The non-genotoxicity to rodents of the potent rodent bladder carcinogens o-anisidine and p-cresidine.

The two potent rodent bladder carcinogens o-anisidine and p-cresidine, and the structurally related non-carcinogen 2,4-dimethoxyaniline, have been extensively evaluated for genotoxicity to rodents and found to be inactive. Most data were generated on o-anisidine, an agent that is also only marginally genotoxic in vitro. The two carcinogens induced methaemoglobinaemia in rodents indicating that the chemicals are absorbed and metabolically oxidized. Despite their total lack of genotoxicity in vivo, the two carcinogens have the hall-marks of being genotoxic carcinogens given that most test animals of both sexes of B6C3F1 mice and F344 rats are reported to have succumbed rapidly to malignant bladder cancer. No reasons for this dramatic conflict of test data are so far apparent. The experiments described involve, in one or other combination, 2 strains of mice (including B6C3F1) and 4 strains of rat (including F344), the use of oral and i.p routes of exposure and observations made after 1, 3 or 6 doses of test chemical. 6 tissues (including the rat bladder) were assayed using 3 genetic endpoints (unscheduled DNA synthesis, DNA single-strand breaks and micronuclei induction). Aroclor-induced rats were employed in one set of experiments with o-anisidine. In the case of one set of mouse bone-marrow micronucleus experiments the same batch of the 3 chemicals as used in the cancer bioassays, and the same strain of mouse, were used. Possible further experiments and the implications of these findings are discussed.     

Chemical carcinogens. A review and analysis of the literature of selected chemicals and the establishment of the Gene-Tox Carcinogen Data Base. A report of the U.S. Environmental Protection Agency Gene-Tox Program

The literature on 506 selected chemicals has been evaluated for evidence that these chemicals induce tumors in experimental animals and this assessment comprises the Gene-Tox Carcinogen Data Base. Three major sources of information were used to create this evaluated data base: all 185 chemicals determined by the International Agency for Research on Cancer to have Sufficient evidence of carcinogenic activity in experimental animals, 28 selected chemicals bioassayed for carcinogenic activity by the National Toxicology Program/National Cancer Institute and found to induce tumors in mice and rats, and 293 selected chemicals which had been evaluated in genetic toxicology and related bioassays as determined from previous Gene-Tox reports. The literature data on the 239 chemicals were analyzed by the Gene-Tox Carcinogenesis Panel in an organized, rational and consistent manner. Criteria were established to assess individual studies employing single chemicals and 4 categories of response were developed: Positive, Negative, Inconclusive (Equivocal) and Inconclusive. After evaluating each of the individual studies on the 293 chemicals, the Panel placed each of the 506 chemicals in an overall classification category based on the strength of the evidence indicating the presence or absence of carcinogenic effects. An 8-category decision scheme was established using a modified version of the International Agency for Research on Cancer approach. This scheme included two categories of Positive (Sufficient and Limited), two categories of Negative (Sufficient and Limited), a category of Equivocal (the evidence of carcinogenicity from well-conducted and well-reported lifetime studies had uncertain significance and was neither clearly positive nor negative), and three categories of Inadequate (the evidence of carcinogenicity was insufficient to make a decision, however, the data suggested a positive or negative indication). Of the 506 chemicals in the Gene-Tox Carcinogen Data Base, 252 were evaluated as Sufficient Positive, 99 as Limited Positive, 40 as Sufficient Negative, 21 as Limited Negative, 1 as Equivocal, 13 as Inadequate with the data suggesting a positive indication, 32 as Inadequate with the data suggesting a negative indication, and 48 Inadequate with the data not suggesting any indication of activity. This data base was analyzed and examined according to chemical class, using a 29 chemical class scheme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemical carcinogens a review and analysis of the literature of selected chemicals and the establishment of the Gene-Tox carcinogen data base: A report of the U.S. environmental protection agency Gene-Tox program

The literature on 506 selected chemicals has been evaluated for evidence that these chemicals induce tumors in experimental animals and this assessment comprises the Gene-Tox Carcinogen Data Base. Three major sources of information were used to create this evaluated data base: all 185 chemicals determined by the International Agency for Research on Cancer to have Sufficient evidence of carcinogenic activity in experimental animals, 28 selected chemicals bioassayed for carcinogenic activity by the National Toxicology Program/National Cancer Institute and found to induce tumors in mice and rats, and 293 selected chemicals which had been evaluated in genetic toxicology and related bioassays as determined from previous Gene-Tox reports. The literature data on the 239 chemicals were analyzed by the Gene-Tox Carcinogenesis Panel in an organized, rational and consistent manner. Criteria were established to assess individual studies employing single chemicals and 4 categories of response were developed: Positive, Negative, Inconclusive (Equivocal) and Inconclusive. After evaluating each of the individual studies on the 293 chemicals, the Panel placed each of the 506 chemicals in an overall classification category based on the strength of the evidence indicating the presence or absence of carcinogenic effects. An 8-category decision scheme was established using a modified version of the International Agency for Research on Cancer approach. This scheme included two categories of Positive (Sufficient and Limited), two categories of Negative (Sufficient and Limited), a category of Equivocal (the evidence of carcinogenicity from well-conducted and well-reported lifetime studies had uncertain significance and was neither clearly positive nor negative), and three categories of Inadequate (the evidence of carcinogenicity was insufficient to make a decision, however, the data suggested a positive or negative indication). Of the 506 chemicals in the Gene-Tox Carcinogen Data Base, 252 were evaluated as Sufficient Positive, 99 as Limited Positive, 40 as Sufficient Negative, 21 as Limited Negative, 1 as Equivocal, 13 as Inadequate with the data suggesting a positive indication, 32 as Inadequate with the data suggesting a negative indication, and 48 Inadequate with the data not suggesting any indication of activity.This data base was analyzed and examined according to chemical class, using a 29 chemical class scheme. The major chemical classes represented were: acyl, alkyl and aryl halides (38 chemicals); alcohols and phenols (28 chemicals); alkyl and aryl epoxides (20 chemicals); amines, amides and sulfonamides (70 chemicals); aromatic azo, azide, azoxy, diazo, hydrazo and nitrile chemicals (28 chemicals); aziridines, nitrogen and sulfur mustards (25 chemicals); carbamates, dicarboximides, thioureas and ureas (21 chemicals); metals and organometallics (41 chemicals); nitroalkanes, nitroaromatics, nitrofurans, nitroimidazoles and nitroquinolines (23 chemicals); nitrosamines (19 chemicals); and polycyclic aromatic hydrocarbons and dihydrodiol derivatives (57 chemicals). The Gene-Tox Carcinogen Data Base provides a basis for future in-depth analyses of genetic toxicology bioassay systems with regard to their ability to predict the carcinogenic effects of chemicals.     

Evaluation of genetic alterations in cancer-related genes in lung and brain tumors from B6C3F1 mice exposed to 1,3-butadiene or chloroprene

1,3-Butadiene and chloroprene are multisite carcinogens in B6C3F1 mice with the strongest tumor response being the induction of lung neoplasms in females. Incidence of brain tumors in mice exposed to 1,3-butadiene was equivocal. This article reviews the efforts of our laboratory and others to uncover the mechanisms of butadiene and chloroprene induced lung and brain tumor responses in the B6C3F1 mouse. The formation of lung tumors by these chemicals involved mutations in the K-ras cancer gene and loss of heterozygosity in the region of K-ras on distal chromosome 6, while alterations in p53 and p16 were implicated in brain tumorigenesis.     

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC publication No. 13. The need for biological risk assessment in reaching decisions about carcinogens

The prudent assumption that carcinogen bioassays in rodents predict for human carcinogenicity is examined. It is suggested that in certain cases, as for example the induction of tumors against a high incidence in controls, or in situations in which high dose toxicity may be a critical factor in the induction of cancer, the probability that animal bioassays predict for humans may be low. The term 'biological risk assessment' is introduced to describe that part of risk assessment concerned with the relevance of specific animal results to the induction of human cancer. Biological risk assessment, which is almost entirely dependent on an understanding of carcinogenesis mechanisms, is an important addition to present mathematical modeling used to predict the effects of animal carcinogens that have been demonstrated after high dose exposure, to the effects of the much smaller doses to which humans are perceived to be exposed. Evidence for the conclusions reached by biological risk assessment may sometimes be supported by a careful review of human epidemiological data.     

Implications for genetic toxicology of the chromosomal breakage syndromes

The activation of oncogenes and our knowledge of the chromosome breakage syndromes show that both intragenic mutations and chromosomal aberrations are important in carcinogenesis. Each suggests that an agent could produce genetic changes in a tissue without producing cancer there, if the types of genetic change do not match: chromosomal aberrations may be irrelevant in the mammary epithelium but be very significant in the bone marrow, and vice versa. This has vital implications for genetic toxicology: (1) both gene mutations and chromosomal aberrations should be measured, and (2) carcinogens may be mutagenic in tissues in which they are not carcinogenic. One might therefore expect in vivo assays for mutagenicity to correlate rather well with cancer bioassays; unfortunately, the bioassays themselves seem faulty. If cancer bioassays are valid, they would be reproducible. If bioassays are reproducible, they would be internally consistent. The information supplied by Tennant et al. (1987) for their validation of in vitro assays gives data from both sexes in rats and mice for 70 chemicals. When the data are analyzed site-by-site, positive results were not replicated in the other sex or in the other species much of the time: in half the cases the other sex does not give the same result; in two-thirds of the cases the other species does not give the same result. There are 3 potential explanations for these differing results: (1) genuine sex-specific carcinogens are common, (2) genuine species-specific carcinogens are common, or (3) the bioassay does not replicate well, i.e., is erratic. The third possibility best explains the data. The apparent inability of short-term in vitro tests to discriminate well between carcinogens and non-carcinogens may be more a reflection of the cancer bioassays that were used to determine which chemicals were carcinogenic than any defect in the assays. In this situation in vivo assays can scarcely be expected to do better even if they are better.     

An analysis of the Gene-Tox Carcinogen Data Base

The Gene-Tox Carcinogen Data Base is an evaluated source of cancer data on 506 chemicals selected in part for their previous assessment in genetic toxicology bioassays. This data base has been analyzed for the distribution of these chemicals into chemical classes. The major chemical classes (6% or greater of the total data base) are: acyl-, alkyl-, and aryl-halides; alcohols and phenols; aliphatic and aromatic amines, amides, and sulfonamides; benzene-ring-containing chemicals; organo-lead, -mercury, -phosphorous compounds, metals and derivatives, phosphoric acid esters, and phosphoramides; and polycyclic aromatic hydrocarbons. Cancer studies representing a subset of the Gene-Tox Carcinogen Data Base, 199 chemicals which were rated as Sufficient Positive/Negative or Limited Positive/Negative, were examined for distribution of those studies by animal species, gender, route of chemical administration, duration of study, major tumor sites, and major tumor types. These analyses revealed that the Gene-Tox Carcinogen Data Base contains a large number of lifetime studies involving the rat and mouse treated by oral routes of administration. The major organs that were targets were: liver, lung, skin, forestomach, bladder, and mammary gland, while the major tumor types were: carcinoma, sarcoma, papilloma, and adenoma. Chemicals in the data base have been assessed for species-specific carcinogenic effects, and these results indicate that for mice and rats there is a high correspondence (85%). This number is higher than that (71%) reported by Tennant et al. (1986) based on the recent results of 72 chronic cancer bioassays performed by the National Toxicology Program. This difference is probably based on the nature of the chemicals selected for inclusion in both data bases. Although the absolute value of this correspondence is unknown, it would seem to be within this range. When chemicals in the Gene-Tox Carcinogen Data Base were examined for their previous evaluation in 73 genetic toxicology bioassays, only 26 of these bioassays had 30 or more chemicals. In these 26 bioassays, the prevalence of positive chemicals was generally greater than 80-90%. This suggests that a thorough evaluation of genetic toxicology bioassays in regard to their ability to predict carcinogenic effects in animals is premature at this time.     

In vivo short-term assays for tumor initiation and promotion in the glandular stomach of Fischer rats

Here we summarize the data on 55 compounds tested in in vivo short-term assays for tumor-initiating and tumor-promoting activity in the glandular stomach of male Fischer (F344) rats. Most of the data has been previously published. Tumor-initiating activity was assayed by measuring the induction of unscheduled DNA synthesis (UDS) and DNA single strand scission; tumor-promoting activity was assayed by measuring the induction of ornithine decarboxylase (ODC) activity, increased replicative DNA synthesis (RDS), and of c-fos and c-myc oncogene expression. The compounds were orally administered. Twenty-nine compounds were tested for UDS. Eight were positive, including 5 glandular stomach carcinogens; 16 were negative, including 5 liver carcinogens; and 5 were equivocal. Twenty compounds were tested for DNA single strand scission. Twelve were positive, including 6 glandular stomach carcinogens; 7 negative, including 2 liver carcinogens; and 1 was equivocal. Thirty-two compounds were tested for RDS. Twenty-six were positive, including 8 glandular stomach carcinogens and 6 glandular stomach tumor-promoters; 4 were negative, including 3 liver carcinogens and a stomach irritant; and 2 were equivocal. Forty-five compounds were tested for ODC. Thirty-seven were positive, including 8 glandular stomach carcinogens and 6 glandular stomach tumor promoters; 7 were negative, including 3 liver carcinogens; and one was equivocal. All glandular stomach carcinogens and tumor-promoters examined were positive in both RDS and ODC. Two compounds were tested for c-fos and c-myc expression; one was a glandular stomach carcinogen and one was a glandular stomach tumor promoter, and both were positive. In addition, 2 compounds inhibited the increase in RDS induced by the tumor promoter NaCl, suggesting anti-tumor-promoter activity. Thus these assays are useful for assessing potential tumor-initiating and tumor-promoting activity in the rat glandular stomach.     

Definitive relationships among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by the U.S. NTP.

An analysis is presented in which are evaluated correlations among chemical structure, mutagenicity to Salmonella, and carcinogenicity to rats and mice among 301 chemicals tested by the U.S. NTP. Overall, there was a high correlation between structural alerts to DNA reactivity and mutagenicity, but the correlation of either property with carcinogenicity was low. If rodent carcinogenicity is regarded as a singular property of chemicals, then neither structural alerts nor mutagenicity to Salmonella are effective in its prediction. Given this, the database was fragmented and new correlations sought between the derived sub-groups. First, the 301 chemicals were segregated into six broad chemical groupings. Second, the rodent cancer data were partially segregated by target tissue. Using the previously assigned structural alerts to DNA reactivity (electrophilicity), the chemicals were split into 154 alerting chemicals and 147 non-alerting chemicals. The alerting chemicals were split into three chemical groups; aromatic amino/nitro-types, alkylating agents and miscellaneous structurally-alerting groups. The non-alerting chemicals were subjectively split into three broad categories; non-alerting, non-alerting containing a non-reactive halogen group, and non-alerting chemical with minor concerns about a possible structural alert. The tumor data for all 301 chemicals are re-presented according to these six chemical groupings. The most significant findings to emerge from comparisons among these six groups of chemicals were as follows: (a) Most of the rodent carcinogens, including most of the 2-species and/or multiple site carcinogens, were among the structurally alerting chemicals. (b) Most of the structurally alerting chemicals were mutagenic; 84% of the carcinogens and 66% of the non-carcinogens. 100% of the 33 aromatic amino/nitro-type 2-species carcinogens were mutagenic. Thus, for structurally alerting chemicals, the Salmonella assay showed high sensitivity and low specificity (0.84 and 0.33, respectively). (c) Among the 147 non-alerting chemicals less than 5% were mutagenic, whether they were carcinogens or non-carcinogens (sensitivity 0.04).     

Epigenetic processes and cancer risk assessment

The U.S. Environmental Protection Agency's Guidelines for Carcinogen Risk Assessment encourages the use of mechanistic data in the assessment of human cancer risk at low (environmental) exposure levels. The key events that define a particular mode of action for tumor formation have been concentrated to date more on mutational responses that are broadly the result of induced DNA damage and enhanced cell proliferation. While it is clear that these processes are important in terms of tumor induction, other modes that fall under the umbrella of epigenetic responses are increasingly being considered to play an important role in susceptibility to tumor induction by environmental chemicals and as significant modifiers of tumor responses. Alterations in gene expression, DNA repair, cell cycle control, genome stability and genome reprogramming could be the result of modification of DNA methylation and chromatin remodeling patterns as a consequence of exposure to environmental chemicals. These concepts are described and discussed.     

Mutagenic activity of carcinogens detected in transgenic rodent mutagenicity assays at dose levels used in chronic rodent cancer bioassays

Data on transgenic rodent mutagenicity of five human carcinogens were summarised and compared with the results from rodent carcinogenicity studies. Four out of five carcinogens showed mutagenic activity already at daily dose levels which induced cancer in long-term rodent bioassays in at least one target tissue of carcinogenesis. In several of these studies, even single dose applications were sufficient to significantly increase the mutation frequency in vivo. Other genotoxic carcinogens required application of multiple dosing at dose-levels used in rodent cancer bioassays to show their in vivo mutagenicity. A rodent respiratory tract carcinogen, 1,2-dibromoethane (DBE), following inhalation exposure, displayed no mutagenic activity, neither in lung nor in nasal mucosa, at a single 2-h exposure to 30 ppm, which is below the highest concentration used in a NTP cancer bioassay. In contrast, after multiple treatment for 10 days at the same daily doses, a significant increase of the mutation frequency in nasal mucosa was apparent. We conclude, that especially when studying new chemicals in these transgenic rodent mutation assays, a multiple dosing protocol should be preferred. For dose selection, the same criteria could be applied as for chronic rodent bioassays.     

How many high production chemicals are rodent carcinogens? Why should we care? What do we need to do about it?

High production volume (HPV) chemicals are produced in or imported to the US in amounts greater than 1 million pounds per chemical per year. The EPA has identified thousands of HPVs. Due to their abundance, such chemicals bring a substantial risk for human exposure, and as a result some level of adverse consequences to health are to be expected. In order to examine the potential for cancer risk associated with HPVs, this paper examines HPVs that have been tested in the National Toxicology Program's rodent cancer bioassay. The chemicals tested in the bioassay represent a small sample of the universe of environmental chemicals to which people are exposed. Unexpectedly, 60% of the 128 HPVs evaluated in the bioassay proved to be rodent carcinogens. This value compares to a predicted prevalence of only 16.5% carcinogens in a previous study. The previous study concluded that HPVs were less likely to be toxic by a variety of other test criteria as well. However, the approach involved identifying putative carcinogens using quantitative chemical structure-activity relationships (QSAR) in contrast to the direct tabulation of bioassay test results performed here. Detailed examination of bioassay results reveals that test outcomes depend heavily on route of administration as well as on dose level, sex, strain, and species used. Since most of these factors have little or no apparent relationship to chemical structure, results of this study suggest that QSAR, as well as virtually all other alternative methods, may not generally provide accurate predictions of carcinogenic potential. As we wait for efficient and effective methods for predicting carcinogens to be developed, people continue to be exposed to environmental carcinogens. Progress on regulating environmental carcinogens as well as on developing more effective test methods has been minimal since "war on cancer" began approximately 30 years ago. The present study questions whether the current strategy for dealing with environmental carcinogens will ever be successful. Close examination of rodent cancer test results seems to suggest that almost all chemicals may have carcinogenic potential in some genotypes under some exposure circumstances. If this hypothesis is correct, it would explain the general lack of progress in developing methods to differentiate carcinogens from noncarcinogens. A completely new strategy for dealing with cancer caused by exposures to environmental chemicals seems to be needed.     

Predictive assay for rodent carcinogenicity using in vivo biochemical parameters: operational characteristics and complementarity.

111 chemicals of known rodent carcinogenicity (49 carcinogens, 62 noncarcinogens), including many promoters of carcinogenesis, nongenotoxic carcinogens, hepatocarcinogens, and halogenated hydrocarbons, were selected for study. The chemicals were administered by gavage in two dose levels to female Sprague-Dawley rats. The effects of these 111 chemicals on 4 biochemical assays (hepatic DNA damage by alkaline elution (DD), hepatic ornithine decarboxylase activity (ODC), serum alanine aminotransferase activity (ALT), and hepatic cytochrome P-450 content (P450)) were determined. Composite parameters are defined as follows: CP = [ODC and P450), CT = [ALT and ODC), and TS = [DD or CP or CT]. The operational characteristics of TS for predicting rodent cancer were sensitivity 55%, specificity 87%, positive predictivity 77%, negative predictivity 71%, and concordance 73%. For these chemicals, the 73% concordance of this study was superior to the concordance obtained from published data from other laboratories on the Ames test (53%), structural alerts (SA) (46%), chromosome aberrations in Chinese hamster ovary cells (ABS) (48%), cell mutation in mouse lymphoma 15178Y cells (MOLY) (52%), and sister-chromatid exchange in Chinese hamster ovary cells (SCE) (60%). The 4 in vivo biochemical assays were complementary to each other. The composite parameter TS also shows complementarity to all 5 other predictors of rodent cancer examined in this paper. For example, the Ames test alone has a concordance of only 53%. In combination with TS, the concordance is increased to 62% (Ames or TS) or to 63% (Ames and TS). For the 67 chemicals with data available for SA, the concordance for predicting rodent carcinogenicity was 47% (for SA alone), 54% (for SA or TS), and 66% (for SA and TS). These biochemical assays will be useful: (1) to predict rodent carcinogenicity per se, (2) to 'confirm' the results of short-term mutagenicity tests by the high specificity mode of the biochemical assays (the specificity and positive predictivity are both 100%), and (3) to be a component of future complementary batteries of tests for predicting rodent carcinogenicity.     

Rodent tumor profiles, Salmonella mutagenicity and risk assessment

The tumorigenesis profiles of 116 chemicals, which proved to induce cancer in the NCI/NTP experimentation, were studied by multivariate data analysis methods. Three main patterns of tumor induction were evident. One chemical (benzene) was not classifiable in any of the 3 clusters of chemicals. The carcinogen classes based on patterns of tumor induction did not reflect a repartition between Ames-positive and Ames-negative chemicals. Therefore any classification of carcinogens as either 'primary' (genotoxic, hence assumed to pose a greater risk) or 'secondary' (presumably carcinogenic via non-genotoxic mechanisms) would seem to be a subject for research and speculation, and, for the present, an unsuitable basis for risk assessment.     

Transgenic tumor models for carcinogen identification: the heterozygous Trp53-deficient and RasH2 mouse lines

Genetically altered mouse models (GAMM) for human cancers have been critical to the investigation and characterization of oncogene and tumor suppressor gene expression and function and the associated cancer phenotype. Similarly, several of the mouse models with defined genetic alterations have shown promise for identification of potential human carcinogens and investigation of mechanisms of carcinogen-gene interactions and tumorigenesis. In particular, both the B6.129N5-Trp53 mouse, heterozygous for a p53 null allele, and the CB6F1-RasH2 mouse, hemizygous for the human H-ras transgene, have been extensively investigated. Using 26-week exposure protocols at or approaching the maximum tolerated dose, the summary results to date indicate the potential for GAMM to identify and, possibly, classify chemicals of potential risk to humans using short-term carcinogenicity experiments. This IWGT session focused on: (1) the development of recommendations for genetic/molecular characterization required in animals, tissues, and tumors before and after treatment for identification of presumptive human carcinogens based on the current state of knowledge, (2) identification of data gaps in our current state of knowledge, and (3) development of recommendations for research strategies for further development of our knowledge base of these particular models. By optimization of protocols and identification of significant outcomes and responses to chemical exposure in appropriate short-term mechanism-based genetically altered rodent models, strategies for prevention and intervention may be developed and employed to the benefit of public health.     

Evaluation of the alkaline elution/rat hepatocyte assay as a predictor of carcinogenic/mutagenic potential

We have recently developed an alkaline elution/rat hepatocyte assay to sensitively measure DNA single-strand breaks induced by xenobiotics in non-radiolabeled rat hepatocytes. Here we have evaluated this assay as a predictor of carcinogenic/mutagenic activity by testing 91 compounds (64 carcinogens and 27 non-carcinogens) from more than 25 diverse chemical classes. Hepatocytes were isolated from uninduced rats by collagenase perfusion, exposed to chemicals for 3 h, harvested, and analyzed for DNA single-strand breaks by alkaline elution. DNA determinations were done fluorimetrically. Cytotoxicity was estimated by glutamate-oxaloacetate transaminase release or by trypan blue dye exclusion. The assay correctly predicted the reported carcinogenic/non-carcinogenic potential of 92% of the carcinogens tested and 85% of non-carcinogens tested. The assay detected a number of compounds, including inorganics, certain pesticides, and steroids, which give false-negative results in other short-term tests. Only 2 rat liver carcinogens were incorrectly identified; the other carcinogens incorrectly identified are weakly or questionably carcinogenic (i.e., they cause tumors only in one species, after lifetime exposure, or at high doses). Some chemicals cause DNA damage only at cytotoxic concentrations; of 16 such compounds in this study, 12 are weak carcinogens suggesting a link between DNA damage caused by cytotoxicity and carcinogenesis. Our data indicate that this assay rapidly, reproducibly, sensitively, and accurately detects DNA single-strand breaks in rat hepatocytes and that the production of these breaks correlates well with carcinogenic and mutagenic activity.     

Distinction between octachlorostyrene and hexachlorobenzene in their potentials to induce ethoxyphenoxazone deethylase and cause porphyria in rats and mice

The potentials of octachlorostyrene (OCS) and hexachlorobenzene (HCB) to induce liver microsomal ethoxyphenoxazone deethylation (an indicator of induction of 3-methylcholanthrene and beta-naphthoflavone-like cytochrome P-450 monoxygenase activity) and cause porphyria in male C57BL/6 and C57BL/10 mice and female F344 rats were compared. Ethoxyphenoxazone deethylation was induced much more by HCB than by OCS in both of these strains of mice (although neither OCS nor HCB greatly induced deethylation in the DBA/2 strain). In rats ethoxyphenoxazone deethylase was induced 26-fold by HCB but only four-fold by OCS, whereas dealkylation of pentoxyphenoxazone (an indicator of phenobarbital-like induction) increased 43- and 36-fold, respectively. Both chemicals were poor inducers of dealkylation of pentoxyphenoxazone in mice. When fed HCB continuously but not when given OCS, C57BL/6 and C57BL/10 mice (both after pretreatment with iron) and F344 rats developed porphyria with a depression of hepatic uroporphyrinogen decarboxylase activity. The results illustrate that in these species OCS and HCB cannot be considered as equally efficient agents for inducing ethoxyphenoxazone deethylation or causing porphyria. If these effects are mediated through binding to the aromatic hydrocarbon responsiveness (Ah) receptor, HCB would appear to have a much greater affinity than OCS despite the face that neither chemical possesses a structure currently considered to be necessary for efficient binding.     

Understanding the role of xenobiotic-metabolism in chemical carcinogenesis using gene knockout mice

Most chemical carcinogens require metabolic activation to electrophilic metabolites that are capable of binding to DNA and causing gene mutations. Carcinogen metabolism is carried out by large groups of xenobiotic-metabolizing enzymes that include the phase I cytochromes P450 (P450) and microsomal epoxide hydrolase, and various phase II transferase enzymes. It is extremely important to determine the role P450s play in the carcinogenesis and to establish if they are the rate limiting and critical interface between the chemical and its biological activities. The latter is essential in order to validate the use of rodent models to test safety of chemicals in humans. Since there are marked species differences in expressions and catalytic activities of the multiple P450 forms that activate carcinogens, this validation process becomes especially difficult. To address the role of P450s in whole animal carcinogenesis, mice were produced that lack the P450s known to catalyze carcinogen activation. Mouse lines having disrupted genes encoding the P450s CYP1A2, CYP2E1, and CYP1B1 were developed. Mice lacking expression of microsomal epoxide hydrolase (mEH) and NADPH-quinone oxidoreductase (NQO1) were also made. All of these mice exhibit no gross abnormal phenotypes, suggesting that the xenobiotic-metabolizing enzymes have no critical roles in mammalian development and physiological homeostasis. This explains the occurrence of polymorphisms in xenobiotic-metabolizing enzymes among humans and other mammalian species. However, these null mice do show differences in sensitivities to acute chemical toxicities, thus establishing the importance of xenobiotic metabolism in activation pathways that lead to cell death. Rodent bioassays using null mice and known genotoxic carcinogens should establish whether these enzymes are required for carcinogenesis in an intact animal model. These studies will also provide a framework for the production of transgenic mice and carcinogen bioassay protocols that may be more predictive for identifying the human carcinogens and validate the molecular epidemiological studies ongoing in humans that seek to establish a role for polymorphisms in cancer risk.     

Prevention of cancer and other chronic diseases worldwide based on sound mechanisms

The transformation of normal cells by DNA reactive, genotoxic carcinogens and the growth promotion and development of mutated cells by enhancing factors is involved in the overall basic mechanism of cancer induction. Thus, discrimination between genotoxic carcinogens and nongenotoxic chemicals is essential. The dose-response curves, reversibility, and organ-and species specificity are distinct. Genotoxic carcinogens are mutagenic, form DNA adducts, induce DNA repair, and form hydroxy radicals and inappropriate peroxidation reactions that antioxidants such as those in vegetables, fruits, and tea can decrease. In contrast, promoters do not form DNA adducts, but raise cell duplication rates, among other attributes. In the USA, about 35% of known cancers are associated with tobacco use and about 55% with inappropriate nutritional habits. Cancer induction can be decreased by avoiding the formation of carcinogens, reducing their metabolic activation, or increasing their detoxification. Excessive dietary salt, and heterocyclic arylamines formed in cooking of meats or fish, and high intake of 40% of calories in fats are health risks, but vegetables, fruits, tea, soy products, and fibers are protective. We review nutritional factors involved in cancer and chronic disease causation and prevention.