Proc. Nati. Acad. Sci. USA Vol. 74, No. 5, pp. 1875-1878, May 1977 Biochemistry Regulation of heme pathway enzymes and cellular glutathione content by metals that do not chelate with tetrapyrroles: Blockade of metal effects by thiols (metal toxicity/trace elements/cytochrome P450/renal metabolism/Ni and Pt) MAHIN D. MAINES AND ATTALLAH KAPPAS The Rockefeller University, New York, New York 10021 Communicated by E. H. Ahrens, Jr., February 11, 1977 ABSTRACT The trace metals nickel and platinum, which metals appear capable of altering heme metabolism (3) it are not substrates for ferrochelatase and thus do not form heme seemed likely that the binding of metals with such functional in biological systems, were found to act similarly to cobalt, and groups is involved in their ability to perturb cellular heme heme itself, in regulating heme metabolism in liver and kidney. These metals induced heme oxygenase activity in both organs metabolism. with the peak of induced enzyme activity reached approxi- This question was examined in the present study by utilizing mately 16 hr after single injections in rats. Both metals caused cysteine and glutathione (GSH) to increase the biological transient depression of cellular glutathione content followed availability, in animals, of binding sites for the complexing of by increases above normal after 12 hr in liver. Nickel and plat- ionic metals. The results indicate that an exogenous supply of inum were more potent inducers of heme oxygenase in kidney binding sites for metal ions can compete with endogenous than in liver (10-13 times normal versus 5-6 times normal). At regulatory binding sites for these elements and thus can render high concentrations, they inhibited heme oxygenase [heme, hydrogen-donor:oxygen oxidoreductase (a-methene-oxidizing, metals incapable of depleting cellular heme and hemoprotein. hydroxylating), EC 1.14.99.31 in vitro. Both were active in reg- Cysteine and glutathione thus entirely block the toxic actions ulating heme metabolism only when administered in the ionic of metals on heme metabolism and cytochrome P-450 func- form. Complexing of the metals with sulfhydryl agents com- tion. pletely blocked their actions on heme metabolism. Adminis- tration of cysteine orally prior to or shortly after administration METHODS of the metals had a similar blocking effect. Nickel and platinum produced depression of 6-aminolevuli- In Vivo Treatment. Male Sprague-Dawley rats (150-180 nate synthase [succinyl-CoA:glycine c-succinyltransferase (de- carboxylating), EC 2.3.1.371 activity in liver, but neither inhib- g) were treated subcutaneously with (i) NiCl2.6H20 (Ni2+) or ited this rate-limiting enzyme for heme synthesis in vitro. Fur- CoCl2-6H20 (Co2+) at 250 ,umol/kg or PtK2CI4-6H20 (Pt2+) thermore, despite the substantial decreases in cellular heme and or PtK2CI6.6H20 (Pt4+) at 125 ,Amol/kg or (ii) the metals hemoprotein contents mediated by the metal, production of complexed with cysteine or GSH at a 1:3 or 1:4 molar ratio of 6-aminolevulinate synthase did not undergo the compensatory the metal to the amino acid or tripeptide. In other experiments, increase that would be expected if there were a direct reciprocal animals were given 1-2 ml of 1.0 M cysteine orally 45 min be- feedback relationship between cellular heme level and synthesis of this enzyme. These findings indicate that it is not necessary fore or 15 min after injection of the metal. At various times as for metal ions to be chelated in the porphyrin ring in order to indicated, the animals were decapitated and the livers and regulate the enzymes of heme synthesis and heme oxidation. kidneys were perfused in situ with isotonic saline until fully Accordingly, it is suggested that the iron atom of heme is the blanched. The organs were homogenized in 0.05 M Tris-HCl proximately active regulator of 5-aminolevulinate synthase and buffer, pH 7.5, containing 0.25 M sucrose. heme oxygenase-actions generally ascribed to the iron-te- Whole cell preparations were used for the determination of trapyrrole complex itself-and that the tetrapyrrole moiety of the complex functions primarily as a means of transport of the total GSH content by the fluorometric method of Cohn and metal to regulatory sites in cells. Lyle (6) and of the activity of 3-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), Heme plays a central role in aerobic cellular processes, in- EC 2.3.1.371 (Alev synthase) by the method of Marver et al. (7). cluding those cytochrome P-450-dependent oxidative trans- Microsomal fractions prepared as described elsewhere (2) were formations that result in the biological inactivation and de- used for the determination of heme content by the method of toxification of numerous drugs and other foreign chemicals. In Paul et al. (8) and of cytochrome P-450 content by the method recent studies (1-5), we have defined new and potent actions of Omura and Sato (9). Microsomal heme oxygenase [heme, of trace metals on enzymes of heme metabolism, involving both hydrogen-donor:oxygen oxidoreductase (a-methene-oxidizing, synthesis and degradation, which result in substantial depletion hydroxylating), EC 1.14.99.3] activity was assayed by the of cellular contents of heme and hemoprotein and thus of cel- method of Maines and Kappas (2). At least three rats were used lular capacity to carry out cytochrome P-450-dependent de- for each experimental value reported. Results were statistically toxification reactions (2, 4). analyzed by the Student t test; P < 0.05 denoted signifi- Metals that markedly alter heme metabolism include certain cance. heavy metals and a series of transition elements which have in In Vitro Studies. Whole cell homogenate and the micro- common the ability to bind a number of functional groups in- somal fractions from hepatocytes of untreated animals were cluding sulfhydryl and carbonyl groups and, to a lesser extent, used. A small volume (10-50 Al) of a solution of metal or of carboxyl and amino groups. Because only the ionic forms of the metal complexed with GSH or cysteine was added to various assay media to obtain a 1 mM final concentration of the metal Abbreviations: GSH, glutathione; Alev synthase, 6-aminolevulinate (the pH of the medium was not changed by these additions). synthase. The assays were then carried out as described above. 1875 Downloaded by guest on September 27, 2021 1876 Biochemistry: Maines and Kappas Proc. Natl. Acad. Sci. USA 74 (1977) 500 400 300[ c 200 a- 100 80 60( 0 1 6 12 24 40 Time ( hour) 1 LfS f 1 3 12 16 24 FIG. 1. Time course of the effect of Ni2+ on hepatocyte content Pt4+ Time (hour) of GSH in rats. Co2+ and Pt4+ showed similar effects. FIG. 3. Time course of response to Pt4+ of heme pathway en- zymes in rat liver. Rats were treated with PtK2Cl6 and then killed at RESULTS indicated intervals. Various assays were performed as described in Metal Effects on Hepatic Cell GSH Content. The time the Methods section. The control values for the measured parameters were within +10-20% of the control values given in the legend of Fig. course of the effects of Co2+, Ni2+, and Pt4+ on hepatocyte 2, and the same symbols are used. content of GSH was investigated. Administration of these metals produced a 30-50% decline in liver content of GSH within 6 sponses of microsomal heme and cytochrome P-450 content and hr; by 12 hr this decline had reversed, and between 12 and heme oxygenase and Alev synthase activities to Ni2+ and Pt4+ 24 hr-the total liver content of GSH had more than doubled over administration are shown in Figs. 2 and 3. Both metals produced the control value (Fig. 1). [The pattern of depletion of GSH a transient depression of heme oxygenase followed by a marked followed by a rebound is also produced by metal administration induction of this enzyme activity. When heme oxygenase ac- to cultured avian hepatocytes (10).] The depletion of fluo- tivity was highly elevated (12t24 hr), microsomal heme and rometrically detectable free GSH at 6 hr in liver may reflect cytochrome P-450 contents had diminished substantially. These metal complexing with the tripeptide rather than an inhibition decreases occurred in the presence of either normal (Fig. 3) or of the synthesis of GSH, because this depletion of GSH content low levels (Fig. 2) of Alev synthase activity. In the latter cir- is observed in the absence of inhibition of cellular protein cumstance it is of considerable interest that Alev synthase, an synthesis (10). However, the mechanism by which liver cells enzyme with a short half-life and with a rate of formation restore GSH to above normal levels is not known. It is of interest presumed to be reciprocally related to cellular heme content that this pattern of response mimics that exhibited by Alev (12), did not display a rebound induction response when cellular synthase in response to cobalt (2, 5) and heme (11) in whole heme levels were maximally depressed. This finding strongly animals. suggests that Ni2+ is itself capable of directly repressing Alev Ni2+ and Pt4+ Effects On Heme Oxygenase, Microsomal synthase activity, as does heme (12). Pt4+ similarly directly Heme and Cytochrome P450, and Mitochondrial Alev inhibited Alev synthase formation but to a lesser degree. Neither Synthase In Vivo and In Vitro. The time course of the re- metal inhibited this enzyme in vitro (Table 1), indicating that the inhibition observed in vivo reflects diminished enzyme formation or enhanced degradation. It is of interest that the 600 pattern of response of heme oxygenase to Ni2+ and Pt4+ re- sembled that seen with hepatocyte content of GSH after metal 500 400- Table 1.