COOPERATIVE EXTENSION UNIVERSITY OF CALIFORNIA
ENVIRONMENTAL TOXICOLOGY NEWSLETTER

Vol. 6 No. 4 July 28, 1986

ANOTHER PERSPECTIVE

Introduction

Dr. Bruce Ames, known for his work in the development of short-term mutagenicity tests, gave a seminar on the Davis Campus on March 1, 1986. I wrote the following article based on notes I took during the seminar. This article was edited by Dr. Ames and he added some information I had missed during his seminar. He also sent a copy of the last article in this newsletter (Water Pollution, Pesticide Residues and Cancer) and graciously assented to its inclusion here. I would like to thank him for his contributions to this special edition. Please bear in mind throughout the following article this message Dr. Ames conveyed to the audience when comparing natural chemicals to human made chemicals; "I did this comparison not to show that these natural chemicals are dangerous, but to put things into perspective".

I.__Cancer_death_rates_in_humans.__1930-1985.

Dr. Ames started by presenting a slide showing age adjusted death rates for various human cancers during the last 50 odd years. He noted that the only one that showed a dramatic increase over time was lung cancer, and this is directly linked to smoking. (He also noted that lung cancer death rates for women are catching up to those for men and is a reflection of the increased numbers of women that smoke.) Stomach cancer death rates have decreased as have uterine and liver cancer death rates. Death rates from breast and prostate cancer have changed little over the last 50 years.

The point he made is that whatever is responsible for these cancers has been with us for a long time, even well before 1930 even, if we accept that it takes 10-30 years for carcinogens to produce their effect. He also expressed optimism that we are making progress in identifying the risk factors that are linked to these cancers so that we may be able to prevent them.

II.__The_development_of_tests_for_carcinogens_and_mutagens.

Dr. Ames pointed out that the rat and mouse carcinogenicity bioassays were first developed to help prove the link between occupational exposure to chemicals and cancer and also to help identify the chemicals that might cause cancer in workers exposed to high levels of industrial chemicals. He also stated that for that reason, the bulk of the chemicals tested in the carcinogenicity bioassay are man-made, not naturally occurring.

He spoke about his work in the development of the short-term in vitro assay that bears his name. The carcinogenicity bioassay in rats and mice takes about 3 years and may cost upwards of 1/2 million dollars. There was a need for a short-term, inexpensive test to screen thousands of compounds. Based on the assumption that carcinogenesis has to have a genetic component, he developed a test for mutagenicity which has a good correlation with the carcinogenesis bioassay results (not perfect, but pretty good). As 3000 laboratories are using his test, many naturally occurring chemicals have been screened and a world of mutagens have been discovered.

III.__Sources_of_mutagens_in_our_diet.

Dr. Ames went on to point out that there are manifold natural mutagens and other types of toxins in our diet and that we produce more mutagens by cooking foods. Coffee has hydrogen peroxide and methyl glyoxal both of which are carcinogens. Cola and beer have formaldehyde (also found in shrimp and fresh baked bread) which is also a carcinogen. Potatoes contain solanine (a toxic acetylcholinesterase inhibitor). Mustard and horseradish contain allyl isothiocyanate (a carcinogen). Alfalfa sprouts contain up to 2% by weight canavanine, an arginine analog (arginine is an amino acid) that is incorporated into proteins, and has been shown to be associated with the development of lupus erythematosis (an autoimmune disease) in monkeys. Black pepper contains a carcinogen, perhaps the piperine which is 1% of its weight. He presented quite a list of foods and the toxicants found in them and then mentioned that pesticide residues may also be found in foods. Using the U.S. market basket survey he said he calculated that the average daily intake of pesticide residues in the U.S. would be about 150 ug. He felt that this quantity is 10,000 fold less than the levels of naturally occurring pesticides in foods (such as solanine in potatoes). He expressed concern that there is something wrong in our perspective when we concentrate so much attention on this 150 ug and think nothing of the world of natural mutagens and carcinogens present in coffee and cooked foods in mg quantities.

IV.__Mutagens_produced_by_cellular_metabolism.

Dr. Ames discussed the production of mutagenic chemicals by cells as they go through the chemical reactions of metabolism (also known as life). In metabolism, oxygen is the electron acceptor and ultimately ends up as water. In this process however, H2O2 (hydrogen peroxide), .OH (hydroxyl radical), and O2- (superoxide) are formed. All of these are highly reactive mutagens, are known to be produced in cells and are also the active products of radiation. How do the cells protect themselves? Plants have beta-carotene which can scavenge radicals and singlet oxygen, another active form of oxygen formed by light. In addition, cells have enzyme systems (superoxide dismutase catalases and peroxidases) to detoxify these mutagens. In addition to these protective mechanisms, there are other protectants like vitamin C, uric acid, beta-carotene, and vitamin E which scavenge these reactive chemicals.

The point is that cells can and do have protective mechanisms in place to detoxify these natural mutagens. But they are not always effective, and DNA damage does occur. What then? Then DNA repair mechanisms come into play (and these are not 100% effective either).

Dr. Ames and his colleagues have developed means to measure the excretion of damaged DNA bases in the urine. (These would be the bases "hit" by the oxygen mutagens). Their calculations show that on the average, there are about 1000 "hits" per cell per day in humans, and about 15,000 hits/cell/day in rats. Rats have a much higher metabolic rate and age specific cancer rate and shorter lifespans than humans. He related this to current theories of aging that postulate that cells essentially "burn out" after expending so much metabolic energy and undergoing the associated damage caused by the reactive byproducts.

V.__Carcinogen_"hazard"_comparisons.

(Sometimes referred to in our department as the Peanut Butter Sandwich Toxicity or Chlorinated Tap Water Toxicity Index).

Dr. Ames then discussed a project he has worked on to help develop perspective about human exposure to carcinogens in relation to the doses used in rat and mouse carcinogenicity bioassays.

They started by doing intensive literature reviews to collect data on all carcinogenicity bioassays that have been completed. They then calculated a TD50 (tumor dose for 50%) of the test animals for each chemical; that is, the daily dose in mg/kg that would give 50% of the animals cancer. Using this data they then calculated the human exposure on a mg/kg basis, and expressed it as a percentage (x1000) of the rat TD50. Thus 100,000 would indicate a dose equivalent on a weight basis equal to the rat TD50. Any value lower than 100,000 is less than the rat TD50. (Keep in mind that the TD50 is for a daily dose every day of the rats lives.)

Based on this index, a glass of chlorinated tap water has an index (based on it's content of chloroform, a trihalomethane) of about 0.3. A glass of well water contaminated with trichloroethylene at about 3 ppm (the worst well in Silicon Valley), had an index of about 1 mainly due to the fact that TCE is substantially weaker than chloroform as a carcinogen. He stated "I'm just very skeptical of water being a serious contender for any kind of carcinogenic risk". Eating grains containing EDB had an index of about 0.4, about equivalent to a glass of chlorinated tap water. A few strips of bacon (nitrosamines) had an index of 9, and a peanut butter sandwich (aflatoxins at average 2 ppb) an index of 28. (FDA allows 10x this.) Dr. Ames also had good news for the beer drinkers of the world. Ethyl alcohol (in its various forms) is considered a human carcinogen of the liver, mouth and esophagus. It has recently been shown to cause cancer in rats which would give a daily bottle of beer on the scale an index of 2700 based on the rat data.

Dr. Ames also looked at 75 different pesticide residues and totaled them for their contributions based on the market basket survey. He reports an index of about 0.3 (similar to a glass of chlorinated tap water) for a few of those which have caused concern such as EDB, PCBs, DDE-DDT. After giving numerous examples using this index he asked; "Can we really take all this seriously?" He then stressed that the point he was trying to make is that we should worry less about pollution by man-made chemicals, considering the variety and amounts of natural carcinogens we ingest daily.

He then went on to stress that we really do need to be concerned about occupational, high level exposure to man-made chemicals (EDB for example).

VI.__Relevance_of_carcinogenicity_bioassay.

Dr. Ames explained that of all the chemicals tested in the cancer bioassay, over 50% were positive (carcinogenic). Only 42% were positive in both rats and mice. He then asked "What are we measuring in these tests?" Chemicals are tested at maximum tolerated doses (MTD) which are not necessarily non-toxic to other systems. There is a lot of evidence that chemicals stimulating cell proliferation (tissue damage does this) can increase the development of tumors. (This is true in the liver, thyroid, reproductive and respiratory tracts.) It is possible that many "carcinogens" only active at high doses, cause chronic tissue damage and repair. (At the SOT meeting in February of this year I attended a symposium during which a paper was presented on some preliminary data showing a definite correlation between acute toxicity (LD50) and the dose of chemical needed to produce cancer in experimental animals. This is not conclusive, but is surprising.)

WATER POLLUTION, PESTICIDE RESIDUES, AND CANCER

Adapted from November 11, 1985, Testimony of Professor Bruce N. Ames to Art Torres, California Senate Committee on Toxics and Public Safety Management

The carcinogens and pesticide residues currently being found in California water supplies, such as in Silicon Valley, are present in extraordinarily tiny amounts that, except in rare cases, are trivial relative to the background level of carcinogens in Nature. Therefore, I am convinced that such water pollution is irrelevant as a cause of human cancer.

The main current fallacy in our approach to such pollution consists in believing that carcinogens are rare and that they are mostly man-made chemicals. Quite the contrary is the case. My estimate is that over 99.99% of the carcinogens Californians ingest are from natural (e.g., substances normally present in food) or traditional sources (e.g., cigarettes, alcohol, and chemicals formed by cooking food).

Every meal has many carcinogens and when one compares the level of carcinogens in contaminated water or pesticide residues in food to the level of natural carcinogens also present in the diet, it is clear that water pollution or pesticide residues represent a trivial exposure by comparison.

Water pollution and pesticide residues are almost always present in the ppb (parts per billion) range. One part per billion (i.e., 1 person in all of China) is an extraordinarily small amount. By comparison, the carcinogens in a few common drinks are listed below. Every common drink contains carcinogens.

a) Coffee contains the known natural carcinogens hydrogen peroxide and methylglyoxal, each at about 4,000 ppb. b) Tap water contains the carcinogen chloroform at 83 ppb (U.S. average), as a consequence of chlorinating the water. c) Cola drinks contain the carcinogen formaldehyde at 7,900 ppb, though this is not much higher than human blood, which averages about 3,000 ppb in formaldehyde from normal metabolism. d) Beer contains nitrosamines, formaldehyde (700 ppb), and alcohol (50 million ppb, or 5%), all known carcinogens. Alcohol consumption is a known cause of human cancer (3% of U.S. cancer) and ethyl alcohol is a carcinogen in rats. e) Milk contains a high percentage of fat, and high fat consumption has been implicated in human breast and colon cancer and rodent cancer (though milk is an important source of calcium, which may be important as an anticarcinogen). And f) fruit juices may have various amounts of carcinogenic mold toxins, depending on how many moldy fruits were processed.

Calculating a possible hazard to humans from information obtained from a cancer test on rats must take into account the potency of the carcinogen in rats as well as the human dose. We are just completing a study where we compare possible hazards for humans due to typical daily intake of carcinogens, adjusting for the potency of each carcinogen from the animal data. This adjustment is necessary because the potency of carcinogens varies over a million-fold, e.g., aflatoxin, a mold carcinogen that is present in small amounts in peanut butter (2 ppb U.S. average) or in corn products such as tortillas, requires about a million times smaller dose to cause the same incidence of cancer in test animals as trichloroethylene, which was the main contaminant in Silicon Valley wells. I give some comparisons below.

Contaminated_water. The level of carcinogens in contaminated well water (e.g., trichloroethylene in Silicon Valley or Woburn, Massachusetts) only rarely involves a possible hazard more than that of ordinary chlorinated tap water. Of 35 private wells shut down in Silicon Valley because of their supposed carcinogenic hazard in an EPA study, only two were of greater possible hazard than ordinary tap water (well water usually lacks the chloroform present in chlorinated tap water), and the most polluted well (2,800 ppb trichloroethylene) is still at least 1,000 times less of a possible hazard than an equal volume of cola, beer, or wine. This is because trichloroethylene is an extremely weak carcinogen. It is comparable to saccharin and only 10 times more potent than alcohol, which is present at about 50 million ppb in a beer. Given that the consumption of tap water is only about 1 or 2 liters per day, it seems unlikely that man-made water pollution in the trace amounts typically seen is causing more than a minimal hazard.

Pesticide_Residues. Man-made pesticide residues present in our food amount to about 100 ppb on the average; most of these residues are composed of non-carcinogenic compounds. The man- made carcinogenic residue of most interest in food is likely to be DDT and its metabolite DDE. The possible hazard of the DDT/DDE of the average U.S. daily intake is equivalent to that of the chloroform in 1 glass of tap water and is insignificant compared to natural carcinogens in our diet. Even an occasional highly DDT/DDE- or PCB-contaminated fish (e.g., 100 times the average level) would contribute a possible hazard that is comparable to the average peanut butter sandwich and is small compared to other very common minimal risks such as a glass of beer.

Nature's_pesticides. We are ingesting natural pesticides in our diet in amounts at least 10,000 times more than man-made pesticide residues. Natural pesticides are natural toxic chemicals, which are present in all plants, usually making up 5- 10% of a plant's weight. They have an enormous variety of chemical structures, though only a few are present in each plant species. Their function is protection against fungi, insects and animal predators. Thus, a major aspect of evolution of plants is chemical warfare. There has been relatively little research in the toxicology or carcinogenicity of these compounds until quite recently, and so, very few of the large number present in the human diet have been tested in animal cancer bioassays. A fair percentage of those few that have been tested have turned out to be carcinogens in rats or mice. They include estragole (in basil), safrole (in herbs), symphitine (in comfrey tea), psoralens (in parsley and celery), hydrazines (in mushrooms), and allyl isothiocyanate (in mustard). The possible carcinogenic hazard of Nature's pesticides completely overshadow the traces of man-made pesticide residues found in the daily diet. Plants also contain anticarcinogens and valuable nutrients, so I believe that even these possible hazards seem too small to worry about, particularly given the points made below.

Skepticism_about_extrapolating_risks_from_rodents. All calculations of human risk based on rat and mouse cancer tests, both from natural and man-made carcinogens, are hypothetical. Thus, they should be taken with a great dose of skepticism, unlike known human carcinogens such as smoking (400,000 deaths from cancer, heart disease, etc., per year in the U.S.) or alcoholic beverages (100,000 deaths from cancer, etc. per year). There are many new reasons for being skeptical of uncritical low- dose extrapolation of risk to humans from animal data obtained by feeding enormous doses, which can't be discussed here. This is reinforced by the studies of cancer epidemiologists who are making considerable progress in understanding the smoking, dietary, hormonal, viral, and occupational contributions to human cancer, but who are finding remarkably little solid evidence for any significant contribution from pollution.

Since we now know that carcinogens are common, not rare (over half of the chemicals tested in rats or mice were judged carcinogens), we must of necessity ignore the trivia if we wish to deal with the important causes of cancer. We might possibly eliminate every trace of man-made carcinogen from our water or food supply, but it would cost an enormous amount of California's wealth, be of minimal relevance to causes of human cancer, and distract health workers from real, more important risks. Thus, one can either chase after parts per billion of every man-made carcinogen that turns up or have some sensible regulations about pollution. We should also remember that, except for the increase in cancer due to smoking, age-adjusted cancer death rates are decreasing (stomach, uterus, liver) or have not increased, and that life expectancy increases every year.

WATER, Vol. 27, Number 2 (Summer 1986).

Arthur L. Craigmill
Toxicology Specialist
U.C. Davis