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British Journal of Industrial Medicine 1991;48:435-444 The toxicity of benzene and its metabolism and molecular pathology in human risk assessment 437 A Yardley-Jones, D Anderson, D V Parke Abstract reflecting a concern for the risk of neoplasia. Benzene, a common industrial chemical and a The American Conference of Governmental component of gasoline, is radiomimetic and Industrial Hygienists (ACGIH) has even more exposure may lead progressively to aplastic recently recommended that, as benzene is con- anaemia, leukaemia, and multiple myeloma. sidered an A1 carcinogen, the threshold limit Although benzene has been shown to cause value (TLV) should be decreased to 0.1 ppm. many types of genetic damage, it has consis- Only one study in man, based on nine cases of tently been classified as a non-mutagen in the benzene associated fatal neoplasia, has been Ames test, possibly because of the inadequacy considered suitable for risk assessment. of the S9 microsomal activation system. The Recent re-evaluation of these data indicated metabolism of benzene is complex, yielding that past assessments may have overestimated glucuronideand sulphate conjugatesof phenol, the risk, and different authors have considered quinol, and catechol, L-phenylmercapturic that lifetime exposure to benzene at 1ppm acid, and muconaldehyde and trans, trans- would result in an excess of leukaemia deaths muconic acid by ring scission. Quinol is of 0.5 to 1.0 per 1000. Although in this study, oxidised to p-benzoquinone, which binds to deaths at low levels of benzene exposure were vital cellular components or undergoes redox associated with multiple myeloma and a long cycling to generate oxygen radicals; muconal- latency period, instead of leukaemia, which dehyde, like p-benzoquinone, is toxic through might justify further lowering of the exposure depletion of intracellular glutathione. limit, the risk assessment model has been Exposure to benzene may also induce the found to be non-significant for response at low microsomal mixed function oxidase, cyto- levels of exposure. The paucity of data for man, chrome P450 IIE1, which is probably responsi- the complexity of the metabolic activation of ble for the oxygenationof benzene, but also has benzene, the interactive and synergistic a propensity to generate oxygen radicals. The mechanisms of benzene toxicity and carcino- radiomimetic nature of benzene and its ability genicity,the different diseaseendpoints (aplas- to induce different sites of neoplasia indicate tic anaemia, leukaemia, and multiple that formation of oxygen radicals is a major myeloma), and different individual suscep- cause of benzene toxicity, which involves tibilities, all indicate that in such a complex multiple mechanisms including synergism scenario, regulators should proceed with cau- between arylating and glutathione-depleting tion before making further changes to the reactive metabolites and oxygen radicals. The exposure limit for this chemical. occupational exposure limit in the United Kingdom (MEL) and the United States (PEL) was 10 ppm based on the associationof benzene Benzene, a common industrial chemical, a compon- exposure with aplastic anaemia, but recently ent of gasoline, and a constituent of engine emissions was lowered to 5 ppm and 1 ppm respectively, and tobacco smoke,' has a production rate of 15 million tonnes per year, with a total global cycle of 32 million tonnes per year. Benzene is ubiquitous in the Department of Biochemistry, University of Surrey, environment, and chronically exposed populations Guildford, Surrey GUZ SXH, U K include petrochemical workers, petrol station atten- A Yardley-Jones,D V Parke The British Industrial Biological Research Associa- tion, Woodmansterne Road, Carshalton. Surrey SM5 4DS D Anderson dants, and smokers.* Benzene is a radiomimetic chemical, with heavy exposure resulting in progressive degeneration of the bone marrow, aplastic anaemia, and leukaemia, and in dysfunction of the Burmah Castrol Trading Ltd. Burmah Castrol immune svstem.' In 1971 the United States House, Pipers Wag. Swmaon. Wiitshire SN3 I R E A Yardlev-iones Occupational Safety and Health Administration (OSHA` inrroduced a permissible exposure limit (PEL) of 10 ppm, based on the concern for benzene causing aplastic anaemia and myelosuppression.` which was later lowered to 1 ppm based on the concern for the risk of benzene associated neoplasia.` The American Conference of Governmental Industrial Hygienists (ACGIH) has recently proposed that benzene be listed as an A1 confirmed human carcinogen with an eight hour time weighted average threshold limit value (TLV) of 0.1 ppm and a skin notation (1990-1991 T L V Notice of Intended Changes).' Benzene toxicity Since Delore and Borgomano in 1928' first suggested an association between occupational exposure to benzene and the development of leukaemia, it has been generally agreed that a causal relation exists between high exposures to benzene and the development of pancytopaenia, aplastic anaemia, and acute 'mylogenous leukaemia; a pattern consistent with toxic myelosuppression.` A review of the relevant clinical publications concluded that benzene related haemotoxicity in man was associated with benzene concentrations in the workplace of >50 pprn; no evidence of haemotoxicity was reported to be associated with prolonged exposure to benzene concentrations of 20-25 ppm.Y As well as its haemotoxicity, benzene is considered to be a Group I carcinogen; sufficient evidence of carcinogenicity in man and in laboratory animals.'o The association between long term exposure to benzene and the occurrence of leukaemiae was supported by the epidemiological evidence of Vigliani" and more recently by the Bologna experiments, which have shown that benzene is a multipotential carcinogen increasing the incidence of a wide variety of neoplasms in rats and mice.'* Studies by the National Cancer Institute and in the National Toxicology Program have shown that benzene induces a larger number of unique sites of neoplasia than any other chemical.` These findings of neoplasia, haemotoxicity, and myelosuppression are indicative of the radiomimetic properties of benzene, and consequently of its potential for oxygen radical generation. T h e firm evidence of carcinogenic potential in rodents` led OSHA to lower the permissible level of occupational exposure from ID ppm to 1 PPm. Benzene has produced many types of genetic damage, but although it has caused chromosomal aberrations in animals and man and sister chromatid exchanges and micronuclei both in vitro and in vivo, and has also produced aneuploidy in dividing cells, it has not consistently produced point mutations in genotoxicity test systems.' I3 Benzene itself has not consistently produced mutagenic effects in conventional agar plate techniques for bacterial mutagens. This is possibly because or the inaaequacy of Aroclor inducrion to producc an appropriate spectrum of the cytochromes P450 to metabolically activate benzene to mutagenic products.' The benzene metabolites, quinol, p-benzoquinone, and trans, rrans-muconaldehyde (the most cytotoxic metabolites of benzenei4,? showed very weak mutagenic efiFects in bacteria, but were strongly mutagenic in Chinese hamster V79 cells, mouse L5178Y cells," and cultured human lymphocyres,16I' and induced an increase in sister chromatid exchanges and a decrease in mitotic index in mice in vivo.`"' The little known metabolite of benzene, the anti-diol epoxide, which is a poor substrate for epoxide hydrase, showed, however, a broad spectrum of genotoxicity in bacterial and mammalian cells.'O Studies of developmental toxicity have generally failed to show any significant adverse effects of exposure to benzene in rodents or rabbits. Metabolism of benzene T h e earliest studies of the metabolism of benzene, undertaken by Baumann and others more than a century ago, showed that it was metabolised mostly by oxidation to phenol, quinol, and catechol, which were excreted in the urine as sulphates and glucuronides." Later studies showed that L-phenylmercapturic acid and trans-trans-muconic acid were minor metabolites found in urine. Following the synthesis of "C-benzene, and the preparation of pure benzene for the first time in 1952, Parke and Williamsz2 undertook a quantitative study of the metabolism of this chemical in rabbits. They confirmed that the major metabolites, excreted in the urine, were phenylsulphate and phenylglucuronide, with smaller amounts of the sulphate and glucuronide conjugates of quinol and catechol, and even smaller amounts of phenylmercapturic acid and trans-trans-muconic acid. They also found evidence for the further oxidation of the ring scission products to two-carbon fragments that became incorporated into the animal tissues, and were also completely oxidised to respiratory CO,.They concluded that the metabolism of benzene is a multistep process and fig 1 shows the major pathways. Incubation of 14C-benzene with mouse liver microsomes in the presence of NADPH resulted in its metabolism by ring opening to give trans, rransmuconaldehyde," a direct acting alkylating agent," which is further metabolised to trans, rrans-muconic acid.z5trans, trans-Muconic acid is a good indicator of benzene exposure, and may be quantified in urine by high pressure liquid chromatography, with a sensitivity of 0.1 mg/l; exposure of workers to benzene at 5 ppm resulted in urine concentrations of 38 mgj1.2b "C-Benzene is metabolised by rat liver microsomes to reactive intermediates that bind - The toxicity of benzene and its metabolism and molecular pathology in human risk assessment ao-HHO \ OH 439 Glucuronide and Sulphate NHCOCH, L - Phenylmercapturic acid 1,2,4 - Trihydroxybenzene Conjugates GSH 10:1 t 0:-:Conjugates Phenyl premercapturic acid Catechol 0- ooHt t [Do-] -Conjugates Benzene Benzene oxide Benzene diol Phenol I HO \ Quinol -Conjugates -trans - t-rans - Muconaldehyde -trans - t-rans - Muconic acid p-Benzoquinone Finure I The major pathwa-vs of benzene merabolim. Formulae in parenrheses are postulated intermediates. irreversibly to microsomal protein.*' Prostaglandin H synthase, an enzyme with both peroxidase and cyclooxygenase activity, which is known to oxidatively activate many carcinogens and toxic chemicals-for example, benzoiajpyrene and paracetamol-in a process known as co-oxidation, is also capable of oxidising phenolic metabolites of benzene to reactive metabolites, which bind to protein and DNA.*' The cytochrome P450 dependent oxidation of benzene to phenol and its activation to covalently binding reactive intermediates are mediated by hydroxyl radicals; biphenyl is also formed indicating the formation of a hydroxycyclohexadienyl radical as an ir1termediate.2~Benzene is a substrate of cytochrome P450IIE1, a microsomal cytochrome with a propensity for generating reactive c 440 Yaraiei+Jones. .-inaerson. Parke oxygen radicals, which is believed to effect the oxygenation of substrates by the generation of hydroxyl radical^.'^ Ethanol is also metabolised by this particular cytochrome and, like benzene and other substrates, leads to induction of this cytochrome with consequent increase in oxygen radical generation that may account for the radiomimetic toxicity of this chemical. Metabolism of '"C-benzene to phenol, quinol, and their conjugates by liver slices and microsomal preparations occurred at similar rates with tissue from mouse, rat, and man, but covalent binding of I4C-reactive metabolites to microsomal protein was in the order man > mouse > rat." In mice, metabolism to quinol glucuronide and rrans, rransmuconic acid (markers of toxic pathways of benzene metabolism) was proportionately greater at lower dosage; in rats a similar trend was seen, and although quinol glucuronide is a minor metabolite in rats at all doses, rrans, rrans-muconic acid formation was proportionately greater at lower doses." T h e pathways of detoxication of benzene in rodents are characterised by low affinity and high capacity, whereas the pathways leading to putative toxic metabolites have high affinity and low capacity, so that the use of high dose metabolism studies in rodents for assessment of the health risk to man of exposure to low doses of benzene could lead to an undere~timate.~' Molecular pathology of benzene poisoning Hypotheses as to the mechanism of benzene toxicity have been the subject of much research and debate, but there seems little doubt that multiple mechanisms are involved; these may include synergism between different metabolites, such as quinol and muconaldehyde as suggested by Snyder er al" or synergism between glutathione-depleting metabolites of benzene and hydroxyl radicals. Indeed, the formation of DNA adducts from reactive metabolites of benzene may be of minor importance, as the indications are that benzene and its metabolites are only weakly genotoxic, at least in man.3435 Benzene is metabolically activated via phenol to quinol and then to its oxidation products p-benzosemiquinone and p-benzoquinone, which may covalently bind to glutathione, proteins, or other cellular macromolecules (fig 2).36The bone marrow toxicity and micronucleus formation in mice treated with benzene were inhibited by simultaneous administration of the cyclooxygenase inhibitor, indomethacin, indicating a role for prostaglandin H synthase in benzene myelotoxicity possibly by co-oxygenation of phenolic metabolites to p-benzoquinone." T h e metabolism of benzene by ring opening yields trans, trans-muc~naldehyde~w' hich is a direct alkylating agent interacting with cellular thiol and amino groups, and is a potent bone marrow toxin in micez438 :fig 7'.. Increased production of hvdroxvl radicals has been shown to occur in rats dosed with benzene,'" and may arise from redox cycling of p-benzoquinone. induction of cytochrome P450IIE1, which has a propensity to generate oxygen radicals, or depletion of endogenous glutathione by p-benzoquinone or rrans. rrans-muconaldehyde (fig2 ) . Although Snyder er al" have been able to show the formation of DNA adducts of benzene metabolites in the bone marrow of rats dosed orally with benzene (1 mli'kg daily for four days) by a "P-post-labelling technique, a nuclease 3ZP-post-labellingassay with a sensitivity of detecting one adduct in 10"' DNA nucleotides was able to show only four lesions per 10' DNA nucelotides in rats dosed with 200-500 mg/kg of benzene daily for five days a week for up to 10 weeks, and this only in the Zymbal gland; no adduct formation was detected in liver, kidney, bone marrow, or mammary gland in this more detailed study, and no benzene metabolite led to DNA adduct formation in any tissue."' It has been suggested that DNA damage results from peroxidase oxidation of phenol and quinol to p-benzoquinone, which is known to damage DNA, and to result in induction of micronuclei in human lymphocytes in vitro." Benzene has also been shown to activate protein kinase c, an enzyme playing a pivotal part in signal transduction, which is involved in cell transformation and tumour promotion." Thus both genotoxic and nongenotoxic mechanisms of carcinogenicity are evoked by benzene and its metabolites, so providing mechanistic evidence for the potential tumorigenicity of benzene. D N A synthesis in a mouse lymphoma cell line was inhibited by p-benzoquinone > quinol > 1,2,4benzenetriol > catechol > phenol, but not by benzene itself, by a redox-type mechanism; the ease of oxidation of the benzene metabolites correlated with their ED,, values for inhibition of DNA synthesis (fig 2).42Benzene and its metabolites, phenol, quinol, and p-benzoquinone have also been shown to inhibit RNA synthesis in macrophages and may thus inhibit haematopoiesis." Other studies, notably by Irons et al" show that benzene and its metabolites can effect cell maturation in that the metabolites are able to suppress the synthesis of various cellular proteins as well as DNA and RNA. Benzene shares certain characteristics with colchicine and the vinca alkaloids, which interfere with microtubular assembly and mitotic spindle formation and result in an arrest of cycling cells in the G2/ M phase of the cell cycle. Benzene toxicity is associated with the dihydroxy metabolites, catechol and quinol, which concentrate in the bone-marrow; quinol and its oxidation product, p-benzoquinone, interfere with microtubule assembly and react with nucleophilic sulphydryl groups that are essential for the binding of guanosine triphosphate to tubulin, The toxicity of benzene and its metabolism and molecular pathology in human risk assessment 441 Cvtochrorne P450 l l E l Covalent binding lo macromolecules t 4m / = f /I Biphenyl Covalent binding to CSH and macromolecules t II`OH t IIOH tt ` y HHydroxycryacdliochael udiene Benzene OH pBenzoscrniquinone 0 pBenzoquinone Benzene oxide Benzene !a1~-1,2diol OH Benzenetriol anti-Diol epoxide / Covalent binding IO GSH and Macromolecules Quinones &Muconic acid Covalent binding to CSH and Macromolcculn Figure 2 Molecular mechanism of benzene toxicityjcarcinogenicity.p-Benzoquinone, p-benzosemiquinone, other quinones, malondialdehvde, and radical metabolites may covalently bind to glutathione and intracellular macromolecules. Oxygen radicals may be produced by redox cycling wirh the benzoquinones, orfrom cytochrome P450 I I E I , which is induced by benzene. resulting in the arrest of cell division and suppression of lymphocyte blast~genesis.~B~enzene and its metabolites kill cells undergoing cell division, but spare resting cells by a mechanism as yet not defined; however, it seems plausible that the cytogenetic effects associated with benzene, which are primarily chromosomal gaps and chromosomal breaks, may play a part.13 The pattern of benzene associated bone marrow toxicity has been reproduced by the coadministration of two benzene metabolites-namely, phenol and quinol-although neither of these compounds administered alone produced significant myelosuppression,46thus confirmingthe previous observations of Tunek et aL4' When phenol and quinol were coadministred to mice by a "continuous" regimen, bone mamow cellularity decreased initially, with gradual recovery beginning at the second week despite continued treatment. When the mice were treated by a "discontinuous" regimen, however, bone marrow cellulariry decreased profoundly, with no evidence of recovery during the treatment period. This apparent paradox, in which the lower total dose administered in the ``discontinuous" protocol was more toxic than the higher dose of the "continuous" regimen, supports the view that the toxic effect of benzene metabolites on the marrow was cycle depen- dent. Supportive evidence of this came from the studies of Luke et who found that a three day exposure regimen produced more micronucleated polychromatic erythrocytes than did five days of exposure. Finally, a series of studies have shown that benzene affects the function of the cellular and hormonal regulators of blood formation, in particular, the function of the stromal cell.5051 This effect appears to be due to a selective suppression of IL-1 released by macrophages, which in the case of the benzene- associated suppression of pre-B lymphocytes, results in a reduction of IL-1 dependent release of IL-4 by marrow fibroblast^.^' Thus the haemotoxic effects of henzene, and its cytotoxicity, genotoxicity, and car- f 442 Yardiev- iones. .-lnuerson. f a r k e cinogenicity, are clearly the consequences of 3 number of highly complex. interactive biological processes. Risk assessment The PEL in the United States and the United Kingdom for many years was 10 ppm (32 mgjm'), but this was recently lowered to 1 pprn in the United States and 5 ppm in the United Kingdom. Benzene is no longer used as a general solvent in the United States, the United Kingdom, and other European countries, but it is still widely used as an industrial solvent in China where the present exposure limit is 40 mg/m' (12.5 ppm); of more than 50 000 work places in China the mean concentration of benzene was 5.5 ppm. I n more than 500 000 workers in China exposed to benzene or benzene mixtures the prevalence of benzene poisoning was 0.5%." In a 35 year longitudinal study of the haematological surveillance records of 459 rubber workers exposed to benzene, strong positive correlations were found between blood count fluctuations and fluctuations in exposure to benzene for the earlier periods of observation (1940-8, average exposures were 75 ppm) but not for later years (1948-75, average exposures were 15-20 ~ p m ) ~e"st;imates of the risk of leukaemia related to occupational exposure to benzene for a working lifetime (40 to 45 years) were 9.5174cases/1000workers for exposures to 10 ppm, and 5-14 cases/1000 workers for exposures to 1 ppm. T h e lifetime cancer risk recently calculated from a pharmacokinetically derived risk assessment based on rodent data with scaling across species gave 6-14 cases/1000 workers for exposure to benzene at 10 From a consideration of benzene metabolism to model the internal dose, from the administered dose in animal studies, and fitting a multistage risk assessment model, Bailer and H o e P calculated that a 1 ppm lifetime exposure would result in 0.7-1.0 excess cancers per 1000 persons exposed. In a recent review and update of leukaemia risk associated with exposure to benzene it was concluded that the cohort of Rinsky et al" provides the best basis for estimation of the benzene associated risk of leukaemia, and that no other available study is suitable for assessment of risk.58Re-evaluation of the data indicates that past assessments may have overestimated the risk by a factor of three to 24 and, based on the data of Rinsky et a1" and exposure matrices of Crump and Allen59an estimate of 7.9 excess leukaemia deaths per 1000workers exposed for 45 years to 10 ppm of benzene, and an excess of 0.5 leukaemia deaths per 1000 workers exposed for 45 years to 1 ppm, were proposed.5RExamination of the data of Rinsky et al" which are based on nine leukaemia related deaths, shows that three cases had cumulative exposure between 470-640 ppm-years, two cases had cumulative exposure between 250 and 260 ppm- years, and the remaining four cases had cumuiative exposures of 99, 50. 10, and 0.1 ppm-years. that is. effectively three different data points. Rinsky er ai'' stated that the shape of the best fit model was linear. If che observed and prelcted probabilities of leukaemia are examined, however, the data are seen to be polarised into three areas, with the response at lower levels being relatively flat. Thus, in the risk assessment of Rinsky et ~ 1it is, th~e th~ree highest exposure cases that drive the model. When using a conditional logistic regression to predict the probability of leukaemia at a given level of exposure, with the three high cases included, the model was significant (;cz = 13.3, p < 0.01); after removal of the three cases (and their control), however, the model was not (x'significant = 1.4, p = 0.23). Furthermore, the estimates of exposure, as highlighted in the study, were considered to be an ~nderestimate,~a' nd underestimation of exposure would increase the predicted risk at any given exposure. A further confusing factor is that multiple myeloma was the cause of death in four members of the Rinsky cohort of benzene workers,ii three of the four were among the group with the lowest cumulative exposure to benzene ( <40 ppm-years), and all four required an exceptionally long latency period (>20 years).b0 These two factors indicate a possibility that low cumulative exposure to benzene may result in well differentiated malignancy such as multiple myeloma, whereas higher exposures lead to leukaemia.m This observation confuses even further the risk assessment procedure for occupational and environmental exposure to benzene, for if true it might justify the recent ACGIH proposal for a decrease in the T L V for benzene to 0.1 ppm. The risk assessment for exposure of man to low concentrations of benzene is based on few cases (nine).5RFurthermore, it is difficult to explain the observed different disease end points in relation to low level exposure and duration of such exposure, as described by Rinsky,wunless differentmechanisms of toxicity/carcinogenicity at different levels and durations of exposure are postulated. This complicates any assessment of risk and is likely to lead to overestimation of such risks. Examination of relevant, non-epidemiological experimental data shows a negative effect of benzene-but not of its toxic metabolites-in the h n e s and other genotoxicity tests that depend on activation systems such as the Aroclor-induced rat h e r S9 mix, possibly indicating the involvement of cytochromes other than the Aroclor-induced P450IA1 and P450IIB in the metabolic activation of benzene. The data are further complicated by the known induction by benzene of P450IIE 1, an oxygen radical generator, the high susceptibility of rodents to oxygen,6' and the vulnerability of isolated cell systems to oxygen toxicity because of depletion of glutathione and o h e r The toxicity of benzene a n d its metabolism a n d molecular pathology in human risk assessmenr 443 components of the biological anti-oxidant defence system. The reasons for toxicity and carcinogenicity of benzene are thus complex, involving several different mechanisms (p-benzoquinone covalent binding, trans, trans-muconaldehyde depletion of glutathione, oxygen radical production by redox cycling from p-benzosemiquinone, and induction of cytochrome P450I IE) all possibly associated with different disease endpoints (haemotoxicity, leu- kaemia, multiple myeloma), and probably related to individual susceptibility (genetic, dietary, smoking, alcohol consumption). Given that this is the case, then regulators should proceed with caution when making judgements on acceptable limits of exposure, based on an inadequate data base in man and using such epidemiologicalrisk models as described in this review. Requests for reprints to: Professor D V Parke, Department of Biochemistry, University of Surrey, Guildford, Surrey GU2 5XH, UK. 1 Kalf GF. Recent advances in the metabolism and toxicity of benzene. CRC Crir Rev Toxicol 1987;18:141-59. 2 Howard PH, Durkin PR. Sourcesof contamination,ambient levels andfare of benzene in the environment.Washington, D C U.S. Environmental Protection Agency, 1974. (EPA 560/5-75005.) 3 Snyder R, Longacre SL, Wiuner CM, Kocsis JJ. Biochemical toxicology of benzene. Reviews of Biochemical Toxicology 1981;3:123-53. 4 Huff JE, Haseman JK, De Marini DM, er al. Multiple-site carcinogenicity of benzene in Fischer 344 Rats and B6 C3F1 mice. Environ Healrh Perspecr 1989;82:125-63. 5 Anon. Notice of lntended changes-Benzene. 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In: Occuparional Carcinogenesis. Ann A' Y Acaa` Sei 1976; 271:143-51, 12 Maltoni C, Cilibeni A. Corti G. Conti B, Belpoggi F. Benzene, an experimental multipotential carcinogen: Results of the long-term bioassays performed at the Bologna Institute of Oncologv. Envirim Health Perspect 1989;82:109-24. 13 Dean BJ. Recent findings on the genetic toxicology of benzene, toluene. xvlenes and phenols. Mutar Res 1985;154:153-81. 14 Glatt H. W i k G. Studies on the induction of gene mutations in bacrenal and mammalian cells by the ring-opened benzene metabolites tram, trans-muconaldehyde and rrans, rransmuconic acid. Muta~enesis1990;5:263-6. 15 Pellack-Walker P. Blumer TL. DNA damaac in LjIi'SY cells following exposure to benzene metaboh& Mol Pharmacol 1983:30:42-7 16 Monmoto K. Wolff S . Koizum~A. Inductionofsisrer-chromatid exchanges in human lvmpbocvteshy microsomal activation of benzene metabolites. Mutar Res 1963;119:355-60. 17 Smith M T , Yager JW, Steinmetz KL, Easunond DA. 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Formation of muconalde- hyde, an open-ring metabolite of benzene, in mouse liver microsomes: An additional pathway for toxic metabolites. Proc Kat Acad Sci USA 1986;83:8356-60. 24 W i n G, Latriano L, Goldstein BD. Metabolism and toxicity of rrans, trans-muconaldehyde, an open-ring microsomal metabolite of benzene. Environ Health Perspecr 1989;82: 19-22. 25 W i n G, Maniara W, Mylavarapu V, Goldstein GD. Com- parative metabolism of benzene and trans, rrans-muconalde- hyde to trans, trans-muconic acid in DBA/ZN and C57BL/6 mice. Biochem Pharmacol1990;40:1275-80. 26 lnoue 0,SeijiK, Nakatsuka H, et al. Urinary t,t-muconic acid as an indicator of exposure to benzene. Br J Ind Med 1989; 46:122-7. 27 Tunek A, Platt KL, Bentley P, Oesch F. Microsomal metabol- ism of benzene to species irreversibly binding to microsomal protein and effects of modifications of this system. Mol Pharm a d 1978;14:920-9. 28 Schlosser MJ, Shurina RD, Kalf GF. Metabolism of phenol and hydroquinone to reactive products by macrophage peroxidase -or ourified DrostaglandinH svnthase. Environ Health Perspect 1989;82:229-37. -29 lohansson 1. Ineelman-Sundbere M. Hvdroxvl radicalmediated c.btoch;ome P-450-depkdent mGabol2 activation of benzene in microsomes and reconstituted systems from rabbit liver. J Biol Chem 1983;258:7311-16. 30 Brodfuehrer J I , Chapman DE, Wilke TJ, Powis G. Comparative studies of the in vitro metabolism and covalent binding of "C- benzene by liver slices and microsomal fraction of mouse, rat and human. Drug Merab Dispos 1990;18:20-7. 31 Sabourin PJ, Bechtold WE, Griffith WC, Birnbaum LS, Lucier G, Henderson RF. Effect of exposure concentration, exposure rate, and route of administration on metabolism of benzene by F34.1 rats and B6C3F1 mice. Toxicol Appl Pharmacol 1989; 99:4214. 32 Henderson RF, Sabourin PJ, Bechtold WE, et ai. 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Roles of cytochromes P-450 in mouse liver tumour production. In: Stevenson DE, McClain RM, Popp JA, Slaga TJ, Ward JM, Pitot HC, eds. Mouse liver carctnogenests: mechanisms and speciescomparisons. New York: Alan R Liss, 1990:215-30. Accepted 14 January 1991 I Destruction ofmanuscripts I From 1 July 1985 articles submitted for publication will not be returned. Authors whose papers are rejected will be advised of the decision and the manuscripts will be kept under security for three months to deal with any inquiries and then ' destroyed. 1