Chronic human exposure to benzene has been linked to several hematopoietic disorders, including leukemia and lymphomas. Certain benzene metabolites, including benzoquinone (BQ), are genotoxic and mutagenic. Bone marrow stem cells are targets for benzene-induced cytotoxicity and DNA damage that could result in changes to the genome of these progenitor cells, thereby leading to hematopoietic disorders and cancers. Human bone marrow CD34(+) hematopoietic progenitor cells (HPC) were exposed in vitro to 1,4-BQ to assess cytotoxicity, genotoxicity, and DNA damage responses and the molecular mechanisms associated with these events. CD34(+) HPC from 10 men and 10 women were exposed to 0, 1, 5, 10, 15, or 20 microM of 1,4-BQ and analyzed 72 h later. Apoptosis and cytotoxicity were dose-dependent, with exposure to 10 microM 1,4-BQ resulting in approximately 60% cytotoxicity relative to untreated controls. A significant increase in the percentage of micronucleated CD34(+) cells was detected in cultures treated with 1,4-BQ. In addition, the p21 mRNA level was elevated in 1,4-BQ-treated cells, suggesting that human CD34(+) cells utilize the p53 pathway in response to 1,4-BQ-induced DNA damage. However, there were no significant changes in mRNA levels of the DNA repair genes ku80, rad51, xpa, xpc, and ape1 as well as p53 following treatment with 1,4-BQ. Although interindividual variations were evident in the cellular response to 1,4-BQ, there was no gender difference in the response overall. These results show that human CD34(+) cells are sensitive targets for 1,4-BQ toxicity that use the p53 DNA damage response pathway in response to genotoxic stress. Human CD34(+) HPC will be useful for testing the toxicity of other benzene metabolites and various hematotoxic chemicals.
Chronic exposure to benzene results in progressive decline of hematopoietic function and may lead to the onset of various disorders, including aplastic anemia, myelodysplastic syndrome, and leukemia. Damage to macromolecules resulting from benzene metabolites and misrepair of DNA lesions may lead to changes in hematopoietic stem cells (HSCs) that give rise to leukemic clones. We have shown previously that male mice exposed to benzene by inhalation were significantly more susceptible to benzene‐induced toxicities than females. Because HSCs are targets for benzene‐induced cytotoxicity and genotoxicity, we investigated DNA damage responses in HSC from both genders of 129/SvJ mice after exposure to 1,4‐benzoquinone (BQ) in vitro or benzene in vivo. 1,4‐BQ is a highly reactive metabolite of benzene that can cause cellular damage by forming protein and DNA adducts and producing reactive oxygen species. HSCs cultured in the presence of 1,4‐BQ for 24 hours showed a gender‐independent, dose‐dependent cytotoxic response. RNA isolated from 1,4‐BQ–treated HSCs and HSCs from mice exposed to 100 ppm benzene by inhalation showed altered expression of apoptosis, DNA repair, cell cycle, and growth control genes compared with unexposed HSCs. Rad51, xpc, and mdm‐2 transcript levels were increased in male but not female HSCs exposed to 1,4‐BQ. Males exposed to benzene exhibited higher mRNA levels for xpc, ku80, ccng, and wig1. These gene expression differences may partially explain the gender disparity in benzene susceptibility. HSC culture systems such as the one used here will be useful for testing the hematotoxicity of various substances, including other benzene metabolites.
Enzymes involved in benzene metabolism are likely genetic determinants of benzene-induced toxicity. Polymorphisms in human microsomal epoxide hydrolase (mEH) are associated with an increased risk of developing leukemia, specifically those associated with benzene. This study was designed to investigate the importance of mEH in benzene-induced toxicity. Male and female mEH-deficient (mEH-/-) mice and background mice (129/Sv) were exposed to inhaled benzene (0, 10, 50, or 100 ppm) 5 days/week, 6 h/day, for a two-week duration. Total white blood cell counts and bone marrow cell counts were used to assess hematotoxicity and myelotoxicity. Micronucleated peripheral blood cells were counted to assess genotoxicity, and the p21 mRNA level in bone marrow cells was used as a determinant of the p53-regulated DNA damage response. Male mEH-/- mice did not have any significant hematotoxicity or myelotoxicity at the highest benzene exposure compared to the male 129/Sv mice. Significant hematotoxicity or myelotoxicity did not occur in the female mEH-/- or 129/Sv mice. Male mEH-/- mice were also unresponsive to benzene-induced genotoxicity compared to a significant induction in the male 129/Sv mice. The female mEH-/- and 129/Sv mice were virtually unresponsive to benzene-induced genotoxicity. While p21 mRNA expression was highly induced in male 129/Sv mice after exposure to 100-ppm benzene, no significant alteration was observed in male mEH-/- mice. Likewise, p21 mRNA expression in female mEH-/- mice was not significantly induced upon benzene exposure whereas a significant induction was observed in female 129/Sv mice. Thus mEH appears to be critical in benzene-induced toxicity in male, but not female, mice.
The acute toxicity of tetravalent platinum was studied in vitro by use of rabbit alveolar macrophages and human lung fibroblasts (strain WI-38). Alveolar macrophages were exposed in tissue culture for 20 hr to platinum dioxide (PtO2) or platinum tetrachloride (PtCl4). There was no evidence of dissolution of PtO2 and no decrease in viable cells at concentrations as high as 500 mug/ml. PtCl4 was soluble in the macrophage system and after a 20-hr exposure, resulted in loss of viability in 50% of the cells originally present at a concentration of 0.30mM (59 mug Pt/ml). After a 20-hr exposure, rapidly growing human lung fibroblasts were rendered nonviable by PtCl4 at comparable concentrations. A decrease in total cellular ATP was observed at lower concentrations in macrophages and fibroblasts along with a reduction in phagocytic activity of macrophages as compared to controls. With the fibroblasts, a 50% decrease in incorporation of 14C-thymidine was observed after a 22-hr exposure to PtCl4 at a concentration of 0.007mM; higher concentrations were required to inhibit the incorporation of 14C-uridine and 14C-leucine. Time-course studies indicated that the inhibition of 14C-thymidine incorporation was nearly complete (90%) after 7 hr in the presence of 0.06mM PtCl4. Under the same conditions, there was little inhibition (15%) of 14C-leucine incorporation and moderate inhibition (50%) of 14C-uridine incorporation. Higher concentrations of PtCl4 were required to inhibit 14C-thymidine incorporation into the acid-soluble fraction than were required to inhibit incorporation into the acid-precipitable fraction. Hence, the preferential inhibition of DNA synthesis by PtCl4 may result from an impairment of the incorporation process.
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