Critical effective methods to detect genotoxic carcinogens and neoplasm-promoting agents.

Neoplasia in fish can result from contamination of waters with carcinogens and promoters. Cancer in fish, therefore, is a possible indicator of cancer risk to man and serves as a guide to the need for preventive approaches involving improved means of waste disposal and environmental hygiene. Moreover, cancer in fish indicates that this important food source may be contaminated. Detection of genotoxic carcinogens to which fish are exposed can be achieved quickly and efficiently by carefully selected batteries of complementary in vitro and in vivo bioassays. One such battery consists of the Ames test, a reverse mutation assay in prokaryotic Salmonella typhimurium, and the Williams test, involving DNA repair in freshly explanted metabolically highly competent liver cells from diverse species, including humans. Determination of DNA-carcinogen adducts by varied techniques, including 32P-postlabeling, as well as DNA breakage, mammalian cell mutagenicity, chromosome aberrations, sister chromatid exchange, or cell transformation represent additional approaches, each with its own advantages and disadvantages. More research is needed on systems to apprehend neoplasm promoters, but tests to determine interruption of intercellular communications through gap junctions appear promising. Other approaches rely on measurement of enzymes such as ornithine decarboxylase and protein kinase C. Approaches to the definition of risk to fish or humans require characterization of the genotoxic or nongenotoxic properties of a chemical, relative potency data obtained in select, limited rodent bioassays, and knowledge of prevailing environmental concentrations of specific carcinogens.


Introduction
Specific types of cancer are the major premature killing diseases in many parts ofthe world (1)(2)(3)(4)(5). In the Western World, the high incidence of cancer of the lung, pancreas, kidney, and bladder can be mainly attributed to cigarette smoking. Cancer of the oral cavity and esophagus are associated with tobacco chewing in the Western World and in India where the traditional chewing of a mixture of tobacco and betel nut has led to oral cavity cancer as the major neoplastic disease. In China and Japan, however, the customary intake of salted and pickled food, particulary fish, leads to risk for cancer of the esophagus and stomach, and in part, ofthe liver. In many parts ofAfrica, liver cancer is a major problem, with food mycotoxins and the hepatitis B antigen as causative agents. In the Western Wrld, the customary intake of appreciable amounts of fat has been associated with cancers of the breast, colon, ovary, endometrium, and pancreas. Thus, in many parts of the world, changes in lifestyle to avoid defined cancer risks have been recommended.
Historically, however, cancer in man was first documented to be due to an environmental cause through the study of cancer related to specific occupations, such as the scrotal cancers observed by Pott or the bladder cancers recorded by Rehn (3).
With increasing industrialization, especially with the growth of the chemical industry, questions arose as to whether the limited occupational cancers seen by Rehn occurred at an increasing rate due to contamination. Indeed, careless handling of chemicals, with consequent contamination of water with toxic agents, has led to serious adverse effects in sizable numbers ofpeople, such as in the case of Minamata, Japan (6), or more recently, the accidental mixing of polybrominated biphenyls with animal food in Michigan that led to the extensive occurrence ofthis toxicant in milk and in food reaching humans (7). Incidents ofdeliberate addition oftoxic agents to comestible oils in Spain and Turkey are other examples of undesirable and indeed criminal contamination ofthe human environment with toxic agents (8). In the U.S., the question ofhuman neoplasia stemming from water has been considered (9)(10)(11)(12).
Any toxic effect is the outcome ofthe occurrence in the human environment ofagents at dosages and chronicity ofexposure sufficient to lead to the syndromes observed. Concentrations most likely are highest directly at the site ofproduction or use, as for example, in the case of polybrominated biphenyls (PBBs) and congeners (13). Also, a critical review of the literature dealing with the occurrence of angiosarcoma of the liver in factory workers exposed to vinyl chloride has demonstrated that only reactor cleaners exposed chronically to several hundred parts per million or more had a high risk of cancer (14). Workers not exposed to such high concentrations so far have not displayed adverse effects.
In the context ofchemical production, a problem often unfortunately neglected is the disposal of raw or partially processed waste. One improper means of disposal has been to use bodies of water, rivers, lakes, or ocean estuaries. A number of studies in the last 25 years have reported that fish from rivers or harbors contaminated by industrial effluents displayed evidence of cancer, whereas similar fish caught in clean control rivers did not, as reported in detail at this and preceding conferences (15)(16)(17)(18)(19). Therefore, fish are indicators ofpotential problems for humans (20). At this time, in view of our extensive knowledge in toxicology and cancer causation, industries and municipalities must avoid the needless contamination of water with any waste product. Indeed, rational management in industry, supported by wise government actions, will find it not only safer but also profitable to make the investment in recycling waste products and producing valuable new materials. Alternatively, high temperature incineration yielding carbon dioxide, water, and acids such as nitric, sulfuric, and hydrochloric that can be absorbed by bases yielding marketable salts is a proper technique to be used anywhere in the world. Special facilities, including mobile ship-borne facilities have been engineered to accomplish such disposal safely. Governments and political bodies need to encourage such effective means of waste disposal. The world's growing population, generating increasing volumes of wastes, demands urgent effective disposal methods such as high temperature incineration, that in part return cost in the form of energy. The question often bandied about relative to the generation of dangerous dioxins (fortunately not by informed individuals) is not based on documented emissions and harms the effective implementation of reliable destruction ofhuman waste materials. Burial certainly is not sound, as has been shown by the broad contamination of bodies of water.
Monitoring systems need to be established to detect and quantitate carcinogens in the effluent from factories and private or public waste treatment plants. Since the limits of sensitivity of biological detection impose restrictions on the ability to prevent contamination ofthe environment, it will be important to monitor at points where the most concentrated contaminants arise or design means ofconcentrating potentially harmful products. For example, Hayatsu (21) has developed specific absorbent procedures for certain mutagens and carcinogens.

Mechanisms of Carcinogenesis and Rational Selection of Bioassay Systems
In the last few decades, it has been established that neoplastic diseases arise through a complex series of steps, beginning with the transformation ofnormal cells to abnormal cells at the genetic level through specific alterations of DNA (22). Cancer results from a somatic mutation. Rapid in vitro and in vivo bioassays have been developed to detect chemicals or radiation that can alter DNA and thus act as genotoxic carcinogens (23). Most human cancers due to occupational exposure (a small and declining proportion) and those due to lifestyle (the great majority) are caused by genotoxic carcinogens (24,25). In many instances, however, nongenotoxic epigenetic enhancing or promoting factors play an important role in eliciting invasive, metastatic neoplasms. The overall complex processes are outlined in Figure 1, and the ensuing logical classification ofcarcinogens is presented in lTble 1, but the reader is referred to more specialized reviews for details (22). Because most human cancers are caused by genotoxic carcinogens, knowing whether carcinogens are present in the environment makes the reliable detection and quantitation of genotoxic carcinogens an essential component of cancer prevention. Basically 'This assay has fewer false negatives or positives than other in vitro or in vivo-in vitro bioassays. bAniline, weakly carcinogenic at high dose levels because of slow poisoning of the hematopoietic system. C4-Acetylaminofluorene is unreliably positive in this and also in the Ames test. Carcinogenicity tests negative, but true carcinogenic risk unknown. dDiphenylnitrosanmne, considered a classic noncarcinogen, at high dose levels induced a small yieldofurinary bladder cancer in rats, through unknown mechanisms.
genotoxic potential: those using prokaryotic organisms and those using eukaryotic cell systems. An initial review of chemical structure (probable activity, or lack thereof) provides important background information and guidance to the selection of bioassay systems (22,23). A systematic decision point approach, providing qualitative and semiquantitative tests of increasing complexity, has been developed ( Table 2).

Prokaryotic Test Systems
The most widely practiced test in prokaryotic organisms is the reverse mutation in several strains of Salmonella typhimurium developed by Ames. Previously, Rosenkranz had demonstrated the use of repair-deficient E. coli (23). A large number of chemicals has been tested, especially in the Ames test (23). The readily performed standard tests, such as the Ames test, require an exogenous liver cell S-9 fraction to provide for metabolism, since most environmental carcinogens are procarcinogens and promutagens that must be metabolized to the reactive genotoxic product (26). However, the metabolic system ofthis liver fraction is inherently deficient in detoxification enzymes, which are available in vivo. Therefore, the Ames test presents a number of false positives. It is also not uniformly sensitive to all genotoxic agents, again, most likely because of the inadequacy of the S-9 fraction used. Even so, the Ames test is an economic, rapid, and valuable component of screening batteries. It has been used to study the occurrence of Ames-positive mutagens in water or in concentrates of water (27)(28)(29). Thus, a positive finding in the Ames test is essentially a warning that a potential, although certainly not an actual, cancer risk is present. Because ofthe occurrence of false positives, such as the plant component quercetin, which is positive in the Ames test but negative in other tests and negative in all carcinogen bioassays (30), the Ames test is not by itselfa predictor ofcancer risk but acts as a warning, calling for further exploration.

Eukaryotic Test Systems
Among the systems using eukaryotic cells, a reliable indicator for genotoxic carcinogens rests on the fact that such carcinogens damage DNA, leading to DNA repair. Williams (31) has used the broad metabolic competence of freshly explanted liver cells from rodent and human livers and the simultaneous presence in the cell of indicator DNA to develop a hepatocyte DNA repair test, using a cell system that metabolically resembles the in vivo situation (26,32,33). Thus, this test accurately mimicks the metabolic conditions to detect potential human risk factors (Table 3). A battery composed of the readily performed Ames test and the Williams test is a suitable set ofcomplementary tests to determine whether or not a given chemical or extract is genotoxic and thus may constitute a possible human cancer risk. The Williams test has also been adapted to an in vivo-in vitro situation where animals are given a chemical followed by excision ofthe liver and the determination of DNA repair in such livers (23,34). Some tests such as the determination of sister-chromatid exchange (SCE) present the advantage that they can be applied to the study of pre-exposed humans. Cell transformation tests, or others such as the lymphoma test, suffer from difficulties in execution and scoring (cell transformation) or lack of accurate responses with genotoxic agents, demonstrating too many false positives or negatives (lymphoma test). Any test system should be evaluated with known carcinogens of various chemical types and related noncarcinogens. Problems with those bioassay approaches and the underlying mechanisms have been critically reviewed (35).

Promoters and Enhancers
Many types ofhuman cancer including cancer ofthe lung from cigarette smoking and cancer ofthe breast or colon in individuals consuming high-fat diets involves not only the action ofgenotoxic carcinogens but also ofnongenotoxic, epigenetic, enhancing, and promoting elements that play crucial roles in the development of important human cancers. For example, tobacco smoke contains an acidic fraction composed ofphenolic substances that are not carcinogenic but have enhancing properties. These have significant functions since tobacco smoke contains relatively small amounts ofgenotoxic carcinogens (36,37). Likewise, in the nutritionally linked cancers, dietary fat translates to metabolic effects such as control of bile acid levels that promote colon cancer or effects on the endocrine system that enhance the risk for breast cancer. Here also, the putative genotoxic carcinogens are present in small amounts, so thatpromong elements are critical (38,39).
The mechanism of promotion is only partially understood.
Nonetheless, tion is highly dose dependent and reversible. This is the rationale for the lower lung cancer risk upon cessation of smoking; it is the basis for encouraging Western people to lower their total fat intake to lower their risk for the nutritionally linked diseases.
The occurrence ofcancer in fish unquestionably involves the presence ofgenotoxic carcinogens. For example, the neoplasms in gills may relate to contamination of water and sediments by polycyclic aromatic hyroarbons and similar products. Aflatoxin B, has been the main carcinogen incriminated in causing hepatocellular carcinoma in species such as trout. It is not yet known whether promotion operates in any type of fish or some types of fish, or not at all. Contamination of harbors and estuaries with complex petroleum wastes from ships and other sources may not only be the source ofpolycyclics but also promoting substances. The effect in fish, however, is not clear. For example, phenobarbital is a good promoter in the development of primary liver cancer in rats, but not in hamsters treated previously with a genotoxic carcinogen such as nitrosodiethylamine or 2-acetylaminofluorene. Future research, therefore, will need to delineate the role ofpromotion in carcinogenesis in fish. Promotion is often target-organ specific, a fact that needs to be considered in designing appropriate approaches. Such studies are important because a number of the water and especially bottom contaminants such as polychlorinated biphenyls (PCBs), chloroform, other halolkanes, trichloroethylene, or phenols most likely operate by a promoting mechanism.
Enhancement ofcarcinogenesis may stem from a cytotoxic action of a given chemical, leading to regeneration. This means there is increased DNA synthesis and mitosis, conditions favoring cancer production in the presence ofa genotoxic carcinogen. This type ofenhancement should not be defined as promotion but rather co-carcinogenesis due to cytotoxicity. Obviously, dose levels that are not cytotoxic are also not cocarcinogenic.
Promoters can exert their action and therefore can be tested tirough a number ofspecific mechanisms, such as dtose involving membrane effects or tirough the interruption ofcell-to-cell communication via gapjunctions (lable 4). The reader is referred to more specialized literature for detailed mehds (26,40,41).

Rodent Bioasays
The traditional chrnic bioassay in rodents is an importnt tool to examine whether or not a given chemical represents a cancer risk for man (42,43 in the treated animals represents the promotion phenomenon on an organ that already has the cellular genetic structure typical of a transformed cell documented by genetic analyses (44,45). Thus, even chronic bioassays in rodents need to be analyzed carefully as to underlying mechanisms. This in turn requires in vitro bioassays through the Ames and Williams test to assign genotoxicity or absence thereof. More fundamental, precise studies on DNA binding, DNA lability, and chromosomal changes aid in defining the genotoxic properties of a given chemical (46)(47)(48)(49)(50). Thus, the in vitro tests and biochemical studies necessarily precede a chronic rodent bioassay so as to be in a position to design the bioassay in the light of the findings made (Si). The chronic bioassay would serve to provide semiquantitative informationon the potency ofa given agent, once it has been established to be genotoxic. This is important, for in the absence of genotoxicity, quantitative risk assessment needs totally different parameters, including the question of dose-response relationships, the probable existence of a threshold with nongenotoxic agents, and above all, the reversibility of effects of such agents (22,51,52).
In relation to the question ofneoplasms found in fish growing and living in waste-contaminated waters, discussed at this conference, bioassays in specific types offish are ofgreat relevance.
Several previous recent reports (15)(16)(17)(18)(19)(53)(54)(55) have dealt with the problem, as well as with the necessary species-related and controlled biochemical activation of procarcinogens to reactive genotoxins through metabolism, demonstating that types offish studied differ from rodents and humans in this respect. Anders and associates (56) have provided interesting new concepts as to gene rearrangements and amplification in neoplasia tirough their detailed study of hybrids of Xiphophorus, a tropical fish originally found in Central America that develops melanomas and other neoplasms.

Conclusions
In summar, in the overall context ofcancer prevention, it is important to adjust lifestyle to avoid conditions with demonstrated adverse effects such as that oftobacco or excessive fat intake, obesity, or the relative deficiency of cereal fiber and vegetable consumption. Also, methods have been developed, based on sound knowledge ofthe mechanisms ofcarcnogenesis, that rapidly and accurately give qualitative information as to whether or not a given environmental chemical or mixture is genotoxic or has promoting potential. This permits improved control measures to be instituted and also effective designs for chronic aninul studies that will provide the basis for risk assessment and risk control.
In the context ofthis conference, it is also important to realize that among the sources ofprotein available to man, fresh or salt water fish represent one ofthe best nutritional resources available to humans (Ikble 5) (57-a. Another reason, doumented in the last 15 years, is at thetypeoffat present in seafood itself, namely, omega-3 fatty acids, is highly beneficial in maintaining desirable plasma cholesterol levels and thus avoiding heart diase risk, high blood pressure and stroke, and controlling the clotting process and avoiding emboli. It behooves all concerned to avoid contamination of rivers, lakes, and oceans with chemicals that would adversely affect such a valuable food resource and make it potentially hazardous to humans. Health Thble S. Benefb of ish 'm health promotion. promotion not only requires accurate knowledge ofenvironmental carcinogens, cocarcinogens, and promoters affecting fish and man, but also appropriate recycling and disposal ofhuman and animal wastes, not by burial and water disposal, but by effective high temperature combustion and simultaneous use of heat generated for electricity production, and recovering ofvaluable metal and glass. Medical and engineering research has provided sound facts and methods. It is essential and urgent that current knowledge be translated to a cleaner, more wholesome environment to ensure man's survival.
This investigation was suppoted by US PHS grants CA-17613, CA-24217, CA-42381, and CA-45720. We are grateful to C. Horn for excellent editorial assistance.