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.     

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.     

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.     

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     

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.     

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.     

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.     

Benzene's toxicity: a consolidated short review of human and animal studies by HA Khan

Khan's review is a brief summary of the complex field of study revolving around bone marrow toxicity and leukemogenesis observed in people chronically exposed to benzene. These comments are intended to demonstrate the use of the Kahn review as a launching pad for an in-depth analysis of the several related areas that must be fully explored to understand benzene-related diseases. The accumulated evidence demonstrates that benzene-induced bone marrow damage results from the production of hematotoxins that are metabolic products of benzene metabolism. The metabolism of benzene is described with respect to the formation benzene metabolites with emphasis on phenol and hydroquinone, which are the major metabolites, the significance of the formation of glutathione conjugates, the activity of NAD(P)H:quinone oxidoreductase (NQO1), and the ring opening products. Results are shown suggesting that oxidative stress induced by benzene metabolites is likely to be a significant factor in damaging DNA in bone marrow cells. Although a variety of effects on bone marrow can be demonstrated it is not yet clear which metabolites are most important in either benzene-induced aplastic anemia or leukemia. Benzene metabolism alone is insufficient to fully describe benzene toxicity. The impact of benzene metabolites on bone marrow cells must be fully explored to determine how benzene exposure can result in decreased viability or genetic toxicity to cells in the bone marrow.     

Comparative studies of the in vitro metabolism and covalent binding of 14C-benzene by liver slices and microsomal fraction of mouse, rat, and human

Metabolism of benzene by the liver has been suggested to play an important role in the hepatotoxicity of benzene. The role of the different benzene metabolites and the causes of species differences in benzene hepatotoxicity are, however, not known. The metabolism and covalent binding of 14C-benzene by liver microsomal fractions and liver slices from rat, mouse, and human subjects have been studied. Rat microsomal fraction formed phenol at a rate of 0.32 nmol/min/mg of protein; mouse microsomal fraction formed phenol at 0.64 nmol/min/mg and hydroquinone at 0.03 nmol/min/mg; and human microsomal fraction formed phenol at 0.46 nmol/min/mg and hydroquinone at 0.07 nmol/min/mg. Covalent binding of 14C-benzene metabolites to rat, mouse, and human liver microsomal protein was 29, 113, and 169 pmol/min/mg of protein, respectively. The rates of metabolite formation from benzene by liver slices in nmol/min/g of tissue were: rat, phenol 0.15, hydroquinone 0.26, and phenylsulfate 1.22; mouse: phenol 0.13, hydroquinone 0.29, phenylsulfate 1.37, and phenylglucuronide 1.34; and human: phenol 0.16, hydroquinone 0.27, phenylsulfate 0.83, and phenylglucuronide 0.52. trans,trans-Muconic acid formation was not detected with liver slices of any species. Covalent binding of 14C-benzene metabolites to rat, mouse, and human liver slices was 8.2, 79.7, and 27.3 pmol/min/g liver, respectively. There was no correlation between ascorbic acid levels in the human liver slices and covalent binding of 14C-benzene metabolites. The results show that phenol and hydroquinone found in extrahepatic tissues, including bone marrow, of animals exposed to benzene could originate from the liver. There was no evidence for the release of highly reactive benzene metabolites such as trans,trans-muconaldehyde or p-benzoquinone from liver cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Erythroid progenitor cells that survive benzene exposure exhibit greater resistance to the toxic benzene metabolites benzoquinone and hydroquinone

Benzene is a well known hematotoxicant which induces hematopoietic dyscrasias of varying intensities in different individuals and even in different strains of the same experimental animal species. Although there is ample evidence that diverse responses to benzene are related to differences in benzene metabolism, we have recently provided evidence implicating differences in host target cell susceptibility to these diverse responses to benzene. The present study extends our previous work and concerns strain-specific differences in marrow progenitor cells that survive benzene exposure. Two mouse strains (Swiss-Webster and C57Bl/6J) which respond to benzene exposure with different intensities of bone marrow cytotoxicity were used. Bone marrow cells from benzene-exposed and untreated mice were cultured with one of five benzene metabolites: 1,4-benzoquinone (BQ), catechol (C), hydroquinone (HQ), muconic acid (MA) or phenol (P) and the abilities of these cells to produce erythroid (CFU-e) or granulocyte/macrophage colonies (GM-CFU-c) were assessed. In both strains, marrow cells isolated from benzene-exposed mice showed a higher percentage of plated CFU-e surviving culture with BQ, HQ or MA than marrow cells isolated from control mice. In contrast, both strains of benzene-exposed mice displayed decreased percentages of plated CFU-e surviving culture with catechol than cells isolated from control mice. Only one condition (the culturing of cells with HQ under GM-CFU-c forming conditions) showed any strain-specific difference in plating efficiency. In all, 20 possible combinations of benzene metabolites and cell types were examined (5 metabolites × 2 progenitor cell types × 2 strains). With seven of these combinations, the colony-forming efficiencies were higher for plated cells isolated from benzene-exposed mice than from untreated mice. With three combinations, the colony-forming efficiencies were lower for cells from benzene-exposed mice, and for ten combinations, there were no changes in plating efficiencies. Possible mechanisms for an acquired resistance to the toxicities of benzene metabolites were explored by measuring the concentrations of hepatic and bone marrow sulfhydryl (SH) groups in cells isolated from benzene-exposed and untreated mice. In both strains, benzene exposure induced no changes in hepatic SH concentrations, but the SH content of bone marrow was more than doubled after benzene exposure in both strains. These results suggest that a fraction of hematopoietic progenitor cells are able to survive severe benzene exposure and produce progeny because of a marked increase in marrow SH groups which react with electrophilic benzene metabolites. Moreover, this protective mechanism occurs in two mouse strains with differing susceptibilities to benzene. Received: 23 November 1993/Accepted: 26 April 1994     

Phenolic metabolites of benzene induced caspase‐dependent cytotoxicities to K562 cells accompanied with decrease in cell surface sialic acids

Benzene‐induced erythropoietic depression has been proposed to be due to the production of toxic metabolites. Presently, the cytotoxicities of benzene metabolites, including phenol, catechol, hydroquinone, and 1,2,4‐benzenetriol, to erythroid progenitor‐like K562 cells were investigated. After exposure to these metabolites, K562 cells showed significant inhibition of viability and apoptotic characteristics. Each metabolite caused a significant increase in activities of caspase‐3, ‐8, and ‐9, and pretreatment with caspase‐3, ‐8, and ‐9 inhibitors significantly inhibited benzene metabolites‐induced phosphatidylserine exposure. These metabolites also elevated expression of Fas and FasL on the cell surface. After exposure to benzene metabolites, K562 cells showed an increase in reactive oxygen species level, and pretreatment with N‐acetyl‐l ‐cysteine significantly protected against the cytotoxicity of each metabolite. Interestingly, the control K562 cells and the phenol‐exposed cells aggregated together, but the cells exposed to other metabolites were scattered. Further analysis showed that hydroquione, catechol, and 1,2,4‐benzenetriol induced a decrease in the cell surface sialic acid levels and an increase in the cell surface sialidase activity, but phenol did not cause any changes in sialic acid levels and sialidase activity. Consistently, an increase in expression level of sialidase Neu3 mRNA and a decrease in mRNA level of sialyltransferase ST3GAL3 gene were detected in hydroquione‐, catechol‐, or 1,2,4‐benzenetriol‐treated cells, but no change in mRNA levels of two genes were found in phenol‐treated cells. In conclusion, these benzene metabolites could induce apoptosis of K562 cells mainly through caspase‐8‐dependent pathway and ROS production, and sialic acid metabolism might play a role in the apoptotic process. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1437–1451, 2014.     

Toxic effects of benzene and benzene metabolites on granulopoietic stem cells and bone marrow cellularity in mice

Mice were injected subcutaneously with benzene or one of three of its metabolites (phenol, hydroquinone, or 1,2-dihydro-1,2-dihydroxybenzene). The adverse effects on the concentration of granulopoietic stem cells (measured as number of colony-forming units per tibia or per 10⁵ cells) and on the bone marrow cellularity in tibia were measured. Benzene had strong toxic effects. Thus, 0.7 mg benzene/kg body wt injected daily on 6 consecutive days gave detectable effects on stem cell concentration, and 3.5 mg/kg/day affected also cellularity. Six daily injections of 440 mg benzene/kg reduced cellularity and number of colony-forming units per tibia by 86–95%. None of the benzene metabolites tested could reproduce the strong effects of benzene when injected subcutaneously, although phenol slightly but significantly affected stem cell concentration. Toluene, a competitive inhibitor of benzene metabolism, significantly alleviated the effects of benzene. Regeneration of the bone marrow after benzene injections occurred rapidly during the first week, then at a slower rate for the next 4 weeks. At this time cellularity and granulopoietic stem cell concentration were restored, but the fraction of stem cells in S phase was still higher than in controls, indicating a still elevated proliferation rate.     

Alterations in the morphology and functional activity of bone marrow phagocytes following benzene treatment of mice.

Benzene is a well-established hematotoxin that affects developing leukocytes and erythrocytes as well as bone marrow stromal cells. In the present studies we analyzed the effects of benzene on the morphology and functional activity of bone marrow phagocytes. Male Balb/c mice were treated with benzene (660 mg/kg) once per day for 3 days. Bone marrow cells were then isolated and fractionated by density gradient centrifugation. Using highly sensitive techniques in flow cytometry/cell sorting, we found that we could separate three distinct populations of bone marrow cells that differed with respect to size and density. Monoclonal antibody binding and cell sorting revealed a large, dense population that consisted predominantly of granulocytes, a smaller, less dense population of lymphocytes, and a population of intermediate size and density consisting of mononuclear phagocytes and precursor cells. Differential staining of sorted mononuclear phagocytes revealed that benzene treatment of mice caused a marked increase in the number of mature, morphologically activated macrophages in the bone marrow. Benzene treatment of mice also resulted in enhanced chemotaxis and production of hydrogen peroxide by bone marrow granulocytes and mononuclear phagocytes. In contrast, treatment of mice with the combination of hydroquinone and phenol (50 mg/kg each, 1 x/day, 3 days), two metabolites of benzene, resulted in a significant (p < or = 0.02) depression of granulocyte chemotaxis and had no effect on hydrogen peroxide production by bone marrow phagocytes compared to cells from control animals. Taken together these results demonstrate that benzene causes increased differentiation and/or activation of phagocytes in the bone marrow.     

Prevention of benzene-induced myelotoxicity and prostaglandin synthesis in bone marrow of mice by inhibitors of prostaglandin H synthase

Administration of benzene to mice causes bone marrow toxicity and elevations in prostaglandin E2 (PGE2), a negative regulator of myelopoiesis. In these experiments, benzene (400 mg/kg; 2 x/day for 2 days) administered to DBA/2 or C57Bl/6 mice decreased bone marrow cellularity and myeloid progenitor cell development (measured as colony-forming units per femur) by 40%. When inhibitors of the cyclooxygenase component of prostaglandin H synthase (PHS) (either indomethacin, 2 mg/kg; aspirin, 50 mg/kg; meclofenamate, 4 mg/kg) were coadministered with benzene, myelotoxicity and the elevation in bone marrow PGE level were prevented. Additionally, when indomethacin (1 microM) was added to cultures of bone marrow cells from benzene-treated mice, myeloid progenitor cell development was the same as the controls. The doses of indomethacin used had no affect on the hepatic conversion of benzene to its major metabolite, phenol. Using purified PHS, indomethacin (10 microM) inhibited the arachidonic acid-dependent oxidation of hydroquinone to p-benzoquinone, a putative reactive metabolite of benzene. Indomethacin (10 microM) had no effect on the H2O2-driven oxidation of hydroquinone catalysed by either PHS-peroxidase or myeloperoxidase. Coadministration of the benzene metabolites, phenol and hydroquinone, has been reported previously to reproduce the myelotoxicity of benzene. In our studies, phenol and hydroquinone (50 mg/kg each; 2 x/day for 2 days) decreased bone marrow cellularity by 40%; however, coadministration of indomethacin (2 mg/kg) or meclofenamate (4 mg/kg) with these metabolites did not prevent the decrease in bone marrow cell number. Our results implicate marrow PHS in mediating the short-term myelotoxicity of benzene.     

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.     

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.     

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.     

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.     

Gender- and Age-Specific Cytotoxic Susceptibility to Benzene Metabolites in Vitro

Benzene, a widely used compound, is a known carcinogen and hematopoietictoxicant. Several studies have shown gender and age differencesin the responses to benzene-induced hematotoxic-ity. It is notknown if these differences in response are due to age-or gender-associatedmetabolic differences or to age- or gender-associated differencesin the susceptibilities of the target cells. In order to addressthis issue, mouse colony-forming units-erythroid (CFU-e, anerythroid precursor cell particularly susceptible to benzenetoxicity) were cultured in the presence of either individualbenzene metabolites or binary mixtures of these metabolites.CFU-e were obtained from unexposed age-matched adult male andfemale (both virgin and pregnant) Swiss Webster (SW) mice andfrom SW male and female 16-day fetuses. The metabolites usedwere phenol, hydroquinone, catechol, benzoquinone, and trans,trans-muconic acid. The concentrations of the individual metabolitesused were 10, 20, and 40 µM. Binary mixtures of metaboliteswere prepared using the lowest concentrations of the individualmetabolites that caused cytotoxicity. These concentrations were10 µM for hydroquinone, catechol, and benzoquinone, and40 µM for phenol and muconic acid. In general, the CFU-efrom adult females (both virgin and pregnant) were more resistantto the toxic effects of the individual metabolites than CFU-efrom other subjects. CFU-e from adult males were more susceptibleto the cytotoxic effects of hydroquinone and benzoquinone thanCFU-e from other subjects and CFU-e from both male and femalefetuses were highly sensitive to the toxic effects of catechol.On the other hand, CFU-e from adult males were less susceptibleto the cytotoxic effects of catechol than CFU-e from other subjects.Similar results were observed with binary mixtures of metabolites.CFU-e from adult males were more susceptible to the binary mixturesthan CFU-e from virgin females and CFU-e from fetal males weremore susceptible than CFU-e from fetal females. In addition,CFU-e from fetuses were more resistant than CFU-e from adultsto the cytotoxic effects of those binary mixtures that did notcontain catechol. In contrast, binary mixtures containing catecholwere more toxic to fetal cells than to adult cells. These resultssuggest that differences in benzene hematotoxicity associatedwith gender and age may be due, at least in part, to intrinsicfactors at the level of the target cell rather than solely toage- or gender-related differences in the metabolism of benzene.     

Species comparison of hepatic and pulmonary metabolism of benzene

Benzene is an occupational hazard and environmental toxicant found in cigarette smoke, gasoline, and the chemical industry. The major health concern associated with benzene exposure is leukemia. Studies using microsomal preparations from human, mouse, rabbit, and rat to determine species differences in the metabolism of benzene to phenol, hydroquinone and catechol, indicate that the rat is most similar, both quantitatively and qualitatively, to the human in pulmonary microsomal metabolism of benzene. With hepatic microsomes, rat is most similar to human in metabolite formation at the two lower concentrations examined (24 and 200 microM), while at the two higher concentrations (700 and 1000 microM) mouse is most similar in phenol formation. In all species, the enzyme system responsible for benzene metabolism approached saturation in hepatic microsomes but not in pulmonary microsomes. In pulmonary microsomes from mouse, rat, and human, phenol appeared to competitively inhibit benzene metabolism resulting in a greater proportion of phenol being converted to hydroquinone when the benzene concentration increased. The opposite effect was seen in hepatic microsomes. These findings support the hypothesis that the lung plays an important role in benzene metabolism, and therefore, toxicity.