COOPERATIVE EXTENSION
UNIVERSITY OF CALIFORNIA
ENVIRONMENTAL TOXICOLOGY NEWSLETTER
Vol. 2 No. 4 March 17, 1982
GENETIC TOXICOLOGY
Introduction
Last fall the Food Protection and Toxicology Center at the University of California, Davis held a symposium entitled "Genetic Toxicology: An Agricultural Perspective". During the winter quarter the Department of Environmental Toxicology at the University of California, Davis, has hosted a series of seminar speakers in the area of chemical mutagenesis and carcinogenesis.
This newsletter represents a distillation of much of the information presented at the symposium and in some of the seminars this winter. I would like to thank Dr. Dennis Hsieh, Professor of Environmental Toxicology for his assistance with the preparation of this article.
Genetic toxicology is the study of the toxic effects of chemicals on genetic material. The focus of genetic toxicology is on heritable defects in somatic (body) cells and germ cells (reproductive cells). Changes in gene structure and function in somatic cells is widely accepted to be a basic mechanism for the production of cancer (oncogenesis). Changes in structure or function of reproductive cells may result in:
Genetic changes can be classified according to the type of change that occurs to the genetic material. The first type of genetic toxicity is mutation. Mutations occur when a base pair in DNA is deleted, added, or substituted. These mutations are sometimes called point mutations because they affect single units of DNA structure. Slight mutational events may be very difficult to analyze unless the consequences are severe. The second type of genetic toxicity is clastogenesis. Clastogenesis occurs when segments of chromosomes containing many base pairs are added, deleted, or substituted. Clastogenic changes may be picked up by carefully studying chromosomal banding patterns in cell materials. The third type of genotoxic effect is aneuploidization which results when intact chromosomes are added or deleted. One of the best known examples of aneuploidy is Down's Syndrome (mongolism) in humans which results from an extra chromosome 21.
Genotoxic effects may also be classified as: (1) chromosomal abnormalities (2) dominant gene mutations (3) recessive gene mutations or (4) polygenic mutations. Chromosomal abnormalities are one of the most easily recognized forms of genotoxic effects because they can be seen under the microscope as changes in the number or structure of chromosomes. The incidence of chromosomal abnormalities in the normal human population is approximately 0.5%, or 1 in 200 persons. Dominant gene mutations are easily recognized because they are always expressed in the individual that carries that gene. There are over 1000 different recognized types of dominant gene mutations in humans and the total incidence is approximately 0.5%. Recessive gene mutations are not expressed in offspring unless each parent contributed a recessive gene to the child. There are over 1000 recessive genetic abnormalities in humans and it is estimated that the incidence of recessive genes in the general population is from 5 to 7.5%. Polygenic disorders depend on a number of genes for expression. Diabetes and epilepsy are two human diseases that are thought to have polygenic components. These are the most difficult to chart out because so many may be involved.
Because the basic mechanism of cancer involves genetic changes in somatic cells, genotoxicity testing is currently used as a screening procedure for potential carcinogenic effects. We already seem to have a high incidence of genetic damage under natural conditions, thus we would not want to introduce any chemical agent into our environment which might increase the rate of mutations in humans. The incidence of birth defects in the human population is approximately 7% of all live births. Some teratologists estimate that 80% of birth defects may be genetic and that 50% of all spontaneous abortions may be due to genetic defects that result in intrauterine death of the fetus. It would be impossible to document human genetic damage due to chemical exposure until long after the exposure had occurred. For this reason it is advisable to screen compounds for potential genetic effects in order to prevent exposure to genotoxic agents whose effects might not appear for two to three generations.
Genotoxicity Tests
The genotoxicity tests that will be discussed here are short-term tests that have been found useful for indicating chronic toxicity measured in long-term tests.
The types of tests that are used to detect genotoxic effects fall into 3 categories: (1) bacterial (2) mammalian cell culture (3) whole animal. The most well known bacterial genotoxicity test is the Ame's Assay. In the Ame's Assay well characterized strains of bacteria are exposed to chemicals and the mutation rate in the presence and in the absence of chemicals compared. Because bacteria may reproduce many times in a single day, the generation time is extremely short and mutations can be discovered easily by examining their growth characteristics. In order to better mimic the biochemical conditions in mammals, mixtures of mammalian enzymes are sometimes introduced along with the chemical in order to determine if the chemical is activated by metabolism to a mutagenic agent. If a genotoxic effect does occur, the affected bacteria will grow differently from the original untreated bacteria. One of the advantages of the Ame's Assay is that it is inexpensive and allows the researcher to experiment with many different dose levels of chemicals and compare their potency with respect to one another. It should be stressed that the potency of a compound in the Ame's Assay does not necessarily correlate with its potency in other genotoxic testing systems. A positive result in the Ame's Assay does not necessarily indicate carcinogenic activity or mutagenic activity in other test systems.
Another in vitro test system that is used is the measurement of DNA repair in non-replicating mammalian cells, a process called unscheduled DNA synthesis. In normal cells, when DNA is damaged by a mutagen, it is often repaired. The amount of repair may be used as a measure of the amount of genetic damage inflicted. Radioactive bases are added to mammalian cell cultures along with suspect genotoxic agents. If a suspect agent damages DNA in the cells, the repair process will incorporate the radioactive material into the new segment of DNA, and the amount of radioactivity can then be used as a measure of DNA repair. The fact that DNA can be repaired indicates that genotoxic effects can be repaired by normal cells. In order to increase the sensitivity of the above-mentioned Ame's Assay, the strains of bacteria used are those which cannot repair their DNA after it is damaged.
Mammalian cells can be cultured in a way similar to bacteria and the effects of genotoxic chemicals studied on these cells in vitro. Mammalian test systems certainly have more relevance than bacterial systems due to the fact that they are animal and not plant cells. Mammalian cell cultures can be exposed to different doses of suspect genotoxic agents and examined for unscheduled DNA synthesis, chances in chromosome structure, and changes in cell structure. These tests are more expensive and harder to perform, and are usually not performed unless a positive result is obtained in bacterial systems. If a positive result is also obtained in mammalian cell cultures, suspect chemicals may then be tested in whole animals.
In vivo genotoxicity tests are usually done using insects or mammals. The most common insect test system is the fruit fly because its genetics are so well known. Mammalian test systems which utilize whole animals must be considered the most valid test systems for predicting potential genotoxic effects in humans. They are also the most expensive to perform and complex to analyze. One well known mammalian test system is the dominant lethal test. In this test, male rats are exposed to doses of a suspect genotoxic agent and are then sequentially mated to numerous female rats. These female rats are then killed after 12.5 days gestation and the number of viable and resorbed fetuses is counted. In rats when a fetus dies it is not aborted but it is resorbed into the mother and it is easy to determine the number of resorbed fetuses by opening the uterus and looking for the resorbtion sites. The theory behind the dominant lethal assay is that increased numbers of resorbed fetuses should indicate genotoxic effects to the male rats spermatozoa which result in intrauterine fetal death. Because this is a whole animal test there are numerous factors which can influence the outcome of it and for this reason, it is often called into question with respect to its accuracy in predicting genotoxic effects in humans.
Whole animals may also be treated with various doses of suspect genotoxic agents, their cells removed, and the genetic material inside them examined. One such method that is often used to measure genetic damage in humans is the number of chromosome breaks in white blood cells. This is a simple test which requires removal of a small quantity of blood, separation of the white blood cells, and culturing in supportive media. Special preparations of these white blood cells can then be made so that the chromosomes in them can be examined microscopically. Chromosome breaks and chromosome banding patterns can be examined and compared to normal patterns for other humans. Single chromosome breaks are virtually meaningless. Multiple chromosome breaks may be significant since they can lead to rearrangements of genetic material in cells. Unfortunately, the relationship of chromosomal breaks to human health effects is very tenuous. Therefore, increased numbers of chromosomal breaks may be of little significance with respect to the health of the person involved.
Another type of test that can be performed using mammalian cells is the sister chromatid exchange (SCE test). SCE does not result in morphological changes in chromosome structure. It simply represents the exchange of old and new arms during the duplication of the chromosomes. The newly synthesized chromosomal material can be stained and easily differentiated from the old. Increased numbers of sister chromatid exchanges can result from genotoxic agents, however, the relationship between SCE and cancer or birth defects is very tenuous. The mechanism of sister chromatid exchange is unknown, but it is a relatively sensitive test for genotoxic agents, however, its relationship to human health is even more tenuous than chromosome breaks.
Another way to monitor genetic damage in humans is through monitoring sperm morphology. Sperm counts do not necessarily relate to genotoxic effects, however, sperm morphology is a better indication of genotoxic effects in mammals. In addition, sperm morphology does not correlate with spontaneous abortion rates. Some of the problems with measuring abnormal sperm morphology is the fact that there is a great variation between observers and raters of sperm morphology, and the fact that it is often difficult to find cooperative donors. It may be a very useful indicator of genotoxic effects in humans if it could be followed over long periods of time starting before exposure to suspect chemicals.
Evaluation of Bacterial Genotoxicity Tests
Genotoxic action in a bacterial test system does not definitely indicate genotoxicity in mammalian test systems. Positive results in bacterial assays are useful in pointing out the necessity of further testing. Negative genotoxicity in bacterial test systems does not necessarily mean that a chemical will be negative in mammalian test systems. All of these genotoxicity test systems are screening tests and therefore will have false positives and false negatives. Genotoxicity data's greatest use currently is for indicating carcinogenic potential and the necessity for further carcinogenicity tests. Because of the number of chemicals in the environment, the in vitro procedures are best used to prioritize chemicals which may require further testing. Regulatory agencies in the United States and other countries recognize the need for screening procedures of this nature. As time goes by, these tests become more validated with respect to their usefulness in predicting carcinogenicity. At this time, it is still necessary to perform chronic feeding trials on animals for pesticides and other chemicals that enter the environment.
(Taken from the NTP (National Toxicology Program) Technical Bulletin, January 1982, #6)
RAPID IN VITRO TEST CAPABILITIES -- Short-term (bacterial (microbial) genotoxicity) tests and combinations of tests have been proposed to predict the potential carcinogenicity of chemicals. However, current data are not sufficient, particularly across chemical classes, to correlate short-term test results with known carcinogenicity so that the limits of predictability can be accurately judged. A primary emphasis of the Cellular and Genetic Toxicology Branch (of the National Toxicology Program) is to develop, evaluate, and validate test systems which will contribute to a data base that should permit clear decisions about the relative value of a proposed test. To serve the broader objectives of the NTP, it is important to provide short-term test information which can be used by the experimental design groups and for establishing the priority of chemicals to be entered into long-term carcinogenicity bioassays. The five broad classes of in vitro short-term tests are:
DNA synthesis (rat hepatocytes)
This group of tests was chosen because the basic categories of genotoxic effects can be detected, the tests are readily available, and the accepted protocols have been subjected to some form of evaluation or validation. At present there is inadequate evidence to select or justify any specific assay system as a prescreen for carcinogens or mutagens. The use of the above group should not be interpreted as a position on the part of the NTP of endorsing any specific battery of biologically based tests. Rather, it is intended that the results provide a reasonable profile of the genetic toxicity of a chemical.
ERRATA
Furadan Toxicity in Pennsylvania Cattle
A group of 20 dairy heifers were being raised for herd replacements. The owner acquired an open, partly-full bag with a commercial mineral mix label from a neighbor. Assuming the contents to be a mineral mix, he top dressed 2-3 tablespoonfuls per head on grain and corn silage in a feed bunk.
The following day he found 8 animals dead and several others showing bloat, blood-tinged froth on the muzzle, salivation, tremors and diarrhea. The attending veterinarian made a tentative diagnosis of organophosphate or carbamate toxicity.
Analysis of the "mineral mix" revealed that the material was carbofuran, FuradanR.
This incident illustrates the importance of positively identifying all feed ingredients and all form chemicals.
-LJH, Extension Veterinarian, PSU
(Penn. State Univ., Coop. Ext. Veterinary News - Vol. 82, No. 1, January 1982)
AMPHIBIAN HIGHS .... Booze and Newts Don't Mix. A recent issue of the Journal of the American Medical Association reported two cases of poisoning from the Oregon rough-skinned Newt. A 29-year-old man died after drinking whiskey and then swallowing a newt on a dare. After about two hours, the daring young man went into cardiopulmonary arrest. He was resuscitated, but died later in the day. The skin of the Oregon rough-skinned newt contains "Tetrodotoxin", a powerful neurotoxin that blocks the conduction of nerve signals, leading to a muscular paralysis and respiratory arrest--usually the cause of death. In 1971 a similar incident occurred in Brookings, Oregon when a 26-year-old man swallowed five newts. He had been drinking beer and consumed the amphibians on a bet. Fortunately he vomited and survived the poisonings. One study on the toxicity of the newt reported that a 10 gr newt specimen contained enough neurotoxin to kill 1500 white mice. Residents of Northern California and Oregon should not be too surprised if they find newt-infested streams and ponds posted: WARNING: Amphibians may be intoxicating and harmful to your health. Do not use with alcohol or while driving or operating machinery.
(The Toxicology Newsletter - Vol. 8, No. 3, December 1981)
Use of Lead Tetroxide as a Folk Remedy for Gastrointestinal Illness
In June 1981, a 4-month-old Mexican-American infant was admitted to Olive View Medical Center in Los Angeles County with a 12-hour history of vomiting and diarrhea. Initially, the stools were watery and green; however, later bouts of diarrhea contained fresh blood.
The mother repeatedly denied having given any medicinal substance to the baby but when the potential danger of this unknown substance was explained, she admitted that a baby healer had given the infant an orange powder known in Mexico as azarcon.
The bloody diarrhea gradually subsided over a period of 4 days and when the baby showed no other evidence of poisoning, he was discharged on June 10.
At 2 local county clinics, patients and their families were questioned about azarcon. Many were aware of the substance, and related that it is used in small doses for empacho (chronic indigestion) and other gastrointestinal illnesses. It is readily available and a sample purchased recently in Tijuana, Mexico, was identified as lead tetroxide by FDA analysis.
It is unknown at this time how common the use of azarcon is. A survey is currently in progress to determine its availability and use in Los Angeles County.
(Morbidity and Mortality Weekly Report - Vol. 30, No. 43, November 6, 1981)
Notice: Some "subscribers" to this newsletter will have received mailing list update postcards. These were not sent to most specialists and farm advisors and they will continue to receive the newsletter. Starting with the next newsletter, the updated mailing list will be in effect.
Arthur L. Craigmill, Ph.D.
Extension Toxicologist
Environmental Toxicology and Veterinary Extension
University of California
Davis, CA 95616
(530)752-1142