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The use of brominated flame retardants and incineration of bromine-containing materials has lead to an increase in polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) in the environment. Measurable amounts of PBDD/Fs have been detected in soil, seafood, and human breast milk and serum. Studies indicate that the relative potencies of some PBDD/Fs based on enzyme induction are equivalent to those of some polychlorinated dibenzo-p-dioxins and dibenzofurans. To assess the humoral immunity relative potencies of PBDD/Fs and compare them to their chlorinated analogs, female B6C3F1/N mice received a single oral exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrabromodibenzofuran (TBDF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentabromodibenzofuran (1PeBDF), 1,2,3,7,8-pentachlorodibenzofuran (1PeCDF), 2,3,4,7,8-pentabromodibenzofuran (4PeBDF), 2,3,4,7,8-pentachlorodibenzofuran (4PeCDF), 2,3-dibromo-7,8-dichlorodibenzo-p-dioxin (DBDCDD), or 2,3,7-tribromodibenzo-p-dioxin (TriBDD). Inhibition of the immunoglobulin M (IgM) antibody forming cell response was measured 4 days following immunization with sheep red blood cells. The data were fit to a Hill model to estimate the ED50 for inhibition. Expression of xenobiotic metabolizing enzyme (XME) and thyroxine transport protein (Ttr) genes in liver was measured by PCR to assess aryl hydrocarbon-mediated responses. TCDD, TBDF, TCDF, 1PeBDF, 4PeBDF, 4PeCDF, and DBDCDD suppressed the IgM antibody response and Ttr gene expression, and upregulated phase I XME genes. 1PeCDF suppressed the IgM antibody response but only upregulated phase I XME genes; TriBDD had no effect on antibody response. The rank order of potency (ED50) for these chemicals was TCDD>TBDF>4PeBDF>TCDF/4PeCDF/1PeBDF>1PeCDF. Whereas TCDD was the most potent compound tested, the brominated analogs were more potent than their chlorinated analogs, suggesting that these compounds should be considered in toxic equivalency factor evaluation and risk assessment.

The dose-response relationship for the induction of micronuclei (MN) and the impact of glutathione (GSH) detoxication on naphthalene-induced cytotoxicity and genotoxicity were investigated in human TK6 cells. TK6 cells were exposed to 10 concentrations ranging from 0.0625 to 30μM naphthalene in the presence of β-naphthoflavone- and phenobarbital (βNP/PB)-induced rat liver S9 with a nicotinamide adenine dinucleotide phosphate-generating system. Three approaches were used to identify a no-observed-effect level (NOEL) for naphthalene-induced genotoxicity: (1) laboratory criteria of ≥ twofold increase over the concurrent solvent controls (NOEL = 10μM), (2) ANOVA with Bonferroni correction (NOEL = 2.5μM), and (3) the benchmark dose approach (BMCL(10) = 3.35μM). The NOEL and point of departure micronucleus frequency for naphthalene-induced MN are between the tested naphthalene concentrations of 2.5-10.0μM in this experimental system. Supplementation of the exposure system with physiological relevant concentrations of 5mM GSH eliminated naphthalene-induced cytotoxicity and genotoxicity; no increased cytotoxicity or genotoxicity was observed at concentrations of up to 500μM naphthalene in the presence of GSH compared with 2.5-10.0μM in the absence of GSH. Naphthalene bioactivation by βNP/PB-induced rat liver S9 exhibits a nonlinear dose-response for the induction of MN in TK6 cells with a NOEL of 2.5-10μM that in the presence of GSH is shifted upward greater than 50- to 200-fold. These data demonstrate a nonlinear dose-response for naphthalene-induced genotoxicity that is eliminated by GSH, and both observations should be considered when assessing human risk from naphthalene exposures.

Acrylamide is an industrial chemical used in polymer manufacture. It is also formed in foods processed at high temperatures. It induces chromosome aberrations and micronuclei (MN) in somatic cells of mice, but not rats, and mutations in transgenic mice. This study evaluated the low-dose MN response in mouse bone marrow and the shape of the dose-response curve. Mice were treated orally with acrylamide for 28 days using logarithmically spaced doses from 0.125 to 24 mg/kg/day, and MN were assessed in peripheral blood reticulocytes (RETs) and erythrocytes by flow cytometry. Liver glycidamide DNA adducts and acrylamide and glycidamide N-terminal valine hemoglobin adducts were also determined. Acrylamide produced a weak MN response, with statistical significance at 6.0 mg/kg/day, or greater, in MN-RETs and at 4.0 mg/kg/day or greater in MN normochromatic erythrocytes (NCEs). The MN responses at the lower doses were indistinguishable from the concurrent and historical controls. The adducts increased at a much different rate than the MN. When the MN-NCE values were compared to administered dose, the response was consistent with a linear model. However, when hemoglobin or DNA adducts were used as the dose metric, the response was significantly nonlinear, and models that assumed a threshold dose of 1 or 2 mg/ kg/day provided a better fit than a linear model. The MN-RET dose-response had greater variability than the MN-NCE response and was consistent with linearity and with a threshold at 1 or 2 mg/ kg/day, regardless of the dose metric. These data suggest a threshold for acrylamide in the MN test.

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.

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.

Benzene, a carcinogen that induces chromosomal breaks, is strongly associated with leukemias in humans. Possible genetic determinants of benzene susceptibility include proteins involved in repair of benzene-induced DNA damage. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), encoded by Prkdc, is one such protein. DNA-PKcs is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Here we compared the toxic effects of benzene on mice (C57BL/6 and 129/Sv) homozygous for the wild-type Prkdc allele and mice (129/SvJ) homozygous for a Prkdc functional polymorphism that leads to diminished DNA-PK activity and enhanced apoptosis in response to radiation-induced damage. Male and female mice were exposed to 0, 10, 50, or 100 ppm benzene for 6 h/d, 5 d/week for 2 weeks. Male mice were more susceptible to benzene toxicity compared with females. Hematotoxicity was evident in all male mice but was not seen in female mice. We observed similar, large increases in both micronucleated erythrocyte populations in all male mice. Female mice had smaller but significant increases in micronucleated cells. The p53-dependent response was induced in all strains and genders of mice following benzene exposure, as indicated by an increase in p21 mRNA levels in bone marrow that frequently corresponded with cell cycle arrest in G2/M. Prkdc does not appear to be a significant genetic susceptibility factor for acute benzene toxicity. Moreover, the role of NHEJ, mediated by DNA-PK, in restoring genomic integrity following benzene-induced DSB remains equivocal.

Transgenic mouse mutation assays, such as Muta Mouse (lacZ, CD2F1) and Big Blue® (lacI, B6C3F1), afford the opportunity to evaluate the mutagenic potential of chemicals in any target organ in vivo. This paper discusses published data collected from the analysis of the skin, stomach and lung DNA after topical, oral and inhalation exposure, respectively. These data indicate that both Muta Mouse and Big Blue® should play an important part in the evaluation of genotoxicity in vivo, particularly where the endpoint or target tissue available in the more conventional tests is inappropriate. It is concluded that there is a distinct role for this type of assay in genetic toxicology testing. For substances applied to the skin or dosed orally or by inhalation and which are unlikely to reach either the bone marrow or the liver, then data derived from these assays may be more relevant to an assessment of possible risk to man than the currently used unscheduled DNA synthesis in liver and cytogenetics assays in bone marrow or peripheral blood.

1,3-Butadiene (BD) is an indirect-acting mutagen that is bioactivated in laboratory animals to at least two mutagenic metabolites, 1,2-expoxy-3-butene (EB) and 1,2,3,4-diepoxybutane (DEB). In the present study, the cytotoxicity, mutagenicity and mutational spectrum at hprt were determined after EB-exposure of human TK6 lymphoblastoid cells (TK6 cells). EB was cytotoxic at concentrations ranging from 200 to 1000 microM x 24 h; at 400 microM x 24 h, the cell survival relative to unexposed controls was approximately 10%. Exposure of TK6 cells to EB (400 microM x 24 h) resulted in a 5-9-fold increase in the hprt mutant frequency. Molecular characterization of EB-induced hprt mutants indicated that 78% of the mutations at hprt were single base substitutions. A significant (P T:A transversions was observed compared with spontaneous hprt mutants isolated during these studies. All of the A:T-->T:A transversions in EB-induced mutants occurred with the A in the non-transcribed strand. These data indicate that a primary mode of genotoxicity induced by EB in human TK6 cells is the induction of single base substitutions.

1,3-Butadiene (BD) is a carcinogen in both rats and mice with mice being substantially more sensitive than rats. It is not known if BD poses a carcinogenic risk for humans. Findings from exposure assessment studies indicate that potential industrial exposure to BD in monomer, polymer, and end-user industries is typically