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.     

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     

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.     

An interaction of benzene metabolites reproduces the myelotoxicity observed with benzene exposure

Benzene-induced myelotoxicity can be reproduced by the coadministration of two principal metabolites, phenol and hydroquinone. Coadministration of phenol (75 mg/kg) and hydroquinone (25-75 mg/kg) twice daily to B6C3F1 mice for 12 days resulted in a significant loss in bone marrow cellularity in a manner exhibiting a dose-response. One explanation for this potentiation is that phenol stimulates the peroxidase-dependent metabolism of hydroquinone. Addition of phenol to incubations containing horseradish peroxidase, H2O2, and hydroquinone resulted in a stimulation of both hydroquinone removal and benzoquinone formation. Stimulation occurred with phenol as low as 100 microM and with very low concentrations of horseradish peroxidase. When boiled rat liver protein was added to identical incubations containing [14C]hydroquinone, the level of radioactivity recovered as protein bound increased by 37% when phenol was added. Similar results were observed when [14C]hydroquinone was incubated in the presence of activated human leukocytes. Hydroquinone binding was increased by approximately 70% in the presence of phenol. Phenol-induced stimulation of hydroquinone metabolism and benzoquinone formation represents a likely explanation for the bone marrow suppression associated with benzene toxicity.     

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.     

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.     

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.     

Influence of strain differences in mice on the metabolism and toxicity of benzene

Chronic benzene exposure results in a progressive depression of bone marrow function and is thought to be caused by a metabolite of benzene (Snyder and Kocsis, 1975; Goldstein and Laskin, 1977). Several reports concerning differences in xenobiotic metabolism and toxicity among inbred strains of mice prompted us to study benzene metabolism and toxicity in C57BL/6 and DBA/2 mice. DBA/2 mice were more susceptible to benzene than C57BL/6 mice. No differences in the total amount of urinary benzene metabolites produced were found between the strains; however, differences in the relative amounts of specific metabolites were noted. DBA/2 mice produced more phenylglucuronide but less ethereal sulfate conjugates than C57BL/6 mice. Hydrolysis of the urinary conjugates revealed that DBA/2 mice excreted more phenol, but less hydroquinone than C57BL/6 mice. Multiple dose studies revealed that the more resistant C57BL/6 mice contained less water soluble benzene metabolites in bone marrow, liver, kidney, blood, spleen, and lung than DBA/2 mice. C57BL/6 mice also contained less covalently bound metabolites in bone marrow, blood, spleen, and muscle than DBA/2 mice following multiple doses of benzene. V_max values for UDPGA utilization in C57BL/6 mice were almost six times the V_max values observed for DBA/2 mice. Furthermore, V_max values for phenylsulfate formation in C57BL/6 mice were three times the V_max values for DBA/2 mice. It was concluded that the difference in susceptibility to benzene between C57BL/6 and DBA/2 mice was not the result of a single factor, buth rather, the sum total of a number of metabolic events.     

Benzene disposition in the rat after exposure by inhalation.

Little information is available on benzene disposition after exposure by inhalation despite the importance of this route in man. Benzene metabolites as a group have been measured in bone marrow, but quantitation of individual metabolites in this target tissue has not been reported. Male Fischer-344 rats were exposed to 500 ppm benzene in air and the uptake and elimination was followed in several tissues. Concentrations of free phenol, catechol, and hydroquinone in blood and bone marrow were also measured. Steady-state concentrations of benzene (11.5, 37.0, and 164.0 μg/g in blood, bone marrow, and fat, respectively) were achieved within 6 hr in all tissues studied. Benzene half-lives during the first 9 hr were similar in all tissues (0.8 hr). A plot of amount of benzene remaining to be excreted in the expired air was biphasic with $\text{t}\text{1}\text{2}$ values for the α and β phases of 0.7 and 13.1 hr, respectively. Phenol was the main metabolite in bone marrow at early times (peak concentration, 19.4 μg/g). Catechol and hydroquinone predominated later (peak concentrations, 13.0 and 70.4 μg/g, respectively). Concentrations of these two metabolites declined very slowly during the first 9 hr. These data indicate that free catechol and hydroquinone persist in bone marrow longer than benzene or free phenol.     

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.     

Identification of N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine as a urinary metabolite of benzene, phenol, and hydroquinone

The metabolite 2-(S-glutathionyl)hydroquinone is formed when a microsomal incubation mixture containing either benzene or phenol is supplemented with glutathione. This metabolite is derived from the conjugation of benzoquinone, an oxidation product of hydroquinone. However, neither the glutathione conjugate or its mercapturate, N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine, have been identified as metabolites resulting from in vivo metabolism of benzene, phenol, or hydroquinone. To determine if a hydroxylated mercapturate is produced in vivo, we treated male Sprague-Dawley rats with either benzene (600 mg/kg), phenol (75 mg/kg), or hydroquinone (75 mg/kg) and collected the urine for 24 hr. HPLC coupled with electrochemical detection confirmed the presence of a metabolite that was chromatographically and electrochemically identical to N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine. The metabolite was isolated from the urine samples and treated with diazomethane to form the N-acetyl-S-(2,5-dimethoxyphenyl)-L-cysteine methyl ester derivative. The mass spectra obtained from these samples were identical to that of an authentic sample of the derivative. The results of these experiments indicate that benzene, phenol, and hydroquinone are metabolized in vivo to benzoquinone and excreted as the mercapturate, N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine.     

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.     

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)     

Identification of benzene metabolites in dermal fibroblasts as nonphenolic: regulation of cell viability, apoptosis, lipid peroxidation and expression of matrix metalloproteinase 1 and elastin by benzene metabolites

The skin is exposed to benzene and its derivatives, prevalent environmental chemicals. They may impair the structural integrity of the skin by increased expression of matrix metalloproteinase 1 (MMP-1; degrades structural collagen) and elastin, synthesized primarily by the dermal fibroblasts. We examined the metabolism of benzene in dermal fibroblasts and identified the benzene metabolites as toluene, benzaldehyde, aniline and benzoic acid. These metabolites were not toxic to the cells with regard to cell viability, apoptosis and lipid peroxidation, unlike the phenolic benzene metabolites (hydroquinone, t-butyl hydroquinone and phenol) or hydrogen peroxide. Toluene and phenol, which compose cigarette smoke, and benzaldehyde stimulated MMP-1 and/or elastin expression. In summary, the dermal fibroblasts metabolize benzene to nonphenolic metabolites that are less toxic to the cellular components than the phenolic benzene derivatives. Toluene, benzaldehyde and phenol can directly cause facial wrinkling and impaired structural integrity by upregulating MMP-1 and/or elastin.     

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.     

Modulation of mast cell and basophil functions by benzene metabolites

Benzene is a carcinogenic compound used in industrial manufacturing and a common environmental pollutant mostly derived from vehicle emissions and cigarette smoke. Benzene exposure is associated with a variety of clinical conditions ranging from hematologic diseases to chronic lung disorders. Beside its direct toxicity, benzene exerts multiple effects after being converted to reactive metabolites such as hydroquinone and benzoquinone. Mast cells and basophils are primary effector cells involved in the development of respiratory allergies such as rhinitis and bronchial asthma and they play an important role in innate immunity. Benzene and its metabolites can influence mast cell and basophil responses either directly or by interfering with other cells, such as T cells, macrophages and monocytes, which are functionally connected to mast cells and basophils. Hydroquinone and benzoquinone inhibit the release of preformed mediators, leukotriene synthesis and cytokine production in human basophils stimulated by IgE- and non IgE-mediated agonists. Furthermore, these metabolites reduce IgE-mediated degranulation of mast cells and the development of allergic lung inflammation in rats. Both in vitro and in vivo studies indicate that benzene metabolites alter biochemical and functional activities of other immunocompetent cells and may impair immune responses in the lung. These inhibitory effects of benzene metabolites are primarily mediated by interference with early transduction signals such as PI3 kinase. Together, currently available studies indicate that benzene metabolites interfere by multiple mechanisms with the role of basophils and mast cells in innate immunity and in chronic inflammation in the lung.     

CYP2E1-dependent benzene toxicity: the role of extrahepatic benzene metabolism

Benzene, a ubiquitous environmental pollutant, is haematotoxic and myelotoxic. As has been shown earlier, cytochrome P450 2E1 (CYP2E1)-dependent metabolism is a prerequisite for the cytotoxic and genotoxic effects of benzene, but which of the benzene metabolites produces toxicity is still unknown. The observed differences between the toxicity of benzene and that of phenol, a major metabolite of benzene, could be explained by alternative hypotheses. That is, whether (1) toxic benzene effects are caused by metabolites not derived from phenol (e.g. benzene epoxide, muconaldehyde), which are formed in the liver and are able to reach the target organ(s); or (2) benzene penetrates into the bone marrow, where local metabolism takes place, whereas phenol does not reach the target tissue because of its polarity. To further investigate hypothesis 2, we used various strains of mice (AKR, B6C3F1, CBA/Ca, CD-1 and C57Bl/6), for which different toxic responses have been reported in the haematopoietic system after chronic benzene exposure. In these strains, CYP2E1 expression in bone marrow was investigated and compared with CYP2E1 expression in liver by means of two independent methods. Quantification of CYP2E1-dependent hydroxylation of chlorzoxazone (CLX) by high-performance liquid chromatography (HPLC; functional analysis) was used to characterize specific enzymatic activities. Protein identification was performed by Western blotting using CYP2E1-specific antibodies. In liver microsomes of all strains investigated, considerable amounts of CYP2E1-specific protein and correspondingly high CYP2E1 hydroxylase activities could be detected. No significant differences in CYP2E1-dependent enzyme activities were found between the five strains (range of medians, 4.6–12.0 nmol 6-OH-CLX/[mg protein × min]) in hepatic tissue. In the bone marrow, CYP2E1 could also be detected in all strains investigated. However, chlorzoxazone hydroxylase activities were considerably lower (range of medians, 0.2–0.8 × 10⁻³ nmol 6-OH-CLX/[mg protein × min]) compared with those obtained from liver microsomes. No significant (P > 0.05) interstrain differences in CYP2E1 expression in liver and/or bone marrow could be observed in the mouse strains investigated. The data obtained thus far from our investigations suggest that strain-specific differences in the tumour response of the haematopoietic system of mice chronically exposed to benzene cannot be explained by differences in either hepatic or in myeloid CYP2E1-dependent metabolism of benzene. Received: 7 September 1998 / Accepted: 13 April 1999     

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.     

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.     

Suppression of bone marrow stromal cell function by benzene and hydroquinone is ameliorated by indomethacin

Administration of benzene to mice will inhibit bone marrow stromal cell-supported hemopoiesis in culture. Hydroquinone, a major metabolite of benzene, will cause a similar inhibition of stromal cell function in vitro. Stromal cells produce both an inducer (colony-stimulating factor) and an inhibitor (prostaglandin E2; PGE2) of hemopoiesis. This research was conducted to determine if prostaglandin synthesis is involved in the suppression of stromal cell function by benzene and hydroquinone. Male B6C3F1 mice were administered benzene (100 mg/kg), indomethacin (1 mg/kg), or benzene plus indomethacin twice a day for 4 consecutive days. On Day 5 bone marrow cells were removed to determine the effect of treatment. In a second series of experiments mouse bone marrow stromal cells in culture were treated with hydroquinone (10(-7) to 10(-4) M), indomethacin (10(-6) M), or a combination of hydroquinone plus indomethacin. Stromal cell function was based on the ability of the treated stromal cells to support granulocyte/monocyte colony development in coculture. The results demonstrated that preadministration of indomethacin in vivo ameliorated benzene-induced inhibition of bone marrow stromal cell function. In vitro, indomethacin ameliorated hydroquinone toxicity to stromal cell function. Benzene administration in vivo induced elevated PGE2 in bone marrow samples which were prevented by preadministration of indomethacin. However, hydroquinone in vitro did not induce a consistent increase in PGE2 levels. These results suggested that toxicity to stromal cells was not due solely to increased prostaglandin synthetase activity.