Short-term tests for carcinogens and mutagens

We studied the efficiency with which two chemical mutagens, ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) can induce mutations at different stages of spermatogenesis in zebrafish (Brachydanio rerio). Both EMS and ENU induced mutations at high rates in post-meiotic germ cells, as indicated by the incidence of F1 progeny mosaic for the albino mutation. For pre-meiotic germ cells, however, only ENU was found to be an effective mutagen, as indicated by the frequencies of non-mosaic mutant progeny at four different pigmentation loci. Several mutagenic regimens that varied in either the number of treatments or the concentration of ENU were studied to achieve an optimal ratio between the mutagenicity and toxicity. For the two most mutagenic regimens: 4 x 1 hr in 3 mM ENU and 6 x 1 hr in 3 mM ENU, the minimum estimate of frequencies of independent mutations per locus per gamete was 0.9-1.3 X 10(-3). We demonstrate that embryonic lethal mutations induced with ENU were transmitted to offspring and that they could be recovered in an F2 screen. An average frequency of specific-locus mutations of 1.1 X 10(-3) corresponded to approximately 1.7 embryonic lethal mutations per single mutagenized genome. The high rates of mutations achievable with ENU allow for rapid identification of large numbers of genes involved in a variety of aspects of zebrafish development.

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Efficient recovery of ENU-induced mutations from the zebrafish germline.

The covalent binding of chemical carcinogens to DNA of mammalian organs is expressed per unit dose, and a 'Covalent-Binding Index', CBI, is defined. CBI for various carcinogens span over 6 orders of magnitude. A similar range is observed for the carcinogenic potency in long-term bioassays on carcinogenicity. For the assessment of a risk from exposure to a carcinogen, the total DNA dmaage can be estimated if the actual dose is also accounted for. A detailed description is given for planning and performing a DNA-binding assay. A complete literature survey on DNA binding in vivo (83 compounds) is given with a calculation of CBI, where possible, 153 compounds are listed where a covalent binding to any biological macromolecule has been shown in vivo or in vitro. Recent, so far unpublished findings with aflatoxin M1, macromolecule-bound aflatoxin B1, diethylstilbestrol, and 1,2-epithiobutyronitrile are included. A comparison of CBI for rat-liver DNA with hepatocarcinogenic potency reveals a surprisingly good quantitative correlation. Refinements for a DNA-binding assay are proposed. Possibilites and limitations in the use of DNA binding in chemical carcinogenesis are discussed extensively.

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In vivo covalent binding of organic chemicals to DNA as a quantitative indicator in the process of chemical carcinogenesis.

Induced chromosomal and gene mutations play a role in carcinogenesis and may be involved in the production of birth defects and other disease conditions. While it is widely accepted that in vivo mutation assays are more relevant to the human condition than are in vitro assays, our ability to evaluate mutagenesis in vivo in a broad range of tissues has historically been quite limited. The development of transgenic rodent (TGR) mutation models has given us the ability to detect, quantify, and sequence mutations in a range of somatic and germ cells. This document provides a comprehensive review of the TGR mutation assay literature and assesses the potential use of these assays in a regulatory context. The information is arranged as follows. (1) TGR mutagenicity models and their use for the analysis of gene and chromosomal mutation are fully described. (2) The principles underlying current OECD tests for the assessment of genotoxicity in vitro and in vivo, and also nontransgenic assays available for assessment of gene mutation, are described. (3) All available information pertaining to the conduct of TGR assays and important parameters of assay performance have been tabulated and analyzed. (4) The performance of TGR assays, both in isolation and as part of a battery of in vitro and in vivo short-term genotoxicity tests, in predicting carcinogenicity is described. (5) Recommendations are made regarding the experimental parameters for TGR assays, and the use of TGR assays in a regulatory context.

Detailed review of transgenic rodent mutation assays

An integrative theory is proposed in which environmental carcinogenesis is viewed as a process by which the genetic control of cell divzsion and differentiation is altered by carcinogens. In thzs theory, carcinogens include physical, chemical, and viral "mutagens," as well as chemical and viral gene modulators. Existing explanations of carcinogenesis can be considered either as somatic mutation theories or as epigenetic theories. Evidence seems to support the hypothesis that both mutations and epigenetic processes are components of carcinogenesis. The mutational basis of cancer is supported by the clonal nature of tumors, the mutagenicity of most carcinogens, high mutationfrequencies in cells of cancer-prone human fibroblasts lacking DNA repair enzymes, the correlation of in vitro DNA damage and in vitro mutation and transformationfrequencies with in vivo tumorigenesis, age-related incidences of various hereditary tumors, and the correlation between photoreactivation of DNA damage and the biological amelioration of UV-induced neoplasms. Since both mutagens and gene modulators can be carcinogenic it may be that carcinogens affect genes which control cell division. An integration of the mutation and epigenetic theories of cancer with the "two-stage" theory and Comings's general theory of carcinogenesis is proposed. This integrative theory postulates that carcinogens can affect regulatory genes which control a series of "transforming genes. A general hypothesis is advanced that involves a common mechanism of somatic mutagenesis via error-prone repair of DNA damage which links carcinogenesis, teratogenesis, atherosclerosis and aging. Various concepts are presented to provide aframework for evaluating the scientific, medical, and social implications of cancer. "This innerfragmentation of man mirrors his view of the world 'outside' which is seen as a multitude of separate objects and events. The natural environment is treated as if it consisted of separate parts to be exploited by different interest groups. The fragmented view is further extended to society which is split into different nations, races, religious and political groups. The belief that all thesefragments - in ourselves, in our environment and in our society - are really separate can be seen as the essential reason for the present series of social, ecological, and cultural crises. It has alienated us from nature andfrom our fellow human beings. It has brought a grossly unjust distribution of natural resources creating economic and political dzsorder; an ever rising wave of violence both spontaneous and

An integrative model

An unscheduled DNA synthesis has been clearly demonstrated in meiotic and postmeiotic germ cell stages of the male mouse after in vivo treatment with a 250 mg/kg dose of ethyl methanesulfonate. The germ cell stages showing unscheduled DNA synthesis range from early to middle meiotic prophase stages through early to middle spermatid stages. The initiation of this synthesis, taken to be repair of chemically damaged DNA in these germ cells, is rapid, beginning within 1 hour after the injection of ethyl methanesulfonate. Unscheduled DNA synthesis has not been detected in the most mature germ cell stages, which give rise to dominant lethal mutations, nor does it occur in germ cell stages where protamine has replaced the chromosomal histones.

https://www.pnas.org/content/pnas/71/12/4955.full.pdf

Unscheduled DNA synthesis in the germ cells of male mice exposed in vivo to the chemical mutagen ethyl methanesulfonate.

This study shows that 2 commonly held explanations for mosaics induced by treating postmeiotic spermatozoa with chemical mutagens are incosistent with an analysis of the percent of mutant tissue produced in the F 1 . The first model tested assumes that mosaics are the results of altering one strand of the DNA double helix so that there will be an equal number of mutant and nonmutant nuclei following semiconservative replication. The second model assumes that alkylation of a base in DNA will cause a fixed probability of base pairing mistakes and may produced a mutation at any replication. In phage the probability of mispairing following alkylation is low so that a mutation likely occurs only once in 8 replications; therefore, most of the mutations are fixed after the first 2 divisions. If Drosophila is similar to phage, the mean frequency of mutant nuclei and the frequency distribution of mosaics by class of percent mutant tissue will be distinctly different for the 2 models. Males were fed ethyl methanesulfonate and mutations were detected in the F 1 by the specific locus method at the yellow ( y ) and white w loci. Agreement between this experiment and previous work with mosaics produced by chromosome aberrations was found in that the germ line developed from a small sample of poorly mixed nuclei that was independent of the phenotype of head and thorax. The latter finding allowed a selection of mosaics to be made independent of the germ line, and a method of analysis was developed for flies selected because they were mosaic in the head or thorax. Analyses of results from the eye mosaics in this experiment and from 3 experiments with the dumpy (dp) locus previously reported by others but not analyzed in this was were inconsistent with both original models. The average percent of mutant phenotype among the yellow mosaic observed in this experiment was also found inconsistent with both original models, and a frequency distribution of mosaics classified by percent of mutant tissue was inconsistent with both original models and a combination of the two; therefore, both of our initial models and a combination of the two were rejected. The data are consistent with delayed mutation in which an alkylated base has about a 50% probability of mispairing at each replication and producing a fixed mutation or with a multistranded model in which the postmeiotic eukaryote chromosome contains 2 parallel DNA molecules for each locus.

Chemically induced mutations observed as mosaics in Drosophila melanogaster.

Summary A method for determining the chemical dose of ethyl methanesulfonate (EMS) to the DNA of Drosophila melanogaster sperm cells has been developed. Males fed as larvae on media containing [ Me - 3 H]thymidine were fed0.025 M , 0.0125 M and 0.008 M concentrationd of [I- 14 C]ethyl methanesulfonate ([ 14 ]EMS) as adults. Our feeding procedures and concentrations were chosen so as to duplicate as nearly as possible the treatments other workers have used. The amount of 3 H per sperm cell was determined by microscopically counting sperm cells, washing them with cold trichloroacetic acid (TCA) onto a MIllipore filter, and counting radioactivity with a liquid scintillation spectrometer. 14 C and 3 H counts were determined for oligodeoxynucleotides released from labeled sperm DNA by electrophoretically purified deoxyribonuclease. From these two counts, the specific activity of [ 14 C]EMS and the amount of 3 H per cell, the number of ethylations per sperm cell DNA was found to range from 1.10 7 to 3.10 8 . Estimates for the number of nucleotides per sperm cell DNA of Drosophila are found from the literature to range from 1.6· 10 8 to 4.5 · 10 8 . It has, therefore, been concluded that the concentrations of [ 14 C]EMS used in these experiments were able to ethylate between 3% and 100% of the nucleotides in the sperm cell DNA of Drosophila, assuming one ethylation per nucleotide. The results of our chemical dosimetry experiments are also discussed in relation to comparative mutagenesis studies with other genetically well studied organisms, such as the mouse. On the basis of the extensive DNA ethylation found in Drosophila sperm combined with previously published reports on sex-linked recessive lethal frequencies and production of mosaics following EMS treatment of Drosophila, it is hypothesized that ethylation of a rare site or combination of sites in the sperm DNA is responsible for the mutagenic action of EMS. Previous models models suggesting the production of mutations by incorrect base pairing of 7-ethylguanine following treatment by EMS cannot explain mutagenesis in Drosophila.

Dosimetry of the chemical mutagen ethyl methanesulfonate in spermatozoan DNA fromDrosophila melanogaster

This report describes a model for producing quantitative genetic risk assessments for human populations. The model is patterned after current methods used in cancer risk analysis. The risk to humans is expressed as the number of additional dominant genetic disease added to the existing genetic burden, in the offspring of the exposed individuals.

Gary A. Sega

Papers

Unscheduled DNA synthesis in the germ cells of male mice exposed in vivo to the chemical mutagen ethyl methanesulfonate.

Chemically induced mutations observed as mosaics in Drosophila melanogaster.

Dosimetry of the chemical mutagen ethyl methanesulfonate in spermatozoan DNA fromDrosophila melanogaster

Genetic risk extrapolation from animal data to human disease: A taskgroup report

Mutations produced by transmutation of phosphorus-32 to sulfur-32 within Drosophila DNA.