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A possible role for metabolic activation of 2,2',5, 5'-tetrachlorobiphenyl (TCB) to quinonoid metabolites was investigated in vitro in rat liver microsomes and in vivo in male Sprague-Dawley rats. Incubation of TCB with phenobarbital-induced rat liver microsomes resulted in metabolism of TCB to 3-hydroxy-TCB (3-OH-TCB) and 3,4-dihydroxy-TCB (3,4-diOH-TCB), which were further oxidized to form a reactive intermediate that bound to liver proteins. The predominant species observed in the Raney nickel assay for cysteinyl adducts was identified as 3,4-diOH-TCB, consistent with an adduct having the structure 5-cysteinyl-3,6-dichloro-4-(2', 5'-dichlorophenyl)-1,2-benzoquinone. This adduct may arise via the Michael addition of the sulfhydryl group of cysteine to 3, 6-dichloro-4-(2',5'-dichlorophenyl)-1,2-benzoquinone (Cl(4)PhBQ). Metabolism of 3-OH-TCB by phenobarbital-induced microsomes in the presence of either NADPH or cumene hydroperoxide as a cofactor resulted in the formation of adducts. Dose-dependent formation of cysteinyl adducts was observed in liver cytosolic protein from rats treated with a single dose of TCB (0-200 mg/kg) by gavage. By regression analysis, the TCB adducts decayed with a half-life of 2. 03 +/- 0.131 days (mean +/- SE), which is approximately 2.5-fold shorter than the endogenous half-life for liver cytosolic protein in rat liver, suggesting adduct instability. Saturable formation of TCB adducts was observed in liver cytosolic protein of rats receiving multiple doses of TCB over 5 days. The levels of Cl(4)PhBQ-derived adducts were 2.1-fold greater than the estimated steady-state levels predicted by the single-dose treatment [97.7 +/- 13.2 vs 45.7 +/- 3. 73 (pmol/g)/(mg/kg of body weight)], suggesting induction of metabolism. A single cysteinyl adduct, inferred to be 5-cysteinyl-3, 6-dichloro-4-(2',5'-dichlorophenyl)-1,2-benzoquinone, was detected in brain cytosolic protein of rats treated with multiple doses of TCB with levels of 15.2 (pmol/g)/(mg/kg of body weight). Implied involvement of a reactive quinone in the liver and brain of TCB-treated rats supports the idea that quinonoid metabolites may be important contributors to PCB-derived oxidative damage to genomic DNA.

Five cysteinyl adducts (including one with multiple isomeric forms) of hemoglobin (Hb) and albumin (Alb) have been characterized in the blood of Sprague-Dawley rats following administration of pentachlorophenol (PCP). Three of these adducts were formed by multiple substitution reactions of tetrachloro-1,4-benzoquinone (Cl4-1,4-BQ) and its products, and two arose from reactions of tetrachloro-1,4-benzosemiquinone (Cl4-1,4-SQ) and tetrachloro-1,2-benzosemiquinone (Cl4-1,2-SQ). Adducts of tetrachloro-1,2-benzoquinone (Cl4-1,2-BQ) were not observed. Regarding adducts of Cl4-1,4-BQ and its products, specific structures were assigned to monosubstituted, disubstituted, and trisubstituted adducts of Hb and Alb following modification of rat blood with Cl4-1,4-BQ (0-45 microM) in vitro and after metabolism of PCP (0-40 mg/kg body weight) in Sprague-Dawley rats, in vivo. The formation of all adducts was linear over the ranges tested, with Alb adducts being more abundant than Hb adducts. The levels of the adducts measured were in the following order: monosubstituted > disubstituted > trisubstituted. The observation that Cl4-1,4-BQ can produce multisubstituted adducts with proteins suggests that protein-protein cross links may be formed, with inherent toxicological implications. Regarding adducts of the semiquinones (detected only in vivo), linear production of Hb and Alb adducts was observed with increasing dosage of PCP for adducts of both Cl4-1,4-SQ and Cl4-1,2-SQ. Higher levels of the semiquinone adducts were observed in Hb than in Alb, in contrast to the results with the quinone adducts. In a separate in vivo experiment (20 mg PCP/kg body weight), where animals were sacrificed at intervals up to 336 h postadministration, adducts were eliminated at rates which were comparable among the different adducts of a given protein.

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Tetrachloro-1,4-benzoquinone (Cl4BQ), a metabolite of pentachlorophenol (PCP), is believed to play a role in the genotoxicity of PCP. We have developed a method to measure the adducts of Cl4BQ with cysteine residues of hemoglobin (Hb) and albumin (Alb). This method employs the use of Raney nickel to selectively cleave the sulfur-bound adducts. Adducts of Hb and Alb with Cl4BQ were measured following modification of rat blood with Cl4BQ (0-90 microM) in vitro. The formation of both Hb and Alb adducts was linear over the entire range with second-order rate constants of 6.89 and 167 L mol-1 h-1, respectively. The proportions of the concentrations of these Hb and Alb adducts to those of all covalently-bound products were estimated to be 0.053 and 0.178, respectively, at initial Cl4BQ concentrations between 3 and 90 microM. The overall rate of reaction of Cl4BQ in rat blood (in vitro) was pseudo-first-order with an estimated half-time of 4.35 h. Hb and Alb adducts of Cl4BQ were also measured in vivo following oral administration of PCP to rats (0-20 mg/kg body wt). Linear production of Hb and Alb adducts was observed over the entire range of dosages, with slopes of 0.09 and 8.22 pmol of adduct (g of protein)-1 [(mg of PCP)/(kg body wt)]-1, respectively. On the basis of production of Hb adducts in vitro and in vivo, it is estimated that 2.7 x 10(-7) mol of Cl4BQ was released to the blood of rats per mole of PCP administered.(ABSTRACT TRUNCATED AT 250 WORDS)

The objectives of this investigation were to examine whether airborne particles induce DNA damaging and estrogen-disrupting effects and to explore the relationships between them. In this study, airborne particulate was collected at an urban, a suburban, and a rural site in central Taiwan. The organic solvent extracts of airborne particulate were examined in human MCF-7 and T47D-KBluc breast cancer cells. We observed significant increases in reactive oxygen species (ROS) generation in MCF-7 cells after treatment with the particulate extracts whereas aryl hydrocarbon receptor (AhR) antagonist blocked the particulate-induced ROS generation in cells. Further, induction of CYP1A1 protein expression was confirmed by immunoblots in cells treated with airborne particles, suggesting the roles of AhR status in mediating the particulate-induced toxicity. In addition, we observed that at non-cytotoxic concentration (∼0.01 m(3) air equivalent), airborne particles induced decreases in intracellular NAD(P)H and NAD(+) in MCF-7 cells. These decreases were completely blocked by three types of poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors. Moreover, we demonstrated increases in the number of DNA strand breaks in MCF-7 cells exposed to airborne particles as measured by the single-cell gel electrophoresis (Comet) assay. Overall, this evidence confirms that airborne particles induce decreases in intracellular NAD(P)H and NAD(+) through PARP-1 activation mediated by formation of DNA strand breaks. Furthermore, we confirmed that with series dilution airborne particles (∼10(-7)-10(-2) m(3) air equivalent) possess both estrogenic and anti-estrogenic activities as determined by the ERα-mediated reporter gene assay in human T47D-KBluc breast cancer cells. In conclusions, we confirmed that the DNA-damaging activity and estrogenicity of airborne particles varied considerably with concentration (air equivalent). Our findings add further support to the theme that ROS formation is a significant determinant factor in mediating the induction of oxidative DNA damage and repair in human breast cancer cells exposed to airborne particles and that oxidative stress and the subsequent induction of DNA damage may, in part, contribute to airborne particle-induced carcinogenesis.