Benzene and its phenolic metabolites produce oxidative DNA damage in HL60 cells in vitro and in the bone marrow in vivo.

Benzene, an important industrial chemical, is myelotoxic and leukemogenic in humans. It is metabolized by cytochrome P450 2E1 to various phenolic metabolites which accumulate in the bone marrow. Bone marrow contains high levels of myeloperoxidase which can catalyze the further metabolism of the phenolic metabolites to reactive free radical species. Redox cycling of these free radical species produces active oxygen. This active oxygen may damage cellular DNA (known as oxidative DNA damage) and induce genotoxic effects. Here we report the induction of oxidative DNA damage by benzene and its phenolic metabolites in HL60 cells in vitro and in the bone marrow of C57BL/6 x C3H F1 mice in vivo utilizing 8-hydroxy-2'-deoxyguanosine as a marker. HL60 cells (a human leukemia cell line) contain high levels of myeloperoxidase and were used as an in vitro model system. Exposure of these cells to phenol, hydroquinone, and 1,2,4-benzenetriol resulted in an increased level of oxidative DNA damage. An increase in oxidative DNA damage was also observed in the mouse bone marrow in vivo 1 h after benzene administration. A dose of 200 mg/kg benzene produced a 5-fold increase in the 8-hydroxydeoxyguanosine level. Combinations of phenol, catechol, and hydroquinone also resulted in significant increases in steady state levels of oxidative DNA damage in the mouse bone marrow but were not effective when administered individually. Administration of 1,2,4-benzenetriol alone did, however, result in a significant increase in oxidative DNA damage. This represents the first direct demonstration of active oxygen production by benzene and its phenolic metabolites in vivo. The conversion of benzene to phenolic metabolites and the subsequent production of oxidative DNA damage may therefore play a role in the benzene-induced genotoxicity, myelotoxicity, and leukemia.

Metabolism of hydroquinone by human myeloperoxidase: mechanisms of stimulation by other phenolic compounds.

Hydroquinone, a metabolite of benzene, is converted by human myeloperoxidase to 1,4-benzoquinone, a highly toxic species. This conversion is stimulated by phenol, another metabolite of benzene. Here we report that peroxidase-dependent hydroquinone metabolism is also stimulated by catechol, resorcinol, o-cresol, m-cresol, p-cresol, guaiacol, histidine, and imidazole. In order to gain insights into the mechanisms of this stimulation, we have compared the kinetics of human myeloperoxidase-dependent phenol, hydroquinone, and catechol metabolism. The specificity (Vmax/Km) of hydroquinone for myeloperoxidase was found to be 5-fold greater than that of catechol and 16-fold greater than that of phenol. These specificities for myeloperoxidase-dependent metabolism inversely correlated with the respective one-electron oxidation potentials of hydroquinone, catechol, and phenol and suggested that phenol- and catechol-induced stimulation of myeloperoxidase-dependent hydroquinone metabolism cannot simply be explained by interaction of hydroquinone with stimulant-derived radicals. Phenol (100 microM), catechol (20 microM), and imidazole (50 mM) did, however, all increase the specificity (Vmax/Km) of hydroquinone for myeloperoxidase, indicating that these three compounds may be stimulating hydroquinone metabolism by a common mechanism. Interestingly, the stimulation of peroxidase-dependent hydroquinone metabolism by other phenolic compounds was pH-dependent, with the stimulating effect being higher under alkaline conditions. These results therefore suggest that the interaction of phenolic compounds, presumably by hydrogen-bonding, with the activity limiting distal amino acid residue(s) or with the ferryl oxygen of peroxidase may be an important contributing factor in the enhanced myeloperoxidase-dependent metabolism of hydroquinone in the presence of other phenolic compounds.

Metabolism of phenylhydroquinone by prostaglandin (H) synthase: possible implications in o-phenylphenol carcinogenesis.

o-Phenylphenol (OPP) and its sodium salt sodium ortho-phenylphenate (NaOPP) are broad spectrum fungicides and antibacterial agents. Both are urinary bladder and renal carcinogens in the Fischer 344 rat. OPP is converted by mixed-function oxidases in the liver to phenylhydroquinone (PHQ). Since appreciable amounts of prostaglandin (H) synthase (PGS) are found in rat bladder and kidney-medullary papilla, the target sites of OPP- and NaOPP-induced tumors, we hypothesized that a secondary PGS-mediated activation of PHQ to phenylbenzoquinone (PBQ) may occur in the bladder and kidney. We have studied the metabolism of PHQ by PGS in the presence of arachidonic acid and hydrogen peroxide as co-factors. These studies showed that PHQ is indeed metabolized to a product having identical spectral and electrochemical properties to PBQ. The disappearance of PHQ with time was stoichiometric to the formation of PBQ. Less than 10% of PHQ was converted to PBQ in the absence of enzyme, indicating that auto-oxidation may play only a minor role in the conversion of PHQ to PBQ. Similar results were obtained when PGS was replaced with either myeloperoxidase or horseradish peroxidase and hydrogen peroxide as co-factor. These studies suggest that the peroxidative metabolism of PHQ by PGS to the reactive PBQ could play an important role in OPP-induced urinary bladder and kidney carcinogenesis in rats.

Hydroxylation of phenol to hydroquinone catalyzed by a human myeloperoxidase-superoxide complex: possible implications in benzene-induced myelotoxicity.

Benzene, a known human myelotoxin and leukemogen is metabolized by liver cytochrome P-450 monooxygenase to phenol. Further hydroxylation of phenol by cytochrome P-450 monooxygenase results in the formation of mainly hydroquinone, which accumulates in the bone marrow. Bone marrow contains high levels of myeloperoxidase. Here we report that phenol hydroxylation to hydroquinone is also catalyzed by human myeloperoxidase in the presence of a superoxide anion radical generating system, hypoxanthine and xanthine oxidase. No hydroquinone formation was detected in the absence of myeloperoxidase. At low concentrations superoxide dismutase stimulated, but at high concentrations inhibited, the conversion of phenol to hydroquinone. The inhibitory effect at high superoxide dismutase concentrations indicates that the active hydroxylating species of myeloperoxidase is not derived from its interaction with hydrogen peroxide. Furthermore, catalase a hydrogen peroxide scavenger, was found to have no significant effect on hydroxylation of phenol to hydroquinone, supporting the lack of hydrogen peroxide involvement. Mannitol (a hydroxyl radical scavenger) was found to have no inhibitory effect, but histidine (a singlet oxygen scavenger) inhibited hydroquinone formation. Based on these results we postulate that a myeloperoxidase-superoxide complex spontaneously rearranges to generate singlet oxygen and that this singlet oxygen is responsible for phenol hydroxylation to hydroquinone. These results also suggest that myeloperoxidase dependent hydroquinone formation could play a role in the production and accumulation of hydroquinone in bone marrow, the target organ of benzene-induced myelotoxicity.

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

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

Peroxidase/hydrogen peroxide--or bone marrow homogenate/hydrogen peroxide--mediated activation of phenol and binding to protein.

1. 14C-Phenol was metabolized by rat bone marrow homogenate and H2O2. The homogenate catalyst, however, was inactivated by preincubation with H2O2, presumably due to inactivation of the enzyme(s) involved in phenol metabolism. 2. The majority of the metabolized 14C-phenol was bound to bone marrow proteins. o,o'-Biphenol and p,p'-biphenol were the principal non-protein-bound products. Ascorbate was unable to remove phenol oxidation products bound to protein, although o,o'-biphenol recovery from the reaction mixture was markedly enhanced. Prior alkylation of protein thiols with N-ethylmaleimide decreased the binding of 14C-phenol oxidation products to bone marrow proteins by only 10-20%. 3. 14C-Phenol (200 microM) metabolism by horseradish peroxidase (10 micrograms) and H2O2 (200 microM) also resulted in extensive binding to externally added bovine serum albumin. The absorption spectrum of 14C-phenol oxidation products bound to bovine serum albumin was similar to that of bound oxidation products of o,o'-biphenol but not of p,p'-biphenol. 4. Protease digestion of bovine serum albumin bound 14C-phenol oxidation products, followed by ethyl acetate extraction, extracted 75% of the 14C, indicating that most of the binding is probably non-covalent. Up to 32% of the 14C-phenol oxidation products binding to bovine serum albumin may be covalent, since derivation with dinitrofluorobenzene and extraction under acid, but not alkaline, conditions extracted the 14C. The percentage of metabolites covalently bound to bovine serum albumin was increased to 59% when horseradish peroxidase concentration was decreased to 0.2 micrograms. 5. The thiol groups of bovine serum albumin were unaffected by o,o'-biphenol oxidation products, slightly decreased by phenol oxidation products, but were completely depleted by p,p'-biphenol oxidation products. 6. These results indicate that o,o'-biphenol oxidation products are responsible for much of the 14C-phenol binding to protein.

Phenol-induced stimulation of hydroquinone bioactivation in mouse bone marrow in vivo: possible implications in benzene myelotoxicity.

The coadministration of phenol and hydroquinone has been shown to produce myelotoxicity in mice similar to that observed following benzene exposure. One explanation of this phenomenon may be that phenol enhances the peroxidase-dependent metabolic activation of hydroquinone in the mouse bone marrow. Here we report that radiolabeled [14C]hydroquinone and [14C]phenol bind covalently to tissue macromolecules of blood, bone marrow, liver and kidney, when administered intraperitoneally to the mouse in vivo. Substantially more radiolabeled hydroquinone was covalently bound 18 h after administration as compared with that bound after 4 h. Phenol, when administered together with [14C]hydroquinone, significantly stimulated the covalent binding of [14C]hydroquinone oxidation products to blood (P less than 0.001) and bone marrow (P less than 0.05) macromolecules, but had no significant effect on covalent binding of [14C]hydroquinone oxidation products to liver and kidney macromolecules (P greater than 0.05). Catechol, on the other hand, had no effect on the binding of [14C]hydroquinone oxidation products in either bone marrow, kidney or liver (P greater than 0.05). When hydroquinone was administered together with [14C]phenol, a stimulation of the covalent binding of phenol oxidation products to bone marrow macromolecules also occurred (P less than 0.05). In addition, hydroquinone co-administration increased the covalent binding of [14C]phenol oxidation products in kidney and blood (P less than 0.05), but significantly decreased the covalent binding in liver (P less than 0.05). These results suggest that altered pharmacokinetics play a major role in the hydroquinone-dependent stimulation of covalent binding of [14C]phenol oxidation products to extrahepatic tissue macromolecules in vivo. The mechanism underlying the phenol-induced stimulation of binding of [14C]hydroquinone by phenol in blood and bone marrow remains unclear, but stimulation of peroxidase-mediated hydroquinone metabolism may be responsible. The latter may therefore play an important role in benzene-induced myelotoxicity.

Bone marrow stromal cell bioactivation and detoxification of the benzene metabolite hydroquinone: comparison of macrophages and fibroblastoid cells.

Bone marrow stroma consists predominately of two cell types, macrophages and fibroblastoid stromal cells, which regulate the growth and differentiation of myelopoietic cells via the production of growth factors. We have previously shown that macrophages are more sensitive than fibroblastoid stromal cells (LTF cells) to the toxic effects of the benzene metabolite hydroquinone. In this study, the role of selective bioactivation and/or deactivation in the macrophage-selective effects of hydroquinone was examined. LTF and macrophage cultures were incubated with 10 microM [14C]hydroquinone to examine differential bioactivation. After 24 hr, the amount of 14C covalently bound to acid-insoluble macromolecules was determined. Macrophages had 16-fold higher levels of macromolecule-associated 14C than did LTF cells. Additional experiments revealed that hydroquinone bioactivation to covalent-binding species was hydrogen peroxide dependent in macrophage homogenates. Covalent binding in companion LTF homogenates was minimal, even in the presence of excess hydrogen peroxide. These data suggest that a peroxidative event was responsible for bioactivation in macrophages and, in agreement with this, macrophages contained detectable peroxidase activity whereas LTF cells did not. Bioactivation of [14C]hydroquinone to protein-binding species by peroxidase was confirmed utilizing purified human myeloperoxidase in the presence of hydrogen peroxide and ovalbumin as a protein source. High performance liquid chromatographic analysis of incubations containing purified myeloperoxidase, hydroquinone, and hydrogen peroxide showed that greater than 90% of hydroquinone was removed and could be detected stoichometrically as 1,4-benzoquinone. 1,4-Benzoquinone was confirmed as a reactive metabolite formed from hydroquinone in macrophage incubations using excess GSH and trapping the reactive quinone as its GSH conjugate, which was measured by high performance liquid chromatography with electrochemical detection. The activity of DT-diaphorase, a quinone reductase that has been invoked as a protective mechanism in quinone-induced toxicity, was 4-fold higher in LTF cells than macrophages. These data suggest that the macrophage-selective toxicity of hydroquinone results from higher levels of peroxidase-mediated bioactivation and/or lower levels of DT-diaphorase-mediated detoxification.

Bioactivation of catechol in rat and human bone marrow cells.

o-Benzoquinone-glutathione (GSH) conjugate formation and covalent binding of [14C]catechol to protein were utilized as probes of bioactivation of catechol in both rat and human white bone marrow cell systems. Conjugate formation and binding occurred in the absence of exogenous hydrogen peroxide, but were markedly stimulated by its addition. Protein-binding and conjugate formation using rat cells in the presence of exogenous peroxide were increased by the presence of phenol whereas GSH and hydroquinone inhibited binding. Similarly, protein-binding in the absence of exogenous peroxide was inhibited by GSH and exacerbated by phenol. Prostaglandin synthase, the peroxidatic function of which may also utilize hydrogen peroxide as a substrate, appeared on the basis of experiments using arachidonic acid to play only a minor role in bioactivation of catechol in rat bone marrow cells. These results show that peroxide-dependent bioactivation of catechol occurs in rat and human bone marrow cells and that hydroquinone and GSH inhibit whereas phenol stimulates bioactivation.

Oxidation of catechol by horseradish peroxidase and human leukocyte peroxidase: reactions of o-benzoquinone and o-benzosemiquinone.

The metabolism of secondary phenolic metabolites of benzene, such as catechol, by peroxidases represents one possible mechanism underlying benzene-induced myelotoxicity. The oxidation of catechol by horseradish peroxidase and peroxidases present in human leukocytes was therefore examined. Peroxidatic oxidation resulted in o-benzoquinone production, which was characterized as its bromothiophenol adduct. o-Benzoquinone-glutathione conjugates were formed during peroxidatic oxidation of catechol in the presence of glutathione. Both mono- and diglutathione conjugates were detected. As much as 80% of catechol removed during peroxidatic oxidation could be recovered as glutathione conjugates of o-benzoquinone. Glutathione had no inhibitory effect on the removal of catechol during peroxidatic oxidation. In the presence of divalent cations (Mg2+, Zn2+), however, which slow the rate of o-semiquinone disproportionation, glutathione was found to inhibit catechol removal. This suggests that in the absence of stabilizing metal, reduction of the o-benzosemiquinone radical by glutathione cannot compete with other rapid reactions of the radical such as disproportionation. No interaction of the o-benzosemiquinone radical with oxygen could be detected even in the presence of stabilizing metals or superoxide dismutase which inhibits the reverse reaction of the SQ + O2 in equilibrium Q + O.2 equilibrium. Thus, under physiological conditions, glutathione and oxygen would not be expected to reduce or oxidize respectively the o-benzosemiquinone radical. These data show that the generation of thiol conjugates of o-benzoquinone can be used as probes of peroxidatic oxidation of catechol.

Glutathione oxidation during peroxidase catalysed drug metabolism.

The peroxidase catalyzed oxidation of certain drugs in the presence of glutathione (GSH) resulted in extensive oxidation to oxidized glutathione (GSSG). Extensive oxygen uptake ensued and thiyl radicals could be trapped. Only catalytic amounts of drugs were required indicating a redox cycling mechanism. Active drugs included phenothiazines, aminopyrine, p-phenetidine, acetaminophen and 4-N,N-(CH3)2-aminophenol. Other drugs, including dopamine and alpha-methyl dopa, did not catalyse oxygen uptake, nor were GSSG or thiyl radicals formed. Instead, GSH was depleted by GSH conjugate formation. Drugs of the former group, e.g. acetaminophen, aminopyrine or N,N-(CH3)2-aniline have also been found by other investigators to form GSSG and hydrogen peroxide when added to hepatocytes or when perfused through an isolated liver. Although cytochrome P-450 normally catalyses a two-electron oxidation of drugs, serious consideration should be given for some one-electron oxidation resulting in radical formation, oxygen activation and GSSG formation.

Oxygen activation during drug metabolism.

The peroxidase-H2O2 catalyzed oxidation of certain drugs in the presence of GSH resulted in extensive oxidation to thiyl radicals and GSSG. NADH or arachidonate in place of GSH was also readily oxidized. Extensive oxygen uptake ensued resulting in the formation of superoxide radicals and H2O2. Only catalytic amounts of drugs and low peroxide levels were required, indicating a radox cycling mechanism. Active drugs included morphine, phenothiazines, aminopyrine, p-phenetidine, acetaminophen and 4-N,N-(CH3)2-aminophenol. Other drugs, including dopamine and methyl-alpha-dopa, did not catalyze oxygen uptake, nor was GSH oxidized to GSSG. Instead, GSH was depleted by GSH conjugate formation. Drugs of the former group, e.g. acetaminophen, aminopyrine or N,N-(CH3)2-aniline, have also been found by other investigators to form GSSG and hydrogen peroxide when added to hepatocytes or when perfused through an isolated liver. Although cytochrome P-450 normally catalyzes a two-electron oxidation of drugs, serious consideration should be given to some one-electron oxidation occurring as well and resulting in radical formation, oxygen activation and GSSG formation.

Peroxidase-catalyzed-3-(glutathion-S-yl)-p,p'-biphenol formation.

Oxidation of p,p'-biphenol with horseradish peroxidase (HRP)-hydrogen peroxide in the presence of bovine serum albumin or with bone marrow cell homogenate-hydrogen peroxide resulted in the formation of reactive products that conjugate with protein. Glutathione prevented the protein binding. Glutathione readily reacted with p,p'-biphenoquinone, the principal oxidation product of p,p'-biphenol in the HRP-hydrogen peroxide system and resulted in the formation of several glutathione conjugates, p,p'-biphenol and small amounts of oxidized glutathione. The major glutathione conjugate was identified as 3-(glutathion-S-yl)-p,p'-biphenol by high field nuclear magnetic resonance and fast atom bombardment mass spectrometry. The same conjugate was formed in the bone marrow homogenate-hydrogen peroxide system. p,p'-Biphenoquinone reduction by glutathione to p,p'-biphenol without glutathione oxidation was explained by the rapid reduction of p,p'-biphenoquinone by 3-(glutathion-S-yl)-p,p'-biphenol.

Peroxidase catalysed oxygen activation by arylamine carcinogens and phenol.

Peroxidase catalysed the formation of active oxygen in the presence of NADH or GSH and traces of H2O2 and arylamine or phenolic substrates. Some oxygen activation occurred with some arylamines even in the absence of NADH or GSH. Oxygen consumption was proportional to the NADH oxidized or GSSG formed. Approximately 0.80 and 0.40 mol of oxygen were consumed per mole of NADH or GSH oxidized respectively. The requirement for trace amounts of hydrogen peroxide and arylamine or phenolic substrates suggest that redox cycling resulted in H2O2 formation. It is proposed that initially formed phenoxy radicals or arylamine cation radicals oxidize NADH or GSH to radicals which react with oxygen to form superoxide radicals and H2O2.

Phenol oxidation product(s), formed by a peroxidase reaction, that bind to DNA.

Phenol oxidation by horseradish peroxidase/H2O2 initially results in p,p'-biphenol and o.o'-biphenol formation and subsequently results in polymer formation. o,o'-Biphenol is the major product formed, but it is rapidly oxidized to the polymer, particularly in the presence of phenol. p,p'-Biphenol is very rapidly oxidized to p,p'-biphenoquinone which can also be involved in polymer formation. Extensive binding of 14C-phenol oxidation products to DNA occurs if the DNA is present in the reaction mixture. However, enzymic hydrolysis of DNA releases the bound polymers. p,p'-Biphenol, however, did not bind to DNA following peroxidase-catalysed oxidation, but o,o'-biphenol readily binds to DNA following peroxidase-catalysed oxidation. Enzymic hydrolysis of the oxidized o,o'-biphenol-bound DNA also resulted in the release of the polymers.

Peroxidase-catalysed binding of [U-14C]phenol to DNA.

14C-Phenol binds irreversibly to calf-thymus DNA in the presence of horseradish peroxidase and hydrogen peroxide, approximately 65% of the added phenol was bound to DNA. Binding was maximal at an equimolar concentration of hydrogen peroxide. Binding also occurred to homopolyribonucleotides polyadenylic acid, polyguanylic acid, polycytidylic acid and polyuridylic acid, and suggests that binding is relatively non-specific with respect to the nucleotide bases. p,p'-Biphenol, p,p'-biphenoquinone, o,o'-biphenol and two unidentified products were formed by the oxidation of phenol, in the presence and in absence of DNA. DNA accelerated phenol oxidation four fold and prevented the polymerization of oxidized phenol products, but was found to have no effect on the range of ethyl acetate-extractable products. Phenol accelerated the metabolism of o,o'-biphenol but had no effect on p,p'-biphenol metabolism. The mechanism of phenol activation is not clear, but p,p'-biphenoquinone binds to protein and not to DNA. DNA binding was prevented by glutathione, N-acetyl-cysteine and ascorbate, and the mechanism was shown to involve reduction of the activated phenol intermediates and the formation of conjugates with glutathione and N-acetyl-cysteine. DNA binding was not inhibited by lysine and proline.

Peroxidase-mediated irreversible binding of arylamine carcinogens to DNA in intact polymorphonuclear leukocytes activated by a tumor promoter.

Addition of the tumor promoter phorbol myristate acetate to polymorphonuclear leukocytes results in the oxidation of the arylamine carcinogens; [14C]benzidine, N-[14C]methylaminoazobenzene and [14C]aminofluorene to reactive intermediate(s) that bind irreversibly to the leukocyte DNA. The binding was dependent on oxygen and was decreased by sulfhydryl inhibitors and phenolic antioxidants that inhibit the respiratory burst triggered by the phorbol myristate. Both the binding and the respiratory burst were increased by azide, presumably as a result of intracellular catalase inhibition. However higher concentrations of azide and cyanide prevented binding without affecting the respiratory burst indicating that myeloperoxidase is a catalyst for the binding. Granules isolated from the activated leukocytes and H2O2 catalyzed a cyanide sensitive benzidine binding to calf thymus DNA. Myeloperoxidase and H2O2 also catalysed extensive binding of these arylamines to calf thymus DNA. The leukocytes appear to be a useful model cell for studying one electron oxidation-catalyzed carcinogen activation.