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Induction of hepatic heme oxygenase activity by bromobenzene
ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 196, No. 1, August, pp. 178-185, 1979
Induction of Hepatic Heme Oxygenase Activity by Bromobenzene1>2 PHILIP
S. GUZELIAN
Liver Study Unit, Departments
of Medicine
NABIL A. ELSHOURBAGY
AND
and Pharmacology,
Virginia
Commonwealth
University,
Medical College of Virginia, Richmond, Virginia 23298 Received December 18, 1978; revised March 5, 1979 Hepatic heme oxygenase, an enzyme which converts heme to carbon monoxide and bile pigment in vitro, is inducible by heme but also by large “toxic” doses of such nonheme substances as hormones, endotoxin, and heavy metal ions. When we gave rats a single hepatotoxic dose of ally1 alcohol, ethionine, acetaminophen, furosemide, or endotoxin, hepatic heme oxygenase activity rose modestly (two- to fivefold) after 20 h. In contrast, administration of bromobenzene (5 mmol/kg) induced heme oxygenase in the liver an average of 15-fold after 20 h but was without effect on the enzyme in the kidney or spleen. The change in heme oxygenase was accompanied by a loss in cytochrome P-450 concentration and, in rats labeled with 5-6-amino[14C]levulinic acid, an increased rate of degradation of hepatic [‘%]heme to %O. Induction of heme oxygenase by bromobenzene was blocked by cycloheximide, an inhibitor of protein synthesis, but not by actinomycin D, an inhibitor of RNA synthesis. This suggests that bromobenzene stimulates de nova enzyme synthesis at the step of translation. Subtoxic doses of bromobenzene (less than 1 mmol/kg) gave proportionately greater induction of heme oxygenase. Furthermore, induction of the enzyme remained unaffected when bromobenzene hepatotoxicity was blocked by pretreatment of rats with SKF-525A, 3-methylcholanthrene, or cysteine (which supplements liver sulfhydryl content), or when hepatotoxicity was enhanced by pretreatment with phenobarbital or with diethylmaleate (which depletes hepatic glutathione). These data suggest that with induction of heme oxygenase by bromobenzene, neither liver cell necrosis nor alteration in hepatic sulfhydryl metabolism is indispensible. The latter characteristic differs from induction of the enzyme by metal ions in which depletion of sulfhydryl-containing constituents has been thought to be essential. We conclude that bromobenzene is a novel inducer of heme oxygenase activity in the liver, differing from other nonheme substances in potency and specificity for the liver, and in utilizing mechanism(s) which require neither production of hepatotoxicity, depletion of hepatic glutathione, nor sensitivity to actinomycin D.
Degradation of heme in the liver is mediated by microsomal heme oxygenase, an enzyme which converts heme in vitro to carbon monoxide and bile pigment (1). An important role of heme oxygenase in heme turnover is inferred from the fact that the enzyme is inducible by its substrate, heme. 1 Supported by NIH Grant lPOlAM18976. Dr. Guzelian is the recipient of a Clinical Investigator Award in Gastroenterology from the NIH. Dr. Elshourbagy is supported by an NIH Training Grant in Gastroenterology, 5T32AM07150. ’ Unless specified otherwise, the terms “induction” or “stimulation,” when applied to heme oxygenase, are used to indicate increase in enzyme activity without implying a specific mechanism for the increase. ooo3-9861/79/090178-03$02.00/0 Copyright 0 1979 by Academic F~ess, Inc. All rights of reproduction in any form reserved.
1’78
Intravenous administration of heme in the form of hemoglobin (2, 3), or of methemalbumin (4, 51, increases heme oxygenase activity in rat liver parenchymal cells. These findings suggest that heme oxygenase activity, and hence the capacity of the liver to degrade circulating heme, may be regulated by the level of extrinsic heme entering the liver (2). A new perspective on the control of heme degradation has been afforded by the discovery that heme oxygenase is induced also by diverse agents other than heme. These nonheme inducers which include epinephrine (6), glucagon (6), zymosan (2), endotoxin (5), or cobalt and other heavy metal ions
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OF HEPATIC HEME OXYGENASE
(7), are not believed to promote intravascular hemolysis (8). Indeed, the magnitude of enzyme induction by some of these agents (‘7) is greater than that caused by administered hemoglobin or methemalbumin. Furthermore, induction of heme oxygenase occurs spontaneously in rat hepatocytes cultured in serum-free medium (9) and in cultures of avian liver cells treated with cobalt (10). Whereas these findings demonstrate that nonheme compounds affect the liver directly, the mechanism of heme oxygenase induction by these substances remains unanswered and controversial. Maines and Kappas have championed the concept that heavy metal ions are the proximate regulators of heme oxygenase (7). They postulate that these metals deplete the liver of glutathione and other key sulfhydryl-containing constituents by forming metal-thiol complexes, and this in some manner directly induces heme oxygenase (11). As a consequence of the rise in heme oxygenase, the degradation of intrinsic hepatic heme is accelerated (12) and the concentration of cytochrome P-450, the predominant hepatic hemoprotein, declines (7). In contrast to this view, Bissell and Hammaker in the course of their detailed studies of endotoxin in rats noted that a common property of all nonheme inducers of heme oxygenase studied, and indeed of heme itself, is that high, “toxic” doses must be administered for maximal effect on the enzyme (5). They concluded that the toxicity of these agents labilizes cytochrome P-450, promoting dissociation of heme from its apoprotein (13, 14). This increases the size of a regulatory pool of “free” heme which secondarily induces heme oxygenase (5). In preliminary investigations of these questions, we surveyed a series of hepatotoxic agents and found that bromobenzene is an exceedingly potent stimulator of hepatic heme oxygenase activity (15). Bromobenzene was selected for further study because the mechanism of its toxicity to the liver parenchymal cell is well known to involve metabolic conversion of the chemical to electrophilic products capable of combining with glutathione and other sulfhydrylcontaining substances (16). Using experimental manipulations previously reported
to modulate the metabolism of bromobenzene, and hence, its toxicity, we have found that bromobenzene induces heme oxygenase activity by a unique mechanism which appears to rely upon neither depletion of glutathione nor necrosis of liver cells. MATERIALS
AND METHODS
Male Sprague-Dawley albino rats, weighing 180 to 220 g, were purchased from Flow Laboratories (Dublin, Va.) and were maintained in wire-bottom cages with free access to chow and water until the time of study. The animals were fasted overnight prior to killing. The timing of injections of test substances was adjusted according to protocol so that all rats were killed between 8:00 and 900 AM. Bromobenzene was purchased from Eastman Fine Chemical Company (Rochester, N. Y.) and was administered intraperitoneally as a 50% (v/v) solution in corn oil. For studies of the relationships among bromobenzene activation, hepatic sulfhydryl metabolism, and heme oxygenase activity, the experimental protocol of Jollow et al. (17) was adopted. Where indicated, rats received intraperitoneal injections of phenobarbital (75 mgikg, daily for 3 days), 3-methylcholanthrene (20 mg/kg, daily for 3 days), or the appropriate vehicle (saline or corn oil), and were used experimentally 24 h after the last injection. Diethylmaleate (0.6 ml/kg) was administered intraperitoneally 30 min before bromobenzene. Cysteine (150 mgikg) was administered intraperitoneally 5 min before and 60 min after bromobenzene. SKF-525A (p-diethylaminoethyl diphenylpropylacetate) (75 mgikg ip) was dissolved in saline and adminstered 1 h before bromobenzene and at 8-h intervals thereafter. Control animals received a volume of corn oil equal to that used to dissolve the respective dose of bromobenzene. In other experiments, actinomycin D and cycloheximide were given intraperitoneally 1 h prior to bromobenzene. A single dose of cobaltous chloride was given with two subcutaneous injections. Endotoxin from Salmonella typhimurium was purchased from Difco (Detroit, Mich.). Hepatotoxic doses of ally1 alcohol (50 &kg) (18), ethionine (200 mg/kg) (19), acetaminophen (750 mg/kg) (20), furosemide (400 mg/kg) (21), and endotoxin (2 mg/kg) (5) were given in a single intraperitoneal injection. Preparation of hepatic mierosomes. Animals were killed by decapatation. The liver was perfused through the hepatic veins with iced phosphate-buffered saline (pH 7.4) in sufficient volume to achieve complete blanching of the organ. Next, the liver was excised and homogenized in 4 vol of 0.1 M potassium phosphate buffer (pH 7.4). The homogenate was filtered through two layers of gauze and was centrifuged at IS,0009 for 20 min. The supernatant was transferred to a Beckman L5-50 ultracentrifuge and was centrifuged
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at 105,OOOgfor 1 h. The supernatant was removed significant (P < 0.05) rise in heme oxyand the microsomal pellet was resuspended in 0.1 M genase activity within 2 h (Fig. 1). Therepotassium phosphate buffer. after, enzyme activity increased progresAnalytical procedures. Measurement of [%]heme sively, rising to a peak at 20 h, which degradation in tiwo was carried out using the averaged 15 times the mean control level protocol of Bissell and Hammaker (5). In order to (Fig. 1) with values ranging as high as radiolabel hepatic heme to the exclusion of hemoglobin 28-fold over controls in some experiments. heme, pairs of rats were given 6 j&i of 5-&aminoAt 48 and 72 h after bromobenzene, heme [‘*C]levulinic acid (New England Nuclear Corporation, Boston, Mass.) by injection in the tail veins. Since oxygenase activity had gradually declined this heme precursor labels the bridge carbons of the toward the normal range (Fig. 1). Acporphyrin ring, 1 mol of %O is released upon companying the rise in heme oxygenase degradation of each mole of heme. Sixteen hours later activity was a decline in the level of cyto[when the bulk of the labeled heme in the liver is chrome P-450 in the liver to approximately associated with cytochrome P-450 (5)], control collec50% of its initial level after 20 h (Fig. 1). tions of expired air were made by placing each By several criteria, the heme oxygenase animal in a metabolic cage connected to a “‘CO activity measured in hepatic microsomes trapping system (5). When such measurements confirmed that the two rats were excreting similar prepared from bromobenzene treated rats amounts of 14C0 [which indicates that total cyto- was the same as that obtained from controls chrome P-450 [‘%]heme is similar in the two animals or from animals given methemalbumin. (5)], one rat was given bromobenzene, the other, corn Thus, the enzyme required NADPH (l), oil. The rate of excretion of ‘*CO was followed produced a product spectrally the same as hourly for 9 h. authentic bilirubin (l), and was inhibited by Heme oxygenase activity was measured by a slight antibody directed against purified porcine modification of the method of Tenhunen et al. (1). NADPH-dependent cytochrome c reductase A typical reaction mixture contained 1.67 ml of 0.1 M potassium phosphate buffer (pH 7.4), 0.2 ml of microsomes containing approximately 1 mg of protein, 0.4 ml of a methemalbumin solution (5), and, as a source of biliverdin reductase, 0.05 ml of 100,OOOg supernatant prepared from homogenates of normal rat liver. The final concentration of heme in the assay was 40 pM. The solution was divided equally between two l-ml masked cuvettes and these were incubated for 1 to 2 min at 3X, while baseline absorbance at 468 nm was measured using an Aminco DW-2A dual beam spectrophotometer in split beam mode (American Instrument Company, Silver Spring, Md.). The reaction “““““’ ob ‘1’ “’ Jo was initiated by addition of NADP (160 pg in 0.04 ml) 0 8 16 24 32 40 48 56 64 72 HOURS AFTER BROMOBENZENE to the sample cuvette and an equal volume of phosphate buffer to the reference cuvette. The rate FIG. 1. Effect of bromobenzene on hepatic heme of formation of bilirubin was estimated from the linear oxygenase activity and cytochrome P450 concentration. increase in absorbance using an extinction coefficient In each experiment, a group of 12 rats of similar age of 60 mM-’ cm-’ (22). and weight received intraperitoneal injections of either Cytochrome P-450 was assayed as the carbon bromobenzene (5 mmol/kg) or corn oil as control. At the monoxide binding hemoprotein in the dithionite reduced indicated times, pairs of animals were killed, liver midifference spectrum as described by Omura and Sato crosomes were prepared from each animal, and heme (23). Protein was measured by the methd of Schacterle oxygenase activity and cytochrome P-450 concentraand Pollack (24) using crystalline bovine albumin as tion were measured as outlined under Materials and standard. Methods. Cytochrome P-450 concentration was calculated as nmol/mg of microsomal protein, and the results RESULTS were expressed as percentage of the average value in controls. The present results were obtained by comAdministration of bromobenzene to rats bining four experiments, and each data point repreat a dose (5 mmol/kg) which produces both sents the mean f SEM of at least eight animals. The histologic (25) and biochemical (26) evidence shaded region represents the average (*SD) of heme of liver cell injury after 24 h, produced a oxygenase activity in controls.
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OF HEPATIC
(27) (a generous gift of Dr. Masters, Dallas, Tex.) (data not shown). In other experiments, additions of bromobenzene to a reaction mixture containing microsomes from control animals had no effect on heme oxygenase activity. Dose-response relationships between bromobenzene and heme oxygenase activity are illustrated in Fig. 2. Doses higher than 5.0 mmol/kg (approaching the LD,, of 10 mmol/kg) (26) were not studied because most of the animals appeared agonal. By inspection of the curve for analyses made 20 h following bromobenzene, it appeared that the rise in heme oxygenase was proportionately greater for small doses (less than 1.0 mmol/kg). Sensitivity of the enzyme to small doses was more readily apparent when measurements were made 9 h after bromobenzene (Fig. 2), a time at which detectable increases were noted with as little as 0.05 mmoYkg (Fig. 2). Comparison of the two curves (Fig. 2) revealed that heme oxygenase activity tended to be greater at 9 h than at 20 h with small doses of bromobenzene (P < 0.05 for 1.0 and 0.2 mmol/kg), whereas the reverse was true for 5.0 mmoYkg. The difference in enzyme induction between these times may be due to the rapid decline in bromobenzene concentration in the liver 8-12 h after administration of small doses (16, 25).
012345 DOSE OF BROMOBENZENE (mmol I kg)
FIG. 2. Relationship between dose of bromobenzene and induction of heme oxygenase activity. Groups of rats received various doses of bromobenzene or corn oil as control and were killed either 9 or 20 h later. Hepatic microsomes were prepared, and heme oxygenase activity was measured as outlined under Materials and Methods. Results were expressed as percentage of the control value. Each data point represents the mean % SEM for at least four animals.
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The effect of bromobenzene on the rate of degradation of cytochrome P-450 heme in viva was examined by measurement of 14C0 excretion in rats with hepatic heme radiolabeled by administration of 5-&amino[14C]levulinic acid (5). At a dose of 5 mmol/kg, bromobenzene produced an increase in the rate of 14C0excretion which was detectable within 2 to 4 h, rose to a peak at 6-7 h, and returned to the control rate after 9 h, similar to the effect reported for methemalbumin (5). The cumulative increase in excretion of 14C0was 34 + 3% (SEM, N = 5) above the control value. A lower dose of bromobenzene (0.5 mmol/kg) produced a similar pattern of change in 14C0 excretion, although the magnitude of the increase [22 + 4% (SEM, N = 5)] was significantly less (P < 0.05). These data suggest that bromobenzene stimulates catabolism of cytochrome P-450 [14C]heme in parallel with dose-dependent changes in heme oxygenase activity. To investigate the mechanism of induction of heme oxygenase by bromobenzene, rats were pretreated with cycloheximide or actinomycin D, compounds which block synthesis of protein or RNA, respectively. Cycloheximide blocked completely the induction of heme oxygenase, whereas actinomycin D was without effect (Table I). In performing a positive control experiment for the latter result, we found that actinomycin D partially inhibited induction of heme oxygenase by cobaltous chloride (Table I), confirming previous reports (28). The effect of bromobenzene was specific for the liver. In a single experiment, three rats were given either 0.5, 3.0, or 5.0 mmollkg bromobenzene while a fourth served as control. After 9 h, heme oxygenase activity in the liver rose to 5.9-, 15.7-, and 20.0-fold, respectively, over the control value, whereas heme oxygenase activity in kidney or spleen microsomes from these animals was the same as that in the control. Because heme oxygenase was inducible with low doses of bromobenzene previously found to be “nontoxic” (25), we tested the hypothesis that induction of the enzyme might be unrelated to production of hepatocellular necrosis. By comparison with bromobenzene, other necrogenic agents (ethionine,
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furosemide, ally1 alcohol, endotoxin, or acetaminophen) produced only a modest (two- to fourfold) stimulation of heme oxygenase activity (data not shown). Furthermore, pretreatment of rats with SKF-525A had no effect on the induction of heme oxygenase by bromobenzene (Table I), despite the fact that this compound abolishes the hepatotoxicity in rats receiving 20 times more bromobenzene (17, 25). SKF-525A blocks the metabolic activation of bromobenzene and the subsequent loss of hepatic glutathione (17). Likewise, phenobarbital pretreatment had little effect on induction of heme oxygenase (Table I), even though phenobarbital enhances liver cell necrosis by augmenting the rates of bromobenzene metabolism and of glutathione depletion (17). Although liver cell necrosis from bromobenzene can be either intensified by pretreatment with diethylmaleate-a substance which rapidly reduces the levels of
intercellular glutathione (29>-or prevented by pretreatment with cysteine which supplements stores of sulfhydryl groups in the liver (l’i’), these agents failed to exert similar effects on induction of heme oxygenase by bromobenzene (Table II). Indeed, diethylmaleate appeared to impair induction of heme oxygenase (Table II), whereas diethylmaleate alone stimulated the enzyme modestly as has been reported previously (11). Finally, even though the metabolism of bromobenzene is diverted to nontoxic products by 3-methylcholanthrene pretreatment, heme oxygenase induction was unaffected. These experiments demonstrate that induction of heme oxygenase need not be accompanied by loss of hepatocellular sulfhydryl content or viability. DISCUSSION
The present studies demonstrate that bromobenzene administration initiates marked changes in the homeostasis of hepatic heme. TABLE I These include a rise in heme oxygenase EFFECT OF ACTINOMYCIN D OR CYCLOHEXIMIDEON activity measured in hepatic microsomes INDUCTIONOF HEME OXYGENASEBY in vitro, accelerated conversion of the heme BROMOBENZENEORCOBALTOUS in hepatic hemoproteins (cytochrome P-450) CHLORIDES to physiologic degradation products in vivo, and a decline from steady-state levels in Heme oxygenase activity the concentration of cytochrome P-450. (nmol bilirubini Similar manifestations of accelerated catabTreatment 10 minimg protein) olism of cytochrome P-450 heme are produced Corn oil + saline also by such nonheme substances as cobalt (Control) 0.139 * 0.011 (7), and endotoxin (5), as well as by heme Bromobenzene 1.081 2 0.056 itself (5). The effects of these compounds Actinomycin D 0.156 2 0.014 undoubtedly involve the hepatocyte since Bromobenzene liver sinusoidal cells contain only trace + actinomycin D 0.997 k 0.076 amounts of cytochrome P-450 and less than Cycloheximide 0.109 r 0.014 10% of heme oxygenase activity in the liver Bromobenzene (3). Because the individual steps in the + cycloheximide 0.105 + 0.015 pathway for catabolism of hepatic hemoCobaltous chloride 1.169 2 0.049 Cobaltous chloride proteins have not been identified, it is + actinomycin D 0.483 + 0.032 uncertain whether stimulation of a common rate-controlling mechanism underlies the ‘I Rats were given either actinomycin D (0.8 mg/kg), accelerated breakdown of intrinsic hepatic cycloheximide (2 mg/kg), or the appropriate vehicle heme by these “toxic” agents. Bromo(corn oil or saline) and, 1 h later, the animals received benzene appears to be useful for probing a second injection containing either bromobenzene (2.0 this question because the characteristics of mmolikg), cobaltous chloride (60 mgikg), or the approheme oxygenase induction by this chemical priate vehicle. The rats were killed 20 h following the differ in several important aspects from last injection and liver microsomes were prepared for those reported for heme or for other nonmeasurement of heme oxygenase. Results represent the average (rSEM) of three animals. heme inducers of the enzyme.
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OF HEPATIC HEME OXYGENASE TABLE II
EFFECT OF VARIOUS PRETREATMENTS ON STIMULATION OF HEME OXYGENASE BY BROMOBENZENE~
Dose of bromobenzene (mmol/kg)
0.5 0.5 0.5 2.0 2.0 2.0 2.0 2.0
Treatment
Heme oxygenase activity (% of control f SEM)
Control Bromobenzene SKF-525A Bromobenzene + SKF-525A Diethylmaleate Bromobenzene + diethylmaleate Bromobenzene Phenobarbital Bromobenzene + phenobarbital 3-Methylcholanthrene Bromobenzene + 3-methylcholanthrene Bromobenzene + diethylmaleate Cysteine Bromobenzene + cysteine
100 471 2 39 102 2 4.1* 429 k 47 346 2 26 395 k 38 694 zt 54 97 * 7.5* 512 f 31 89 k 13* 628 2 44 412 + 28 94 + 8* 785 2 31
n Rats received the indicated treatment as described under Materials and Methods and were killed 20 h after administration of bromobenzene. Hepatic microsomes were prepared for measurement of heme oxygenase activity. Results were calculated as percentage of the activity in the respective control animals. The average of at least four values is nresented. All treatments other than those indicated by an asterisk are significantly greater than controls (P -< 0.05).
Bromobenzene was found to be among the most potent inducers of hepatic heme oxygenase yet described, a single dose producing an average E-fold increase in enzyme activity (Fig. 1). Increases ranging between two- and sixfold are commonly encountered with metabolic alterations (6), intravascular hemolysis (2), or following single large doses of methemalbumin (4), or one of several hepatotoxins (see results), whereas large doses of metal ions are somewhat more effective inducers of heme oxygenase (6- to lo-fold) (7). The effect of bromobenzene is selective in that prominant stimulation of heme oxygenase occurs at times (as early as 2 h) when bromobenzene is without effect on the level of other microsomal enzymes [e.g., glucose-6-phosphatase or NADPH neotetrazolium reductase activity (31)] or on the rate of general protein synthesis (31). Our data also suggests that neither the rate of bromobenzene metabolism nor the formation of a particular metabolite is a key event in the induction process. Thus, inhibition of bromobenzene metabolism by SKF-525A, acceleration of bromobenzene metabolism by pretreatment with pheno-
barbital, or alteration of the profile of bromobenzene metabolites by pretreatment with 3-methylcholanthrene had no effect on the stimulation of heme oxygenase activity. Since liver cell necrosis parallels bromobenzene metabolism (1’7),it follows that induction of heme oxygenase does not depend upon production of overt hepatotoxicity. In view of these results, the suggestion (5) that induction of heme oxygenase by nonheme substances involves production of toxicity to the animal may require modification. Although modest stimulation of heme oxygenase activity may be associated with hepatotoxicity, the remarkably potent effect of bromobenzene strongly implicates the involvement of other, apparently more selective mechanisms. Maines and Kappas have proposed that cobalt and other metals capable of forming complexes with sulfhydryl groups directly regulate hepatic heme oxygenase (11). They reported that 2 to 6 h after injection of cobalt or other metals to rats, the level of glutathione in the liver was significantly reduced in association with increased heme oxygenase activity (32). Furthermore, pre-
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treatment with diethylmaleate, which depletes glutathione in the liver, augmented induction of heme oxygenase by cobalt (ll), whereas supplementation of liver sulfhydryl stores with cysteine reduced the cobalt-mediated induction of the enzyme (11). These authors concluded that metals combine with glutathione and with specific receptor sites in the liver (presumably containing key sulfhydryl groups), the latter event triggering induction of heme oxygenase activity (11). If this concept were correct, then bromobenzene and metals might both be expected to affect heme oxygenase through this common mechanism because there is conclusive evidence that metabolism of bromobenzene produces short-lived, highly reactive products which combine with glutathione or with other sulfhydryl-containing constituents (16, 17, 26). However, neither the modifications in the rate of bromobenzene metabolism mentioned in the preceeding paragraph, nor pretreatment with diethylmaleate or cysteine had the effects on bromobenzene-mediated induction of heme oxygenase (Table I) predicted by this hypothesis. If changes in sulfhydryl content are necessary for heme oxygenase induction, then bromobenzene metabolites may combine preferentially with a different subspecies of sulfhydryl-containing components than do metals. An alternative possibility is that the assumed cause and effect relationship between the capacity of metals to bind sulfhydryl groups and to stimulate heme oxygenase may be invalid. In support of this idea, Maines and Kappas were unable to demonstrate a decrease in glutathione in association with induction of heme oxygenase by cobalt in cultures of avian hepatocytes (10). More important is the recent report of Sasame and Boyd who reexamined the effects of cobaltous chloride over a wide range of doses in rats and found that hepatic glutathione concentration increased significantly within 2 h and rose continuously thereafter (33). They observed that other divalent metal inducers of heme oxygenase had various effects on glutathione concentration, including large increases, slight decreases, or no change (33). In view
of the present studies with bromobenzene and the conflicting reports regarding the effects of metals on glutathione, it appears unlikely that alteration in hepatic sulfhydry1 content is the proximate event controlling heme oxygenase induction. The mechanism of heme oxygenase induction by bromobenzene has been characterized only indirectly. Whereas administered hemoglobin (34) or metal ions (35) induce heme oxygenase in both the liver and kidneys, bromobenzene stimulates the liver enzyme specifically. The latter event is blocked by cycloheximide, a finding which suggests the requirement for protein synthesis, possibly de novo enzyme formation. Since actinomycin D, an inhibitor of RNA synthesis, had no effect on inducticn of heme oxygenase, the effect of bromobenzene may be localized to translation. Induction of heme oxygenase by methemalbumin similarly is resistent to actinomycin D (4), whereas cobalt is sensitive to this inhibitor (26). These facts point to different mechanism(s) underlying induction of heme oxygenase by bromobenzene, metals, or heme. Nevertheless, an intimate relationship is maintained between heme oxygenase activity and hepatic hemoprotein turnover. Because detectable changes in degradation of cytochrome P-450 [14C]heme in vivo (14C0 excretion) and a detectable rise in heme oxygenase activity both occur within 2 to 4 h following endotoxin, methemalbumin, or bromobenzene (5), the present techniques do not resolve the question of whether induction of heme oxygenase preceeds accelerated catabolism of hepatic heme or whether induction of the enzyme is a consequence of stimulated hemoprotein turnover. Recent progress in purification of heme oxygenase (36) may speed the development of specific inhibitors of the enzyme which, together with stimulators of heme oxygenase, should facilitate advances in defining the pathways of hepatic heme degradation. ACKNOWLEDGMENTS The authors wish to thank Joyce Barwick for assistance in conducting some of these experiments. Careful typing of the manuscript was provided by Marcia Tetlak.
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H. A., AND POTTER, W. Z. (1976) Chem.Biol. Interact. 15, 139-147. VOGT, M. T., AND FARBER, E. (1970) Arch. Biochem. Biophys. 141, 162-173. MITCHELL, J. R., JOLLOW, D. J., POTTER, W. Z., DAVIS, D. C., GILLETTE, J. R., AND BRODIE, B. B. (1973) J. Pharmacol. Exp. Ther. 187, 185-194. MITCHELL, J. R., NELSON, W. L., POTTER, W. Z., SASAME, H. A., AND JOLLOW, D. J. (1976) J. Pharmacol. Exp. Ther. 199, 41-52. BISSELL, D. M., HAMMAKER, L. E., AND SCHMID, R. (1972) J. Cell Biol. 54, 107-119. OMURA, T., AND SATO, R. (1964) J. Biol. Chem. 239,2370-2378. SCHACTERLE, G. R., AND POLLACK, R. L. (1973) Anal. Biochem. 51, 654-655. REID, W. D., CHRISTIE, B., KRISHNA, G., MITCHELL, J. R., MOSKOWITZ, J., AND BRODIE, B. B. (1971) Pharmacology 6, 41-55. KOCH-WESER, D., DE LA HUERGA, J., YESINICK, C., AND POPPER, H. (1953) Metabolism 2, 248-260. SCHACTER, B. A., NELSON, E. B., MARVER, H. S., AND MASTERS, B. S. S. (1972) J. Biol. Chem. 247, 3601-3607. MAINES, M. D., AND KAPPA& A. (1975) J. Biol. Chem. 250, 4171-4177. BOYLAND, E., AND CHASSEAUD, L. F. (1970) Biothem. Pharmacol. 19, 1526-1528. ZAMPAGLIONE, N., JOLLOW, D. J., MITCHELL, J. R., STRIPP, B., HAMRICK, M., ANDGILLETTE, J. R. (1973) J. Pharmacol. Exp. Ther. 187, 218-227. REYNOLDS, E. F. (1972) Biochem. Pharmacol. 21, 2555-2561. MAINES, M. D., AND KAPPAS, A. (1977) Proc. Nat. Acad. Sci. USA 74, 1875-1878. SASAME, H. A., AND BOYD, M. R. (1978) J. Pharmacol. Exp. Ther. 205, 718-724. PIMSTONE, N. R., ENGLE, P., TENHUNEN, R., SEITZ, P. T., MARVER, H. S., AND SCHMID, R. (1971) J. Clin. Invest. 50, 2042-2050. KAPPAS, A., AND MAINES, M. D. (1976) Science 192, 60-62. YOSHIDA, T., AND KIKUCHI, G. (1978) J. Biol. Chem. 253,4224-4229.
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 196, No. 1, August, pp. 178-185, 1979
Induction of Hepatic Heme Oxygenase Activity by Bromobenzene1>2 PHILIP
S. GUZELIAN
Liver Study Unit, Departments
of Medicine
NABIL A. ELSHOURBAGY
AND
and Pharmacology,
Virginia
Commonwealth
University,
Medical College of Virginia, Richmond, Virginia 23298 Received December 18, 1978; revised March 5, 1979 Hepatic heme oxygenase, an enzyme which converts heme to carbon monoxide and bile pigment in vitro, is inducible by heme but also by large “toxic” doses of such nonheme substances as hormones, endotoxin, and heavy metal ions. When we gave rats a single hepatotoxic dose of ally1 alcohol, ethionine, acetaminophen, furosemide, or endotoxin, hepatic heme oxygenase activity rose modestly (two- to fivefold) after 20 h. In contrast, administration of bromobenzene (5 mmol/kg) induced heme oxygenase in the liver an average of 15-fold after 20 h but was without effect on the enzyme in the kidney or spleen. The change in heme oxygenase was accompanied by a loss in cytochrome P-450 concentration and, in rats labeled with 5-6-amino[14C]levulinic acid, an increased rate of degradation of hepatic [‘%]heme to %O. Induction of heme oxygenase by bromobenzene was blocked by cycloheximide, an inhibitor of protein synthesis, but not by actinomycin D, an inhibitor of RNA synthesis. This suggests that bromobenzene stimulates de nova enzyme synthesis at the step of translation. Subtoxic doses of bromobenzene (less than 1 mmol/kg) gave proportionately greater induction of heme oxygenase. Furthermore, induction of the enzyme remained unaffected when bromobenzene hepatotoxicity was blocked by pretreatment of rats with SKF-525A, 3-methylcholanthrene, or cysteine (which supplements liver sulfhydryl content), or when hepatotoxicity was enhanced by pretreatment with phenobarbital or with diethylmaleate (which depletes hepatic glutathione). These data suggest that with induction of heme oxygenase by bromobenzene, neither liver cell necrosis nor alteration in hepatic sulfhydryl metabolism is indispensible. The latter characteristic differs from induction of the enzyme by metal ions in which depletion of sulfhydryl-containing constituents has been thought to be essential. We conclude that bromobenzene is a novel inducer of heme oxygenase activity in the liver, differing from other nonheme substances in potency and specificity for the liver, and in utilizing mechanism(s) which require neither production of hepatotoxicity, depletion of hepatic glutathione, nor sensitivity to actinomycin D.
Degradation of heme in the liver is mediated by microsomal heme oxygenase, an enzyme which converts heme in vitro to carbon monoxide and bile pigment (1). An important role of heme oxygenase in heme turnover is inferred from the fact that the enzyme is inducible by its substrate, heme. 1 Supported by NIH Grant lPOlAM18976. Dr. Guzelian is the recipient of a Clinical Investigator Award in Gastroenterology from the NIH. Dr. Elshourbagy is supported by an NIH Training Grant in Gastroenterology, 5T32AM07150. ’ Unless specified otherwise, the terms “induction” or “stimulation,” when applied to heme oxygenase, are used to indicate increase in enzyme activity without implying a specific mechanism for the increase. ooo3-9861/79/090178-03$02.00/0 Copyright 0 1979 by Academic F~ess, Inc. All rights of reproduction in any form reserved.
1’78
Intravenous administration of heme in the form of hemoglobin (2, 3), or of methemalbumin (4, 51, increases heme oxygenase activity in rat liver parenchymal cells. These findings suggest that heme oxygenase activity, and hence the capacity of the liver to degrade circulating heme, may be regulated by the level of extrinsic heme entering the liver (2). A new perspective on the control of heme degradation has been afforded by the discovery that heme oxygenase is induced also by diverse agents other than heme. These nonheme inducers which include epinephrine (6), glucagon (6), zymosan (2), endotoxin (5), or cobalt and other heavy metal ions
REGULATION
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OF HEPATIC HEME OXYGENASE
(7), are not believed to promote intravascular hemolysis (8). Indeed, the magnitude of enzyme induction by some of these agents (‘7) is greater than that caused by administered hemoglobin or methemalbumin. Furthermore, induction of heme oxygenase occurs spontaneously in rat hepatocytes cultured in serum-free medium (9) and in cultures of avian liver cells treated with cobalt (10). Whereas these findings demonstrate that nonheme compounds affect the liver directly, the mechanism of heme oxygenase induction by these substances remains unanswered and controversial. Maines and Kappas have championed the concept that heavy metal ions are the proximate regulators of heme oxygenase (7). They postulate that these metals deplete the liver of glutathione and other key sulfhydryl-containing constituents by forming metal-thiol complexes, and this in some manner directly induces heme oxygenase (11). As a consequence of the rise in heme oxygenase, the degradation of intrinsic hepatic heme is accelerated (12) and the concentration of cytochrome P-450, the predominant hepatic hemoprotein, declines (7). In contrast to this view, Bissell and Hammaker in the course of their detailed studies of endotoxin in rats noted that a common property of all nonheme inducers of heme oxygenase studied, and indeed of heme itself, is that high, “toxic” doses must be administered for maximal effect on the enzyme (5). They concluded that the toxicity of these agents labilizes cytochrome P-450, promoting dissociation of heme from its apoprotein (13, 14). This increases the size of a regulatory pool of “free” heme which secondarily induces heme oxygenase (5). In preliminary investigations of these questions, we surveyed a series of hepatotoxic agents and found that bromobenzene is an exceedingly potent stimulator of hepatic heme oxygenase activity (15). Bromobenzene was selected for further study because the mechanism of its toxicity to the liver parenchymal cell is well known to involve metabolic conversion of the chemical to electrophilic products capable of combining with glutathione and other sulfhydrylcontaining substances (16). Using experimental manipulations previously reported
to modulate the metabolism of bromobenzene, and hence, its toxicity, we have found that bromobenzene induces heme oxygenase activity by a unique mechanism which appears to rely upon neither depletion of glutathione nor necrosis of liver cells. MATERIALS
AND METHODS
Male Sprague-Dawley albino rats, weighing 180 to 220 g, were purchased from Flow Laboratories (Dublin, Va.) and were maintained in wire-bottom cages with free access to chow and water until the time of study. The animals were fasted overnight prior to killing. The timing of injections of test substances was adjusted according to protocol so that all rats were killed between 8:00 and 900 AM. Bromobenzene was purchased from Eastman Fine Chemical Company (Rochester, N. Y.) and was administered intraperitoneally as a 50% (v/v) solution in corn oil. For studies of the relationships among bromobenzene activation, hepatic sulfhydryl metabolism, and heme oxygenase activity, the experimental protocol of Jollow et al. (17) was adopted. Where indicated, rats received intraperitoneal injections of phenobarbital (75 mgikg, daily for 3 days), 3-methylcholanthrene (20 mg/kg, daily for 3 days), or the appropriate vehicle (saline or corn oil), and were used experimentally 24 h after the last injection. Diethylmaleate (0.6 ml/kg) was administered intraperitoneally 30 min before bromobenzene. Cysteine (150 mgikg) was administered intraperitoneally 5 min before and 60 min after bromobenzene. SKF-525A (p-diethylaminoethyl diphenylpropylacetate) (75 mgikg ip) was dissolved in saline and adminstered 1 h before bromobenzene and at 8-h intervals thereafter. Control animals received a volume of corn oil equal to that used to dissolve the respective dose of bromobenzene. In other experiments, actinomycin D and cycloheximide were given intraperitoneally 1 h prior to bromobenzene. A single dose of cobaltous chloride was given with two subcutaneous injections. Endotoxin from Salmonella typhimurium was purchased from Difco (Detroit, Mich.). Hepatotoxic doses of ally1 alcohol (50 &kg) (18), ethionine (200 mg/kg) (19), acetaminophen (750 mg/kg) (20), furosemide (400 mg/kg) (21), and endotoxin (2 mg/kg) (5) were given in a single intraperitoneal injection. Preparation of hepatic mierosomes. Animals were killed by decapatation. The liver was perfused through the hepatic veins with iced phosphate-buffered saline (pH 7.4) in sufficient volume to achieve complete blanching of the organ. Next, the liver was excised and homogenized in 4 vol of 0.1 M potassium phosphate buffer (pH 7.4). The homogenate was filtered through two layers of gauze and was centrifuged at IS,0009 for 20 min. The supernatant was transferred to a Beckman L5-50 ultracentrifuge and was centrifuged
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at 105,OOOgfor 1 h. The supernatant was removed significant (P < 0.05) rise in heme oxyand the microsomal pellet was resuspended in 0.1 M genase activity within 2 h (Fig. 1). Therepotassium phosphate buffer. after, enzyme activity increased progresAnalytical procedures. Measurement of [%]heme sively, rising to a peak at 20 h, which degradation in tiwo was carried out using the averaged 15 times the mean control level protocol of Bissell and Hammaker (5). In order to (Fig. 1) with values ranging as high as radiolabel hepatic heme to the exclusion of hemoglobin 28-fold over controls in some experiments. heme, pairs of rats were given 6 j&i of 5-&aminoAt 48 and 72 h after bromobenzene, heme [‘*C]levulinic acid (New England Nuclear Corporation, Boston, Mass.) by injection in the tail veins. Since oxygenase activity had gradually declined this heme precursor labels the bridge carbons of the toward the normal range (Fig. 1). Acporphyrin ring, 1 mol of %O is released upon companying the rise in heme oxygenase degradation of each mole of heme. Sixteen hours later activity was a decline in the level of cyto[when the bulk of the labeled heme in the liver is chrome P-450 in the liver to approximately associated with cytochrome P-450 (5)], control collec50% of its initial level after 20 h (Fig. 1). tions of expired air were made by placing each By several criteria, the heme oxygenase animal in a metabolic cage connected to a “‘CO activity measured in hepatic microsomes trapping system (5). When such measurements confirmed that the two rats were excreting similar prepared from bromobenzene treated rats amounts of 14C0 [which indicates that total cyto- was the same as that obtained from controls chrome P-450 [‘%]heme is similar in the two animals or from animals given methemalbumin. (5)], one rat was given bromobenzene, the other, corn Thus, the enzyme required NADPH (l), oil. The rate of excretion of ‘*CO was followed produced a product spectrally the same as hourly for 9 h. authentic bilirubin (l), and was inhibited by Heme oxygenase activity was measured by a slight antibody directed against purified porcine modification of the method of Tenhunen et al. (1). NADPH-dependent cytochrome c reductase A typical reaction mixture contained 1.67 ml of 0.1 M potassium phosphate buffer (pH 7.4), 0.2 ml of microsomes containing approximately 1 mg of protein, 0.4 ml of a methemalbumin solution (5), and, as a source of biliverdin reductase, 0.05 ml of 100,OOOg supernatant prepared from homogenates of normal rat liver. The final concentration of heme in the assay was 40 pM. The solution was divided equally between two l-ml masked cuvettes and these were incubated for 1 to 2 min at 3X, while baseline absorbance at 468 nm was measured using an Aminco DW-2A dual beam spectrophotometer in split beam mode (American Instrument Company, Silver Spring, Md.). The reaction “““““’ ob ‘1’ “’ Jo was initiated by addition of NADP (160 pg in 0.04 ml) 0 8 16 24 32 40 48 56 64 72 HOURS AFTER BROMOBENZENE to the sample cuvette and an equal volume of phosphate buffer to the reference cuvette. The rate FIG. 1. Effect of bromobenzene on hepatic heme of formation of bilirubin was estimated from the linear oxygenase activity and cytochrome P450 concentration. increase in absorbance using an extinction coefficient In each experiment, a group of 12 rats of similar age of 60 mM-’ cm-’ (22). and weight received intraperitoneal injections of either Cytochrome P-450 was assayed as the carbon bromobenzene (5 mmol/kg) or corn oil as control. At the monoxide binding hemoprotein in the dithionite reduced indicated times, pairs of animals were killed, liver midifference spectrum as described by Omura and Sato crosomes were prepared from each animal, and heme (23). Protein was measured by the methd of Schacterle oxygenase activity and cytochrome P-450 concentraand Pollack (24) using crystalline bovine albumin as tion were measured as outlined under Materials and standard. Methods. Cytochrome P-450 concentration was calculated as nmol/mg of microsomal protein, and the results RESULTS were expressed as percentage of the average value in controls. The present results were obtained by comAdministration of bromobenzene to rats bining four experiments, and each data point repreat a dose (5 mmol/kg) which produces both sents the mean f SEM of at least eight animals. The histologic (25) and biochemical (26) evidence shaded region represents the average (*SD) of heme of liver cell injury after 24 h, produced a oxygenase activity in controls.
REGULATION
OF HEPATIC
(27) (a generous gift of Dr. Masters, Dallas, Tex.) (data not shown). In other experiments, additions of bromobenzene to a reaction mixture containing microsomes from control animals had no effect on heme oxygenase activity. Dose-response relationships between bromobenzene and heme oxygenase activity are illustrated in Fig. 2. Doses higher than 5.0 mmol/kg (approaching the LD,, of 10 mmol/kg) (26) were not studied because most of the animals appeared agonal. By inspection of the curve for analyses made 20 h following bromobenzene, it appeared that the rise in heme oxygenase was proportionately greater for small doses (less than 1.0 mmol/kg). Sensitivity of the enzyme to small doses was more readily apparent when measurements were made 9 h after bromobenzene (Fig. 2), a time at which detectable increases were noted with as little as 0.05 mmoYkg (Fig. 2). Comparison of the two curves (Fig. 2) revealed that heme oxygenase activity tended to be greater at 9 h than at 20 h with small doses of bromobenzene (P < 0.05 for 1.0 and 0.2 mmol/kg), whereas the reverse was true for 5.0 mmoYkg. The difference in enzyme induction between these times may be due to the rapid decline in bromobenzene concentration in the liver 8-12 h after administration of small doses (16, 25).
012345 DOSE OF BROMOBENZENE (mmol I kg)
FIG. 2. Relationship between dose of bromobenzene and induction of heme oxygenase activity. Groups of rats received various doses of bromobenzene or corn oil as control and were killed either 9 or 20 h later. Hepatic microsomes were prepared, and heme oxygenase activity was measured as outlined under Materials and Methods. Results were expressed as percentage of the control value. Each data point represents the mean % SEM for at least four animals.
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OXYGENASE
181
The effect of bromobenzene on the rate of degradation of cytochrome P-450 heme in viva was examined by measurement of 14C0 excretion in rats with hepatic heme radiolabeled by administration of 5-&amino[14C]levulinic acid (5). At a dose of 5 mmol/kg, bromobenzene produced an increase in the rate of 14C0excretion which was detectable within 2 to 4 h, rose to a peak at 6-7 h, and returned to the control rate after 9 h, similar to the effect reported for methemalbumin (5). The cumulative increase in excretion of 14C0was 34 + 3% (SEM, N = 5) above the control value. A lower dose of bromobenzene (0.5 mmol/kg) produced a similar pattern of change in 14C0 excretion, although the magnitude of the increase [22 + 4% (SEM, N = 5)] was significantly less (P < 0.05). These data suggest that bromobenzene stimulates catabolism of cytochrome P-450 [14C]heme in parallel with dose-dependent changes in heme oxygenase activity. To investigate the mechanism of induction of heme oxygenase by bromobenzene, rats were pretreated with cycloheximide or actinomycin D, compounds which block synthesis of protein or RNA, respectively. Cycloheximide blocked completely the induction of heme oxygenase, whereas actinomycin D was without effect (Table I). In performing a positive control experiment for the latter result, we found that actinomycin D partially inhibited induction of heme oxygenase by cobaltous chloride (Table I), confirming previous reports (28). The effect of bromobenzene was specific for the liver. In a single experiment, three rats were given either 0.5, 3.0, or 5.0 mmollkg bromobenzene while a fourth served as control. After 9 h, heme oxygenase activity in the liver rose to 5.9-, 15.7-, and 20.0-fold, respectively, over the control value, whereas heme oxygenase activity in kidney or spleen microsomes from these animals was the same as that in the control. Because heme oxygenase was inducible with low doses of bromobenzene previously found to be “nontoxic” (25), we tested the hypothesis that induction of the enzyme might be unrelated to production of hepatocellular necrosis. By comparison with bromobenzene, other necrogenic agents (ethionine,
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furosemide, ally1 alcohol, endotoxin, or acetaminophen) produced only a modest (two- to fourfold) stimulation of heme oxygenase activity (data not shown). Furthermore, pretreatment of rats with SKF-525A had no effect on the induction of heme oxygenase by bromobenzene (Table I), despite the fact that this compound abolishes the hepatotoxicity in rats receiving 20 times more bromobenzene (17, 25). SKF-525A blocks the metabolic activation of bromobenzene and the subsequent loss of hepatic glutathione (17). Likewise, phenobarbital pretreatment had little effect on induction of heme oxygenase (Table I), even though phenobarbital enhances liver cell necrosis by augmenting the rates of bromobenzene metabolism and of glutathione depletion (17). Although liver cell necrosis from bromobenzene can be either intensified by pretreatment with diethylmaleate-a substance which rapidly reduces the levels of
intercellular glutathione (29>-or prevented by pretreatment with cysteine which supplements stores of sulfhydryl groups in the liver (l’i’), these agents failed to exert similar effects on induction of heme oxygenase by bromobenzene (Table II). Indeed, diethylmaleate appeared to impair induction of heme oxygenase (Table II), whereas diethylmaleate alone stimulated the enzyme modestly as has been reported previously (11). Finally, even though the metabolism of bromobenzene is diverted to nontoxic products by 3-methylcholanthrene pretreatment, heme oxygenase induction was unaffected. These experiments demonstrate that induction of heme oxygenase need not be accompanied by loss of hepatocellular sulfhydryl content or viability. DISCUSSION
The present studies demonstrate that bromobenzene administration initiates marked changes in the homeostasis of hepatic heme. TABLE I These include a rise in heme oxygenase EFFECT OF ACTINOMYCIN D OR CYCLOHEXIMIDEON activity measured in hepatic microsomes INDUCTIONOF HEME OXYGENASEBY in vitro, accelerated conversion of the heme BROMOBENZENEORCOBALTOUS in hepatic hemoproteins (cytochrome P-450) CHLORIDES to physiologic degradation products in vivo, and a decline from steady-state levels in Heme oxygenase activity the concentration of cytochrome P-450. (nmol bilirubini Similar manifestations of accelerated catabTreatment 10 minimg protein) olism of cytochrome P-450 heme are produced Corn oil + saline also by such nonheme substances as cobalt (Control) 0.139 * 0.011 (7), and endotoxin (5), as well as by heme Bromobenzene 1.081 2 0.056 itself (5). The effects of these compounds Actinomycin D 0.156 2 0.014 undoubtedly involve the hepatocyte since Bromobenzene liver sinusoidal cells contain only trace + actinomycin D 0.997 k 0.076 amounts of cytochrome P-450 and less than Cycloheximide 0.109 r 0.014 10% of heme oxygenase activity in the liver Bromobenzene (3). Because the individual steps in the + cycloheximide 0.105 + 0.015 pathway for catabolism of hepatic hemoCobaltous chloride 1.169 2 0.049 Cobaltous chloride proteins have not been identified, it is + actinomycin D 0.483 + 0.032 uncertain whether stimulation of a common rate-controlling mechanism underlies the ‘I Rats were given either actinomycin D (0.8 mg/kg), accelerated breakdown of intrinsic hepatic cycloheximide (2 mg/kg), or the appropriate vehicle heme by these “toxic” agents. Bromo(corn oil or saline) and, 1 h later, the animals received benzene appears to be useful for probing a second injection containing either bromobenzene (2.0 this question because the characteristics of mmolikg), cobaltous chloride (60 mgikg), or the approheme oxygenase induction by this chemical priate vehicle. The rats were killed 20 h following the differ in several important aspects from last injection and liver microsomes were prepared for those reported for heme or for other nonmeasurement of heme oxygenase. Results represent the average (rSEM) of three animals. heme inducers of the enzyme.
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OF HEPATIC HEME OXYGENASE TABLE II
EFFECT OF VARIOUS PRETREATMENTS ON STIMULATION OF HEME OXYGENASE BY BROMOBENZENE~
Dose of bromobenzene (mmol/kg)
0.5 0.5 0.5 2.0 2.0 2.0 2.0 2.0
Treatment
Heme oxygenase activity (% of control f SEM)
Control Bromobenzene SKF-525A Bromobenzene + SKF-525A Diethylmaleate Bromobenzene + diethylmaleate Bromobenzene Phenobarbital Bromobenzene + phenobarbital 3-Methylcholanthrene Bromobenzene + 3-methylcholanthrene Bromobenzene + diethylmaleate Cysteine Bromobenzene + cysteine
100 471 2 39 102 2 4.1* 429 k 47 346 2 26 395 k 38 694 zt 54 97 * 7.5* 512 f 31 89 k 13* 628 2 44 412 + 28 94 + 8* 785 2 31
n Rats received the indicated treatment as described under Materials and Methods and were killed 20 h after administration of bromobenzene. Hepatic microsomes were prepared for measurement of heme oxygenase activity. Results were calculated as percentage of the activity in the respective control animals. The average of at least four values is nresented. All treatments other than those indicated by an asterisk are significantly greater than controls (P -< 0.05).
Bromobenzene was found to be among the most potent inducers of hepatic heme oxygenase yet described, a single dose producing an average E-fold increase in enzyme activity (Fig. 1). Increases ranging between two- and sixfold are commonly encountered with metabolic alterations (6), intravascular hemolysis (2), or following single large doses of methemalbumin (4), or one of several hepatotoxins (see results), whereas large doses of metal ions are somewhat more effective inducers of heme oxygenase (6- to lo-fold) (7). The effect of bromobenzene is selective in that prominant stimulation of heme oxygenase occurs at times (as early as 2 h) when bromobenzene is without effect on the level of other microsomal enzymes [e.g., glucose-6-phosphatase or NADPH neotetrazolium reductase activity (31)] or on the rate of general protein synthesis (31). Our data also suggests that neither the rate of bromobenzene metabolism nor the formation of a particular metabolite is a key event in the induction process. Thus, inhibition of bromobenzene metabolism by SKF-525A, acceleration of bromobenzene metabolism by pretreatment with pheno-
barbital, or alteration of the profile of bromobenzene metabolites by pretreatment with 3-methylcholanthrene had no effect on the stimulation of heme oxygenase activity. Since liver cell necrosis parallels bromobenzene metabolism (1’7),it follows that induction of heme oxygenase does not depend upon production of overt hepatotoxicity. In view of these results, the suggestion (5) that induction of heme oxygenase by nonheme substances involves production of toxicity to the animal may require modification. Although modest stimulation of heme oxygenase activity may be associated with hepatotoxicity, the remarkably potent effect of bromobenzene strongly implicates the involvement of other, apparently more selective mechanisms. Maines and Kappas have proposed that cobalt and other metals capable of forming complexes with sulfhydryl groups directly regulate hepatic heme oxygenase (11). They reported that 2 to 6 h after injection of cobalt or other metals to rats, the level of glutathione in the liver was significantly reduced in association with increased heme oxygenase activity (32). Furthermore, pre-
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treatment with diethylmaleate, which depletes glutathione in the liver, augmented induction of heme oxygenase by cobalt (ll), whereas supplementation of liver sulfhydryl stores with cysteine reduced the cobalt-mediated induction of the enzyme (11). These authors concluded that metals combine with glutathione and with specific receptor sites in the liver (presumably containing key sulfhydryl groups), the latter event triggering induction of heme oxygenase activity (11). If this concept were correct, then bromobenzene and metals might both be expected to affect heme oxygenase through this common mechanism because there is conclusive evidence that metabolism of bromobenzene produces short-lived, highly reactive products which combine with glutathione or with other sulfhydryl-containing constituents (16, 17, 26). However, neither the modifications in the rate of bromobenzene metabolism mentioned in the preceeding paragraph, nor pretreatment with diethylmaleate or cysteine had the effects on bromobenzene-mediated induction of heme oxygenase (Table I) predicted by this hypothesis. If changes in sulfhydryl content are necessary for heme oxygenase induction, then bromobenzene metabolites may combine preferentially with a different subspecies of sulfhydryl-containing components than do metals. An alternative possibility is that the assumed cause and effect relationship between the capacity of metals to bind sulfhydryl groups and to stimulate heme oxygenase may be invalid. In support of this idea, Maines and Kappas were unable to demonstrate a decrease in glutathione in association with induction of heme oxygenase by cobalt in cultures of avian hepatocytes (10). More important is the recent report of Sasame and Boyd who reexamined the effects of cobaltous chloride over a wide range of doses in rats and found that hepatic glutathione concentration increased significantly within 2 h and rose continuously thereafter (33). They observed that other divalent metal inducers of heme oxygenase had various effects on glutathione concentration, including large increases, slight decreases, or no change (33). In view
of the present studies with bromobenzene and the conflicting reports regarding the effects of metals on glutathione, it appears unlikely that alteration in hepatic sulfhydry1 content is the proximate event controlling heme oxygenase induction. The mechanism of heme oxygenase induction by bromobenzene has been characterized only indirectly. Whereas administered hemoglobin (34) or metal ions (35) induce heme oxygenase in both the liver and kidneys, bromobenzene stimulates the liver enzyme specifically. The latter event is blocked by cycloheximide, a finding which suggests the requirement for protein synthesis, possibly de novo enzyme formation. Since actinomycin D, an inhibitor of RNA synthesis, had no effect on inducticn of heme oxygenase, the effect of bromobenzene may be localized to translation. Induction of heme oxygenase by methemalbumin similarly is resistent to actinomycin D (4), whereas cobalt is sensitive to this inhibitor (26). These facts point to different mechanism(s) underlying induction of heme oxygenase by bromobenzene, metals, or heme. Nevertheless, an intimate relationship is maintained between heme oxygenase activity and hepatic hemoprotein turnover. Because detectable changes in degradation of cytochrome P-450 [14C]heme in vivo (14C0 excretion) and a detectable rise in heme oxygenase activity both occur within 2 to 4 h following endotoxin, methemalbumin, or bromobenzene (5), the present techniques do not resolve the question of whether induction of heme oxygenase preceeds accelerated catabolism of hepatic heme or whether induction of the enzyme is a consequence of stimulated hemoprotein turnover. Recent progress in purification of heme oxygenase (36) may speed the development of specific inhibitors of the enzyme which, together with stimulators of heme oxygenase, should facilitate advances in defining the pathways of hepatic heme degradation. ACKNOWLEDGMENTS The authors wish to thank Joyce Barwick for assistance in conducting some of these experiments. Careful typing of the manuscript was provided by Marcia Tetlak.
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HEME
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