Marcus A. Jackson

A survey of EPA/OPP and open literature on selected pesticide chemicals: II. Mutagenicity and carcinogenicity of selected chloroacetanilides and related compounds

With this effort, we continue our examination of data on selected pesticide chemicals and their related analogues that have been presented to the U.S. Environmental Protection Agencys (USEPAs) Office of Pesticide Programs (OPP). This report focuses on a group of selected chloroacetanilides and a few related compounds. As part of the registration process for pesticidal chemicals, interested parties (registrants) must submit toxicity information to support the registration including both mutagenicity and carcinogenicity data. Although this information is available to the public via Freedom of Information (FOI) requests to the OPP, publication in the scientific literature allows greater dissemination and examination of the data. For this Special Issue, graphic profiles have been prepared of the mutagenicity and carcinogenicity data available in the submissions to OPP. Also, a discussion is presented about how toxicity data are used to help establish tolerances (limits of pesticide residues in foods). The mutagenicity results submitted by registrants are supplemented by data on these chemicals from the open literature to provide a full perspective of their genetic toxicology. The group of chloroacetanilides reviewed here display a consistent pattern of mutagenic activity, probably mediated via metabolites. This mutagenic activity is a mechanistically plausible factor in the development of tumors seen in experimental animals exposed to this class of chemicals.

Activity profiles of antimutagens: in vitro and in vivo data

In this review, retinol, chlorophyllin, and N-acetylcysteine are examined and compared with regard to their antimutagenic activity against some promutagens and a group of direct-acting alkylating agents. The promutagens included aflatoxin B1, certain polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene), and certain heterocyclic amines (e.g., food pyrolysates). Results of antimutagenicity testing selected from data surveyed in the published literature are displayed graphically as activity profiles of antimutagens showing both the doses tested and the extent of inhibition or enhancement of mutagenic activity. All three antimutagens are discussed in terms of their putative mechanisms of action in vitro and in vivo with emphasis on the xenobiotic metabolizing enzyme systems.

The performance of short-term tests in identifying potential germ cell mutagens: a qualitative and quantitative analysis

A retrospective analysis was undertaken to assess the performance of selected short-term tests in the discrimination of mammalian germ cell mutagens and nonmutagens using data derived from the U.S. Environmental Protection Agency/International Agency for Research on Cancer Genetic Activity Profile (EPA/IARC GAP) and EPA GENE-TOX databases. The short-term tests selected were gene mutation in Salmonella (S. typhimurium), cultured mammalian cell gene mutation and chromosomal aberrations, and mammalian bone marrow cytogenetics (micronucleus and chromosomal aberrations). These are the first level tests used in the EPA mutagenicity testing guidelines. The results of this analysis showed good sensitivity of short-term in vitro tests for mammalian cell gene mutation (96%) or chromosomal aberrations (92%) in identifying germ cell mutagens, while the sensitivity of tests for gene mutation in S. typhimurium was lower (79%). Bone marrow micronucleus or chromosomal aberration assays in vivo each displayed a sensitivity of 96%. Thus, both the in vitro and in vivo tests may be used effectively to screen chemicals for potential germ cell mutagenicity. In contrast, the in vitro tests mentioned above performed poorly in discriminating putative germ cell nonmutagens, giving results for specificity at or below what is expected due to chance alone (50-11%). The bone marrow assays were more efficient in this regard, the micronucleus test yielding a specificity of 63% and the chromosomal aberrations assay 64%. The mouse bone marrow micronucleus test also performed well on a quantitative basis, responding at or below the lowest effective doses tested in the mouse dominant lethal assay. Regression analysis of the mean lowest effective doses of chemicals evaluated in vivo showed approximately 1:1 linear correlations for mouse germ cell assays (heritable translocation vs dominant lethal or specific locus tests) as well as for mouse bone marrow assays (micronucleus vs chromosomal aberration). The results suggest the value of the bone marrow micronucleus test as an assay for potential germ cell mutagenicity and the dominant lethal test as a relatively inexpensive choice for confirmation of germ cell damage. The sensitivity of the in vitro assays investigated and the discriminatory capability of the in vivo bone marrow assay affirmed the utility of these tests within the framework of the EPA mutagenicity testing guidelines.

A survey of EPA/OPP and open literature on selected pesticide chemicals. III. Mutagenicity and carcinogenicity of benomyl and carbendazim.

The known aneuploidogens, benomyl and its metabolite, carbendazim (methyl 2-benzimidazole carbamate (MBC)), were selected for the third in a series of ongoing projects with selected pesticides. Mutagenicity and carcinogenicity data submitted to the US Environmental Protection Agencys (US EPAs) Office of Pesticide Programs (OPP) as part of the registration process are examined along with data from the open literature. Mutagenicity and carcinogenicity profiles are developed to provide a complete overview and to determine whether an association can be made between benomyl- and MBC-induced mouse liver tumors and aneuploidy. Since aneuploidogens are considered to indirectly affect DNA, the framework adopted by the Agency for evaluating any mode of action (MOA) for carcinogenesis is applied to the benomyl/MBC data. Both agents displayed consistent, positive results for aneuploidy induction but mostly negative results for gene mutations. Non-linear dose responses were seen both in vitro and in vivo for aneuploidy endpoints. No evidence was found suggesting that an alternative MOA other than aneuploidy may be operative. The data show that by 14 days of benomyl treatment, events associated with liver toxicity appear to set in motion the sequence of actions that leads to neoplasms. Genetic changes (as indicated by spindle impairment leading to missegregation of chromosomes, micronucleus induction and subsequent aneuploidy in bone marrow cells) can commence within 1-24h after dosing, well within the time frame for early key events. Critical steps associated with frank tumor formation in the mouse liver include hepatotoxicity, increased liver weights, cell proliferation, hypertrophy, and other steps involving hepatocellular alteration and eventual progression to neoplasms. The analysis, however, reveals weaknesses in the data base for both agents (i.e. no studies on mouse tubulin binding, no in vivo assays of aneuploidy on the target tissue (liver), and no clear data on cell proliferation relative to dose response and time dependency). The deficiencies in defining the MOA for benomyl/MBC introduce uncertainties into the analysis; consequently, benomyl/MBC induction of aneuploidy cannot be definitively linked to mouse liver carcinogenicity at this time.

Differences in detection of DNA adducts in the 32P-postlabelling assay after either 1-butanol extraction or nuclease P1 treatment

The use of nuclease P1 treatment and 1-butanol extraction to increase the sensitivity of the 32P-postlabelling assay for DNA adducts have been compared. Although similar results were obtained with the two methods for standard adducts formed with benzo[a]pyrene diol epoxide I (BPDE-I), nuclease P1 treatment resulted in a significant reduction in detection of major adducts from 1-amino-6-nitropyrene (1-amino-6-NP), 1-amino-8-nitropyrene (1-amino-8-NP), 2-aminofluorene (2-AF), 2-naphthylamine (2-NA) and 4-aminobiphenyl (4-ABP) modified DNAs, but not following the 32P-postlabelling analysis of 2-acetylaminofluorene (2-AAF) modified DNA. These results suggest that, at least initially, both modifications of the 32P-postlabelling assay should be used for the detection of unknown adducts or for adducts derived from nitroaromatics and aromatic amines.

A review of the genetic and related effects of 1,3-butadiene in rodents and humans

In this paper, the metabolism and genetic toxicity of 1,3-butadiene (BD) and its oxidative metabolites in humans and rodents is reviewed with attention to newer data that have been published since the latest evaluation of BD by the International Agency for Research on Cancer (IARC). The oxidative metabolism of BD in mice, rats and humans is compared with emphasis on the major pathways leading to the reactive intermediates 1,2-epoxy-3-butene (EB), 1,2:3, 4-diepoxybutane (DEB), and 3,4-epoxy-1,2-butanediol (EBdiol). Results from recent studies of DNA and hemoglobin adducts indicate that EBdiol may play a more significant role in the toxicity of BD than previously thought. All three metabolites are capable of reacting with macromolecules, such as DNA and hemoglobin, and have been shown to induce a variety of genotoxic effects in mice and rats as well as in human cells in vitro. DEB is clearly the most potent of these genotoxins followed by EB, which in turn is more potent than EBdiol. Studies of mutations in lacI and lacZ mice and of the Hprt mutational spectrum in rodents and humans show that mutations at G:C base pairs are critical events in the mutagenicity of BD. In-depth analyses of the mutational spectra induced by BD and/or its oxidative metabolites should help to clarify which metabolite(s) are associated with specific mutations in each animal species and which mutational events contribute to BD-induced carcinogenicity. While the quantitative relationship between exposure to BD, its genotoxicity, and the induction of cancer in occupationally exposed humans remains to be fully established, there is sufficient data currently available to demonstrate that 1,3-butadiene is a probable human carcinogen.

Genetic activity profiles of anticancer drugs

The results from short-term tests for genetic and related effects, abstracted from the open literature for 36 anticancer drugs, are examined in this review. Data for 27 of these agents are available in the EPA/IARC Genetic Activity Profile (GAP) database. Data summaries, including data listings and activity profiles, are presented for nine anticancer drugs added to the GAP database for this analysis. Genetic toxicity data from the recent literature are included for the additional agents to provide a broader representation of the categories of drugs being evaluated. These categories, based on the chemical mode of action, are covalent and noncovalent DNA-binding drugs, topoisomerase II inhibitors, antimetabolites, mitotic spindle inhibitors, and drugs which affect endocrine function. The qualitative data for all 36 drugs are summarized in this report and findings are presented from pair-wise matching of genetic activity profiles, based on test results in common, for some chemical analogs. The significance of germ cell test results for some of these drugs and their implication in assessing risk of heritable genetic disease are discussed.

The genetic toxicology of putative nongenotoxic carcinogens

This report examines a group of putative nongenotoxic carcinogens that have been cited in the published literature. Using short-term test data from the U.S. Environmental Protection Agency/International Agency for Research on Cancer genetic activity profile (EPA/IARC GAP) database we have classified these agents on the basis of their mutagenicity emphasizing three genetic endpoints: gene mutation, chromosomal aberration and aneuploidy. On the basis of results of short-term tests for these effects, we have defined criteria for evidence of mutagenicity (and nonmutagenicity) and have applied these criteria in classifying the group of putative nongenotoxic carcinogens. The results from this evaluation based on the EPA/IARC GAP database are presented along with a summary of the short-term test data for each chemical and the relevant carcinogenicity results from the NTP, Gene-Tox and IARC databases. The data clearly demonstrate that many of the putative nongenotoxic carcinogens that have been adequately tested in short-term bioassays induce gene or chromosomal mutations or aneuploidy.

Databases applicable to quantitative hazard/risk assessment—Towards a predictive systems toxicology

The Workshop on The Power of Aggregated Toxicity Data addressed the requirement for distributed databases to support quantitative hazard and risk assessment. The authors have conceived and constructed with federal support several databases that have been used in hazard identification and risk assessment. The first of these databases, the EPA Gene-Tox Database was developed for the EPA Office of Toxic Substances by the Oak Ridge National Laboratory, and is currently hosted by the National Library of Medicine. This public resource is based on the collaborative evaluation, by government, academia, and industry, of short-term tests for the detection of mutagens and presumptive carcinogens. The two-phased evaluation process resulted in more than 50 peer-reviewed publications on test system performance and a qualitative database on thousands of chemicals. Subsequently, the graphic and quantitative EPA/IARC Genetic Activity Profile (GAP) Database was developed in collaboration with the International Agency for Research on Cancer (IARC). A chemical database driven by consideration of the lowest effective dose, GAP has served IARC for many years in support of hazard classification of potential human carcinogens. The Toxicological Activity Profile (TAP) prototype database was patterned after GAP and utilized acute, subchronic, and chronic data from the Office of Air Quality Planning and Standards. TAP demonstrated the flexibility of the GAP format for air toxics, water pollutants and other environmental agents. The GAP format was also applied to developmental toxicants and was modified to represent quantitative results from the rodent carcinogen bioassay. More recently, the authors have constructed: 1) the NIEHS Genetic Alterations in Cancer (GAC) Database which quantifies specific mutations found in cancers induced by environmental agents, and 2) the NIEHS Chemical Effects in Biological Systems (CEBS) Knowledgebase that integrates genomic and other biological data including dose-response studies in toxicology and pathology. Each of the public databases has been discussed in prior publications. They will be briefly described in the present report from the perspective of aggregating datasets to augment the data and information contained within them.

Genetic pathways to colorectal cancer.

The colorectal cancer paradigm explains how genetic and histological changes lead normal epithelial cell to transform into pre-malignant adenomas then progress to malignant carcinomas. Using the Genetic Alterations in Cancer Knowledge System intragenic allele loss and gene mutation data from approximately 9000 colorectal tumors were compared to the model of colorectal tumor development. The distribution of mutations along the TP53 codons as a function of tumorigenesis also was analyzed. Alterations of APC, KRAS and TP53 were observed in a higher percentage of adenocarcinomas compared to adenomas (P<0.05) indicating that the alterations accumulated with malignancy. Alterations in BRAF, CTNNB, HRAS and NRAS were infrequent regardless of morphology. Differences were observed in the distribution of TP53 mutations with tumorigenesis. Mutations (single base substitutions) occurred most frequently at codons 175 and 273 in both tumor types; however, in adenocarcinomas the mutation incidence at codon 248 was approximately three times that reported in adenomas. It is proposed that the higher incidence of mutation at codon 248 is a later event in colorectal tumorigenesis that occurs as the tumors become malignant.