Relationships between benzo(a)pyrene-DNA adduct levels and genotoxic effects in mammalian cells.

The effectiveness of benzo(a)pyrene [B(a)P]-DNA binding as an internal dosimeter was evaluated. Data were obtained from concurrent studies, measuring B(a)P induced genotoxic effects and DNA adducts in several short-term bioassay systems: cytotoxicity, gene mutation, and sister chromatid exchange in Chinese hamster V79 cells; cytotoxicity, gene mutation, and chromosome aberrations in mouse lymphoma L5178Y TK+/-; cytotoxicity and enhanced virus transformation in Syrian hamster embryo cells; and cytotoxicity and morphological transformation in C3H10T1/2CL8 mouse embryo fibroblasts. Both total B(a)P-DNA binding and specific B(a)P-DNA adducts were measured. N2-(10 beta-[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl)deoxyguanosine [BPDE I-dGuo] was one of the major adducts identified in all bioassay systems. DNA binding and genotoxic responses varied significantly between bioassays. Each genetic end point was induced with a differing efficiency on a per adduct basis. However, the relationships between frequency of genetic effect or morphological transformation and B(a)P-DNA binding or BPDE I-dGuo were linear within a given assay. In order to compare biological end points of diverse frequencies in diverse biological systems, a doubling adduct level, expressed as the number of BPDE I-dGuo adducts per unit of DNA required to double the induced frequency of biological response, was applied to the data.

Genotoxicity studies of benz[l]aceanthrylene.

The genotoxicity of the cyclopenta-fused polycyclic aromatic hydrocarbon, benz[l]aceanthrylene (B[l]A), was evaluated in vitro using the L5178Y/TK+/- mouse lymphoma assay and in vivo using the mouse peripheral blood lymphocyte (PBL) culture system. The mutagenicity and sister chromatid exchange (SCE) inducing potential of B[l]A was then compared to that of benzo[a]pyrene (B[a]P). B[l]A appeared to be slightly less mutagenic than B[a]P at the TK locus, and each compound produced both small and large colony mutants indicating that they are clastogenic as well as mutagenic. Gross chromosome aberration analysis of treated L5178Y/TK+/- mouse lymphoma cells confirmed the clastogenicity of B[l]A in vitro. In the mouse PBL system, after administration by gavage, B[l]A was more cytotoxic and produced a sharper elevation in SCE frequency than B[a]P.

Genotoxicity of acrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate, and ethyl methacrylate in L5178Y mouse lymphoma cells.

A series of monomeric acrylate/methacrylate esters (methyl acrylate, ethyl acrylate, methyl methacrylate, and ethyl methacrylate) as well as acrylic acid were examined for genotoxic activity in L5178Y mouse lymphoma cells without exogenous activation. All five compounds induced concentration-dependent increases in mutant frequency. Small-colony, trifluorothymidine-resistant mutants were primarily induced, which suggests that these compounds may act via a clastogenic mechanism. This prediction was confirmed by the finding that all five compounds produced gross chromosome aberrations in mouse lymphoma cells. The two acrylates were much more potent in their response than acrylic acid. Methyl acrylate (22 micrograms/ml, survival = 18%) induced 385 mutants/10(6) survivors (total mutant frequency less the spontaneous mutant frequency) and 45 chromosome aberrations/100 cells analyzed (total aberrations less the spontaneous background). Ethyl acrylate (37.5 micrograms/ml, survival = 15%) induced 683 mutants/10(6) survivors and 48 aberrations/50 cells analyzed. Acrylic acid (500 micrograms/ml, survival = 22%) induced 245 mutants/10(6) survivors and 37 aberrations/100 cells analyzed. The two methacrylates required higher concentrations to induce a positive response. Methyl methacrylate (2,799 micrograms/ml, survival = 11%) induced 230 mutants/10(6) survivors and 29 aberrations/200 cells analyzed. Ethyl methacrylate was extremely difficult to test because of a plateau in the dose response, over which the toxicity fluctuated from 2% to 37% survival. Positive responses (twice the spontaneous background) were only obtained at toxicity levels with less than approximately 20% survival. A concentration of 1,626 micrograms/ml (survival = 16%) induced 83 mutants/10(6) survivors and 11 aberrations/200 cells analyzed. The evidence suggests that the genotoxicity of these compounds is most likely due to a clastogenic mechanism.

Cytogenetic effects of phosphine inhalation by rodents. I: Acute 6-hour exposure of mice.

Phosphine (PH3) is a highly toxic grain fumigant that can be produced from the reaction of metal phosphides with water. To determine the in vivo cytogenetic effects of inhalation of PH3, male CD-1 mice were exposed to either 0, 5, 10, or 15 ppm target concentrations of PH3 for 6 hr. Twenty hours after the termination of exposure, the spleens of the mice were removed, macerated, and the splenocytes cultured for analyses of sister chromatid exchanges, chromosome aberrations, and micronuclei in cytochalasin B-induced binucleated cells. In addition, bone marrow smears were made for the analysis of micronuclei in polychromatic erythrocytes. No increase in any of the cytogenetic endpoints was found at any of the concentrations examined. The only statistically significant response was a concentration-related slowing of the cell cycle in the splenocytes.

Interspecies cytogenetic comparisons: studies with X-radiation and bleomycin sulfate.

A series of in vitro experiments were conducted to determine if there are innate differences in the sensitivity of peripheral blood lymphocytes (PBLs) from different mammalian species to clastogens. Mouse, rat, and human whole blood samples were exposed to either 0, 0.38, 0.75, 1.5, or 3.0 Gy x-radiation or 0, 5, 10, 20, 40, or 80 micrograms/ml bleomycin for 4 hr. Bromodeoxyuridine-containing cultures were initiated and the PBLs stimulated to divide with phytohemagglutinin. All cultures were harvested following a 3-hr colcemid treatment. Slides were made and differentially stained, and first-division metaphases were scored for chromosome aberrations. In the x-radiation studies human PBLs were significantly more sensitive than mouse PBLs which were in turn more sensitive than rat PBLs as measured by either the total percent aberrant cells or the number of dicentrics. Data from all three species could be fitted to a linear-quadratic model. Results with bleomycin suggest that the mouse and human PBLs are equally sensitive to the clastogenic effects of bleomycin. Both appeared to be more sensitive than the rat PBLs, but the variation between experiments was such that the results among species were not significantly different. These results indicate that there may be inherent differences in sensitivity among PBLs of mammalian species; however, more studies are needed to determine if the differences presented here hold for other agents.

Cytogenetic effects of butadiene metabolites in rat and mouse splenocytes following in vitro exposures.

As a first step in investigating the genotoxic effects of the principal metabolites of 1,3-butadiene (BD) in both rats and mice, splenocytes (which have little mixed function oxidase activity) from each specimen were exposed to a series of concentrations of either 3,4-epoxy-1-butene (EB) (20 to 931 microM) or 1,2:3,4-diepoxybutane (DEB) (2.5 to 160 microM) for 1 h. The splenocytes were then washed, cultured, and stimulated to divide with concanavalin A, and metaphases were analyzed for the induction of sister chromatid exchanges (SCEs) and chromosome aberrations (CAs). In addition, cells from some experiments were taken after exposure but before culture, and subjected to the single cell gel (SCG) assay to measure DNA damage in the form of DNA strand breakage and/or alkaline-labile sites. Initial studies indicate that EB does not induce cytogenetic damage in either rat or mouse G0 splenocytes. However, DEB was an extremely potent SCE- and CA-inducer in both species with no species differences apparent. Neither DEB nor EB produced any statistically significant DNA-damaging effects as measured by the SCG assay.

Mutagenicity and clastogenicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in L5178Y/TK+/(-)-3.7.2C mouse lymphoma cells.

3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) was tested without exogenous activation in L5178Y/TK+/(-)-3.7.2C mouse lymphoma cells for mutation at the thymidine kinase locus and for clastogenicity. At a concentration of 0.75 micrograms/ml, the induced mutant frequency was 1027 per 10(6) survivors (survival = 11%). A concentration-related increase of large and small colony mutants was observed, but the majority of the MX induced mutants formed small colonies, consistent with the positive clastogenic response that was observed. MX primarily induced chromatid breaks and rearrangements (30 chromatid and 4 chromosome aberrations per 100 cells) at the 0.75 microgram/ml dose. These studies indicate that MX induces a broad spectrum of genetic damage.

Cell cycle specificity of cytogenetic damage induced by 3,4-epoxy-1- butene.

3,4-epoxy-1-butene (EB), a primary metabolite of butadiene, is a direct-acting "S-dependent" genotoxicant that can induce sister chromatid exchanges (SCEs) and chromosome aberrations (CAs) in cycling cells in vitro. However, EB is almost inactive when splenic or peripheral blood lymphocytes are exposed at the G(0) stage of the cell cycle. To investigate whether repair of DNA lesions is responsible for the lack of cytogenetic responses seen after G(0) treatments, we used cytosine arabinoside (ara-C) to inhibit DNA polymerization during DNA repair. If enough repairable lesions are present, double-strand breaks should accumulate and form chromosome-type ("S-independent") deletions and exchanges. This is exactly what occurred. EB induced chromosome deletions and dicentrics at the first division following treatment, when the EB exposure was followed by ara-C. Without ara-C treatment, there was no induction of CAs. These experiments indicate that the relatively low levels of damage induced by EB in G(0) lymphocytes are removed by DNA repair prior to DNA synthesis and thus, before the production of SCEs or chromatid-type aberrations.

Relative genotoxic potency of arsenic and its methylated metabolites.

Arsenic is one of the few identified human carcinogens that has yet to be shown to cause cancer in rodents when the standard bioassay protocols are used. The reasons for this apparent interspecies difference are unclear but may be related to differences between humans and rodents in their detoxification capabilities. Detoxification of arsenic may occur through a methylation pathway. If, in fact, methylation does detoxify arsenic, one would predict that the methylated arsenicals might be less genotoxic than the inorganic arsenicals. To evaluate the hypothesis that the inorganic arsenicals are more mutagenic than the organic arsenicals, we tested sodium arsenite, sodium arsenate, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) for their relative mutagenic and clastogenic potentials. We used the L5178Y/TK+/- mouse lymphoma assay which allows the detection of chemicals inducing a broad spectrum of different types of genetic damage. Sodium arsenite and sodium arsenate were active at concentrations of 1-2 micrograms/ml and 10-14 micrograms/ml, respectively. MMA was active between 2500-5000 micrograms/ml; while DMA required almost 10000 micrograms/ml to induce a genotoxic response. The organic arsenicals are thus much less potent as mutagenic agents than the inorganic arsenicals. All four of these arsenicals appear to act by mechanisms that cause chromosomal mutations.

Micronucleus, chromosome aberration, and small-colony TK mutant analysis to quantitate chromosomal damage in L5178Y mouse lymphoma cells.

In testing the hypothesis that the small-colony thymidine kinase-deficient mutants of L5178Y/TK+/- -3.7.2C mouse lymphoma cells represent an estimate of the clastogenicity of test chemicals, we have been performing gross aberration analysis. The present study was initiated to determine if the cytokinesis block method of micronucleus analysis could be performed in mouse lymphoma cells and to compare 3 different endpoints of clastogenicity: the number of metaphases with aberrations, number of binucleates with micronuclei, and small-colony TK mutant frequency. In this study, 12 compounds having varying clastogenic potencies were evaluated. As would be expected, the 3 endpoints vary in the relative magnitude of the quantitated response. This difference likely results from the types of clastogenic damage detected by each endpoint. Of the 3 endpoints tested, only the small-colony TK mutant frequency measures events compatible with long-term cell survival.

Mutagenicity of three disinfection by-products: di- and trichloroacetic acid and chloral hydrate in L5178Y/TK +/- (-)3.7.2C mouse lymphoma cells.

The disinfection of water, required to make it safe for human consumption, leads to the presence of halogenated organic compounds. Three of these carcinogenic 'disinfection by-products', dichloroacetic acid (DCA), trichloroacetic acid (TCA) and chloral hydrate (CH) have been widely evaluated for their potential toxicity. The mechanism(s) by which they exert their activity and the steps in the etiology of the cancers that they induce are important pieces of information that are required to develop valid biologically-based quantitative models for risk assessment. Determining whether these chemicals induce tumors by genotoxic or nongenotoxic mechanisms (or a combination of both) is key to this evaluation. We evaluated these three chemicals for their potential to induce micronuclei and aberrations as well as mutations in L5178Y/TK +/- (-)3.7.2C mouse lymphoma cells. TCA was mutagenic (only with S9 activation) and is one of the least potent mutagens that we have evaluated. Likewise, CH was a very weak mutagen. DCA was weakly mutagenic, with a potency (no. of induced mutants/microgram of chemical) similar to (but less than) ethylmethanesulfonate (EMS), a classic mutagen. When our information is combined with that from other studies, it seems reasonable to postulate that mutational events are involved in the etiology of the observed mouse liver tumors induced by DCA at drinking water doses of 0.5 to 3.5 g/l, and perhaps chloral hydrate at a drinking water dose of 1 g/l. The weight-of-evidence for TCA suggest that it is less likely to be a mutagenic carcinogen. However, given the fact that DCA is a weak mutagen in the present and all of the published studies, it seems unlikely that it would be mutagenic (or possibly carcinogenic) at the levels seen in finished drinking water.

Cytogenetic studies of three triazine herbicides. II. In vivo micronucleus studies in mouse bone marrow.

Atrazine, simazine, and cyanazine are widely used preemergence and postemergence triazine herbicides that have made their way into the potable water supply of many agricultural communities. Although there are several contradictory genotoxicity studies in the literature, our previous in vitro studies with human lymphocytes showed that atrazine, simazine, and cyanazine did not induce sister chromatid exchanges (SCEs) or chromosome aberrations (CAs) up to the limits of solubility in aqueous medium using 0.5% dimethyl sulfoxide. To expand upon these results and to ensure that our in vitro findings could be replicated in an in vivo system, mice were treated with each triazine by two intraperitoneal injections, 24h apart. The animals were sacrificed and the bone marrow removed for micronucleus (MN) analysis, 24h after the last injection. Two to four independent trials were performed for MN analysis in polychromatic erythrocytes, and in some trials the spleen was removed, cultured, and analyzed for SCEs and CAs. None of the triazines investigated induced MN in the bone marrow, even at doses that caused significant bone marrow suppression and/or death. These results indicate that atrazine, simazine, and cyanazine are not genotoxic as measured by the bone marrow MN assay in mice following high dose exposures.

Genotoxicity of 2-amino-6-N-hydroxyadenine (AHA) to mouse lymphoma and CHO cells.

2-Amino-6-N-hydroxyadenine (AHA) treated L5178Y/TK (+/-)-3.7.2C mouse lymphoma cells were evaluated for mutations at the tk, hgprt, and Na+/K+ ATPase loci, as well as for gross chromosome aberrations and induction of micronuclei. In addition, AHA was evaluated for its ability to induce HGPRT mutants in CHO cells. AHA was found to induce mutations at all evaluated loci and in both cell types. The TK mutants were primarily large colonies although a few small colonies were also induced, particularly at the higher concentrations. Preliminary cytogenetic analysis of AHA-treated mouse lymphoma cells indicated that some gross aberrations but not micronuclei were induced. The 20 small-colony TK mutants evaluated by banded karyotype indicate that only a small fraction (2 of 20) showed chromosome 11 abnormalities. From these studies, it appears that AHA may be one of a very few chemicals that is capable of inducing multi-locus point mutations, with only slight clastogenic activity. Particularly at the higher concentrations, some of the mutants may contain multi-locus point mutations that result in slow growth.

Cytogenetic effects in mice of divinylbenzene-55 inhalation.

Male B6C3F1 mice (8 weeks of age) were exposed by inhalation to divinylbenzene-55 (DVB-55), at target concentrations of 0, 25, 50 and 75 ppm for 6 h per day for 3 days. Following exposure the animals were killed blood smears were prepared for micronucleus (MN) analysis, and the spleens were removed and cultured for sister chromatid exchange (SCE) and chromosome aberration (CA) analyses. DVB-55 induced a dose dependent increase in SCE with the two highest doses reaching statistical significance. Similarly, there was a statistically significant although less pronounced increase in the frequency of CAs in splenocytes and MN in polychromatic erythrocytes. There was no indication of toxicity as measured by cell cycle kinetics in the splenocytes or the percentage of polychromatic erythrocytes in the peripheral blood smears. Thus, DVB-55 appears to be a weak genotoxicant in vivo.

Mutagenicity of actinomycin D in mammalian cells due to clastogenic effects.

Actinomycin D was clastogenic and mutagenic in L5178Y/TK +/- -3.7.2C mouse lymphoma cells. The majority of the mutants were small colonies, indicating that actinomycin D acts primarily by a clastogenic mechanism.

Mutagenicity and clastogenicity of teniposide (VM-26) in L5178Y/TK +/- -3.7.2C mouse lymphoma cells.

The antitumor drug teniposide (VM-26) is a potent inducer of DNA breaks (Long et al., Cancer Res., (1985) 45, 3106), but it is only weakly mutagenic at the hprt locus in CHO cells (Singh and Gupta, Cancer Res., (1983) 43, 577). In the present study, the mutagenic and clastogenic activities of teniposide were evaluated in L5178Y/TK +/- -3.7.2C mouse lymphoma cells. Although teniposide is a weak mutagen at the hprt locus, it is a potent mutagen at the tk locus, with as little as 0.5 ng/ml producing 220 TK mutants/10(6) survivors at 96% survival (background = 100/10(6) survivors). This same dose of teniposide induced 38 aberrations per 100 metaphases (background = 7/100 cells). At 7 ng/ml, teniposide induced approximately 2700 TK mutants/10(6) survivors at approximately 10% survival. At the highest dose sampled for aberration analysis (5 ng/ml), teniposide induced 44 aberrations/100 cells. Most of the aberrations were chromosomal rather than chromatid events. As expected for a compound acting primarily by a clastogenic mechanism, most of the TK mutants were small colonies. Thus, teniposide is a potent clastogen, and it is a potent mutagen at the tk locus but not at the hprt locus. These results support the hypothesis that the location of the target gene affects the ability of the assay to detect both intragenic events and events causing functional multilocus effects. Thus, a heterozygous locus (like tk) but not a functionally hemizygous locus (like hprt) may permit the detection of mutagens that act primarily by a clastogenic mechanism. Because teniposide induces topoisomerase II-associated DNA breaks, and because there is evidence that teniposide may not interact directly with DNA, we discuss the possibility that the potent clastogenic/mutagenic activity of teniposide may be mediated by topoisomerase II.

Mutagenicity and clastogenicity of adriamycin in L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells.

Adriamycin was found to be both mutagenic and clastogenic to L5178Y/TK(+/-)-3.7.2C mouse lymphoma cells. A dose of only 5 ng/ml (survival = 62% or 67%) gave an induced TK mutant frequency of 307 or 296 per 10(6) survivors in two separate experiments. This dose was also clastogenic, inducing 20 chromosome aberrations/100 cells analyzed. The majority of the mutants were small-colony mutants, indicating that adriamycin likely acts primarily by a clastogenic mechanism.

Cytogenetic studies of three triazine herbicides. I. In vitro studies.

Atrazine, simazine, and cyanazine are widely used pre-emergence and post-emergence triazine herbicides that have made their way into the potable water supply of many agricultural communities. Because of this and the prevalence of contradictory cytogenetic studies in the literature on atrazine, simazine, and cyanazine, a series of in vitro experiments was performed to investigate the ability of these three triazines to induce sister chromatid exchanges (SCEs) and chromosome aberrations (CAs) in human lymphocyte cultures. Our results showed that all three triazines failed to produce any significant increases in SCEs or CAs up to the limits of solubility [using 0.5% dimethyl sulfoxide (DMSO)]. Our results are discussed in light of contradictory results in the literature.

Mutagenicity and clastogenicity of proflavin in L5178Y/TK +/- -3.7.2.C cells.

We evaluated the ability of proflavin to induce specific-locus mutations at the heterozygous thymidine kinase (tk) locus of L5178Y/TK +/- -3.7.2C mouse lymphoma cells, which appears to permit the recovery of mutants due to single-gene and chromosomal mutations. Proflavin was highly mutagenic at the tk locus, producing 724-965 TK mutants/10(6) survivors (background = 56-85/10(6); survival = 29-32%). Most of the mutants were small colonies, which suggested that proflavin may induce chromosomal mutations. The potent clastogenicity of proflavin was confirmed by cytogenetic analysis for chromosomal aberrations. At the highest dose analyzed (1.5 micrograms/ml), proflavin produced 82 aberrations/100 metaphaes (background = 2/100). The large-colony TK mutant frequency produced by proflavin (48-109/10(6) survivors; background = 23/10(6); survival = 57-61%) was similar to published HPRT mutant frequencies produces by proflavin in L5178Y and CHO cells (50-100/10(6) survivors; background = 2-50/10(6); survival = 50-62%). These results lead to the conclusion that proflavin is a potent clastogen and induces a high frequency of small-colony TK mutants; however, it induces a low frequency of HPRT mutants and a low frequency of large-colony TK mutants.

Genotoxicity of three pyridine compounds to L5178Y mouse lymphoma cells.

The L5178Y mouse lymphoma assay was used to examine the potential mutagenicity of three halogenated pyridine compounds. Position effects of the halogen moiety and the role of metabolic activation were analyzed based on induced mutant frequency, gross chromosome aberrations, and micronuclei. Without activation, 2-chloropyridine, 3-chloropyridine, and 2-chloro-5-trifluoromethylpyridine produced a small increase in mutant frequency; only the 2-chloropyridine activity was significantly increased with activation. All three compounds were also clastogenic as demonstrated by increases in chromosome aberrations and micronuclei (except for 2-chloro-5-trifluoromethylpyridine which did not induce micronuclei either with or without activation).