Document JzXaX0zZnvMwDv33wqR4VwZ

i Free Rcdicul Biology & Medicine. Vol. 11. pp. 495-515. 1991 Rinrcd in UIC USA. All righu resewed. 0891-5849/91 $3.00 + .OO COpyrighlQ 1991 pertumnRcrs PIC +* Review Article POTENTIAL ROLE OF FREE RADICALS IN BENZENE-INDUCED MYELOTOXICITY AND LEUKEMIA VANGALVA. SUBRAHMANYADMAV,I*D Ross,f DAVID A. EASTMONaDnd~ MARTYNT.S m * *Department of Biomedical and Environmental Health Sciences. School of Public Health, University of California, Berkeley, CA 94720, U S A . tMolecular Toxicology and Environrnenlal Hcalth Sciences Rogrm. School of Pharmacy, University of Colorado, Boulder, CO 80309. U.S.A. *Environmental Toxicology Graduate R o p m , Depanment of Entomology, University of California. Riverside. CA 92521, U.S.A. (Received 4 Februa? 1991; Revised and Accepted 10 June 1990) Abmct-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 effccts. Benzene exposm in humans and animals has also k e n shown to result in structural and numerical chromosomal akmtions 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 incnased frec radical activity in beniceneinduced myelotoxic and leukemogenic effects. Benzene administration to rodents has been associated with increased lipid penxidation in liver, plasma, and bone marrow, as shown by an increase in the formation of thiobarbituric-acid reactive products that absorb at 535 nm. Bmzcne administration to rodents also results in incrca.4 pmstaglandin 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 conrains 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 superoNde radicals. respectively. These metabolites, upon co-administration also produce a myelomxicity similar to that observed with k n m e . Recently. we have found that exposure of human promyelocytic leukemia (HL-60) cells (a cell h e rich in myelaperoxidaK). to the benzene metabolites, hydroquinone and 1.2.4-benzenetriol results in increased stcady-state levels of 8-hydrOxydeo~yguanosine a marker of oxidative DNA damage. Peroxidatic metabolism of benzene's phenolic metabolites m y therefore be responsible for the increased fm radical activity and toxicity produced by benzene in bone marrow. We thus hypothesize that fm radicals contribute, at least in part, to the toxic and leukemogenic effects of benzene. Keywords-Benzene. Fme radicals. Lipid peroxidation. Oxidative DNA damage. Genotoxicity, Myeloroxicity, Leukemia Address all correspondence to Manyn T. Smith Depamnent of Biomedical and Environmental Health Sciences. School of Public H 4 t h . University of California. Berkeley, CA 94720. Vangala Subrahmanyam obtained hls Ph.D. in 1986 from Memorial University of Newfoundland, Canada. Aftcr two yuvs of post- Qcloral studies at the School of Pharmacy. University of Colorado, Boulder, CO, he joined the Department of Biomedical and Environ- m n u l Health Sciences. School of Public Health. University of Cali- fornia, Berkeley. CA. where he is currently appointed as Assistant Specialist Scientist. David Ross received hls Ph.D. in 1982 from the University of Aston at Birmingham in England. After postdoctoral sndies at the Department of Forensic Medicine. Karolinska Institute. stockboim. Sweden. and the School of Public Health,University of cllifamia. Berkeley. CA. he moved to the School of Warmacy, University of Colorado. Boulder, CO. w h m he is currently Associ*fmfeuar of Tox~cology.David Eastmond received his Ph.D. in - l9-8.7-fnrm the School of Public Health. University of c;llifornia, Bahdy. CA. He spem rwu years as a AJeXaodrr Hollacndrr Dis- a a g a h e d w f d h *a I~WIUILCi vC~ n n o rN~ational Labnrqir,CA. md now is aa Assistant Rofessw of Toxicology at I k U a i v u s i t y of W d Rivaside. Martyn Smith obtaurcd his - -pkI).-knn mC University of London in 1980, was a postdoctoral INTRODUCTION Benzene (BZ) is an ubiquitous environmental pollutant and an important industrial chemical. the annual production of which has been estimated to be over one billion gallons in the U.S.alone.' BZ is a constituent of gasoline and is also used in the manufacture of a wide variety of consumer goods such as plastic con- tainers, radios, toys, sporting goods, furniture, appliances, automobiles, tires, adhesives, textiles, dyes, drugs, pesticides, lubricants, solvents and cleaning products. In addition, BZ m a y be found as a contaminant in drinking water, ambient air, and certain types of foods. Extensive epidemiological studies have linked occupational exposure to BZ with the incidence of bone marrow toxicity and a variety of leukemias in human~.'- ~However, despite extensive research, the mo- k i b w t mC K a ~ ~ l i n sInhaimte urd is now Associate Professor of Tb#k&gy m me school of Public wth.univasity of caiifornia. P l j . CA.All /- g - r d i u lM -d q inveqatom . h ve m a x Lmg-sMckng icify i . nt n a. dw. in ~ oxy. lecular mechanisms responsible for the induction of bone marow toxicity and leukemia by BZ have yet to be fully elucidated. It is generally accepted that BZ re- 495 4% V. V. S m m m m i n u et al. quires metatmiism to induce its toxic effects.''-'4 Metabolism of BZ is very complex (Fig. 1) and is not completely understood. Numerous in vivo and in vitro studies have shown that BZ is primarily metabolized to phenol (PH). hydroquinone (HQ), catechol (CAT) and 1,2,4-benzenemol (BT).lSm The majority of these phenolic metabolites arc excreted in urine as glucuronide and sulfate conjugates. Some of the HQ and CAT, however, is further metabolized to reactive 1.4-benzoquinone (1.4-BQ) and 1.2-benzoquinone ( 1,2-BQ). respectively. Some investigators believe that these quinones are responsible for BZ-induced toxic effect~.'**~B'Z has also been shown to be metabolized to a ring-opened oxidation product, rans.trans-muconic acid, which has been detected in both bone marrow and urine after administration of BZ to mice. The corresponding dialdehyde trans,trans-muconaldehyde (t,tMA) has also been proposed to be one of BZ's toxic metabolites. Although this aldehyde has not yet been detected in vivo, studies by Lamano et al.32 have demonstrated that mouse liver microsomes can metabolize BZ to a product, which co-elutes with r,t-MA. The myelotoxic effects of BZ have also been associated with free radical formation, either as BZ metab- olites or oxygen radicals and lipid peroxidation products. 33-35Several reviews of BZ-induced myelotoxic effects in relation to its metabolism have dealt with quinones andor t.t-MA as the ultimate toxic metabolites. Little attention has been paid, however, to the toxicological significance of free radicals as ultimate toxic species. In this review we have attempted to compile the available evidence on free radical formation and associated damage during the oxidation of BZ and its metabolites and discuss their role in BZ- induced myelotoxicity and carcinogenicity. FREE RADICALS A free radical has been defined as any molecule that contains an odd number or unpaired A free radical is usually depicted as R', where the dot repre- sents the unpaired electron. Most free radicals are highly reactive and are short lived. The formation and reactions of free radicals are classified as three steps namely initiation, propagation, and termination. Initia- tion of free radical formation in biological systems may occur during enzymatic- or auto-oxidation or during reduction. The extent of propagation and termination reactions depends upon the reactivity of the initiated free radical and the environment in which the radical is formed. The biological threat from free radicals has been thought to be due to their interaction with cellular di- oxygen. Lipids, thiols, proteins. and DNA.-' The reactions of frcx d d s with dioxygen may eilber d t in the formation of peroxyl rarliFals or supcroxidt radi- cal anions (02.-)T.he latter arc known to dismutate. either spontaneously or catalyzed by supemxi& cfismu- tase (SOD),to hydrogen peroxide (Hz-02).Eurtlurmore, reaction of H202and 0," in the presence of iron or other transition metals could result in the formation of hydroxyl radicals (HO3. In addition. upon supply of energy, ground state dioxygen may be activated into reactive singlet oxygen (IO?)species. The activated oxygen species HO' and '0,may at- tack cellular DNA resulting in DNA-strand scission,43** and hydroxylation of bases.4547 Such processes have been implicated both in the initiation and promotion phases of c a r ~ i n o g e n e s i s I.n~ ~ad~dition, reactions of drug-derived radicals or oxygen radicals with cellular lipids may initiate lipid peroxidation.JOFree radicals of all types may interact with cellular macromolecules such as DNA, proteins. and carbohydrates and initiate or promote toxic and carcinogenic processes. BENZENE-INDUCED FREE RADICAL FORMATION IN VIVO Administration of BZ has been shown to induce the formation of thiobarbituric acid (TBA)-reactive products (having an absorbance maximum of 535 nm) in liver.33 plasma,33 and bone marrow34 of rats, suggesting the formation of malondialdehyde-like products (TBA-MDA adducts: E = 1.4 x 10SM-'cm-'). The authors of these studies have suggested that BZ-induces lipid peroxidation. Lamano and coworker^^*"-^^ have recently demonstrated that t,r-MA, the open-ring oXidation product of BZ also forms an adduct with TBA having an absorbance maximum at 490 nm (TBA-f.tMA-adduct: E = 7.3 X lo") with little or no absorbance at 535 nm. It therefore appears unlikely that these TBA-reactants absorbing at 535 nm in rats following BZ administration are due to t.t-MA. Since these studies did not record the whole spectrum of TBA-reactants, the formation of r,t-MA-TBA adducts, in BZ-exposed animals cannot be excluded. It is surprising, however, although the hplc techniques to separate TBA-t,t-MA adducts from TBA-t,t-MDA adducts have been available for some time," these techniques have not been applied to detect the formation of t,t-MA adducts in BZ exposed animals. Nevertheless, Gaido and WierdaJ3 have shown that BZ administration increases PGE, levels in bone m m w indicating increased arachidonic acid peroxidation. Laskin et al." have demonstrated an increase in the phagocytic activity of bone maxrow macrophages and granulocytes of Balbk mice treated with BZ (880 mg/kg) relative to i F m radicals in benzene myelotoxicity ~ 491 LJ t.t-rmcar;c add lA4-knunlrw l.L-buPog*#n Fig. 1. Metabolic pathways of benzene (adapted from refs. 13, 14). the control animals treated with saline or corn oil. They The above studies suggest that free radical forma- also indicated that a similar increase in phagocytic ac- tion during the metabolism of BZ in bone marrow tivity of bone marrow is observed following combined could play a role in BZ-induced myelotoxicity. Further- exposure of mice to HQ (50 mgkg) and PH (50 mg/ more free radical formation could play a role in the kg). Khan et al.35 showed that the S-9 fractions isolated from bone marrow of female albino rats following carcinogenicity of HQ to mouse liver.60 The following sections present the evidence for free radical formation the adminisnation of BZ (0.5 mUkg), had higher ca- during the in vitro metabolism of BZ and its phenolic pacity to oxidize dimethyl sulfoxide (a HO' scaven- metabolites in order to understand the exact mecha- ger), to formaldehyde and deoxyribose to thiobarbituric nisms by which BZ-exposure could lead to the for- acid-reactive products, indicating that BZ may increase mation of free radicals in bone marrow. We have the HO' production in bone marrow. Anwar et ai? also reviewed the literature on the mechanisms of in- have shown that dimethyl sulfoxide, can indeed reduce teraction of free radical metabolites of BZ with di- the formation of micronuclei in erythrocytes of BZ-ex- oxygen and cellular targets such as DNA, GSH, pro- posed mice. presumably by scavenging HO'. These teins and lipids and the potential significance of such authors, however, suggested that this reduction of BZ- reactions in BZ-induced myelotoxicity and leukemia induced genotoxic effects by dimethyl sulfoxide, may will be discussed. be due to the inhibition of HO `-mediated BZ oxidation to genotoxic r , r - M A , but mechanism for dimethyl sulfoxide effects remain unclear. Taken together, these studies indicate that administration of BZ to rodents increases the generation of oxygen radicals and lipid FREE RADICAL FORMATION DURING ENZYMATIC METABOLISM OF BENZENE AND ITS METABOLITES W VITRO peroxidation in bone marrow. Interest in free radical reactions of BZ metabolites Recently. Ekstrom et aLS6demonstrated excretion of stems primarily from the presence of high levels of malondialdehyde in urine of male Sprague-Dawley rats heme-protein peroxidase enzymes in bone marrow and given HQ (100 or 200 mgkg) by stomach tube. In a their ability to oxidize various phenolic compounds to follow-up study," this p u p demonstrated that HQ free radicals." Myeloperoxidase (MPO) and eosinophil administration to these rats also results in the depletion peroxidase (Em)enzymes constitute the majority of - of hepatic glutathione (GSH). These authors suggested the peroxidase activity detected in bone marrow.61 ttrat the malondjaldehyie excretion in urine thar occurs However, some investigators have suggested that the following ingestion of HQ to rats is presumably due to peroxidase activity of prostaglandin (H) synthase (PGS) lipid peroxidation that occurs following a depletion may also conmbute to the bioactivation of phenolic of hepatic GSH. similar to that observed in isolated metabolites of BZ in bone m a r r ~ w . T~his~ s.ec~- ~ ~ hepamcyres.58-59 tion focuses mainlv on MPO- and PGS-catalyzed me- - +- c. _ 398 v. v. sum-AM et d. N-N 0 Fig. 2. Catalytic cycle of horseradish peroxidase (adaptcd from ref. 66). RH and R' rrpresent electron donor and its free radial product ~ p c t i v e l y P. henolic metabolites (phenol. hydroquinone and catcchol) of bcnzene could also serve as elcctron donors to compounds I and tabolism of phenolic metabolites of BZ. The relative roles of these enzymes in catalyzing the bioactivation of BZ and its metabolites to free radical intermediates is unclear. Studies performed with neutrophils, bone marrow cells. and homogenates and the specific cell types of bone marrow are also described to indicate that peroxidase(s) present in these preparations can catalyze the bioactivation of BZ's phenolic metabolites similar to purified enzymes. A. Mechanisms of peroxidase-catalyzed oxidations The mechanisms of peroxidase-catalyzed oxidations of electron donors have been studied by several inves- tigators using horseradish peroxidase (HRP),a model plant peroxidase. Peroxidase enzymes contain an iron- porphyrin complex at the active site that can reduce peroxidese (Fig. 2). During this process the resting enzyme which contains an Fec3 heme iron is oxidized to compound I, which is believed to contain two-oxi- dizing equivalents above the resting state (Fecs).67 More recent studies have demonstrated that only one oxidizing equivalent is present in the iron moiety. The other oxidizing equivalent resides on the porphyrin moiety as a porphyrin n-cation Compound I can be reduced by a variety of electron donors to compound II. Compound II has been shown to be at F e f 4 oxidation state like compound I, but does not contain the porphyrin II-cation Compound II can also be reduced by electron donors releasing the native enzyme (FeC3). During the reduction of com- pound I and compound 11 by electron donors, highly reactive free-radical metabolites of electron donors can be formed.67 Pioncering work of Y d ef d.%" demonstrated that phenolic compoands were excclkm e l e m n donors for the higher oxidatim states of HRP. BZ. however. is not an electron donor for higher oxi- dation states of HRP formed from inuraaitm wnh hy- drogen peroxide (Submhmyam. V.V.. unpublished). Unformnately, the chemistry of heme-prosthetic p u p in MPO is not well understood. but recent studies by Dugad et indicate that the 'H Nh4R spectrum and the nuclear Overhauser effect (NOE)of cyanide-ligated femc complex of MPO (MPO-CN) were remarkably similar to the prosthetic group of lactoperoxidase. Nicholl and coworkers7' showed that the prosthetic group of lactoperoxidase is an iron-porphyrin thiol. Whatever the s t r u c m of the heme prosthetic group of MPO is, it forms spectral complexes with H20:- similar to that of "his indicates that MPO is potentidy a good candidate in catalyzing the phenolic metabolites of BZ to free radicals. B. Myeloperoxidase-catalyzedmetabolism of the phenolic metabolites of benzene Using the purified human MPO we studied the me- tabolism of PH.'la3 HQ,12*8a2nd*8C3AT83*84to de- termine the mechanisms of their oxidation by MFQ. Our studies show that HQ has the highest specificity for MPO followed by CAT and PH indicating that HQ is a better substrate for MPO than either CAT or PH. PH metabolism by human MPO results in the formation of 2.2'-biphenol and 4.$'-biphenol initially, which are further metabolized rapidly by MPO (Fig. 3). Studies with HRP as a model peroxidase indicate that 2.2'biphenol is the major metabolite of peroxidase-mediated oxidation of PH.8s48 The formation of biphenolic metabolites is indicative of initial phenoxy1 radical formation. the dimerization of which results in the formation of biphenols. Biphenols are also substrates for pemxidases. Our studies with both MPO and HRP show that further metabolism of 2.2'-biphenol results in the formation of insoluble melanin-like polymers as dark brown colored granular specks." 4,4'-Biphenol metabolism results in the formation of 4,4'-biphenoquinone, which in the presence of GSH results in the formation of GSH-conjugates.'' *89 Catechol metabolism by human MPO and H202re- sults in the formation of 1,2-BQ.80 Kalyanaraman and associate^^.^' have shown that CAT metabolism by HRP and H20Zproceeds through initial 1.2-benzosemi- quinone radical (1.2-SQ") formation. The fate of 1.2-SQ'- appears to be its spontaneous dispropomonation to 1.2-BQ and CAT. Using HPLC analysis, we characterized 1.2-BQ as its bromothiophenol adduct Free radicals in benzcne myclotoxicity 499 2.2'-oiptnd I Ino' I I 1 I*[-= l+?zyjProdwt. POlymWlC *Caolino Peroxieso RodWt. 7 I I 00- 4.4l'-- ! ! Fig. 3. Mechanisms of metabolic activation of phenol by human myeloperoxidase (adapted from ref. 12). ! during the metabolism of CAT by MPO/H,O, or HRP/ be its spontaneous disproportionation to 1,4-BQ and I H20,.80 1,2-BQ could not be isolated and purified due HQ. similar to that of 1,2-SQ'-. However, 1.4-SQ'I to its instability. Subsequently, we analysed GSH-con- may, under certain conditions, also react with dioxy- jugates formed during CAT metabolism by MPO/H202 gen resulting in the generation of 02'-,which will be I or HRP/H,02 in the presence of GSH. Two GSHcon- discussed later in this article. jugates were formed, the major conjugate appears to be I the mono GSH-conjugate of 1.2-BQ (Fig. 4). The fare C . Metabolism of benzene and its phenolic metabolites I of GSH-conjugates of CAT, if formed in bone marrow, is unclear. by neutrophils Neutrophils typically make up 50-7096 of the white I Hydroquinone metabolism by human MPO and H20, cell fraction of human peripheral blood.92 Large num- I results in the formation of 1.4-BQ as the major metab- olite. " Yamazaki and coworker^^^-^^ have demon- bers of immature neutrophils also reside in the BM.93-96 MPO comprises 5 8 of the dry weight of neutrophil^.^^ I strated that the oxidation of HQ by HRP and H20' Neutrophils also possess the ability to be stimulated by I proceeds through 1.4-benzosemiquinone radical (1,4- several particulate and nonparticulate agents resulting I SQ'-) intermediate. The fate of 1.4-SQ'- appears to in oxygen radical generati~n.~T'his process, known as the "oxidative burst" is due to the activation of a membrane-bound NADPH oxidase which is dormant in resting cells and becomes activated during phagocyto- sis or following interaction with suitable stimulants. Flavoproteins, cytochrome bSs8 and ubiquinone have been proposed to be the components of this oxidase- system. The presence of ubiquinone is, however, con- troversial. Many of the stimulants of the oxygen radical I activity in neutrophils appears to mediate their actions by activating protein kinase C, either by a direct inter- action or indirectly by receptor-triggered cascade (Fig. 5).97 The receptor-triggered cascade involves the acti- vation of phospholipase C, which acts on phosphatidyl - inositol (P-inositol) and hydrolyses it to diaclyglycerol. The compounds having structural similarity to diacyl- .glycerol. such as phorbol myristate acetate (PMA; stim- ulant 4, Fig. 5 ) have been shown to directly activate protein kinase c . p~rotein kinase C appears to regu- ;. _- .. ....,- . .. :. V.V.Surrwrunrurrnd SflYULANT : (0.0. F W P ) J. Rocoptor 4CTP-bindlng protoin STIWLAWT 2 (0.0. comardln A) J- Roeoptor '"itP-Inositol I .--- -phoiphollpaio C) I/ Diacyiglycorol STIYUUNT 4 ST(0lM.9U. LACON"l S Y +[ca**], (0.9. PUA) lonophoror) Protein Phosphorylation. conformational modiflcotlonr and/or arromblr of NADPH -J. <oxidoro complox STIMULANT 5 (0.9. oxogonour arachidonic acid) 0, 0,- Fig. 5. pathways for stimulation of NADPH-oxib activity (adapted from ref. 98). Benzene may also stimulate NADPH oxidase activity r h r ~ g hprotein lrinase c activation.'m*'o' late the activity of NADPH oxidase and result in the stimulation of oxidative burst in neutrophils. Recent studies by Castagna and colleagues'oo~lolhave shown that BZ can activate protein kinase C activity in intact -platelets isolated from rabbit blood. but very high concentrations of BZ (0.8 4.8%) are required. The physiological relevance of this phenomenon is doubtful. PMA is known to activate protein kinase C in human promyelocytic leukemia (HL-60) cells and also results in the differentiation of these cells to macrophag= 102-106 m-60cells, however, do not possess oxidative burst, but do develop the oxidative burst during their differentiation to macrophages.1o3 Recent studies by Kalf and colleagues have shown that incubation of HL-60 cells with BZ induces differentiation of these cells into macrophages,1M presumably by activating protein kinase C. In contrast, preincubation of HL-60 cells with HQ (0.5-5 pM) inhibits the PMA-induced differentiation of HL-60 cells'08 suggesting that HQ or its metabolite 1.4-BQ may act by inhibiting protein kinase C. It is interesting to note that BZ and its metab- olite HQ have contrasting effects on HL-60 cell differentiation. The relevance of these studies in BZ- induced myelotoxic effects is not clear. Kalir et ai.'09 have shown that addition of high concentrations of BZ (1.3 mh4) to human neutrophils results in a marked decrease in the number of cellular granules, a decrease in the amount of heterochromatin and a rarefication (decreased density) of ribosomes. These effects of BZ were prevented in the incubations containing 2-aminoethylthiosuic acid, a free radical scavenger. These authors concluded a free radical metabolite of BZ was responsible for the observed toxic effects on neutrophils, but did not detemine the nature of the free radical metabolite. We have shown that PH and HQ are activated to DNA and protein-binding metabolites by intact neutrophils stimulated with the tumor promoter PMA.l'o-l'z Inhibitors of MPO such as azide and cyanide inhibited the PH-"' and HQ-'" induced DNA- and protein- binding, respectively, in neutrophils, indicating that MPO is involved in the activation of these phenolics in neutrophils. SOD,which increases the dismutation of ~ F m radicals in benzene myelotoxicity 501 02*t-o H202, increased the covalent binding of PH to neutrophil DNA. Conversely, catalase, which decomposes H,02 to dioxygen and water, decreased the covalent binding of PH to neutrophil DNA.'" These results indicated that H202 is required for the activation of PH and HQ to products that covalently bind to neutrophil DNA and protein. Since H202 is required for peroxidatic metabolism of BZ's phenolic metabolites, we have attempted to determine whether they activate neutrophil NADPHoxidase activity similar to PMA and thus generate H20, required for their metabolism by MPO. Our efforts using manometric studies (Subrahmanyam, V.V. and O'Brien. P.J.,unpublished) and cytochrome c reduction (Eastmond, D.A. and Smith, M.T.,unpub- lished), to determine whether the phenolic metabolites of BZ could stimulate the oxidative burst in neuuophils were negative but inconclusive. It is possible that these techniques are not sufficiently sensitive to detect small increases in oxidative burst activity in neutrophils. Al- ternatively, it is possible that BZ and its metabolites could prime the NADPH oxidase activity for activation by endogenous stimulants. In support of this, Laskin et al." have recently demonstrated an increase in phagocytic activity of bone marrow cells of mice adminis- tered with BZ or PH and HQ, indicating that BZ or phenolic metabolites of BZ may act by priming the NADPH-oxidase activity of bone marrow cells. Unfortunately. whether such priming of NADPH oxidase activity. by BZ or its phenolic metabolites, occurs in vitro and neunophils or bone marrow cells has not been tested. D . Metabolism of benzene and its phenolic metabolites by bone marrow cells and homogenates Benzene metabolism in bone mmow homogenates is mediated by the cytochrome P450 monooxygenase system.1'3-'15 This enzyme system has been shown to hydroxylate BZ to PH. The significance of cytochrome P450, however, in the hydroxylation of BZ in situ in bone mmow is not clear and requires further work. The mechanism(s) of BZ hydroxylation to PH has been reviewed extensively in the recent years and therefore we suggest that interested readers consult those reviews, ".'' We have shown that bone marrow cells and homogenates of rat. mouse. and human have the capacity to metabolize PH.95."6 HQgSand CAT'" in the pres- ence of H20, as cofactor. In mouse bone marrow cells sindin_pof '"c-cAT metabolites to protein (20nmobmg protein) was found to be twice that obtained with '*C-HQ (10 nmoYmg protein) and twelve-fold higher than that obrained with '"C-PH (1.5 nmol/mg prorein;.95 01 I eon ~ 0 . am, ~ 01 I Fig. 6. Arachidonic acid metabolism by prostaglandin H synthase. RN and R' rrprcsent electron donor and its free radical product. re- spectively. Phenolic metabolites (phenol. hydroquinone. and catcchol)of benzene could also m e as electron donors for the pcroxidasc activity of prosraplandin H Metabolism of PH by rat bone marrow homogenate in the presence of H,O, has been shown to result in the formation of 2.2'- and 4,4'-biphenol as the major indicating the peroxidative nature of the metabolism. Extensive protein binding of PH-equivalents to bone marrow homogenate also occurred. Using HRP and hydrogen peroxide as a model system, we have characterized the nature of products binding to bovine serum albumin. This protein-binding appears to result from a nonconvalent interaction of polymers (further oxidation products of 2,2'-biphenol) as well as a covalent interaction of 4.4'4iphenoquinone (fur- ther oxidation product of 4,4'-biphenol) with sulfhydryl groups of albumin). The binding of the polymers to protein is so strong that normal procedures for establishing covalent binding to protein were not effective in removing all of these polymers from protein. Enzymic digestion of protein, however, resulted in the release of the polymers from protein. A charge-transfer complex formation between the polymers and the protein has been tentatively suggested as an explanation for this noncovalent. yet strong interaction of these polymers with protein. The toxicological si@icance of this type of interaction is not clear, but such an ex- - 502 v. v. -AM tensive protein-binding could result in the inactivation of various cellular enzymes and conmbute to cytotoxic events. Extensive noncovalent binding of polymeric oxidation products of PH with DNA has also bem ob- served during a peroxidatic metabolism. which will be described in later sections. Metabolism of CAT by rat and human bone marrow cells also results in the formation of reactive metabolites that bind to protein.'17 Protein binding could be inhibited by GSH. We have shown that the GSH-conjugates are formed from the reaction of GSH with 1,Z-BQ formed during the metabolism of CAT. Although H,O?-dependent metabolism of HQ and CAT in bone marrow could be catalyzed by either peroxidases or cytochrome P-450, indirect evidence using inhibitors suggest that cytochrome P-450 plays little role in peroxidatic bioactivation in bone marrow. 'I7 E. Metabolism of phenolic metabolites of benzene by specific cell ypes of bone marrow Bone m m w stroma primarily consists of two cell types (macrophages and fibroblastoid stromal cells) which appear to control the differentiation and maturation of myelopoietic cells via the production of growth factors."6 Studies from Wierda's"' laboratory have shown that macrophages are more sensitive than fibroblastoid stromal cells to the toxic effects of HQ. Interestingly, bone m w macrophages contain appreciable peroxidase activity while fibroblastoid cells do not.'** Hydrogen peroxide-dependent bioactivation of HQ occumd readily in bone m m w macrophages, but not in fibroblastoid stromal cells, indicating macrophages contain an active peroxidase. The selective toxicity of HQ to bone marrow macrophages rather than fibroblastoid stromal cells could be a result of both increased peroxidative activation of HQ to 1.4-BQ in the macrophage and an increased deactivation of 1.4-BQ in the fibroblast by the obligate two electron reductase, DT-diaphorase. Inhibition of DT-diaphorase by dicumarol in the stromal fibroblast, but not in the stromal macrophage, has been shown to lead to increased covalent binding of HQ to protein.'*' These data and previous work,iu suggest that activation and deactivation mechanisms, such as peroxidase and DT-diaphorase, respectively, in individual cell types are an important determinant of the cell specific toxicity of phenolic metabolites of BZ. F. Prostaglandin (H)synthase-catalyzed metabolism of phenolic metabolites of benzene Prostaglandin (H) synthase (PGS), another heme- protein enzyme that is mainly involved in arachidonic ef d. acid (AA) metabolism to genenut pSi#hdins, has also been implicated in CWltidBticm of several camno- gens and drugs to reactive species (Fig. 6).i3*'24This enzyme has two distinct activities, being both a cy- clooxygenase and a peroxidase. It has been established that AA is oxygenated by the cyclooxygenasc actlwty of this enzyme to generate the endopcroxy hydroperox- ide (PGG,).Two molecules of oxygen are incorporated into AA during this process. This hydroperoxide is subsequently reduced to a hydroxy derivative of AA (PGH?) by the peroxidase activity of PGS. This perox- idase activity of PGS is similar to the peroxidase activ- ity of other peroxidases and cooxidizes a wide variety of electron donors to free radical intermediates.i23*124 Indomethacin, a nonsteroidal anti-inflammatory drug has been shown to inhibit the cyclooxygenase activity of PGS but has no effect on its peroxidase activity.'= Prostaglandins. such as PGE?. have been shown to down-regulate granulocytehonocyte colony formation and may contribute to the myelotoxic effects of BZ. Wierda and his associates53have demonstrated that in- traperitoneal administration of BZ (200 mg/kg; twice a day for four days) to male B6C3F1 mice elevates PGE, levels in bone marrow. This increase was prevented by pretreatment of mice with indomethacin (2 mg/kg). Further studies by Kalf et al.62*63have demonstrated that indomethacin (2 mgkg) can alleviate the genotoxic effects (micronucleus formation)62 and bone marrow depression63 induced in bone marrow following in- traperitoneal administration of BZ (0-1200 mg/kg twice daily for two days) to Swiss-Webster male mice. These results led Kalf et al.62.63to suggest that PGS activity in bone marrow may be involved in the myelotoxic effects of BZ. The major problem with this hypothesis is that in- domethacin is assumed to be a specific inhibitor of PGS and to act only in the bone marrow. Indometha- cin may have profound effects on the toxicokinetics of BZ and its metabolites. It can also inhibit MPO," BZ-dihydrodiol dehydrogenaseiz6 and diglyceride li- pase.lZ7 and ingestion of indomethacin (5 mg/kg) has also been shown to reduce intestinal peroxidase activ- ity in rats.'28*''9 Moreover, PGS has never been purified from bone marrow. To date there are only two major peroxidases isolated from bone marrow, Le., MPO and Recently, Zilletti et al.I3O have shown that HRP and bovine milk lactoperoxidase are capable of oxidizing AA to prostaglandins, but this activity has yet not been demonstrated with either MPO or PO. However, they did show that indomethacin inhibited the peroxidase-dependent formation of PGE, from AA with an IC,, of 3 pM. Thus. it is possible, that the prostaglandins detected in bone marrow after BZ ad- ministration are the result of peroxidase activity and not that of PGS. -. L ,- I1- I 8. Free radicals in benzene rnyclotoxicity 503 Early studies by Christ and Van Dorp13' indicated that guinea pig bone marrow contains no detectable PGS activity. Ziboh et al.132 incubated bone m m w microsomes (isolated from Sprague-Dawley rats) with 14C-AA and found that PGE, and PGF, (another prostaglandin metabolite of AA) were the major metabolites. These studies contained at least 6-mg microsomal protein in 2 ml of reaction mixture. The total conversion of AA into the products was found to be less than 2%. These incubations also contained HQ (0.55 mM), presumably to prevent the rapid inactivation of cyclooxygenase activity by AA metabolites. However, no studies were performed by these authors to determine the fate of added HQ. Kojima et al.133incubated "C-AA with 15 mg of whole bone marrow homogenate (prepared from male Wistar rats) in 0.5 ml of incubation mixture and found that approximately 80% of the added AA was converted into three products. The major product, which contained approximately 60% of the incubated AA, was not identified and was assumed to be a nonprostaglandin derivative due to its distinct chromatographic properties. The other major product, which contained approximately 20% of the incubated AA, was tentatively identified as prostaglandin D2. Formation of these metabolites from AA is completely inhibited by indomethacin (25 FM). The requirement of large amounts of bone marrow protein to study the metabolism of AA by bone marrow suggests that bone marrow contains very low levels of prostaglandin biosynthetic capability. Ziboh et al.132 reported that microsomes isolated from hyperplastic bone marrow (turpentine-induced) contained 2.5fold higher prostaglandin biosynthetic activity compared to the activity in normal bone marrow microsomes. However, the total formation of PGE, and PGF, in hyperplastic bone mmow represented only 4.2% of incubated AA. Gaido and Wierdas3 suggested that macrophages present in adherent bone marrow stromal cells could be the source of PGS activity in bone marrow. These au- thors found that incubation of HQ with adherent bone marrow stromal cell cultures induced PGE, synthesis in these cells. They also showed that the stromal cells incubated with HQ also prevented granulocyte/monocyte colony formation in co-culture. The presence of indomethacin in these incubations ameliorated the toxic effects of HQ on granulocyte/monocyte colony formation. Interestingly. indomethacin was not able to reduce the PG& levels induced by HQ in these c u l m s . These authors concluded that HQ-induced stromal cell toxicity was not solely due to incnased PGS activity. Kalf et al..a2-as however, maintain that bioactivation of phenolic metabolites of BZ by PGS is an important step in BZ-induced bone marrow myelmxicity. These authors conducud a series of exptnments usmg puri- fied PGS (from ram seminal vesicles) and isolated peritoneal macrophages (from the peritoneal cavity of male C57BU6 m i ~ e ) . T~h.ei~r ~experiments showed that purified PGS. either in the presence of AA or H202, readily catalyzes the bioactivation of 14C-PH and 14C-HQ to reactive protein-binding metabolites. These authors also showed that a similar H,O,-dependent bioactivation of HQ and PH occurs in peritoneal macrophages, indicating a peroxidase is responsible. The observation that purified PGS can catalyze activation of phenolics via cooxidative mechanisms is not surprising, and the critical question is the role of PGS in bioactivation of phenolics in bone marrow cells in general and macrophages in particular. Using white bone marrow cell fractions from either mouse (Ross, D., unpublished) or rat'" we have not been able to demonstrate a major role for PGS in bioactivation of phenolics to protein binding species. Studies in purified bone marrow cell types are of more relevance, however, and using bone marrow macrophages, rather than peritoneal macrophages, we have shown that they are indeed capable of peroxidatic bioactivation of HQ to 1,4-BQ, which can either form a GSH-conjugate andor covalently bind to protein.120 We have been unable to demonstrate arachidonate stimulated covalent binding of "C-HQ to protein, which would implicate PGS in the binding process, in bone mmow macrophages (Ganaosis,L. and Ross,D., unpublished). Schlosser and Kalf,64*65however, have reported such a stimulation, albeit marginal, in peritoneal macrophages. Michel et al.'" found that mouse bone marrow macrophages can catalyze the synthesis of PGD, from AA that is associated with endogenous peroxidase activity. The nature of peroxidase present in the bone marrow macrophages may therefore be MPO or an, as yet, uncharacterized peroxidase. Since peroxidases other than PGS have also been shown to transform AA to prostaglandins, it remains to be determined whether the prostaglandin biosynthetic activity of bone marrow macrophages is due to PGS or other peroxidases. Clearly much more work needs to be done to determine whether PGS plays a role in modulating BZ toxicity. Whether the role, if any, of PGS in BZ toxicity is at the level of peroxidatic activation of phenolic metabolites to reactive species or is a result of altered levels of eicosanoids in bone marrow due to the presence of BZ metabolites remains to be determined. MOLECLZAR AN?) CELLULAR EFFECTS OF BENZENE AND ITS METABOLITES In order to understand the mechanisms by which BZ exerts its toxic and leukemogenic effects, numerous studies have attempted to identify the cellular and mo- 504 V. V. SlowWmm et d. Table 1 . A Comparison of HRP/H,O,-Mediated Binding of Oxidation Roducu of l%phcnol. `%atecho1 and `%hydroquinone to Calf Thymus DNA Condition' `v-bound to DNA (nmoUmg) Phosphate Buffer (0.1 M , pH 7.4) W-PH. HRP. HzO,, DNA minus HRP I4C-CAT. HRP,Hz02. DNA minus HRP `%-HQ. HRP.Hz02. DNA minus HRP Tris HCI Buffer (0.1 M; pH 7.4) "C-PH, HRP.H202, DNA minus HRP "C-CAT. HRP, HZOZ. DNA minus HRP I`C-HQ, HRP.H202, DNA minus HRP 66.8 s 15.7" 0.6 2 0.2 14.6 2 1.2 0.5 2 0.2 0.56 2 0.18 0.46 2 0.2 42.1 2 8.4 0.6 2 0.1 4.89 2 0.12 0.38 2 0.04 0.33 2 0.08 0.24 2 0.05 `Incubations of either phosphate or ds-HC1 buffer, contained phenolic substrate (200@). HRP (10 pg) DNA (1 mg). The rextions were initiated with the addition of H202 (400 @) and incubated for 30 min at room temperature. DNA was purified as described previously by Subtahmanyam and O ' B ~ i e n . ~ ' . ~ ~ bvalues represent mean -c S.E.M. (n = 3). lecular effects that occur following exposure to BZ and its metabolites. Most research has focused on the effects of 1,4-BQ and BZ's phenolic metabolites due to evidence for their involvement in myelotoxicity, their commercial availability and their stability in aqueous solutions. Exposure to other reactive BZ metabolites such as 1,2-BQ, t,t-MA, or BZ-epoxide may induce similar effects although much less data is available on the adverse effects produced by these compounds. Indeed, W i a et al.I3' have shown that t,t-MA is a potent myelotoxicant in CD-1 male mice. The adverse effects of the benzoquinones and t,t-MA are primarily related to their ability to react with nucleophilic sulfhydryl and amino groups at key functional sites on proteins and with nucleophilic sites on DNA. In addition, the ability of quinones to undergo cyclic reduction and oxidation reactions (redox cycling), producing oxygen- and lipid-derived radicals, has also been implicated in the induction of pathological effects by these compounds. The following sections will overview the research efforts in five areas, DNA damage, protein damage, oxygen activation, GSH oxidation and lipid peroxidation, which have been proposed as being involved in the hematotoxic effects of BZ. A. DNA-damage Due to an association between neoplastic development and the ability of many chemical carcinogens to bind to DNA,136considerable research has focused on the interaction of BZ and its metabolites with DNA. Initial studies by Lua and Scfilattef3' r e c o v d radio- activity bound to rat liver DNA following the admini.ctration of mtiared and "C-BZ to male rats. Additional studies by Gill and A b ~ n e d 'an~d~Arfellini et d.139 demonstrated that BZ (or metabolites) was capable of binding to bone marrow DNA following in vivo administration. More recently, a series of investigations has focused on the ability of the individual BZ metabolites to bind to deoxynucleotides and DNA in vitro.L40-L4s These studies have indicated that 1.4-BQ, HQ, PH. BT. t.r-MA. and CAT are all capable of forming ad- ducts with deoxynucleotides and DNA during in vitro incubations. However, the nature of the binding in the bone marrow and the identity of the reactive species involved remain largely unknown. We have performed extensive in vitro studies to de- termine whether free radicals or other oxidation products of BZ could bind to the DNA. Table 1 shows a comparison of binding of reactive metabolites of `"CPH, I4C-CAT, and 14C-HQ formed during a peroxidase-catalyzed reaction. The reactions were performed in phosphate buffer as well as in ms-HC1 buffer at pH 7.4.Extensive binding of PH and CAT oxidation prod- ucts to DNA was observed. The binding of "C-PH, or `"C-CAT oxidation products to DNA is better in phosphate buffer than in ms-HC1 buffer. Oxidation of "C- HQ, either in phosphate buffer or in ms-HC1-buffer, however, resulted in no significant increase in the binding of I4C-HQ oxidation products to DNA com- pared to that in the absence of enzyme. Nonenzymic binding to DNA, of HQ-autooxidation products, is also not significantly different in both buffers. Previous studies aimed at determining the ability of 1.4-BQ to bind DNA and RNA. during microsomal activation of 14C-BZ or `"C-PH, have resulted in contradictory results. The studies by Subrahmanyarn and O'Brieng8and Tunek et a l . ' ~fa~i~led to detect any covalent binding of 14C-PH oxidation products with DNA or RNA, during microsome-mediated activation although PH metabolism occurred. Arfellini et al..I3' however, found a significant association of radioactivity with DNA dur- ing microsomal activation of "C-BZ, which was amibuted to a covalent interaction of 1.4-BQ with DNA. In addition, Snyder and c ~ w o r k e r s 'f~ou~nd' ~th~at~1.4-BQ covalently interacts with DNA following their incubation together. While our studies were performed in tris-HC1 buffer.88 Arfellir~i,'a~nd~ groups performed their studies in phosphate buffer. The data presented in Table 1, however, shows that there is no significant difference in the DNA-binding of 14C-HQ oxidation products, in either buffer. Therefore, it is clear that the differences in our results with the other groups were not due to the different buffers used. Nevertheless. the data in Table I indicated that some oxidation products of 14C-HQ are capable of binding A F m radicals in benzene rnyelotoxicity 505 to DNA. presumably due to autooxidation, since the peroxidatic metabolism of 14C-HQ did not increase this binding that occurred in the absence of the enzyme. On the other hand, the reason for the increased binding of 14C-PH or I4C-CAT oxidation products in phosphate buffer is not clear and remains to be determined. How- ever, an analysis of the DNA-binding of PH oxidation products revealed that most of the binding is due to a noncovalent, charge-transfer complex formation between the melanin-like polymers and DNA!' DNA had no effect on the formation of biphenols which indicated that phenoxy radicals were not capable of binding to DNA. HRP-catalyzed oxidation of CAT also resulted in the formation of black melanin-like products, the formation of which was not inhibited by the presence of DNA (Howell, M.; Ross, D., and Subrahmanyam, V.V., unpublished). Similarly, the peroxidatic metabolism of the bladder carcinogens benzidine, I phenylenediamineI6 and methylaminoazobenzene,la~nd the uterine carcinogen diethylstilbestrol.14' have been shown to form polymeric oxidation products that interact noncovalently with DNA. Peroxidatic metabolism of acetaminophen has also shown to result in the formation of polymers,'48 but whether these products bind to DNA has not been investigated. The interactions of polymers with DNA may result in irrepairable mutagenic changes. but this remains to be established. But, if this type of binding occurs in the cells, repair may be difficult and the cell may die readily. Cell proliferation may be initiated in neighbouring less damaged cells. Snyder and coworkersL4'*'" have isolated DNA adducts formed following chemical oxidation of HQ using FeCl, in the presence of DNA or following the incubation of 1.4-BQ with DNA and have identified these adducts as (3'OH) benzethenol(1,N2)deoxyguanosine. These investigators have recently reported, using a 32~-postlabellingtechnique, formation of several DNA adducts in the liver DNA of rabbits following the in vivo administration of BZ.'49 However, neither the chemical structure of thls adduct nor its ability to co-chromatograph with the known standard 1,rlBQ-adducts was determined. In contrast, Reddy and associates.150.151 using a nuclease PI-enhanced 32Ppost-labelling technique, have been unable to detect DNA adducts in the liver. kidney, bone mamow, and mammary gland of rats mated with BZ or PH and HQ. Some preliminary evidence of the formation of adducts in the Zymbal @and of BZ-treated rats was observed Qrring these experiments. Studies by I\iorpotb et al. have identified N'-phenylguanine in the urine of rats following the intraperitDneal administration of BZ at 330-400 m e g . These autburs suggested mat this adduct was formed h u g n the reaction of BZepoxide with guamne residues in DNA and that its presence in the urine was the result of the excision repair of this DNA adduct. In spite of the considerable effort which has been made to identify DNA binding following BZ administration and to characterize the nature of the DNA adducts, there is no direct evidence for the involvement of these events in BZ-induced leukemia. The degree of covalent DNA binding recovered following BZ administration is low and rankings of carcinogenic agents by DNA binding generally rank BZ among the weakest initiating agents, approaching agents that act through indirect genotoxic mechanisms.136a139 Subrahmanyam and O'Brien,87 several years ago, found that an in vitro peroxidative metabolism of PH leads to the formation of polymeric products that strongly bind to DNA but do not form covalent adducts. The binding of these polymers to DNA is so strong that normal procedures for establishing covalent binding to DNA were not effective in removing all of these polymers from DNA. Therefore, a strong association of some radioactivity with DNA, following administration of radio-labelled BZ to animals, should not be taken as indicating cova- lent interaction of BZ metabolites with DNA. In addition, BZ and its metabolites are weakly or nomutagenic in specific gene mutation assays.153These observations suggest that other types of molecular events are likely to be involved in the development of leukemia by BZ. B. Protein-damage One consistent observation that has been made in BZ-exposed humans and animals is the appearance of structural and numerical chromosomal aberrations and sister chromatid exchanges in lymphocytes and bone marrow cells indicating that BZ is g e n o t o x i ~ . ' ~ ~ ' ~ ~ Chromosome and chromatid deletions, gaps, chromatid exchanges, hyperdiploidy and micronuclei induction, all have been detected to some extent. Since the induction of chromosomal aberrations in humans have been associated with a variety of neoplastic development,158.159 these chromosomal aberrations could also contribute to the myelotoxic and leukemogenic effects of BZ in humans. BZ administration to animals is also accompanied with an inhibition of DNA, RNA, and protein synthesis (reviewed".' '). BZ metabolites interfere with DNA replication and transcription in rat liver and BM mitochondria in vitro that has been attributed to the inhibition of DNA polymerase gamma activity by HQ and 1.4-BQ. Studies by Lee and coworkers16o have confiied that an inhibition of DNA synthesis occurs in bone marrow following the in vivo administration of BZ to mice and have indicated that inhibition occurred at doses that had no inhibitory effect on the synthesis of protein or heme. These authors reported that BZ itselfhad the ability to inhibit DNA sq-nthesis in vitro in 506 v. v. slarurrunuc ct d. cell-free incubations following the addition of BZ. Ad- ditional studies by these investigators studying the ef- fects of individual BZ metabolites indicated that CAT, BT, HQ, and 1.4-BQ were able to inhibit nuclear DNA synthesis in cultured mouse bone marrow cells. 16' How- ever, only 1.4-BQ and BT were able to inhibit DNA synthesis in a cell-free system. This effect appeared to be due to an interaction of these compounds with DNA polymerase-alpha. The inhibition of DNA polymerase-alpha or -gummu activity by HQ and 1.4-BQ was suggested to be due to the arylation of 1.4-BQ with a critical sulfhydryl group on these proteins. Due to the arylating activity of quinones with sulfhydryl groups, ~ r o n s ~s'everal years ago postulated that quinone metabolites of BZ may act as spindle poisons during cell division and disrupt microtubule assembly. This may cause lagging chromosomes and result in numerical chromosomal ab- errations (aneuploidy). Indeed, Irons and colleagues'62 have demonstrated that administration of BZ to animals results in the block of cell division in the G2 or M phase of the cell cycle, supporting a role for disruption of microtubule assembly. These investigators also found that HQ and 1,CBQ are highly effective inhibitors of microtubule assembly in mouse lymphocyte^'^^ and isolated microtubule preparations. 16J The mechanism of this inhibition appeared to involve the 1.4-BQdepen- dent arylation of extremely nucleophilic sulfhydryl groups on tubulin, which function in GTP binding and serve as a control site for the regulation of microtubule as- sembly. The inhibition of microtubule assembly by l&BQ appeared to parallel the inhibition of cell growth and microtubule assembly induced by the T suppressor celI lymphokine, soluble immune response suppressor (SIRS)suggesting that 1,4-BQ may act by mimicking this intracellular messenger in vivo.'6o Although the spindle-poisoning effects by BZ metabolites appears to be a likely mechanism for the induction of numerical chromosomal aberrations, it cannot explain the induc- tion of structural chromosomal aberrations that have been observed following BZ exposure. Such events probably occur via DNA damage, either through oxi- dative mechanisms or adduct formation. C.Active oxygen formation Ground state dioxygen itself is a radical species since it has two unpaired electrons with the same spin number (i.e., mplet state). Supply of energy can activate the triplet oxygen to highly reactive singlet oxygen in which the two-electrons have opposite signs. Other active oxygen species such as 02'-H,202, and HO' can also be generated during successive one-electron reduction of dioxygen to water [eqn (l)]?' e! o,+ e-=- e- - e II f ---.H,OZ--rtIO - + H O . 4 0 - . IH.0 (1) Oxygen radicals m a y iittacic ceiluiar DNA resuiring in DNA-strand scission"*w and base d a ~ n a g e . ~ " ~ . ~ ~ Some of the basedarnaged products arc hydroxyked bases which include 8-hydroxydeoxyguanosine (8- OHdG). thymine glycol (5.6dihydroxydihydrothymine) and hydroxymethyluracil. Presence of 8-OHdG in DNA has been recently shown to cause DNA-polymerase to miscode nucleotide incorporation in the replicated strand and result in mutagenesis. 166.167 Some xenobiotic free radicals have the ability to in- teract with dioxygen, resulting in 02'- formation.16' However, oxidation of PH, CAT. or HQ by peroxidase and H.0. which results in the formation of phenoxyl- or 1.2-SQ'- or 1,CSQ'- radicals, respectively, did not result in any oxygen consumption.167This indicates that these radicals may not be reactive enough to inter- act with dioxygen. Sawada et have, however, demonstrated increased 1,CBQ formation during per- oxidase-catalyzed oxidation of HQ in the presence of SOD. They suggested that the reaction of 1.4SQ.- with dioxygen could indeed occur if the 02'-are re- moved from the reaction. This appears to be due to faster back reaction, i.e., reaction of l,.IBQ and 02'- forming 1.4-SQ'- and dioxygen [eqn (2)] 1.4-SQ'- + 0 2 =1,4-BQ + 0 2 ' - . (2) Our studiesw confirmed Sawada et al.'~'~' study showing an increased production of 1.4-BQ during peroxidatic metabolism of HQ in the presence of SOD. Interestingly, we found no increased production of 1,2-BQ during peroxidatic metabolism of CAT in the presence of SOD.@ It has been shown, however, by Kasai and Ni~himura'~th' at incubatim of either HQ or CAT with H2O2. femc iron (Fec3) and deoxyguanosine results in the formation of 8-OHdG, presumably due to the addition of HO' to deoxyguanosine. Recently, Iwahashi et ale172 showed that a variety of catechols, including CAT in the presence of femc iron, EDTA and H,O,, could result in the formation of HO'. The mechanism appears to be due to the initial reduction of iron from the femc (Fef3)state to ferrous (Fec2) state by CAT. The reaction of Fec2 with H202 thus generates the reactive HO' as shown below: + +CAT FeC3--f 1.2-SQ'- Fec2, (3) +FeC2 + H202+ HO' + OH- FeC3. (4) Similarly, HO' formation was described by Rao and za 0.4 n -0, =L 0.3 \ Free radicals in benzene myeiotoxicity -- BT(1OpM) HQ(10pM) QD I =o 15 30 45 60 75 Time (min) Fig. 7. Formation of 8-hydroxydeoxyguanosinein DNA of human promyelocytic leukemia cells cells exposed to benzene metabolites. Pandya,'73 who showed that incubation of HQ or BT with DNA in the presence of copper ions ( C U + ~r)e- sults in the formation of thiobarbituric acid reactive products. This process was inhibited to some extent by mannitol, a HO' scavenger, and also by catalase and SOD, which prevent the HO' formation by decompos- ing H202 and removing 02*-re,spectively. In con- trast, Kawanishi et al.174did not detect HO' formation using electron spin resonance during auto-oxidation of BT in the presence of copper ions, although they de- tected HO' formation in the presence of iron (Fe+3). They indicated that the DNA-damage induced by BT and copper ions in their studies may not be due to HO'. Lewis et have shown that DNA-damage also occurred during the auto-oxidation of HQ and BT at physiological pH but only the damage induced by the latter was inhibited by scavengers of 02'-,Hz02, and HO'. It is likely that the differences in these stud- ies are due to differences in the concentrations em- ployed. The relevance of metal-catalyzed oxygen radical production by BZ metabolites in myelotoxicity induced by BZ is not known, but, it is worth emphasizing that iron accumulation in bone marrow has been observed following administration of BZ to rats.3J.176This iron accumulation may be due to the induction of heme ox- ygenase,"' which also occurs in rats exposed to BZ. Heme oxygenase is an enzyme responsible for heme d e m o n to biliverdin.'78.179Biliverdin can be fur- ther reduced to bilirubin by biliverdin reductase. Iron may be released from heme during its degradation. It is also interesting to note that biliverdin and bilirubin arc considered to be good antioxidants.178Therefore, it is difficult ax this point to emphasize whether induction of heme oxygenase by BZ in bone marrow increases mytlotoxicity by rrleasing iron from heme or prevents the free radical &om in bow marrow by increasing the levels of the antioxidants, biliverdin, and bilirubin. Pellack-Walker et al.'80 have shown that HQ,BT, CAT, and PH are cytotoxic to L5178YS cells, inhibit DNA synthesis and induce DNA-strand breaks in these cells. The inhibition of DNA synthesis by these BZ metabolites correlated inversely with their respective one-electron redox potentials: HQ is most potent followed by BT, CAT, and PH. These authors suggested that oxygen radicals play a major role in the cytotoxic- ity and DNA strand breaks induced by these BZ metabolites in these cells. Human promyelocytic leukemia (HL-60) cells contain appreciable levels of MP0.'81 We found that incubation of HL-60 cells with HQ (10 pM) or BT (10 pM) can result in alkylation of cellular proteins and cytotoxicity.182.183 This cytotoxicity and alkylation of HL-60 cells was potentiated in the presence of hydrogen peroxide, indicating that MPO may be involved in the bioactivation of HQ to the reactive 1,CBQ. Formation of 1.4-BQ was confirmed by the presence of a GSH-conjugate that co-elutes with the standard 1.4- BQ-GSH conjugate during HPLC analysis. We also found that incubation of HL-60 cells with HQ (10 pM) or BT (10 pM) results in a 2- and Cfold increase in 8-OHdG formation in DNA, re~pectively'~(F~ig. 7). The increase in 8-OHdG formation, however, is transient, and prolonged incubation of these cells results in a drop in 8-OHdG levels in DNA to steady-state levels, indicating that these cells are also equipped with an efficient repair mechanism for oxidative DNA damage. These results suggest that alkylation of cellular proteins and oxidative DNA-damage induced by BZmetabolites in HL-60 cells may contribute to cytotoxic and gentoxic events in these cells. They also provide the first direct evidence that BZ metabolites could produce murations via oxidative modification of DNA. I V. V. Smmawmm sd. .. GSSG .> -os- 4 O:,R*+ GSH ---+ R + G S GSSG'- GSSG + "2 0 , 1 Fig. 8. Interaction of free radicals (denoted as R' in figure) with glutathione and funher reactions of the glutathionyl radical. Sirmlarly. phenoxy radials could act as R' initiating glutathione oxidation and subsequent oxygen activation.'w (Adapted from ref. 185. Set text for a complete description of the figure.) D . Glutathione oxidation Glutathione (GSH; y-glutamylcysteinylglycine)is the major cellular soluble thiol. It is involved in the detoxification of electrophilic or free radical metabolites of xenobiotics.1B4-186 The detoxification of electrophilic metabolites by GSH appears to involve direct conjugation of the nucleophilic sulfhydryl group with the electrophilic metabolite, which may requireGSH-transferases. GSH-conjugates of electrophilic metabolites of toxic chemicals that are formed in the liver are further metabolized to mercapturic acids and excreted in urine.187 BZ administration to rabbits results in the excretion of phenylmercapturic acid, which suggests that BZ-ep oxide conjugation with GSH occurs in the liver of these animals.16 Jerina et al.Ia8 have shown that BZ-epoxideGSHconjugates can be formed in vitro. Studies in vitro also revealed that 1,4-BQ,22*'6 1,2-BQ,84 and 1,4-diphenoq~inone,~al~l*r~e~act with GSH, forming one or more GSH-conjugates. A mercapturate derivative of HQ has recently been isolated as one of the urinary metabolites of BZ, PH, and HQ, indicating that 1,CBQ-GSH conjugate formation also occurs in vivo. The detoxification of certain free radical metabolites of xenobiotics was shown to be due to the interaction of xenobiotic-derived free radicals with GSH forming thiyl radicals (GS3185*1(8F6ig. 8). These GS' can readily interact with dioxygen. forming a peroxy sulfenyi radical (GSOO';1, Fig. 8). in the presence of excess GSH result in the formation of dimerized radical (GSSG'-; 2, Fig. 8) or dimerize to yield GSSG (3, Fig. 8). The reaction of dimerized thiyl radicals with oxygen yields O2*- , which subsequently dismutates to H202. Further reactions of GSOO' with excess GSH also eventually results in the formation of GSSG.The GSSG and H202can subsequently be detoxified by GSH peroxidasdGSSG reductase system. The oxidation of PH by HRP and H202results in the formation of phenoxy1 radical^.^' These radicals in the presence of GSH are reduced back to PH. resulting in the formation of thiyl radicals.8J*169.'wExtensive oxygen uptake occurs during this process (Table 2). which results in the formation of superoxide radi- c a l ~Ca.tal~ytic~am~oun~ts of~ PH~ and H202 oxidize large excess of GSH to GSSG. The amount of GSH oxidation to GSSG was increased in the presence of Table 2. Phenol-induced Glutathione Oxidation and Arachidonate Peroxidation During PeroxidativeMetabolism of Phenol Phenolic Subsnte ~~ Oxygen Uptakc Oxygen Uptake ( +(FGMSH))' Thiyl Radical (a) Formationb ( + arachidonate)' ~~ ~ None F'hcnol Catechol Hydroquinone 0.05 f 0.05 124 f 14 0.5 f 0.5 0.5 2 0.5 + - 0.6 2 0.5 13 2 2 0.6 2 0.5 0.6 2 0.5 'Reamon system contained 3 ml of 10-mM aicHCl buffer (pH a).7.4). phenolic substrate (100 pM), HRP (10 pg) and GSH (200 FM) or AA (500 The reactions were initiated with the addition of HZ02 (50 pM). %yI radicals were trapped with 5.5-dimethyl-l-pyrroline-N-o~- ide (DMPO: 0.1 M) in a reaction system containing phenolic sub- suau (100 pM) in 1 ml of 0.1-M nis-HC1 buffer containing I-mm EDTA (pH 8.0). ESR spectra were recorded on a Varian E9 spec- mmeter. Fne radicals in knzcne myetotoxicity 509 Eg.9. Hypothetical scheme for the possible pathways of fm radical formation during benzene metabolism that induce oxygen activation. lipid peroxidation. glutathione oxidation and DNMprotein damage. (See Abbreviations for the exact names of the abbreviatcd f o m used in this figure.) SOD.This may be due to the increased dismutation of E . Lipid peroxidation O,*- to H202. This H202can efficiently oxidize more PH by peroxidase. No PH removal occurs until all the The peroxidation of lipids is commonly described as GSH is converted to GSSG. HQ and CAT oxidation an oxidative, oxygen-dependent deterioration of fats, by the HRP/H,O, system in the presence of GSH. particularly unsaturated fatty a ~ i d s . ' ~ L~i.p'id~ p~eroxi- however, do not result in oxygen activation, indicating dation can be initiated by abstraction of an H atom that the semiquinone radicals of HQ and CAT may not from unsaturated fatty acids. This can be achieved ei- react readily with GSH. ther enzymatically as described earlier for AA oxygen- The physiological significance of the reactions of ation by PGS, or by lipoxygenase or by reaction with phenoxy1 radicals with GSH and the subsequent redox another free radical. The addition of oxygen yields a cycling of thiyl radicals, in BZ-induced toxicity re- lipid peroxyl radical, which is considered a hallmark mains to be determined. We found that red blood cell of peroxidizing lipids. The peroxy radical combines fiee bone marrow cells contain low levels of GSH (1 with the hydrogen atom that it abstracts to give a lipid nmol/106cells)."' Smart and Z a ~ o x ~fiou~n~d 'that the total GSH content of guinea pig bone marrow (unper- hydroperoxide, R-OOH. An alternative fate of peroxyl radicals is to form cyclic peroxides. Pun: lipid hydro- fused)is approximately in khc range of 2&25 mnol/mg protein. which is similar -to -the hepatic GSH levels peroxides an fairly stable molecules at physiologic temperatures, but they rapidly decompose in the pres- ( 2 7 - W m g pra&I). This hukatcs that the majlnily ofbone mamw33m lrray be armc* lad withthe a c e of transition-metal complexes. This results in the fission of 0-0 bond to form an alkoxy1 radical. xed Moad ail hctiaa It is also possible that species ~exist*~-ftvefsof~marrow. WhiChIUlUlhStObe&Ql-hG- fi. -- ._---- -*-- -- R-0-0-H+ Fe"-complex -+ Fe+3-complex + OH' + R-O'. (5) -~ - -- . . . .- - .. _ _ _ .. .. . 510 V . V . - n d . . _. Peroxyl radicals seem to be less reactive rhan alkoxy1 radicals.194 In both cases, however, the formanon of radical products will stimulate the chain reaction of lipid petoxidation by causing more initiation. b-Scission. a well-known reaction of alkoxyl radicals. leads somal aberrations that have been obsaved in humans following exposure to BZ and the subsequent devefop a m t of leukemia. Free radical formatioa in liver. a secondary target o r p for BZ-toxicity, can also occur provided cellular defenses such as GSH arc depleted. to the production of compounds conraining the carbonyl group C=O, particularly aldehydes such as malondialdehyde and 4hydroxy nonenal. These aldehydes can also readily attack macromolecules such as DNA and protein, which then results in covalent binding as well as intra- and inter-molecular crosslinks of DNA -Ackmwledgemenrr Supported by NM P42 ES04705 and P30 ES01896 (MTS)and ROES 04112 tDR) aad thc Umvmlty of California. Tobacco-related Discase Research Program (DR.We thank Dr. P. Kolachana for analyzing the HL-60 cells for the p s - ence of 8-OHdG in their DNA. We also thank P. Doane Seam for drawing figures with the usc of Slidcwrite 3.1 graphics software. and proteins. 192*193-195-tI9n8 addition. lipid peroxida- tion products such as PGE2 are potent regulators of REFERENCES hematopoiesis.53 It is interesting that HRP-catalyzed oxidation of PH in the presence of AA results in rapid oxygen uptake (Table 2) and conjugated-diene formation.alO.lw However, HQ and CAT were found to be inactive in this system. It appears that the phenoxy1 radicals formed in this system can efficiently abstract an hydrogen atom from AA and induce lipid peroxidation. Baumann et recently analyzed the products of arachidonic acid in this reaction and found that 90% of the products co-chromatographed with 15-HPETEand 15-HETE. resembling a 15-lipoxygenase-cataiyzedpathway. Benzene administration to rats has been shown to induce lipid peroxidation in both liver and bone marrow of rats.33*34*5I3t is possible that the phenoxy radicals andor oxygen radicals generated during BZ metabolism could contribute to the observed lipid peroxidation following BZ administration. CONCLUSIONS This review indicates that a good deal of informa- tion is already available in the literature that documents the ability of BZ to induce free radical formation in vivo. However, the role these free radicals play in BZinduced myelotoxicity and carcinogenesis remains to be determined. Figure 9 summarizes the possible mechanisms of free radical formation, during BZ metabolism that can result in damage to the cellular macromolecules. Free radical formation in bone marrow may occur either during peroxidatic metabolism (catalyzed by MPO, PO,and perhaps PGS) or during autosxidation of BZ's phenolic metabolites. 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ABBREVIATIONS AA - hd'lidonic acid -1.2-BO 1.2-Benzwuinone in^^^1, ~ B Q- 1 -BT 1.2,CBenzenemol -BZ - Benzene CAT Catechol DMPO - 5.5-Dimethyl-I-pyrroline-N-oxide DNA - Deoxyribonucleic acid PO - Eosinophil peroxidase GSH - Reduced glutathione GSSG - Oxidized glutathione HL-60 - Human promyelocytic leukemia cells -Hz02 Hydrogen peroxide -HPLC High pressure liquid chromotography -HQ Hydroquinone -HRP Horseradish peroxidase -HBQ 2-hydroxy, 1.4-benzoquinone HSQ - 2-hydroxy, 1.4-benzosemiquinone anion radical -MPO Myeloperoxidase .- -1.2-SQ'- - 1,2-Benzosemiquinoneanion radical 1.4-SQ 1.CBenzosemiquinont anion radical -r,t-MA - trans,trans-muconaldehyde NADPH Reduced nicotinamide adenine dinucleotide phosphate NO - Nuclear overhausser effect 02'-- Superoxide anion radical -HO ' Hydroxyl radical lo2- Singlet oxygen 8-OHdG - 8-hydroxy-:!'-deoxyguanosine PGG2 - 15-hydroperoxy-9,ll-endoperoxide --PGH, - 15-hvdroxv-9.11-endowroxide PGS Prosta&nd:m H synthase PMA - Phorbol myrismte acetate RNA - Ribonucleic acid SIRS - Soluble immune response suppressor SOD - Superoxide dismutase TBA - Thiobarbituric acid