David Brusick

An evaluation of the Escherichia coli WP2 and WP2uvrA reverse mutation assay

The methodology and status of the Escherichia coli WP2 reverse mutation system as it applies to chemical screening were reviewed using the available published literature. 163 documents were reviewed by the Working Group. These included abstracts, research articles, review articles and publicly available contract and grant final reports. From this group, 115 documents were rejected for critical evaluation by the Working Group. 48 documents were reviewed and the test results summarized. The general conclusion of the Working Group was the the E. coli WP2 reverse mutation system is a valuable tool for mutagenesis research, but that there is no evidence from a review of the literature that this assay will contribute significantly to the results obtainable from careful application of the Ames Salmonella assay. Another review of the role of this system in general screening may be warranted after more research and development with the plasmid-containing WP2 derivatives.

Implications of treatment-condition-induced genotoxicity for chemical screening and data interpretation.

Ionic and pH alterations appear to be directly responsible for the induction of genotoxic effects in cultured mammalian cells. In vivo studies also associate high ion concentrations and pH changes with tumor enhancement of the glandular stomach and urinary bladder of rats. The implications of these findings are directly relevant to the design of in vitro and in vivo tests and to the interpretation of results from tests using materials likely to produce alterations in ionic and/or pH levels.

The genetic properties of beta-propiolactone

Abstract BPL is a highly active molecule with numerous biological properties. The chemical reacts rapidly with nucleophilic centers such as proteins, particularly sulfur containing amino acids, and nucleic acids. The product of these reactions is an alkylated moiety which in the case of DNA results in the induction of mutations and chromosome aberrations. The predominant alkylated base in DNA exposed to BPL is 7-(2-carboxyethyl) guanine. Adenine is alkylated to a lesser extent at the 3-position [44]. Alkylation of guanine can lead to either depurination or an ionized form of the molecule capable of mis-hydrogen bonding with thymidine. Depurination can also lead to numerous transition or transversion types of base-pair substitutions. Conversely, alkylguanine-specific repair reduces the DNA-modifying effects of BPL. Anomalous base-pairing between guanine and thymine would be expected to result in GC to AT transition base-pair substitution mutations, and a substantial amount of the mutation data obtained from phage, bacteria and yeast indicate but the predominant mechanism is base-pair substitutions of the GC to AT type. In addition to base-pair substitutions, BPL was shown to produce multilocus deletions in phage, Neurospora and probably yeast. This information, plus the data showing DNA cross-linking in phage, clearly indicates the high degree of reactivity and the multiplicity of mechanisms by which mutagenic effects can be induced by BPL. The rapid and extensive binding of the active form of BPL to nucleophilic centers is also obvious from its in vivo toxicity. Administration of BPL to mice or rats does not result in either germ cell or bone marrow effects as found with other slower reacting alkylating agents (e.g., triethylene melamine). The biological half-life of BPL in vivo is probably too short for substantial quantities of the alkylating form to reach either target site. The lack of systemic neoplasia from either subcutaneous or oral exposure of animals to BPL is consistent with these data. As a result of this type of chemical reactivity, it is difficult to determine the impact of BPL on humans. Risk assessments using normal methods of exposure, such as drinking water, feed or i.p. or i.m. injection would be expected to show little or no mutagenic effects in acute or chronic studies. Yet, the potent mutagenic properties of this chemical are clearly obvious in plant, microbial or tissues exposed at the site of application but has little carcinogenic activity for systemic targets, even though DiPaolo et al. [22] reported transplacental transport and malignant transformation by BPL administered i.p. to pregnant hamsters. The significance of this finding is not clear in the light of normal human exposure to BPL. Because of the wealth of chemical, physical and biological information available on BPL, this chemical appears to be an excellent material for use in studies aimed at elucidating the mechanisms of mutagenesis and carcinogenesis and the functional relationship linking these two phenomena.

In Vitro Mutagenesis Assays as Predictors of Chemical Carcinogenesis in Mammals

In vitro microbial mutagenesis assays coupled with mammalian activation systems offer promising technique to screen chemicals for their potential carcinogenic activity. The correlation between mutagenic and carcinogenic properties for a large array of chemicals is approximately 0.9. The best correlation exists for those carcinogens which are themselves highly electrophilic or produce electrophilic metabolites. Correlation between mutagenicity and carcinogenicity for hormonal, metallic, or physical carcinogens has been disappointing but not unexpected based on their proposed mechanisms of action. In addition to the application of in vitro mutagenesis techniques to screening chemicals for the identification of potential carcinogens, they are useful tools for investigating genetic, biochemical, and pharmacologic properties of different animal species. Studies with the chemical carcinogen dimethylnitrosamine have been conducted and show a functional relationship between mutagenesis and carcinogenesis. The assays can also be conducted using activation systems prepared from the tissues of any mammalian species. This permits a direct assessment of phylogenic extrapolation by comparing the metabolic activation capabilities of tissues from several mammalian species, including human samples. The advantages of mutagenicity testing are the short period of time required for results, the high sensitivity of the assay (microgram of nanogram quantities of chemicals can be used), and the fact that the ultimate agent can be detected biologically without first necessitating chemical identification and isolation. It appears from current studies that in vitro mutagenesis techniques may well open new avenues of investigation into some old toxicologic problems.

Genotoxicity hazard assessment of Caramel Colours III and IV

Results from a battery of short-term tests in vitro and in vivo used to assess the genotoxicity of caramel colours are presented and discussed in relation to reports from the literature. No evidence of genotoxicity was found in the Salmonella plate incorporation test using five standard strains or in the Saccharomyces cerevisiae gene conversion assay using strain D4, either with or without S-9 for activation. A weak clastogenic effect for a sample of Caramel Colour III in CHO cells was abolished in the presence of S-9. Two samples of Caramel Colour IV were not clastogenic in CHO cells. Salmonella pre-incubation tests without S-9 also failed to reveal any mutagenic activity for any of the caramel colours tested. The Caramel Colour III sample that showed clastogenic activity in CHO cells in vitro did not induce micronuclei when evaluated in a mouse bone marrow assay. These results are in general agreement with reports in the literature regarding the genotoxicity of caramel colours, and support the conclusion that caramel colours do not pose a genotoxic hazard to humans.

MUTAGENICITY AND CARCINOGENICITY CORRELATIONS BETWEEN BACTERIA AND RODENTS

Detection of mutation in bacteria has acquired the status of an accepted procedure in genetic toxicology programs. The methods presently employed in such programs include both forward and reverse mutation-induction techniques in strains of Salmonella typhimurium and Escherichia coli. The specific strains used in these techniques have been selected over the years on the basis of their sensitivity to a broad range of chemical mutagens. In addition, it has been reported that chemical carcinogens can be presumptively identified on the basis of these assays, and bacterial testing has been generally considered the front-line test procedure for the identification of presumptive mutagenic carcinogens. An analysis of correlative studies both retrospective and cross-sectional shows a range of predictive capabilities depending on features such as chemical class, carcinogenic mechanism, and requirements for specific metabolic toxification processes. The greatest limitations associated with the use of bacteria mutation testing is the real and/or perceived issue of the test or a misinterpretation of the correlation coefficients under conditions of routine application. Concerns related to the performance (reliability, reproducibility, and predictability) and relevance of bacteria assays perpetuate controversy surrounding their application to hazard assessment. A review of several studies comparing mutation induction and tumor induction indicates that the Ames test can be useful in screening large numbers of chemicals, but the true correlation coefficient is only about 80% when compared to tumor responses in mice and rats.

Genotoxicity of theobromine in a series of short-term assays

Theobromine (3,7-dimethylxanthine) was evaluated for genotoxic activity in a series of in vitro assays. Theobromine was not mutagenic in the Ames assay up to a maximum concentration of 5000 micrograms/plate either with or without S9 activation. The compound also failed to induce significant levels of chromosome aberrations in CHO cells (with and without S9 activation) or transformation in Balb/c-3T3 cells. At the maximum tolerated concentration theobromine increased the frequency of TK-/- mutants in mouse lymphoma L5178Y cells. Increased frequencies were observed both with and without S9 activation and they were reproducible in 2 independent experiments. Statistically significant increases in SCEs were obtained in human lymphocytes and in CHO cells under nonactivation test conditions. The spectrum of results in this battery of tests indicate that theobromine treatment results in the expression of genotoxic potential in some assays and the observed activity appears qualitatively and quantitatively similar to that of caffeine, a closely related methylxanthine.

EVALUATION OF CHRONIC RODENT BIOASSAYS AND AMES ASSAY TESTS AS ACCURATE MODELS FOR PREDICTING HUMAN CARCINOGENS

The question “What percentages of mammalian carcinogens and noncarcinogens can be predicted by bacterial mutation tests?” probably cannot be answered for the following two reasons: 1.) There seems to be no universal agreement upon what criteria are absolutely necessary to unequivocally establish a carcinogen for mammals. Consequently, the bull’s eye of the target for which the bacterial tests must aim continually shifts, causing the results of comparisons conducted at one point in time to be adjusted at a different point in time. 2.) Carcinogen results are not obtained for “mammals”, they are obtained in unique mammalian species which often differ among themselves as to whether a particular chemical is a carcinogen or not. Bacterial tests might hopefully match the results in a single mammalian model but cannot be expected to match the combined results from several mammalian species which fail to agree among themselves as to the correct answer.

Evaluation Of The Mutagenic Potential Of Corn (ZEA HAYS L.) Grown In Untreated AND Atrazine (AATREX®) T Treated Soil In The Field

Bacterial assays using extracts from field corn plants (harvested at one month, silage and mature stages) do not indicate that soil treatment with atrazine, at its maximum use rate, alters the endogenous mutagens present in these extracts, nor that atrazine itself is degraded to mutagenic products. Extracts of corn grown in soil treated with AAtrex were equally mutagenic with those of corn grown in untreated soil when tested in Salmonella typhimurium TA-100 by a reversion assay or in Salmonella typhimurium TM-677 in a forward mutation assay. Higher concentrations of histidine in corn grown in AAtrex treated soil may interfere with the reversion assay, but do not affect the forward mutation assay. The nature of the agent(s) responsible for the positive response was not determined. The mutagenicity may be due to natural plant constituents, an artifact of the sample preparation, or mycotoxins from some unrecognized plant infection. The experimental results in these field studies do not show that atrazine is degraded or metabolized by corn plants to mutagens in this sensitive bacterial assay.

Genetic Screening of Compounds Used in Drug Abuse Treatment I. Naltrexone Hydrochloride

Several compounds used clinically in drug abuse therapy were evaluated for genetic activity in a series of in vitro assays. This initial report describes the results for one of these compounds, Naltrexone. Nalrexone is a relatively nontoxic drug antagonist related to Naloxone which appears to be effective in diminishing the euphoria and dependence upon heroin in clinical studies. With the exception of weak nonspecific DNA damage observed in an E. coli DNA repair test and possibly with WI-38 cells as well, Naltrexone did not demonstrate significant potential for the induction of gene mutations or chromosomal aberrations under the conditions of this evaluation.