Lecture Outline: Environmental & Chemical Mutagens
Background information
About 10% of human disease is caused by inherited defects; these arose from
mutations and any increase in mutation will increase the frequency of genetic
defects.
There are over 70,000 man made chemicals available commercially, including
fertilizers, preservatives, pesticides herbicides, drugs, etc. that we may ingest.
Many natural compounds that may occur in our air, food and water are also
potential mutagens.
Until recently, chemicals were not tested before being released; the Delaney
Amendment assures that any that are to be used as food additives must be
tested for carcinogenicity, and EPA regulations require environmental impact
assessments, so at least toxicity may be detected.
When tested, about 90% of proven carcinogens are mutagenic, and
as we will see in a later lecture, mutations in certain genes can
cause cancer.
Even essential compounds may be carcinogenic at high doses.
examples: Calcium at >5X the recommended dose can increase
mutations;
selenium at >5 ppm is carcinogenic, although low levels are
anticarcinogenic.
Why is it so difficult to identify potential mutagens/carcinogens?
Mutations do occur spontaneously.
Each of the 4 bases in DNA will briefly exist as a rare tautomer
where the single and double bonds in the ring structure rearrange.
Each rare tautomer pairs with the wrong partner purine or
pyrimidine.
Since mutations can occur spontaneously in the absence of added mutagens,
it is virtually impossible to say that any one mutation was the result of a
specific mutagen; however it is possible to prove that some chemicals
increase the number of mutations.
Dose responses may not be linear
Tests are often made on 50 male and/or 50 female rats given
very high doses of the compound, and extrapolations are then
made to low doses.
The actual response may not be linear, and may even have a
"threshold level" below which damage is not found.
Normal repair mechanisms may break down when overloaded.
Unexpected, non-chemical effects may occur:
Example; rats fed 5% of their diet as MSG (ACCENT) tended to
develop kidney tumors.
Autopsies showed that at high doses, needle-like crystals of MSG
were present in kidneys; these damaged cells, induced more cell
divisions etc. Almost any mitogen (agent that increases mitosis)
leads to increased rate of cancer.
Other chronic irritants and inflammatory agents such as asbestos
fibers and hepatitis B virus may act this way to increase risk of
cancer.
Interactions may be important
BHT, a food preservative (see your next Twinkies wrapper) is not
mutagenic, but if it is added to a test system along with a known
mutagen, many more mutations often result
Compounds that inhibit an error prone DNA repair system may
decrease mutation rates, but inhibition of accurate repair system
while leaving error-prone repair active will increase mutation
rates.
Metabolism can alter effective doses and compounds
Normal enzymes that help protect our cells by breaking down
toxic compounds can produce highly reactive intermediates.
AHH (aryl hydrocarbon hydrolase), an inducible liver enzyme
functions in association with C y t o c h r o m e P 4 5 0 to break down
many long chain and ring-structured compounds that may be
toxic but not necessarily mutagenic by first making an epoxide
ring; the epoxides are very reactive and can damage DNA.
Examples:
Aflatoxins, produced by fungi that are often found in moldy
grain, may end up in peanut butter and beer for example.
Persons who eat lots of raw peanuts have an increased risk of
stomach cancer.
Benzpyrene, a byproduct common in tobacco smoke is also
present in almost any burned biological material (biopsies of
lungs from cancer patients at MD Anderson showed that 76 0f
78 had very high levels of AHH; cause or effect?)
Compounds that are mutagenic may be rapidly broken down to
harmless compounds so can be less harmful than predicted
Metabolic differences may be important
In rat tests, about 25% of the animals converted cyclamates, the
artificial sweetener used before saccharin, to cyclohexamines, that
are very mutagenic (as detected by broken chromosomes in tissue
culture tests). If humans do the same, many people would be at
increased risk, so the product was banned. About 1991, further
tests did not find any evidence for conversion of cyclamates to
cyclohexamines in humans
Effects may not be apparent for years
DES) was used as a drug during pregnancy in
Diethyl stilbestrol (D
the 1950s to prevent miscarriage and premature births.
Epidemiological data revealed that daughters exposed in utero had
much increased risk of cervical and ovarian cancer in their 20's
Exposed male fetuses started showing an increased frequency of
testicular cancer 40 years after exposure.
Rats metabolize and age at a higher rate (7X) than humans, so
effects may show up in a few years.
A Canadian study using a cancer prone strain of rats (repair
defective), fed saccharin to male and female rats at .05% to 5% of
diet;
In the second generation of lifetime feeding, sons of exposed
mothers had increased leukemias and lymphomas.
(Males metabolic rate > females)
Rats with increased % of diet as saccharin had low blood sugar,
so ate more rat chow and did not lose weight.
Extrapolation of the rat study data to the human population
based on the average amount of saccharin consumed at the
time, would suggest 4/1000 might be affected; this ignores that
the tests were made on a cancer prone strain
This is why saccharin now carries a warning label.
Overall, about 333/1000 person will eventually get some form
of detectable cancer; > 100 of these can be attributed to
smoking.
Improved tests for detecting mutagenicity
Now, a tier of tests, beginning with a bacterial test called the "Ames test" (Bruce
Ames developed the test) is generally employed first. Only chemicals that pass
this test are likely to be tested on tissue culture cells or rodents if the chemical
will be used in a way that will to lead to public exposure. (Potential drugs are
carried on if not too risky in comparison to potential good. For example,
isoniazid used for treatment of TB is a weak mutagen.)
His- strains of S. typhimurium are used. His- strains are histidine
auxotrophic mutants that require histidine, one of the 20 amino
acids needed for making protein in order to grow. Normal
Salmonella cells can make histidine. These mutant bacteria die
unless histidine is added to the growth medium, or unless they
have a second mutation that restores them to his+.
H i s - cells will not grow on minimal medium, only
the "mutant" cells that revert to his+ can grow
Different strains test different types of point mutations:
All types of transitions or transversions can be defined
TA base pair to AT
AT base pair to GC
Add or subtract one base to counter opposite type of mutation
(frameshift)
Strains are available with or without cell walls to see if the
compound can't get in the cell
tests can be made with and without rat liver extract to see if
AHH-type enzymes cause conversion to mutagenic compounds (or
destroy mutagenicity)
The test is made by mixing the his- bacteria (≈109/ml) and the
compound, or simply spreading the bacteria evenly on minimal
medium and adding a drop of the compound: any increase of the
number of colonies that grow compared to control plates is usually
obvious
1
2
3
4
Presence of excess numbers of colonies on a plate of medium where
there is no histidine indicates a back mutation has occurred. In the
example above, the high number of colonies seen around test tab two
shows it contains a mutagenic compound.
Examples of results of AMES tests
"TRIS", a flame retardant chemical that was added to infant
garments to satisfy Ralph Nader's consumer safety group, was
found to be mutagenic; it was absorbed through the skin and
sucked out of the garment by the infants to the level that
mutagenicity could be detected in the urine.
Dioxin, a contaminant of Agent Orange used in Viet Nam as a
defoliant is not mutagenic in the Ames test (A related byproduct,
TCDD, is very carcinogenic). Many tests and lawsuits have
resulted. Dioxin is extremely toxic to some organisms and less to
others. Contact causes chloracne, i.e. disfigured skin that takes a
long time to heal and dioxin is absorbed and stored in fat if
ingested. Air Force men who handled Agent Orange the most
have higher levels in their bodies than those less exposed, but do
not have increased rates of cancer, at least so far in ongoing tests.
(Some tests have twins where one was exposed and other not).
Despite lack of evidence for mutagenicity, millions of $ have been
awarded to "victims", and whole towns have been evacuated and
abandoned because of presence of dioxin in road oil.
More recently, dioxin has been shown to be present in some paper
products, including milk cartons.
Natural food components, if extracted and tested alone, may be
mutagenic
piperine from pepper, caffeic acid from fruits (the amount in an
apple is 500X as mutagenic, on a per apple dose, if tested alone,
than the ALAR that can no longer be applied and caused such a
scare (Neither is very mutagenic)
Ames estimates that 99.9% of the chemical mutagens we are
exposed to are natural compounds and this does not include Radon
gas
antimutagenic" compounds that reduce
Foods also contain "a
mutation rates; most are antioxidants such as ascorbate, carotenoids,
alpha-tocopherol etc found in fruits and vegetables (it is estimated
that high fruit and vegetable diets (5 servings per day) cuts the
lifetime risk of cancer in half)
Reactive oxygen compounds created during metabolic reactions in
our cells damage about 10,000 bases/cell in our DNA every day.
Most are obviously repaired, but this is thought to be a factor in
aging
h t t p : / / p o t e n c y . b e r k e l e y . e d u / t e x t / d r u g m e t r e v . h t m l for an
See
interesting discussion of comparative mutagenic risks.
A photograph of a plate usid in an Ames test can be seen at:
http://people.ne.mediaone.net/jkimball/BiologyPages/A/AmesTest.html
Proven Chemical Mutagens:
D i r e c t a c t i n g c h e m i c a l s react with DNA to alter bases and thus
their pairing properties
Example 1; H N O 2 = n i t r o u s a c i d = N A
removes the amino group on the ring of purines and pyrimidines
and replaces it with =O For example, if an A is subjected to
oxidative deamination, it is converted to hypoxanthine, which
pairs with C. After repair or replication the net effect is to change
a base pair from AT to GC.
NA can cause transitions in either direction ie AT
GC
where the purine in one strand is replaced by the other purine
NA is also involved in indirect mutagnicity:
At low pH, as in the stomach, nitrites are equivalent to NA and
interact with secondary amines, including the amino acid proline,
to form nitroso-amines
The nitrosoamines formed are broken down by P450 type
enzymes and can form very reactive, highly mutagenic
intermediates.
Nitrite is used as a meat preservative; in red meat, it reacts with
hemoglobin (Hb) to make metHb which keeps the meat looking
red and fresh.
In cultures that use a lot of nitrite, especially to preserve fish
which does not have much Hb to bind the nitrite, rates of stomach
cancer are higher than in other cultures
So why use it? It is very effective against Clostridium botulinum,
the bacterium that produces the toxin that causes botulism
poisoning, which is often fatal; it is a trade off! Allowable limits
have been greatly reduced in recent years
Some vegetables and natural water supplies also have nitrites
present; However most of the nitrite most of us ingest probably is
formed in our saliva by bacteria that convert nitrates to nitrites!
Example 2; alkylating agents such as mustard gas, EMS, and
nitrosoguanidine (NG)
Alkylating agents react with bases to add a methyl (CH3-) group
to the ring
http://dtp.nci.nih.gov/docs/static_pages/compounds/37364.html
can cause transitions and transversions,- where a purine is
replaced by a pyrimidine in the DNA
Mustard gas was the first proven chemical mutagen. This was
shown during WWII by Charlotte Auerbach in England, but since
it was being used as a weapon, the discovery not announced until
later (it is very caustic and destroys lungs if breathed).
NG is one of the most potent mutagens ever discovered; although it is not very
toxic, it usually causes many base changes instead of one. It is also extremely
carcinogenic; I do not allow it in my lab!
http://ntp-server.niehs.nih.gov/htdocs/8_RoC/RAC/MNNG.html
B ase analo g s: Compounds that are so similar to the normal purines
and pyrimidines that they can be mistakenly be inserted into DNA
during replication or repair can also lead to increased rates of
mutation. The best known example is 5-bromo uracil (5BU). This is
a pyrimidine that is almost identical to T, but has a Br attached to the
5' carbon instead of CH3-. The eletronegative bromine atoms allow
rearrangements of the double and single bonds in the 5BU to occur
more readily than they do in T. When 5BU is in the rare tautomer
form - maybe 1% of the time, it is actually more like a C and will pair
with G and not A.. Thus if 5BU can be incorporated into DNA as
either a T or a G, and it can lead to later changes too. Compounds like
5BU are often used in chemotherapy for cancer; the idea is that they
will only be incorporated into the DNA of actively replicating cells eg
cancer cells primarily, and thus will tend to cause defects in theses
cells, hopefully leading to cell death. Of course other dividing cells
can also be sensitive and thus we see side effects such as the loss of
hair.
A c r i d i n e d y e s : Acridines are planar molecules that seem to mimic
a base pair, not just one base. They tend to intercolate between the
base pairs of a DNA double helix, but are not attached to the
phosphate sugar backbone. This seems to confuse repair enzymes
into sometimes adding a real base pair or removing a real base pair,
so that the primary effect of acridine mutagenicity is the addition or
deletion of a single base pair. Acridines differ in their side chains.
One of the best known is ethidium bromide which fluoresces pink
under UV light when it is intercolated into a DNA double helix.
Another is ICR 171, a compound that has been used as an anti-tumor
agent.
You can see the structure of compounds mentioned by going to
http://chem.sis.nlm.nih.gov/chemindex.html
Click on ChemIDplus and and then search for the compound by name
(ex. ICR 171, 5-Bromo Uracil or class (acridine). Once it appears, you
can enlarge it and view it as a "3D" structure.