DNA damage in L5178YS cells following exposure to benzene metabolites

Because DNA modification may be a prerequisite for chemical carcinogenesis, the DNA-damaging potential of benzene and its metabolites was examined in order to identify the proximate DNA-damaging agent associated with benzene exposure. A DNA synthesis inhibition assay previously identified p-benzoquinone as the most potent overall cellular toxin and inhibitor of DNA synthesis, but failed to discriminate among the hydroxylated metabolites. Therefore, the ability of benzene and its metabolites to induce DNA strand breaks in the mouse lymphoma cell line, L5178YS, was examined in order to provide a more accurate indication of the DNA damage associated with benzene and its metabolites. Cells were exposed to benzene, hydroquinone, catechol, phenol, 1,2,4-benzenetriol, or p-benzoquinone over a 1000-fold concentration range (1.0 microM-1.0 mM). Concentrations of benzene, phenol, or catechol as high as 1.0 mM did not increase the percentage of single-stranded DNA observed. Concentrations of hydroquinone as high as 0.1 mM were also ineffective. In contrast, both p-benzoquinone and 1,2,4-benzenetriol produced DNA breaks in a dose-related fashion. Of the two, benzoquinone proved to be more potent with an ED50 of approximately equal to 2.5 microM compared with 55.0 microM for benzenetriol. The DNA damage induced by 6.0 microM benzoquinone was maximal within 3 min of exposure and yielded approximately 70% single-stranded DNA after alkaline denaturation. By contrast, the single-stranded DNA observed after benzenetriol exposure required 60 min of exposure to achieve the same extent of damage as that found with benzoquinone. These results suggest that the benzene metabolites, benzenetriol and benzoquinone, may cause DNA damage and that the mechanisms responsible for the damage associated with these two compounds may be different.     

Relationship between the oxidation potential of benzene metabolites and their inhibitory effect on DNA synthesis in L5178YS cells

The effects of benzene and its metabolites on the rate of DNA synthesis were measured in the mouse lymphoma cell line, L5178YS. The direct toxicity of benzene could be distinguished from that of its metabolites since bioactivation of benzene in L5178YS cells was not observed. Cells were exposed to benzene, phenol, catechol, hydroquinone, p-benzoquinone, or 1,2,4-benzenetriol over the range of 1.0 X 10(-7) to 1.0 X 10(-2) M for 30 min, and the rate of DNA synthesis was measured at various times after chemical washout. Cell viability and protein synthesis were determined by trypan blue dye exclusion and [3H]leucine incorporation, respectively. Effects were designated as "DNA specific" when DNA synthesis was inhibited in the absence of discernible effects on cell membrane integrity and protein synthesis. Concentrations of benzene as high as 1 mM had no effect on DNA synthesis. Comparison of the effects at the maximum nontoxic dose for each compound showed that catechol and hydroquinone were the most effective, inhibiting DNA synthesis by 65%. Phenol, benzoquinone, and benzenetriol inhibited DNA synthesis by approximately 40%. Maximum inhibition was observed 60 min after metabolite washout in each case. Benzoquinone was the most potent inhibitor of DNA synthesis, followed by hydroquinone, benzenetriol, catechol, and phenol with ED50 values of 5 X 10(-6), 1 X 10(-5), 1.8 X 10(-4), 2.5 X 10(-4), and 8.0 X 10(-4), respectively. Cyclic voltammetric experiments were performed on the hydroxylated metabolites of benzene to assess the possible involvement of a redox-type mechanism in their inhibition of DNA synthesis. The ease of oxidation of these metabolites correlated with their ED50 values for inhibition of DNA synthesis (r = 0.997). This suggests that oxidation of phenol or one of its metabolites may be necessary for production of the species involved in inhibition of DNA synthesis.     

In vitro effects of benzene metabolites on mouse bone marrow stromal cells

Benzene exposure can result in bone marrow myelotoxicity. We examined the effects of benzene metabolites on bone marrow stromal cells of the hemopoietic microenvironment. Male B6C3F1 mouse bone marrow adherent stromal cells were plated at 4 X 10(6) cells per 2 ml of DMEM medium in 35-mm tissue culture dishes. The growing stromal cell cultures were exposed to log 2 doses of five benzene metabolites: hydroquinone, benzoquinone, phenol, catechol, or benzenetriol for 7 days. The dose which caused a 50% decrease in colony formation (TD50) was 2.5 X 10(-6) M for hydroquinone, 17.8 X 10(-6) M for benzoquinone, 60 X 10(-6) M for benzenetriol, 125 X 10(-6) M for catechol, and 190 X 10(-6) M for phenol. We next examined the effect of benzene metabolites on the ability of stromal cells to influence granulocyte/monocyte colony growth (G/M-CFU-C) in a coculture system. Adherent stromal cells were plated and incubated for 14 days and then exposed to a benzene metabolite. After 3 days the medium and metabolite were removed and an agar:RPMI layer containing 10(6) fresh bone marrow cells was placed over the stromal layer. After incubation for 7 days the cultures were scored for G/M colony formation. Hydroquinone and benzoquinone were most toxic, while catechol and benzenetriol inhibited colony growth only at high doses. These results indicate that injured bone marrow stromal cells may be a significant factor in benzene-induced hemotoxicity.     

The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells

Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-l-methionine or its demethylated product S-adenosyl-l-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-l-homocysteine partly explained catechol-enhanced erythroid differentiation.     

Enhanced expression of membrane-associated sialidase Neu3 decreases GD3 and increases GM3 on the surface of Jurkat cells during etoposide-induced apoptosis

We previously reported that, in Jurkat human T cells, the topoisomerase II inhibitor etoposide enhances sialidase activity and reduces cell surface sialic acid levels at an early stage of apoptosis and that the decreases in sialic acid are suppressed by the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid [Azuma Y., et al., Glycoconj. J., 17, 301-306 (2000)]. In the current studies, we treated Jurkat cells with etoposide and examined the changes in the cell surface levels of gangliosides GM1, GM2, GM3, GD1a, and GD3 at physiological pH using anti-ganglioside antibodies. We also examined the sialidase activity on the cell surface using 4-methylumbelliferyl N-acetylneuraminic acid and measured the mRNA expression of the plasma membrane-associated sialidase Neu3 and the lysozomal Neu1 using real-time PCR. We found an increase in GM3 and a decrease in GD3 during the early stage (4 h) of etoposide-induced apoptosis that preceded the increase in cell surface exposure of phosphatidylserine (4 to 6 h). The caspase 3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde significantly suppressed changes in GM3 and GD3 and blocked the enhanced cell surface sialidase activity. Furthermore, etoposide caused a gradual up-regulation of Neu3 mRNA expression but not Neu1 mRNA expression. Enhanced Neu3 mRNA expression was suppressed in the presence of caspase 3 inhibitor. These results indicate that Neu3 is up-regulated in Jurkat cells undergoing etoposide-induced apoptosis through intracellular signaling events downstream of caspase 3 activation and that enhanced Neu3 activity is closely related to the changes of cell surface ganglioside composition.     

l‐Carnitine protects against 1,4‐benzoquinone‐induced apoptosis and DNA damage by suppressing oxidative stress and promoting fatty acid oxidation in K562 cells

Widespread occupational and environmental exposure to benzene is unavoidable and poses a public health threat. Studies of potential interventions to prevent or relieve benzene toxicity are, thus, essential. Research has shown l ‐carnitine (LC) has beneficial effects against various pathological processes and diseases. LC possesses antioxidant activities and participates in fatty acid oxidation (FAO). In this study, we investigated whether 1,4‐benzoquinone (1,4‐BQ) affects LC levels and the FAO pathway, as well as analyzed the influence of LC on the cytotoxic effects of 1,4‐BQ. We found that 1,4‐BQ significantly decreased LC levels and downregulated Cpt1a, Cpt2, Crat, Hadha, Acaa2, and Acadvl mRNA expression in K562 cells. Subsequent assays confirmed that 1,4‐BQ decreased cell viability and increased apoptosis and caspase‐3, ‐8, and ‐9 activities. It also induced obvious oxidative stress and DNA damage, including an increase in the levels of reactive oxygen species and malondialdehyde, tail DNA%, and olive tail moment. Additionally, the mitochondrial membrane potential was significantly reduced. Cotreatment with LC (500 μmol/L) relieved these alterations by reducing oxidative stress and increasing the protein expression levels of Cpt1a and Hadha, particularly in the 20 μmol/L 1,4‐BQ group. Thus, our results demonstrate that 1,4‐BQ causes cytotoxicity, reduces LC levels, and downregulates the FAO genes. In contrast, LC exhibits protective effects against 1,4‐BQ‐induced apoptosis and DNA damage by decreasing oxidative stress and promoting the FAO pathway.     

Inhibition of erythropoiesis by benzene and benzene metabolites

Mice were injected sc with benzene or one of its metabolites, phenol, catechol, or hydroquinone. The ability of these compound to inhibit erythropoiesis was quantified by measuring the incorporation of ⁵⁹Fe into developing erythrocytes. Benzene decreased ⁵⁹Fe incorporation into developing erythrocytes in a dose-dependent manner. Maximum inhibition was observed when benzene was administered 48 hr prior to initiation of the ⁵⁹Fe uptake test. The three metabolites of benzene also significantly inhibited ⁵⁹Fe incorporation when they were administered 48 hr prior to initiation of ⁵⁹Fe uptake assay. The degree of inhibition observed with the metabolites was not as great as that observed with benzene. Coadministration of the microsomal mixed-function oxidase inhibitor, 3-amino-1,2,4-triazole, abolished the erythropoietic toxicity of benzene and phenol but had no effect on the catechol- or hydroquinone-induced toxicity.     

Modifications in the metabolic pathways of benzene in streptozotocin-induced diabetic rat

Benzene is a ubiquitous environmental pollutant primarily metabolized by a cytochrome P-450 (CYP-450) isoenzyme, CYP-450 IIE1. A consistent induction of CYP450 IIE1 has been observed in both rat and human affected by diabetes mellitus. The aim of this study was to evaluate whether streptozotocin (STZ)-induced diabetes determines modifications in the metabolic pathways of benzene in rat. Benzene (100 mg/kg per day, dissolved in corn oil) was administered i.p. once a day for 5 days. Urine samples were collected every day in STZ-treated and normoglycaemic animals, treated and untreated with benzene (n = 10). Urinary levels of trans,trans-muconic acid and of phenol, catechol and hydroquinone (free and conjugated with sulphuryl and glucuronic group) were measured by high-performance liquid chromatography (HPLC). In normoglycaemic rats during the 5 days of treatment with benzene we observed a progressive and significant decrement in the urinary excretion of phenol, phenyl sulphate and glucuronide, catechol, catechol glucuronide, hydroquinone, hydroquinone glucuronide and t,t-muconic acid (P < 0.05). In the diabetic animals, conversely, the same metabolites showed progressively increasing urinary levels (P < 0.05). Catechol sulphate and hydroquinone sulphate levels were below the instrument's detection limit. In the comparison between diabetic and normoglycaemic benzene treated rats, the inter-group difference was significant (P < 0.05) from day 3 of treatment for t,t-muconic acid, and from day 1 for free and conjugated phenol, free and glucuronide catechol and free hydroquinone. In the normoglycaemic rat exposed to benzene the decreasing trend observed in urinary excretion of free and conjugated metabolites may be due to their capability to reduce cytochromial activity. Conversely, in the diabetic rat, urinary levels of benzene metabolites tended to increase progressively, probably due to the consistent induction of CYP-450 IIE1 observed in diabetes, which would overwhelm the inhibition of this isoenzyme caused by phenolic metabolites. Furthermore, the metabolic switch towards detoxification metabolites observed after administration of high doses of benzene is not allowed in the diabetic because of reduced glutathione-S-transferase activity. As a consequence, higher levels of hydroquinone, phenol and catechol, considered the actual metabolites responsibles for benzene toxicity, will accumulate in the diabetic rat. Extrapolating these data to human, we may thus suggest that occupational exposure to benzene of a diabetic subject poses a higher risk level, as his metabolism tends to produce and accumulate higher levels of reactive benzene catabolites. Received: 14 December 1998 / Accepted: 23 March 1999     

Inhibitory effect of benzene metabolites on nuclear DNA synthesis in bone marrow cells

Effects of endogenously produced and exogenously added benzene metabolites on the nuclear DNA synthetic activity were investigated using a culture system of mouse bone marrow cells. Effects of the metabolites were evaluated by a 30-min incorporation of [3H]thymidine into DNA following a 30-min interaction with the cells in McCoy's 5a medium with 10% fetal calf serum. Phenol and muconic acid did not inhibit nuclear DNA synthesis. However, catechol, 1,2,4-benzenetriol, hydroquinone, and p-benzoquinone were able to inhibit 52, 64, 79, and 98% of the nuclear DNA synthetic activity, respectively, at 24 microM. In a cell-free DNA synthetic system, catechol and hydroquinone did not inhibit the incorporation of [3H]thymidine triphosphate into DNA up to 24 microM but 1,2,4-benzenetriol and p-benzoquinone did. The effect of the latter two benzene metabolites was completely blocked in the presence of 1,4-dithiothreitol (1 mM) in the cell-free assay system. Furthermore, when DNA polymerase alpha, which requires a sulfhydryl (SH) group as an active site, was replaced by DNA polymerase I, which does not require an SH group for its catalytic activity, p-benzoquinone and 1,2,4-benzenetriol were unable to inhibit DNA synthesis. Thus, the data imply that p-benzoquinone and 1,2,4-benzenetriol inhibited DNA polymerase alpha, consequently resulting in inhibition of DNA synthesis in both cellular and cell-free DNA synthetic systems. The present study identifies catechol, hydroquinone, p-benzoquinone, and 1,2,4-benzenetriol as toxic benzene metabolites in bone marrow cells and also suggests that their inhibitory action on DNA synthesis is mediated by mechanism(s) other than that involving DNA damage as a primary cause.     

Effects of benzene and its metabolites on global DNA methylation in human normal hepatic l02 cells

Benzene is an important industrial chemical that is also widely present in cigarette smoke, automobile exhaust, and gasoline. It is reported that benzene can cause hematopoietic disorders and has been recognized as a human carcinogen. However, the mechanisms by which it increases the risk of carcinogenesis are only partially understood. Aberrant DNA methylation is a major epigenetic mechanism associated with the toxicity of carcinogens. To understand the carcinogenic capacity of benzene, experiments were designed to investigate whether exposure to benzene and its metabolites would change the global DNA methylation status in human normal hepatic L02 cells and then to evaluate whether the changes would be induced by variation of DNA methyltransferase (DNMT) activity in HaeIII DNMT‐mediated methylation assay in vitro. Our results showed that hydroquinone and 1,4‐benzoquinone could induce global DNA hypomethylation with statistically significant difference from control (p < 0.05), but no significant global DNA methylation changes were observed in L02 cells with benzene, phenol, and 1,2,4‐trihydroxybenzene exposure. Benzene metabolites could not influence HaeIII DNMT activity except that 1,4‐benzoquinone shows significantly inhibiting effect on enzymatic methylation reaction at concentrations of 5 μM (p < 0.05). These results suggest that benzene metabolites, hydroquinone, and 1,4‐benzoquinone can disrupt global DNA methylation, and the potential epigenetic mechanism by which that global DNA hypomethylation induced by 1,4‐benzoquinone may work through the inhibiting effects of DNMT activity at 10 μM (p < 0.05). © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 108–116, 2014.     

A proposed mechanism of benzene toxicity: Formation of reactive intermediates from polyphenol metabolites

Male Fischer-344 rats were given 100 μCi (14 mg/kg) [¹⁴C]catechol or [¹⁴C]hydroquinone by injection into the lateral tail vein. For a period of at least 24 hr, soluble radioactivity associated with either compound was retained in the bone marrow, but not in the liver or thymus. The amount of covalently bound radioactivity increased with time in all tissues examined and was significantly depressed in liver, white blood cells, and bone marrow in rats pretreated with Aroclor 1254, a regimen which protects against benzene toxicity. Potential enzymatic and nonenzymatic activation pathways for catechol, hydroquinone, and other known benzene metabolites were examined. In air-saturated 50 mm phosphate buffer (pH 7.4) at 37°C, only hydroquinone and 1,2,4-benzenetriol autoxidized. The oxidation product of hydroquinone had an uv absorption maximum (248 nm) identical to that of benzoquinone. With 250 units superoxide dismutase, hydroquinone autoxidation increased fivefold, whereas the oxidation of 1,2,4-benzenetriol was inhibited (4% of control). Epinephrine autoxidation, an indirect measure of superoxide anion generation, was stimulated by 1,2,4-benzenetriol and hydroquinone, but was barely detectable in the presence of catechol. Of the compounds studied, only benzoquinone augmented the oxidation of NADPH by a 3000g rat bone marrow supernatant. These data support a mechanism for benzene toxicity in which the formation of potentially cytotoxic metabolites, semiquinone, and quinone oxidation products and superoxide radicals, result from autoxidation of at least two polyphenol metabolites of benzene, hydroquinone, and 1,2,4-benzenetriol.     

Induction of G2/M phase arrest and apoptosis by a novel indoloquinoline derivative,IQDMA, in K562 cells

The indoloquinoline, IQDMA (N′‐(11H‐indolo[3,2‐c]quinolin‐6‐yl)‐N,N‐dimethylethane‐1,2‐diamine), was identified as a novel antineoplastic agent with broad spectrum of antitumor activities against several human cancer cells. IQDMA‐induced G2/M arrest was accompanied by up‐regulation of the cyclin‐dependent kinase inhibitors (CDKIs), p21 and p27, and down‐regulation of Cdk1and Cdk2. IQDMA had no effect on the levels of cyclin A, cyclin B1, cyclin D3, or Cdc25C. IQDMA also increased apoptosis, as characterized by apoptotic body formation, increase of the sub G₁ population and poly (ADP‐ribose) polymerase (PARP) cleavage. Further mechanistic analysis demonstrated that IQDMA upregulated FasL protein expression, and kinetic studies showed the sequential activation of caspases‐8, ‐3, and ‐9. Both caspase‐8 and caspase‐3 inhibitors, but not a caspase‐9‐specific inhibitor, suppressed IQDMA‐induced cell death. These molecular alterations provide an insight into IQDMA‐caused growth inhibition, G2/M arrest, and apoptotic death of K562 cells. Drug Dev. Res. 67:743–751, 2006. © 2006 Wiley‐Liss, Inc.     

Effects of co-exposure to extremely low frequency (ELF) magnetic fields and benzene or benzene metabolites determined in vitro by the alkaline comet assay

In the present study, we investigated in vitro the possible genotoxic and/or co-genotoxic activity of 50 Hz (power frequency) magnetic fields (MF) by using the alkaline single-cell microgel-electrophoresis (comet) assay. Sets of experiments were performed to evaluate the possible interaction between 50 Hz MF and the known leukemogen benzene. Three benzene hydroxylated metabolites were also evaluated: 1,2-benzenediol (1,2-BD, catechol), 1,4-benzenediol (1,4-BD, hydroquinone), and 1,2,4-benzenetriol (1,2,4-BT). MF (1 mT) were generated by a system consisting of a pair of parallel coils in a Helmholtz configuration. To evaluate the genotoxic potential of 50 Hz MF, Jurkat cell cultures were exposed to 1 mT MF or sham-exposed for 1h. To evaluate the co-genotoxic activity of MF, the xenobiotics (benzene, catechol, hydroquinone, and 1,2,4-benzenetriol) were added to Jurkat cells subcultures at the beginning of the exposure time. In cell cultures co-exposed to 1 mT (50 Hz) MF, benzene and catechol did not show any genotoxic activity. However, co-exposure of cell cultures to 1 mT MF and hydroquinone led to the appearance of a clear genotoxic effect. Moreover, co-exposure of cell cultures to 1 mT MF and 1,2,4-benzenetriol led to a marked increase in the genotoxicity of the ultimate metabolite of benzene. The possibility that 50 Hz (power frequency) MF might interfere with the genotoxic activity of xenobiotics has important implications, since human populations are likely to be exposed to a variety of genotoxic agents concomitantly with exposure to this type of physical agent.     

Metabolism of benzene in rat hepatocytes. Influence of inducers on phenol glucuronidation

Alterations of benzene metabolism in liver markedly influence benzene toxicity at extrahepatic target tissues. Therefore, generation of 11 phase I and II metabolites of benzene (including phenol, hydroquinone, catechol, benzene-1,2-dihydrodiol, their sulfates and glucuronides, and phenylglutathione) was compared in hepatocytes from 3-methylcholanthrene (MC)- or phenobarbital-treated rats and from untreated controls. At 0.1 mM benzene, total metabolism appeared to be unchanged by treatment with inducers. Phenylsulfate (35%), phenylglucuronide (15%), and phenylglutathione (12%) represented the major metabolites in hepatocytes from untreated controls. With hepatocytes from MC-treated rats, a pronounced shift from phenylsulfate to phenylglucuronide (increase to 34%) was observed, while the formation of unconjugated phenol, hydroquinone, and catechol was decreased (from 16 to 10%). A similar shift from sulfation to glucuronidation was seen in similar studies with phenol. Lineweaver-Burk analysis of microsomal phenol UDP-glucuronosyltransferase activity suggested that MC-treatment induced a high affinity isozyme (KM = 0.14 mM), in addition to the low affinity isozyme (KM = 3.1 mM) present in liver microsomes from untreated and phenobarbital-treated rats. It is concluded that induction by MC of a high affinity hepatic phenol UDP-glucuronosyltransferase effectively shifts benzene metabolism toward formation of less toxic metabolites. This shift may reduce toxic risks at extrahepatic target tissues.     

Evidence for strain-specific differences in benzene toxicity as a function of host target cell susceptibility

It has long been recognized that benzene exposure produces disparate toxic responses among different species or even among different strains within the same species. There is ample evidence that species- or strain-dependent differences in metabolic activity correlate with the disparate responses to benzene. However, bone marrow cells (the putative targets of benzene toxicity) may also exhibit species- or strain-dependent differences in susceptibility to the toxic effects of benzene. To investigate this hypothesis, two sets of companion experiments were performed. First, two strains of mice, Swiss Webster (SW) and C57B1/6J (C57), were exposed to 300 ppm benzene via inhalation and the effects of the exposures were determined on bone marrow cellularity and the development of bone marrow CFU-e (Colony Forming Unit-erythroid, an early red cell progenitor). Second, bone marrow cells from the same strains were exposed in vitro to five known benzene metabolites (1,4 benzoquinone, catechol, hydroquinone, muconic acid, and phenol) individually and in binary combinations. Benzene exposure, in vivo, reduced bone marrow cellularity and the development of CFU-e in both strains; however, reductions in both these endpoints were more severe in the SW strain. When bone marrow cells from the two strains were exposed in vitro to the five benzene metabolites individually, benzoquinone, hydroquinone, and catechol reduced the numbers of CFU-e in both strains in dose-dependent responses, phenol weakly reduced the numbers of the C57 CFU-e only and in a non-dose-dependent manner, and muconic acid was without effect on cells from either strain. Only benzoquinone and hydroquinone exhibited differential responses to CFU-e from the two strains and both of these metabolites were more toxic to SW cells than to C57 cells. Six of the ten possible binary mixtures of metabolites were differentially toxic to the CFU-e from the two strains and five of these mixtures were more toxic to SW cells than to C57 cells. Thus, SW mice were more susceptible to the toxic effects of inhaled benzene and their bone marrow cells were more severely affected by in vitro exposure to benzene metabolites. The binary combinations containing phenol produced little or no enhancement of the toxic effects of the non-phenol metabolites. The weak toxic response induced by phenol, whether delivered alone or in binary mixtures, suggests that little metabolism occurred during the 48 h of the in vitro exposures since benzoquinone and hydroquinone, which were clearly toxic when added to the CFU-e culture system, are formed by further metabolic oxidation of phenol. Thus, strain-dependent differential metabolism appeared to play a minimal role in the disparate toxicity observed in the in vitro studies, implying that the diverse responses were due to inherent differences in the susceptibilities of the CFU-e to the toxic action of the benzene metabolites.     

Homologous recombination initiated by benzene metabolites: a potential role of oxidative stress.

Benzene is a ubiquitous pollutant and known human leukemogen. Benzene can be enzymatically bioactivated to reactive intermediates that can lead to increased formation of reactive oxygen species (ROS). ROS formation can directly induce DNA double-strand breaks, and also oxidize nucleotides that are subsequently converted to double-strand breaks during DNA replication that can be repaired through homologous recombination, which is not error-free. Therefore increased DNA double-strand-break levels may induce hyper-recombination, which can lead to deleterious genetic changes. To test the hypothesis that benzene and its metabolites can initiate hyper-recombination and to investigate the potential role of ROS, a Chinese hamster ovary (CHO) cell line containing a neo direct repeat recombination substrate (CHO 3-6), was used to determine whether benzene or its metabolites phenol, hydroquinone, catechol, or benzoquinone initiated increased homologous recombination and whether this increase could be diminished by the coincubation of cells with the antioxidative enzyme catalase. Results demonstrated that cells exposed to benzene (1, 10, 30, or 100 micro M) for 24 h did not exhibit increased homologous recombination. Increased recombination occurred with exposure to phenol (1.8-, 2.6-, or 2.9-fold), catechol (1.9-, 2-, 5-, or 3.2-fold), or benzoquinone (2.7-, 5.5-, or 6.9-fold) at 1, 10, and 30- micro M concentrations, respectively, and with exposure to hydroquinone at 10 and 30 micro M concentrations (1.5-1.9-fold; p < 0.05). Studies investigating the effects of catalase demonstrated that increased homologous recombination due to exposure to phenol, hydroquinone, catechol, or benzoquinone (10 micro M) could be completely abolished by the addition of catalase. These data support the hypothesis that increased homologous recombination mediates benzene-initiated toxicity and supports a role for oxidative stress in this mechanism.     

Cell-specific metabolism in mouse bone marrow stroma: studies of activation and detoxification of benzene metabolites.

Two of the major cell types in bone marrow stroma, macrophages and fibroblasts, have been shown to be important regulators of both myelopoiesis and lymphopoiesis. The enzymology relating to cell-specific metabolism of phenolic metabolites of benzene in isolated mouse bone marrow stromal cells was examined. Fibroblastoid stromal cells had elevated glutathione-S-transferase (4.5-fold) and DT-diaphorase (4-fold) activity relative to macrophages, whereas macrophages demonstrated increased UDP-glucuronosyltransferase (UDP-GT, 7.5-fold) and peroxidase activity relative to stromal fibroblasts. UDP-GT and glutathione-S-transferase activities in macrophages and fibroblasts, respectively, were significantly greater than those in unpurified white marrow. Aryl sulfotransferase activity could not be detected in either bone marrow-derived macrophages or fibroblasts, and there were no significant differences in GSH content between the two cell types. Because UDP-GT activity is high in macrophages, these data suggest that DT-diaphorase levels would be rate limiting in the detoxification of benzene-derived quinones in bone marrow macrophages. The peroxidase responsible for bioactivation of benzene-derived phenolic metabolites in bone marrow macrophages is unknown but has been suggested to be prostaglandin H synthase (PGS). Hydrogen peroxide, but not arachidonic acid, supported metabolism of hydroquinone to reactive species in bone marrow-derived macrophage lysates. These data do not support a major role for PGS in peroxidase-mediated bioactivation of hydroquinone in bone marrow-derived macrophages, although PGS mRNA could be detected in these cells. Similarly, hydrogen peroxide, but not arachidonic acid, supported metabolism of hydroquinone in a human bone marrow homogenate. Peroxidase-mediated interactions between phenolic metabolites of benzene occurred in bone marrow-derived macrophages. Bioactivation of hydroquinone to species that would bind to acid-insoluble cellular macromolecules was increased by phenol and was markedly stimulated by catechol. Bioactivation of catechol was also stimulated by phenol but was inhibited by hydroquinone. These data define the enzymology and the cell-specific metabolism of benzene metabolites in bone marrow stroma and demonstrate that interactions between phenolic metabolites may contribute to the toxicity of benzene in this critical bone marrow compartment.