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The anti-oestrogen tamoxifen is very effective in the treatment and prevention of breast cancer. Tamoxifen is not a pure antagonist, but possesses weak oestrogenic activity that may contribute to a slightly increased risk of endometrial cancer. Whilst this can be incorporated into risk-benefit analysis for the use of the drug, residual concerns exist over the exact mechanism of formation of these tumours. Tamoxifen is a potent hepatocarcinogen in the rat, probably via a genotoxic mechanism. Whilst tamoxifen does not appear to cause liver tumours in humans, DNA adducts have been found in endometrial tissue of women receiving the drug. Hence, there is still a need to establish the mechanism of formation of these tumours. We have therefore determined the molecular nature of mutations induced in vitro by alpha-hydroxytamoxifen, the putative proximate genotoxic metabolite, in a mammalian cell line (V79-rHSTa) with stable expression of rat hydroxysteroid sulfotransferase a, which catalyses the further metabolism of alpha-hydroxytamoxifen to its ultimate genotoxic product. DNA sequence alterations were examined at the Hprt gene in 50 mutant clones. Simple base substitutions, mainly GC-->TA transversions, predominated. However, single G:C base pair deletions and partial/complete exon skippings were also observed. All but one of the mutations involved guanine bases on the non-transcribed strand, probably indicating preferential repair of alpha-hydroxytamoxifen-induced guanine adducts from the transcribed strand. Nearest neighbour analysis of the mutations (on the non-transcribed strand) indicated that thymines (20/40) followed by guanines (13/40) were the most frequent 5' neighbours, with adenines or guanines the most frequent 3' neighbours. Many of the mutations occurred at TTGA/G sequences. Three mutational hot spots accounted for 11 GC-->TA transversions and another site for two single G:C base pair deletions. A search for these characteristic mutations in tumour-related genes of treated rats and humans should help in understanding the mechanism(s) of tamoxifen-induced carcinogenicity.

The natural hormone estradiol (E2) induces tumors in rodents and various types of DNA damage in vitro and in vivo, but has not been mutagenic in bacterial or mammalian assays. Recent reports of chromosomal and genetic lesions induced by E2 has led us to re-examine the mutation frequency and molecular alterations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene in Chinese hamster V79 cells. E2 at both physiological and pharmacological concentrations (10-11, 10-10, and 10-7, 10-6 M) significantly increased the mutation frequency of the hprt gene by 2. 57-, 3.45-, 2.63-, and 8.78-fold, respectively, compared to the controls, while 10-13, 10-12, 10-9, or 10-8 M E2 induced little change (< or =0.93-fold). PCR and a molecular analysis of the hprt coding sequence identified genetic lesions in the cDNA and/or genomic DNA in 15 of the 21 picked E2-induced mutants (71%). Simple base substitutions, such as Tright curved arrow G or Tright curved arrow A transversions, were the most common mutations (8/21 or 38%) and frequently occurred at 122 bp or 407 bp of the hprt coding sequence. Deletion mutations were detected in 6 of the 21 clones (29%). An Aright curved arrow G and a Cright curved arrow T transition and a four-base insertion (TATT) were identified each in one mutant clone. A RT-PCR analysis demonstrated an abundant expression of the estrogen receptor-alpha (ERalpha). However, ICI 182,780, an antagonist of ERalpha, acted in an additive manner with E2 and increased the hprt mutation frequency. In conclusion, E2 induces a low frequency of mutations (deletions and point mutations) in V79 cells, which is consistent with the weak carcinogenic activity of this hormone. The mutagenic effects of E2 in V79 cells are not mediated by the ERalpha.

This article addresses the evidence that trichloroethylene (TCE) or its metabolites might mediate tumor formation via a mutagenic mode of action. We review and draw conclusions from the published mutagenicity and genotoxicity information for TCE and its metabolites, chloral hydrate (CH), dichloroacetic acid (DCA), trichloroacetic acid (TCA), trichloroethanol, S-(1, 2-dichlorovinyl)-l-cysteine (DCVC), and S-(1, 2-dichlorovinyl) glutathione (DCVG). The new U.S. Environmental Protection Agency proposed Cancer Risk Assessment Guidelines provide for an assessment of the key events involved in the development of specific tumors. Consistent with this thinking, we provide a new and general strategy for interpreting genotoxicity data that goes beyond a simple determination that the chemical is or is not genotoxic. For TCE, we conclude that the weight of the evidence argues that chemically induced mutation is unlikely to be a key event in the induction of human tumors that might be caused by TCE itself (as the parent compound) and its metabolites, CH, DCA, and TCA. This conclusion derives primarily from the fact that these chemicals require very high doses to be genotoxic. There is not enough information to draw any conclusions for trichloroethanol and the two trichloroethylene conjugates, DCVC and DCVG. There is some evidence that DCVC is a more potent mutagen than CH, DCA, or TCA. Unfortunately, definitive conclusions as to whether TCE will induce tumors in humans via a mutagenic mode of action cannot be drawn from the available information. More research, including the development and use of new techniques, is required before it is possible to make a definitive assessment as to whether chemically induced mutation is a key event in any human tumors resulting from exposure to TCE.

The genotoxic effect of dioxidine (300, 100, 30, and 10 mg/kg, i.p.) on the liver cells of outbred male rats was studied by fluorometric analysis of DNA unwinding (FADU) using the cell DNA samples taken from animals sacrificed 1, 6, 18, and 24 h after drug introduction. At a dose of 10 mg/kg, dioxidine produced no statistically significant increase in the amount of DNA damage relative to control. Increase in the dioxidine dose to 30 mg/kg resulted in a significant DNA damage 1 h after drug administration, while a dose of 100 or 300 mg/kg reliably caused damage at all times of exposure.

We have investigated the mutagenicity of benzo[a]pyrene (B[a]P) in small intestine using the Dlb-1 locus assay in the mouse. Administration of B[a]P by the oral and i.p. routes had markedly different effects on the number of Dlb-I mutations and the pattern of induction of cytochrome P-4501A1 (CYP1A1). In Ahr-responsive animals i.p. injection resulted in marked induction in crypt cells along the length of the small intestine, with some induction in the villus cells. In contrast, after oral administration, CYP1A1 induction was evident only in the villus cells, and this declined distally. The intensity and speed of induction in Ahr-responsive animals was such that the genotoxic effect of a single injection of B[a]P could not be augmented by prior treatment with non-genotoxic inducers such as beta-napthoflavone and TCDD. Oral B[a]P treatment resulted in a decrease in the number of mutations when compared with the i.p. route. Studies in congenic Ahr-non-responsive versus Ahr-responsive mice indicated that induction of CYP1A1 was associated with increased numbers of Dlb-1 mutations. Mutation induction in Ahr-non-responsive mice in the absence of detectable CYP1A1 in either liver or small intestine indicates that an appreciable portion of B[a]P activation to a genotoxin must be by other than a CYP1A1 mediated route. These data show that B[a]P is a potent small intestinal mutagen at the Dlb-1 locus.

It is known that DNA repair is heterogeneous in human cells since open chromatin, active genes and their transcribed strands are preferentially repaired. It is thus expected that DNA repair is clustered in chromosomes with high gene density. We have employed a DNA repair inhibitor, cytosine arabinoside (Ara-C), to convert ethyl methane sulfonate (EMS)-induced excision repairable lesions to chromosomal breaks, to check for the existence of heterogeneity of repair at the chromosome level. Chromosome staining by fluorescence in situ hybridization (FISH) was used to analyze breakage in chromosomes with diverse gene densities. These chromosomes were identified by means of the CpG island distribution after FISH with a CpG island-rich probe isolated from total human genomic DNA. Thus, three chromosomes with very high gene density (numbers 1, 19 and 20) were compared with two chromosomes with very low gene density (numbers 4 and 18) for clastogenicity and sensitivity to co-treatment with Ara-C and EMS. Our data indicate that those chromosome with higher gene density are more sensitive to a combination treatment with Ara-C and EMS, indicating that the level of excision repair synthesis is higher in those chromosome. It is therefore concluded that DNA excision repair is preferentially directed to chromosomes with high gene density. The implications of this finding in human biomonitoring using FISH techniques are discussed.

The role of the nucleotide excision repair (NER) pathway in removal of DNA ethylation damage was investigated by means of hprt mutational spectra analysis in the NER-deficient Chinese hamster ovary cell line UV5, which lacks ERCC2/XPD, and in its parental cell line AA8. Both cell lines were exposed to ethyl methanesulfonate (EMS) or N-ethyl-N-nitrosourea (ENU). EMS gave a similar dose-dependent increase in hprt mutants in UV5 compared with AA8. In both cell lines EMS-induced mutations in the hprt coding region consisted almost exclusively of GC-->AT transitions, probably due to the direct miscoding lesion O6-ethylguanine. ENU, an agent that in addition to O6-ethylguanine also induces other O-alkylation products, was significantly more mutagenic in UV5 than in AA8. Mutational spectra analysis showed that the proportions of ENU-induced GC-->AT, AT-->TA and AT-->GC base pair changes were similar for both cell lines. ENU-induced DNA lesions that may be involved in GC-->AT transitions are O6-ethylguanine and O2-ethylcytosine, the latter being a chemically stable DNA lesion of which the miscoding properties and repair characteristics are largely unknown. ENU-induced AT-->TA transversions are probably caused by O2-ethylthymine, which mispairs with thymine. In AA8 thymines in ENU-induced AT-->TA transversions were exclusively located in the non-transcribed strand of the hprt gene, whereas in UV5 30% of these thymines were found in the transcribed strand. Together, these results indicate that O6-ethylguanine is a poor substrate for NER in rodent cells and that O2-ethylpyrimidines are preferentially removed from the transcribed strand of the hprt gene by NER.

The paper reviews the main characteristics of the cytogenetic indicators most frequently used in monitoring early biological effects in groups exposed to genotoxic agents, i.e. chromosome aberrations (CA), sister chromatid exchanges (SCE) and micronuclei (MN) in lymphocytes. The results of the numerous studies on CA and/or SCE performed on hospital or industry personnel potentially exposed to antineoplastic drugs are conflicting, probably due to the different degrees of exposure in different occupational settings, to different sensitivity of the indicators or to their different persistence. However, the studies carried out on groups of subjects who have always worked in protected conditions have given negative results for all indicators.

DNA alterations induced in V79 cells treated with UV light or methyl-nitro-nitrosoguanidine were analyzed by the mutagenicity test at the hprt locus and by DNA fingerprint analysis. Treated and control cells were seeded in the presence or absence of 6-thioguanine to determine mutant frequency and cell survival. From clonal cultures of the same cell populations we isolated a number of clones and grew them up individually to obtain appropriate amounts of DNA. High molecular weight DNA was digested with HinfI or HaeIII and hybridized with 32P-labelled 33.15 multilocus probe. The induction of 6-thioguanine resistant cells depended on the mutagen dose. The highest value of mutant frequency obtained was 7475 x 10(-6) (MNNG, 27 microM), corresponding to 0.7 percent of clonable cells. DNA fingerprint analysis carried on the same treated cells showed that DNA rearrangements occurred at minisatellites much more frequently than in transcribed sequences. UV irradiation produced the highest frequency of variation, modifying minisatellite patterns in about 50 percent of the analyzed clones.

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.

A total of 15 caramel colours were examined for genotoxic activity using the Salmonella typhimurium plate incorporation assay (Ames test). Five bacterial strains, TA1535, TA1537, TA1538, TA98 and TA100 were used in all the plate incorporation tests. Caramel colours can be divided into four classes, classification depending on the preparative method used. In this study, representatives of all four classes of caramel colour were tested for genotoxic potential in the Ames test, some of the caramel colours being tested both with and without a pre-incubation stage. None of the 15 caramel colours tested exhibited genotoxic potential in any of the five bacterial tester strains. The last two caramel colours tested, in the series of 15 [203-23-4 (Class II) and 311 (Class III)] were also assessed for clastogenic potential. For this test, cultures of CHO cells were exposed to the two caramel colours and metaphase preparations from these cultures examined for evidence of chromosomal aberrations. No evidence of chromosome damaging activity was observed.

A battery of three short-term tests in vitro and one in vivo was used to determine the genotoxicity of Caramel Colour I. The results of the bacterial mutation assay, using five strains of Salmonella typhimurium, and the mouse micronucleus assay in vivo showed no evidence of genotoxic activity. Results from both the cytogenetics assay in vitro, using CHO cells, and the mouse lymphoma assay indicated that there was some genotoxic activity associated with Caramel Colour I but only in the absence of S-9 and at very high dose levels.

The mutagenic activation of benzo[a]pyrene (BaP) after exposure to aorta smooth muscle cells of different origin was examined. Three test systems with different genetic endpoints--sister-chromatid exchange (SCE), gene mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus and unscheduled DNA synthesis (UDS)--were used. Treatment of rat and rabbit aorta smooth muscle cells with BaP (1-6 micrograms/ml) resulted in a significant increase of SCEs, HGPRT mutations and UDS. So smooth muscle cells are capable of converting BaP to metabolites with a DNA-damaging action. In order to investigate the relation between the formation of mutagenic BaP metabolites and the susceptibility to atherosclerosis we compared the mutagenic potential of BaP using aorta smooth muscle cells of different species (rat, rabbit) and locations (thoracic and abdominal aorta). Rabbits and abdominal aortas are more susceptible to atherosclerosis than rats and thoracic aortas. The SCE, HGPRT and UDS assays revealed that smooth muscle cells of different origin possessed the same metabolic potential towards BaP. There was no correlation between the mutagenic potency of BaP metabolites and the susceptibility to atherosclerosis. As smooth muscle cells have a low metabolic capacity towards BaP, probably other factors in addition to the metabolic capacity of smooth muscle cells are responsible for species and tissue differences in susceptibility to atherosclerosis.

A variation of the mouse lymphoma (L5178Y TK+/(-)-3.7.2c) assay has been developed using a microtiter cloning technique instead of the standard agar method. The cell line has been used to detect both gene mutations (at the Na+/K+ ATPase and thymidine kinase loci) and chromosome damage (micronucleus induction) in the same experiment. The system was validated using gamma-irradiation (a known clastogen), 2 direct-acting mutagens, ethyl and methyl methanesulphonate and an indirect-acting mutagen, benzo[a]pyrene. Using the assay, 1-methoxy-1,3,5-cycloheptatriene was shown to be a clastogenic mutagen in the presence of S9, since a clear dose-dependent increase in micronuclei was observed, mainly small colony thymidine kinase mutants were observed, and no ouabain-resistant mutants were induced, a profile very similar to gamma-irradiation. The results suggest that metabolic activation potential explains the results in the accompanying paper (Asquith et al., 1990). The implications for mutagenicity testing are discussed.

The predominant test system uses a near-diploid L5178Y mouse lymphoma cell line and is based on the quantitation of forward mutations occurring at the heterozygous thymidine kinase (TK) locus (TK+/- leads to TK-/-). (Other markers, such as ouabain- or methothrexate-resistance and alanine independence, in other L5178Y mouse lymphoma cells were also examined, but our criteria for the acceptability of data or the paucity of data considerably reduced the value of these mutagenesis test systems to this study.) The biochemical basis for the L5178Y/TK+/- assay depends on the ability of TK-competent cells to phosphorylate 5-bromo-2'-deoxyuridine or trifluorothymidine. The phosphorylated product or its metabolites kill these cells, thus, medium containing 5-bromo-2'-deoxyuridine or trifluorothymidine is capable of selecting for cells that are lacking the TK enzyme (TK-/-). TK-/- cells eventually give rise to a bimodal distribution of colony sizes. The relative proportion of small and large colonies appears to be characteristic of the mutagen, its dose, and the length of the expression period. A total of 108 references were reviewed and 48 chemical agents were evaluated. Of these, in vivo carcinogenicity data existed for 44 and covered a wide variety of chemical classes (43 compounds) and a complex mixture. In this system, 39 agents were positive, 1 was negative, and 4 yielded inconclusive results. The 44 test substances evaluated were insufficient to single out agents or agent classes for which the assay was particularly well suited; however, with the exception of thymidine analogs, the system seems to be versatile. The correlation of the TK locus assay results with the carcinogenicity data revealed that 2 agents were definite false positives (sodium azide and methotrexate) and 1 agent was a definite false negative (1,1-dimethylhydrazine). Further evaluation suggested that 4-acetylaminofluorene and diphenylnitrosamine were questionable false positives, while benzo[e]pyrene was a questionable false negative. (The term questionable was used to imply uncertainties concerning the mutagenicity and/or carcinogenicity data). Thus, the assay is of value in the battery approach to mutagenicity/carcinogenicity screening.

Chinese hamster cells were made use of to study the effect of low doses of benz(a)pyrene on gene mutations during metabolic activation. The HGRPT mutant clones were selected on the basis of their resistance to 8-azaguanine. The raising of benz(a)pyrene concentration (from 5 g/ml) was followed by the enhanced mutagenic effect which was approximately 7-10 times greater than the spontaneous level at a concentration of 20-25 micrograms/kg.

Genetic control of somatic mutagen sensitivity was studied in a strain of Drosophila melanogaster containing mutations in the second chromosome. The method used allowed to identify the determinants of different kind of sensitivity in the same experiment. It has been shown that the second chromosome of the strain analysed carries two independent mutations, one of them determining supersensitivity of Drosophila larvae to ionizing radiation, the other--to methyl methane-sulfonate. Mutant loci have been located at 59,9 +/- 1,1 and 73,8 +/- 2,5 units of the chromosome genetic map. They have been called rad(2)201G1 and mus(2)201G1, respectively.

Physarum displays two vegetative cell types, uninucleate myxamoebae and multinucleate plasmodia. Mutant myxamoebae of Physarum resistant to the antitubulin drug methylbenzimidazole-2-yl-carbamate (MBC) were isolated. All mutants tested were cross-resistant to other benzimidazoles but not to cycloheximide or emetine. Genetic analysis showed that mutation to MBC resistance can occur at any one of four unlinked loci, benA, benB, benC or benD. MBC resistance of benB and benD mutants was expressed in plasmodia, but benA and benC mutant plasmodia were MBC sensitive, suggesting that benA and benC encode myxamoeba-specific products. Myxamoebae carrying the recessive benD210 mutation express a beta-tubulin with novel electrophoretic mobility, in addition to a beta-tubulin with wild-type mobility. This and other evidence indicates that benD is a structural gene for beta-tubulin, and that at least two beta-tubulin genes are expressed in myxamoebae. Comparisons of the beta-tubulins of wild-type and benD210 strains by gel electrophoresis revealed that, of the three (or more) beta-tubulin genes expressed in Physarum, one, benD, is expressed in both myxamoebae and plasmodia, one is expressed specifically in myxamoebae and one is expressed specifically in plasmodia. However, mutation in only one gene, benD, is sufficient to confer MBC resistance on both myxamoebae and plasmodia.

Crude extracts from maize plants treated with varying concentrations of AAtrex, an s-triazine herbicide, were tested for the induction of gene conversion in a diploid strain of Saccharomyces cerevisiae heteroallelic at two loci. These extracts were then subjected to analysis by higher pressure liquid chromatography (HPLC), and the fractions collected in water, 50% methanol, and 100% methanol were similarly tested. Dose-dependent convertogenic activity observed in the 50% methanol fractions suggested that the mutagen(s) is recovered in this fraction.

The trifunctional alkylating mutagen Trenimon interferes with the genetic material of a variety of organisms and test systems with respect to the induction of point and chromosomal mutations, sister-chromatid exchanges, recombination phenomena and phage induction. Beneath these mutagenic effects several biochemical and cell physiological aspects have been investigated. In this review we discuss chemical and cell physiological effects of Trenimon, aspects of cancer therapy with Trenimon and genetic effects induced by Trenimon. The available data on mutagenic effects of Trenimon are presented according to organisms or test systems. A short discussion on a possible genetic load by therapy with Trenimon in man concludes this review. DNA damage, especially the induction of cross-linkings, seems to represent the common reason for most of the described effects of Trenimon on cells and organisms.

For certain organic solvents, such as benzene, vinyl chloride, styrene, technical grade trichloroethylene, and acrylonitrile, the available studies provide convincing evidence to demonstrate activity in short-term genetic assays. For a few solvents, such as phenol, vinyl toluene, ethanol, and tetrachloroethylene, the evidence is limited to a certain test system and/or test organism. For most of the solvents reviewed, studies are either lacking or they are so inadequate that no final evaluation on the mutagenic activity of the solvents can be made.

2,4-Diaminotoluene, a hepatocarcinogenic aromatic amine, was tested for mutagenic potential at both the autosomal tk locus and the sex-linked hgprt locus of both L5178Y 3.7.2C mouse lymphoma cells and Chinese hamster ovary (CHO) AT3-2 cells. This compound was mutagenic in both cell types at the tk locus but not at the hgprt locus. Mutagenic activity was observed in L5178Y cells only in the absence of exogenous metabolic activation, but was observed in CHO-AT3-2 cells both with and without activation.

A benzene metadiamine derivative, 2-(2', 4' diaminophenoxy) ethanol proved to be interesting in hairdyeing. Its genotoxicity was investigated in various tests. The ingredient showed no mutagenicity in the different assays which indicates a correlation among the battery of tests. It also appears that within a chemical category, similarity of structure does not always imply similar genetic response.

Three antihypertensive hydrazine derivatives (hydralazine, dihydralazine, and endralazine) were found to be genotoxic in four in vivo or in vitro short-term test systems. a) In mice, a single ip administration of the LD50 of the three drugs caused a small but statistically significant increase over controls in DNA elution rate, ie, a modest amount of DNA fragmentation, in three of the four organs (liver, lung, kidney, and spleen) tested, DNA damage being absent in lung for hydralazine and endralazine and in liver for dihydralazine. Only for hydralazine DNA lesions were always repaired within 12 hr, in agreement with the constant lack of cumulative effects in mice given five successive daily doses. The rank of potencies was hydralazine greater than dihydralazine greater than endralazine. b) In mice bone marrow cells, all three hydrazine derivatives induced a modest but statistically significant increase over controls in the frequency of sister chromatid exchanges, the rank of potencies being in this case dihydralazine greater than endralazine greater than hydralazine. c) In the Ames reversion test all three drugs behaved as direct-acting mutagens of low potency, whose activity was not influenced by rat liver nor by mouse liver or lung S-9 fractions. Hydralazine and dihydralazine elicited mixed genetic mechanisms of mutations, while endralazine exclusively induced frameshift errors in Salmonella DNA. The recently developed strain TA97 was the most efficient in revealing frameshift errors with all three drugs. d) The selective lethality assays in a battery of two S typhimurium and five E coli strains confirmed the direct genotoxicity of hydralazine, dihydralazine, and endralazine, in order of potency. Potency was evaluated by means of a sensitive and reliable micromethod procedure. Among those investigated, the recA recombination repair and the lexA post-replication repair ("SOS functions") and, to a lesser extent, also the polymerase I mechanism, appeared to contribute to the specific DNA repair with all three drugs, while excision repair systems (uvrA and uvrB) did not appear to be involved.

Experiments are performed to compare the mutagenic properties of the three phosphamide esters of nitrogen mustard, cyclophosphamide (CP), ifosfamide (IF), and trofosfamide (TF), in different bacterial systems. The systems include forward mutations leading to resistance against 5-methyltryptophan (MTR) and from galR-s18 to gal-+ in Escherichia coli 343/113, back mutations from arg56 to arg-+ in Escherichia coli 343/113 and back mutations from hisG46 to his-+ in Salmonella typhimurium TA1535. CP, IF, and TF are not mutagenic per se. After biotransformation through isolated rodent liver homogenates (S-9 fraction) all three compounds exhibit mutagenic activity in the order CP smaller than IF smaller than TF. Specific activating potential of mouse liver extracts is higher than that of rat liver. Except for back mutations in S. typhimurium TA1535, all mutation systems tested show a similar pattern of induction after treatment with CP, IF, and TF. However, because gal-+ mutations are not induced by CP under conditions where arg-+ and MTR are induced, it is suggested that more than one mutational system be used in routine mutagenicity testing.

The mutational specificity of the monofunctional alkylating agent N-propyl-N'-nitro-N-nitrosoguanidine (PNNG) has been determined through the DNA sequence characterization of 109 LacI- mutations of Escherichia coli. The predominant mutation induced was the G:C----A:T transition (73%), presumably the result of O6-propylguanine damage. Transversions constituted 18% of the mutants, almost entirely due to G:C----T:A (9%) and A:T----C:G (8%) events. Two identical deletions, one single base pair frameshift and a tandem double base change were also recovered. In contrast, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was previously found to induce only transitions, again predominantly G:C----A:T events (98%). Moreover, the site specificity observed for PNNG-induced G:C----A:T transitions is quite distinct from that induced by MNNG. G:C----A:T transitions recovered following PNNG treatment do not appear to be influenced by neighbouring base sequence to the extent seen for MNNG.

The ability of sorbic acid and its potassium and sodium salts to induce chromosome aberrations, sister chromatid exchanges (SCE) and gene mutations in cultured Chinese hamster V79 cells was examined. Sodium sorbate caused significant induction of chromosome aberrations and SCE, and also induced 6-thioguanine-resistant mutations in a dose-dependent manner. The clastogenic potency of sodium sorbate was found to be less than one hundredth of that of the potent clastogen N-methyl-N'-nitro-N-nitrosoguanidine. The induction of SCE by sodium sorbate was twice the control level, whereas that by methyl methanesulphonate, a potent inducer of SCE, was 14 times the control level. The mutagenic potency of sodium sorbate was less than one-tenth that of ethyl methanesulphonate, a potent inducer of mutation, when compared at an equitoxic level. Sorbic acid and its potassium salt induced chromosome aberrations, but only at the highest doses tested. These compounds also induced 1.2 times the control level of SCE, but neither compound induced 6-thioguanine-resistant mutations. The cytogenetic activity of sodium sorbate was concluded not to be due to the effect of osmotic pressure or an impurity. These results indicate that sodium sorbate is a genotoxic agent, although its potency seems to be weak, and that sorbic acid and potassium sorbate are less genotoxic than the sodium salt.

Extensive tests to detect transmitted genetic effects of hycanthone in mammals have been made to provide the most pertinent information for estimating a possible mutagenic hazard of this compound. The tests include dominant lethal studies in males and females, X chromosome loss study in offspring of treated females, and a search for deficiencies and gene mutations in the offspring of treated males whose germ cells were exposed in spermatogonial and postspermatogonial stages. In most tests, no genetic effect of hycanthone was observed even at dosages much higher than the clinical level. In the few cases where the results are possibly or definitely positive, there is none where the magnitude of the effect estimated, with maximizing assumptions, at the clinical level reaches as high as 10% of the spontaneous mutation rate or 10% of the frequency in controls.

Ethyl methanesulfonate (EMS) is a monofunctional ethylating agent that has been found to be mutagenic in a wide variety of genetic test systems from viruses to mammals. It has also been shown to be carcinogenic in mammals. Alkylation of cellular, nucleophilic sites by EMS occurs via a mixed SN1/SN2 reaction mechanism. While ethylation of DNA occurs principally at nitrogen positions in the bases, because of the partial SN1 character of the reaction, EMS is also able to produce significant levels of alkylation at oxygens such as the O6 of guanine and in the DNA phosphate groups. Genetic data obtained using microorganisms suggest that EMS may produce both GC to AT and AT to GC transition mutations. There is also some evidence that EMS can cause base-pair insertions or deletions as well as more extensive intragenic deletions. In higher organisms, there is clear-cut evidence that EMS is able to break chromosomes, although the mechanisms involved are not well understood. An often cited hypothesis is that DNA bases ethylated by EMS (mostly the N-7 position of guanine) gradually hydrolyze from the deoxyribose on the DNA backbone leaving behind an apurinic (or possibly an apyrimidinic) site that is unstable and can lead to single-strand breakage of the DNA. Data also exist that suggest that ethylation of some chromosomal proteins in mouse spermatids by EMS may be an important factor in causing chromosome breakage.

The present paper reviews the currently available in vivo systems for detection of chemically induced mutations and chromosome aberrations and summarizes the data of the relevant tests for mammalian germ-cell mutations (specific-locus test and heritable translocation test). The value of in vivo screening tests (somatic mutations and sperm abnormalities) for predicting specific-locus mutations is illustrated by comparing doubling doses. The results from the mammalian germ-cell mutation tests (specific-locus test and heritable translocation test) constitute the base-line for an assessment of predictability. Radiation and chemically induced specific-locus mutations differ in a number of respects, suggesting a need for caution in making risk estimates for chemical mutagen exposures in terms of radiation-equivalent doses. In vivo nondisjunction tests are discussed. Finally, unsolved problems and difficulties in generalizing qualitative and quantitative correlations between test systems are outlined. It is concluded that even qualitative predictions from data on somatic cells to germ cells are at best insecure because germ-cell specificity cannot be foretold, not to mention the fact that quantitative extrapolations from the results of in vivo screening tests, in general, are fraught with even more uncertainties. There is an acute need for collection of more data from studies involving germ cells.

Using oligonucleotide-directed mutagenesis, we are trying to define the features of the protein structure that are important for the DNA and c-AMP binding by CAP from E. coli, the enzymic activity and putative DNA binding of dihydrofolate reductase of L. casei, and the functionally important regions of the self-splicing RNA of the r-RNA intron of Tetrahymena thermophila. We have used a modification of the method described by Norris et al. [1]. A mutagenic primer and an M13 universal sequencing primer are annealed simultaneously to a template from an M13 clone containing the DNA to be mutagenised and, after DNA strand extension, the fragment is cut out and recloned into either M13 or plasmid vectors. We have analysed the effect on the frequency of mutation of: the temperature used for strand extension; the class of base change attempted; the host mismatch repair system. A recently developed system for phenotypic detection of mutations in the Tetrahymena intron aided in determining mutation frequencies.

Dose-response relationships and determination of dose of mutagens and carcinogens are summarized and discussed on the basis of conceptual and kinetic aspects. Different dose definitions may be referred to steps in the chain of events from exposure (or emission) to observed effects. A system is applied to show the influence of various processes on the kinetics of the transfers between consecutive steps. The same system illustrates processes influenced by protraction and fractionation of dose, synergists, comutagens/cocarcinogens, heritable factors, etc. The response at a given dose is expected to depend on the product of consecutive transfer functions. An application of general rules of chemical kinetics shows that when a chemical is introduced at a sufficiently low level, all processes affecting the transfers and therefore the transfer functions themselves become first-order, provided the induction status of enzymes and the cell-division rate remain constant. Under the same conditions, dose-response relationships are expected to be linear, i.e. without "safe" thresholds. However, present knowledge of the kinetics of repair at low levels of DNA damage and of the kinetics of induction of repair functions is not enough complete to be decisive. These considerations and the fact that observed dose-response data in some cases indicate the existence of thresholds but in others appear able to reject the threshold hypothesis lead to the conclusion that, generally, dose-response curves are most probably linear down to dose zero. However, certain mutagens/carcinogens give rise to lesions repaired so effectively that quasi-thresholds appear in certain subpopulations or organs.

A genetic study was undertaken in Drosophila with N-alpha-acetoxymethyl-N-methylnitrosamine, a precursor of the alpha-hydroxymethyl derivative of N,N-dimethylnitrosamine, to assess the role of alpha-carbon oxidation in toxicological activation. Genetic activity was measured for the whole testicular tissue with respect to the general response of the X chromosome (recessive lethals and visibles), as well as certain specific genic sites, including representatives of the RNA genes. The biological activity of the acetoxy compound proved to be considerably higher than that of the parent amine with respect to both cytotoxicity and mutagenicity. At low and equitoxic molarities of the 2 agents (0.1 to 1.0 and 1.0 to 10.0 mM, respectively), dose-dependence for all the investigated genetic functions followed identical patterns, which were best described by quadratic dose curves. However, the regression coefficients for the acetoxy derivative were at least 1 order of magnitude higher than the corresponding values for the amine, indicating a consistent level of mutagenic activation as a result of the alpha-acetoxymethyl substitution. At mutagenically equivalent doses, the 2 compounds gave statistically comparable frequencies of mosaicism among corresponding mutational classes and equal ribosomal DNA selectivity indices, indicating identical molecular mechanisms of mutagenesis. The higher mutagenicity of N-alpha-acetoxymethyl-N-methylnitrosamine compared to N,N-dimethylnitrosamine was paralleled by its higher carcinogenicity which would suggest that the same effective metabolites might be involved in both processes.