with altered mutated tissue.

(i) The direct and exact evidence suppor­ting the triplet code concept was provided by Crick et al. (1961) based on their experiments on a virus, T4 bacteriophage (Fig. 15.2). They found, that the treatment with a chemical called pro-flavin either added or removed a base in its DNA molecule, thus damaging the virus and resulting in an altered or mutant form of the virus.

An addition followed by a deletion of base close by resulted in the restoration of the original virus. This implied that the normal sequences of bases in the DNA molecule had been restored by the second change.

A deletion or insertion completely upsets the reading frame as may be seen from the example of the base sequence GTCCAGACC. Normally the sequence will be read as GTC, CAG, ACC, …, but with the insertion of a new base T between the first and second nucleotides, it yields the sequence GTTCCAGACC … and leads to reading in the groups GTT, CCA, GAC, C …, and specifies wrong amino acids.

A similar con­sequence results from a deletion. Crossing between an addition and deletion will restore the correct reading frame of the sequence except in the region between them. It is easy to see that the combinations of two mutants in the form of two insertions or two deletions will still produce a misplaced reading frame.

Crick (1961) found that three additions or deletions of adjacent nucleotides resulted in the production of the normal virus, due to the restoration of the normal base sequence in DNA.

Thus experiments demonstrating that a combina­tion of three insertions or deletions produced a bacteriophage of perfectly normal appearance and that recombinants containing insertions or deletions in numbers not multiples of three pro­duce only nonfunctional or wrong protein, pro­vided strong evidence that the genetic code operates as a triplet code or that one triplet of nucleotides constitutes a codon.

(ii) The triplet nature of the code was fur­ther confirmed through the research work of Nirenberg and Leder (1965) who found that although little binding of tRNA was possible in the presence of dinucleotide messengers, it occurred preferentially with trinucleotides.

They were able to stimulate binding of different amino acids through different sequences of the same three bases, once again giving credence to the existence of a triplet code.

Code is Non-Overlapping:

In nature, there is always a tendency towards economy. As suggested by Gamow, in his ‘over­lapping’ coding hypothesis, the code is in the form of triplets, but not arranged in a straight chain. It is overlapping in the regions where a particular nucleotide serves in more than one coding unit.

Gamow suggested overlapping code on the basis of two characteristics:

(a) Distance between two bases in a DNA molecule is 3.4A

(b) In a protein molecule also, the distance between two adjacent amino acids is 3.4A.

This can be explained in cases of mono-coding as well as overlapping coding but this is quite impro­bable in a straight chain triplet coding. In the non-overlapping code six nucleotides would code for two amino acids, while in case of over­lapping code up-to four (Fig. 15.3).

In the non- overlapping code each letter Is read only once while in the overlapping code it would be read three times, each time as a part of different words. Mutational changes in one letter would affect only one word in the non-overlapping code while it would affect three words in the overlapping code.

There are evidences of non- overlapping nature of genetic code.

(i) The experimental evidence by Crick (1961) compellingly argued against an over­lapping code and through their research substan­tiated the arguments provided by earlier scien­tists in favour of a non-overlapping code. They started with a messenger of known triplet sequence and used this to synthesize a particular protein.

On adding a nucleotide to it, the parti­cular protein could no longer be synthesized. The result remained unaltered even with the addition of a second necleotide. The proper function of the nucleotide was restored, how­ever, on introduction of a third nucleotide.

A given nucleotide sequence ACTACTAC- TACT bears the codons ACT, ACT, ACT, ACT under the non-overlapping coding systems. An insertion of a nucleotide G between the first C and the first T, under such a system will change the nucleotide sequence to ACGTACTACTACT and codon sequences to ACG, TAG, TAG, TAG, T.

The synthesis of original protein will not take place after the addition of a nucleotide. Instead the altered amino acid chain will be producing an altogether different protein. A second inser­tion of another nucleotide G between the first C and first G of the previously altered nucleotide chain results into a new nucleotide sequence ACGGTACTACTACT and the corresponding codon sequence ACG, GTA, CTA, CTA, CT.

The particular protein still cannot be synthesized. A third nucleotide addition, an insertion of nucleotide G, in the beginning of the nucleotide chain available after the last step causes it to read as GAGGGTACTACTACT and the corresponding codon chain available is GAC, GGT, ACT, ACT, ACT.

The third addition has restored most of the original triplet sequence. The deletion of bases from DNA has the same effect as that of deletion. The third deletion will, however, restores most of the reading frame and allow a sequence of amino acids, differing slightly from its original one. This suggests that the code is non-overlapping.

(ii) Another evidence supporting the exis­tence of a non-overlapping code is provided by the effect of single-site mutations.

A single muta­tion in an overlapping coding system would invariably affect two or more adjacent amino acids in the nucleotide chain. A mutation from the first G to C in the nucleotide sequence ATGATGATG will cause change in one codon only in the case of a non-overlapping code. The original codon sequence of ATG, ATG, ATG will result into a codon sequence ATC, ATG, ATG after single mutation.

However, if the code was an overlapping one, the original codon sequence ATG, TGA, GAT, ATG, TGA, GAT, ATG will change into the codon sequence ATC, TGA, CAT, ATC, TGA, GAT, .ATG. As a result of single mutation, three changes take place. In the codon sequence when the overlapping code is in ope­ration.

Only one change would be expected in case of a non-overlapping code. Since only sin­gle amino acid changes have been observed in the experimental studies of single-site mutation, this evidence reinforces the existence of non-overlapping code.

(iii) Brenner (1957), on the basis of all the published data on the studies of the sequence of amino acids in proteins, concluded that there were no forbidden zones in proteins, and neigh­bouring amino acids were invariably coded by unrelated groups of nucleotides.

It was further established that no specific amino acid will always have the same nearest neighbours and the amino acid sequences appear to be almost completely at random. Such revelations would not have been feasible had the code been of an overlapping nature.

(iv) Yanofsky (1963) provided perhaps the most convincing evidence available that excludes any overlapping code. In his studies of both mutation and recombination through transduc­tion technique, he found that in each protein with a different amino acid at a given position, the amino acids on either side remained unchanged.

Code is Degenerate:

Sometimes three or four triplet codons code for a particular amino acid. Such a genetic code where there are more than one triplet (codon) codes for a single amino acid is known as degen­erate code. Out of possible 64 different codons, 61 codons code for different amino acids.

As there are 20 amino acids, so it is obvious that more than one codon or triplet codes for one amino acid. If each amino acid is coded by a single codon, 44 codons out of 64 will be useless or nonsense codons.

Numerous evidences indicate that the genetic code is degenerate.

(i) If twenty triplets only would have made sense and the remaining forty four remained non­sense, then in a chromosome length mutations could occur only at very limited sites representing one-third of the length and not throughout its entire length.

But the rate of spontaneous muta­tion as well as the results of induced mutation through X-rays has shown that nearly the entire chromosome site is capable of undergoing muta­tion. It is possible if only when the code is degene­rate. However, though the degenerate nature of the code has been established, the presence of high number of repeated sequences may make major segments of chromosomes non-mutable.

(ii) When two bases U and C, in a 3:1 pro­portion are synthesized into in RNA, the possible triplets and their frequency can be mathemati­cally determined :

UUU = 3/4 x 3/4 x 3/4 = 27/64 UUC = 3/4 x 3/4 x 1/4 =9/64 UCU = 3/4 X 1/4 X 3/4 = 9/64 CUU = 1/4 x 3/4 x 3/4 = 9/64 UCC = 3/4 x 1/4 X 1/4 = 3/64 CUC = 1/4 x 3/4 X 1/4 = 3/64 CCU = 1/4 x 1/4 x 3/4 = 3/64 CCC = 1/4 X 1/4 X 1/4 = 1/64.

mRNA of this compo­sition should guide the incorporation of eight amino acids but in fact only four amino acids were actually detected in the protein chain indi­cating the degenerate nature of the code, i.e., some of the codons in this case have directed the incorporation of the same amino acid.

(iii) According to the wobble hypothesis of Crick (1966), the first two bases of the triplet codon pair according to the set rules, i.e., A with U and G with C but the third base having much more freedom of movement than the other two, wobbles and permits more than one type of pair­ing at that position. Thus the wobble hypothesis explains the degeneracy of the code to some extent.

It is sometimes argued that the third base of a code is not very important and that specificity of a codon is particularly determined by the first two bases. It has been shown that the same tRNA can recognise more than one codons differing only at the third posi­tion. This paring is not very stable and is allowed due to wobbling in base pairing at this third posi­tion.

Crick in 1965 proposed a hypothesis called wobble hypothesis to explain this phenomenon. He discovered that if U is present at first position of anticodon, it can pair with either A or G at the third position of codon. Similar is the case with G, found in anticodon, which can pair with either C or U of codon (Table 15.1 A).

The wobble hypothesis visualizes that many codons are able to tolerate mutations at the third base site because of the non-restrictive spatial limitations for the corresponding base in the anti- codon. The third nucleotide in many codons was better tolerated and could be substituted without damage.

The corresponding base in the anticodon would wobble and accommodate. This kind of wobbling allows economy of the number of tRNA molecules since several codons meant for same amino acid are recognized by same tRNA.

Code is Comma-less:

A comma-less code means that no punctua­tion marks are needed between two words. In other words, we can say that after one amino acid is coded, the second amino acid will be automatically coded by the next three letters and no letters are wasted (Fig. 15.4).

However, the code for an entire polypeptide having several amino acids is always terminated by a nonsense codon which servers as full stop in the coding terminology.

If the genetic code functions with commas, a specific nucleotide serves as a punc­tuation mark. Through experiments it has been established that poly-A (AAA) codes for lysine, poly-C (CCC) for proline, and poly-U (UUU) for phenylalanine, which implies that the commas are not made up of A, C and U.

Code is Non-Ambiguous:

Ambiguity denotes that a single codon may code for more than one amino acid. Non- ambiguous means that there is no ambiguity about a particular codon. A particular codon will always code for the same amino acid.

The genetic code is generally non-ambiguous, can be experimentally confirmed using a specific single triplet-ribosome complex which directs the binding of specific tRNA. For example, UUU triplet-ribosome complex directs the binding of phenylalanine-tRNA and AAA triplet-ribosome complex directs the binding of the lysine-tRNA.

In the similar manner, by using the triplets of known sequence, the codons for valine, cysteine, leucine and some other amino acids were determined, thus clearly establishing the non-ambiguous nature of the genetic code under natural physiological conditions.

Code is Universal:

The genetic code is universal. It means that the same codon codes for the same amino acid in all the organisms, from human beings to virus.

Universal nature of genetic code has been experimentally evidenced.

(i) The crucial point in the genetic code is the fitting of tRNA with specific anticodon into the codon of the mRNA.

Thus if mRNA is taken from an eukaryote and tRNA from a prokaryote and protein synthesis could be carried as coded in the mRNA, then it can be proved that code is universal, if mRNA and ribosome are taken from E. coli, and amino acid and tRNA from rat, pro­tein synthesis can be carried out as coded in the mRNA of E. coli. This is true also the other way round.

Von Ehrenstein and Lipmann found that E. coli tRNA to which labeled amino acids were added would form haemoglobin when incubated with the mRNA and ribosomes of rabbit reticulo­cytes.

The precision with which this interspecific attachment occurs was shown by converting cysteine into alanine in amino acid-activated tRNAcys and then observing that this alanine was now inserted into peptide positions ordinari­ly occupied by cysteine, in other words, the anti- codon of the cysteine-tRNA of a bacterial species recognized the cysteine codon of mammalian mRNA in spite of the fact that the tRNA was carrying an alanine amino acid.

(ii) The tRNA from E. coli, Xenopus laevis and guineapig bind to the same trinucleotides as shown by Nirenberg et al., indicates the univer­sality of the code.

(iii) Studies of Merril and co-workers (1971) revealed that a bacterial enzyme X-D-galactose -1 phosphate uridyl transferase which catalyses the metabolism of galactose sugars is produced in human tissue culture cells, previously unable to make it, after infection by a virus carrying the E. coli gal + gene. This provides strong evidence in favour of the universality of the code.

(iv) The correlated nucleotide and amino acid sequences in the overlapping genes of the DNA bacteriophage ф x 174 and in the capsid protein coding gene of RNA bacteriophage MS2 indicates that the genetic code is universal.

(v) Uniformity in amino acid sequence of homologous proteins, e.g., cytochrome c collec­ted from widely divergent species like human, horse, chickens, yeast and bacteria displayed universality of the genetic code.

(vi) Finally genes from human and other organisms have been expressed in E. coli and those from bacteria and other organisms in plants. In each such case, the polypeptide produced by a gene in the new organism was identical with the one it produced in the orga­nism of its origin.

Exceptions of Genetic Code:

A triplet codon demands its own tRNA with a complementary anticodon or a single tRNA responds to both members of a codon pair or to all (or at least some) of the four members of a codon family. Often one tRNA can recognise more than one codon, i.e., codon is degenerate.

This means that the base in the first position of the anticodon must be able to partner alternative bases in the corresponding third position of the codon. In such cases there may be differences in the efficiencies of the alternative recognition reactions (as a general rule, codons that are com­monly used tend to be more efficiently read).

In addition to the constructions of a set of tRNAs able to recognise all the codons, there may be multiple tRNAs that respond to the same codon. The predictions of wobble pairing accord very well with the observed abilities of almost all tRNAs. But there are exceptions in which the codons recognized by a tRNA differ from those predicted by the wobble rules.

Such effects pro­bably result from the influence of neighbouring bases and/or the conformation of the anticodon loop in the overall tertiary structure of the tRNA. Indeed, the importance of the structure of anti­codon loop is inherent in the idea of the wobble hypothesis itself.

Further support for the influ­ence of the surrounding structure is provided by the isolation of occasional mutants in which a change in a base in some other region of the molecule alters the ability of the anticodon to recognize codons.

Another unexpected pairing reaction is pre­sented by the ability of the bacterial initiator, fMet-tRNA ƒmet to recognize both AUG and GUG. This misbehavior involves the third base of the anticodon. Though the genetic code is non-ambiguous, but GUG codes for methionine when used as initiator codon, but it codes for valine if present at the intercalary position, indi­cating its ambiguous nature.

The universality of the genetic code is stri­king, but some exceptions exist. They tend to affect the codons involved in initiation or termi­nation and result from the production (or absence) of tRNAs representing certain codons. Almost all of the changes found in principal genomes affect termination codons.

In the prokaryote Mycoplasma capricolum, UGA is not used for termination, instead codes for tryptophan. In fact, it is the predominant Trp codon, and UGG is used only rarely. Two Trp-tRNA species exist, with the anticodons UCA (reads UCA and UGG) and CCA (reads only UGG).

Some ciliates (unicellular protozoa) read UAA and UAG as glutamine instead of termina­tion signals. Tetrahymena thermophile, one of the ciliates, contains three tRNAglu species. One recognises the usual codons CAA and CAG for glutamine, one recognises both UAA and UAG (according to wobble hypothesis), and the last recognizes only UAG.

We assume that the release factor eRF has a restricted specificity, compared with that of other eukaryotes.

In another ciliate (Euplotes octacarinatus), UGA codes for cysteine. Only UAA is used as a termination codon, and UAG is not found. The change in meaning of UGA might be accom­plished by a modification in the anticodon of tRNAcys to allow it to read UGA with the usual codon UGU and UGC.

The only substitution in coding for amino acids occurs in a yeast (Candida), where CUG means serine instead of leucine (and UAG is used as a sense codon).

All of these changes are sporadic, which is to say that they appear to have occurred indepen­dently in specific lines of evolution. They may be concentrated on termination codons, because these changes do not involve substitution of one amino acid for another. Thus the divergent uses of the termination codons could represent their ‘capture’ for normal coding purposes.

Exceptions to the universal genetic code also occur in the mitochondria from several species.

The earliest change was the employment of uni­versal stop codon UGA to code for tryptophan which is common to all (non-plant) mitochon­dria. It is not likely that UGA coded for trypto­phan in the universal code, but was changed to termination in cytoplasmic translation, because it is a stop codon in bacteria, plant mitochondria and nuclear genomes.

Departures from the universal code, all in non-plant mitochondria, are CUN (leucine) for threonine (in yeasts), AAA (lysine) for asparagine (in Platyhelminthes and echinoderms), UAA (stop) for tyrosine (in Planaria), and AGR (arginine) for serine (in several animal orders and for stop (in vertebrates) [N = A, U, G or C R = A or G) (Table 15.1B).

The mitochondria of plants and protozoans differ in importing and utilizing tRNAs encoded by the nuclear as well as the mitochondrial genome, whereas in animal mitochondria, all the tRNAs are encoded by the organelle.

The small number of tRNAs encoded by the mitochondrial genome highlights an important feature of the mitochondrial genetic system — the use of a slightly different genetic code, which is distinct from the universal code used by both prokaryotic and eukaryotic cells.

Some of these changes make the code simpler, by-replacing two codons that had different meanings with a pair that has a single meaning. Pairs treated like this include UGG and UGA both Trp instead one Trp and one termination) and AUG and AUA (both Met instead of one Met and other lie).

The changes are typically prece­ded by loss of a codon from all coding sequences in an organism or organelle, often as a result of directional mutation pressure, accompanied by loss of the tRNA that translates the codon.

The code reappears later by conversion of another codon and emergence of a tRNA that translates the reappeared codon with a different assign­ment. Changes in release factors also contribute to this revised assignment. Thus the genetic code, formerly thought to be frozen, is now known to be in a state of evolution.

Decipherence of Genetic Code:

It was not possible to say which codon of the possible 64 codons should code for which of the 20 amino acids until the first clue to this problem came when M.W. Nirenberg used in vitro sys­tem for the synthesis of a polypeptide using an artificially synthesized mRNA molecule.

In 1961 Nirenberg and Mathaei characterized the first specific coding sequences, which helped in analysis of genetic code.

Their success on decipherence of code was dependent on two experimental systems:

(i) In vitro (cell free) protein synthesizing system,

(ii) An enzyme, polynucleotide phosphorylase which allowed the synthesis of synthetic mRNAs. These mRNAs served as templates for polypeptide synthesis in the cell free system.

The enzyme polynucleotide phosphorylase functions metabolically in bacteria to degrade RNA, but with high concentrations of ribo­nucleotide diphosphates, the reaction can be ‘forced’ in the opposite direction to synthesize RNA.

Like RNA polymerase it does not require any DNA template, each addition of ribo­nucleotide is random based on the relative concentration of the four ribonucleoside diphos­phates added to the reaction mixtures. The probability of insertion of a specific ribonucleo­tide is proportional to the availability of that molecule, relative to other available ribonucleo­tides.

The cell free system for protein synthesis and the availability of synthetic mRNAs provided a means of deciphering the ribonucleotide compo­sition of various triplets encoding specific amino acids.

Homopolymers Technique (Poly U Experiment):

In their initial experiments, Nirenberg and Mathaei, synthesized RNA homopolymers, each consisting of only one type of ribonucleotide, i.e., the produced mRNA in the in vitro system is either UUUUU …, AAAAA …, CCCCC … or GGGGG … In testing each mRNA, it was very much easy to determine which amino acid was incorporated in the polypeptide chain.

Different amino acids were labelled by using 14 C and tested separately by radioactive counting. In the synthesized RNA using only uracil, there was no other base all along the length of mRNA and the only possible triplet was UUU.

When such a poly-U (RNA) was used in the synthesis of a polypeptide (using all extracts from E. coli, and supplying all the required components of protein synthesizing machinery), only polyphenylalanine was synthesized, meaning that the only amino acid coded was phenylalanine.

It was, therefore, immediately concluded that the input UUU coded for the amino acid phenylalanine. Subsequently, poly A gave polylysine and poly C gave poly-proline. Therefore, UUU was assigned to phenylalanine, AAA to lysine and CCC to pro­line. But the poly G did not serve as template as it gets folded backs on itself, for this assignment other method had been followed.

Heteropolymers (Random): Mixed Copolymers Technique:

The study of polynucleotides were further extended with copolymers as synthetic messen­gers containing two or more bases in definite proportion in cell free system. These randomly synthesized polynucleotides resulted in direct incorporation of amino acids into protein in a manner which indicated that a number of different code words are involved in the binding of different amino acids.

In cell free culture, with these synthetic polyribonucleotide’s, the different amino acids incorporated in a messenger could be clearly correlated with the expected variations in the frequency of different triplets in the synthetic copolymers. Thus this experiment showed the way of deriving nucleotide composition of triplets for each of the amino acids.

Nirenberg, Mathaei and Ochoa did their experiments using the RNA heteropolymers in this technique two or more different ribonucleoside diphosphates were added in combination to form the artificial message. The frequency of a particular triplet codon on the synthetic mRNA depended on the relative proportion of ribo­nucleotide addition in the cell free system.

The percentage of incorporation of particular amino acid in the polypeptide chain could be used for prediction against a particular triplet codon.

For example, in a system A and C are added in a ratio of 1 A: 5C. Now, the insertion of a ribonu­cleotide at any position along the RNA molecule during its synthesis is determined by the ratio of A:C. Therefore, there is a 1/6 possibility for an A and a 5/6 chance for a C to occupy each position.

On this basis, we can calculate the frequency of any given triplet appearing in the message. For AAA, frequency is (1/6) 3 or 0.4%. For AAC, ACA and CAA, the frequencies are identical (1/6) 3 x 5/6 or 2.3%, all three together it is 6.9%. In the same way 1A:2C is calculated which is 1/6 x (5/6) 2 or 11.6% or all together 34.8%, whereas CCC is (5/6)3 or 57.9% of the triplets.

Now by examining the percentage of any given amino acid incorporated into the protein synthesized under the direction of this message, it is possible to propose probable base composi­tion. As because proline appears 69%, it can be deduced that proline is likely to be coded by CCC (57.9%) and also by one of the triplet code 1A : 2C variety (11.6%), i.e., 57.9 + 11.6.

Histidine incorporation percentage is 14% which is probably coded by one 1A:2C category and another 1C:2A category (11.6+2.3)%. Threonine shows 12% incorporation, i.e., likely to be coded by one 1A:2C category. Asparagine and glutamine appear to be coded by one of the 1C:2A triplets and lysine appears to be coded by AAA.

Using as many as all four ribonucleotides to construct this kind of random heteropolymers of synthetic mRNA, the composition of triplet code words corresponding to all 20 amino acids could be determined (Table 15.2).

Heteropolymers (Ordered): Repea­ting Copolymers Technique:

In early 1960s H.G. Khorana could chemi­cally synthesize long RNA molecule consisting of short sequences repeated many times. The short sequences were of di-, tri- or tetra-nucleotides, which were replicated many a times and finally joined enzymatically to form the long polynu­cleotides.

The dinucleotide repeats will be trans­lated for two different amino acids trinucleotide repeats will be converted into 3 potential triplets, depending on the point at which initiation occurs and a tetra-nucleotide creates four repea­ting triplets.

When these synthetic mRNAs were added to a cell free system and amino acid incorporation is matched, the conclusions can be drawn from the composition assignment and triplet binding, and specific assignments were possible.

When the repeating dinucleotide sequence is UCUCUCUC…, it produces the triplets UCU and CUC — they can incorporate leucine and serine into the polypeptide. When the repeating trinucleotide sequence is UUCUUCUUC…, the possible triplets are of three kinds: UUC, UCU and CUU depending on the initiation point and they can incorporate phenylalanine, serine and leucine.

From the above two results it can be concluded that UCU and CUC encode for serine and leucine and also either UUC or CUU encodes for serine or leucine, while the other encodes for phenylala­nine. Further, when the tetra-nucleotide sequence UUAC is repeated then it produces the UUA, UAC, ACU and CUU.

Here the incorporated amino acids are leucine, threonine and tyrosine. In the above two cases, the common code is CUU and common amino acid incorporated is leucine, so it can be concluded that CUU encodes for leucine.

Now from these experiments logically it can be determined that UCU encodes for serine and the rest UUC encodes for phenylalanine and also the CUC encodes for leucine (Table 15.3).

Like this way, by logical interpretations, Khorana reaffirmed triplets that were already deciphered and filled in gaps left from other approaches (Table 15.4).

Triplet Binding Technique:

Nirenberg and Leder in 1964 found that if a synthetic tri-nucleotide for a known sequence is used with ribosome and a particular aminoacyl- tkNA, these will form a complex provided that the used codon codes for the amino acid attached to the given aminoacyl-tRNA.

In order to work out the code for all 20 amino acids, all the possible 64 triplets had to be tried in cell free culture.

In the experiment, 20 samples of the mixture of all 20 amino acids were taken and in each sample, one amino acid was made radioactive in such a manner that each and every amino acid is radioactive in one sample or the other, and no two samples have same radioactive amino acid. For instance, in one set valine has been labelled and the rest 19 remained unlabelled.

Similarly, in another set lysine was labelled and the rest 19 remained un-labelled. Then the tRNAs and ribosomes are mixed with each of these samples and the same codon is used for all sets. When the mixture is poured on the nitro­cellulose membrane, radioactivity on membrane will be observed only when the radioactive amino acid is taking part in the formation of complex.

Since in each sample the radioactive amino acid is known, it would be possible to detect the amino acid coded by a given codon by the presence of radioactivity on the membrane. Such a treatment was given to all 64 synthetic codons, and their respective amino acids were identified.

Codon Dictionary:

The base sequence in mRNA and the resul­ting amino acid sequence in protein reveals the code for each amino acid. All the 64 codons, along with their amino acids, are represented in Table 15.5.

An examination of the code table reveals the following characteristics:

i. Each codon consists of three nucleo­tides, i.e., the code is triplet. 61 codons represent 20 amino acids. Three represent (UAA, UAG, UGA) punctuation marks for termination of pro­tein synthesis.

ii. Almost all amino acids are coded by more than one codon, except methionine and tryptophan which have only one codon. Phenylalanine, tyrosine, histidine, glutamine, asparagine, lysine, aspartic acid, glutamic add and cysteine are the nine amino acids which are represented by two codons each. Three amino acids, i.e., arginine, serine and leucine have Six codons each. The table indicates the degeneracy of the genetic, code.

iii. If an amino acid has more than one codon, the first two nucleotides are identical and the third nucleotide can be either cytosine or uracil. Adenine and guanine are also similarly interchangeable at the third position. For example, UUU and UUC, both code for phenylalanine, and UCU, UCC, UGA and UCG code for serine.

However, there are some exceptions to the equi­valence rule of the first two nucleotides, as AGU and AGC also code for serine apart from UCU, UCC, UCA and UCG.

Similarly, the amino acid leucine is also coded- by six codons, i.e., UUA, UUG, CUU, CUC, CUA and CUG.

The frequent interchange of cytosine and uracil or guanine and adenine suggests that great variations can occur in AT/GC ratio in certain organisms without affecting large changes in the relative proportions of amino acids present in them, as for almost every amino acid there is one codon that carries G or C and another that carries A or U as its third nucleotide.

The two organisms carrying the same protein sequence information in their DNA, by selecting one or the other kind of synonym codon, can show different AT/GC ratios.

iv. The genetic code has a definite structure in the sense that the synonyms for the same amino acid are not randomly dispersed over the table but are usually found together. The only exceptions are the codons, six each for arginine, serine, and leucine, which are spread over the table.

v. Multiple codons for an amino acid show in general the similarity in first two nucleotides and it is the third nucleotide which varies.

AUG is the initiation codon, i.e., the polypeptide chain starts with methionine. This amino acid is the formulated form of methionine. The initiation codon binds to fmet-tRNA having an anticodon 3′ UAC 5′ which is identical to that of met-tRNA, i.e., both met- tRNA and fmet-tRNA are coded by AUG but the signal for the starting amino acid is much more complex than the signal for all other amino acids.

According to Stent, there exist two separable species of tRNA capable of accepting methionine. Methionine of only one of these is concerned into formyl methionine by the action of the special formulation enzyme. The other or ordinary met- tRNA incorporates methionine into the interior of the growing polypeptide chain and responds to the codon AUG only.

Formyl-met-tRNA initiates the polypeptide chain and responds to GUG (valine codon) also. The GUG while present at the initiation point, codes for methionine whereas in the intercalary position, it codes for valine. The anticodon of this species of tRNA seems to be per­missive with respect to the first nucleotide base of the codon and selective with respect to the second and third nucleotide bases.

UAA, UAG and UGA are the chain termination codons. They do not code for any of the amino acids but serve as stop codon. These codons do not have any tRNA but are read by specific proteins called release fac­tors. These codons are also called nonsense codons.

A mutation from a sense to nonsense codon in the middle of a genetic message results in the release of immature or incomplete polypeptides which do not have any biological activity. Nonsense mutations can be induced by mutagens. UAG was formerly known as amber, UAA as ochre and UGA as opal.

A Short History Of Conformal Therapy

One of the most important pioneers in “conformation therapy” was Shinji Takahashi, who described many of the important concepts of conformal therapy delivery and 3D treatment planning in a 1965 monograph. 1 Takahashi’s innovations included early multileaf collimators, automated (mechanical) conformal beam shaping, dynamic conformal treatments, orthogonal light beams to identify the machine isocenter, and 3D tumor models based on early tomography. 1 Other notable work in this area was performed by Harold Perry and colleagues 2 in Detroit and by Proimos, Wright, and Trump at the Massachusetts Institute of Technology (MIT)–Lahey Clinic. 3 – 7 Another early approach to conformal therapy, known as the Tracking Cobalt Project, 8, 9 was led by Green, Jennings, and others at the Royal Northern and Royal Free Hospitals in England. First reported in the late 1950s, 10 and summarized by Jennings, 8 a series of mechanical, electronic, and, finally, computer-controlled treatment machines were developed to track disease spread, particularly along lymph node chains. By 1980, the computer-controlled version of the tracking system was in clinical use, 11 although Brace summarized the major limitations to the delivery technique: “The major obstacle to the routine use of conformation therapy is treatment planning.” 11 Finally, workers at the Joint Center for Radiation Therapy (JCRT) in Boston added computer control to a modern linear accelerator, so that the treatment table, gantry, collimator, collimator jaws, dose rate, and other parameters could be controlled dynamically while the beam was in use. The JCRT achieved the delivery of what is now called “dynamic conformal therapy,” 12 – 14 15 a modern basis for computer-controlled conformal therapy.

With the widespread implementation of CT-based planning, it became possible to make use of continuing improvements in computer technology and new software developments to create fully 3D treatment planning systems that incorporated 3D graphics and the “beam’s-eye view” (BEV), a 3D graphic reconstruction of the patient anatomy projected into the divergent geometry used by the x-rays in the radiation beam 22 – 26 (Fig. 15-2). Using BEV displays to design field shaping 27 and evaluate coverage of tumor and sparing of normal tissues is perhaps one of the most effective concepts in the entire 3D planning paradigm. Routine clinical use of 3D radiation treatment planning (RTP) began in 1986, 28 and many academic centers began development and then use of 3D planning systems in their clinics. 29 – 32

The capabilities of computer control and MLC systems have made possible the delivery of very complicated plans, including those that make use of modulated intensities (a beam with different intensities in different parts of the field). Intensity modulation created using multiple segments 40 – 43 ,44 ,45 or dynamic MLC motions 33, 46, 47 and computer plan optimization (inverse planning) 48, 49 – 51 ,52 have been integrated into IMRT. 53 The basic concepts of IMRT were described in 1987 by Brahme 33, 48, 54 and a practical implementation was described by Bortfeld soon after. 55 The combination of the flexibility of computer-controlled IMRT delivery with sophisticated plan optimization techniques has made IMRT an extremely powerful tool that can be used to perform conformal therapy.

The initial commercial IMRT implementation by NOMOS in 1992 56 was a form of IMRT now called serial tomotherapy, in which patients were treated slice by slice (as with early CT scanners) by the machine rotating around the patient and a special multileaf collimator (MIMIC) that performed the intensity modulation. Within a few years, all major vendors had implemented MLC systems with leaf widths varying from 1 cm to a few millimeters that could perform IMRT using either dynamic motions of the MLC leaves (DMLC) or a number of static segments (shapes), now called SMLC. 53 A more sophisticated implementation of tomotherapy based on helical delivery of IMRT, helical tomotherapy, 57 also became widely disseminated. In the last several years, a number of vendors have now developed a rotational MLC-based IMRT technique (IMAT), which was originally described by Yu, 58 and is now called VMAT (volumetric modulated arc therapy). 59, 60 Inverse planning has also developed substantially during this time. Though much of this optimization makes use of quadratic weighted sum cost functions and simple gradient-based search algorithms, there have also been developments of sophisticated cost functions 61 and the use of more biologically related costs such as normal tissue control probability (NTCP) models and equivalent uniform dose (EUD). Most systems use the weighted sum cost functions, but there has been development of sophisticated multicriteria methods 62, 63 that more directly take into account the numerous optimization goals involved in a typical clinical radiotherapy treatment plan.

One of the developments that made the conformal therapy revolution possible was the development of amorphous silicon flat panel imagers, 64 which allowed effective electronic portal imaging verification of the accuracy of these newly conformal fields. This technology then was further developed, first for kilovoltage (diagnostic quality) imaging and then to provide cone beam CT (CBCT) capability using kilovoltage imaging systems mounted directly on the treatment machine. 65 The availability of these high-quality CBCT or kilovoltage imaging modalities directly on the treatment machine led to the development of image-guided radiation therapy (IGRT), in which diagnostic imaging was used to correct patient setup and positioning for treatment every day. IGRT processes have greatly increased the delivery accuracy possible and have led to the possibility of much smaller margins for setup errors, as well as enough confidence in targeting accuracy that stereotactic body radiation therapy (SBRT) is now used routinely to give very high doses (as high as 20 Gy/fraction) to well-localized targets in the liver, lung, and other sites. The use of IMRT and the proper handling of patient motion, respiration, and other “4D” issues is a major thread of much research and development at the current time.


We report here the effects of PCZ, PAC, and UCZ treatment on the phenotypes of na2-1, d5, and wild-type siblings at maturity. PCZ treatment of wild-type plants was able to recapitulate the phenotype of na2-1, except for the POPIT phenotype (Table 1, Table 2, and Figure 4b–c). However, a previous study used PCZ at twice the concentration (500 μM) that we used in this study and they did observe POPIT in PCZ-treated wild-type plants (Hartwig et al., 2011 ). Off-target and non-specific effects of inhibitors are a greater concern as concentrations increase, and we sought to minimize these with lower concentrations. The observation that PCZ treatment even at a lower concentration decreased plant height and increased POPIT in na2-1 mutants (Table 1) suggests that residual BR activity is present in na2-1 mutants. Indeed, we observe substantial year-to-year and environmental suppression of POPIT in the na2 and na1 mutants indicating that BR activity is near the threshold at which POPIT occurs. Selection of 250 μM PCZ as our treatment dose appears to have defined the lower boundary of BR inhibition at which POPIT is affected in wild-type maize and indicates that plant height is more sensitive than floral organ persistence to a loss of BR. These results, and our unpublished observations that brd1 mutants and na1/na2 double mutants are more severely dwarfed than na1 or na2 single mutants, suggest that disruption of this gene may not be a complete knockout of brassinosteroid biosynthesis. Rice brassinosteroid biosynthetic mutants accumulate C29/28 and C27 bioactive BR not present at detectable concentrations in wild-type plants due to non-linearity and the existence of bypass pathways which may also exist in maize (Hong et al., 2005 ). Targeted metabolic analysis of all possible BR intermediates in the maize na1, na2, and brd1 mutants is required to determine whether this is the case.

Brassinosteroid and GA interact to control plant development, and the concordance or discordance of PCZ, PAC, and UCZ with the phenotypes presented in the mutants of BR and GA biosynthesis is shown in Figure 7. This summarizes the hypothesis test central to this study, that these inhibitors achieved their phenotypes via the specific inhibition of BR or GA biosynthesis. UCZ treatment to wild-type plants was able to phenocopy d5 mock-treated plants, and UCZ-treated na2-1 plants displayed all the interactions observed following the loss of both GA and BR biosynthesis in na2-1/d5 double mutants (Best, Hartwig et al., 2016 ) (Table 1, Table 3, and Figure 7). UCZ reduced plant height, promoted tiller outgrowth, induced anther persistence in the ear, and suppressed POPIT induced by the loss of BR biosynthesis in the na2-1 mutant (Table 1, Table 3, and Figure 7). These results are consistent with UCZ affecting maize growth via specific inhibition of GA biosynthesis at the concentrations employed. However, the suppression of tiller outgrowth induced by a loss of na2 was the least effective of all treatment and genetic combinations observed (Table 1). Our experiments do not rule out UCZ inhibition of P450 enzymes outside of GA biosynthesis. Indeed, it was established that UCZ inhibits ABA catabolism in Arabidopsis at concentrations lower than employed here and that specific inhibition of recombinant CYP707A occurs with an IC50 of 68 nM (Figure 2d) (Saito et al., 2006 ). This reaction contributes to the formation of phaseic acid which has been demonstrated to have additional biological activities of its own (Figure 2d) (Hill et al., 1992 Sharkey & Raschke, 1980 ). The perfect agreement of the developmental phenotypes caused by UCZ and the GA mutants was somewhat surprising in light of these additional modes of action (Figure 2a–d). It is possible that the weak suppression of tiller formation by na2-1 in UCZ treatments may result from inhibition of other P450s. It is formally possible, although we have no evidence, that UCZ might affect P450s in strigolactone biosynthesis or that ABA might enhance tillering in maize. UCZ also delayed tassel emergence in wild-type, d5, and na2-1 plants (Tables 1 and 3). No effect on node number was observed in wild-type and d5 plants treated with UCZ as compared to controls (Table 3). However, UCZ-treated na2-1 displayed fewer nodes per plant than UCZ- and mock-treated wild types (Table 1 and Figure 8). The simplest interpretation of our results is that UCZ affects all of the traits we measured via inhibition of GA, for which there is ample evidence (Rademacher, 2000 ). Further research, particularly work comprehensively evaluating the binding and catalytic inhibition of recombinant P450s by UCZ, as was done for ABA catabolism and UCZ, is needed to test these hypotheses and establish the specificity of these triazole inhibitors.

Paclobutrazol treatment was unable to phenocopy all of the effects of GA-deficient mutants in maize (Figure 7). PAC treatment of wild type and na2-1 did not promote anther retention in the ear florets nor did it suppress POPIT in na2-1 plants (Table 1). This contrasts with both UCZ treatments and our previous genetic results (Best, Hartwig et al., 2016 ). The concentration of PAC used was the same as UCZ, but it may be that PAC is a weaker inhibitor as has been demonstrated in sunflower, Impatiens walleriana, Salvia splendens, Tagetes erecta L., and Petunia hybrid (Barrett & Nell, 1992 Whipker & Dasoju, 1998 ). If PAC is effectively inhibiting GA biosynthesis, this would contradict maize double-mutant analyses where GA biosynthetic mutants were epistatic to BR biosynthetic mutants for the expression of POPIT (Best, Hartwig et al., 2016 ). More likely the discordance between PAC and genetic ablation resulted from incomplete inhibition of GA biosynthesis by our repeated root drenches with 60 μM PAC. The effects of treatment on height and stimulation of tiller outgrowth in wild-type plants, and suppression of outgrowth by na2-1, matched the effects of reduced GA biosynthesis (Tables 1 and 3). The inability of PAC treatment to induce anther-ear in any genotype, except for d5, or suppress POPIT in na2-1 may be due to the concentration used, unspecific inhibition of other pathways besides GA biosynthesis (Figure 2), or combination of the two. The observation that PAC growth arrest can be reversed by application of sterols raises the possibility that PAC achieves some height reduction by affecting both BR and GA levels (Haughan et al., 1988 ). If BR biosynthesis is inhibited by PAC treatment, this could increase the penetrance of POPIT in the na2-1 mutant, as was observed in PCZ treatment, and require greater reduction in GA to prevent floral organ persistence. However, the lack of anthers in ear florets in PAC treatments, which were observed in all maize GA mutants, suggested that PAC might simply be a weaker GA biosynthesis inhibitor.

The combined effects of loss of na2 and any of the inhibitor treatments on plant height resulted in extreme dwarfism. The ability of PCZ to further inhibit the growth of na2-1 mutants, and to enhance the POPIT phenotype of na2-1 plants, is consistent with residual BR activity in na2-1. The presence of BR activity in mutants of the rice ortholog of na2-1 (Hong et al., 2005 ) is consistent with pathway non-linearity and accumulation of BR intermediates with limited biological activity in single mutants. The height reduction in na2-1 treated with PCZ (7.1 cm Table 1) was indistinguishable from na2-1 treated with UCZ (6.5 cm) and both of these were significantly shorter than na2-1 treated with PAC (9.7 cm). In previous studies of Arabidopsis seedlings, GA biosynthesis was under the control of BR signaling where BR signaling promoted GA biosynthesis (Tong et al., 2014 Unterholzner et al., 2015 ). Our previous study of the interactions between the na2 and d5 mutants on floral organ retention in maize suggested that the opposite of this might be true in maize tassel florets where GA synthesis was downstream of a loss of BR (Figure 7). The finding that PAC was less effective than UCZ at suppressing both plant height and POPIT in the na2-1 BR-deficient background suggested that a complete inhibition of GA biosynthesis is required to recapitulate the genetic interactions with inhibitor treatments. The failure of UCZ and PAC to affect elongation beyond the effect of PCZ suggests that there is little to no compensation for the loss of BR by increased GA biosynthesis in maize stems. Measurements of BR and GA levels and of the expression of signaling outputs such as DELLA-dependent and BZR1/BES1-dependent gene expression in the mutants and inhibitor treatments are required to clarify the relationships between these hormones and resolve the apparent differences between tissues within maize and the difference between maize tassels and Arabidopsis seedlings. The rice GRAS-domain transcription factor DWARF AND LOW-TILLERING (DLT) affects both BR and GA (Li et al., 2010 Tong et al., 2009 , 2012 ), resulting in a suppression of tiller formation much like the na2 mutants of maize. We expect, based on the phenotypes in rice and Arabidopsis, that double mutants between na2-1 and loss-of-function alleles at the maize DLT ortholog (AC234164.1_FG004) will exhibit increased POPIT due to an increase in GA biosynthesis and that double mutants between d5 and loss-of-function alleles at the maize DLT ortholog will suppress tiller outgrowth affected by loss of GA biosynthesis in d5.

The number of nodes was decreased in na2-1 treated with PAC and UCZ however, d5 treated with PCZ showed no effect on total nodes (Figure 8a). The discordance between these may stem from the incomplete inhibition of BR biosynthesis by PCZ. If so, the loss of both BR and GA results in a synergistic reduction of total nodes per plant (Figure 8a). Days to tassel emergence was significantly longer for na2-1 and d5 (Tables 1 and 3) compared to their wild-type siblings, similar to our earlier work (Best, Hartwig et al., 2016 ). The tassels of wild-type siblings treated with PCZ, PAC, or UCZ emerged from the leaf whorl later than mock-treated wild-type controls. Every mutant–triazole combination further increased days to tassel emergence, and this difference was always statistically significant. The delay in tassel emergence in triazole-treated na2-1 mutants was associated with the production of fewer nodes per plant despite the 2–3 extra weeks of growth before tassel emergence. This difference in node number itself was significantly different than na2-1 mock-treated controls for only the PAC treatment, but the delay in flower emergence without additional node production suggests the plastochron depends on BR and GA in maize. The plastochron is the time it takes to initiate each lateral organ from the meristem (Erickson & Michelini, 1957 Meicenheimer, 2014 ). We quantified total number of nodes and time to tassel emergence, so we may overestimate the effect of these treatments on flowering time as decreased elongation should delay emergence of the tassel. However, the average increase in days to tassel emergence for na2-1 or d5 treated with any inhibitor was 19.0 days and 13.4 days, respectively (Tables 1 and 3). Furthermore, PAC and UCZ treatment of na2-1 mutants caused the top ear to reside on a lower node (Table 1) thus, the developmental stage of these plants was different from controls even when observed at the same number of nodes. This could result from BR and GA effects on developmental rate and flowering time or inhibition of another cytochrome P450 by these triazoles. Overexpression of the maize plastochron1 gene, which encodes cytochrome P450 CYP78A1, increased leaf number but decreased plastochron (Sun et al., 2017 ). Loss-of-function CYP78A1 mutants exhibited slower leaf elongation, but interpretation is limited by the presence of multiple CYP78A paralogs in the maize genome. An inhibitor of all CYP78A paralogs would circumvent the need to isolate loss-of-function mutants in all paralogs. As yet, we do not know the phenotype of low CYP78A activity on maize plastochron or the identity of the bioactive metabolite affected by CYP78A enzymes in plants.

We measured traits in this study that were not previously assessed in na2-1/d5 double mutants. Thus, we cannot use these observations as a test of a genetic model. Rather, we hypothesize the BR–GA interactions, or lack thereof, based on this inhibitor study to be tested in future genetic experiments. No interaction between BR and GA for length of the upper and lower leaves was evident. na2-1 and treatment with PAC or UCZ additively reduced both traits (Figure 8b and c). A similar additive effect was observed for d5 treated with PCZ (Figure 8b and c). Leaf width did not consistently respond in mutants, treatments, and their combination (Figure 8b and c). The width of the upper leaf was not significantly different for na2-1 and d5 as compared to their respective wild types. Mutants of na2-1 treated with either GA inhibitor and d5 mutants treated with PCZ had narrower upper leaves than mock-treated mutants, but only the UCZ-treated na2-1 was significantly different from mock-treated mutants (Tables 1 and 3). Lower leaf width was not consistently affected by na2-1 or PCZ (Table 1). The width of the lower leaf of d5 mutants and PAC- and UCZ-treated wild-type siblings were significantly wider than mock-treated wild-type plants (Table 3). Taken together, this suggests that loss of GA widened the lower, but not upper, leaf and that loss of BR and GA synergistically resulted in narrower upper leaves (Figure 8c). Similar to the effect on leaf widths, there were no consistent effects of BR and GA on upper leaf angle (Tables 1 and 3). Lower leaves were more upright in both na2-1 and d5 than wild-type siblings. Treatment of d5 with PCZ and na2-1 with PAC or UCZ did not make the leaves more upright. Therefore, we propose that BR and GA act on the same pathway to increase lower leaf inclination (Figure 8c). Like plant height, tassel length was additively reduced by a loss of BR and GA (Figure 8d) in comparisons between mock-treated mutant controls and na2-1 treated with PAC and UCZ or d5 treated with PCZ (Tables 1 and 3).

Another finding of our experiments was an effect of GA biosynthesis on tassel branch numbers, particularly in the na2-1 background. The d5 mutants had fewer primary tassel branches than wild-type siblings, but na2-1 mutant primary tassel branch numbers were unaffected in both the results of this study (Tables 1 and 3) and our previous work (Best, Hartwig et al., 2016 ). Treatment of wild-type plants with PAC or UCZ also reduced primary tassel branch numbers (Tables 1 and 3) consistent with GA promotion of tassel branching (Figure 8d). All three triazoles severely reduced branching in na2-1 and d5 mutants (Tables 1 and 3). The wild-type siblings of d5 mutants, but not the wild-type siblings of na2-1, exhibited fewer primary tassel branches following PCZ treatment. That na2-1 mutants did not have fewer branches than wild type in mock treatments may result from these genetic backgrounds differentially responding to BR reduction. As was the case for POPIT (Table 1), the treatment of na2-1 mutants with PCZ did reduce tassel branch numbers (Table 1). Thus, we propose that GA and BR additively promote tassel branching (Figure 8d), although a synergistic interaction or off-target effects of the inhibitors are possible. Residual BR biosynthetic activity in all na2 alleles is suggested by the stronger phenotype of brd1 mutants and na1/na2 double mutants (Dilkes and Best, data not shown) and it may be that the residual BR are sufficient to maintain normal tassel branching in na2-1 mutants (Makarevitch et al., 2012 ). Alternatively, PCZ might have effects on regulators of tassel branching encoded by P450s outside of BR biosynthesis. In our previous work, exogenous application of GA to na2-1, d5, and wild-type growing apices did not alter tassel branch numbers (Best, Hartwig et al., 2016 ). Additional work determining the physiological basis of triazole application on tassel branching may uncover as yet unknown impacts of GA on tassel architecture, or implicate triazoles in the inhibition of other branch-promoting signals.

We repeatedly treated plants with PGRs, and this continuous treatment was required to observe the dramatic decrease in plant height. This requirement suggests that maize can catabolize or inactivate these PGRs over the course of a few days. Reduction in the concentrations of bioactive PGRs should permit the biosynthesis of phytohormones to return and promote growth in responsive cells. We ceased treatment at tassel emergence during the elongation of the uppermost internodes, and the lengths of these internodes were not as affected in our treated plants (Table 2 and Figure 3). We propose that PGR inactivation rather than phytohormone-independent growth underlies these differences in internode responses.

The ability to combine chemical and genetic inhibition of phytohormone biosynthesis to assess interactions and combinatorial effects is limited only by resource availability. The discovery of compounds with additional modes of action would enable work on the effects of all phytohormones on plant development. An additional triazole was identified by Ito et al. ( 2010 ) that inhibits SL biosynthesis. This inhibitor, TIS13, effectively reduced SL levels in roots and root exudates of rice and increased tiller outgrowth of the second tiller. In these tests, PAC and UCZ had no effect on SL levels in root or root exudates, eliminating off-target inhibition of SL biosynthesis as a possible side effect of PAC and UCZ treatments in rice. SL control tiller outgrowth in maize (Chou Guan et al., 2012 ). If SL also affects tassel branching, we would expect treatment of maize with TIS13 or GR24, a synthetic SL, to cause a decrease or increase in primary tassel branching, respectively. It may be that loss of GA affects tillering and reduced tassel branching via SL accumulation or signaling. A similar study to this one, using TIS3 application to GA and BR biosynthetic mutants of maize as well as UCZ, PCZ, and PAC treatment of the carotenoid cleavage dioxygenase8 mutant of maize, should provide a test of this hypothesis (Chou Guan et al., 2012 ). Similarly, identification of specific inhibitors of cytochrome P450s in other metabolic pathways would permit combinatorial assessment with available mutants to explore metabolite function in maize.

In conclusion, PCZ treatment of wild-type siblings largely phenocopied na2-1 mutants and PAC or UCZ treatment predominantly phenocopied d5 mutants (Figure 7). PAC did not affect sexual organ persistence, unlike GA biosynthetic mutants or UCZ treatment. There was no strong evidence of non-specific growth inhibition by these three triazole inhibitors in maize. Unexpectedly, primary tassel branch number was reduced in na2-1 or d5 treated with PCZ, PAC, or UCZ. Thus, either all three triazoles affect another pathway controlling tassel branching or BR and GA biosynthetic pathways interact to affect tassel branching. Days to tassel emergence was synergistically increased by PCZ, PAC, or UCZ treatment of both na2-1 and d5. The mechanism for this is unknown, and we did not measure flowering time or days to tassel emergence in na2-1/d5 double mutants in our previous study (Best, Hartwig et al., 2016 ). The similarity of the effects on plant height, tiller outgrowth, and floral organ persistence in the mutant analyses and inhibitor treatments was consistent with the genetic model proposed previously (Best, Hartwig et al., 2016 ). The concordance of phenotypes increased the confidence of interpretation for UCZ and PCZ as GA and BR inhibitors, respectively. Using these inhibitors in the greenhouse via simple repeated pot drench will allow for rapid assessment of phytohormone function, even in the absence of genetic resources. Confidence in the interpretation of phenotypes resulting from inhibitor application should encourage exploration of BR and GA effects on growth and development in grass species such as Sorghum bicolor, Panicum virgatum, Saccharum officinarum, Miscanthus sinensis, and Setaria viridis.

The endocrine system relies on hormones to elicit responses from target cells. These hormones are synthesized in specialized glands at a distance from their target, and travel through the bloodstream or inter-cellular fluid. Upon reaching their target, hormones can induce cellular responses at a protein or genetic level.

This process takes significantly longer than that of the nervous system, as endocrine hormones must first be synthesized, transported to their target cell, and enter or signal the cell. However, although hormones act more slowly than a nervous impulse, their effects are typically longer lasting.

Additionally, the target cells can respond to minute quantities of hormones and are sensitive to subtle changes in hormone concentration. For example, the growth hormones secreted by the pituitary gland are responsible for sustained growth during childhood.


Light meter


-Carbon dioxide

-Osmosis occurs in liquid medium only while Diffusion occurs in gases and liquids
-Osmosis occurs naturally in living organisms while diffusion occurs in living and non-living organisms

–There is absence of scent
–Flowers are usually dull coloured

It produces an oil called sebum that keeps skin moist

(i) Xerophyte:
-Possession of deep root structures
-Possession of thin or small leaves and waxy surfaces to retain moisture

(ii) Hydrophyte:
-Possession of large flat leaves on surface plants for floatation
-Possession of thin cuticle


-It is used for detection of vibration and pressure in water

-Shortage of food

-Back pain
-Dark urine

(a) Onion bulb: Bulb
(b) Cassava: Cutting

A – Queen termite
B – Soldier termite

I – Compound eye
II – Spiracle
III – Abdomen
IV – Hardened cuticle
V – Antenna
VI – Mandible

Under A:
-Large abdomen
-Small head
-Patches of hardened cuticle present
-Absence of Mandible

Under B (Soldier)
-Small abdomen
-Large head
-Patches of hardened cuticle absent
-Present of powerful mandible

Phylum: Arthropoda
Class: Insecta

-Body covered with exoskeleton made of chitin
-Three pairs of jointed legs
-Abdomen is segmented
-Presence of spiracles on abdomen

-It helps in aeration of the soil by burrowing inside the soil
-It is a good source of protein

Incomplete metamorphosis


The Human Eye (vertical section)

I – Sclera
II – Rectus muscle
III – Retina
IV – Yellow sport
V – Optic nerve
VI – Blind spot
VII – Ciliary body
VIII – Iris
IX – Lens
X – Conjuctiva

II – It controls eye movement
III – It has light-sensitive cells called photoreceptors, which transforms these into light rays into electrical impulses.
(V) It transmits impulse to the brain
(VII) It holds lens in place
(IX) It helps to refracts and focuses light rays that enters the eye

(i) Bright light: Muscles of the iris contract, making pupil small and reduce the amount of light entering the eye.
(ii) Dim light: It tends to expand in dim light

(i) Short–sightedness (myopia)
(ii) Long–sightedness (hypermetropia)

(i) Short sightedness is corrected by wearing spectacles fitted with concave or diverging lenses
(ii) Long sightedness is corrected by using spectacles fitted with suitable convex or converging lenses

(i) Use clean water to wash face always
(ii) Avoid staying in areas containing fumes or smoke that can irritate eye

(i) Root
(ii) Bulb
(iii) Leaves
(iv) Tuber

(i) Root
(ii) Leaves
(iii) Stem
(iv) Stem

(i) Adventitious Root
(ii) Stem
(iii) leaves

I – Stem cutting
II – Bulb
III – Rhizomes

Anaerobic respiration in yeast

Incase of overflow of the reaction

10 degree C – 40 degree C

Because the rate of reaction has decreased as a result of bridge in minimun and optimum temperature

(i) Parents: Black coat colors
(ii) Offspring: 3 black and 1 white

PLS NOTE: Draw circles (Here, my 0 is none shaded circe while @ is a shaded circle, so draw in your book, dont write this instruction ooo):
Parent – 00 00
Offspring – 00 [email protected]

(i) The genotype of the gamates are Bb and Bb
(ii) The genotypes of the offspring are BB, Bb, Bb and bb

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15.1B: Metabolism - Biology

Facts about mercury and Dental Amalgam with Medical Study Reverences

Toxic metals such as mercury, lead, cadmium, etc. have been documented to be neurotoxic, immunotoxic, reproductive/developmental toxins that according to U.S. Government agencies cause adverse health effects and learning disabilities to millions in the U.S. each year, especially children and the elderly(105,160). Exposure of humans and animals to toxic metals such as mercury, cadmium, lead, copper, aluminum, arsenic, chromium, manganese, etc. is widespread and in many areas increasing. . The U.S. Center for Disease Control(276) ranks toxic metals as the number one environmental health threat to children. According to an EPA/ATSDR assessment, the toxic metals mercury, lead, arsenic, and cadmium are all ranked in the top 7 toxics having the most adverse health effects on the public based on toxicity and current exposure levels in the U.S., with nickel and chromium also highly listed. While there are large numbers of neurological and immune conditions among adults, the incidence of neurotoxic or immune reactive conditions in infants such as autism, schizophrenia, ADD, dyslexia, learning disabilities, etc. have been increasing especially rapidly in recent years(2,276,409,441). A recent report by the National Research Council found that 50% of all pregnancies in the U.S. are now resulting in prenatal or postnatal mortality, significant birth defects, or otherwise chronically unhealthy babies(441). Exposure to toxic chemicals or environmental factors appear to be a factor in as much as 28 percent of the 4 million children born each year(441), with 1 in 6 having one of the neurological conditions previously listed. EPA estimates that over 3 million of these are related to lead or mercury toxicity(2,276,409).

While there is considerable commonality to the health effects commonly caused by these toxic metals, and effects are cumulative and synergistic in many cases, this paper will concentrate on the health effects of elemental mercury from amalgam fillings. Studies have found considerable genetic variability in susceptibility to toxic metals as well. The public appears to be generally unaware that considerable scientific evidence supports that mercury is the metal causing the most widespread adverse health effects to the public, and amalgam fillings have been well documented to be the number one source of exposure of mercury to most people, with exposure levels often exceeding Government health guidelines and levels documented to cause adverse health effects.

II. Toxicity and Health Effects of Mercury
1. Dental amalgam contains about 50 % mercury, as well as other toxic metals such as tin,copper,nickel, palladium, etc. The average filling has 1 gram of mercury and leaks mercury vapor continuously due to mercury's low vapor pressure along with loss due to galvanic action of mercury with dissimilar metals in the mouth (182,192,292,348,349), resulting in significant exposure for most with amalgam fillings(see Section III). Mercury vapor is transmitted rapidly throughout the body, easily crosses cell membranes, and like organic methyl mercury has significant toxic effects at much lower levels of exposure than other inorganic mercury forms(38,281,287,304,329). According to the U.S. EPA & ATSDR, mercury is among the top 3 toxic substances adversely affecting large numbers of people(217), and amalgam is the number one source of exposure for most people(see III).

2. Mercury is the most toxic of the toxic metals. Mercury (vapor) is carried by the blood to cells in all organs of the body where it:
(a) is cytotoxic(kills cells) (2,21,27,36,56,147,148,150,160,210,259,295,333/333)
(b) penetrates and damages the blood brain barrier(311), resulting in accumulation of mercury and other toxic substances in the brain(14,20,25,85, 99,175,273,301/262,274) also accumulates in the motor function
areas of the brain and CNS(48,291,327,329).
© is neurotoxic(kills brain and nerve cells): damages brain cells and nerve cells (19,27,34,36,43,69,70,147, 148,175,207,211,273, 291,295,327,329,301,303,395/39,262,274,303) generates high levels of reactive oxygen species(ROS) and oxidative stress, depletes glutathione and thiols causing increased neurotoxicity from interactions of ROS, glutamate, and dopamine(13,56,98,102,126,145,169,170,184,213,219,250,257, 259,286,290,291,302,324,326,329,424,442) kills or inhibits production of brain tubulin cells (66,67,161,166, 207,300) inhibits production of neurotransmitters by inhibiting: calcium-dependent neurotransmitter release(372,432), dihydroteridine reductase(27,122,257,333), nitric oxide synthase(259), blocking neurotransmitter amino acids(412), and effecting phenylalanine, serotonin, tyrosine and tryptophan transport to neurons (34,122,126,257,285, 288,333,372,374,412/255,333)
(d) is immunotoxic(damages and inhibits immune T-cells, B-cells, neutrophil function, etc.) (17,27,31,38,44,45,46,60,127,128,129,130,152,155,165,181,226,252,270,285,316,355/272) and induces ANA antibodies and autoimmune disease(38,43,45,59,60,118,131,181,234,269,270,313,314,334,342,343,35)
(e) is nephrotoxic(toxic to kidneys) (14,20,203,223,254,260,268,334,438)
(f) is endocrine system-disrupting chemical(accumulates in pituitary gland and damages or inhibits pituitary glands hormonal functions at very low levels(9,19,20,25,85,99,105,273,312,327,348,369/274), adrenal gland function(84,369,381), thyroid gland function(50,212,369,382,459,35), and disrupts enzyme production processes at very low levels of exposure (9,13,33,35,56,111,194,348,355,410-412)
(g) exposure to mercury vapor (or methyl mercury) causes rapid transmittal through the placenta to the fetus (20,22-24,27,38,39,61,112,186,281,287,304,311,338,339,348,361,366,20/4,22,37,39,41,42) and significant developmental effects-much more damage to the fetus than for maternal exposure to inorganic mercury and at lower exposure levels than for for organic mercury(287,304,etc.).
(h) reproductive and developmental toxin (2,4,9,10,22,23,24,31,37,38,41,61,105,149,160,275,276,281,305, 338,361,367,381,20/4,39,55,149,162,255,308,339,357) damages DNA(296,327,272,392,142,38,41,42,35) and inhibits DNA & RNA synthesis(114,35/149) damages sperm, lowers sperm counts and reduces motility. (4,37,104.105,159,160,35/4,55,162) causes menstrual disturbances (9,27,146) reduces bloods ability to transport oxygen to fetus and transport of essential nutrients including amino acids, glucose, magnesium, zinc and Vit B12(43,96,198,263,264,338,339,347,427) depresses enzyme isocitric dehydrogenase (ICD) in fetus, causes reduced iodine uptake & hypothyroidism(50,91,212,222,369,382,459, 35) & learning deficits causes learning disabilities and impairment, and reduction in IQ (1,3,38,110,160,285c,263,264/39), causes infertility (4,9,10,24,38,121,146,357,365,367/4,10,55,162), causes birth defects (23,35,37,38,110,142,241,338c/241).
(i) prenatal/early postnatal exposure affects level of nerve growth factor in the brain,impairs astrocyte function, and causes imbalances in development of brain(38,119,131,161,175,194,305,458/175,255,39)
(j) causes cardiovascular damage and disease: including damage to vascular endothelial cells, damage to sarcoplasmic reticula, sarcolemma, and contractile proteins, increased white cell count, decreased oxyhemoglobin level, high blood pressure, tachycardia, inhibits cytochrome P450/heme synthesis(84,35), and increased risk of acute myocardial infarction (35,59,202,205,212,232,306,310,351/201,308).
(k) causes immune system damage resulting in allergies, asthma, lupus,chronic fatigue syndrome(CFS),and multiple sensitivities(MCS) (8,17,26,35,45,46,52,60,75,86,87,90,95,97,101,128,129,131,132,154,156,168, 181,212, 226, 228,230,234,265,267,296,313,342,375,388,445,446/272) and neutrophil functional impairment(285,404/59,etc.).
(l) causes interruption of the cytochromeC oxidase system/ATP energy function(43,84,232,338c,35) and blocks enzymes needed to convert porphyrins to adenosine tri phosphate(ATP) causing progressive porphyrinuria, resulting in low energy, digestive problems, and porphyrins in urine (34,35,69,70,73,210,212,226,232,260)
(m) inhibition of immune system facilitates increased damage by bacterial, viral, and fungal infections (17,45,59,129,131,251,296,350,40), and increased antibiotic resistance(116,117,161,258,389,53).
(n) mercury causes significant destruction of stomach and intestine epithelial cells, resulting in damage to stomach lining which along with mercury's ability to bind to SH hydroxyl radical in cell membranes alters permeability(338,35) and adversely alters bacterial populations in the intestines causing leaky gut syndrome with toxic, incompletely digested complexes in the blood(222,228b,35) and accumulation of heliobacter pylori, a suspected major factor in stomach ulcers and stomach cancer(256) and candida albicans, as well as poor nutrient absorption.
(o) forming strong bonds with and modification of the-SH groups of proteins causes mitochondrial release of calcium (1,21,35,38,43,329,333,432),as well as altering molecular function of amino acids and damaging enzymatic process(33,96,111,194,252,338,410-412) resulting in improper cysteine regulation(194), inhibited glucose transfer(338,254), damaged sulfur oxidation processes(33,338), and reduced glutathione availability (necessary for detoxification)(13,126,54).

3. Mercury has been well documented to be an endocrine system disrupting chemical in animals and people, disrupting function of the pituitary gland, thyroid gland, enzyme production processes, and many hormonal functions at very low levels of exposure . Mercury (especially mercury vapor) rapidly crosses the blood brain barrier and is stored preferentially in the pituitary gland, hypothalamus, and occipital cortex in direct proportion to the number and extent of dental amalgam surfaces (1,14,16,19,20,25,34,38,61,85,99,162,211,273,274,287, 327,348,360,366,369) Thus mercury has a greater effect on the functions of these areas. The pituitary gland controls many of the body's endocrine system functions and secretes hormones that control most bodily processes, including the immune system and reproductive systems . One study found mercury levels in the pituitary gland ranged from 6.3 to 77 ppb(85), while another(348) found the mean level to be 30ppb- levels found to be neurotoxic and cytotoxic in animal studies.
Mercury blocks thyroid hormone production by occupying iodine binding sites and inhibiting hormone action even when the measured thyroid level appears to be in proper range(35). The thyroid and hypothalamus regulate body temperature and many metabolic processes including enzymatic processes that when inhibited result in higher dental decay(35) . Mercury damage thus commonly results in poor bodily temperature control, in addition to many problems caused by hormonal imbalances such as depression. Such hormonal secretions are affected at levels of mercury exposure much lower than the acute toxicity effects normally tested, as previously confirmed by hormonal/reproductive problems in animal populations(104,381). Mercury also damages the blood brain barrier and facilitates penetration of the brain by other toxic metals and substances(311).

4. Mercury's biochemical damage at the cellular level include DNA damage, inhibition of DNA and RNA synthesis(4,38,41,42,114,142,197,272,296,392/149) alteration of protein structure(33,111,114,194,252/114) alteration of the transport of calcium(333,43,96,254,329,432) inhibitation of glucose transport(338,254), and of enzyme function and other essential nutrients(96,198,254,263,264,338,339,347,410-412) induction of free radical formation(13,54), depletion of cellular gluthathione(necessary for detoxification processes) (111,126), inhibition of glutathione peroxidase enzyme(13), endothelial cell damage(202), abnormal migration of neurons in the cerebral cortex(149), and immune system damage (34,38,111,194, 226,252,272,316,325,355). Oxidative stress and reactive oxygen species(ROS) have been implicated as major factors in neurological disorders including stroke, PD, Alzheimer's, ALS, etc.(13,56,84,98,145,169,207b,424,442,453). Mercury induced lipid peroxidation has been found to be a major factor in mercury's neurotoxicity, along with leading to decreased levels of glutathione peroxidation and superoxide dismustase(SOD)(13). Only a few micrograms of mercury severely disturb cellular function and inhibit nerve growth(175,147,175,226,255,305). Exposure to mercury results in metalloprotein compounds that have genetic effects, having both structural and catalytic effects on gene expression(114,241,296). Some of the processes affected by such metalloprotein control of genes include cellular respiration, metabolism, enzymatic processes, metal-specific homeostasis, and adrenal stress response systems. Significant psysiological changes occur when metal ion concentrations exceed threshold levels. Such metalloprotein formation also appears to have a relation to autoimmune reactions in significant numbers of people(114,60,313,342,368,369). Of a population of over 3000 tested by the immune lymphocyte reactivity test(MELISA,60,275), 22% tested positive for inorganic mercury and 8% for methyl mercury .

A direct mechinism involving mercury's inhibition of cellular enzymatic processes by binding with the hydroxyl radical(SH) in amino acids appears to be a major part of the connection to allergic/immune reactive conditions such as autism(408-414,439,33,160), schizophrenia(409,410), lupus(113,234,330,331), eczema and psoriasis(323,375,385,419,455,33), and allergies(26,46,60,95,132,152,156,271,313,330,331,445,446). For example mercury has been found to strongly inhibit the activity of dipeptyl peptidase (DPP IV) which is required in the digestion of the milk protein cassein(411,412) as well as of xanthine oxidase(439). Studies involving a large sample of autistic and schizophrenic patients found that over 90 % of those tested had high levels of the milk protein beta-casomorphin-7 in their blood and urine and defective enzymatic processes for digesting milk protein(410). Elimination of milk products from the diet has been found to improve the condition. Such populations have also been found to have high levels of mercury and to recover after mercury detox(413,60,313). As mercury levels are reduced the protein binding is reduced and improvement in the enzymatic process occurs. Additional cellular level enzymatic effects of mercury's binding with proteins include blockage of sulfur oxidation processes(33,114,412), enzymatic processes involving vitimins B6 and B12(418), effects on the cytochrome-C energy processes (43,84,232,338c,35), along with mercury's adverse effects on cellular mineral levels of calcium, magnesium, zinc, and lithium(43,96,119,198,333,386,427,432, 38). And along with these blockages of cellular enzymatic processes, mercury has been found to cause additional neurological and immune system effects in many through immune/autoimmune reactions (60,313,314).

But the effect on the immune system of exposure to various toxic substances such as toxic metals and environmental pollutants has also been found to have additive or synergistic effects and to be a factor in increasing eczema, allergies, asthma, and sensitivity to other lesser allergens. Most of the children tested for toxic exposures have found high or reactive levels of other toxic metals, and organochlorine compounds(413,313,415). Much mercury in saliva and the brain is also organic (220,272), since mouth bacteria and other organisms in the body methylate inorganic mercury to organic mercury(51, 81,225). Bacteria also oxidize mercury vapor to the water soluble, ionic form Hg(II) (431).

5. Because of the extreme toxicity of mercury, only ½ gram is required to contaminate a 10 acre lake to the extent that a health warning would be issued by the government to not eat the fish(151,160). Over half the rivers and lakes in Florida have such health warnings(160). Some Florida panthers that eat birds and animals that eat fish containing very low levels of mercury(about 1 part per million) have died from chronic mercury poisoning(104,160). Since mercury is an estrogenic chemical and reproductive toxin, the majority of the rest cannot reproduce. The average male Florida panther has higher estrogen levels than females, due to the estrogenic properties of mercury(105,160). Similar is true of some other animals at the top of the food chain like alligators, which are affected by mercury and other hormone disrupting chemicals..

6. Mercury accumulates in the pituitary glands, ovaries, testes, and prostrate gland(35,99,9 19,20,25,85,273).
In addition to having estrogenic effects, mercury has other documented hormonal effects including effects on the reproductive system resulting in lowered sperm counts, defective sperm cells, damaged DNA, aberrant chromasome numbers rather than the normal 46, chromasome breaks, and lowered testosterone levels in males menstrual disturbances and infertility in women and increased neurological problems related to lowered levels of neurotransmitters dopamine, serotonin, and noreprenephrine (4,9,35,38,104,105,107,140,141,275,276,288, 290,296,365,367,372,381,432,412). Some of the effect on depression is related to mercury's effect of reducing the level of posterior pituitary hormone(oxytocin). The pituitary glands of a group of dentists had 800 times more mercury than controls(99). This may explain why dentists have much higher levels of emotional problems, depression, suicide,etc(Section VIII.). Low levels of pituitary function are associated with depression and suicidal thoughts, and appear to be a major factor in suicide of teenagers and other vulnerable groups. Amalgam fillings, nickel and gold crowns are major factors in reducing pituitary function(35,50,369,etc.). Supplentary oxytocin extract has been found to alleviate many of these mood problems(35), along with replacement of metals in the mouth(Section VI.). The normalization of pituitary function also often normalizes menstrual cycle problems, endometriosis, and increases fertility(35,9).

7. An average amalgam filling contains over ½ gram of mercury, and the average adult had at least 5 grams of mercury in fillings(unless most has vaporized). Mercury in solid form is not stable, having low vapor pressure and being subject to galvanic action with other metals in an oral environment(182,192,292,348,349),so that within 10 years up to half has been found to have been transferred to the body of the host(34,35,182, & section III).

8. Elemental mercury vapor is more rapidly transmitted throughout the body than most other forms of mercury and has more much toxic effects on the CNS and other parts of the body than inorganic mercury due to its much greater capacity to cross cell membranes, according to the World Health Organization and other studies (38,183, 282,287,360,section III). Mercury vapor rapidly crosses the blood-brain barrier(14,85,311) and placenta of pregnant women (20,22-24,27,38,105,162,186,231,281,287,304,308, 311,361) Developmental, learning, and behavioral effects have been found from mercury vapor at much lower levels than for exposure to methyl mercury(287,304). Similarly for inhibition of some essential cellular processes(333,338,329).

9. Running shoes with ½ gram of mercury in the heels were banned by several states, because the amount of mercury was considered dangerous to public health and created a serious disposal problem. Mercury from dental offices and human waste from people with amalgam fillings has much higher levels and is a major source of mercury in Florida waters. One study found dental offices discharge into waste water between 65 and 842 milligrams per dentist per day(231), amounting to several hundred grams per year per office. This is in addition to air emissions. Additionally cremation of those with amalgam fillings adds to air emissions and deposition onto land and lakes. A study in Switzerland found that in that small country, cremation released over 65 kilograms of mercury per year as emissions, often exceeding site air mercury standards(420), while another Swiss study found mercury levels during cremation of a person with amalgam fillings as high as 200 micrograms per cubic meter(considerably higher than U.S. mercury standards). The amount of mercury in the mouth of a person with fillings was on average 2.5 grams, enough to contaminate 5 ten acre lakes to the extent there would be dangerous levels in fish(151). A Japanese study estimated mercury emissions from a small crematorium there as 26 grams per day(421). A study in Sweden found significant occupational and environmental exposures at cremetoria, and since the requirement to install selenium filters mercury emission levels in crematoria have been reduced 85%(422).

10. Studies have found that levels of exposure to the toxic metals mercury, cadmium, and lead have major effects on classroom behavior, learning ability, and also in mental patients and criminals behavior(3,160).
Studies have found that both genetic susceptability and environmental exposures are a factor in xenobiotic related effects and disease propagation. Large numbers of animal studies have documented that genetically susceptable strains are more affected by xenobiotic exposures than less susceptable strains (234,336,425,526,etc.). Some genetic types are susceptable to mercury induced autoimmunity and some are resistant and thus much less affected(234,336,425,383). Studies found that mercury causes or accelerates various systemic conditions in a strain dependent manner, and that lower levels of exposure adversely affect some strains but not others, including inducing of autoimmunity. Also when a condition has been initiated and exposure levels decline, autoimmune antibodies also decline in animals or humans(233,234c,60,368,405). One genetic factor in Hg induced autoimmunity is major histocompatibility complex(MHC) linked. Both immune cell type Th1 and Th2 cytokine responses are involved in autoimmunity(425c). One genetic difference found in animals and humans is cellular retention differences for metals related to the ability to excrete mercury(426). For example it has been found that individuals with genetic blood factor type APOE-4 do not excrete mercury readily and bioaccumulate mercury, resulting in susceptability to chronic autoimmune conditions such as Alzheimer's, Parkinsons, etc. as early as age 40, whereas those with type APOE-2 readily excrete mercury and are less susceptable. Those with type APOE-3 are intermediate to the other 2 types(437,35). The incidence of autoimmune condtions have increased to the extent this is now one of the leading causes of death among women(450).

11. Long term occupational exposure to low levels of mercury can induce slight cognitive deficits, lability, fatigue, decreased stress tolerance, etc. Higher levels have been found to cause more serious neurological problems (119,128,160,285,457,etc.). Occupational exposure studies have found mercury impairs the body's ability to kill Candida albicans by impairment of the lytic activity of neutrophils and myeloperoxidase in workers whose mercury excretion levels are withing current safety limits(285,404). Such levels of mercury exposure were also found to inhibit cellular respiratory burst. A population of plant workers with average mercury excretion of 20 ug/ g creatinine was found to have long lasting impairment of neutrophil function(285,404). Another study(59) found such impairment of neutraphils decreases the body's ability to combat viruses such as those that cause heart damage, resulting in more inflamatory damage. Another group of workers with average excretion rates of 24.7 ug/ g creatinine had long lasting increases in humoral immunological stimulation of IgG, IgA, and IgM levels. Other studies(285b,g,395) found that workers exposed at high levels at least 20 years previous(urine peak levels above 600 ug/L demonstrated significantly decreased strength, decreased coordination, increased tremor, decreased sensation, polyneuropathy, etc. Significant correlations between increasing urine mercury concentrations and prolonged motor and sensory distal latencies were established(285g). Elemental mercury can affect both motor and sensory peripheral nerve conduction and the degree of involvement is related to time-integrated urine mercury concentrations. Thirty percent of dentists with more than average exposure were found to have neuropathies and visuogrphic dysfunction(395).
Another study found that many of the symptoms and signs of chronic candidiasis, multiple chemical sensitivity and chronic fatigue syndromes are identical to those of chronic mercurialism and remit after removal of amalgam combined with appropriate supplementation and gave evidence to implicate amalgam as the only underlying etiologic factor that is common to all(404).

Other studies(285c) found that mercury at levels below the current occupational safety limit causes adverse effects on mood, personality, and memory- with effects on memory at very low exposure levels.
More studies found that long term exposure causes increased micronuclei in lymphocytes and significantly increased IgE levels at exposures below current safety levels(128), as well as maternal exposure being linked to mental retardation(110) and birth defects(23,35,37,38,142,241,361,338c/241).

III. Systemic Mercury Intake Level from Amalgam Fillings

1. The tolerable daily exposure level for mercury developed in a report for Health Canada is .014 micrograms/kilogram body weight(ug/kg) or approximately 1 ug/day for average adult(217). The U.S. EPA Health Standard for elemental mercury exposure(vapor) is 0.3 micrograms per cubic meter of air(2). The U.S. ATSDR health standard(MRL) for mercury vapor is 0.2 ug/ M3 of air, and the MRL for methyl mercury is 0.3 ug/kg body weight/day(217). For the average adult breathing 20 M3 of air per day, this amounts to an exposure of 4 or 6 ug/day for the 2 elemental mercury standards. The EPA health guideline for methyl mercury is 0.1 ug/kg body weight per day or 7 ug for the average adult(2), or approx. 14 ug for the ATSDR acute oral toxicicity standard. Since mercury is methylized in the body, some of both types are present in the body. The older World Health Organization(183) mercury health guideline(PTWI) is 300 ug per week total exposure or approx. 42 ug/day.

2. Mercury in the presence of other metals in the oral environment undergoes galvanic action, causing movement out of amalgam and into the oral mucosa and saliva(174,192,436,179,199). Mercury in solid form is not stable due to low vapor pressure and evaporates continuously from amalgam fillings in the mouth, being transferred over a period of time to the host(15-19,26,31,36,79,83,211,182,183,199,298,299,303,332,335,371). The daily total exposure of mercury from fillings is from 3 to 1000 micrograms per day, with the average exposure being above 10 micrograms per day and the average uptake over 5 ug/day (183,199,209,18,19,77,83, 85,100,335,352,371,etc.). (see further details continued)

A large study was carried out at the Univ. Of Tubingen Health Clinic in which the level of mercury in saliva of 20,000 persons with amalgam fillings was measured(199). The level of mercury in unstimulated saliva was found to average 11.6 ug Hg/L, with the average after chewing being 3 times this level. Several were found to have mercury levels over 1100 ug/L, 1 % had unstimulated levels over 200 ug/L, and 10 % had unstimulated mercury saliva levels of over 100 ug/L.. The level of mercury in saliva has been found to be proportional to the number of amalgam fillings, and generally was higher for those with more fillings. The following table gives the average daily mercury exposure from saliva alone for those tested, based on the average levels found per number of fillings and using daily saliva volumes of 890 ml for unstimulated saliva flow and 80 ml for stimulated flow (estimated from measurements made in the study and comparisons to other studies). It also gives the 84th percentile mercury exposure from saliva for the 20,000 tested by number of fillings. Note that 16% of all of those tested with 4 amalgam fillings had daily exposure from their amalgam fillings of over 17 ug per day, and even more so for those with more than 4 fillings.

Table: Average daily mercury exposure in saliva by number of amalgam fillings(199)

Number of fillings 4 5 6 7 8 9 10 11 12 13 14 15 16
Av. Daily Hg(ug) 6.5 8 9.5 11 12.4 14 15.4 16.9 18.3 19.8 21.3 22.8 24.3
84th percentile(ug) 17 23.5 26 30.5 35 41.5 43.8 48.6 50.3 46.7 56.6 61.4 64.5

Saliva tests for mercury are commonly performed in Europe, and many other studies have been carried out with generally comparable results(292,315,79,9b,335,179,317,352). Another large German study(352) found significantly higher levels than the study summarized here, with some with exposure levels over 1000 ug/day.
Three studies that looked at a population with more than 12 fillings found generally higher levels than this study, with average mercury level in unstimulated saliva of 29 ug/L(18), 32.7 ug/L (292c), and 175 ug/day(352). The average for those with 4 or less fillings was 8 ug/L(18). While it will be seen that there is a significant correlation between exposure levels and number of amalgam surfaces and exposure generally increases as number of fillings increases, there is considerable variability for a given number of fillings. Some of the factors that will be seen to influence this variability include composition of the amalgam, whether person chews gum or drinks hot liquids, bruxism, oral environmental factors such as acidity, type of tooth patse used, etc. Chewing gum or drinking hot liquids can result in 10 to 100 times normal levels of mercury exposue from amalgams during that period(15,35).
The Tubingen study did not assess the significant exposure route of intraoral air and lungs. One study that looked at this estimated a daily average burden of 20 ug from ionized mercury from amalgam fillings absorbed through the lungs(191), while a Norwegian study found the average level in oral air to be 0.8 ug/M3(176). Another study at a Swedish University(335) measured intraoral air mercury levels from fillings of from 20 to 125 ug per day, for persons with from 18 to 82 filling surfaces. Another study found similar results(83), and some individuals have been found to have intraoral air mercury levels above 400 ug/ M3 (319). Most of those whose intraoral air mercury levels were measured exceeded Gov't health guidelines for workplace exposure(2).
The studies also determined that the number of fillings is the most important factor related to mercury level, with age of filling being much less significant(319b). Different filling composition/manufacturer can also make a difference in exposure levels( as will be further discussed). The authors of the Tubingen study calculated that based on the test results with estimates of mercury from food and oral air included, over 40 % of those tested in the study received daily mercury exposure higher than the WHO standard(PTWI). As can be seen most people with several fillings have daily exposure exceeding the Health Canada TDE and the U.S. EPA and ATSDR health guideline for mercury(2,209,199,etc.), and many tested in past studies have exceeded the older and higher WHO guideline for mercury(183), without consideration of exposure from food, etc..

3. The main exposure paths for mercury from amalgam fillings are absorption by the lungs from intraoral air vapor absorbed by saliva or swallowed amalgam particles swallowed and membrane, olfactory, venous, and neural path transfer of mercury absorbed by oral mucosa, gums, etc.
(6,17,18,31,34,77,79,83,94,133,174,182,209,211,216,222,319,335,348,364,436) A study at Stockholm Univ.(335) made an effort to determine the respective parts in exposure made by these paths. It found that the majority of excretion is through feces, and that the majority of mercury exposure was from elemental vapor. Daily exposure from intraoral air ranged from 20 to 125 ug of mercury vapor, for subjects with number of filling surfaces ranging from 18 to 82. Daily excretion through feces amounted to from 30 to 190 ug of mercury, being more variable than other paths. Other studies had similar findings(6,15,16,18,19,25,31,36,77,79,80,83,115, 196,386.) Most with several amalgams had daily fecal excretion levels over 50 ug/day.

The feces mercury was essentially all inorganic with particles making up at most 25%, and the majority being mercury sulfuhydryl compounds- likely originating as vapor. Their study and others reviewed found that at least 80% of mercury vapor reaching the lungs is absorbed and enters the blood from which it is taken to all other parts of the body(335,348,349,363). Elemental mercury swallowed in saliva can be absorbed in the digestive tract by the blood or bound in sulfhydryl compounds and excreted through the feces. A review determined that approx.20 % of swallowed mercury sulfhydryl compounds are absorbed in the digestive tract, but approx 60% of swallowed mercury vapor is absorbed(292,335,348). At least 80% of particle mercury is excreted. Approx. 80% of swallowed methyl mercury is absorbed(335,199,etc.)e, with most of the rest being converted to inorganic forms apparently. The primary detoxification/excretion pathway for mercury absorbed by the body is as mercury-glutathione compounds through the liver/bile loop to feces(111,252), but some mercury is also excreted though the kidneys in urine and in sweat. The range of mercury excreted in urine per day by those with amalgams is usually less than 15 ug(6,49,83,138,174,335,etc.), but some patients are much higher(93). A large NIDH study of the U.S. military population(49) with an average of 19.9 amalgam surfaces and range of 0 to 60 surfaces found the average urine level was 3.1 ug/L, with 93% being inorganic mercury. The average in those with amalgam was 4.5 times that of controls and more than the U.S. EPA maximum limit for mercury in drinking water(218). The avergage level of those with over 49 surfaces was over 8 times that of controls. The same study found that the average blood level was 2.55 ug/L, with 79 % being organic mercuy. The total mercury level had a significant correlation to the number of amalgam fillings, with fillings appearing to be reponsible for over 75% of total mercury. From the study results it was found that each 10 amalgam surfaces increased urine mercury by approx. 1 ug/L. A study of mercury species found blood mercury was 89% organic and urine mercury was 87% inorganic(349b), whicle another study(363) found on average 77% of the mercury in the occipital cortex was inorganic. In a population of women tested In the Middle East(254), the number of fillings was highly correlated with the mercury level in urine, mean= 7 ug/L. Nutrient transport and renal function were also found to be adversely affected by higher levels of mercury in the urine.

As is known from autopsy studies for those with chronic exposure such as amalgam fillings (1,14,17,20,31,34, 85,94), mercury also bioaccumulates in the brain/CNS(301,273,274,327,329,348,18,19,85),liver, kidneys(85,273), (14,85)heart(59,205,348)), and oral mucosa(174,192,436) with the half life in the brain being over 20 years. Elemental mercury vapor is transmitted throughout the body via the blood and readily enters cells and crosses the blood-brain barrier, and the placenta of pregnant women(38,61,287,311,361), at much higher levels than inorganic mercury and also higher levels than organic mercury. Significant levels are able to cross the blood brain barrier, placenta, and also cellular membranes into major organs such as the heart since the oxidation rate of Hg0 though relatively fast is slower than the time required by pumped blood to reach these organs(290,370). Thus the level in the brain and heart is higher after exposure to Hg vapor than for other forms(360,370). While mercury vapor and methyl Hg readily cross cell membranes and the blood-brain barrier, once in cells they form inorganic mercury that does not readily cross cell membranes or the blood brain barrier readily and is responsible for the majority of toxicity effects. Thus inorganic mercury in the brain has a very long half life(85,273,274,etc.).

4. The average amalgam filling has approximately 0.5 grams(500,000 ug) of mercury. As much as 50% of mercury in fillings has been found to have vaporized after 5 years and 80% by 20 years(182,204). Mercury vapor from amalgam is the single largest source of systemic mercury intake for persons with amalgam fillings, ranging from 50 to 90 % of total exposure.
(14,16,17,19,36,57,61,77-83,94,129,130,138,161,167,183, 191, 196,211,216,273,292,303,332,), averaging about 80% of total systemic intake. After filling replacement levels of mercury in the blood, urine, and feces typically temporarily are increased for a few days, but levels usually decline in blood and urine within 6 months to from 60 to 85% of the original levels(57,79,82,89,196,303). Mercury levels in saliva and feces usually decline between 80 to 95% (79,196,335,386)

5. Having dissimilar metals in the teeth(e.g.-gold and mercury) causes galvanic action, electrical currents, and much higher mercury vapor levels and levels in tissues. (182,192,292,348,349,390,19,25,27,29,30,35,47,48,100) Average mercury levels in gum tissue near amalgam fillings are about 200 ppm, and are the result of flow of mercury into the mucous membrane because of galvanic currents with the mucous membrane serving as cathode and amalgam as cathode(192). Average mercury levels are often 1000 ppm near a gold cap on an amalgam filling due to higher currents when gold is in contact with amalgam (30,25,35,48). These levels are among the highest levels ever measured in tissues of living organisms, exceeding the highest levels found in chronically exposed chloralkali workers, those who died in Minamata, or animals that died from mercury poisoning. German oral surgeons have found levels in the jaw bone under large amalgam fillings or gold crowns over amalgam as high as 5760 ppm with an average of 800 ppm(436). These levels are much higher than the FDA/EPA action level for prohibiting use of food with over 1 ppm mercury. Likewise the level is tremendously over the U.S. Dept. Of Health/EPA drinking water limit for mercury which is 2 parts per billion(218). Amalgam manufacturers, Government health agencies such as Health Canada,dental school texts, and dental materials researchers advise against having amalgam in the mouth with other metals such as gold(446,35), but many dentists ignore the warnings.

Concentrations of mercury in oral mucosa for a population of patients with 6 or more amalgam fillings taken during oral surgery were 20 times the level of controls(174). Studies have shown mercury travels from amalgam into dentin, root tips, and the gums, with levels in roots tips as high as 41 ppm(192). Studies have shown that mercury in the gums such as from root caps for root canaled teeth result in chronic inflammation, in addtion to migration to other parts of the body(200,47,35). Mercury and silver from fillings can be seen in the tissues as amalgam "tatoos", which have been found to accumulate in the oral mucosa as granules along collagen bundles, blood vessels, nerve sheaths, elastic fibers, membranes, striated muscle fibers, and acini of minor salivary glands. Dark granules are also present intracellularly within macrophasges, multinucleated giant cells, endothelial cells, and fibroblasts. There is in most cases chronic inflammatory response or macrophagic reaction the the metals(47), usually in the form of a foreingn body granuloma with multinucleated giant cells of the foreign body and Langhans types(192).

The periodontal ligament of extracted teeth is often not fully removed and results in incomplete jawbone regrowth resulting in a pocket where mouth bacteria in anerobic conditions along with similar conditions in the dead tooth produce extreme toxins similar to botulism which like mercury are extremely toxic and disruptive to necessary body enzymatic processes at the cellular level, comparable to the similar enzymatic disruptions caused by mercury and previously discussed(35,437).

The component mix in amalgams has also been found to be an important factor in mercury vapor emissions. The level of mercury and copper released from high copper amalgam is as much as 50 times that of low copper amalgams(191). Studies have consistently found modern high copper non gamma-two amalgams have a high negative current and much greater release of mercury vapor than conventional silver amalgams and are more cytotoxic (35,298,299). Clinics have found the increased toxicity and higher exposures to be factors in increased incidence of chronic degenerative diseases(35,etc). While the non gamma-two amalgams were developed to be less corrosive and less prone to marginal fractures than conventional silver amalgams, they have been found to be instable in a different mechanism when subjected to wear/polishing/ chewing/ brushing: they form droplets of mercury on the surface of the amalgams(182,297). This has also been found to be a factor in the much higher release of mercury vapor by the modern non gamma-two amalgams. Recent studies have concluded that because the high mercury release levels of modern amalgams, mercury poisoning from amalgam fillings is widespread throughout the population"(95,199,238). Numerous other studies also support this finding(Section IV).

Amalgam also releases significant amounts of silver, tin, and copper which also have toxic effects, with organic tin compounds formed in the body being even more neurotoxic than mercury(51,222,262).

6. The number of amalgam surfaces has a statistically significant correlation to :
(a) blood plasma mercury level (17,22,23,49,79,89,133,211)(usually not as strong as other measures)
(b) urine mercury level (38,49,57,76,77,79,82,83,134,138,167,176,254,303,332,335)
© oral air(16,18,100,176,335)
(d) saliva and oral mucosa(18,30,77,79,117,179,174,199,211,222,292,315,317)
(e) feces mercury (25,79,80,83,115,117,182,335,386)
(f) pituitary gland (19,20,25,85,99,273/274)
(g) brain occipital cortex (14,16,19,25,34,85,211,273,348,366/274)
(h) renal(kidney) cortex(14,16,19,20,85,254,273,348,366)
(I) liver(14,19,85,366)
(j) motor function areas of the brain & CNS: brain stem, cerebellum, rhombencephalon, dorsal root ganglia, and anterior horn motor neurons (48,291,327,329,442,35.)
(k) fetal and infant liver/brain levels(61,112,186,231,22) related to maternal fillings.

7. A person with amalgam fillings has daily systemic intake from mercury vapor of between 3 and 70 micrograms of mercury, with the average being at least 7 micrograms(ug) per day (18,77,83,85,93,138,183,199,211,292,315,335). In a large German study, the median daily exposure for those with fillings through saliva was approx. 10 ug/day, 4% of those with fillings had daily exposure through saliva of over 80 ug/day, and 1% had over 160 ug/day(199). The methods and results of the Tubingen study(199) were similar to those of other German studies(292,315,9, 138, 317,335). Total intake is proportional to the number and extent of amalgam surfaces, but other factors such as chewing gum, drinking hot liquids, brushing or polishing, and using fluoride toothpaste significantly increase the intake(15,18,28,31,100,134-137,182,183,199,209,211, 292,317,319,348,349,350). Vapor emissions range up to 200 ug/M3 (35) and are much higher after chewing(15,137,319). After chewing, those with amalgams had levels over 50 times higher than those without, and the average level of exposure was 29 ug/day for those with at least 12 occlusal surfaces(18). At least 30% of those having amalgam fillings tested in a large German study had ingested mercury levels exceeding the WHO PTWI mercury standard of 43 ug/day (199,183), and over 50% of those with 6 or more fillings had daily exposures more than the U.S. EPA health guideline level(199) of 0.1 ug/kg body weight/day(199). The median daily exposure through saliva for those with 10 or more fillings was over 10 times that of those with no fillings(199,292,315,318). Mercury level in saliva has been found to give much better indication of body levels than blood or urine levels(36). Most people with fillings have daily exposure levels exceeding the U.S. ATSDR and EPA health guideline levels (2,36,83,89,183,199,209,217,261,292,335,93)

8. The blood and urine mercury load of a person with amalgam fillings is often 5 times that of a similar person without.(14,16,17,79,80,82,93,136,138, 303,315,317,318) The average blood level for one large population was 5 ug/l(176). Normal blood levels are less than 20 ppb, but health effects have been observed in patients in the upper part of this range. A Swedish study estimated the total amount mercury swallowed per day from intra-oral vapor was 10 micrograms per day(177),and a large German study(199) found median exposure through saliva alone for those with fillings to be about 10 ug/day, with many having several fillings with over 10 times that level. Other studies have found similar amounts(18,83,211,183,209).

9. Teeth are living tissue and have massive communication with the rest of the body via blood, lymph, and nerves. Mercury vapor (and bacteria in teeth ) have paths to the rest of the body. (34,etc.) German studies of mercury loss from vapor in unstimulated saliva found the saliva of those with amalgams had at least 5 times as much mercury as for controls(138,199,292,315).

10. Mercury (especially mercury vapor) rapidly crosses the blood brain barrier and is stored preferentially in the pituitary gland, hypothalamus, and occipital cortex in direct proportion to the number and extent of amalgam surfaces.(14,19,20,25,34,38,85,99,273,274,287,348,366) Thus mercury has a greater effect on the functions of these areas. The range in one study was 2.4 to 28.7 ppb(85), and one study found on average that 77% of the mercury in the occipital cortex was inorganic(363).

11. Some mercury entering nasal passages is absorbed directly into the olfactory lobe and brain without coming from blood(34,35,182,222,348,364). Mercury also is transported along the axons of nerve fibres (5,25,34,35,327,329).

12. Mercury has a long half life in the body and over 20 years in the brain, and chronic low level intake results in a slow accumulation in body tissues. (20,34,35,38,85,etc.)

13. Methyl mercury is more toxic to some body processes than inorganic mercury. Mercury from amalgam is methylated by bacteria, galvanic electric currents(35), and candida albicans in the mouth and intestines(51,81,98,182,225). Oral bacteria streptococommus mitior,S.mutans, and S.sanguis were all found to methylate mercury(81). High levels of Vit B12 in the system also have been found to result in increased methyl mercury concentrations in the liver and brain(51). Methyl mercury is 10 times more potent in causing genetic damage than any other known chemical (Ramel, in(35)), and also crosses the blood-brain barrier readily. Once mercury vapor or methyl mercury are converted to inorganic mercury in cells or the brain, the mercury does not readily cross cell membranes or the blood-brain barrier. Thus mercury has a very long half life in the brain. N-acetylcysteine (NAC) has been found to be effective at increasing glutathione levels and chelating methyl mercury(54,126).

14. The level of mercury in the tissue of the fetus, new born, and young children is directly proportional to the number of amalgam surfaces in the mother's mouth. (20,23,61,112,210,361) The level of mercury in umbilical cord blood and placenta was higher than that in mother's blood(22,186). The saliva and feces of children with amalgams have approximately 10 times the level of mercury as children without(25,315,386), and much higher levels in saliva after chewing. A group of German children with amalgam fillings had urine mercury level 4 times that of a control group without amalgams(76), and in a Norwegian group with average age 12 there was a significant correlation between urine mercury level and number of amalgam fillings(167). The level of mercury in maternal hair was significantly correlated to level of mercury in nursing infants(279). One study found a 60% increase in average cord blood mercury level between 1980 and 1990 in Japan(186).

16. The fetal mercury content after maternal inhalation of mercury vapor was found to be higher than in the mother( 4,etc.) Mercury from amalgam in the blood of pregnant women crosses the placenta and appears in amniotic fluid and fetal blood, liver, and pituitary gland soon after placement (20,22,23,31,36,61,162, 186,281,348,366). Dental amalgams are the main source of mercury in breast milk(112,186,304,339,20). Milk increases the bioavailability of mercury(112,304,391) and mercury is often stored in breast milk and the fetus at much higher levels than that in the mother's tissues (19,20,22,23,61,112,186,210, 287,304). The level of mercury in breast milk was found to be significantly correlated with the number of amalgam fillings(61), with milk from mothers with 7 or more fillings having levels in milk approx. 10 times that of amalgam-free mothers. The milk sampled ranged from 0.2 to 6.9 ug/L. Several authors suggest use of early mother's milk as a screen for potenital problems since it is correlated both to maternal and infant mercury levels. The highest level is in the pituitary gland of the fetus which affects development of the endocrine system. Levels for exposure to mercury vapor has been found to be approx 10 times that for maternal exposure to an equivalent dose of inorganic mercury(281,287), and developmental behavioral effects from vapor have been found at levels considerably below that required for similar effects by methyl mercury (20,49,119c,264,287,304,338). The level of total mercury in nursing infants was significantly correlated to total mercury level in maternal hair(22,279).

17. There is a significant correlation between number of amalgam fillings of the mother and the level of the fetus and older infants(20,22,23,61,304), and also with the level in mother's milk (19,20,38,112, 304). Fertile women should not be exposed to vapor levels above government health guidelines(38,61,182,282) the U.S. ATSDR mercury health MRL of 0.2 mcg/M3 (2,217) or have amalgams placed or removed during pregnancy(20,182,231,304,etc.).

IV. Immune System Effects and Autoimmune Disease

1. Many thousands of people with symptoms of mercury toxicity have been found in tests to have high levels of mercury, and many thousands who have had amalgam fillings removed(most) have had health problems and symptoms alleviated or greatly improved(see Section VI). From clinical experience some of the symptoms of mercury sensitivity/mercury poisoning include chronic fatigue, dizziness, frequent urination, insomnia, headaches, chronic skin problems, metallic taste, gastrointestinal problems, asthma(8,97), stuffy nose, drycrusts in nose, rhinitis, plugged ears, ringing ears, chest pain, hyperventilation, diabetes(35), spacy feeling, chilly, chronic skin problems, immune and autoimmune diseases, cardiovascular problems and many types of neurological problems (26,34,35,36,38,45,59,60,69,70,71,75,91,109,148,165,204,212,199,246,255,268-270,290,291,294, 313,343). Amalgam results is chronic exposure rather than acute exposure and accumulation in body organs over time, so most health effects are of the chronic rather than acute in nature, but serious health problems have been documented to be related to amalgam and researchers have attributed some deaths as due to amalgam (356,32,245).

2. Mercury vapor exposure at very low levels adversely affects the immune system(17,27,31,38,45,60,84,118,129, 131,165,226,270,285,296,313,314,355368,369). From animal studies it has been determined that mercury damages T-cells by generating reactive oxygen species(ROS), depleting the thiol reserves of cells, damaging and decreasing the dimension of mitochondria, causing destruction of cytoplasmic organelles with loss of cell membrane integrity, inhibiting ability to secrete interleukin IL-1 and IL-2R, causing activation of glial cells to produce superoxide and nitric oxide, and inactivating or inhibiting enzyme systems involving the sulphydrol protein groups(226,424,442). Mercury caused adverse effects on both neutrophil and macrophage function and after depletion of thiol reserves, T-cells were susceptible to Hg induced cellular death (apoptosis).(226,272,355) Interferon syntheses was reduced in a concentration dependent manner with either mercury or methyl mercury as well as other immune functions(131), and low doses also induce aggregation of cell surface proteins and dramatic tyrosine phosporlation of cellular proteins related to asthma, allergic diseases such as eczema and lupus (234,323,35), and autoimmunity(181,314). One study found that insertion of amalgam fillings or nickel dental materials causes a supression of the number of T-lympocytes(270), and impairs the T-4/T-8 ratio. Low T4/T8 ratio has been found to be a factor in lupus, anemia, MS, eczema, inflamatory bowel disease, and glomerulonephritis. Mercury induced autoimmunity in animals and humans has been found to be associated with mercury's expression of major histocompatibility complex(MHC) class II genes(314,181,226,425c). Both mercuric and methyl mercury chlorides caused dose dependent reduction in immune B-cell production. (316) B-cell expression of IgE receptors were significantly reduced(316,165), with a rapid and sustained elevation in intracellular levels of calcium induced(316,333). Both forms are immontoxic and cytotoxic ant very low levels seen in individuals. Mercury also inhibited B-cell and T-cell RNA and DNA synthesis. The inhibition of these functins by 50 % occurred rapidly at very low levels, in the range of 10 to 25 ug/L. All types of cells exhibited a dose dependent reduct in cellular glutathione when exposed to mercury, inhibiting generation of GSH by lumpocutes and moncytes(252). Workers occupationally exposed to mercury at levels within guidelines have been found to have impairment of lytic activity of neutrophils and reduced abiltiy of neutraphils to kill invaders such as candida(285,404). Immune Th1 cells inhibit candida by cytokine related activation of macrophages and neutraphils. Development of Th2 type immune responses deactivate such defenses(404b). Mercury inhibits macrophage and neutraphil defense against candida by its affects on Th1 and Th2 cytokine effects(181,285). Low doses also induced autoimmuntiy in some species(181,314,404,129,131,43). Another effect found is increase in the average blood white cell count significantly (35). The increased white count usually normalizes after amalgam removal. Mercury also blocks the immune function of magnesium and zinc (198,427,43,38). Several studies found adverse health effects at mercury vapor levels of 1 to 5 mcg/M3 (35). Large numbers of people undergoing amalgam removal have clinically demonstrated significant improvements in the immune system parameters discussed here and recovery and significant improvement in immune system problems in most cases surveryed(Section VI). Antigen specific LST-test was performed on a large number of patients with atopic eczema(323), using T-cells of peripheral blood. 87% showed LST positive reactions to Hg, 87% to Ni, 38% to Au and 40% to Pd They removed LST positive dental metals from the opral cavities of patients. Improvement of symptoms was obtained in 82% (160/196) of the patients within 1-10 months. Similar results have been obtained at other clinics(455).

3. Mercury from amalgam interferes with production of cytokines that activate macrophage and neutraphils, disabling early control of viruses and leading to enhanced infection(131,251). Animal studies have confirmed that
mercury increases effects of the herpes simplex veris type 2 for example(131). Both mercuric and methyl mercury were equally highly toxic at the cellular level and in causing cell volume redcuctions(131). However methyl mercury inhibits macrophage functions such as migration and phagocytosis at lower levels.
4. Body mercury burden was found to play a role in resistant infections such as Chlamydia trachomatis and herpes family viral infections it was found many cases can only be effectively treated by antibiotics after removal of body mercury burden(cilantro tablets were used with followup antibiotics)(251,131). Similar results have been found for treatment of cancer(35).

5. Mercury by its effect of weakening the immune system contributes to increased chronic diseases and cancer(91,180,237,239,222,234,355,35,38,40,etc,). Exposure to mercury vapor causes decreased zinc and methionine availability, depresses rates of methylation, and increased free radicals-all factors in increased susceptability to cancer(14,34,38,43,143,144,180,237,239,251,256,283). Amalgam fillings have also been found to be positively associated with mouth cancer(206,251,403). Mercury from amalgam fillings has also been found to cause increase in white blood cells and in some cases to result in leukemia(35,180). White cell levels decline after total dental revision(TDR) and some have recovered from leukemia after removal of amalgam fillings in a very short time(35,180). Among a group of patients testing positive as allergic to mercury, low level mercury exposure was found to cause adverse immune system response, including effects on vitro production of tumor necrosis factor TNF alfa and reductions in interleukin-1. (131,152)
Nickel and beryllium are 2 other metals commonly used in dentistry that are very carcinogenic, toxic, and cause DNA malformations(35,456). Nickel ceramic crowns and root canals have also been found to be a factor in some breast cancer and some have recovered after TDR, which includes amalgam replacement, replacement of metal crowns over amalgam, nickel crowns, extraction of root canaled teeth, and treatment of cavitations where necessary(35). Similarly nickel crowns and gold crowns over amalgam have been found to be a factor in lupus(456,35,229) and Belle's Palsy from which some have recovered after TDR and Felderkrais exercises(35).

6. A high correlation has been found between patients subjectively diagnosed with CNS & systemic symptoms suggestive of mercury intoxication and immune reactivity to inorganic mercury(MELISA test,118,160) as well as with MRI positive patients for brain damage. Controls without CNS problems did not have such positive correlations. Mercury,nickel,palladium, and gold induce autoimmunity in genetically predisposed or highly exposed individuals(314,234,130,342). Tests have found a significant portion of people to be in this category and thus more affected by exposure to amalgam than others(see section V).

Mercury also interrupts the cytochrome C oxidase system, blocking the ATP energy function (35,43,84,232,338c). These effects along with reductions in red blood cells oxygen carrying capability often result in fatigue and reduced energy levels as well as neurological effects (35,60,119,140,141,182,202,212,232,235,313).
7. People with chronic and immune reacitive problems are increasing findng dental materials are a factor in their problems and getting biocompatiblity tests run to test their immune reactivity to the various dental materials used.
A high percentage of such patients test immune reactive to many of the toxic metals. Of the many thousands who have had the Clifford immune reactivity test(445), the following percentages were immune reactive to the following metals that have very common exposues: aluminum(91%), antimony(36%), arsenic(86%), beryllium(74%), cadmium(63%), chromium(83%), cobalt(78%), copper(32%), lead(68%), mercury(93%), nickel(98%), palladium(32%), silver(25%), tin(32%), zinc(33%).
Toxic/allergic reactions to metals such as mercury often result in lichen planus lesions in oral mucosa or gums and play a roll in pathogenesis of periodontal disease. Removal of amalgam fillings usually results in cure of such lesions(60,75,78,82,86, 87,90,94,101,118,133,168,313). A high percentage of patients with oral mucosal problems along with other autoimmune problems such as CFS have significant immune reactions to mercury, palladium, gold, and nickel(46,60,118,313,81,90,212,313,342,368,369,375,456), including to mercury preservatives such as thimersol. 94% of such patients had significant immune reactions to inorganic mercury(MELISA test) and 72% had immune reactions to low concentrations of HgCl2(<0.5 ug/ml). 61% also had immune reaction to phenylHg, which has been commonly used in root canals and cosmetics(313). 10% of controls had significant immune reactions to HgCl and 8.3% to palladium. Other studies of patients suffering from chronic fatigue found similar results(369,375). Of 50 patients suffering from serious fatigue refered for MELISA test(369), over 70% had significant immune reaction to inorganic mercury and 50% to nickel, with most patients also reactive to one or more other metals such as palladium, cadmium, lead, and methyl mercury.
Mercury has been found to impair conversion of thyroid T4 hormone to the active T3 form as well
as causing autoimmune thyroiditis common to such patients(369,382,459,35). In general immune activation from toxics such as heavy metals resulting in cytokine release and abnormalities of the hypothalamus-pituitary-adrenal axis can cause changes in the brain, fatigue, and severe psycholgical symtoms(379-382,385,369,375,381,382,453, 118, 60) such as profound fatigue, muscosketal pain, sleep disturbances, gastrointestinal and neurological problems as are seen in CFS, fibromyalgia, and autoimmune thyroidititis. Such symptoms usually improve significantly after amalgam removal. Such hypersensitivity has been found most common in those with genetic predisposition to heavy metal sensitivity(368,369,382,60), such as found more frequently in patients with HLA-DRA antigens(375,383). A significant portions of the population appear to fall in this category.

8. Patients with other systemic neurological or immune symptoms such as arthritis, myalgia, eczema, CFS, MS, lupus, ALS, diabetes, epilepsy, Hashimodo's thyroiditis, schleroderma,etc. also often recover or improve significantly after amalgam replacement (12,35,60,113,212,222,229,313,323,342,368,369,375,453,459,section VI). Of a group of 86 patients with CFS symptoms, 78% reported significant health improvements after replacement of amalgam fillings within a relatively short period, and MELISA test found significant reduction in lymphocyte reactivity compared to pre removal tests(342,368,369,375). The improvement in symptoms and lymphocute reactivity imply that most of the Hg-induced lymphocyte reactivity is allergenic in nature. Although patch tests for mercury allergy are often given for unresolved oral symptoms, this is not generally recommended as a high percentage of such problems are resolved irrespective of the outcome of a patch test(87,86,90,101,168,etc.) Also using mercury in a patch test has resulted in some adverse health effects. A group of patients that had amalgams removed because of chronic health problems, was able to detect subjectively when a patch test used mercury salts in a double blind study(373). Mercury inhibits production of insulin and is a factor in diabetes and hypoglycemia, with significant reductions in insulin need after replacement of amalgam filings and normalizing of blood sugar(35).

Of the over 3,000 patients tested for lymphocyte reactivity to metals(60,342,368,375), the following were the percentages testing positive: nickel- 34%, inorganic mercury- 23%, phenol mercury- 13%, gold- 12%, cadmium- 11%, palladium- 11%, silver- 1%. Other studies have also found relatively high rates of allergic reactions to inorganic mercury and nickel(81,35,445,456). For groups with suspected autoimmune diseases such as neurological problems, CFS, and oral lichen planus most of the patients tested positive to inorganic mercury and most of such patients health improved significantly and immune reactivity declined after amalgam removal. In a group of patients tested by MELISA before and after amalgam removal at a clinic in Uppsula Sweden, the patients reactivity to inorganic mercury, palladium, gold and phenyl mercury all had highly significant differences from the control group, with over 20 % being hihgly reactive to each of these metals(375). A high percentage were also reactive to nickel in both groups. After amalgam removal the immune reactivity to all of these metals other than nickel declined significantly, and 76% reported significant long term health improvements after 2 years. Only 2% were worse. The study concluded that immune reactivity to mercury and palladium is common and appears to be allegenic/immune related in nature since immune reactivity declines when exposure levels are reduced. Such studies have also found that deficiencies in detoxification enzymes such as glutathione transfereases cause increased susceptibility to metals and other chemicals(384). Such deficiencies can be due to genetic predisposition, but are also known to be caused by acute or chronic toxic exposures.

For MS and lupus patients, a high percentage tested positive to nickel and/or inorganic mercury(MELISA).

A patch test was given to a large group of medical students to assess factors that lead to sensitization to mercury(132). 13% tested positive for allergy to mercury. Eating fish was not a significant factor between sensitive and non- sensitized students, but the sensitized group had a significantly higher average number of amalgam fillings and higher hair mercury levels. In a population of dental students tested, 44% were positive for allergy to mercury(156).

9. A high correlation has been found between patients subjectively diagnosed with CNS & systemic symptoms suggestive of mercury intoxication and immune reactivity to inorganic mercury(MELISA test,118) as well as with MRI positive patients for brain damage. 81% of the group with health complaints had pathological MRI results including signs of degeneration of the basal ganglia of the brain, but none in the controls. 60% of the symptom group tested positive for immune system reaction to mercury. Controls without CNS problems did not have such positive correlations. The authors concluded that immune reactions have an important role in development of brain lesions ,and amalgam fillings induce immune reactions in many patients (91,118)(270,286). Mercury,nickel,palladium, and gold induce autoimmunity in genetically predisposed or highly exposed individuals(60,314,234,130,342,35). Tests have found a significant portion of people to be in this category and thus more affected by exposure to amalgam than others.

10. Low level mercury exposure(as well as other toxic metals) including exposure to amalgam fillings has been found to be associated with increased autoimmune diseases (19, 27,34,35,44,45,60,215,234,268,269,270, 313,314), including lupus(12,33e,35,60,113,229,233,234,270,323,330,331,456),Chrons Disease, lichen planus(86,87,90,168,313), endometriosis (1,9,38,229). Silver also is released from amalgam fillings and stored in the body and has been shown to cause immune complex deposits, immune reactions and autoimmunity in animal studies (77,78,129,314).

11. Mercury exposure through fillings appears to be a major factor in chronic fatigue syndrome(CFS) through its effects on ATP and immune system(lymphocute reactivity, neutraphil activity, effects on T-cells and B-cells) and its promotion of growth of candida albicans in the body and the methylation of inorganic mercury by candida to the extremely toxic methyl mercury form which like mercury vapor crosses the blood-brain barrier and also damages and weakens the immune system(222,225,226,234,235,265,293,60,313,314,342,368,369, 404), and both inorganic and methyl mercury have been shown in animal studies to induce autoimmune reactions and disease in susceptible types through effects on immune system T cells (226,234,268,269,270,314,425,426/272.)

V. Medical Studies Finding Health Problems Related to Amalgam Fillings (other than immune)

1. Neurological problems are among the most common and serious and include memory loss, moodiness, depression, anger and sudden bursts of anger/rage(434), self-effacement, suicidal thoughts, lack of strength/force to resolve doubts or resist obsessions or compulsions, etc. Many studies of patients with major neurological diseases have found evidence amalgam fillings may play a major role in development of conditions such as depression(94,107,109,212,222,271,294,212,229,233,285e,317,320,322,372,374,453), schizophrenia(34,35,295), memory problems(212,222), and other more serious neurological diseases such as MS, ALS, Parkinson's, and Alzheimer's(see # 25). One mechanism by which mercury has been found to be a factor in aggressiveness and violence is its documented inhibition of the brain neurotransmitter acetylcholinesterase(175,451).

Mercury causes decreased lithium levels, which is a factor in neurological diseases such as depression and Alzheimer's. Lithium protects brain cells against excess glutamate and calcium, and low levels cause abnormal brain cell balance and neurological disturbances (280,294,333,33,56 ). Medical texts on neurology (27,295) point out that chronic mercurialism is often not recognized by diagnosticians and misdiagnosed as dementia or neurosis or functional psychosis or just "nerves". "Early manifestations are likely to be subtle and diagnosis difficult: Insomnia, nervousness, mild tremor, impaired judgment and coordination, decreased mental efficiency, emotional lability, headache, fatigue, loss of sexual drive, depression, etc. are often mistakenly ascribed to psychogenic causes". Very high levels of mercury are found in brain memory areas such as the cerebral cortex and hippocampus of patients with diseases with memory related symptoms (158,34,207,etc.>

Mercury(as well as toxins from root canals and cavitations) interact with brain tubulin and disassembles microtubiles that maintain neurite structure(207b,35,437). Thus chronic exposure to low level mercury vapor can inhibit polymerzation of brain tubulin and creatinine kinase which are essential to formation of microtubles. Studies of mercury studies on animals give results similar to that found the the Alzheimer brain. The effects of mercury with other toxic metals have also been found to be synergistic, having much more effect than with individual exposure(35).

Animal studies of developmental effects of mercury on the brain have found significant effects at extremely low exposure levels, levels commonly seen in those with amalgam fillings or in dental staff working with amalgam. One study(175) found mercury vapor decreased NGF concentration in rat's forebrain at 4 parts per billion(ppb) tissue concentration. Another study(134) found general toxicity effects at 1 micromole(uM) levels in immature cell cultures, increased immunoreactivity for glial fibrillary protein at 1 nanamole (0.2 ppb) concentration, and microglial response at even lower levels. Other animal studies on rodents and monkeys have found brain cellular migration disturbances, behavioral changes, along with reduced learning and adaption capacity after low levels of mercury vapor exposure (210,264,287,149). The exposure levels in these studies are seen in the fetus and newborn babies of mother's with amalgam fillings or who had work involving amalgam during pregnancy(61).

Epidemiological studies have found that human embryos are also highly susceptible to brain damage from prenatal exposure to mercury. Studies have confirmed that there are vulnerable periods during brain and CNS development that are expecially sensitive to neurotoxic exposures and affect development processes and results(429).The fetal period is most sensitive, but neural developement extends through adolescence. Maternal hypothyroidism has been found to cause endocrine system abnormalities in the fetus, and mercury is documented to commonly cause hypothyroidism, both chronically or as a transient condition(458). Some conditions found to be related to such toxic exposures include autism, scizophrenia, ADD, dyslexia, eczema, etc. Prenatal/early postnatal exposure to mercury affects level of nerve growth factor(NGF) in the brain and causes brain damage and imbalances in development of the brain (38,119,181,305,259,210,149,305,24/ 39,175, 255,149). Exposure of developing neuroblastoma cells to sub-cytotoxic doses of mercuric oxide resulted in lower levels of neurofilament proteins than unexposed cells(305). Mercury vapor exposure causes impaired cell proliferation in the brain and organs, resulting in reduced volume for cerebellum and organs and subtle deficiencies(38,305). Exposure to mercury and 4 other heavy metals tested for in a study of school children accounted for 23% of the variation in test scores for reading, spelling and visual motor skills(3). A Canadian study found that blood levels of five metals were able to predict with a 98% accuracy which children were learning disabled(3). Several studies found that mercury causes learning disabilities and impairment, and reduction in IQ(3,21,38,110,264,285c,279). Mercury has an effect on the fetal nervous system at levels far below that considered toxic in adults, and background levels of mercury in mothers correlate significantly with incidence of birth defects and still births (23,38,287,338c,10).

2. Calcium plays a major role in the extreme neurotoxicity of mercury and methyl mercury. Both inhibit cellular calcium ATPase and calcium uptake by brain microsomes at very low levels of exposure (270,288,329,333,432,56,). Protein Kinase C (PKC) regulates intracellular and extra cellular signals across neuronal membranes, and both forms of mercury inhibit PKC at micromolar levels, as well as inhibiting phorbal ester binding(43,432). They also block or inhibit calcium L-channel currents in the brain in an irreversable and concecentration dependent manner. Mecury vapor or inorganic mercury exposure affects the posterior cingulate cortex and causes major neurological effects with sufficient exposure(428,453). Some of the resulting conditions include stomatitis, tremor, ADD, erythism, etc. Metallic mercury is much more potent than methyl mercury in such actions, with 50 % inhibitation in animal studies at 13 ppb(333,329).

Spatial and temporal changes in intracellular calcium concentrations are critical for controlling gene expression and neurotransmitter release in neurons(432,412). Mercury alters calcium homeostasis and calcium levles in the brain and affects gene expression and neurotransmitter release through its effects on calcium, etc.

Mercury inhibits sodium and potassium (N,K)ATPase in dose dependent manner and inhibits dopamine and noreprenephrine uptake by synaptosomes and nerve implulse transfer(288,50,270,35). Mercury also interrupts the cytochrome oxidase system, blocking the ATP energy function (35,43,84,232,338c), lowering immune growth factor IGF-I levels and impairing astrocyte function(119,131). Astrocytes are common cells in the CNS involved in the feeding and detox of nerve cells. Increases in inflamatory cytokines such as caused by toxic metals trigger increased free radical activity and damage to astrocyte and astrocyte function(152). IGF-I protects against brain and neuronal pathologies like ALS, MS, and Fibromyalgia by protecting the astrocytes from this destructive process.

Mercury lymphocyte reactivity and effects on glutamate in the CNS induce CFS type symptoms including profound tiredness, musculoskeletal pain, sleep distubances, gastrointestinal and neurological problems along with other CFS symptoms and fibromyalgia(342,346,368,369,375). Mercury has been found to be a common cause of fibromyalgia(293,346,369). Glutamate is the most abundant amino acid in the body and in the CNS acts as excitory neurotransmitter(346,386,412), which also causes inflow of calcium. Astrocytes, a type of cell in the brain and CNS with the task of keeping clean the area around nerve cells, have a function of neutralizing excess glutamate by transforming it to glutamic acid. If astrocytes are not able to rapidly neutralize excess glutamate, then a buildup of glutamate and calcium occurs, causing swelling and neurotoxic effects(119,131,152,333). Mercury and other toxic metals inhibit astrocyte function in the brain and CNS(119,131), causing increased glutamate and calcium related neurotoxicity(119,152,333,226a) which are responsible for much of the fibromylgia symptoms and a factor in neural degeneration in MS and ALS. This is also a factor in conditions such as CFS, Parkinson's, and ALS(346,416). Animal studies have confirmed that increased levels of glutamate(or aspartate, another amino acid excitory neurotransmitter) cause increased sensitivity to pain , as well as higher body temperature- both found in CFS/fibromyalgia. Mercury and increased glutamate activate free radical forming processes like xanthine oxidase which produce oxygen radicals and oxidative neurological damage(346,142,13). Extremely toxic anerobic bacteria from root canals or cavitations formed at incompletely healed tooth extraction sites have also been found to be common factors in fibromyalgia and other chronic neurological conditions such as Parkinson's and ALS, with condensing osteitis which must be removed with a surgical burr along with 1 mm of bone around it(35,437). Cavitations have been found in 85% of sites from wisdom tooth extractions tested and 55% of molar extraction sites tested(35,437). The incidence is likely somewhat less in the general population. Medical studies and doctors treating fibromylagia have found that supplements which cause a decrease in glutamate or protect against its effects have a positive effect on fibromyalgia and other chronic neurologic conditions. Some that have been found to be effective include Vit B6, methyl cobalamine(B12), L-carnitine, choline, ginseng, Ginkgo biloba,vitamins C and E, nicotine, and omega 3 fatty acids(fish and flaxseed oil)(417).

3. Numerous studies have found long term chronic low doses of mercury cause neurological, memory, behavior, sleep, and mood problems(3,34,60,69,70,71,74,107, 108,109,119,140,141,160,199,212,222,246,255,257, 258, 282,290,453). Neurological effects have been documented at very low levels of exposure(urine Hg< 4 ug/L), levels commonly received by those with amalgam fillings(290). One of the studies at a German University(199) assessed 20,000 people. There is also evidence that fetal or infant exposure causes delayed neurotoxicity evidenced in serious effect at middle age(255,306). Organic tin compounds formed from amalgam are even more neurotoxic than mercury (222,262). Studies of groups of patients with amalgam fillings found significantly more neurological, memory, mood, and behavioral problems than the control groups. (3,34,107,108,109,140,141,160,199,212,222,290,453).

4. Mercury binds to hemoglobin oxygen binding sites in the red blood cells thus reducing oxygen carrying capacity(332,35) and adversely affects the vascular response to norepinepherine and potassium. Mercury's effect on pituitary gland vasopression is a factor in high blood pressure(35). Mercury also increases cytosolic fre calcium levels in lymphocytes in a concentration-dependant manner causing influx from the extracellular medium(270c), and blocks entry of calcium ions into the cytoplasm (1,16,17,21,33,35,333), and at 100 ppb can destroy the membrane of red blood cells(35,22,17,270c) and damage blood vessels- reducing blood supply to the tissues (34,202,306). Amalgam fillings have been found to be related to higher blood pressure, hemoglobin irregularities, tachycardia, chest pains, etc.(201,202,205,212,222,306,310,35). Mercury also interrupts the cytochrome oxidase system, blocking the ATP energy function(35,43,84,232,338c) and impairing astrocyte function(119).. These effects often result in fatigue and reduced energy levels (35,60,119,140,141, 182,202,212,232,235,313). Mercury also accumulates in the heart and damages myocardial and heart valves (Turpayev,in (35)) & (59,201,205,306,351,370). Both mercury and methyl mercury have been shown to cause depletion of calcium from the heart muscle and to inhibit myosin ATPase activity by 50% at 30 ppb(59), as well as reducing NK-cells in the blood and spleen. The interruption of the ATP energy chemistry results in high levels of porphyrins in the urine(260). Mercury,lead, and other toxics have different patterns of high levels for the 5 types of porphyrins, with pattern indicating likely source and the level extent of damage. The average for those with amalgams is over 3 time that of those without, and is over 20 times normal for some severely poisoned people(232,260). The FDA has approved a test measuring porphyrins as a test for mercury poisoning. However some other dental problems such as nickel crowns, cavitations, and root canals also can cause high porphyrins. Cavitations are diseased areas in bone under teeth or extracted teeth usually caused by lack of adequate blood supply to the area. Tests by special equipment(Cavitat) found cavitations in over 90% of areas under root canals or extracted wisdom teeth that have been tested, and toxins such as anerobic bacteria and other toxics which significantly inhibit body enzymatic processes in virtually all cavitations(437). These toxins have been found to have serious systemic health effects in many cases, and significant health problems to be related such as arthritis, MCS, and CFS. These have been found to be factors along with amalgam in serious chronic conditions such as MS, ALS, Alzheimer's, MCS, CFS, etc.(35,204,222,292,437). The problem occurs in extractions that are not cleaned out properly after extraction(437). Supplements such as glucosamine sulfate and avoidance of orange juice and caffein have been found to be benefical in treating arithritic conditions as well(35).

A study funded by the Adolf Coors Foundation(232) found that toxicity such as mercury is a significant cause of abnormal cholesterol levels, increasing as a protective measure against metals toxicity, and that cholesterol levels usually normalize after amalgam replacement. However lowering cholesterol levels by other means below 160 correlates with much higher rates of depression, suicide, cancer, violent deaths, cerebral hemorage, and deaths- all known to be affected by mercury effects(35). The study also found that mercury has major adverse effects on red and white blood cells, oxygen carrying capacity, and porphyrin levels(232), with most cases seeing significant increase in oxyhemogolbin level and reduction in porphyrin levels along with 100% experiencing improved energy.
5. Patch tests for hypersensitivity to mercury have found from 2% to 44% to test positive (87,154,156, 178, 267), much higher for groups with more amalgam fillings and length of exposure than those with less. In studies of medical and dental students, those testing positive had significantly higher average number of amalgam fillings than those not testing positive(and higher levels of mercury in urine(132,156). Of the dental students with 10 or more fillings at least 5 years old, 44% tested allergic. Based on these studies and statistics for the number with 10 or more fillings, the percent of Americans allergic to mercury just from this group would be about 17 million people especially vulnerable to increased immune system reactions to amalgam fillings. However, the total would be much larger and patch tests do not measure the total population getting toxic reactions from mercury. The most sensitive reactions are immune reactions, DNA mutations, developmental,enzyme inhibition, and systemic effects(34,38,61,149,186,226,263,264,270,272,296,305,410-412/357).

6. People with amalgam fillings have an increased number of intestinal microorganisms resistant to mercury and many standard antibiotics(35,116,117,161,389). Mercury is extremely toxic and kills many beneficial bacterial, but some forms of bacteria can alter their form to avoid being killed by adding a plasmid to their DNA making the bacteria mercury resistant. But this transformation also increases antibiotic resistance and results in adversely altered populations of bacteria in the intestines. Recent studies have found that drug resistant strains of bacteria causing ear infections, sinuitis, tuberculosis, and pneumonia more than doubled since 1996, and similar for strains of bacteria in U.S. rivers(53). Studies have found a significant correlation between mercury resistance and multiple antibiotic resistance (116,117,161,369), and have found that after reducing mercury burden antibiotic resistance declines (251,389,40). The alteration of intestional bacterial populations necessary for proper digestion along with other damage and membrane permeability effects of mercury are major factors in creating "leaky gut" conditions with poor digestion and absorption of nutrients and toxic incompletely digested compounds in the bloodstream(338,35,etc.).

7. Mercury from amalgam binds to the -SH (sulphydryl) groups, resulting in inactivation of sulfur and blocking of enzyme function, producing sulfur metobolites with extreme toxicity that the body is unable to properly detoxify(33,114), along with a defeciency in sulfates required for many body functions. Sulfur is essential in enzymes, hormones, nerve tissue, and red blood cells. These exist in almost every enzymatic process in the body. Blocked or inhibited sulfur oxidation at the cellular level has been found in most with many of the chronic degenertive diseases, including Parkinson's, Alzheimer's, ALS, lupus, rheumatoid arthritis, MCS, autism, etc(330,331,33,35,56), and appears to be a major factor in these conditions. Mercury also blocks the metabolic action of manganese and the entry of calcium ions into cytoplasm(333). Mercury from amalgam thus has the potential to disturb all metabolic processes(25,21,33, 35,56,60,111,180,194,197>. Mercury is transported throughout the body in blood and can affect cells in the body and organs in different ways.

8. A large study of 20,000 subjects at a German university found a significant relation between the number of amalgam fillings with periodontal problems, neurological problems, and gastrointestinal problems(199). Allergies and hair-loss were found to be 2-3 times as high in a group with large number of amalgam fillings compared to controls(199,9). Levels of mercury in follicular fluid was significantly higher for those with amalgam fillings (9,146). Based on this finding, a Gynecological Clinic that sees a large number of women suffering from alopecia/hair loss that was not responding to treatment had amalgams replaced in 132 women who had not responded to treatment. 68 % of the women then responded to treatment and alopecia was alleviated(187). In other studies involving amalgam removal, the majority had significant improvement (40,317). Higher levels of hormone disturbances, immune disturbances, infertility, and recurrent fungal infections were also found in the amalgam group. The results of hormone tests, cell culture studies, an intervention studies agree(9,146). Other clinics have also found alleviation of hair loss/alopechia after amalgam removal and detox(40,317). Another study in Japan found significantly higher levels of mercury in gray hair than in dark hair(402).

9. Mercury accumulates in the kidneys with increasing levels over time. One study found levels ranging from 21 to 810 ppb. A study of levels in kidney donars found an average of 3 times higher mercury level in those with amalgams versis those without(14c). Mercury exposure has been shown to adversely affect kidney function in occupational and animal studies (20,203,211,260,438), and also in those with more than average number of amalgam fillings(254).. Inorganic mercury exposure has been found to exert a dose-dependent cytotoxicity by generating extremely high levels of hydrogen peroxide, which is normally quenched by pyruvate and catalase(203). HgCl2 also has been found to impair function of other organelles such s lysomomes that maintain transmembrane proton gradient, and to decrease glutathione peroxidase activity in the kidneys while upregulating heme oxidase function. The Government's toxic level for mercury in urine is 30 mcg/L (189), but adverse effects have been seen at lower levels and low levels in urine often mean high mercury retention and chronic toxicity problems.

10. Amalgam fillings produce electrical currents which increase mercury vapor release and may have other harmful effects(19,27,28,29,30,35,100,192,194). These currents are measured in micro amps, with some measured at over 4 micro amps. The central nervous system operates on signals in the range of nano-amps, which is 1000 times less than a micro amp(28). Negatively charged fillings or crowns push electrons into the oral cavity since saliva is a good electrolyte and cause higher mercury vapor losses(35,192). Patients with autoimmune condtions like MS, or epilepsy, depression, etc. are often found to have a lot of high negative current fillings(35). The Huggins total dental revision(TDR) protocol calls for teeth with the highest negative charge to be replaced first(35). Other protocols for amalgam removal are available from international dental associations like IAOMT(153) and mercury poisoned patients organizations like DAMS(447). For these reasons it is important that no new gold dental work be placed in the mouth until at least 6 months after replacement. Some studies have also found persons with chronic exposure to electromagnetic fields(EMF) to have higher levels of mercury exposure and excretion(28).

11. Mercury from amalgam fillings is transferred to the fetus of pregnant women and children who breast feed at levels often higher than those of the mother(18,19,20,23,31,38,61,112, 186,281). Mercury has an effect on the fetal nervous system at levels far below that considered toxic in adults, and background levels of mercury in mothers correlate significantly with incidence of birth defects and still births(10,23,38,197,210,287,338c,361). Mercury vapor exposure causes impaired cell proliferation in the brain and organs, resulting in reduced volume for cerebellum and organs and subtle deficiencies(38,305).

12. Since mercury(all forms) is documented from studies of humans and animals to be a reproductive and developmental toxin(23,38,61,105,186,224,255,287.305,381,etc.), mercury can reduce reproductive function and cause birth defects and developmental problems in children(2,4,9,10,20,23,24,31,37,38,39,41,55,61,104,146,159, 162,224,255,458). Clinical evidence indicates that amalgam fillings lead to hormone imbalances that can reduce fertility(9,38,55,4,105,146,367). Mercury has been found to cause decreased sperm volume and motility , increased sperm abnormalities and spontaneous abortions, increased uterine fibroids/endometritis, and decreased fertility in animals(4,104,105,162) and in humans(9,10,23,31,37,105,146,159,395,433,27,35,38). In studies of women having miscarriages or birth defects, husbands were found to typically have low sperm counts and significantly more visually abnormal sperm(393). Studies indicate an increase in the rate of spontaneous abortions with an increasing concentration of mercury in the fathers' urine before pregnancy(37). Studies have found that mercury accumulates in the ovaries and testes, inhibits enzymes necessary for sperm production, affects DNA in sperm, causes aberrant numbers of chromosomes in cells, causes chromosome breaks, etc.- all of which can cause infertility, spontaneous abortions, or birth defects(10,31,35,296). Subfertile males in Hong Kong were found to have 40% more mercury in their hair than fertile controls(55). Studies in monkeys have found decreased sperm motility, abnormal sperm, increased infertility and abortions at low levels of methyl mercury(162,365). Researcher's advise pregnant women should not be exposed to mercury vapor levels above government health standards (2,19,25,227,61,100,182,282,366) currently U.S. ATSDR mercury health MRL of 0.2 mcg/M3 which is exceeded by any dental work involving amalgam(Section III). Many governments have bans or restrictions on use of amalgam by women of child-bearing age.

13. Mercury causes breaks in DNA (4,38,41,42,197,272,296). Low non-cytotoxic levels of mercury induce dose dependent binding of mercury to DNA and significantly increased cell mutations (142,4) and birth defects(197,38,105). In addition to effects on DNA, mercury also promotes cancer in other ways. Mercury depletes and weakens the immune system in many ways documented throughout this paper.

14. Mercury has been well documented to be an endocrine system disrupting chemical in animals and people, disrupting function of the pituitary gland, hypothallamus, thyroid gland(50,369,35), enzyme production processes (111,194,33,56), and many hormonal functions at very low levels of exposure (9,105,146, 210, 312,369). The pituitary gland controls many of the body's endocrine system functions and secretes hormones that control most bodily processes, including the immune system and reproductive systems(105,312,381). The hypothallamus regulates body temperature and many metabolic processes. Mercury damage thus commonly results in poor bodily temperature control, in addtion to many problems caused by hormonal imbalances. Such hormonal secretions are affected at levels of mercury exposure much lower than the acute toxicity effects normally tested. Mercury also damages the blood brain barrier and facilitates penetration of the brain by other toxic metals and substances (311). Low levels of mercuric chloride also inhibit ATPase activity in the thyroid, with methyl mercury inhibiting ATP function at even lower levels(50,35). Both types of mercury were found to cause denaturing of protein, but inorganic mercury was more potent. These effects result commonly in a reduction in thyroid production(50) and an accumulation in the thyroid of radiation. Toxic metal exposure's adverse influence on thyrocytes can play a major role in thyroid cancer etiology(144) . Among those with chronic immune system problems with related immune antibodies, the types showing the highest level of antibody reductions after amalgam removal include thyreoglobulin and microsomal thyroid antigens(91)

15. There has been no evidence found that there is any safe level of mercury in the body that does not kill cells and harm body processes(WHO,183,189, etc.). This is especially so for the pituitary gland of the developing fetus where mercury has been shown to accumulate and which is the most sensitive to mercury(2-4,19-24,30,31,36-44,61,186).

16. Low levels of mercury and toxic metals have been found to inhibit dihydroteridine reductase, which affects the neural system function by inhibiting transmitters through its effect on phenylalanine, tyrosine and tryptophan transport into neurons(27,98,122,257,289,372,342,372,412). This was found to cause severe impaired amine synthesis and hypokinesis. Tetrahydrobiopterin, which is essential in production of neurotransmitters, is significantly decreased in patients with alzheimer's, Parkinson's, MS, and autism. Such patients have abnormal inhibition of neurotransmitter production. Such symptoms improved for most patients after administration of
R-tetrahydrobiopterin(412), and some after 5-formyltetrahydrofolate, tyrosine(257), and 5-HTP(412).

17. The level of mercury released by amalgam fillings is often more than the levels documented in medical studies to produce adverse effects and above the U.S. government health guidelines for mercury exposure(see previous text).

18. Many studies of patients with major neurological or degenerative diseases have found evidence amalgam fillings may play a major role in development of conditions such as such as Alzheimers (66,67,158,166,204, 207, 221,238,242,244,257,295,300,35), ALS(92,97,229,325,346,416,423,35), MS(102,163,170,183,184,212,285,291, 302, 324,326,35), Parkinson's(98,169,248,250,258,363,56,84,35),ADD(285e), etc. Mercury exposure causes high levels of oxidative stress/reactive oxygen species(ROS)(13,442), which has been found to be a major factor in neurological disease(56,442). Mercury and quinones form conjugates with thiol compounds such as glutathione and cysteine and cause depletion of glutathione, which is necessary to mitigate reactive damage. Such congugates are found to be highest in the brain substantia nigra with similar congugates formed with L-Dopa and dopamine in Parkinson's disease(56). Mercury depletion of GSH and damage to cellular mitochrondria and the increased lipid perxodation in protein and DNA oxidation in the brain appear to be a major factor in Parkinson's disease(33,346). One study found higher than average levels of mercury in the blood, urine, and hair of Parkinson's disease patients(363). Another study(169) found blood and urine mercury levels to be very strongly related to Parkinson's with odds ratios of approx. 20 at high levels of Hg exposure. Increased formation of reactive oxygen species(ROS) has also been found to increase formation of advanced glycation end products(AGEs) that have been found to cause activation of glial cells to produce superoxide and nitric oxide, they can be considered part of a vicious cycle, which finally leads to neuronal cell death in the substantia nigra in PD(424). Another study (145) that reveiwed occupational exposure data found that occupational exposure to manganese and copper have high odds rations for relation to PD, as well as multiple exposures to these and lead, but noted that this effect was only seen for exposure of over 20 years.
Mercury has been found to accumulate preferentially in the primary motor function related areas such as the brain stem, cerebellum, rhombencephalon, dorsal root ganglia, and anterior horn motor neurons, which enervate the skeletal muscles(48,291,327,329,442). There is considerable indication this may be a factor in ALS development (48,325,405,416,423,442). Mercury penetrates and damages the blood brain barrier allowing penetration of the barrier by other substances that are neurotoxic (20,38,85,105,162,301,311/262). Such damage to the blood brain barrier's function has been found to be a major factor in chronic neurological diseases such as MS(286,289,291,302, 324,326). MS patients have been found to have much higher levels of mercury in cerebrospinal fluid compared to controls (163,35,139). Large German studies including studies at German universities have found that MS patients usually have high levels of mercury body burden, with one study finding 300% higher than controls(271). Most recovered after mercury detox, with some requiring addtional treatment for viruses and intestinal dysbiosis. Studies have found mercury related mental effects to be indistinguishable from those of MS (207,212,222,244,271,289,291,302,183,184,324,326).
Low levels of toxic metals have been found to inhibit dihydroteridine reductase, which affects the neural system function by inhibiting brain transmitters through its effect on phenylalanine, tyrosine and tryptophan transport into neurons(122,257,289,372). This was found to cause severe impaired amine synthesis and hypokinesis. Tetrahydro-biopterin, which is essential in production of nerurotransmitters, is significantly decreased in patients with Alzheimer's's, Parkinson's, and MS. Such patients have abnormal inhibition of neurotransmitter production.(supplements which inhibit breach of the blood brain barrier such as bioflavonoids have been found to slow such neurological damage).
Clinical tests of patients with MND,ALS, Parkinson's, Alzheimer's, Lupus(SLE), rheumatoid arthritis and autsism have found that the patients generally have elevated plasma cysteine to sulphate ratios, with the average being 500%higher than controls(330,331,56,33e), and in general being poor sulphur oxidizers. This means that these patients have insufficient sulfates available to carry out necessary bodily processes. Mercury has been shown to diminish and block sulphur oxidation and thus reducing glutathione levels which is the part of this process involved in detoxifying and excretion of toxics like mercury(33). Glutathion is produced through the sulphur oxidation side of this process. Low levels of available glutathione have been shown to increase mercury retention and increase toxic effects(111), while high levels of free cysteine have been demonstrated to make toxicity due to inorganic mercury more severe(333,194,56,33e). Mercury has also been found to play a part in inducing intolerance and neuronal problems through blockage of the P-450 enzymatic process(84,33e).
In one subtype of ALS, damaged, blocked, or faulty enzymatic superoxide dimutase (SOD) processes appear to be a major factor in cell apoptosis involved in the codition(443). Mercury is known to damage or inhibit SOD actitivity(441,33,111).

19. Mercury at extremely low levels also interferes with formation of tubulin producing neurofibrillary tangles in the brain similar to those observed in Alzheimers patients, with high levels of mercury in the brain (207), and low levels of zinc(363,43). Mercury and the induced neurofibrillary tangles also appear to produce a functional zinc deficiency in the of AD sufferers(242),as well as causing reduced lithium levels which is another factor in such diseases. Lithium protects brain cells against excess glutamate induced excitability and calcium influx(280,56). Also mercury binds with cell membranes interfering with sodium and potassium enzyme functions, causing excess membrane permeability, especially in terms of the blood-brain barrier (155,207,311). Less than 1ppm mercury in the blood stream can impair the blood- brain barrier. Mercury was also found to accumulate in the mitochondria and interfere with their vital functions, and to inhibit cytochrome C enzymes which affect energy supply to the brain(43,84,232,338c,35). Persons with the Apo-E4 gene form of apolipoprotein E which transports cholesterol in the blood, are especially susceptible to this damage(207,221,346), while those with Apo-E2 which has extra cysteine and is a better mercury scavanger have less damage. The majority have an intermediate form Apo-E3. This appears to be a factor in susceptablity to Alzheimer's disease, Parkinson's disease and multiple schlerosis. Ones susceptability can be estimated by testing for this condition. In many cases (many thousand documented)removal of amalgam fillings and treatment for metal toxicity led to "cure' or significant improvement in health(see Section V). Mercury causes an increase in white blood cells, with more created to try to react to a foreign toxic substance in the body. There is evidence that some forms of leukemia are abnormal response to antigenic stimulation by mercury or other such toxics, and removal of amalgam has led to remission very rapidly in some cases(35,38,180,239).

20. Mercury and methyl mercury impair or inhibit all cell functions and deplete calcium stores(96). This can be a major factor in bone loss of calcium(osteoperosis). Mercury(like copper) also accumulates in areas of the eyes such as the endothelial layer of the cornea and macula and is a major factor in chronic and degenerative eye conditions such as iritis, astigmatism, myopia, black streaks on retina, cataracts, macula degeneration, etc. Most of these condtions have been found to improve after amalgam replacement(35,212,271,322,etc.)

VI. Results of Removal of Amalgam Fillings

1. For the week following amalgam removal, body mercury levels increase significantly, depending on protective measures taken, but within 2 weeks levels fall significantly.(82,89) Chronic conditions can worsen temporarily, but usually improve if adequate precautions are taken to reduce exposure during removal.

2. Removal of amalgam fillings resulted in a significant reduction in body burden and body waste product load of mercury(75,82,88,89,93,95,115). Total reduction in mercury levels in blood and urine is often over 80% within a few months(79,82,89,93,115,57).

3. For the following case studies of amalgam replacement, some clinics primarily replaced amalgam fillings using patient protective measures and supportive supplements, whereas some clinics do something comparable to Hal Huggins total dental revision where in addition to amalgam replacement, patients gold or nickel crowns over amalgam are replaced by biocompatible alternatives, root canals extracted and cavitations checked for and cleaned. There are extensive documented cases (many thousands) where removal of amalgam fillings led to cure or significant improvement of serious health problems such as periodontal diseases(35,40,46,57,60,75,78,82,86, 87,90,94,95,100,101,115,133,168,212,222,233,271,313,317,321,322,376), oral keratosis(pre cancer)(87,251), immune system/ autoimmune problems (8,35,60,222,270,271,313,323,368,91,212,229,291,452), allergies(8,26, 35,40,46,94,95,97,165,212,222,228,229,233,271,317,322,349,376), asthma(8,75,97,222,228,271,322), chronic headaches/ migraines (5,34,35,95,212 222,229,233,271,317,322,349,354,115, 376,440,453), multiple chemical sensitivities (26,35,95,222, 229,232,233,115,313,368), epilepsy (5,35,309,229), blood conditions(212,222,232, 233,271,35,95), eczema (60,212,222, 271,313,317,323,94,376,341,459), chron's disease(222,229), stomach problems (35,95,212,222,228,229,233,271,317, 322,440,35), lupus(12,35,113,222, 229,233,323), dizzyness/vertigo(40,95,212, 222,271,322,376,453), arthritis(35,95,103,212, 222,271,313,322,358), MS(94,95,102,170,212,222,271,291,302, 34, 35,229), ALS(97,229,423,405,35), Parkinson's/ muscle tremor (222,248,229,271,212,94,98,35), Alzheimer's(204,35), muscular/joint pain/fibromyalgia (35,222,293,317, 322, 369,440,94), infertility(9,35,38, 229,367), depression (94,107,222,271,294,212,229,233,285e,317,322,376, 453,35,40), schizohprenia (294,34,35), insomnia(35,94,212,222,271,317,322,376), anger(212,233,102,35), anxiety & mental confusion (94,212,222, 229,233,271,317,322,440,453,35,57), susceptability to infections (35,40,222,251, 317,349,350), antibiotic resistant infection(251), endometriosis (229,35,38), Chronic Fatigue Syndrome (8,35,60,212,293,229,222, 232,233,271, 313,317,323,368,369,375,376,440), tachycardia and heart problems (205,35,59,94,115,212,222,232,233,271,306, 310, 212), memory disorders (35,94,212,222,440,453), cancer(breast,etc./ leukemia( 35,38,94,180), neuropathy/paresthesia (35,94,212,222,322), alopecia/hair loss (40,187,271,317,322,349),sinus problems (35,40,94, 222,271,322), tinnitus(35,94,222,271,349,376), chronic eye conditons: inflamation/iritis/ astigmetism/myopia /cataracts/macula degeneration (35,222,271,322), vision disturbances(35,212,271,322), psoriasis(323,385,375,408,459), skin conditions (212,222), urinary/prostrate problems(212,222), hearing loss(102,35), candida(26,35,404,etc.), PMS(35,6,etc.) diabetes(35,etc.), etc. The above over 60,000 cases of cure or significant improvements were not isolated cases of cures the clinical studies indicated a large majority of most such type cases treated showed significant improvement. Details available and case histories. Some of the above cases used chemical or natural chelation to reduce accumulated mercury body burden in addtion to amalgam replacement. Some clinics using DMPS for chelation reported over 80% with chronic health problems were cured or significantly improved(222,271,359). Other clinics reported similar success. But the recovery rate of those using dentists with special equipment and training in protecting the patient reported much higher succes rates than those with standard training and equipment, 97% versis 37 to 88%(435). The Huggins TDR protocol includes testing teeth with metal for level of galvanic current caused by the mixed metals, and removal of the teeth with highest negative galvanic current first(35). This has been found to improve recovery rate for chronic conditions like epilepsy and autoimmune conditions. Metals are being pushed into the body from negatively charged metal dental work with saliva as electrolyte and the highest charged teeth lose the most metal to the body(35).

Clinical studies have found that patch testing is not a good predictor of success of amalgam remvoal, as a high percentage of those testing negative also recovered from chronic conditions after rplacement of fillings(86,87,168,etc.).

The Huggins Clinic using TDR has suceessfully treated over a thousand patients with chronic autoimmune conditions like MS, Lupus, ALS, AD, diabetes, etc.(35), including himself with the population of over 600(approx. 85%) who experienced significant improvement in MS. In a large German study of MS patients after amalgam revision, extraction resulted in 85% recovery rate versis only 16% for filling replacement alone (222,302). Other cases have found that recovery from serious autoimmune diseases, dementia, or cancer may require more agressive mercury removal techniques than simple filling replacement due to body burden. This appears to be due to migration of mercury into roots & gums that is not eliminated by simple filling replacement. That such mercury(and simiarly bacteria) in the teeth and gums have direct routes to the brain and CNS has been documented by several medical studies(34,325,etc.).

Among those with chronic immune system problems with related immune antibodies, the types showing the highest level of antibody reductions after amalgam removal include glomerular basal membrane, thyreeoglobulin, and microsomal thyroid antigens(91). TDR and other measures used in metals detox have been found to increase T-cells and immune function in AIDS patients(35).

Swedish researchers have developed a sophisticated test for immune/autoimmune reactions that has proved sucessful in diagnosing and treating environmetally caused diseases such as lichen planus, CFS,MS, etc. related to mercury and other immunotoxics(60,313,etc.).

Interviews of a large population of Swedish patients that had amalgams removed due to health problems found that virtually all reported significant health improvements and that the health improvements were permanent(233). (study period 17 years) A compilation of an even larger population found similar results(212,282). For example 89% of those reporting allergies had significant improvements or total elimination extrapolated to U.S. population this would represent over 17 million people who would benefit regarding allergies alone.

VII. Tests for Mercury Level or Toxicity and Treatmenst

1. Feces is the major path of excretion of mercury from the body, having a higher correlation to systemic body burden than urine or blood, which tend to correlate with recent exposure level (35,36,79,80,183, 278). For this reason many researchers consider feces to be the most reliable indicator of daily exposure level to mercury or other toxics. The average level of mercury in feces of those with fillings is over 1 ppm and approx. 10 times that of a similar group without fillings (79,80,83,335,386,25,), with significant numbers of those with several filings having over 10 ppm and 170 times those without fillings(80). For those with several fillings daily fecal mercury excretion levels range between 20 to 200 ug/day. The saliva test is another good test for daily mercury exposure, done commonly in Europe and representing one of the largest sources of mercury exposure.

There is only a weak correlation between blood or urine mercury levels and body burden or level in a target organ(36,157,183,278,11,etc.). Mercury vapor passes through the blood rapidly(half-life in blood is 10 seconds, 370) and accumulates in other parts of the body such as the brain, kidneys, liver, thyroid gland, pituitary gland, etc. Thus blood test measures mostly recent exposure. As damage occurs to kidneys over time, mercury is less efficiently eliminated (11,36,57,183, 216,260), so urine tests are not reliable for body burden after long term exposure. Some researchers suggest hair offers a better indicator of mercury body burden than blood or urine(279), though still not totally reliable and may be a better indicator for organic mercury than inorganic. In the early stages of mercury exposure before major systemic damage other than slight fatigue results you usually see high hemoglobin, hemocrit, alkatline phosphatase, and lactice dehydroganese in later states you usually see marginal hemoglobin, hemocrit, plus low oxyhemoglobine(35).

Hair was found to be significantly correlated with fish consumption, as well as with occupational dental exposure and to be a good medium for monitoring internal mercury exposure, except that external occupational exposure can also affect hair levels. Mercury hair level in a population sampled in Madrid Spain ranged from 1.3 to 92.5 ppm. This study found a significant positive correlation between maternal hair mercury and mercury level in nursing infants. Hair mercury levels did not have a significant correlation with urine mercury in one study(340) and did not have a significant correlation to number of fillings(350). One researcher suggests that mercury levels in hair of greater than 5 ppm are indicative of mercury intoxication.

A new test approved by the FDA for diagnosing damage that has been caused by toxic metals like mercury is the fractionated porphyrin test(260,35), that measures amount of damage as well as likely source. Mercury blocks enzymes needed to convert some types of porphyrins to hemoglobin and adenosine tri phosphate(ATP). The pattern of which porphyrins are high gives an indication of likely toxic exposure, with high precoproporphyrin almost always high with mercury toxicity and often coproporphyrin.

Provocation challenge tests after use of chemical chelators such as DMPS or DMSA also are effective at measuring body burden(57,58), but DMPS can be dangerous to some people- especially those still having amalgam fillings or those allergic to sulfur drugs or sulfites. Many studies using chemical chelators such as DMPS or DMSA have found post chelation levels to be poorly correlated with prechelation blood or urine levels(57,115,303), but one study (340) found a significant correlation between pre and post chelation values when using DMPS. Challange tests using DMPS or DMSA appear to have a better correlation with body burden and toxicity symptoms such as concentration , memory, and motor deficits(290)- with many studies finding a significant correration between post chelation mercury level and the number of amalgam surfaces(57,172,173,222,290,292,273,303). Several doctors use 16 ug/L as the upper bound for mercury after DMPS challange, and consider anyone with higher levels to have excess body burdern(222,352). However one study(290) found significant effects at lower levels. Some researchers believe DMSA has less adverse side effects than DMPS and prefer to use DMSA for chelation for this reason. Some studies have also found DMSA as more effective at removing mercury from the brain(58). A common protocol for DMSA(developed to avoid redistribution effects) is 50 mg orally every 4 hours for 3 days and then off 11 days.

Another chelator used for clogged arteries, EDTA, forms toxic compounds with mercury and can damage brain function(307). Use of EDTA may need to be restricted in those with high Hg levels. N-acetylcystein(NAC) has been found to be effective at increasing cellular glutathione levels and chelating mercury(54). Experienced doctors have also found additional zinc to be useful when chelating mercury(222) as well as counteracting mercury's oxidative damage(43). Zinc induces metallothionein which protects against oxidative damage and increases protective enzyme activities and glutathione which tend to inhibit lipid peroxidation and suppress mercury toxicity(430). Also lipoic acid has been found to dramatically increase excretion of inorganic mercury(over 12 fold), but to cause decreased excretion of organic mercury(54) and copper. Lipoic acid has a protective effect regarding lead or inorganic mercury toxicity through its antioxidant proprties, but should not be used with high copper. Zinc is a mercury and copper antgonist and can be used to lower copper levels and protect against mercury damage.

2. Tests suggested by Huggins/Levy(35) for evaluation and treatment of mercury toxicity:
(a) hair element test(386) (low hair mercury level does not indicate low body level)(more than 3 essential minerals out of normal range indicates likely metals toxicity)
(b) CBC blood test with differential and platelet count
© blood serum profile
(d) urinary mercury (for person with average exposure with amalgam fillings, average mercury level is 3 to 4 ppm lower test level than this likely means person is poor excretor and accumulating mercury, often mercury toxic(35)
(e) fractionated porphyrin(note test results sensitive to light, temperature, shaking)
(f) individual tooth electric currents(replace high negative current teeth first)
(g) patient questionnaire on exposure and symptom history
(h) specific gravity of urine(test for pituitary function, s.g>1.022 normal s.g.< 1.008 consistent with depression and suicidal tendancies(35)>

3. Note: during initial exposure to mercury the body marshalls immune system and other measures totry to deal with the challange, so many test indicators will be high after prolonged exposure the body and immune system inevitably lose the battle and measures to combat the challange decrease- so some test indicator scores decline. Chronic conditions are common during this phase. Also high mercury exposures with low hair mercury or urine mercury level usually indicates body is retaining mercury and likely toxicity problem(35). In such cases where (calcium> 1100 or < 300 ppm) and low test mercury,manganese,zinc,potassium mercury toxicity likely and hard to treat since retaining mercury.

Test results indicating mercury/metals toxicity(35):
(a) white blood cell count >7500 or < 4500
(b) hemocrit > 50% or < 40%
© lynphocyte count > 2800 or < 1800
(d) blood protein level > 7.5 gm/100 ml
(e) triglycerides > 150 mg %ml
(f) BUN > 18 or < 12
(g) hair mercury > 1.5 ppm or < .4 ppm
(h) oxyhemoglobin level < 55% saturated
(I) carboxyhemoglubin > 2.5% saturated
(j) T lymphocyte count < 2000
(k) DNA damage/cancer
(l) TSH > 1 ug
(m) hair aluminum > 10 ppm
(n) hair nickel > 1.5 ppm
(o) hair manganese > 0.3 ppm
(p) immune reactive to mercury, nickel, aluminum, etc.
(q) high hemoglobin and hemocrit and high alkaline phosphatase(alk phos) and lactic dehydrogenese(LDA) during initial phases of expsoure with low/marginal hemoglobin and hemocrit plus low oxyhemoglobin during long term chronic fatigue phase.

4. Huggins Total Dental Revision Protocol(35):
(a) history questionnaire and panel of tests.
(b) replace amalgam fillings starting with filling with highest negative current or highest negative quadrant, with supportive vitamin/mineral supplements.
© extract all root canaled teeth using proper finish protocol.
(d) test and treat cavitations and amalgam tattoos where relevant
(e) supportive supplementation, periodic monitoring tests, evaluate need for further treatment(not usually needed).
(f) avoid acute exposures/challanges to the immune system on a weekly 7/14/21 day pattern.

note: after treatment of many cases of chronic autoimmune conditions such as MS, ALS, Parkinson's, Alzheimer's, CFS, Lupus, Rheumatoid Arthritis, etc., it has been observed that often mercury along with root canal toxicity or cavitation toxicity are major factors in these conditions, and most with these condtions improve after TDR if protocol is followed carefully(35). Other measures in addition to TDR that have been found to help in treatment of MS in clinical experience are avoidance of milk products, get lots of sunlight, supplementation of calcium AEP(448) and alpha lipoic acid(448b). Progesterone creme has been found to promote regrowth of myelin sheaths in animals(448c).

VIII. Health Effects from Dental Personnel Exposure to Mercury Vapor

1. It is well documented that dentists and dental personnel who work with amalgam are chronically exposed to mercury vapor, which accumulates in their bodies to much higher levels than for most non-occupationally exposed. Adverse health effects of this exposure including subtle neurological effects have also been well documented that affect most dentists and dental assistants, with measurable effects among those in the lowest levels of exposure. Mercury levels of dental personnel average at least 2 times that of controls for hair(397-401), urine(25d,57,64,69,99,123,124,138,171,173,222,249,290,362,397-399) and for blood (124,195,253,249,397). Sweden, which has banned use of mercury in fillings, is the country with the most exposure and health effects studies regarding amalgam, and urine levels in dental professionals from Swedish and European studies ranged from 0.8 to 30.1 ug/L with study averages from 3.7 to 6.2 ug/L (124,172,253,64,68). The Swedish safety guideline for mercury in urine is 5.6 nmol Hg/mmol(11.6 ug/L). Study averages for other countries ranged from 3.3 to 36 microgram/liter(ug/L)(69,70,171,290,397). A large survey of dentists at the Norwegian Dental Assoc. meeting(171) found that the mean mercury level in 1986 was 7.8 ug/L with approx. 16% above 13.6ug/L, and for 1987 found an average of 8.6 ug/L with approx. 15% above 15.8 ug/L, with women having higher levels than men in general. A U.S. national sample of dentists provided by the American Dental Association had an average of 5.2 ug/L (70,290). In that large sample of dentists, 10% of dentists had urine mercury levels over 10.4 ug/L and 1% had levels over 33.4ug/L(290,25c), indicating daily exposure levels of over 100 ug/day. Mercury excretion levels were found to have a positive correlation with the number of amalgams placed or replaced per week, the number of amalgams polished each week, and with the number of fillings in the dentist(171,172,173). In one study, each filling was found to increase mercury in the urine approx. 3%, though the relationship was nonlinear and increased more with larger number of fillings(124). Much higher accumulated body burden levels in dental personnel were found based on challenge tests than for controls(303), with excretion levels after a dose of a chelator as high as 10 times the corresponding levels for controls(57,69,290,303). Autopsy studies have found similar high body accumulation in dental workers, with levels in pituitary gland and thyroid over 10 times controls and levels in renal cortex 7 times controls(99,363,38). Autopsies of former dental staff found levels of mercury in the pituitary gland averaged as high as 4,040 ppb. They also found much higher levels in the brain occipital cortex(as high as 300 ppb), renal cortex(as high as 2110 ppb) and thyroid(as high as 28,000 ppb. In general dental assistants and women dental workers showed higher levels of mercury than male dentists (171,172,173,253,303,362).

Mercury levels in blood of dental professionals ranged from 0.6 to 57 ug/L, with study averages ranging from 1.34 to 9.8 ug/L (124,195,253,249). A review of several studies of mercury level in hair or nails of dentists and dental workers found median levels were 50 to 300% more than those of controls(38, p287-288,& 10,16,178). A group of dental students taking a course involving work with amalgam had their urine tested before and after the course was over. The average urine level increased by 500% during the course(63). Allergy tests given to another group of dental students found 44% of them were allergic to mercury(156). Studies have found that the longer time exposed, the more likely to be allergic. Another group of dental students had similar results(362), while another group of dental student showed comprimized immune systems compared to medical students. The total lympocyte count, total T cell numbers(CD3), T helper/ inducer(CD4+CD8-), and T suppressor/cytotoxic(CD4-CD8+) numbers were singinficantly elevated in the dental students compared to the matched control group(408). Similar results have been seen in other studies as well(408).

Urinary porphyrin profiles were found to be an excellent biomarker of level of body mercury level and mercury damage neurological effects, with coprorphyrin significantly higher in those with higher mercury exposure and urine levels(70,260). Coproporphyrin levels have a higher correlation with symptoms and body mercury levels as tested by challenge test(69,303), but care should be taken regarding challenge tests as the high levels of mercury released can cause serious health effects in some, especially those who still have amalgam fillings or high accumulations of mercury. Screening test that are less burdensome and less expensive are now available as first morning void urine samples have been found to be highly correlations to 24 hour urine test for mercury level or porphyrins(73).

2. The average dental office exposure affects the body mercury level at least as much as the workers on fillings(57,64,69,123,138,171,173,303), with several studies finding levels approximately the same as having 19 amalgam fillings(123,124,173). Many surveys have been made of office exposure levels(1,6,7,10, etc.) The level of mercury at breathing point in offices measured ranged form 0.7 to over 300 micrograms per cubic meter(ug/M3) (120,172,253,249). The average levels in offices with reasonable controls ranged from 1.5 to 3.6 ug/M3, but even in Sweden which has had more office environmental controls than others spot levels of over 150 ug/M3 were found in 8 offices(172). Another study found spot readings as high as 200 ug/M3 in offices with few controls that only used saliva extractor(120). OSHA surveys find 6-16% of U.S. dental offices exceed the OSHA dental office standard of 50 ug/M3, and residual levels in equipment sterilizers often exceed this level(454). The U.S. ATSDR mercury vapor exposure MRL for chronic exposure is much lower, 0.2 ug/M3 (217) (giving approx. 4 ug/day exposure), similar to U.S. EPA and Health Canada guidelines(2,209). Thus most office mercury levels were found to far exceed the U.S. guidelines for chronic mercury exposure.

Use of high speed drill in removal or replacement has been found to create high volume of mercury vapor and respirable particles, and dental masks to only filter out about 40 % of such particles(219,247). This produces high levels of exposure to patient and dental staff. Use of water spray, high velocity evacuation and rubber dam reduce exposure to patient and dental staff significantly, as seen in previous discussion. In addition to these measures researchers also advise all dental staff should wear face masks and patients be supplied with outside air(120,153). Some studies note that carpeting in dental offices should be avoided as it is a major repository of mercury(188,7)

Use of such measures along with a Clean-UpTM aspirator tip was found to reduce exposure to patient and staff approximately 90%(397).

3. Dentists were found to score significantly worse than a comparable control group on neurobehavioral tests of motor speed, visual scanning,and visuomotor coordination(69,70,123,249,290,395,1b), concentration , verbal memory, visual memory(68,69,70,249,290,395,1b), and emotional/mood tests(70,249,290,395,1b). Test performance was found to be proportional to exposure/body levels of mercury(68,70,249,290,395,1b). Significant adverse neurobehavioral effects were found even for dental personnel receiving low exposure levels(less than 4 ug/l Hg in urine)(290). This study was for dental personnel having mercury excretion levels below the 10th percentile of the overall dental population. Such levels are also common among the general population of non- dental personnel with several fillings. This study used a new methodology which used standard urine mercury levels as a measure of recent exposure, and urine levels after chelation with a chemical, DMPS, to measure body burden mercury levels. Thirty percent of dentists with more than average exposure were found to have neuropathies and visuogrphic dysfunction(395).
Chelators like DMPS have been found after a fast to release mercury from cells in tissue to be available for excretion. This method was found to give enhanced precision and power to the results of the tests and correlations. Even at the low levels of exposure of the subjects of this study, there were clear demonstrated differences in test scores involving memory, mood, and motor skills related to the level of exposure pre and post chelation(290). Those with higher levels of mercury had deficits in both memory, mood, and motor function compared to those with lower exposure levels. And the plotted test results gave no indication of there existing a theshhold below effects were not measurable. Mood scores including anger were found to correlate more strongly with pre chelation urine mercury levels while toxicity symptoms, concentration, memory(vocabulary,word), and motor function correlated more strongly with post-chelation mercury levels.

Several dentists have been documented to suffer from mercury poisoning(72,74,193,246,247,248,369), other than the documented neurological effects. One of the common effects of chronic mercury exposure is chronic fatigue due to immune system overload and activation. Many studies have found this occurs frequently in dentists and dental staff along with other related symtoms- lack of ability to concentrate, chronic muscular pain, burnout, etc.(249,369,377,378,1b). In a group of dentists and dental workers suffering from extreme fatigue and tested by the immune test MELISA, 50% had autoimmune reaction to inorganic mercury and immune reactions to other metals used in dentistry were also common(369). Tests of controls did not find such immune reactions common.

In another study nearly 50 % of dental staff in a group tested had positive autoimmune ANA titers compared to less than 1 % of the general population(35).

One dentist with severe symptoms similar to ALS improved after treatment for mercury poisoning(246), and another with Parkinson's disease recovered after reduction of exposure and chelation(248). Similar cases among those with other occupational exposure have been seen. A survey of over 60,000 U.S. dentists and dental assistants with chronic exposure to mercury vapor and anesthetics found increased health problems compared to controls, including significantly higher liver, kidney, and neurological diseases(99,193). Other studies reviewed found increased rates of brain cancer and allergies(99,193). Swedish male dentists were found to have an elevated standardized mortality ratio compared to other male academic groups(284). Dental workers and other workers exposed to mercury vapor were found to have a shortening of visual evoked potential latency and a decrease in amplitude, with magnitudes correlated with urine excretion levels(190). Dentists were also found to have a high incidence of radicular muscular neuralgia and peripheral sensory degradation(190,395).

4. Both dental hygienists and patients get high doses of mercury vapor when dental hygienists polish or use ultrasonic scalers on amalgam surfaces(240,400). Pregnant women or pregnant hygienist especially should avoid these practices during pregnancy or while nursing since maternal mercury exposure has been shown to affect the fetus and to be related to birth defects, SIDS, etc.(10,23,31c,37,38,110,142,146,401,19,31). Amalgam has been shown to be the main source of mercury in most infants and breast milk, which often contain higher mercury levels than in the mother's blood (20,61,112,186,287). Because of high documented exposure levels when amalgam fillings are brushed(182,222,348) dental hygienist are advised not to polish dental amalgams when cleaning teeth. Face masks worn by dental workers filter out only about 40% of small dislodged amalgam particles from drilling or polishing, and very little mercury vapor(247). Dental staff have been found to have significantly higher prevalence of eye problems, conjunctivitis, atopic dermatitis, and contact urticaria(247,156,74).

An epidemiological survey conducted in Lithuania on women working in dental offices(where Hg concentrations were < 80 ug/M3) had increased incidence of spontaneous abortions and breast pathologies that were directly related to the length of time on the job(277a). A large U.S. survey also found higher spontaneous abortion rate among dental assistants and wives of dentists(193), and another study found an increased risk of spontaneous abortions and other pregnancy complications among women working in dental surguries(277b). A study of dentist and dental assistants in the Netherlands found 50% higher rates of spontaneous abortions, stillbirths, and congenital defects than for the control group(394), with unusually high occurance of spina bifida.

A study in Poland also found a significant positive association between mercury levels and occurrence of reproductive failures and menstrual cycle disorders, and concluded dental work to be an occupational hazard with respect to reproductive processes(401).

5. Body burden increases with time and older dentists have median mercury urine levels about 4 times those of controls, as well as higher brain and body burdens(1,34, 68-74,99), and poor performance on memory tests(68, 69,70,249,290) Some older dentists have mercury levels in some parts of the brain as much as 80 times higher than normal levels(14,34,99). Dentists and dental personnel experience significantly higher levels of neurological, memory, musculoskeletal, visiomotor, mood, and behavioral problems, which increase with years of exposure (1,34,68-73,88,123,188,246,247,248,249,290,369,395). Even dental personnel with relatively low exposure(urine Hg<4 ug/l) were found to have significant neurological effects(290) and was found to be correlated with body burden of mercury. Most studies find dentists have increased levels of irritability and tension(1), high rates of drug dependancy and disability due to psychological problems(15,1b), and higher suicide rates than the general white population (284,1b), but one study found rates in same range as doctors.

6. Female dental technicians who work with amalgam tend to have increased menstrual disturbances (275,401,10,38), significantly reduced fertility and lowered probability of conception (10,24,38,121), increased spontaneous abortions (10,31,38,277,433), and their children have significantly lower average IQ compared to the general population (1,279,38,110). Populations with only slightly increased levels of mercury in hair had decreases in academic ability(3). Effects are directly related to length of time on the job(277). The level of mercury excreted in urine is significantly higher for female dental assistants than dentists due to biological factors (171,172,173,247,124a). Several dental assistants have been diagnosed with mercury toxicity and some have died of related health effects(32,245,246,247,248). From the medical register of births since 1967 in Norway, it can be seen that dental nurse/assistants have a clearly increased risk of having a deformed child or spontaneous abortion(433). Female dentists have increased rates of spontaneous abortion and perinatal mortality (193,38,10,433)),compared to controls. A study in Poland found a much higher incidence of birth defects among female dentist and dental assistants than normal(10). A chronically ill dental nurse diagnosed with mercury sensitivity recovered after replacement of fillings and changing jobs(60), and a female dentist recovered from Parkinson's after mercury detox(248). Some studies have found increased risk of lung, kidney, brain, and CNS system cancers among dental workers(14,34,99,143,283).

7. Many homes of dentists have been found to have high levels of mercury contamination used by dentists bringing mercury home on shoes and clothes(188).

IX. Scientists and Government Panels or Bodies That Have Found Amalgam Fillings to be Unsafe.

1. A World Health Organization Scientific Panel concluded that there is no safe level of mercury exposure(183,189,208). The Chairman of the panel, Lars Friberg stated that "dental amalgam is not safe for everyone to use(208,238). A study of dental personnel having very low levels of mercury excretion found measurable neurological effects including memory, mood, and motor function related to mercury exposure level as measured by excretion levels(290). and found no threshhold level below which effects were not measurable.. Other studies have found measurable effects to the immune, cardiovascular, hormonal, and reproductive systems from common levels of exposure(Section IV). Studies have found significant measurable adverse health effects at levels far below current government regulatory levels for mercury(290).

2. In 1987 the Federal Dept. of Health in Germany issued an advisory warning against use of dental amalgam in pregnant women(61). Most major countries other than the U.S. have similar or more extensive bans or health warnings regarding the use of amalgam, including Canada, Great Britain, France, Austria, Norway, Sweden, Japan, Australia, New Zealand, etc.(164,435) A Swedish National Mercury Amalgam Review Panel and a similar Norwegian panel found that "from a toxicological point of view, mercury is too toxic to use as a filling material"(164,435). Both countries have indicated plans to ban or phase out use of amalgam. A major amalgam manufacturer, Caulk Inc., advises that amalgam should not be used as a base for crowns or for retrograde root fillings as is commonly done in some coutries(387). A Swedish medical panel unanimously recommended to the government "discontinuing the use of amalgam as a dental material"(282). The U.S. EPA found that removed amalgam fillings are hazardous and must be sealed airtight and exposed of as hazardous waste(214). Most European countries require controls on dental waste amalgam emissions to sewers or air. A Canadian Government study for Health Canada concluded that any person with any number of amalgam fillings receives exposure beyond that recommended by the USPHS Standard(209). Many of those researching amalgam related health effects including several very prominent scientists have concluded that the health effects are widespread and serious so that mercury should not be used as a filling material (1,18,19,20, 36,38,57,60,61,88,94,99,125,148, 153,164,170,183,208, 209,210,212,222, 227,236, 238,282).

3. The Legislature of the State of California passed a law, Proposition 65, that requires all dentists in the state to discuss the safety of dental materials with all patients and to post the following warning about use of amalgam on the wall of their office:
"This office uses amalgam filling materials which contain and expose you to a chemical known to the State of California to cause birth defects and other reproductive harm".

Essentials of Human Anatomy & Physiology. Chapter 15. The Urinary System. Slides Lecture Slides in PowerPoint by Jerry L.

3 Functions of the Urinary System Regulate aspects of homeostasis Water balance Electrolytes Acid-base balance in the blood Blood pressure Red blood cell production Activation of vitamin D Slide 15.1b

4 Organs of the Urinary system Kidneys Ureters Urinary bladder Urethra Figure 15.1a Slide 15.2

5 Location of the Kidneys Against the dorsal body wall At the level of T 12 to L 3 The right kidney is slightly lower than the left Attached to ureters, renal blood vessels, and nerves at renal hilus Atop each kidney is an adrenal gland Slide 15.3

6 Coverings of the Kidneys Renal capsule Surrounds each kidney Adipose capsule Surrounds the kidney Provides protection to the kidney Helps keep the kidney in its correct location Slide 15.4

7 Regions of the Kidney Renal cortex outer region Renal medulla inside the cortex Renal pelvis inner collecting tube Figure 15.2b Slide 15.5

8 Kidney Structures Medullary pyramids triangular regions of tissue in the medulla Renal columns extensions of cortexlike material inward Calyces cup-shaped structures that funnel urine towards the renal pelvis Slide 15.6

9 Blood Flow in the Kidneys Figure 15.2c Slide 15.7

10 Nephrons The structural and functional units of the kidneys Responsible for forming urine Main structures of the nephrons Glomerulus Renal tubule Slide 15.8

11 Glomerulus A specialized capillary bed Attached to arterioles on both sides (maintains high pressure) Large afferent arteriole Narrow efferent arteriole Figure 15.3c Slide 15.9a

12 Glomerulus Capillaries are covered with podocytes from the renal tubule The glomerulus sits within a glomerular capsule (the first part of the renal tubule) Figure 15.3c Slide 15.9b

13 Renal Tubule Glomerular (Bowman s) capsule Proximal convoluted tubule Loop of Henle Distal convoluted tubule Figure 15.3b Slide 15.10

14 Types of Nephrons Cortical nephrons Located entirely in the cortex Includes most nephrons Figure 15.3a Slide 15.11a

15 Types of Nephrons Juxtamedullary nephrons Found at the boundary of the cortex and medulla Figure 15.3a Slide 15.11b

16 Peritubular Capillaries Arise from efferent arteriole of the glomerulus Normal, low pressure capillaries Attached to a venule Cling close to the renal tubule Reabsorb (reclaim) some substances from collecting tubes Slide 15.12

17 Urine Formation Processes Filtration Reabsorption Secretion Figure 15.4 Slide 15.13

18 Filtration Nonselective passive process Water and solutes smaller than proteins are forced through capillary walls Blood cells cannot pass out to the capillaries Filtrate is collected in the glomerular capsule and leaves via the renal tubule Slide 15.14

19 Reabsorption The peritubular capillaries reabsorb several materials Some water Glucose Amino acids Ions Some reabsorption is passive, most is active Most reabsorption occurs in the proximal convoluted tubule Slide 15.15

20 Materials Not Reabsorbed Nitrogenous waste products Urea Uric acid Creatinine Excess water Slide 15.16

21 Secretion Reabsorption in Reverse Some materials move from the peritubular capillaries into the renal tubules Hydrogen and potassium ions Creatinine Materials left in the renal tubule move toward the ureter Slide 15.17

22 Formation of Urine Figure 15.5 Slide 15.18

23 Characteristics of Urine Used for Medical Diagnosis Colored somewhat yellow due to the pigment urochrome (from the destruction of hemoglobin) and solutes Sterile Slightly aromatic Normal ph of around 6 Specific gravity of to Slide 15.19

24 Ureters Slender tubes attaching the kidney to the bladder Continuous with the renal pelvis Enter the posterior aspect of the bladder Runs behind the peritoneum Peristalsis aids gravity in urine transport Slide 15.20

25 Urinary Bladder Smooth, collapsible, muscular sac Temporarily stores urine Figure 15.6 Slide 15.21a

26 Urinary Bladder Trigone three openings Two from the ureters One to the urethrea Figure 15.6 Slide 15.21b

27 Urinary Bladder Wall Three layers of smooth muscle (detrusor muscle) Mucosa made of transitional epithelium Walls are thick and folded in an empty bladder Bladder can expand significantly without increasing internal pressure Slide 15.22

28 Urethra Thin-walled tube that carries urine from the bladder to the outside of the body by peristalsis Release of urine is controlled by two sphincters Internal urethral sphincter (involuntary) External urethral sphincter (voluntary) Slide 15.23

29 Urethra Gender Differences Length Females 3 4 cm (1 inch) Males 20 cm (8 inches) Location Females along wall of the vagina Males through the prostate and penis Slide 15.24a

30 Urethra Gender Differences Function Females only carries urine Males carries urine and is a passageway for sperm cells Slide 15.24b

31 Micturition (Voiding) Both sphincter muscles must open to allow voiding The internal urethral sphincter is relaxed after stretching of the bladder Activation is from an impulse sent to the spinal cord and then back via the pelvic splanchnic nerves The external urethral sphincter must be voluntarily relaxed Slide 15.25

32 Maintaining Water Balance Normal amount of water in the human body Young adult females 50% Young adult males 60% Babies 75% Old age 45% Water is necessary for many body functions and levels must be maintained Slide 15.26

33 Distribution of Body Fluid Intracellular fluid (inside cells) Extracellular fluid (outside cells) Interstitial fluid Blood plasma Figure 15.7 Slide 15.27

34 The Link Between Water and Salt Changes in electrolyte balance causes water to move from one compartment to another Alters blood volume and blood pressure Can impair the activity of cells Slide 15.28

35 Maintaining Water Balance Water intake must equal water output Sources for water intake Ingested foods and fluids Water produced from metabolic processes Sources for water output Vaporization out of the lungs Lost in perspiration Leaves the body in the feces Urine production Slide 15.29

36 Maintaining Water Balance Dilute urine is produced if water intake is excessive Less urine (concentrated) is produced if large amounts of water are lost Proper concentrations of various electrolytes must be present Slide 15.30

37 Regulation of Water and Electrolyte Reabsorption Regulation is primarily by hormones Antidiuretic hormone (ADH) prevents excessive water loss in urine Aldosterone regulates sodium ion content of extracellular fluid Triggered by the rennin-angiotensin mechanism Cells in the kidneys and hypothalamus are active monitors Slide 15.31

38 Maintaining Water and Electrolyte Balance Figure 15.9 Slide 15.32

39 Maintaining Acid-Base Balance in Blood Blood ph must remain between 7.35 and 7.45 to maintain homeostasis Alkalosis ph above 7.45 Acidosis ph below 7.35 Most ions originate as byproducts of cellular metabolism Slide 15.33a

40 Maintaining Acid-Base Balance in Blood Most acid-base balance is maintained by the kidneys Other acid-base controlling systems Blood buffers Respiration Slide 15.33b

41 Blood Buffers Molecules react to prevent dramatic changes in hydrogen ion (H + ) concentrations Bind to H + when ph drops Release H + when ph rises Three major chemical buffer systems Bicarbonate buffer system Phosphate buffer system Protein buffer system Slide 15.34

42 The Bicarbonate Buffer System Mixture of carbonic acid (H 2 CO 3 ) and sodium bicarbonate (NaHCO 3 ) Bicarbonate ions (HCO 3 ) react with strong acids to change them to weak acids Carbonic acid dissociates in the presence of a strong base to form a weak base and water Slide 15.35

43 Respiratory System Controls of Acid-Base Balance Carbon dioxide in the blood is converted to bicarbonate ion and transported in the plasma Increases in hydrogen ion concentration produces more carbonic acid Excess hydrogen ion can be blown off with the release of carbon dioxide from the lungs Respiratory rate can rise and fall depending on changing blood ph Slide 15.36

44 Renal Mechanisms of Acid-Base Balance Excrete bicarbonate ions if needed Conserve or generate new bicarbonate ions if needed Urine ph varies from 4.5 to 8.0 Slide 15.37

45 Developmental Aspects of the Urinary System Functional kidneys are developed by the third month Urinary system of a newborn Bladder is small Urine cannot be concentrated Slide 15.38a

46 Developmental Aspects of the Urinary System Control of the voluntary urethral sphincter does not start until age 18 months Urinary infections are the only common problems before old age Slide 15.38b

47 Aging and the Urinary System There is a progressive decline in urinary function The bladder shrinks with aging Urinary retention is common in males Slide 15.39

The digestive system eliminated waste from the digestive tract. But we also need a way to eliminate waste from the rest of the body.

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