Carolyn L Doerr

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.

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.

Oxidation and methylation status determine the effects of arsenic on the mitotic apparatus

We investigated the spindle inhibitory properties of six arsenicals differing in their methylation or oxidation state. Human lymphoblasts were exposed for 6 h to either sodium arsenate (NaAsV), sodium arsenite (NaAsIII), monomethylarsonic acid (MMAV), monomethylarsonous acid (MMAIII), dimethylarsinic acid (DMAV), or dimethylarsinous acid (DMAIII). After exposure slides were prepared, and the mitotic indices (MI) were assessed. We also exposed tubulin directly to each arsenical and spectrophotometrically measured its effect on polymerization. NaAsV caused a small but significant increase in MI. MMAV also caused only a slight increase in MI that just reached statistical significance. In contrast, DMAV caused a significant increase in MI, producing ∼75% the MI of demecolcine and ∼4 times the MI of the control. NaAsIII had no significant effect on MI and was quite toxic. MMAIII induced more than a twofold increase in MI compared to the control, which was about 40% that caused by demecolcine. On a micromolar basis, MMAIII was the most potent of the arsenicals tested. DMAIII gave inconsistent results. None of the pentavalent arsenicals had a substantial effect (either inhibition or enhancement) on GTP-induced polymerization of tubulin. In contrast, NaAsIII inhibited polymerization at concentrations of 1 mM and above and MMAIII and DMAIII at 10 μM and above. Taken together, these results present a complex picture of how arsenicals may affect cells. These studies demonstrate that the metabolites of arsenic are active not only as chromosome breaking and DNA damaging agents but can also interfere with cell division via tubulin disruption.

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.

Comparison of cytogenetic effects of 3,4-epoxy-1-butene and 1,2:3, 4-diepoxybutane in mouse, rat and human lymphocytes following in vitro G0 exposures.

To understand better the species differences in carcinogenicity caused by 1,3-butadiene (BD), we exposed G0 lymphocytes (either splenic or peripheral blood) from rats, mice and humans to 3, 4-epoxy-1-butene (EB) (20 to 931 microM) or 1,2:3,4-diepoxybutane (DEB) (2.5 to 320 uM), two of the suspected active metabolites of BD. Short EB exposures induced little measurable cytogenetic damage in either rat, mouse, or human G0 lymphocytes as measured by either sister chromatid exchange (SCE) or chromosome aberration (CA) analyses. However, DEB was a potent inducer of both SCEs and CAs in G0 splenic and peripheral blood lymphocytes. A comparison of the responses among species showed that the rat and mouse were approximately equisensitive to the cytogenetic damaging effects of DEB, but the situation for the human subjects was more complex. The presence of the GSTT1-1 gene (expressed in the erythrocytes) reduced the relative sensitivity of the lymphocytes to the SCE-inducing effects of DEB. However, additional factors also appear to influence the genotoxic response of humans to DEB. This study is the first direct comparison of the genotoxicity of EB and DEB in the cells from all three species.

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.

Effects of arsenic exposure on the frequency of HPRT-mutant lymphocytes in a population of copper roasters in Antofagasta, Chile: a pilot study.

A pilot biomarker study was conducted to investigate the feasibility of using the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene in peripheral blood lymphocytes as a biomarker for detecting genetic effects of arsenic exposure. Blood and urine samples were obtained from workers highly exposed to arsenic in a copper roasting plant in Antofagasta, Chile. Individuals were classified according to their job titles into three potential exposure groups: high, medium, and low. To confirm exposure, arsenic concentration was determined in urine samples. The HPRT mutant frequencies were measured in lymphocytes from 15 individuals ranging in age from 24 to 66 years. The mean mutant frequencies for the three exposure groups were: low (9 x 10(-6)), medium (11 x 10(-6)), and high (24 x 10(-6)). An increased mutant frequency was observed in the highly exposed group, but the response was so slight that it is not likely that this assay will be capable of providing dose-response information across a range of lower, more typical environmental arsenic levels.

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.

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.