Stimulation of endothelial IL-8 (eIL-8) production and apoptosis by phenolic metabolites of benzene in HL-60 cells and human bone marrow endothelial cells

Benzene toxicity is considered to be elicited by its metabolites and phenolic metabolites of benzene are known to induce apoptosis in leukemia cells in culture and in human bone marrow progenitor cells. One potential mechanism of apoptosis induced by benzene metabolites that has not been examined is the production of pro-apoptotic cytokines such as endothelial IL-8 from endothelial cells in bone marrow stroma. In this study, we utilized HL-60 cells which are known to produce the endothelial form of IL-8 (elL-8) and human bone marrow endothelial cells (HBMEC) as model systems. Hydroquinone (HQ), Catechol (Cat) and benzenetriol (BT) all induced eIL-8 production and apoptosis in HL-60 cells. HQ induced a marked 50-70-fold stimulation of eIL-8 levels and HL-60 cells were shown to have the eIL-8 receptor, CXCR I thus enabling an autocrine pathway of apoptosis. However, treatment with recombinant elL-8 failed to induce apoptosis in HL-60 cells as previously reported and antibodies to either IL-8 or CXCRI did not significantly abrogate benzene metabolite-induced apoptosis. HQ and Cat but not BT also induced stimulation of elL-8 production in HBMEC. These data demonstrate that although metabolites of benzene induce marked stimulation of eIL-8, this is unlikely to be responsible for apoptosis induced in HL-60 cells. Our data also demonstrates that phenolic metabolites of benzene stimulate the production of eIL-8 from HBMEC suggesting that higher levels of endothelial-derived cytokines may occur in bone marrow after benzene exposure.     

Stimulation of endothelial IL-8 (eIL-8) production and apoptosis by phenolic metabolites of benzene in HL-60 cells and human bone marrow endothelial cells

Benzene toxicity is considered to be elicited by its metabolites and phenolic metabolites of benzene are known to induce apoptosis in leukemia cells in culture and in human bone marrow progenitor cells. One potential mechanism of apoptosis induced by benzene metabolites that has not been examined is the production of pro-apoptotic cytokines such as endothelial IL-8 from endothelial cells in bone marrow stroma. In this study, we utilized HL-60 cells which are known to produce the endothelial form of IL-8 (eIL-8) and human bone marrow endothelial cells (HBMEC) as model systems. Hydroquinone (HQ), Catechol (Cat) and benzenetriol (BT) all induced eIL-8 production and apoptosis in HL-60 cells. HQ induced a marked 50-70 fold stimulation of eIL-8 levels and HL-60 cells were shown to have the eIL-8 receptor, CXCR1 thus enabling an autocrine pathway of apoptosis. However, treatment with recombinant eIL-8 failed to induce apoptosis in HL-60 cells as previously reported and antibodies to either IL-8 or CXCR1 did not significantly abrogate benzene metabolite-induced apoptosis. HQ and Cat but not BT also induced stimulation of eIL-8 production in HBMEC. These data demonstrate that although metabolites of benzene induce marked stimulation of eIL-8, this is unlikely to be responsible for apoptosis induced in HL-60 cells. Our data also demonstrates that phenolic metabolites of benzene stimulate the production of eIL-8 from HBMEC suggesting that higher levels of endothelial-derived cytokines may occur in bone marrow after benzene exposure.     

Comparison of Toxicity of Benzene Metabolite Hydroquinone in Hematopoietic Stem Cells Derived from Murine Embryonic Yolk Sac and Adult Bone Marrow

Benzene is an occupational toxicant and an environmental pollutant that potentially causes hematotoxicity and leukemia in exposed populations. Epidemiological studies suggest an association between an increased incidence of childhood leukemia and benzene exposure during the early stages of pregnancy. However, experimental evidence supporting the association is lacking at the present time. It is believed that benzene and its metabolites target hematopoietic stem cells (HSCs) to cause toxicity and cancer in the hematopoietic system. In the current study, we compared the effects of hydroquinone (HQ), a major metabolite of benzene in humans and animals, on mouse embryonic yolk sac hematopoietic stem cells (YS-HSCs) and adult bone marrow hematopoietic stem cells (BM-HSCs). YS-HSCs and BM-HSCs were isolated and enriched, and were exposed to HQ at increasing concentrations. HQ reduced the proliferation and the differentiation and colony formation, but increased the apoptosis of both YS-HSCs and BM-HSCs. However, the cytotoxic and apoptotic effects of HQ were more apparent and reduction of colony formation by HQ was more severe in YS-HSCs than in BM-HSCs. Differences in gene expression profiles were observed in HQ-treated YS-HSCs and BM-HSCs. Cyp4f18 was induced by HQ both in YS-HSCs and BM-HSCs, whereas DNA-PKcs was induced in BM-HSCs only. The results revealed differential effects of benzene metabolites on embryonic and adult HSCs. The study established an experimental system for comparison of the hematopoietic toxicity and leukemogenicity of benzene and metabolites during mouse embryonic development and adulthood.     

Micronucleus induction in mice exposed to diazoaminobenzene or its metabolites, benzene and aniline: implications for diazoaminobenzene carcinogenicity

Diazoaminobenzene (DAAB), a manufacturing intermediate metabolized primarily to the known carcinogens benzene and aniline, has been identified as an impurity in a number of dyes and coloring agents that are components of cosmetics, food products, and pharmaceuticals. Several structural analogs of DAAB are carcinogenic as well. DAAB was selected for metabolism and toxicity studies by the National Toxicology Program (NTP) based on the potential for human exposure, positive Salmonella data, and lack of adequate toxicological data. In the toxicology studies in mice, DAAB exhibited properties similar to benzene and aniline. Because both these metabolites induce micronuclei (MN) in rodent bone marrow erythrocytes, DAAB was tested for induction of micronuclei in male B6C3F₁ mice. DAAB was administered twice by corn oil gavage at 24 h intervals, at doses of 25, 50, and 100 mg/kg per day. In addition, comparative micronucleus tests were conducted with benzene, aniline, and a mixture of benzene plus aniline; doses were based on the respective molar equivalents of each metabolite to DAAB. It was hypothesized that any observed increase in micronuclei seen in DAAB-treated mice would be due primarily to the effects of the benzene metabolite, as benzene is a more potent inducer of chromosomal damage than aniline. Results of this study showed that DAAB and benzene were effective inducers of micronuclei, with stronger responses noted for DAAB at higher doses. Positive results were also obtained with the mixture of benzene and aniline, although the magnitude of the response was lower than for DAAB. Aniline gave a weak positive response at doses exceeding its molar equivalent to 100 mg/kg DAAB. Overall, the data indicated that DAAB is a potent inducer of micronuclei in mice, and its activity appears to be closely related to the activity of benzene, one of its primary metabolites. The results are consistent with a prediction of carcinogenicity for DAAB.     

Micronucleus induction in mice exposed to diazoaminobenzene or its metabolites,benzene and aniline: implications for diazoaminobenzene carcinogenicity

Diazoaminobenzene (DAAB), a manufacturing intermediate metabolized primarily to the known carcinogens benzene and aniline, has been identified as an impurity in a number of dyes and coloring agents that are components of cosmetics, food products, and pharmaceuticals. Several structural analogs of DAAB are carcinogenic as well. DAAB was selected for metabolism and toxicity studies by the National Toxicology Program (NTP) based on the potential for human exposure, positive Salmonella data, and lack of adequate toxicological data. In the toxicology studies in mice, DAAB exhibited properties similar to benzene and aniline. Because both these metabolites induce micronuclei (MN) in rodent bone marrow erythrocytes, DAAB was tested for induction of micronuclei in male B6C3F(1) mice. DAAB was administered twice by corn oil gavage at 24 h intervals, at doses of 25, 50, and 100 mg/kg per day. In addition, comparative micronucleus tests were conducted with benzene, aniline, and a mixture of benzene plus aniline; doses were based on the respective molar equivalents of each metabolite to DAAB. It was hypothesized that any observed increase in micronuclei seen in DAAB-treated mice would be due primarily to the effects of the benzene metabolite, as benzene is a more potent inducer of chromosomal damage than aniline. Results of this study showed that DAAB and benzene were effective inducers of micronuclei, with stronger responses noted for DAAB at higher doses. Positive results were also obtained with the mixture of benzene and aniline, although the magnitude of the response was lower than for DAAB. Aniline gave a weak positive response at doses exceeding its molar equivalent to 100 mg/kg DAAB. Overall, the data indicated that DAAB is a potent inducer of micronuclei in mice, and its activity appears to be closely related to the activity of benzene, one of its primary metabolites. The results are consistent with a prediction of carcinogenicity for DAAB.     

微核直径测试作为非整倍体诱发剂的分析手段

以小鼠骨髓红细胞微核直径测试,比较了秋水仙素（COL）与昆明山海棠（THH）、对苯二酚（HQ）在哺乳动物体细胞中的非整倍体诱发效应。丝裂霉素C（MMC）作为多功能染色体断裂剂引入实验,为诱发非整倍体的阴性对照。结果发现,COL组,71%的微核直径（d）大于或等于所在细胞直径（D）的五分之一（d≥D/5）;THH诱发微核中,54%的微核d≥D/5;HQ及MMC组,分别有47%及14%的微核相对直径达此阈值。暗示THH及HQ具有类似COL的某种非整倍体诱发效应。微核直径测试可作为非整倍体诱发剂检测的辅助手段。 The relative diameters of micronucleus induced by colchicines(COL),Triptergium hypoglaucum(Level)Hutch(THH)and hydroquinone(HQ)were compared to evaluate their aneugenic activities in mouse bone marrow erythrocytes.MitomycinC(MMC)was taken as the negative control in the experiment because it is a multifunctional clastogen without aneugenic potential.Diameters of the cytoplasm(D)and the micronucleus(d)of each micronucleated erythrocytes were measured with a micrometer in a microscope.The frequency of relatively large micronuclei(d≥D/5)was found(71%) in COL treated group.In the THH and HQ treated groups,the relatively large micronuclei were 54% and 47%,respectively.Such micronuclei were infrequent(14%)in the MMC treated group.The results implied that THH and HQ may possess some aneugenic potential like COL.     

Benzene and the genotoxicity of its metabolites. I. Transplacental activity in mouse fetuses and in their dams

Benzene and some of its metabolites (hydroquinone, phenol, catechol, 1,2,4-benzenetriol, p-benzoquinone, o,o'-biphenol, p,p'-biphenol) have been tested for their capability to induce micronuclei in bone marrow cells of pregnant mice and, transplacentally, in fetal liver cells. Dams are scarcely sensitive to the genotoxic activity of benzene and its metabolites while the latter are able to produce only evident toxic effects. Benzene and hydroquinone transplacentally induce micronuclei in fetal liver cells while all other metabolites show weak or negative genotoxicity, although they produce severe cellular toxicity.     

Mechanism of benzene toxicity. Effects of benzene and benzene metabolites on bone marrow cellularity,number of granulopoietic stem cells and frequency of micronuclei in mice

The effects of benzene and benzene metabolites (hydroquinone and catechol) on bone marrow cellularity, number of granulopoietic stem cells and on the frequency of micronuclei in polychromatic erythrocytes were investigated in mice. The dose-effect curve for benzene revealed that there was a threshold dose (approx. 100 mg benzene/kg body wt./day injected subcutaneously on 6 consecutive days) above which severe toxicity occurred in all three parameters. Also hydroquinone gave rise to adverse effects in the parameters studied, but the sequence of occurrence was different from that observed with benzene. These data are interpreted to indicate that hydroquinone is a hemotoxic metabolite of benzene in mice in vivo, but that other metabolites, or benzene itself, also probably contribute to the toxicity. Catechol gave no effects. However, due to acute effects like tremor and convulsions only rather low doses could be tested. Simultaneous administration of toluene dramatically reduced the toxicity of benzene, but gave only a small reduction of the hydroquinone-induced effects.     

Potential role of free radicals in benzene-induced myelotoxicity and leukemia.

Occupational exposure to benzene, a major industrial chemical, has been associated with various blood dyscrasias and increased incidence of acute myelogenous leukemia in humans. It is established that benzene requires metabolism to induce its effects. Benzene exposure in humans and animals has also been shown to result in structural and numerical chromosomal aberrations in lymphocytes and bone marrow cells, indicating that benzene is genotoxic. In this review we have attempted to compile the available evidence on the role of increased free radical activity in benzene-induced myelotoxic and leukemogenic effects. Benzene administration to rodents has been associated with increased lipid peroxidation in liver, plasma, and bone marrow, as shown by an increase in the formation of thiobarbituric-acid reactive products that absorb at 535 nm. Benzene administration to rodents also results in increased prostaglandin levels indicating increased arachidonic acid peroxidation. Other evidence includes the fact that bone marrow cells and their microsomal fractions isolated from rodents following benzene-treatment have a higher capacity to form oxygen free radicals. The bone marrow contains several peroxidases, the most prevalent of which is myeloperoxidase. The peroxidatic metabolism of the benzene metabolites, phenol and hydroquinone, results in arachidonic acid peroxidation and oxygen activation to superoxide radicals, respectively. These metabolites, upon co-administration also produce a myelotoxicity similar to that observed with benzene. Recently, we have found that exposure of human promyelocytic leukemia (HL-60) cells (a cell line rich in myeloperoxidase), to the benzene metabolites, hydroquinone and 1,2,4-benzenetriol results in increased steady-state levels of 8-hydroxydeoxyguanosine a marker of oxidative DNA damage. Peroxidatic metabolism of benzene's phenolic metabolites may therefore be responsible for the increased free radical activity and toxicity produced by benzene in bone marrow. We thus hypothesize that free radicals contribute, at least in part, to the toxic and leukemogenic effects of benzene.     

Benzene and the genotoxicity of its metabolites. II. The effect of the route of administration on the micronuclei and bone marrow depression in mouse bone marrow cells

Benzene (880 mg/kg) and 4 of its metabolites, i.e., phenol (265 mg/kg), hydroquinone (80 mg/kg), catechol (40 mg/kg), and p-benzoquinone (5-20 mg/kg) have been tested for their capability to induce micronuclei in bone marrow cells of male mice after oral administration or intraperitoneal injection. Oral administration of benzene shows more activity than intraperitoneal injection, whereas the metabolites show more activity if administered by the latter method. The respective genotoxic strengths of the benzene metabolites are the following: hydroquinone much greater than phenol greater than catechol = p-benzoquinone. This last is active when administered orally.     

Micronucleus test in bone marrow of mice treated with 1-nitropropane, 2-nitropropane and cisplatin

Micronucleus tests were carried out in bone marrow of mice treated with 1-nitropropane, 2-nitropropane and cisplatin. For 1-nitropropane and 2-nitropropane the results were negative. With cisplatin a dose-dependent increase in the number of polychromatic erythrocytes with micronuclei was observed. The lowest positive dose was 0.1 mg/kg (P less than 0.001, Mann-Whitney-Wilcoxon test). The hepatocarcinogen 2-nitropropane showed clastogenic activity in human lymphocytes in vitro in the presence of S9 (Bauchinger et al., 1987). The negative results in bone marrow suggest that short-lived genotoxic metabolites may be formed in the liver but do not reach the bone marrow.     

Simplified mouse peripheral reticulocyte micronucleus test with dimethylnitrosamine.

The induction of micronuclei by treatment with dimethylnitrosamine was evaluated and compared in peripheral blood and bone marrow cells of male CD-1 mice. Peripheral blood preparations were made on acridine orange (AO)-coated slides and scanned by fluorescence microscopy. A significant increase in micronuclei was observed 24 h after treatment in bone marrow polychromatic erythrocytes, and 24-48 h after treatment in peripheral reticulocytes. The peak frequency of micronuclei in peripheral reticulocytes was delayed by about 24 h relative to bone marrow polychromatic erythrocytes. This micronucleus test using peripheral blood was shown to be easy to do and as sensitive as the test using bone marrow cells. From this result, it is concluded that the method with AO-coated slides and peripheral blood is as suitable as bone marrow cells for the micronucleus assay.     

Interaction of Mesna (2-mercaptoethane sulfonate) with the mutagenicity of cyclophosphamide in vitro and in vivo

The effects of sodium 2-mercaptoethane sulfonate (Mesna) on the mutagenicity of cyclophosphamide (CP) were assessed in vitro by the Ames test and in vivo in rats by analyzing micronuclei in bone marrow and mutagenic activity in urine. Mesna alone was negative in all test systems, while CP gave a positive response in all of them. In a combined treatment there was no significant reduction of the CP-induced mutagenicity in Salmonella. In rats the frequency of bone marrow micronuclei was not diminished when Mesna was given together with CP. May-Grunwald-Giemsa staining and Hoechst-Pyronin fluorescent staining techniques for micronuclei yielded similar results. The urine of rats treated with CP was mutagenic to Salmonella and no significant difference was observed when the rats had received both Mesna and CP. The results give support to the theory that Mesna acts primarily by reducing the toxicity of metabolites of CP, particularly acrolein, in the urinary tract and not by suppressing the mutagenicity of the active metabolites of CP.     

Chromosome breakage is primarily responsible for the micronuclei induced by 1,4-dioxane in the bone marrow and liver of young CD-1 mice

1,4-Dioxane, a widely used industrial chemical and rodent hepatocarcinogen, has produced mixed, largely negative results in the mouse erythrocyte micronucleus assay. In contrast, a recent report has indicated that 1,4-dioxane induces micronuclei in mouse hepatocytes following in vivo treatment. The objective of this study was to confirm these earlier results and identify the origin of the induced micronuclei. Following an initial range-finding study, mice were administered 1,4-dioxane by gavage at doses ranging from 1500 to 3500 mg/kg. The test animals were also implanted with BrdU-releasing osmotic pumps to allow cell proliferation to be measured in the liver and to increase the sensitivity of the hepatocyte assay. Upon sacrifice, the frequency of micronuclei in the bone marrow erythrocytes and in the proliferating BrdU-labeled hepatocytes was determined. Significant dose-related increases in micronuclei were seen in both the liver and the bone-marrow with significant increases being detected at all the tested doses in the bone marrow and at the 2500 and 3500 mg/kg doses in the liver. Using CREST staining or pancentromeric FISH to determine the origin of the induced micronuclei, it was determined that 80-90% of the micronuclei in both tissues originated from chromosomal breakage. Small increases in centromere-containing micronuclei were also seen in the hepatocytes. Decreases in hepatocyte proliferation as well as in the ratio of bone marrow PCE:NCE were also observed. Based on these results, we conclude that at high doses: (i) dioxane exerts genotoxic effects in both the mouse bone marrow and liver; (ii) the induced micronuclei are formed primarily from chromosomal breakage; and (iii) dioxane can interfere with cell proliferation in both the liver and bone marrow.     

Genotoxicity of intermittent co-exposure to benzene and toluene in male CD-1 mice

Benzene is an important industrial chemical. At certain levels, benzene has been found to produce aplastic anemia, pancytopenia, myeloblastic anemia and genotoxic effects in humans. Metabolism by cytochrome P450 monooxygenases and myeloperoxidase to hydroquinone, phenol, and other metabolites contributes to benzene toxicity. Other xenobiotic substrates for cytochrome P450 can alter benzene metabolism. At high concentrations, toluene has been shown to inhibit benzene metabolism and benzene-induced toxicities. The present study investigated the genotoxicity of exposure to benzene and toluene at lower and intermittent co-exposures. Mice were exposed via whole-body inhalation for 6h/day for 8 days (over a 15-day time period) to air, 50 ppm benzene, 100 ppm toluene, 50 ppm benzene and 50 ppm toluene, or 50 ppm benzene and 100 ppm toluene. Mice exposed to 50 ppm benzene exhibited an increased frequency (2.4-fold) of micronucleated polychromatic erythrocytes (PCE) and increased levels of urinary metabolites (t,t-muconic acid, hydroquinone, and s-phenylmercapturic acid) vs. air-exposed controls. Benzene co-exposure with 100 ppm toluene resulted in similar urinary metabolite levels but a 3.7-fold increase in frequency of micronucleated PCE. Benzene co-exposure with 50 ppm toluene resulted in a similar elevation of micronuclei frequency as with 100 ppm toluene which did not differ significantly from 50 ppm benzene exposure alone. Both co-exposures - 50 ppm benzene with 50 or 100 ppm toluene - resulted in significantly elevated CYP2E1 activities that did not occur following benzene or toluene exposure alone. Whole blood glutathione (GSH) levels were similarly decreased following exposure to 50 ppm benzene and/or 100 ppm toluene, while co-exposure to 50 ppm benzene and 100 ppm toluene significantly decreased GSSG levels and increased the GSH/GSSG ratio. The higher frequency of micronucleated PCE following benzene and toluene co-exposure when compared with mice exposed to benzene or toluene alone suggests that, at the doses used in this study, toluene can enhance benzene-induced clastogenic or aneugenic bone marrow injury. These findings exemplify the importance of studying the effects of binary chemical interactions in animals exposed to lower exposure concentrations of benzene and toluene on benzene metabolism and clastogenicity. The relevance of these data on interactions for humans exposed at low benzene concentrations can be best assessed only when the mechanism of interaction is understood at a quantitative level and incorporated within a biologically based modeling framework.     

Benzene induces haematotoxicity by promoting deacetylation and autophagy

Chronic exposure to benzene is known to be associated with haematotoxicity and the development of aplastic anaemia and leukaemia. However, the mechanism underlying benzene‐induced haematotoxicity, especially at low concentrations of chronic benzene exposure has not been well‐elucidated. Here, we found that increased autophagy and decreased acetylation occurred in bone marrow mononuclear cells (BMMNCs) isolated from patients with chronic benzene exposure. We further showed in vitro that benzene metabolite, hydroquinone (HQ) could directly induce autophagy without apoptosis in BMMNCs and CD34⁺ cells. This was mediated by reduction in acetylation of autophagy components through inhibiting the activity of acetyltransferase, p300. Furthermore, elevation of p300 expression by Momordica Antiviral Protein 30 Kd (MAP30) or chloroquine reduced HQ‐induced autophagy. We further demonstrated that in vivo, MAP30 and chloroquine reversed benzene‐induced autophagy and haematotoxicity in a mouse model. Taken together, these findings highlight increased autophagy as a novel mechanism for benzene‐induced haematotoxicity and provide potential strategies to reverse this process for therapeutic benefits.     

Dysregulation of apoptosis by benzene metabolites and their relationships with carcinogenesis

Benzene is a widely recognized human carcinogen, the effect of which is attributed to the production of reactive oxygen species (ROS) from its metabolites. Although there have been many reports on the relationship between DNA damage induced by benzene metabolites and carcinogenesis, only a report approached the subject by examining the benzene-induced dysregulation of apoptosis. Inhibition of apoptosis, aberrantly prolonging cell survival, may contribute to cancer by facilitating the insurgence of mutations and by creating a permissive environment for genetic instability. In this study, we examined the mechanism of antiapoptotic effects by benzene metabolites, p-benzoquinone (BQ) and hydroquinone (HQ), and their relationships with carcinogenesis. BQ and HQ inhibited the apoptotic death of NIH3T3 cells induced by both serum starvation and lack of an extracellular matrix (ECM). An antioxidant agent, N-acetylcysteine, significantly inhibited the antiapoptotic effects induced by benzene metabolites, indicating that the effects were mainly due to the production of ROS. Furthermore, BQ and HQ inhibited the in vitro caspase-3 activation, suggesting that the inhibition of caspase-3 activation due to ROS produced by BQ- and HQ-treatment was related to the suppression of apoptosis. The cells that escaped apoptosis could survive with the addition of serum and attachment to the ECM. Levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine were higher in the cells which survived after BQ- and HQ-treatment than in the normal cells. Furthermore, the cells treated with BQ and HQ showed greater proliferation than normal cells under low-serum conditions and anchorage-independent growth in soft agar. These findings suggested that benzene metabolites induced dysregulation of apoptosis due to caspase-3 inhibition, which contributes to carcinogenesis.     

Genotoxic potential of the organochlorine insecticide lindane (gamma-BHC): an in vivo study in chicks.

The purpose of this work was to evaluate the genotoxic potential of lindane (gamma-isomer of benzene hexachloride (BHC)) in chicken in vivo tests: the bone marrow chromosome aberration and micronucleus tests. With the highest dose (100 mg/kg) a significant enhancement of chromosome aberrations was noticed after 24 and 48 h and with the second highest dose (75 mg/kg) after 24 h. A significant increase in the incidence of micronuclei in bone marrow cells was induced by all three doses (100, 75 and 50 mg/kg) given either intraperitoneally or orally while in peripheral erythrocytes only the two higher intraperitoneal doses (100 and 75 mg/kg) gave significant increases. On the basis of these results, lindane may be considered genotoxic in this test system and it is suggested that the chick in vivo system may be used as an alternative to a mammalian system for screening environmental chemicals for genotoxicity.     

Investigation of benzene oxide in bone marrow and other tissues of F344 rats following metabolism of benzene in vitro and in vivo.

This study examines the initial activation of benzene, exploring key aspects of its metabolism by measurement of benzene oxide (BO) and BO-protein adducts in vitro and in vivo. To assess the potential influence of various factors on the production of BO, microsomes were prepared from tissues that were either targets of benzene toxicity, i.e. the bone marrow and Zymbal glands, or not targets, i.e. liver and kidneys, of control and acetone-treated F344 rats. No BO or phenol was detected in microsomal preparations of bone marrow or Zymbal glands (less than 0.007 nmol BO/mg protein and 0.7 nmol phenol/mg protein). On the other hand, BO and phenol were readily detected in preparations of liver and kidney microsomes and acetone pretreatment resulted in a 2-fold (kidney) increase or 3.7-fold (liver) increase in production of these metabolites. Initial rates of BO production in the liver isolates were 30 (control) to 50 (acetone-treated) times higher than in the corresponding kidney tissues. The estimated half-life of BO in bone marrow homogenates was 6.0 min and the second-order reaction rate constant was estimated to be 1.35 x 10(-3) l (g bone marrow)(-1) (h)(-1). These kinetic constants were used with measurements of BO-bone marrow adducts in F344 rats, receiving a single gavage dosage of 50-400 mg benzene (kg body weight)(-1) (McDonald, T.M., et al. (1994), Cancer Res. 54, 4907-4914), to predict the bone marrow dose of BO. Among the rats receiving 400 mg (kg body weight) (-1), a BO dose of 1.13 x 10(3) nM BO-h was estimated for the bone marrow, or roughly 40% of the corresponding blood dose predicted from BO-albumin adducts. Together these data suggest that, although BO is not produced at detectable levels in the bone marrow or Zymbal glands of F344 rats, BO is rapidly distributed via the bloodstream to these tissues where it may play a role in toxicity.