Document DvEO975N1Xry6Kz6XE39b9nOn

THE ROLE OF HEPATIC METABOLITES OF BENZENE IN BONE MARROW PEROXIDASE-MEDIATED MYELO- AND GENOTOXICITY \ George Kalf, Robert Shurina, John Renz, and Michael Schlosser The Department of Biochemistry and Molecular Biology Jefferson Medical College of Thomas Jefferson University Philadelphia, Pennsylvania, 19107 f ! i ii INTRODUCTION Chronic exposure of humans to benzene causes bone marrow depression leading to pancytopenia and aplastic anemia (Goldstein, B.D., 1983). Benzene also causes genotoxic effects such as structural chromosome aberrations and DNA strand breaks (Dean, B.J., 1985) that might be related to the increased incidence of acute 1 myelogenous leukemia that is associated with chronic exposure (Infante, P.F., White, M.C., 1983; Aksoy, M., 1985; Arp, E.W., et al., 1983). i- Benzene metabolism, which is required for toxicity occurs predominantly in the liver via cytochrome P-450 (Sammett, D., et al., 1979; Tunek, A., 1980). Phenol (P), hydroquinone (HQ) and catechol are hepatic metabolites of benzene, and their production appears necessary for benzene-induced myelotoxicity (Arp, E.W., et al., ._1983; Sammett, D., et al., 1979) but they show no overt toxicity to the liver. These metabolites are transported from the liver to the bone marrow (Rickert, D.E., et al., \ 1979; Greenlee, W.P., et al., 1981) where they are bioactivated in a peroxidasemediated reaction (Sawahata, T., et al., 1985; Irons, R.D., 1985) t o biological reactive f compounds that bind to macromolecules and which have been implicated in mediating the toxic effects of benzene (Sammett, D., et al., 1979; Rickert, D.E., et al., 1979). f I I appears to be an important factor in benzene-induced V.N., et ai., 1976; Gaido, K., et al., 1986). The stromal I ial in the regulation of hematopoiesis, has been implicated as i ed hematotoxicity (Lewis, J.G., et al., 1988; Thomas, D.J., et i secreted less interleukin-1, a monokine capable of i i t Macrophages contain considerable amounts of prostaglandin H synthase (PHS) an me with both cyclooxygenase and peroxidase activities (Scott, W.A., et al., 1980; %& et al., 1979). Since phenol and hydroquinone serve as reducing co-substrates d-Whemdmcs [I:Edrud by C.M. Witmer ei ai. c Nn Y e , 1990 443 on Prostaglandins and Thromlox8nas Figure 1. Postulated mechanism for the role of PHS in mediating benzene- induced bone marrow damage. Chronic exposure to benzene causes the release of arachidonic acid from the membrane lipid of marrow stromal cells particularly the resident stromal macrophage resulting r- - i in constitutive synthesis of prostaglandins. The oxidation of the cosubstrate hydroquinone by PHS-peroxidase converts it to a reactive intermediate which can bind to macromolecules and cause toxicity. Inhibition of PHS-cyclooxygenase with indomethacin i n h i b i t s -g prostaglandin synthesis and prevents bioactivation of hydroquinone. oxidase of PHS (Markey, C.M., et al., 19871, macrophages have the capacity t o oxidize these benzene metabolites to compounds capable of reacting with cellular macromolecules. Indeed, Post et al. have demonstrated that the macrophage can metabolize phenol to protein-binding species (Post, G., et al., 1986). *ole of PHS in benzene-induced myelotoxicity seems particularly relevant, benzene administration elevates bone marrow levels of prostaglandin E2 (Gaido, d., 1987; Kalf, G.P., et al., 1989), a negative regulator of myelopoiesis .s., et al., 1987). Nonsteroidal anti-inflammatory drugs, known inhibitors of cyclooxygenase, have been reported not only to inhibit this rise in prostaglandin, but to prevent benzene-induced myelotoxicity as well (Gaido, K.W., et al., 1987; Kalf, G.P., I et al., 1989). Because nonsteroidal anti-inflammatory drugs i n h i b i t the cyclooxygenase-cat alyzed form ation of prostaglandin G2 (PGG2), treat m ent with these i k agents would eliminate the peroxidase-catalyzed reduction of PGG2, thereby avoiding the oxidation of phenolic co-substrates, if present, to reactive compounds. The research to be described w a s designed to determine whether PHS produces benzene-induced myelo- and genotoxicity by the following proposed mechanism (Figure 1). Benzene is metabolized in the liver and bone marrow to P and HQ. Benzene per Se may act on bone marrow cells to effect the constitutive release of arachidonic acid b from membrane phospholipids via the activation of protein kinase C which is known to activate the arachidonic acid cascade in macrophages (Pfankuche, H.J., et al., 1986). Arachidonic acid would be converted by the cyclooxygenase component of PHS to the ide (PGG2). The hydroperoxide in turn would drive the endoperoxidae !tiVity of the enzyme. The cooxidation of HQ or P during endoperoxidase conversion ' PGGz to PGH2, the immediate precursor molecule for prostaglandins, would result in increased levels of prostaglandins. In the case of HQ, cooxidation would generate P- 444 benzoquinone which is known to cause genotoxic damage in the form of adducts, strand breaks, SCE and micronucleus formation (Dean, B.J., 1985). The inability of remaining viable stem and/or progenitor cells to proliferate due to the constitutive production of high levels of prostaglandins, known to be negative regulators of hematopoiesis, coupled with genotoxic damage from reactive metabolites such 8s p-benzoquinone might explain the benzene-induced myelotoxicity. The experiments described herein demonstrate the arachidonic acid-dependent, indomethacin-sensitive, bioactivation of P and HQ to genotoxic compounds by macrophage peroxidase and purified PHS. Furthermore, we show that inhibition of PHS with indomethacin prevents benzene-induced myelo- and genotoxicity in mice when indomethacin is coadministered with benzene. BESULTS Activation of Phenol and Hydroquinone to Biological Reactive Intermediates in Macrophages The inhibition of the toxic effects of benzene by indomethacin (Kalf, G.F., et al., 1989) suggested that metabolites of benzene such as P and/or HQ might be converted to reactive compounds by PHS-peroxidase. Consequently, we tested the ability of macrophages t o effect the arachidonic acid-dependent metabolism of P and HQ to reactive species that bind to macromolecules. In these experiments mouse peritoneal macrophages or P388D1 cells, which morphologically and functionally resemble macrophages, (Koren, H.S., et al., 1975) were used. Similar results can be obtained with both cell types. Purified peritoneal macrophages incubated with [14C]P in the presence and absence of arachidonic acid metabolize P to reactive compounds that irreversibly bind macromolecules in a reaction that is dependent on the concentration of arachidonic acid (Figure 2). These results suggest that PHS-peroxidase in macrophages might be responsible for the cooxidation of P and/or HQ to reactive species during the benzeneinduced formation of prostaglandins. As can be seen in Figure 3, [14C]HQ is also bioactivated by P388D1 cells to species which bind to macromolecules. The addition of TPA causes the further activation of the arachidonic acid cascade which stimulates the oxidation of HQ to reactive species via PHS and this is prevented by indomethacin, a cyclooxygenase inhibitor. Benzene, which has been shown t o activate protein kinase C (Roghani, M., et al., 1987) and thus the arachidonic acid cascade (Pfankuche, H.J., et al., 1986), also stimulates the bioactivation of HQ in an indomethacin-sensitive reaction. Bioactivation of P or HQ to species which bind to macromolecules was shown to occur in a macrophage lysate. The bioactivation of P or HQ occurred in a time and concentration-dependent manner (data not shown). The binding of P or HQ equivalents to protein was decreased significantly, compared to the complete system, for incubations containing the peroxidase inhibitor aminotriazole and for reactions carried Out in the absence of either the macrophage lysate or H 2 0 2 (Figure 4). The effect of hydroxyl radical scavengers was investigated to determine if the macrophage lysate Indirectly activated P and HQ through the generation of hydroxyl radicals. The addition of dimethylsulfoxide (DMSO) or mannitol to standard incubation mixtures unexpectedly resulted in a slight but significant increase in both P and HQ equivalents Pound to TCA-precipitable material (Figure 4). The addition of the cytochrome P-450 Inhibitors, metyrapone and SKF 525A to the macrophage lysate had no affect on the bioactivation of P or HQ (data not shown). These results suggested that adherent macrophages and a macrophage lysate were capable of the bioactivation of P or HQ to covalent binding species and that this w a s most probably catalyzed by PHS-peroxidase. We, therefore, investigated the PHS-catalyzed oxidation of HQ and examined the DNA-damaging effects of the reactive metabolite generated during the reaction. 445 Figure 2. Aracidonic acid-dependent activation of phenol by macrophages. Incubation mixtures contained 2.3 x 106 macrophages, 0.25 mM [14C] phenol (8000 dpm/nmole), 0.1 mM arachidonic acid diluted to a final volume of 1 mL with RPMI-1640 buffered to pH 7.3 with 5 mM HEPES. Reactions were terminated a f t e r 30 min by t h e addition of trichloracetic acid. Irreversible binding of [14C] phenol equivalents was assessed by liquid scintillation counting after extensive washes with acetone/hexane/methanol. Reprinted from reference 26. 300 n 1 %* ?ht 11+1m r'elna+ T PA ['4t1m+ BENZENE Figure 3. C o v a l e n t b i n d i n g of [ 1 4 C ] h y d r o q u i n o n e t o t macromolecules in P388D1 cells. P-388 cells (2x106 p e r incubation) were preincubated f o r 15 min. with e i t h e r 100 mM indomethacin in ethanol or ethanol alone (1%final concentration), then t r e a t e d with (50 pM) [14C]HQ. S o m e incubations included 100 ng TPA or 170 mM b e n z e n e coadministered with t h e hydroquinone. A f t e r a 30 min. incubation at 37C, 5% C02, cells were lysed by freezing and thawing in 10 mM Tris, 1 mM EDTA buffer pH 8.0 containing 2 mm PMSP, 0.1 mM Leupeptin, 0.1 mM pepstatin A and 0.01% Triton X-100. Macromolecules were precipitated in 10% TCA, washed 3 times with a c e t o n e followed by t w o washes in acetone/methanol (1:1, V:V), a n d r a d i o a c t i v e H Q c o v a l e n t l y b o u n d t o macromolecules was determined by scintillation spectrophotometry. Values represent t h e mean f S.D. of three experiments. 446 System nmoiiincubat ion complete a 17.3 f 3.6 - PHS 0.9 k 0.2 - arachidonate 1.0 t 0.1 + indomethacin (10 pM) 4.3 k 0.7 + cysteine (100 pM) CO.1 a Complete system at 37'C contained hydroquinone (100 pM), PHS (5 pg/mL), hematin ( 1 pM), and arachidonic acid (100 pM) in 0.1 M Na+/K+ phosphate buffer, pH 7.0 in a f i n a l volume of 0.5 mL. Reactions were preincubated with indomethacin or 1% ethanol f o r 10 min. p-Benzoquinone was measured using HPLC with electrochemical detection. D a t a is expressed as t h e means f standard deviations. -- -_ time. Similar results were obtained when mtDNA was used in place of calf thymus DNA (Table 3). mtDNA-binding was dependent on enzyme and was inhibited by indomethacin. Thus [14C]HQ, functioning as a co- substrate f o r PHS-peroxidase, is cooxidized in an arachidonic acid- and time-dependent reaction to compound(s) that bind to mtDNA. I14CIHQ-derived mtDNA adducts w e r e isolated as deoxyribonucleoside adducts by hydrophobic chromatography on a n LH-20 column following sequential digestion of radiolabeled mtDNA with nucleases (data not shown). A 14C-labeled adduct was found to co-migrate on a thin layer cellulose chromatographic plate (Figure 6) with a 2'-deoxyguanosine (2'-dG) adduct standard. The s t r u c t u r e of t h e 2'dG adduct, (3'OH) benzetheno (1,NZ) deoxyguanosine has been reported (Snyder, R., et al., 1987) (Figure 7). Table 2. System Arachidonic acid-dependent Formation of Monocysteinehydroquinone by Prostaglandin H Synthase nmol/incubat ion complete a - PHS 14.6 k 0.2 0.8 f 0.1 - arachidonate - cysteine + indomethacin (10 pM) 0.6 f 0.1 <O.l 6.3 f 0.3 a Complete system at 37.C contained hydroquinone (100 pM), PHS (5 pg/mL), hematin (1 pM), cysteine (100 pM) and arachidonic acid (100 pM) in 0.1 M Na+/K+ phosphate buffer, pH 7.0 in a final volume of 0.5 mL. Reactions were preincubated with indomethacin or 1% ethanol for 10 min. Monocysteine-hydroquinone was measured using HPLC with electrochemical detection. Data is expressed as t h e means f standard deviations. - --* % - .- - -. I L 1 448 1- 1.51.4. I 'I ' 02460 Tlme (mlnutes) I' 1 I 0 Figure 5. Time course for DNA binding by hydroquinone during PHS-catalyzed reactions: (?) contained calf thymus DNA (100 mg/mL), I14C)hydroquinone ( 1 0 0 mM; 1 0 , 0 0 0 dpm/nmol), PHS (5 mg/mL) with hematin (1 mM), and arachidonic acid (10 mM); (?) as above except PHS w a s heat-inactivated. Data points represent the means f standard deviations of triplicate determinations. I HQ has been reported to produce strand breaks in DNA (Sawahata, T., et al., Wing an LKB laser scan densitometer, it was found that the intensity of the open circle band in the complete system containing active enzyme (lane 3) was 88% above the complete system that had been inhibited by indomethacin (lane 6). The Role of PHS in the induction of Myelo- and Genotoxicity in Mice by Benzene: The Effect of Indomethacin on Benzeneinduced Bone Marrow Depression and Micronucleus Formation in C57B1/6 Mice f HQ to p-benzoquinone, the formation of a [14C]HQ-derived he ability of p-benzoquinone to nick supercoiled plasmid DNA omethacin. These results suggest that PHS-peroxidase plays a of HQ to p-benzoquinone, a putative genotoxic metabolite of Table 3. Prostaglandin H Synthase- Catalyzed Activation of Hydroquinone to DNA-binding Metabolite(s) System nmoVincubation complete a - PHS + indomethacin (10 mM) 5.11 f 0.55 1.98 f 0.46 2.07 f 0.45 a Complete s y s t e m contained [14Clhydroquinone (100 pM; 10,000 dpmhmol), PHS (5 pg/mL), hematin (1 pM), arachidonic acid (10 pM), and mtDNA (100 pg/mL) in 0.1 M K+ phosphate buffer, pH 7.0 in a final volume of 0.5 mL. Reactions were preincubated with indomethacin or 1% ethanol for 1 min. Radioactivity bound to mtDNA w a s measured by liquid scintillation spectrometry. D a t a is expressed 89 t h e means f standard deviations. Figure 6. Autoradiogram of t h e TLC plate. Arrow indicates t h e position of t h e radiolabeled [14C] p- benzoquinone- mtDNA deoxynucleoside adduct (Rf=0.22). T h e [14C] pbenzoquinone-deoxyguanosine adduct standard appears in lane 2 at a Rf of 0.22. Lane 1 represents [14C] hydroquinone alone. 450 Depression of bone m determine benzene toxicity. a day for two days caused a as a d e c r e a s e in nucleated dependent and there from the femur (data weight) with benzene Indomethacin alone had no effect. Meclofenamate (4 mg/kg) or aspirin (50 which inhibit cycloxygenase activity of PHS by mechanisms different f r o m t indomethacin, also significantly prevented benzene-induced bone marrow depression (data not presented). Under conditions where benzene causes significant myelotoxicity in t h e form of decreased bone marrow cellularity, i t also c a u s e s genotoxicity measured by an increase in t h e frequency of micronucleus formation in peripheral blood polychromatic erythrocytes (PCE). I t c a n be s e e n in Table IV t h a t benzene at 600 mg/kg body weight also c a u s e d a 5.7-fold i n c r e a s e in t h e frequency of m i c r o n u c l e u s formation. Coadministration of indomethacin with benzene prevented t h e increased frequency of micronucleus formation in PCE without affecting t h e division or maturation of nucleated erythroid precursors (NCE) as indicated by no change in t h e PCE/NCE. The administration of indomethacin alone had no a f f e c t on micronucleus formation. Table 4. Prevention of Benzene-Induced Bone-Marrow Depression and Micronucleus Formation in Mice by Indomethacin Group a Nucleated bone marrow Micronuclei/l03 cells x 106/femur PCE NCE PCE 103 NCE control b + benzene C + benzene + indo d + indo d 11.4 + 1.6 6.2 + 1.6* 9.4 + 0.7 12.4 + 1.8 4.5 + 1.0 1.0 + 0.9 25.5 + 9.2* 0.5 + 0.4 10.0 + 5.0 0.75 + 0.3 5.5 + 3.0 1.7 + 0.8 34.0 + 4.5 24.0 + 10.4 32.4 + 3.0 32.0 + 7.6 a Each group consisted of 4 C57B1/6 male mice. b Control animals received corn oil and/or a solution of 4.2% ethanolic PBSA. C Benzene (600 mg/kg in corn oil) was administred ip twice daily for two days. d Indomethacin ( 2 mg/kg in 4.2% ethanolic PBSA was administered ip t w i c e daily for two days. D a t a are expressed as means f SD * p S 0.01 compared to control and indomethacin- treated groups. DISCUSSION -_ Adherent macrophages bioactivate P in an arachidonic acid-dependent reaction to species capable of covalent binding t o macromolecules (Figure 2). Similar results are obtained with HQ. The addition of TPA or benzene, which cause t h e release of I- arachidonic acid from plasma membrane lipids, stimulate the oxidation of HQ t o T r e a c t i v e species (Figure 3). This is prevented by indomethacin, a cyclooxygenase f inhibitor. Similar results can be obtained with a lysate of adherent macrophages. The H202-dependent activation of P or HQ to biological reactive species is inhibited by . aminOtriaZOle, a peroxidase inhibitor (Figure 4), but not by inhibitors of cytochrome p- - 450 such as metyrapone or SKF 525A, or by hydroxyl radical scavengers (Figure 4)- - T h e arachidonic acid-dependent activation of HQ or P by the macrophage lysate Was ;; not a t t e m p t e d since t h e concentration of CTAB used t o solubilize t h e membrane- 5 ig r 452 bound peroxidase also inhibits the cyclooxygenase activity of PHS (data not shown). These results strongly implicate PHS-peroxidase in the bioactivation of these benzene metabolites to biological reactive compounds in macrophages. Further studies using purified PHS confirmed that the enzyme was capable of the arachidonic aciddependent and indomethacin-sensitive oxidation of HQ to p-benzoquinone (Table 1) which could be measured directly by HPLC or trapped with cysteine as a monocysteine-hydroquinone conjugate (Schlosser, M.J., et al., 1989) (Table 2). The oxidation product(s) of HQ were also able to covalently bind to DNA (Figure 5). [14C] HQ, functioning as a cosubstrate for PHS-peroxidase, was also shown to be oxidized in an arachidonic acid- and time-dependent reaction to compound(s) that interact with DNA to form adducts and strand breaks. A 14C-labeled adduct w a s isolated from mtDNA which was found to co-migrate on a thin layer cellulose chromatographic plate (Fig. 6) with a known Z'-deoxyguanosine adduct standard. The structure of the 2'dG adduct w a s -identified as an adduct of pbenzoquinone with guanine to form (3'OH) benzetheno (1,Nz) deoxyguanosine (adduct 2; Fig. 7). Interaction of HQ with PHS-peroxidase and arachidonic acid in the presence of supercoiled Bluescript plasmid resulted in single strand breaks in the supercoiled DNA converting it to an open circle form. The PHS-catalyzed strand breaks were dependent on native enzyme, HQ, and arachidonic acid and were prevented by indomethacin (Fig. 8). Taken together, these results implicate PHS-peroxidase in the bioactivation of HQ to p-benzoquinone, a genotoxic metabolite of benzene, and suggest that in vivo toxicity to marrow progenitor cells might result from the production of reactive species by PHS. That this may be the case is evidenced by the facts that benzeneinduced depression of bone marrow cellularity and increased frequency of micronucleus formation in mice can be prevented by the coadministration of PHS inhibitors such as indomethacin (Table 4). Indomethacin does not appear t o be modulating an alteration of bone marrow cellularity related to an initial benzeneinduced inflammatory reaction since data obtained in a subchronic exposure study (to be published elsewhere) indicate that indomethacin can prevent the myelo- and genotoxic effects induced by benzene over a 3 week period. The amount of indomethacin used in our protocols had no effect on hepatic microsomal P-450 content or benzene hydroxylase activity (Pirozzi, S., et al., 1989) and the effective level of indomethacin was not sufficient to affect dihydrodiol dihydrogenase, phospholipase A2 or myeloperoxidase, enzymes which might be expected to be involved in benzene metabolism. REFERENCES -.Ahoy, M. (1985). 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