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Biomedical subjects

E R Nestmann

Publications and source records attributed to E R Nestmann.

At least 19 recordsLinked to original sources

Modulators of carcinogenesis.

For decades, it has been known that a number of different factors (e.g., species, metabolism, age, animal husbandry, diet) may exhibit a significant modulating effect on the process of carcinogenesis. Often, however, these modulators have been largely uncontrolled and thus have made uncertain the results of many carcinogenicity bioassays. Fortunately, current research into molecular carcinogenesis is beginning to provide methods, not only to understand the molecular basis of known modulators of carcinogenesis, previously described only in empirical terms, but also to allow genetic modulation of carcinogenesis in experimental systems. An expanding body of knowledge regarding the role of oncogenes and tumor suppressor genes in neoplastic events is leading to a better understanding of carcinogenic mechanisms and points to the use of transgenic animal species in carcinogenicity bioassays. The transgenic animal provides methods to examine the molecular basis of carcinogenesis in experimental systems in addition to enhancing the sensitivity of carcinogen identification and the biological specificity of chemical risk extrapolation.

Age Factors

Mutagenic activity of 3 industrial chemicals in a battery of in vitro and in vivo tests.

3 chemicals were selected for mutagenicity testing from a priority list, based on production volume and available mutagenicity data. Propargyl alcohol (PA), 2-nitroaniline (2NA), and 5-methyl-1H-benzo-triazole (MBT) were selected for testing using the approach recommended in the Health Protection Branch Genotoxicity Guidelines. The battery of tests included the Salmonella/mammalian microsome mutation assay, the in vitro chromosomal aberration assay, and the bone-marrow micronucleus assay. The results indicate that 2 of the 3 chemicals, PA and 2NA, were clastogenic in vitro. Both PA and 2NA induced chromosomal aberrations in CHO cells in vitro with and without metabolic activation, while none induced reverse mutations detectable with the Salmonella/mammalian microsome assay. Because PA and 2NA were found to be in vitro clastogens, they also were tested in the mouse bone-marrow micronucleus assay. 2NA induced a small increase in micronuclei in males but not females. PA did not induce an increase in micronuclei.

1-Propanol

Genetic toxicology evaluation of commercial beers, III. SCE, chromosome aberrations, and forward mutation (HGPRT) of commercial beer products in CHO cells.

Concentrated organic residues extracted from 5 blended aliquots of commercial beers were evaluated for their ability to induce sister chromatid exchange (SCE), chromosomal aberrations and forward mutation in Chinese hamster ovary (CHO) cells. Each extract was prepared by blending 4 commercial beers of similar ingredients and brewing method, passing the beer pool over XAD-2 resin, extracting the resin and concentrating the extract. Studies were performed both with and without metabolic activation using variable amounts of reconstituted residues from 225-fold concentrates of the blended samples. CHO cultures were treated with 0.75 microliters/ml through 10.0 microliters/ml of the concentrates in the SCE assays, 1.0 microliters/ml through 10.0 microliters/ml of the extracts in the aberration assays and 2.5 microliters/ml up to 20 microliters/ml for forward mutation assays. In preliminary screening for SCE as an indicator of potential DNA damage, a significant increase was observed for 3 of 5 concentrated samples; however, no increase in SCE was induced by any of the 5 samples when S9 was added as a source of exogenous metabolic activation. More definitive tests for induction of genetic events, i.e., chromosome aberrations and forward HGPRT mutations, were negative for all 5 extracts whether or not S9 mix was present. Since SCE were not induced in tests with metabolic activation and since there was no concordant aberration or point mutation induction, the preliminary indication of potential DNA damage shown by elevated SCE under conditions without metabolic activation appears to have little biological significance.

Animals

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.

Animals

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: study design.

The most commonly used genotoxicity assays for cultured mammalian cells are mammalian cell mutagenesis, chromosome aberrations/SCE, hepatocyte UDS, and cell transformation. Since their inception, protocols for these assays have been modified in various laboratories. It has been observed that minor but potentially significant method modifications frequently remain unpublished (Swierenga et al., 1983) but should be considered in the development of recommended protocols. The present study was undertaken to determine the current 'state of the art' for these tests. Detailed questionnaires on culture conditions and testing protocols for both stock and test cell populations were designed with the assistance of an international advisory committee and sent to all research and contract laboratories that could be readily identified in Canada, U.S.A. and Europe. Responses from 425 completed questionnaires were analyzed to determine the most commonly used approach and modifications for each procedural step. As expected, the results show a large degree of interlaboratory variation. Detailed protocols for conducting each assay have been prepared and include: stepwise instructions, precautionary measures and practical solutions to common problems associated with each assay; recipes for media and solutions; formulas for quantifying genotoxic responses; reference lists of related assays; guidelines for interpretation; and discussions of the applications, advantages and disadvantages of each test.

Animals

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: I. Unscheduled DNA synthesis assay in rat hepatocyte cultures.

A protocol based primarily on current laboratory practices in the performance of the unscheduled DNA synthesis (UDS) assay with primary rat hepatocyte cultures has been developed. These guidelines were developed using tabulated responses to a detailed questionnaire completed by North American and European governmental, university and contract laboratories involved with the UDS test. This report identifies those modifications to previously described methodologies which are used on a regular basis and also serves to clarify confusing or inconsistent practices. Although this protocol pertains specifically to the use of primary rat hepatocyte cultures, it can be modified to incorporate other types of cells in which certain aspects remain the same.

Animals

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: II. Mutation in Chinese hamster ovary, V79 Chinese hamster lung and L5178Y mouse lymphoma cells.

Laboratory protocols and guidelines have been developed for the performance of point mutation assays using Chinese hamster ovary (CHO) cells, V79 cells, and L5178Y mouse lymphoma cells. Since only minor differences in the treatment of CHO and V79 cells exist, these two assays could be combined in one procedural guideline. A second protocol was developed for the mouse lymphoma assay in order to incorporate concerns and methods specific to that cell type and genetic locus. The protocols were based primarily on current laboratory practices as determined by responses to a detailed questionnaire completed by North-American and European governmental, university and contract laboratories involved with in vitro mutation testing. This report identifies those modifications to previously described methodologies which are being used on a regular basis, provides recommendations, and also serves to clarify confusing or inconsistent practices.

Animals

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: III. Cell transformation in C3H/10T1/2 mouse embryo cell, BALB/c 3T3 mouse fibroblast and Syrian hamster embryo cell cultures.

A standardized protocol and guidelines for the performance of cell transformation testing in mouse embryo (C3H/10T1/2), mouse fibroblast (BALB/c 3T3) and Syrian hamster embryo (SHE) cells have been developed. The protocol is based primarily on current laboratory practices as determined by responses to a detailed questionnaire completed by North American and European governmental, university and contract laboratories involved with cell transformation experimentation. This report identifies those modifications to previously described methodologies which are being used on a regular basis and also serves to clarify confusing or inconsistent practices.

Animals

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories, IV. Chromosome aberration and sister-chromatid exchange in Chinese hamster ovary, V79 Chinese hamster lung and human lymphocyte cultures.

A recommended protocol has been developed for chromosomal aberration and sister-chromatid exchange assays in CHO, V79 and human lymphocyte cultures. The protocol was based on the responses to a detailed questionnaire completed by North-American and European governmental, university, and contract laboratories using these tests. This report identifies those modifications to previously described methods that are used on a regular basis and clarifies confusing or inconsistent practices. These protocols can be modified for use in other types of cells.

Animals

Recommended protocols based on a survey of current practice in genotoxicity testing laboratories: study evaluation.

The approach used in the survey of current methodologies used in mammalian cell genotoxicity testing (unscheduled DNA synthesis, mutation, cell transformation and cytogenetics testing) is discussed. The recommended protocols, described in the preceding papers, were developed using responses to detailed questionnaires. This summary outlines general observations related to the survey methodology and to the protocols themselves. Also discussed are the qualities of an effective survey; the evolution of a survey and of a protocol; and the contributions and limitations of this study.

Animals

The SIMULTEST approach for testing mutagens in the Salmonella microtitre fluctuation assay.

The concept of combining several histidine-dependent Salmonella strains in a single test, the SIMULTEST, has been applied to the microtitre fluctuation test. The activity of five mutagens was determined in strains TA97, TA98, TA100, and TA102 individually as well as in a SIMULTEST mixture. All five compounds were mutagenic in the SIMULTEST, demonstrating the utility of this time and labour-saving approach of combining strains for testing with this method. The microtitre fluctuation SIMULTEST results were quantitatively comparable to those of the SIMULTEST Salmonella/microsome plate test. The microtitre fluctuation test compared with the plate incorporation assay generally showed more favourable "sensitivity" and "quantity" indices in that four of the five chemicals tested in the fluctuation test were mutagenic at lower doses than in the plate test.

Culture Media

Human mutagens: evidence from paternal exposure?

The importance of inherited mutations as a cause of human disease has been established clearly through examples of well-defined genetic anomalies, such as Down syndrome and retinoblastoma. Furthermore, it is suspected that environmental contaminants induce mutations resulting in increased risk for such defects in subsequent generations of persons exposed. The present lack of direct evidence for induced inherited genetic disorders in human beings hampers the development of risk estimation techniques for extrapolation from animal models. The most extensive prospective epidemiologic studies of inherited genetic effects have involved survivors of atomic bomb detonations and patients treated with cancer chemotherapy. In neither case has a significant elevation in inherited genetic effects or cancer been detected in the offspring of exposed individuals. Epidemiologic studies of subjects receiving chronic exposure may be confounded by the effect of maternal exposure during pregnancy. Consideration of only paternal exposure can minimize the confounding influence of teratogenicity, enhancing the resolving power of studies for inherited effects. Using this approach, retrospective (case-control) studies of childhood cancer patients have provided limited but suggestive evidence for inheritance of induced effects. Endpoints, such as congenital malformations and spontaneous abortion following paternal exposure, can also be considered as indicators of heritable mutagenic effects. For example, there is limited evidence suggesting that paternal exposure to anaesthetic gases may cause miscarriage and congenital abnormalities as a result of induced male germ cell mutations. By comparing male-exposure endpoints for which there are human data, as described above, with parallel or similar animal endpoints, such as dominant lethal, inherited cancer and "male teratogenic" effects, it is possible that suitable models for extrapolating to human risk can be developed. In order to establish a clearer relationship between induced mutation and genetic disease, the current surveillance systems should be expanded to include endpoints relevant to genetic study. The relaxation of regulations regarding access to census data could improve the chances of documenting such an association.

Abortion, Spontaneous

The SIMULTEST: a new approach to screening chemicals with the Salmonella reversion assay.

A new Salmonella mutagenicity test method is under development to test a chemical with more than one strain simultaneously (the "SIMULTEST"), that is, different Salmonella typhimurium tester strains are used in combination on the same plate. Strains are combined in two sets: strains with plasmid pKM101 (TA97, TA98, TA100, and TA102) and strains without the plasmid (TA1535, TA1537, and TA1538). The SIMULTEST combinations successfully detect the mutagenic activity of five mutagens in different chemical classes. This approach may be useful in reducing the workload associated with mutagenicity testing with Salmonella.

Mutagenicity Tests

Mutagenicity studies in Salmonella: residues of ozonated and/or chlorinated water fulvic acids.

Aqueous residues of ozonated, chlorinated, and ozonated/chlorinated water fulvic acids (WFA) were tested for induction of His+ reversion in Salmonella typhimurium strain TA100 in fluctuation tests for mutagenicity. The data suggest that ozonation of natural organics present in sources of drinking water can prevent subsequent formation of by-products of chlorination that are mutagenic in bacteria. Ozonation of the WFA at different pH and at varying dose levels produced residues that were not or were only weakly mutagenic. Chlorination of WFA or of previously ozonated WFA led to residues that were highly mutagenic. However, mutagen formation in the ozonated/chlorinated residues could be prevented, depending upon the pH of the WFA solutions during ozonation-mutagenicity decreased as pH increased. This decrease in mutagenicity is associated with previous observations of enhanced ozone decomposition into its highly reactive oxidant species at higher pH. Since ozonation seems to be more effective at alkaline pH, alkaline raw water sources seem to be the best candidates for water treatment that involves ozonation.

Benzopyrans

The effect of 2-micron DNA on survival and mutagenesis in Saccharomyces cerevisiae.

Strains of Saccharomyces cerevisiae, with and without endogenous 2-microns DNA, were studied in experiments designed to determine the effect of this plasmid on survival and mutagenesis in yeast. Comparison of the two strains exposed to ultraviolet light, 4-nitroquinoline oxide, or methyl methanesulfonate (MMS), revealed that the presence of 2-microns DNA slightly enhanced survival after exposure to each agent. Spontaneous frequencies of mutations (histidine reversion, canavanine resistance, and mitochondrial petites, but not adenine auxotrophy) were reduced by the presence of 2-microns DNA. MMS-induced His+ reversion was weak, and both strains responded similarly. No difference was found between the two strains when induced forward mutation to canavanine resistance was examined. The extent of induction of mitochondrial petites was about the same in both strains. Therefore, it appears that under these experimental conditions with these mutagens, 2-microns DNA has an effect on spontaneous mutation and survival after DNA damage but not on induced mutagenesis in S. cerevisiae.

4-Nitroquinoline-1-oxide

Contribution of chlorination to the mutagenic activity of drinking water extracts in Salmonella and Chinese hamster ovary cells.

The production of chlorinated by-products through chlorine disinfection of drinking water has been well documented. Natural organic precursors for these chemicals include fulvic and humic acids, the chlorination of which leads to the production of mutagenic compounds. Comparisons of extracts of raw versus treated waters have confirmed that clorination during water treatment produces mutagenic activity in the Salmonella (Ames) test. Present work on XAD-2 extracts of raw and chlorinated water from six municipalities in the Great Lakes region of Canada has involved a battery of mutagenicity assays for various genetic endpoints: the Salmonella test, the sister-chromatid exchange (SCE), and the micronucleus (MN) induction in Chinese hamster ovary (CHO) cells. All extracts of treated (chlorinated), but none of untreated, water were mutagenic in the Salmonella assay. On the other hand, extracts of both treated and untreated water samples showed activity in the SCE and MN assays, but no consistent pattern of response with regard to treatment (chlorination) was evident. These data show that chlorination contributes mutagens to drinking water and suggest that mammalian in vitro assays may be more sensitive for detecting mutagenicity in water samples than the Salmonella test.

Animals

Solvent interactions with test compounds and recommendations for testing to avoid artifacts.

Reports in the literature describing artifactual results owing to interactions of test materials with solvents are becoming more frequent. The present study was initiated to examine possible interactions of 1,1,3-trichloro-, 1,1,3,3-tetrachloro-, pentachloro- and hexachloroacetones with different solvents, since certain solvent effects with the last compound have been shown previously. Quantitative differences in levels of mutagenic potency were found for tri-, tetra-, and pentachloroacetone, dissolved in acetone or dimethyl sulfoxide (DMSO). On the other hand, hexachloroacetone without solvent was mutagenic, and this activity was enhanced when it was dissolved in DMSO; in acetone it was not mutagenic. A time-dependent reaction of hexachloroacetone with DMSO was the only test material-solvent interaction found in this study. The results show clearly that DMSO is not an appropriate solvent for hexachloroacetone. On the basis of these and previous results, we recommend consideration of the following strategy to help prevent reporting of artifactual results owing to interaction with solvents: Results from initial screening experiments should be confirmed in repeat experiments using a different solvent; and a different solvent should be used in confirming the findings of other investigators. Differences in results would indicate a need for further study.

Acetone