T cell-dependent immune reactions to reactive benzene metabolites in mice

Using the popliteal lymph node (PLN) assay in mice, we studied the sensitizing potential of benzene and its metabolites. Whereas benzene and phenol failed to induce a PLN reaction, catechol and hydroquinone induced a moderate, and p-benzoquinone a vigorous response. Following a single injection of the reactive metabolite p-benzoquinone (100 nmol/mouse), cellularity in the draining PLN was increased >15-fold, and reverted back to normal only after ∼100 days. Although the PLN response was T cell-dependent, flow cytometric analysis revealed that the increased cellularity in the PLN after a single injection of p-benzoquinone was mainly due to an increase in B cells. Mice primed to p-benzoquinone and challenged with a small dose of p-benzoquinone (0.1 nmol/mouse) mounted a secondary PLN reaction, indicating hapten specificity of the reaction; this was confirmed by results obtained in the adoptive transfer PLN assay. An unexpected finding was the secondary PLN response to benzene (1 nmol/mouse) observed in mice primed to p-benzoquinone. This finding suggests that some of the benzene (at least 10%) was locally converted into p-benzoquinone, which then elicited the secondary response observed. In conclusion, the reactive intermediate metabolites hydroquinone and p-benzoquinone can act as haptens and sensitize; their precursors, benzene and phenol, may be considered as prohaptens. Received: 7 December 1998 / Accepted: 9 February 1999     

Effects of the principal hydroxy-metabolites of benzene on microtubule polymerization

The principal hydroxy-metabolites of benzene — phenol, catechol and hydroquinone — possess characteristics and produce toxicity similar to those reported for certain inhibitors of microtubule polymerization. In this study we examined the effects of phenol, catechol and hydroquinone on purified microtubule polymerization and the decay of tubulin-colchicine binding activity. Hydroquinone, but not catechol or phenol, inhibited microtubule polymerization and accelerated the decay of tubulin-colchicine binding activity. The latter effect was shown to be dependent on the concentration of GTP. Hydroquinone did not directly complex with GTP or ATP but bound to the high molecular weight fraction of tubulin. Concentration ratios of hydroquinone to tubulin resulting in altered activity were low, suggesting a specific interaction, presumably at the tubulin-GTP binding site. The acceleration of tubulin-colchicine binding activity decay was completely prevented under anaerobic conditions, indicative of an oxidative mechanism. These studies suggest that hydroquinone, which auto-oxidizes, may interfere with microtubule function, nucleotide binding or both and that this mechanism may be involved in eliciting the wide range of cytoskeletal-related abnormalities observed in cells exposed to benzene in vivo or its metabolites in vitro.     

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.     

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     

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.     

Metabolism of benzene in human liver microsomes: individual variations in relation to CYP2E1 expression

In human liver microsomes the oxidations of benzene, chlorzoxazone, aniline, dimethylformamide, and 4-nitrophenol were significantly correlated with each other and with the level of cytochrome P450 (CYP) 2E1 estimated by immunoblotting. Moreover, benzene oxidation to water-soluble metabolites was suppressed by 0.1 mM diethyldithiocarbamate, supposedly a specific inhibitor of CYP2E1 at this level. None of these metabolic rates correlated with immunochemically determined levels of CYP1A2, 2C9, and 3A4 nor oxidation of 7-ethoxyresorufin, tolbutamide, and nifedipine. Benzene oxidation to water-soluble metabolites was characterized by typical Michaelis-Menten kinetics. The different benzene K _m values seen in individual human microsomal samples were not correlated with the level or activity of CYP1A2, 2C9, 2E1, and 3A4 but could be due to CYP2E1 microheterogeneity. The lowest K _m for benzene oxidation could be related to C/D and/or c1/c2 polymorphism of CYP2E1 gene. Covalent binding of benzene reactive metabolites to microsomal proteins was also correlated with the CYP2E1 metabolic rates and immunochemical levels. At high concentrations of benzene covalent binding was inversely related to benzene concentrations (as well as to formation of water-soluble metabolites) in agreement with the view that secondary metabolites, mainly benzoquinone, are responsible for the covalent binding. Received: 8 September 1998 / Accepted: 24 November 1998     

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.     

Distribution studies in CD-1 mice administered [14C]muconaldehyde

Trans,trans-muconaldehyde (muconaldehyde, MUC), a microsomal hematotoxic ring-opened metabolite of benzene, has been proposed to play a role in benzene hematotoxicity. In the present study, [¹⁴C]-muconaldehyde was administered to CD-1 mice and the distribution of [¹⁴C]muconaldehyde equivalents was investigated. The study was carried out to evaluate whether [¹⁴C]muconaldehyde equivalents could reach the bone marrow. [¹⁴C]Muconaldehyde at a dose of 2 mg/kg was administered intraperitoneally (3.4 μCi/mouse) and by intravenous injection (2.7 μCi/mouse). The amount of [¹⁴C]muconaldehyde equivalents was measured in the bone marrow, blood, liver, lung, kidney and spleen at 0.25, 0.5, 1, 2, 4, and 24 h after [¹⁴C]MUC administration. The results indicate that 0.044% or 0.018% of the total dose administered when given i.v. or i.p., respectively, reached the bone marrow. The elimination of the radioactivity in all organs had at least two phases. The bone marrow, kidney, and lung had a rapid first phase (t _1/2 0.5–1.2 h) and a slower second phase (t _1/2 2.8–15.7 h). In the liver, a slow first phase (t _1/2 3.7 h) was followed by a more rapid second phase (t _1/2 1.5 h). The level of radioactivity in blood and bone marrow was significantly higher when [¹⁴C]muconaldehyde was administered intravenously compared with intraperitoneally, demonstrating that the route of administration affects the distribution of [¹⁴C]muconaldehyde equivalents. Received: 26 March 1996 / Accepted: 17 June 1997     

HIGHLIGHT: Dose-Dependent Metabolism of Benzene in Hamsters, Rats, and Mice

The disposition of oral doses of [¹⁴C]benzene was investigatedusing a range of doses that included lower levels (0.02 and0.1 mg/kg) than have been studied previously in rat, mouse,and in hamster, a species which has not been previously examinedfor its capacity to metabolize benzene. Saturation of metabolismof benzene was apparent as the dose increased, and a considerablepercentage of the highest doses (100 mg/kg) was exhaled unchanged.Most of the remainder of the radioactivity was excreted as metabolitesin urine, and significant metabolite-specific changes occurredas a function of dose and species. Phenyl sulfate was the predominantmetabolite in rat urine at all dose levels (64–73%) ofurinary radioactivity), followed by prephenylmercapturic acid(10–11%). Phenyl sulfate (24–32%) and hydroquinoneglucuronide (27–29%) were the predominant metabolitesformed by mice. Mice produced considerably more muconic add(15%), which is derived from the toxic metabolite muconaldehyde,than did rats (7%) at a dose of 0.1 mg/kg. Unlike both ratsand mice, hydroquinone glucuronide (24–29%) and muconicacid (19–31%) were the primary urinary metabolites formedby hamsters. Two metabolites not previously detected in theurine of rats or mice after single doses, 1,2,4-trihydroxybenzeneand catechol sulfate, were found in hamster urine. These dataindicate that hamsters metabolize benzene to more highly oxidized,toxic products than do rats or mice.     

Immunochemical detection of cytochrome P450 isozymes induced in rat liver byn-hexane, 2-hexanone and acetonyl acetone

Cytochrome P450 isozymes induced in rat liver by treatment withn-hexane, 2-hexanone and acetonyl acetone (given intraperitoneally 5 mmol/kg for 4 days) were investigated using enzyme assays (benzene, toluene, 7-ethoxyresorufin and 7-pentoxyresorufin metabolism) and monoclonal antibodies (anti-P450IA1/2, anti-P450IIB1/2, anti-P450IIC11/6, anti-P450IIE1(91) and anti-P450IIE1(98)).n-Hexane treatment enhanced the activities of low-K _m benzene aromatic hydroxylase and toluene side-chain oxidase, but not 7-ethoxyresorufin O-deethylase or 7-pentoxyresorufin O-depentylase. 2-Hexanone or acetonyl acetone treatment enhanced the activities of low-and high-K _m benzene aromatic hydroxylases, toluene side-chain oxidase and 7-pentoxyresorufin O-depentylase, but not of 7-ethoxyresorufin O-deethylase. Immunoblot analysis showed that anti-P450IA1/2 did not bind liver microsomal protein from either control and treated rats in the region of cytochrome P450s, whereas with anti-P450IIE1(98) a clear-cut band was seen in liver microsomes from control and treated rats, with intensities in the following order: 2-hexanone=acetonyl acetone n-hexane > control > phenobarbital. With anti-P450IIB1/2, a band was detected in microsomes from phenobarbital-treated rats, and to a lesser extent, in microsomes from 2-hexanone-and acetonyl acetone-treated rats. Like the immunoblot analysis, anti-P450IIE1(91) inhibited toluene side-chain hydroxylase activity in all microsomes, except in preparations from phenobarbital-treated rats and anti-P450IIB1 in microsomes from phenobarbital-, 2-hexanone- and acetonyl acetone-treated rats. Anti-P450IIC11/6 also inhibited toluene side-chain hydroxylase activity: the inhibited activity in the five different microsome preparations was as follows:n-hexane=control > acetonyl acetone=2-hexanone=phenobarbital. These results indicate thatn-hexane induces only quantitative alterations in the constitutive cytochrome P450 isozyme (P450IIE1), whereas its metabolites 2-hexanone and acetonyl acetone induce not only quantitative changes in constitutive cytochrome P450 (P450IIE1 and P450IIC11/6) but also a different type of isozyme (P450IIB1/2).     

Glutathione S-transferase (GST) M1 and GST T1 genotypes and hematopoietic effects of benzene exposure

This study investigated whether or not the genotypes glutathione S-transferase θ (GST T1) and μ (GST M1) correlated with low white blood cell (WBC) count found in benzene exposed workers. We found that individuals with genotypes positive for both GST T1 and GST M1 showed the highest prevalence of low WBC [odds ratio (OR) = 4.67, P = 0.046, 95% confidence interval (CI) = 1.02–24.15] when the benzene exposure was high. Multiple logistic regression showed that benzene exposure (OR = 2.81, P = 0.062, 95% CI = 0.96–8.30) was associated with increased OR on low WBC and interactions between the benzene exposure and the genotype of GST T1 were also observed. These observations suggest that GST T1 and GST M1 may play important roles in the biotransformation of benzene, the effect which leads to its hematotoxicity. Received: 17 September 1998 / Accepted: 11 January 1999     

The benzene metabolite <Emphasis Type="Italic">para</Emphasis>-benzoquinone is genotoxic in human,phorbol-12-acetate-13-myristate induced,peripheral blood mononuclear cells at low concentrations

Benzene is one of the most prominent occupational and environmental pollutants. The substance is a proven human carcinogen that induces hematologic malignancies in humans, probably at even low doses. Yet knowledge of the mechanisms leading to benzene-induced carcinogenesis is still incomplete. Benzene itself is not genotoxic. The generation of carcinogenic metabolites involves the production of oxidized intermediates such as catechol, hydroquinone and para-benzoquinone (p-BQ) in the liver. Further activation to the ultimate carcinogenic intermediates is most probably catalyzed by myeloperoxidase (MPO). Yet the products of the MPO pathway have not been identified. If an oxidized benzene metabolite such as p-BQ was actually the precursor for the ultimate carcinogenic benzene metabolite and further activation proceeds via MPO mediated reactions, it should be possible to activate p-BQ to a genotoxic compound in vitro. We tested this hypothesis with phorbol-12-acetate-13-myristate (PMA) activated peripheral blood cells exposed to p-BQ, using the cytokinesis-block micronucleus test. Addition of 20–28 ng/ml PMA caused a significant increase of micronuclei at low and non-cytotoxic p-BQ concentrations between 0.04 and 0.2 μg/ml (0.37–1.85 μM). Thus with PMA or p-BQ alone no reproducible elevation of micronuclei was seen up to toxic concentrations. PMA and p-BQ induce micronuclei when administered jointly. Our results add further support to the hypothesis that MPO is a key enzyme in the activation of benzene.     

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.     

Interethnic and interindividual variabilities of platelet sulfotransferases activity in Italians and Finns

Objective: The aim of this investigation was to see whether there was interethnic variability in the platelet activities of catechol- and phenol sulfotransferases in Italians and Finns. Methods: The activities of catechol- and phenol sulfotransferases were measured in platelets obtained from 103 Italian and 74 Finnish individuals. Blood donors were obtained from healthy volunteers free from drugs and without apparent disease. The activities of catechol- and phenol sulfotransferases were measured with 60 μM dopamine and 4 μM 4-nitrophenol as substrates, respectively Results: The activity of catechol sulfotransferase was not gender dependent and the median estimates (pmol/min/mg) were 9.10 in Italians and 6.37 in Finns (P = 0.0018). The activity of phenol sulfotransferase activity was gender dependent in Finns but not in Italians. The median estimates (pmol/min/mg) were 3.81 in Finnish men and 1.18 in Finnish women (P = 0.0007). In Italian men and women, the median estimates (pmol/min/mg) of phenol sulfotransferase activity were 1.25 and 1.24, respectively (NS). Conclusion: This study shows that platelet catechol sulfotransferase activity is greater in Italians than Finns and that the activity of phenol sulfotransferase is gender regulated in Finns but not in Italians. Thus, interethnic differences exist in platelet sulfotransferases between Italians and Finns. Received: 16 April 1999 / Accepted in revised form: 20 August 1999     

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.     

Correlation between the induction of micronuclei in bone marrow by benzene exposure and the excretion of metabolites in urine of CD-1 mice

Male and female CD-1 mice received single oral doses of benzene (220, 440, and 880 mg/kg) and were pretreated with modifiers of the mixed-function oxidase enzyme activities. Urinary metabolites (MT) (0-24 and 24-48 hr) were quantified by high-performance liquid chromatography. The micronucleus test was performed at 30 h. The following pretreatments were used to correlate micronucleus formation and the excreted benzene MT: 3-Methylcholanthrene and beta-naphthoflavone led to a marked increase in micronuclei (MN) and MT, whereas phenobarbital caused a slight increase, and SKF-525A had no effect. MN and MT were decreased when benzene was administered by the ip route or toluene was given simultaneously. Females had a lower number of MN and excreted more unconjugated phenol than did males. Muconic acid, hydroquinone, and phenol glucuronide and MN correlated well. They were dependent on both the dose and route of administration of benzene, being most inducible by P-448 inducers, in males more than females. The administration of hydroquinone induced MN, but phenol or catechol (200, 250, and 150 mg/kg, po, respectively) did not, and none of these compounds yielded trans, trans-muconic acid, a benzene MT in urine. This study establishes that benzene myeloclastogenicity is a function of its metabolism and that quantification of urinary metabolites could provide reliable correlates of this effect in vivo.     

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.     

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)     

In utero and in vitro effects of benzene and its metabolites on erythroid differentiation and the role of reactive oxygen species

Benzene is a ubiquitous occupational and environmental toxicant. Exposures to benzene both prenatally and during adulthood are associated with the development of disorders such as aplastic anemia and leukemia. Mechanisms of benzene toxicity are unknown; however, generation of reactive oxygen species (ROS) by benzene metabolites may play a role. Little is known regarding the effects of benzene metabolites on erythropoiesis. Therefore, to determine the effects of in utero exposure to benzene on the growth and differentiation of fetal erythroid progenitor cells (CFU-E), pregnant CD-1 mice were exposed to benzene and CFU-E numbers were assessed in fetal liver (hematopoietic) tissue. In addition, to determine the effect of benzene metabolite-induced ROS generation on erythropoiesis, HD3 chicken erythroblast cells were exposed to benzene, phenol, or hydroquinone followed by stimulation of erythrocyte differentiation. Our results show that in utero exposure to benzene caused significant alterations in female offspring CFU-E numbers. In addition, exposure to hydroquinone, but not benzene or phenol, significantly reduced the percentage of differentiated HD3 cells, which was associated with an increase in ROS. Pretreatment of HD3 cells with polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) prevented hydroquinone-induced inhibition of erythropoiesis, supporting the hypothesis that ROS generation is involved in the development of benzene erythrotoxicity. In conclusion, this study provided evidence that ROS generated as a result of benzene metabolism may significantly alter erythroid differentiation, potentially leading to the development of Blood Disorders.     

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.