Some insights into the mode of action of butadiene by examining the genotoxicity of its metabolites.

1,3-Butadiene (BTD) is an important commodity chemical and air pollutant that has been shown to be a potent carcinogen in mice, and to a lesser extent, a carcinogen in rats. To better assess butadiene's carcinogenic risk to humans, it is important to understand its mode of action and how this relates to differences in responses among species. In a series of in vitro experiments, lymphocytes from rats, mice, and humans were exposed to 3,4-epoxy-1-butene (EB) or 1,2:3,4-diepoxybutane (DEB) for 1h at the G(0) stage of the cell cycle, stimulated to divide, and cultured to assess the ability of these metabolites to induce sister chromatid exchange (SCE) and chromosome aberrations (CAs). EB induced no increases in SCEs or CAs in the cells from the three species. DEB was a potent SCE- and CA-inducer, with the results being similar in each rodent species. The response for SCEs seen in the human cells was more complex, with genetic polymorphism for glutathione-S-transferases (GST) possibly modulating the response. The single cell gel electrophoresis assay was used on genetically engineered V79 cell lines to investigate a possible influence of GST status. Experiments were also conducted to investigate the reason for EB's failure to induce SCEs or CAs in G(0) cells. The results indicate that EB-induced DNA damage was repaired before DNA synthesis in unstimulated lymphocytes, but EB caused a large increase in SCEs if actively cycling cells were treated. Thus, the results indicate that DEB damage is persistent in G(0) cells, and DEB is a much more potent genotoxicant than EB. The carcinogenic effect of butadiene will most likely depend on the degree to which DEB is produced and reaches target tissues, and to a lesser extent on the ability of EB to reach actively dividing or repair deficient cells.

Attenuation of cytogenetic damage by 2-mercaptoethanesulfonate in cultured human lymphocytes exposed to cyclophosphamide and its reactive metabolites.

Cyclophosphamide (CP) is metabolized to the reactive intermediates, phosphoramide mustard (PAM) and acrolein (AC), which have generally different molecular binding targets. Sodium 2-mercaptoethanesulfonic acid (MESNA) has been used clinically to alleviate hemorrhagic cystitis caused by CP chemotherapy, has exhibited anticarcinogenic effects in rats exposed to CP during a long-term bioassay, and acts in the urogenital tract by reacting with 4'-OH-CP and AC. The purpose of this study was to: (a) compare the relative abilities of PAM and AC to induce cytogenetic damage and cytotoxicity in cultured human lymphocytes; (b) assess the efficacy of MESNA to attenuate the cytogenetic damage and cytotoxicity induced by CP, AC, PAM, and diethyl-4'-hydroperoxycyclophosphamide (DEHP-CP), an activated AC-generating compound; and (c) determine if concanavalin A-stimulated T-lymphocytes, which differentiate into suppressor cells upon lectin activation, exhibit any heightened cytogenetic sensitivity compared to a variety of cultured mammalian cells during exposure to PAM or AC as reported by other investigators. Purified mononuclear leukocytes were stimulated with concanavalin A and exposed to CP (0.5-2.0 mM) without an exogenous activation system, AC (0.001-40.0 microM), PAM (0.0014-27.1 microM), or DEHP-CP (0.1-100.0 microM) in the presence or absence of MESNA (1, 5, or 10 mM). All four compounds induced significant concentration-related increases in the SCE frequency, but only PAM was clastogenic. On an induced SCE/microM basis, PAM was about 130 and 193 times more potent than were DEHP-CP and AC, respectively. MESNA protected against the cytogenetic damage and cytotoxicity induced by the four compounds, but it was particularly effective against AC and DEHP-CP by abolishing SCE induction completely. SCEs and chromosome aberrations differed considerably in their induction kinetics in lymphocytes exposed to PAM, and these disparities suggested an uncoupling of the two phenomena. Although SCE induction was not consistently associated with cytotoxicity with the four agents, chromosome aberration induction coincided with an inhibition of cell cycle kinetics in PAM-treated cells. The exceptionally high SCE frequency of up to 21 times baseline in cells exposed to PAM indicates that T-suppressor lymphocytes stimulated with concanavalin A may be particularly sensitive to the DNA-damaging effects of PAM. Finally, these data suggest that the anticarcinogenicity of MESNA correlates with its ability to attenuate cytogenetic damage and cytotoxicity induced by reactive CP metabolites.

Effect of acrolein on phosphoramide mustard-induced sister chromatid exchanges in cultured human lymphocytes.

Although phosphoramide mustard (PM) is generally recognized as being the most genotoxic metabolite of cyclophosphamide (CP), the contribution of acrolein to the cytogenetic toxicity of CP is unclear. Besides covalently binding to DNA, acrolein can inactivate critical proteins necessary for replicative DNA synthesis, RNA transcription, cell membrane integrity, and metabolism of xenobiotic and endogenous substrates. Because enzymatic processes are involved in sister chromatid exchange (SCE) formation and DNA excision repair, we hypothesized that acrolein might modulate SCE induction by PM due to acrolein's high binding affinity for proteins and low molecular weight sulfhydryl compounds. Human mononuclear leukocytes were isolated on a Ficoll-Hypaque density gradient, and 10(6) cells were inoculated into 1.9 ml of complete medium. T-cells were stimulated to grow with 4 micrograms concanavalin A/ml, and 5-bromo-2'-deoxyuridine (5 microM) was added 24 h later. The cultures were then treated with PM (0.069 microM) in the absence or presence of diethyl-4'-hydroperoxy-CP (DEHP-CP), an activated acrolein-generating compound, at concentrations of 0.1, 1, or 10 microM for 48 h. Demecolcine (1.35 microM) was added for the final 4 h of culture. PM alone induced about a 2-fold increase in the SCE frequency (PM, 14.1 +/- 0.5 (SD) versus control, 7.7 +/- 0.4) without cell cycle inhibition or reduced mitotic activity. DEHP-CP induced a concentration-related increase in the SCE frequency of up to 1.6-fold without any significant cell cycle inhibition or lowered mitotic activity. When PM and DEHP-CP were combined, SCE induction was additive for all three DEHP-CP concentrations. Except at the highest molar ratio of DEHP-CP:PM (145:1), there was no evidence of cytotoxicity in the other treatment groups. These results suggest that acrolein has a diminished role in mediating the cytogenetic and cytotoxic effects of CP. In addition, enzymes associated with SCE formation and, by inference, DNA excision repair may not be particularly susceptible to acrolein-induced inactivation.

Sister chromatid exchange induction in human lymphocytes exposed to benzene and its metabolites in vitro.

Previous in vivo studies have shown that low-dose benzene exposure (10 to 28 ppm for 4 to 6 h) in mice can induce sister chromatid exchange (SCE) in peripheral blood B-lymphocytes and bone marrow as well as micronuclei in bone marrow polychromatic erythrocytes. Because benzene is metabolized to a variety of intermediate compounds and two of these, catechol and hydroquinone, have been reported to be potent SCE-inducers, it is possible that other known and proposed metabolites could have chromosome-damaging effects in lymphocytes. Induced SCE frequencies, mitotic indices, and cell cycle kinetics were quantitated in human peripheral blood T-lymphocytes exposed to benzene, phenol, catechol, 1,2,4-benzenetriol, hydroquinone, 1,4-benzoquinone, or trans,trans-muconic acid. Three proposed metabolites of phenol, 4,4'-biphenol, 4,4'-diphenoquinone, and 2,2'-biphenol, which can be generated by a phenol-horseradish peroxidase-hydrogen peroxide system were also examined. Benzene, phenol, catechol, 1,2,4-benzenetriol, hydroquinone, and 1,4-benzoquinone induced significant concentration-related increases in the SCE frequency, decreases in mitotic indices, and inhibition of cell cycle kinetics. Based on the slope of the linear regression curves for SCE induction, the relative potencies were as follows: catechol greater than 1,4-benzoquinone greater than hydroquinone greater than 1,2,4-benzenetriol greater than phenol greater than benzene. On an induced SCE per microM basis, catechol was approximately 221 times more active than benzene at the highest concentrations studied. trans,trans-Muconic acid had no significant effect on the cytogenetic parameters analyzed. 2,2'-Biphenol induced a significant increase in SCE only at the highest concentration analyzed, and 4,4'-biphenol caused a significant increase in SCE frequency that was not clearly concentration related. However, both 2,2'- and 4,4'-biphenol caused significant cell cycle delay and mitotic inhibition. 4,4'-Diphenoquinone caused only a significant decrease in mitotic activity. These data indicate that in addition to phenol, di- and trihydroxybenzene metabolites play important roles in SCE induction. Furthermore, the results suggest either that benzene alone can induce SCE or, a more likely possibility, that mononuclear leucocytes have a limited capability to activate benzene.

Sister chromatid exchange induction by diaziquone in human and mouse lymphocytes following both in vivo and in vitro exposures.

Diaziquone (AZQ) (NSC 182986), a lipid-soluble benzoquinone derivative currently being tested as an experimental chemotherapeutic agent, was used to treat mouse and human peripheral blood lymphocytes (PBLs) to determine its genotoxic potential by examination of sister chromatid exchange (SCE) induction. In vitro exposure to AZQ caused a linear increase in SCE in both mouse and human PBLs, with mouse PBLs being about twice as sensitive as the human cells. The lowest in vitro concentration found to induce a significant effect on SCE frequency was 0.3 micrograms/ml in mice and 1.0 micrograms/ml in human PBLs. Mice exposed by either i.p. or i.v. injection showed similar dose-related linear increases in SCE frequencies in their PBLs. After i.v. administration of AZQ, splenocytes from treated mice showed approximately the same SCE frequency as found in the PBLs. In general, AZQ caused a slowing of cell cycling in vivo while giving inconsistent responses in vitro. AZQ did cause a dose-related decrease in the number of recoverable mononuclear lymphocytes in mice treated in vivo. Contrary to the in vitro studies, comparison of SCE responses in mice with those previously observed in brain tumor patients undergoing chemotherapy with AZQ (Kligerman et al., Cancer Res., 47: 631-635, 1987) revealed AZQ was a much more potent SCE inducer in humans than in mice.

Sister chromatid exchange induction in mouse B- and T-lymphocytes exposed to cyclophosphamide in vitro and in vivo.

Cyclophosphamide (CPA) is known to exert greater toxic effects of B- than on T-lymphocytes in vivo. Both in vitro and in vivo CPA treatments were used to assess the possible cytogenetic basis for these observations. First, male C57BL/6 mouse lymphocytes were stimulated to divide in vitro with either phytohemagglutinin (T-cell mitogen) or lipopolysaccharide (B-cell mitogen), and were then treated with CPA (0.05 to 1.0 mM) and 5-bromo-2'-deoxyuridine (2 microM) at 24 hr. Cultures were harvested at 60 hr following a 4-hr treatment with demecolcine (1.35 microM). CPA caused concentration-related increases in sister chromatid exchange (SCE) up to 3 times control frequencies; the resulting SCE induction curves for B- and T-cells were sigmoidal and equivalent. Second, mice were given a single i.p. injection of CPA (0.5, 1.0, or 5.0 mg/kg). Blood was removed 24 hr later and cultured without additional CPA, as described above. Dose-related increases in SCE frequencies were seen for both T- and B-lymphocytes. CPA induced consistently 2.5 to 3.7 more SCEs in B-cells than in T-cells. Thus, B- and T-lymphocytes exhibited an equal sensitivity to CPA in vitro, but B-cells were more susceptible to the genotoxic effects in vivo.

Sister chromatid exchange induction in patients with anaplastic gliomas undergoing treatment with radiation plus diaziquone or 1,3-bis(2-chloroethyl)-1-nitrosourea.

Diaziquone (AZQ) (NSC 182986), a lipid-soluble benzoquinone derivative, is presently being tested in a Phase III clinical trial to determine its efficacy in patients with anaplastic gliomas compared to the more standard 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment following whole-brain irradiation. These patients on single-drug chemotherapy allowed us to evaluate the effects of each agent on sister chromatid exchange (SCE) induction in vivo. Eight weeks following the final radiation treatment, patients were randomly assigned to one of two groups: (a) 200 mg BCNU/m2, i.v., every 8 weeks; of (b) 15 mg AZQ/m2/day, i.v., for 3 consecutive days, every 4 weeks. Blood (5-10 ml) was drawn by venipuncture before treatment, within 10 h after treatment, and for two BCNU-treated patients at various other times. Peripheral blood lymphocytes were cultured by standard techniques for analysis of SCE. Eight weeks after irradiation but before chemotherapy, the mean SCE frequency in the patients' peripheral blood lymphocytes was 9.6 SCEs/metaphase. Following treatment with AZQ or BCNU, the baseline SCE frequency was increased more than 2-fold or 3-fold, respectively. Two months after BCNU treatment, there was less than a 25% reduction in SCE levels compared to samples taken and cultured within 10 h after treatment. These data show that lesions leading to SCE in human peripheral blood lymphocytes are relatively longlived, and that on a mg/m2 basis, AZQ is a more potent inducer of SCE in vivo than is BCNU.

Mutagenesis induced by procarcinogens at the hypoxanthine-guanine phosphoribosyl transferase locus of human fibroblasts cocultured with rat hepatocytes.

The addition of diethylnitrosamine or cyclophosphamide in cultures of hepatocytes overlaid on confluent diploid human fibroblasts resulted in a dose-dependent increase in the frequency of hypoxanthine-guanine phosphoribosyl transferase human fibroblast mutants with both chemicals. Different toxicity patterns for the two cell types were seen. Diethylnitrosamine was more toxic to the hepatocytes, whereas cyclophosphamide was more toxic to the fibroblasts. These data open the possibility of using strains of human fibroblasts as in vitro screens for mutagenicity of procarcinogens.

Liver regeneration studies with rat hepatocytes in primary culture.

Adult rat parenchymal hepatocytes in primary culture can be induced to enter into DNA synthesis and mitosis. The optimal conditions for hepatocyte replication are low plating density (less than 10,000 cells/sq cm) and 50% serum from two-thirds partially hepatectomized rats (48 hr after hepatectomy). Approximately 80% of the hepatocytes enter the cell cycle, and most of these cells go through mitosis. The replicating hepatocytes remain positive for glucose-6-phosphatase and negative for gamma-glutamyl transpeptidase, and they accumulate fat, in analogy to regenerating liver. Most of the replicating hepatocytes enter into multiple consecutive rounds of DNA synthesis. Dose-response studies between control animal serum and hepatocyte labeling index indicate that in unoperated animals the serum contains substances stimulatory as well as inhibitory for hepatic growth, with the inhibitory effect prevailing at high concentrations. After partial hepatectomy, the inhibitory activity disappears whereas the hepatopoietin activity reaches almost 90% of maximal biological effectiveness at 25% serum concentration. Addition of hormones to the system shows that the hepatopoietin activity is not identical to epidermal growth factor, platelet-derived growth factor, thyroxine, glucagon, or hydrocortisone. Norepinephrine abolishes the difference between control and hepatectomized serum but does not restore hepatopoietin activity when added to heat-inactivated serum. The results show that this system of replicating hepatocytes can be used to investigate the trophic factors that control growth of normal and neoplastic hepatocytes.

Formation and persistence of novel benzo(a)pyrene adducts in rat lung, liver, and peripheral blood lymphocyte DNA.

Male CD rats were injected with single i.p. doses of benzo(a)pyrene (B(a)P), and peripheral blood lymphocytes (PBLs), livers, and lungs were removed at various times after administration. DNA adducts were analyzed in each tissue by 32P postlabeling with nuclease P1 enhancement. Sister chromatid exchange frequencies were concomitantly measured in cultured whole blood. B(a)P-DNA adducts were observed in all three tissues from animals sacrificed between 1 and 56 days after injection. Maximal adduction levels occurred at about 4 days after administration, followed by a gradual loss of adducts over the period examined. The apparent half-lives of total DNA adducts were 15 days in liver, 17 days in PBLs, and 22 days in lung. Induced sister chromatid exchanges were linearly related to the amount of DNA adducts remaining in the PBLs at the time of harvest up to 56 days and were significantly elevated above concurrent controls up to 14 days. One of the major adducts found in each tissue was N2-(10 beta-[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a) pyrene]yl)deoxyguanosine. An additional novel major adduct was found in the liver DNA and is derived from the further metabolism of B(a)P-trans-7,8-dihydrodiol. A second major novel B(a)P adduct was found in the DNA of lung tissues and accounts for about 40% of the total adducts present. Experimental evidence suggests that this adduct is derived from a metabolic pathway that includes the formation of 9-hydroxy-B(a)P.

Cytogenetic studies in mice treated with the jet fuels, Jet-A and JP-8.

The genotoxic potential of the jet fuels, Jet-A and JP-8, were examined in mice treated on the skin with a single dose of 240 mg/mouse. Peripheral blood smears were prepared at the start of the experiment (t = 0), and at 24, 48 and 72 h following treatment with jet fuels. Femoral bone marrow smears were made when all animals were sacrificed at 72 h. In both tissues, the extent of genotoxicity was determined from the incidence of micronuclei (MN) in polychromatic erythrocytes. The frequency of MN in the peripheral blood of mice treated with Jet-A and JP-8 increased over time and reached statistical significance at 72 h, as compared with concurrent control animals. The incidence of MN was also higher in bone marrow cells of mice exposed to Jet-A and JP-8 as compared with controls. Thus, at the dose tested, a small but significant genotoxic effect of jet fuels was observed in the blood and bone marrow cells of mice treated on the skin.

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.

Quantitative and temporal relationships between DNA adduct formation in target and surrogate tissues: implications for biomonitoring.

DNA-carcinogen adducts offer a potential dosimeter for environmental genotoxicants reaching the exposed individual. Because the target tissues for many chemical carcinogens are not readily accessible for monitoring adducts in humans, peripheral blood lymphocytes (PBLs) have served as surrogate sources of exposed DNA. Both benzo[a]pyrene (BaP) and benzo[b]fluoranthene (BbF) are widely distributed in the environment as components of complex mixtures, such as automobile exhaust, cigarette smoke, foods, water, and urban air. Thus, human exposure to these chemicals is widespread, and they probably contribute to overall human lung cancer risk. The interpretation of the results of such studies would be enhanced by an understanding of the pharmacokinetics of specific DNA adduct formation and persistence in both target and surrogate tissues. Polycyclic aromatic hydrocarbons (PAHs) were administered to male Sprague-Dawley rats IP at 100 mg PAH/kg body weight. Lung, liver, and PBL tissues were harvested 1, 3, 7, 14, 28, and 56 days after treatment. DNA was extracted from each tissue and 32P-postlabeling analysis of DNA adducts with nuclease P1 enhancement was conducted. In all three tissues, BaP-DNA adducts exhibit a similar pattern, reaching a maximum at 3-4 days, followed by a decrease to 56 days. For BbF, the maximum DNA adduct levels in each tissue were between 5 and 14 days after injection. By 56 days after administration, the total adducts remaining in all tissues were measurable. Correlation analyses of the amount of DNA adducts in lung or liver compared to those found in the PBL of the same animals suggest a range of correlations (R2 = 0.67-0.83).(ABSTRACT TRUNCATED AT 250 WORDS)

Results of in vitro and in vivo genetic toxicity tests on methyl isocyanate.

Methyl isocyanate (MIC) was tested for genetic toxicity in a variety of in vitro and in vivo assays. Negative results were obtained in the Salmonella/mammalian microsome assay using five bacterial strains in a preincubation protocol. The Drosophila sex-linked recessive lethal test also gave negative results in studies that involved three routes of administration: inhalation, feeding, and injection. Positive results were obtained for three endpoints in cultured mammalian cells. Reproducible, dose-related increases in trifluorothymidine-resistant clones were induced in L5178Y mouse lymphoma cells, and the frequencies of both SCE and chromosomal aberrations increased in Chinese hamster ovary cells. These effects were independent of exogenous metabolism. In mice exposed to methyl isocyanate by inhalation, cytogenetic analyses were carried out on bone marrow, blood, and lung cells. A single, 2-hr exposure to concentrations of 0, 3, 10, and 30 ppm MIC produced no evidence of chromosomal effects in the bone marrow, although significant cell cycle delay was observed. In four experiments involving exposures on 4 consecutive days to 0, 1, 3, or 6 ppm, delays in bone marrow cell cycle were again observed. Increases in SCE and chromosomal aberrations were observed in bone marrow cells, and a dose-related increase in SCE occurred in lung cells but not in peripheral blood lymphocytes. A significant increase in micronucleated polychromatic erythrocytes in the peripheral blood was observed in male mice in one experiment. From these results, it appears that methyl isocyanate has the capacity to affect chromosome structure but not to induce gene mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

The persistence of lymphocytes with dicentric chromosomes following whole-body X irradiation of mice.

Thirty-six male C57B1/6 mice were X-irradiated whole body with 3 Gy to generate lymphocytes with dicentric chromosomes to study the persistence of these lymphocytes in the spleen and peripheral blood to estimate the life span of mature B- and T-cells. Peripheral blood and spleen were removed from groups of four mice immediately after radiation exposure and on Days 1, 3, 7, 14, 28, 56, and 112 thereafter. Peripheral blood lymphocytes were cultured with phytohemagglutinin to stimulate T-cell division, and splenic lymphocytes were cultured with either lipopolysaccharide or phytohemagglutinin to stimulate B- or T-cell division, respectively. The initial frequencies of dicentric chromosomes with accompanying fragments observed in splenic T-cells (0.44), splenic B-cells (0.43), and peripheral blood lymphocyte cultures (0.48) initiated on Day 0 were not significantly different. For both splenic and peripheral blood T-lymphocytes, the frequency of cells containing dicentric chromosomes declined in an exponential manner following irradiation, with a 50% reduction in frequency occurring 14 days after exposure. In contrast, the frequency of B-cells containing dicentric chromosomes remained stable through Day 7 but then declined precipitously between Day 7 and Day 14 and remained relatively stable, although slightly above baseline, through Day 112 post-exposure. For both B- and T-cells, less than 5% of the cells contained a dicentric chromosome with accompanying fragments at Day 112. These data indicate that B- and T-lymphocytes with dicentric chromosomes show different decay kinetics and suggest that they may possess different life spans.

A cytogenetic comparison of the responses of mouse and human peripheral blood lymphocytes to 60Co gamma radiation.

Experiments were conducted to compare the chromosome damaging effects of 60Co gamma radiation on mouse and human peripheral blood lymphocytes (PBLs). Either whole blood or isolated and pelleted mononuclear leucocytes (MNLs) were irradiated with a 60Co unit to yield exposures of 1, 2, 3, or 4 Gy. In addition, mice were whole-body irradiated in vivo with the same doses so that an in vitro-in vivo comparison could be made. The results indicate that mouse PBLs irradiated in whole blood, whether in vivo or in vitro, respond similarly to 60Co gamma rays as measured by dicentric chromosome formation. In addition, mouse and human PBLs showed a similar radiosensitivity, but because the mouse PBL data were best fitted to an exponential function and the human PBL data to a quadratic function, direct comparisons were difficult to make. Pelleted MNLs from mice were much less sensitive to the clastogenic effects of gamma radiation than whole blood. This is believed to be due to hypoxic conditions that developed during irradiation and transport. Human PBLs did not show a marked difference whether irradiated in whole blood or as pelleted MNLs in tissue culture medium.

A modified mouse peripheral blood lymphocyte culture system for cytogenetic analysis.

A detailed methodology is presented for culturing mouse peripheral blood lymphocytes isolated on density gradients and stimulated to divide using either phytohemagglutinin, concanavalin A, or lipopolysaccharide. The techniques described yield more than sufficient numbers of mitotic cells for analyzing sister chromatid exchange, chromosome aberrations, and micronuclei following in vitro or in vivo exposure to chemicals or radiation.

Induction of genotoxic damage is not correlated with the ability to methylate arsenite in vitro in the leukocytes of four mammalian species.

Arsenic is a natural drinking water contaminant that impacts the health of large populations of people throughout the world; however, the mode or mechanism by which arsenic induces cancer is unclear. In a series of in vitro studies, we exposed leukocytes from humans, mice, rats, and guinea pigs to a range of sodium arsenite concentrations to determine whether the lymphocytes from these species showed differential sensitivity to the induction of micronuclei (MN) assessed in cytochalasin B-induced binucleate cells. We also determined the capacity of the leukocytes to methylate arsenic by measuring the production of MMA [monomethylarsinic acid (MMA(V)) and monomethylarsonous acid (MMA(III))] and DMA [dimethylarsinic acid (DMA(V)) and dimethylarsonous acid (DMA(III))]. The results indicate that cells treated for 2 hr at the G(0) stage of the cell cycle with sodium arsenite showed only very small to negligible increases in MN after mitogenic stimulation. Treatment of actively cycling cells produced induction of MN with increasing arsenite concentration, with the human, rat, and mouse lymphocytes being much more sensitive to MN induction than those of the guinea pig. These data gave an excellent fit to a linear model. The leukocytes of all four species, including the guinea pig (a species previously thought not to methylate arsenic), were able to methylate arsenic, but there was no clear correlation between the ability to methylate arsenic and the induction of MN.

Bleomycin sulfate-induced micronuclei in human, rat, and mouse peripheral blood lymphocytes.

The sensitivity to micronucleus (MN) induction of human, mouse, and rat peripheral blood lymphocytes (PBLs) exposed to bleomycin sulfate (BLM) in vitro was compared in cytochalasin B-induced binucleated (BN) cells. For the PBLs of each species, either 0, 5, 10, 20, 40, 60, 80, or 160 micrograms/ml BLM was added to 5 ml aliquots of whole blood for 4 hr at 37 degrees C in a 5% CO2 atmosphere. Leukocytes were isolated on a density gradient and cultured in the presence of phytohemagglutinin to stimulate blastogenesis, and cytochalasin B was added to each culture at 21 hr postinitiation to prevent cytokinesis. A total of 4,000 BNs/concentration/species was analyzed for MN in two independent experiments. In addition, multiple-MN-BNs were quantitated, and the nucleation index was determined. Significant increases both in total MN-BNs and multiple-MN-BNs were observed at all concentrations in all species. All three species' concentration-response curves gave good fits (r2 values from 0.87 to 0.95) to either a linear or a square root model (y = mx + b or y = m[x]0.5 + b, respectively; where y = the percentage of MN-BN, m is the slope, and b is the y-intercept). The MN induction in the human and rat PBLs was not statistically different, but both were significantly less sensitive than the response shown by the BLM-exposed mouse PBLs. This difference in MN susceptibility was observed only at BLM test concentrations > or = 20 micrograms/ml. The nucleation index was significantly decreased in all species at either 80 or 160 micrograms/ml.

Cytogenetic analyses of mice exposed to dichloromethane.

Chromosome damage was studied in female B6C3F1 mice exposed to dichloromethane (DCM) by subcutaneous or inhalation treatments. No increase in the frequency of either sister chromatid exchanges (SCEs) or chromosome aberrations (CAs) in bone marrow cells was observed after a single subcutaneous injection of 2,500 or 5,000 mg/kg DCM. Inhalation exposure to DCM for 10 days at concentrations of 4,000 or 8,000 ppm resulted in significant increases in frequencies of SCEs in lung cells and peripheral blood lymphocytes, CAs in lung and bone marrow cells, and micronuclei (MN) in peripheral blood erythrocytes. Lung cell CAs and blood erythrocyte MN reached frequencies of approximately two times control levels. Following a 3-month inhalation exposure to 2,000 ppm DCM, mice showed small but significant increases in lung cell SCEs and peripheral blood erythrocyte MN. These findings suggest that genotoxicity may play a role in the carcinogenicity of DCM in the lungs of B6C3F1 female mice.