Human DNA damage induced by 1,2,4-benzenetriol, a benzene metabolite

Reactivities of benzene metabolites (phenol, catechol, hydroquinone, 1,4-benzoquinone, 1,2,4-benzenetriol) and related polyphenols (resorcinol, pyrogallol, phloroglucinol) with DNA were investigated by a DNA sequencing technique using 32P 5'-end-labeled DNA fragments obtained from human c-Ha-ras-1 protooncogene, and the reaction mechanism was studied by UV-visible and electron-spin resonance spectroscopies. 1,2,4-Benzenetriol caused strong DNA damage even without alkali treatment. Alkali-labile sites induced by 1,2,4-benzenetriol were base residues of guanine and adjacent thymine. Catalase, superoxide dismutase and methional inhibited the DNA damage completely, but sodium formate did not inhibit it. 1,2,4-Benzenetriol-induced DNA damage was inhibited by the addition of a Cu(I)-specific chelating agent, bathocuproine, and was accelerated by the addition of Cu(II). The addition of Fe(III) did not create any significant effects on 1,2,4-benzenetriol-induced DNA damage. Electron-spin resonance studies using spin traps demonstrated that addition of Fe(III) increased hydroxyl radical production during the autoxidation of 1,2,4-benzenetriol, whereas the addition of Cu(II) did not. The results suggest that DNA damage was caused by an unidentified active species which was produced by the autoxidation of 1,2,4-benzenetriol in the presence of Cu(II), rather than by hydroxyl radicals. The possibility that 1,2,4-benzenetriol-induced DNA damage is one of the primary reactions in carcinogenesis induced by benzene is discussed.     

Characterization of micronuclei induced in human lymphocytes by benzene metabolites

Benzene is an established human leukemogen. Workers occupationally exposed to benzene exhibit increased frequencies of both structural and numerical chromosomal aberrations in their peripheral blood lymphocytes. The metabolite(s) responsible for these chromosomal aberrations has not yet been identified. Using a modified micronucleus assay, we have examined the ability of the metabolites of benzene to induce chromosomal damage in human lymphocytes. An antikinetochore antibody was used to distinguish micronuclei that have a high probability of containing a whole chromosome (kinetochore positive) from those containing acentric fragments (kinetochore negative). In vitro treatments with the benzene metabolites hydroquinone, 1,4-benzoquinone, phenol, and catechol resulted in significant increases in micronuclei formation. Phenol, catechol, and 1,4-benzoquinone treatments resulted in moderate (2- to 5-fold) increases in micronuclei, whereas hydroquinone treatments resulted in a larger (11-fold) increase in micronuclei. Significant dose-related increases in kinetochore-positive micronucleated cells were not observed following 1,4-benzoquinone treatment but were observed following treatment with phenol, catechol, and hydroquinone. The higher efficacy of hydroquinone in inducing both total micronuclei and kinetochore-positive micronucleated cells when compared with catechol, phenol, and 1,4-benzoquinone suggests that hydroquinone is a major contributor to the clastogenicity and aneuploidy observed in the lymphocytes of benzene-exposed workers. Other metabolites may also contribute, however, to the genotoxic effects of benzene. Since consistent chromosomal aberrations are often observed in human leukemias, the ability of the phenolic metabolites of benzene to induce chromosomal damage in human cells also implicates them in benzene-induced leukemia.     

Topoisomerase inhibition by phenolic metabolites: a potential mechanism for benzene's clastogenic effects

Exposure to benzene, a human and animal carcinogen, resultsin the formation of structural chromosomal aberrations in thebone marrow and blood cells of animals and humans. The mechanismsunderlying these clastogenic effects are unknown. Inhibitionof enzymes involved in DNA replication and repair, such as topoisomeraseenzymes, by the metabolites of benzene represents a potentialmechanism for the formation of chromosomal aberrations. To testthis hypothesis, the inhibitory effects of various phenolicand qulnone metabolites of benzene on the activity of humantopoisomerases I and II were studied in vitro. No inhibitionof topolsomerase I was seen with any of the tested metabolites.Inhibitory effects on topoisomerase II were not observed forhydroqulnone, phenol, 2,2'-biphenol, 4,4'-biphenol and catecholat concentrations as high as 500 µM. 1,4-Benzoquinoneand 1,2,4-benzenetrlol inhibited topoisomerase II at relativelyhigh 500 and 250 µM concentrations, respectively. Howeverfollowing bioactlvation using a peroxldase/H₂O₂ system, Inhibitoryeffects were seen at concentrations as low as 50 µM forboth phenol and 2,2'-biphenol and 10 µM for 4,4'-biphenol.The addition of reduced glutathione (GSH) to the 4,4'-biphenoland horseradish peroxidase reaction system protected topoisomerase II from inhibition suggesting that diphenoquinone oranother oxidation product formed from 4,4'-biphenol might bethe reactive species. These in vitro results indicate that inhibitionof topolsomerase II may contribute to the clastogenic and carcinogeniceffects of benzene. In addition, metabolites formed from thesephenolic compounds appear to represent several new types oftopolsomerase II-Inhibiting compounds.     

The role of nitric oxide on DNA damage induced by benzene metabolites

Benzene, a tobacco constituent, is a leukemogen in humans and a carcinogen in rodents. Several benzene metabolites generate superoxide anion (O(2)(.-)) and induce nitric oxide synthase in the bone marrow of mice. We hypothesized that the reaction of nitric oxide (*NO) with O(2)(.-) leads to the formation of peroxynitrite as an intermediate during benzene metabolism. This hypothesis was supported by demonstrating that the exposure of mice to benzene produced nitrated metabolites and enhanced the levels of protein-bound 3-nitrotyrosine in the bone marrow of mice in vivo. In the current study, we investigated the influence of nitric oxide, generated from sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, on DNA strand breaks induced by each single or binary benzene metabolite at different doses and compared the levels of the DNA damage induced by each benzene metabolite in the presence of nitric oxide with the levels of DNA strand breaks induced by peroxynitrite at similar doses in vitro. We found that among benzene metabolites only 1,2,4-trihydroxybenzene (BT) can induce significant DNA damage in the absence of nitric oxide. While 1,4-dihydroxybenzene (HQ), 1,4-benzoquinone (BQ) and 1,2-dihydroxybenzene (CAT) require .NO to induce DNA strand breaks, hydroquinone was the most potent DNA-damaging benzene metabolite in the presence of *NO. The order of DNA breaks by benzene metabolites in the presence of *NO is: Peroxynitrite = HQ > BT > BQ > CAT. The *NO and O(2)(.-) scavengers inhibited DNA damage induced by [HQ+*NO]. Benzene, trans,trans-muconaldehyde, and phenol, do not induce DNA strand breaks either in the absence or presence of *NO. However, adding phenol to [HQ+*NO] leads to greater DNA damage than [HQ+*NO] alone. Collectively, these results suggest that nitric oxide is an important factor in DNA damage induced by certain benzene metabolites, probably via the formation of the peroxynitrite intermediate. Phenol, the major benzene metabolite that does not induce DNA damage alone and is inactive in vivo, synergistically enhances DNA damage induced by potent benzene metabolite in the presence of nitric oxide.     

HIF-1α高表达对苯代谢物诱导K562细胞毒性的影响

目的：研究缺氧诱导因子-1α（HIF-1α）高表达对苯代谢物1,4-苯醌、氢醌、苯酚诱导K562细胞毒性的影响。方法：用不同浓度的1,4-苯醌（0、10、20、40、80 μmol/L）、氢醌（0、10、20、40、80 μmol/L）、苯酚（0、1、1.5、2、2.5、5 mmol/L）分别染毒对照组K562细胞及高表达HIF-1α的K562细胞24 h,MTT法检测细胞增殖情况,PI/FITC Annxein V双染结合流式细胞术检测细胞凋亡率,碘化丙啶结合流式细胞术检测细胞周期。结果：MTT实验结果显示,1,4-苯醌染毒后,两株细胞均出现增殖率下降且呈剂量效应关系,1,4-苯醌浓度为80 μmol/L时,高表达HIF-1α的K562细胞增殖率高于对照组K562细胞（P < 0.05）。氢醌和苯酚在较高浓度染毒后,对照组K562细胞亦出现增殖抑制,而高表达HIF-1α的K562细胞增殖无明显抑制。流式细胞术结果显示,1,4-苯醌染毒后,两株细胞的细胞凋亡率增加且呈剂量效应关系。20 μmol/L浓度1,4-苯醌染毒时,高表达HIF-1α的K562细胞较对照组K562细胞凋亡减弱（P < 0.05）。氢醌和苯酚染毒后,两株细胞的凋亡率与未染毒组比较,差异均无统计学意义（P均 > 0.05）,高表达HIF-1α的K562细胞的凋亡率较对照组K562细胞亦无显著差异。细胞周期检测结果显示,3种苯代谢物均可引起K562细胞周期阻滞在G0/G1期或S期。结论：3种苯的代谢物中,1,4-苯醌对K562细胞的毒性大于氢醌和苯酚,HIF-1α高表达可降低苯代谢物引起的细胞毒性,其作用的发挥可能是通过调节细胞增殖及凋亡而实现。     

Modeling human metabolism of benzene following occupational and environmental exposures.

We used natural spline (NS) models to investigate nonlinear relationships between levels of benzene metabolites (E,E-muconic acid, S-phenylmercapturic acid, phenol, hydroquinone, and catechol) and benzene exposure among 386 exposed and control workers in Tianjin, China. After adjusting for background levels (estimated from the 60 control subjects with the lowest benzene exposures), expected mean trends of all metabolite levels increased with benzene air concentrations from 0.03 to 88.9 ppm. Molar fractions for phenol, hydroquinone, and E,E-muconic acid changed continuously with increasing air concentrations, suggesting that competing CYP-mediated metabolic pathways favored E,E-muconic acid and hydroquinone below 20 ppm and favored phenol above 20 ppm. Mean trends of dose-specific levels (micromol/L/ppm benzene) of E,E-muconic acid, phenol, hydroquinone, and catechol all decreased with increasing benzene exposure, with an overall 9-fold reduction of total metabolites. Surprisingly, about 90% of the reductions in dose-specific levels occurred below about 3 ppm for each major metabolite. Using generalized linear models with NS-smoothing functions (GLM + NS models), we detected significant effects upon metabolite levels of gender, age, and smoking status. Metabolite levels were about 20% higher in females and decreased between 1% and 2% per year of life. In addition, levels of hydroquinone and catechol were greater in smoking subjects. Overall, our results indicate that benzene metabolism is highly nonlinear with increasing benzene exposure above 0.03 ppm, and that current human toxicokinetic models do not accurately predict benzene metabolism below 3 ppm. Our results also suggest that GLM + NS models are ideal for evaluating nonlinear relationships between environmental exposures and levels of human biomarkers.     

Albumin adducts of benzene oxide and 1,4-benzoquinone as measures of human benzene metabolism

Albumin adducts of benzene oxide (BO-Alb) and 1,4-benzoquinone (1,4-BQ-Alb) were investigated among 134 workers exposed to benzene and 51 unexposed controls in Tianjin, China. Concentrations of both adducts increased with benzene exposure [range = 0.07-46.6 parts/million (ppm); median = 3.55 ppm] and with urinary cotinine. Adduct levels were less than proportional to benzene exposure, suggesting saturable CYP 2E1 metabolism of benzene. Because the transition from linear to saturable metabolism began at approximately 1 ppm, the common assumption of linear kinetics at much higher benzene exposures could lead to substantial underestimation of leukemia risks. Adduct levels were generally lower in older workers, indicating that CYP 2E1 metabolism diminished with age, at approximately 2%/year of life. The ratio of 1,4-BQ-Alb:BO-Alb decreased with age and coexposure to toluene, and increased with alcohol consumption. This indicates that factors affecting CYP 2E1 metabolism exerted a greater role on production of 1,4-BQ than BO, presumably because of the second oxidation step from phenol to hydroquinone. The adduct ratio was also positively associated with urinary cotinine, suggesting that both benzene and hydroquinone from cigarette smoke affected adduct levels. Results of a limited time course study of 11 subjects indicated moderate chemical instability of 1,4-BQ-Alb (half life = 13.5 days compared with 21 days for normal Alb turnover), whereas no evidence of instability of BO-Alb was observed. This study illustrates that Alb adducts can be used to investigate the dispositions of reactive metabolites of procarcinogens in humans, provided that exposures are adequately characterized in the month preceding blood collection.     

Role of oxygen radicals in induction of DNA damage by metabolites of benzene

Benzene is strongly suspected of being an animal and human carcinogen, but the mechanisms by which benzene induces tumors of lymphoid and hematopoietic organs are unknown. Binding studies in vivo suggest a very low level of covalent binding to the DNA of bone marrow elements. Since several metabolites of benzene have the potential to undergo autooxidation and thereby generate reactive oxygen intermediates, we have tested the hypothesis that benzene metabolites can induce DNA damage through the generation of oxygen radicals. Hydroquinone (HQ), benzoquinone (BQ), catechol, and 1,2,4-benzenetriol (BT) were first tested for their ability to generate O2-. at a physiological pH. BT, and to a lesser extent HQ, were autooxidized and produced significant quantities of O2-.. No detectable O2-. was produced by catechol or BQ. Similarly, BT was very efficient at degrading DNA, and this degradation was inhibited by scavengers of O2-., H2O2 and .OH. HQ did not degrade DNA but did induce single- and double-strand breaks. In contrast to the action of BT, the breakage of DNA by HQ was not inhibited by scavengers of reactive oxygen intermediates. The metabolites which did not produce O2-. (catechol and BQ) did not induce significant breakage of DNA. Taken together, the data support the hypothesis that certain benzene metabolites can induce DNA damage through the production of oxygen radicals; they further suggest that other metabolites may act, via another mechanism, to damage DNA.     

Synergistic increase in chromosomal breakage within the euchromatin induced by an interaction of the benzene metabolites phenol and hydroquinone in mice

The hematopoietic and carcinogenic effects of benzene may resultfrom an interaction of various benzene metabolites. Followingthe co-administration of phenol and hydroquinone, a synergisticincrease in myelotoxicity and genotoxicity has been observedin the bone marrow of mice. To understand the mechanisms underlyingthese synergistic geno-toxic effects we have studied the originof micronuclei (MN) formed in bone marrow erythrocytes followingthe co-administration of these two metabolites. Phenol and hydroquinonewere administered to male CD-I mice by i.p. injection threetimes at 24 h intervals. The frequency of MN was evaluated inbone marrow cells harvested 24 h following the final dose. Amarked increase in MN was observed in mice co-administered phenoland hydroquinone, which was significantly greater than thatobserved with the individual metabolites. Labeling with theCREST antibody and multicolor fluorescence in situ hybridizationwith the mouse major and minor satellite probes indicated thatboth chromosomal loss and breakage had occurred. The major increasein MN induced by the phenol and hydroquinone combination originatedfrom breakage in the dichromatic region of the mouse chromosomes.The origin of MN in mice co-administered phenol and hydroquinonediffered substantially from that induced by hydroquinone alone,but was almost identical to that seen in MN from benzene-treatedmice. These results strongly support the hypothesis that interactiveeffects among benzene metabolites play an important role inthe genotoxic and carcinogenic effects of benzene.     

Protein adducts of 1,4-benzoquinone and benzene oxide among smokers and nonsmokers exposed to benzene in China.

Hemoglobin (Hb) and albumin (Alb) adducts of the benzene metabolites benzene oxide (BO) and 1,4-benzoquinone (1,4-BQ) were analyzed by gas chromatography-mass spectrometry in 43 exposed workers and 44 unexposed controls from Shanghai, China, as part of a larger cross-sectional study of benzene biomarkers. When subjects were divided into controls (n = 44) and workers exposed to 31 ppm (n = 22) of benzene, median 1,4-BQ-Alb adducts were 2110, 5850, and 13,800 pmol/g Alb, respectively (correlation with exposure: Spearman r = 0.762; P < 0.0001); median BO-Alb adducts were 106, 417, and 2400 pmol/g Alb, respectively (Spearman r = 0.877; P < 0.0001); and median BO-Hb adducts were 37.1, 50.5, and 136 pmol/g Hb, respectively (Spearman r = 0.757; P < 0.0001). To our knowledge, this is the first observation that adducts of 1,4-BQ are significantly correlated with benzene exposure. When compared on an individual basis, Alb adducts of 1,4-BQ and BO and Hb adducts of BO were highly correlated with each other and with urinary phenol and hydroquinone (P < 0.0001 for all of the comparisons). Although detectable in the assays, Hb adducts of 1,4-BQ and both Hb and Alb adducts of 1,2-BQ produced erratic results and are not reported. Interestingly, cigarette smoking increased Alb adducts of 1,4-BQ but not of BO, suggesting that benzene from cigarette smoke was not the primary contributor to the 1,4-BQ adducts.     

Inhibition of interferon-alpha/beta induction in L-929 cells by benzene and benzene metabolites

Murine L-929 cells were treated with benzene or a series of benzene metabolites, washed and then interferon-alpha/beta was induced with polyriboinosinic-polyribocytidylic acid. Exposure of the cells to benzene or phenol, a monocyclic metabolite of benzene, did not affect interferon-alpha/beta induction. However, exposure of the cells to p-benzoquinone, hydroquinone or catechol, dihydroxy- and diketo-metabolites of benzene, resulted in a severe inhibition of interferon-alpha/beta production. There was no significant loss of viability of the cell cultures. Additional studies with p-benzoquinone indicated that inhibition of interferon-alpha/beta was reversible and could be abrogated by addition of reduced glutathione to the cell cultures.     

Inhibition of interferon gamma production by benzene and benzene metabolites

We exposed spleen cells from female Swiss/Webster mice to benzene and benzene metabolites to determine the effects of such exposure on interferon gamma induction by concanavalin A. Exposing the cells to benzene or phenol did not affect interferon gamma production, but exposing them to p-benzoquinone, catechol, or hydroquinone significantly inhibited interferon gamma production. Cell viability, as determined by trypan blue viability staining, was not influenced by the chemical treatment. When interferon gamma production was inhibited, the inhibition was dose dependent. The time of optimum production of interferon gamma after exposure to concanavalin A was not affected by treatment of the cells with p-benzoquinone. These data indicate the importance of dihydroxy and diketo metabolites as immunotoxic derivatives of benzene.     

Benzene and phenol metabolism by mouse and rat liver microsomes

Benzene, an important industrial solvent and constituent ofunleaded gasoline, causes leukemia and aplastic anemia in humans.Mice are more sensitive than rats to benzene toxicity, thoughneither species has been shown to respond consistently withbenzene-induced leukemia. Benzene biotransformation in liverto phenol, hydroquinone, catechol and/or muconalde-hyde is thoughtto be necessary for its hematotoxicity and/or genotoxicity.Our goal is to develop a mathematical simulation model capableof describing the pathways and kinetics of benzene metabolismby rat and mouse liver microsomes and to assess the role ofspecies metabolic differences in species sensitivity. Microsomeswere incubated with 4 µM [U-14C]-benzene or 4 µM[U-14C]-benzene or 4 µM [U-¹⁴C]phenol. Metabolite productionwas quantified by extraction into ethyl acetate, HPLC separationand liquid scintillation spectroscopy. After 45 min, mouse livermicrosomes converted 20% of the benzene to phenol, 31% to hydroquinoneand 2% to catechol. Rat liver micro-somes converted 23% of benzeneto phenol, 8% to hydro-quinone and 0.5% to catechol. Productionof hydroquinone and catechol continued for 90 min for mouseliver microsomes, while production by rat liver microsomes hadvirtually ceased by 90 min. Muconic acid production by mouseliver microsomes was <0.2% and <0.04% from benzene andphenol respectively after 90 min. A quantitative simulationmodel was constructed to describe the in vitro metabolism ofbenzene, incorporating the reaction sequences: benzene     

Urinary benzene as a biomarker of exposure among occupationally exposed and unexposed subjects

Urinary benzene (UB) was investigated as a biomarker of exposure among benzene-exposed workers and unexposed subjects in Shanghai, China. Measurements were performed via headspace solid phase microextraction of 0.5 ml of urine specimens followed by gas chromatography-mass spectrometry. This assay is simple and more sensitive than other methods (detection limit 0.016 microg benzene/l urine). The median daily benzene exposure was 31 p.p.m. (range 1.65-329 p.p.m.). When subjects were divided into controls (n = 41), those exposed to < or =31 p.p.m. benzene (n = 22) and >31 p.p.m. benzene (n = 20), the median UB levels were 0.069, 4.95 and 46.1 microg/l, respectively (Spearman r = 0.879, P < 0.0001). A linear relationship was observed between the logarithm of UB and the logarithm of benzene exposure in exposed subjects according to the following equation: ln(UB, microg/l) = 0.196 + 0.709 ln (exposure, p.p.m.) (r = 0.717, P < 0.0001). Considering all subjects, linear relationships were also observed between the logarithm of UB and the corresponding logarithms of four urinary metabolites of benzene, namely t,t-muconic acid (r = 0.938, P < 0.0001), phenol (r = 0.826, P < 0.0001), catechol (r = 0.812, P < 0.0001) and hydroquinone (r = 0.898, P: < 0.0001). Ratios of individual metabolite levels to total metabolites versus UB provide evidence of competitive inhibition of CYP450 enzymes leading to increased production of phenol and catechol at the expense of hydroquinone and muconic acid. Among control subjects UB was readily detected with a mean level of 0.145 microg/l (range 0.027-2.06 microg/l), compared with 5.63 microg/l (range 0.837-26.38 microg/l) in workers exposed to benzene below 10 p.p.m. (P < 0.0001). This suggests that UB is a good biomarker for exposure to low levels of benzene.     

Effects of Combined Treatment with Phenolic Compounds and Sodium Nitrite on Two-stage Carcinogenesis and Cell Proliferation in the Rat Stomach

The effects of combined treatment with NaNO₂ and phenolic compounds on N -methyl- N -nitro- N -nitrosoguanidine (MNNG) stomach Carcinogenesis were investigated in F344 rats. In the first experiment, groups of 15–20 male rats were treated with an intragastric dose of 150 mg/kg body weight of MNNG, and starting 1 wk later, were given 2.0% butylated hydroxyanisole, 0.8% catechol, 2.0% 3-methoxycatechol or basal diet either alone or in combination with 0.2% NaNO₂ in the drinking water until they were killed at week 52. All three antioxidants significantly enhanced forestomach Carcinogenesis without any effect of additional NaNO₂ treatment. However, in the absence of MNNG pretreatment, the grade of forestomach hyperplasia in the catechol and 3-raethoxycatechol groups was significantly increased by the combined treatment with NaNO2. In a second experiment, the combined effects of various phenolic compounds and NaNO₂ on cell proliferation in the upper digestive tract were examined. Groups of 5 rats were given one of 24 phenolic compounds or basal diet either alone or in combination with 0.3% NaNO₂ for 4 weeks and then killed. Particularly strong enhancing effects in terms of thickness of the forestomach mucosa were seen with t -butylhydroquinone (TBHQ), catechol, gallic acid, 1,2,4-benzenetriol, dl -3-(3,4-dihydroxyphenyl)-alanine and hydroquinone in combination with NaNOi. In the glandular stomach, similar enhancing effects were evident in 11 cases, and in the esophagus with phenol, TBHQ and gallic acid. These results demonstrate that NaNO₂ can augment cell proliferation induced in the stomach epithelium by various phenolic compounds.     

Using urinary biomarkers to elucidate dose-related patterns of human benzene metabolism

Although the toxicity of benzene has been linked to its metabolism, the dose-related production of metabolites is not well understood in humans, particularly at low levels of exposure. We investigated unmetabolized benzene in urine (UBz) and all major urinary metabolites [phenol (PH), E,E-muconic acid (MA), hydroquinone (HQ) and catechol (CA)] as well as the minor metabolite, S-phenylmercapturic acid (SPMA), in 250 benzene-exposed workers and 139 control workers in Tianjin, China. Median levels of benzene exposure were approximately 1.2 p.p.m. for exposed workers (interquartile range: 0.53-3.34 p.p.m.) and 0.004 p.p.m. for control workers (interquartile range: 0.002-0.007 p.p.m.). (Exposures of control workers to benzene were predicted from levels of benzene in their urine.) Metabolite production was investigated among groups of 30 workers aggregated by their benzene exposures. We found that the urine concentration of each metabolite was consistently elevated when the group's median benzene exposure was at or above the following air concentrations: 0.2 p.p.m. for MA and SPMA, 0.5 p.p.m. for PH and HQ, and 2 p.p.m. for CA. Dose-related production of the four major metabolites and total metabolites (micromol/l/p.p.m. benzene) declined between 2.5 and 26-fold as group median benzene exposures increased between 0.027 and 15.4 p.p.m. Reductions in metabolite production were most pronounced for CA and PH<1 p.p.m., indicating that metabolism favored production of the toxic metabolites, HQ and MA, at low exposures.     

Use of a mathematical model of rodent in vitro benzene metabolism to predict human in vitro metabolism data.

Benzene, a ubiquitous environmental pollutant, is known to cause leukemia and aplastic anemia in humans and hematotoxicity and myelotoxicity in rodents. Toxicity is thought to be exerted through oxidative metabolites formed in the liver, primarily via pathways mediated by cytochrome P450 2E1 (CYP2E1). Phenol, hydroquinone and trans-trans-muconaldehyde have all been hypothesized to be involved in benzene-induced toxicity. Recent reports indicate that benzene oxide is produced in vitro and in vivo and may be sufficiently stable to reach the bone marrow. Our goal was to improve existing mathematical models of microsomal benzene metabolism by including time course data for benzene oxide, by obtaining better parameter estimates and by determining if enzymes other than CYP2E1 are involved. Microsomes from male B6C3F1 mice and F344 rats were incubated with [(14)C]benzene (14 microM), [(14)C]phenol (303 microM) and [(14)C]hydroquinone (8 microM). Benzene and phenol were also incubated with mouse microsomes in the presence of trans-dichloroethylene, a CYP2E1 inhibitor, and benzene was incubated with trichloropropene oxide, an epoxide hydrolase inhibitor. These experiments did not indicate significant contributions of enzymes other than CYP2E1. Mathematical model parameters were fitted to rodent data and the model was validated by predicting human data. Model simulations predicted the qualitative behavior of three human time course data sets and explained up to 81% of the total variation in data from incubations of benzene for 16 min with microsomes from nine human individuals. While model predictions did deviate systematically from the data for benzene oxide and trihydroxybenzene, overall model performance in predicting the human data was good. The model should be useful in quantifying human risk due to benzene exposure and explicitly accounts for interindividual variation in CYP2E1 activity.     

Hypothesis: phenol and hydroquinone derived mainly from diet and gastrointestinal flora activity are causal factors in leukemia.

High background levels of phenol and hydroquinone are present in the blood and urine of virtually all individuals, but vary widely. Phenol and hydroquinone have been strongly implicated in producing leukemia associated with benzene exposure, because they reproduce the hematotoxicity of benzene, cause DNA and chromosomal damage found in leukemia, inhibit topoisomerase II, and alter hematopoiesis and clonal selection. The widely varying background levels of phenol and hydroquinone in control individuals stem mainly from direct dietary ingestion, catabolism of tyrosine and other substrates by gut bacteria, ingestion of arbutin-containing foods, cigarette smoking, and the use of some over-the-counter medicines. We hypothesize that these background sources of phenol and hydroquinone and associated adducts play a causal role in producing some forms of de novo leukemia in the general population. This hypothesis is consistent with recent epidemiological findings associating leukemia with diets rich in meat and protein, the use of antibiotics (which change gastrointestinal flora make-up), lack of breastfeeding, and low activity of NAD(P)H quinone oxidoreductase which detoxifies quinones derived from phenol and hydroquinone and protects against benzene hematotoxicity. An attractive feature of our hypothesis is that it may explain why many people who have no known occupational exposures or significant smoking history develop leukemia. The hypothesis predicts that susceptibility to the disease would be related to diet, medicinal intake, genetics and gut-flora composition. The latter two of these are largely beyond our control, and thus dietary modification and reduced use of medicines that elevate phenol levels may be the best intervention strategies for lowering leukemia risk.     

Hydroquinone, a benzene metabolite, increases the level of aneusomy of chromosomes 7 and 8 in human CD34-positive blood progenitor cells

Benzene is an established human carcinogen, producing leukemia, hematotoxicity and perhaps lymphoma. Its carcinogenicity is most likely dependent upon its conversion to phenol and hydroquinone, the latter being oxidized to the highly toxic 1,4-benzoquinone in the bone marrow. Exposure of human lymphocytes and cell lines to hydroquinone has previously been shown to cause various forms of genetic damage, including aneusomy and the loss and gain of chromosomes. However, the target cells for leukemogenesis are the pluripotent stem cells or early progenitor cells which carry the CD34 antigen (CD34(+) cells). In this study, human cord blood, which is particularly rich in CD34(+) cells, was exposed to hydroquinone for 72 h in a medium that favored CD34(+) cell survival and growth. CD34(+) and CD34(-) cells were then isolated. Fluorescence in situ hybridization was employed to determine the level of aneusomy of chromosomes 7 and 8 in both cell types. CD34(+) cells were generally more susceptible to aneusomy induction by hydroquinone than CD34(-) cells. Increased trisomy and monosomy of chromosomes 7 and 8 were observed in CD34(+) cells (P(trend) < 0.001), whereas in CD34(-) cells only an increased level of monosomy 7 was detected (P(trend) = 0.002). Particularly striking effects of hydroquinone were observed in CD34(+) cells on monosomy 7 and trisomy 8, two common clonal aberrations found in myeloid leukemias, suggesting that these aneusomies produced by hydroquinone in CD34(+) cells play a role in benzene-induced leukemogenesis.     

Metabolism of 3-tert-butyl-4-hydroxyanisole to 3-tert-butyl-4,5-dihydroxyanisole by rat liver microsomes

3-tert-Butylhydroxyanisole (3-BHA) is an antioxidant which can have a modulating effect on chemical carcinogenesis. Information concerning the metabolism of 3-BHA is incomplete. In the present study, the metabolites formed by incubating 3-BHA with liver microsomes from rats given beta-naphthoflavone by p.o. intubation were studied. Three metabolites were identified, two major metabolites and a minor metabolite. One of the major metabolites was the catechol of 3-BHA, i.e., 3-tert-butyl-4,5-dihydroxyanisole, which has not previously been reported. A characteristic of this compound is its capacity to be oxidized readily. The second major metabolite was tert-butyl hydroquinone which has been reported previously to be a liver microsomal metabolite of 3-BHA. The third metabolite, which occurred in small quantities, was 2,2'-dihydroxy-3,3'-di-tert-butyl-5,5'-dimethoxydiphenyl. 2,2'-Dihydroxy-3,3'-di-tert-butyl-5,5'-dimethoxydiphenyl has been identified previously as a major metabolite of 3-BHA in the rat intestine. An understanding of the metabolism of 3-BHA may assist in elucidating the mechanism(s) of its biological effects.