Induction of 6-Aminolevulinate Synthase and Heme Oxygenase (Hemoproteins/Heme Synthesis/Drug Metabolism/Porphyrins) MAHIN D

Proc. Nati. Acad. Sci. USA Vol. 73, No. 12, pp. 4428-4431, December 1976 Biochemistry

Selenium regulation of hepatic heme metabolism: Induction of 6-aminolevulinate synthase and heme oxygenase (hemoproteins/heme synthesis/drug metabolism/porphyrins) MAHIN D. MAINES AND ATTALLAH KAPPAS The Rockefeller University, 1230 York Avenue, New York, N.Y. 10021 Communicated by George C. Cotzias, September 20, 1976

ABSTRACT Selenium was found to be a novel regulator of effective inducer of both 6-aminolevulinate (AmLev) synthase cellular heme metabolism in that the element induced both the [succinyl-CoA:glycine C-succinyl-transferase (decarboxylating); mitochondrial enzyme 6-aminolevulinate synthase [succinyl- EC 2.3.1.37] and heme oxygenase [heme, hydrogen-donor: CoA:glycine C-succinyltransferase (decarboxylating); EC 2.3.1.37] and the microsomal enzyme heme oxygenase [heme, oxygen oxidoreductase (a-methene-oxidizing, hydroxylating); hydrogen-donor:oxygen oxidoreductase(a-methene-oxidizing, EC 1.14.99.3] activties. AmLev synthase is the rate-limiting hydroxylating); EC 1.14.99.31 in liver. The effect of selenium on enzyme in the heme synthetic pathway (8), and heme oxyge- these enzyme activities was prompt, reaching a maximum nase is the microsomal enzyme that degrades heme to bile within 2 hr after a single injection. Other changes in parameters pigment. The effects of selenium on cellular heme metabolism of he atic heme metabolism occurred after administration of were unusual in several respects, including its rather prompt the element. Thirty minutes after injection the cellular content of heme was significantly increased; however, this value slightly induction of AmLev synthase together with a concomitant in- decreased below control values within 2 hr, coinciding with the duction of heme oxygenase without major changes in cellular period of rapid induction of heme oxygenase. At later periods heme content. These effects are unlike those produced by trace heme content returned to normal values. Selenium treatment metal inducers of AmLev synthase such as cobalt which elicit caused only a slight decrease in microsomal cytochrome P450 a clear biphasic response consisting of a substantial early inhi- content. However, drug-metabolizing activity was severely in- bition of the enzyme that, after several hours, is followed by hibited by higher doses of the element. Unlike other inducers of 5-aminolevulinate synthase, which as a rule are also por- derepression of this enzyme synthesis (9, 10). A steady and phyrinogenic agents, selenium induction of this enzyme was substantial decrease in cellular heme content accompanies these not accompanied by an increase in the cellular content of por- changes in AmLev synthase activity (9). Moreover, in contrast phyrins. Wen rats were pretreated with selenium 90 min before to other chemical inducers of AmLev synthase such as allyli- administration of heme, a potent inhibitor of 6aminolevulinate sopropylacetamide (11) and dicarbethoxydehydrocollidine (12), synthase production, the inhibitory effect of heme on formation which invariably produce a porphyrinogenic response in liver, of this mitochondrial enzyme was completely blocked. Seleni- um, at high concentrations in vitro, was inhibitory to 6-ami- selenium was shown not to be a porphyrinogenic agent despite nolevulinate synthase activity. its ability to induce the enzyme. The combination of these se- It is postulated that selenium may not be a direct inducer of lenium actions on cellular heme metabolism is unique and may heme oxygenase as is the case with trace metals such as cobalt, account for some of the biological functions of this element. but may mediate an increase in heme oxygenase through increased production and cellular availability of "free" heme, MATERIALS AND METHODS which results from the increased heme synthetic activity of hepatocytes. Subsequently, the increased heme oxygenase ac- Male Sprague-Dawley rats (150-180 g) were treated (subcu- tivity is in turn responsible for the lack of increase in the mi- taneously) for various lengths of time with sodium selenite crosomal heme content, thus maintaining heme levels at normal values despite the highly increased activities of both heme ox- (Na2SeO3) solutions at pH 7.4. The dose routinely used was 5 ygenase and b-aminolevulinate synthase. It is further suggested ,umol/100 g; other doses were used in some experiments as in- that the increase in 6-aminolevulinate synthase activity is not dicated. Animals were also treated with a combination of so- due to a decreased rate of enzyme degradation or an activation dium selenite and heme (hematin). In these experiments, the of preformed enzyme, but to increased rate of synthesis of en- animals were first treated with Na2SeO3 90 min before the in- zyme protein. Although selenium in trace amounts has been jection of heme (4 ,umol/100 g, intraperitoneally). The solution postulated to be involved in microsomal electron transfer pro- as elsewhere After the cess, the data from this study indicate that excess selenium can of heme was prepared described (13). substantially inhibit microsomal drug metabolism. last injection food was removed and animals were killed at different intervals as indicated. Livers were perfused in situ Selenium has recently been identified as an essential element (0.9% NaCl) and homogenized in Tris-HCl (0.1 M, pH 7.4) in mammalian nutrition (1). It is believed, with vitamin E, to containing 0.25 M sucrose. Aliquots were removed for assay of function as an anti-oxidant in protection of biological mem- AmLev synthase, 6-aminolevulinate dehydratase [porphobili- branes against lipid peroxidation (2) as well as maintaining nogen synthase; 5-aminolevulinatehydro-lyase (adding 5- normal levels of capillary permeability (3). Selenium deficiency aminolevulinate and cyclizing); EC 4.2.1.24], and total por- causes a diminution in the induction response of the hepatic phyrin. The remainder was used for the preparation of mi- microsomal cytochrome P-450 system to barbiturates (4, 5), and crosomal fractions as described earlier (14). AmLev synthase it has been suggested that the element may participate in activity was determined by the method of Marver et al. (15); electron transfer reactions of some bacterial enzymes (6, 7). In AmLev dehydratase activity was determined as described by the present study, we report a newly defined action of selenium Mauzerall and Granick (16). The total porphyrin content was on liver cell metabolism in which selenium was found to be an determined by the method of Granick et al. (17). Microsomal heme and hemoprotein contents were measured by the methods Abbreviation: AmLev, 6-aminolevulinate. of Paul et al. (18) and Omura and Sato (19), respectively. Oxi- 4428 Downloaded by guest on September 24, 2021 Biochemistry: Maines and Kappas Proc. Natl. Acad. Sci. USA 73 (1976) 4429

Table 1. Selenium effects on cellular heme metabolism Dose of AmLev AmLev Total Microsomal Heme Na2SeO3 synthase dehydratase porphyrin heme oxygenase (gmol/lOO g) (pmol/mg-hr) (nmol/mg-hr) (pmol/mg) (nmol/mg) (nmol/mg-hr) Control 125.8 5.38 3.57 1.50 1.85 1 259.9 4.79 3.30 1.38 3.09 5 207.3 6.21 3.32 1.47 5.55 10 209.4 3.64 3.19 1.36 8.00 Male Sprague-Dawley rats (150-180 g) were treated (subcutaneously) with sodium selenite (Na2SeO3) at the doses indicated. Sixteen hours later they were killed. Livers were perfused and aliquots were removed for AmLev synthetase and AmLev dehydratase activities. Hepatic microsomal heme oxygenase activity and heme content were measured as described in Materials and Methods. Heme oxygenase activity is ex- pressed as the nmol of bilirubin formed per mg of protein per hr.

dative drug metabolizing activity was monitored with ethyl- that was sustained thereafter. The early inhibition of AmLev morphine as substrate in the system described earlier (13) synthase may be due to the direct inactivation of the enzyme measuring the formaldehyde produced (20). Microsomal heme by selenium. As Table 2 shows, at high concentrations, selenium oxygenase activity was determined by the method described in vitro can markedly inhibit AmLev synthase activity. Since earlier (9). The activity.of NADPH-cytochrome c reductase immediately after injection the cellular concentration of the (ADPH:ferricytochrome oxidoreductase; EC 1.6.2.4) was compound is highly elevated, it is possible that the inhibition measured as described by Williams and Kamin (21). Protein of AmLev synthase observed shortly after treatment is analo- content was measured by the method of Lowry et al. (22). The gous to the situation in vitro. Despite the increase in AmLev data were analyzed by the "t" test; values of P < 0.05 were synthase activity, no significant changes in hepatic porphyrin regarded as denoting significance. content were produced by selenium; neither was AmLev dehydratase activity altered. Selenium dioxide (SeO2) produced RESULTS comparable changes in hepatic cell metabolism of heme in Selenium significantly modified cellular heme metabolism. comparable doses (data not shown); at higher doses (>10 Table 1 shows the responses of activities of various heme ,umol/100 g) this selenium compound was toxic to the animals. pathway enzymes and related parameters to sodium selenite Fig. 2 shows the effect of selenium on microsomal content of administration; as shown, the effects on AmLev synthase and heme and on heme oxygenase activity. Thirty minutes after cellular porphyrin and heme contents were not dose-dependent, injection the microsomal heme content was slightly. but sig- at least within the range of 1.0-10.0 ,mol/100 g of body weight. nificantly increased (22%). Four hours after injection there was However, the activity of AmLev dehydratase was somewhat a slight decrease observed in microsomal heme content, which decreased by a high dose of selenium and that of heme oxyge- coincided with the substantial increase of heme oxidation ac- nase showed a dose-dependent increase after its injection. Fig. tivity that had occurred. Despite this increase in heme oxyge- shows the time course of effects of selenium on AmLev syn- nase activity, no further depletion of cellular heme content was thase and AmLev dehydratase activities and on total hepatocyte noted. The induction of heme oxygenase caused by selenium porphyrin content. As shown, 30 min after injection there was that was evident within 2 hr after treatment was not signifi- a moderate inhibition (30%) of AmLev synthase which was cantly altered during the subsequent 14 hr. A transient decrease followed by a rapid increase in the enzyme activity within 2 hr of about 25% was observed in heme oxygenase activity 30 min after selenium injection. As Table 2 shows, when selenium was added to the heme oxygenase and AmLev synthase assay mixtures, there was no stimulation of heme oxygenase observed up to 1.0 mM concentration of selenium although the activity of AmLev synthase was, as noted, inhibited at concentrations greater than 0.1 mM. Therefore, itisapparentthat the inductions

Table 2. Effect of sodium selenite in vitro on AmLev synthase and heme oxygenase activities AmLev Heme synthase oxygenase nM Na2SeO3 (pmol/mg-hr) (nmol/mg-hr) OlI 0.5 2 8 16 0 83.70 1.55 50 88.26 1.70 Time (hr) 100 86.74 1.40 FIG. 1. Time course of selenium effects on activities of AmLev 500 16.13 1.25 synthase and AmLev dehydratase and total porphyrin content in 1000 12.17 1.70 hepatocytes. Rats were treated with a solution of sodium selenite (subcutaneously,55gmol/100 g). At different intervals the animals Whole homogenates were prepared from the perfused livers of were killed. Livers were perfused and then homogenized in Tris.HCl starved rats and were used for the assay of AmLev synthase activity buffer (pH 7.4) containing 0.25 M sucrose. The assays were done as in the presence of indicated concentrations of sodium selenite. The described in Materials and Methods. (-) AmLev synthase activity heme oxygenase activity was determined using the microsomal (pmol/mg-hr) X 102; (----) AmLev dehydratase activity (nmol/mg-hr) fraction of the livers in the presence of above concentrations of X 2; (- --) porphyrin content (pmol/mg). selenium. Downloaded by guest on September 24, 2021 4430 Biochemistry: Maines and Kappas Proc. Natl. Acad. Sci. USA 73 (1976)

Table 4. Effect of pretreatment with selenium on heme inhibition of AmLev synthase 5.0 and heme oxygenase activities AmLev Heme Cyto- synthase Total oxygenase chrome 4.0 (pmol/ porphyrin (nmol/ P-450 Treatment mg-hr) (pmol/mg) mg-hr) (nmol/mg) 3.0 Control 82.3 3.34 2.1 0.76 Selenium 263.9 3.66 7.2 0.65 Heme 40.5 3.29 10.7 0.72 Selenium + heme 242.9 3.35 13.5 0.62 Rats were pretreated with sodium selenite (subcutaneously, 5 1. ,4mol/100 g) 90 min prior to heme treatment (4 ,mol/100 g, intraperitoneally). Other groups of animals received appropriate doses of selenium, heme, or saline. After 14.5 hr, the animals were killed,

0 livers were perfused and homogenized, and aliquots were removed 0.5 2 8 16 for the assay of AmLev synthase and determination of porphyrin Time (hr) content. Microsomal fractions were prepared and used for the assay of heme oxygenase and measurement of cytochrome P-450 and FIG. 2. Time course of selenium effects on hepatic microsomal heme contents. heme oxygenase activity and heme content. Rats were treated as described in the legend of Fig. 1 and the microsomal fractions were prepared. Heme oxygenase activity as well as the total heme content cordingly, in the present study the influence of selenium pre- were measured as described in Materials and Methods. (-) Heme treatment on heme effects on AmLev synthase and heme ox- oxygenase activity (nmol/mg-hr); ---) microsomal heme content ygenase activities was studied. Table 4 shows that pretreatment (nmol/mg). with selenium (90 min) blocked heme inhibition of AmLev synthase formation. Moreover, heme administered to animals by selenium of heme synthetic and heme degradation activities pretreated with selenium augmented induction of hepatic heme were biologically mediated effects rather than being chemical oxygenase activity. Microsomal contents of cytochrome P-450 activation phenomena. were not significantly different in the animals treated with On the basis of the findings from other laboratories (4-7) it selenium alone or selenium plus heme. was presumed that selenium might facilitate drug-metabolizing activity by enzymes of the endoplasmic reticulum. However, DISCUSSION an unexpected finding in this study was that selenium inhibited microsomal drug-metabolizing activity by about 15-50% de- Selenium is an essential nutrient, and a number of functions pending on dose (Table 3) despite the presence of an ample ranging from serving as a biological anti-oxidant (2) to being amount of microsomal cytochrome P-450 and the fact that a constituent of enzymes (6, 7) and possibly of the microsomal NADPH-cytochrome c reductase activity was unchanged. electron transport chain (4, 5) have been assigned to it. In the It was noted above that one reported property of selenium present study new actions of this element on heme metabolism is its ability to exert a "protective" effect on biological mem- were identified. The ability of selenium to induce AmLev branes (2, 3). Therefore, the possibility that increased activities synthase, the rate-limiting enzyme in heme synthesis (8), may of AmLev synthase and heme oxygenase after selenium might be one of its most significant actions since hemoproteins con- be due to a decrease in the rate of turnover of the enzymes stitute a major component of certain biological membranes and rather than to increased synthesis was examined. It is known are directly involved in cellular oxidation reactions. Moreover, that exogenous heme is a potent inhibitor of the synthesis of selenium is a unique compound in the effects it exerts on cel- AmLev synthase and is an inducer of heme oxygenase. Ac- lular heme metabolism. Unlike other inorganic elements, such as cobalt, which induce AmLev synthase after an initial pro- Table 3. Effect of selenium treatment longed and significant inhibition of this enzyme activity (10), on microsomal enzyme activities or organic compounds, such as barbiturate analogues, in which and cytochrome P-450 content an induction of AmLev synthase activity occurs concurrently with an increase in cellular content of porphyrins (10-12), no Dose of NADPH-cyto- increase in porphyrins is observed with selenium induction of Na2SeO3 Ethylmorphine chrome c Cytochrome AmLev synthase. The fact that selenium does not increase (/2mol/ N-demethylase reductase P-450 cellular porphyrin content while inducing AmLev synthase 100 g) (nmol/mg-hr) (nmol/mg-hr) (nmol/mg) indicates that the total amount of porphyrin synthesized is 0 157.6 99.8 0.92 converted to heme which in turn is used for the formation of 5 132.3 99.0 0.83 hemoproteins. This would explain the finding that in the 10 79.2 92.1 0.75 presence of the substantially elevated heme oxidation activity produced by selenium no decrease in cellular heme content was Rats were treated as described in the legend of Table 1. The observed. Conversely, the elevation of heme oxygenase activity hepatic microsomal fractions were prepared and assayed for ethyl- elicited by the element was apparently responsible for the ab- morphine N-demethylase and NADPH-cytochrome c reductase in- activities; cytochrome P-450 content was measured. The procedure sence of an increase in cellular heme content despite the used for these assays were those described in Materials and duction of AmLev synthase. Thus, a steady state was established Methods. after selenium administration in which the excess heme syn- Downloaded by guest on September 24, 2021 Biochemistry: Maines and Kappas Proc. Natl. Acad. Sci. USA 73 (1976) 4431 thesized was degraded at an accelerated rate, maintaining the Family Trust. We thank Mrs. Ilona Scher and Mr. Francis A. Farraye cellular content of heme at normal levels. for their able and devoted technical assistance and Miss Ann Marie Heme oxidation activity is induced by a number of com- Quatela for typing of the manuscript. most are certain pounds (9, 23-27), among which the potent 1. Schwarz, K. & Foltz, C. M. (1958) J. Biol. Chem. 233, 245- transition elements and heavy metals (9, 22-26). Experimental 251. evidence indicates that the induction effect elicited by such 2. Hoekstra, W. G. (1975) Fed. Proc. 34,,2083-2089. metals is most likely a direct one rather than an action mediated 3. Combs, G. F., Jr., Noguchi, T. & Scott, M. L. (1975) Fed. Proc. through alterations in intermediary cell components (28). It is 34,2090-2095. possible that the selenium induction of heme oxygenase, how- 4. Burk, R. F., MacKinnon, A. M. & Simon, F. R. (1974) Biochem. ever, does reflect a cellular response to an intermediary com- Biophys. Res. Commun. 56,431-435. pound-specifically heme, the concentration of which is di- 5. Burk, R. F. & Masters, B. S. S. (1975) Arch. Biochem. Biophys. rectly altered by the element. Selenium, by inducing AmLev 170, 124-131. synthase without causing concomitant increases in cell content 6. Shum, A. C. & Murphy, J. C. (1972) J. Bacteriol. 110, 447- of porphyrin or hemoproteins (i.e., cytochrome P-450), in- 452. 7. Turner, D. C. & Stadtman, T. C. (1973) Arch. Biochem. Biophys. creases transiently the cellular pool of "free" heme (in 30 min, 154,366-370. Fig. 2), and this heme fraction then may cause the induction 8. Granick, S. (1966) J. Biol. Chem. 241, 1359-1375. of heme oxygenase. 9. Maines, M. D. & Kappas, A. (1975) J. Biol. Chem. 250, 4171- This effect of selenium is rather unusual since other com- 4178. pounds such as the barbiturates, which induce AmLev synthase 10. Maines, M. D., Janousek, V., Tomio, J. M. & Kappas, A. (1976) and increase cellular heme content, do not induce heme oxy- Proc. Natl. Acad. Sci. USA 71, 4293-4297. genase. Such compounds, however, concomitantly enhance 11. Schwartz, S. & Ikeda, K. (1955) in Porphyrin Biosynthesis and hemoprotein formation, and it can be considered that the heme Metabolism, Ciba Foundation Symposium, eds. Wolstenholme, synthesized as a cellular response to agents such as the barbi- G. E. W. & Millar, E. C. P. (Churchill, London), p. 209. 12. Solomon, H. M. & Figge, F. H. J. (1959) Proc. Soc. Exp. Biol. Med. turates is "committed" heme rather than "free" heme in that 100,583-591. synthesis of a binding protein (apo-cytochrome) is simulta- 13. Maines, M. D. & Kappas, A. (1975) J. Biol. Chem. 250, 2363- neously induced by the agent. The heme of the formed cyto- 2369. chromes does not induce heme oxygenase, presumably because 14. Maines, M. D. & Anders, M. W. (1973) Mol. Pharmacol. 9, of the higher affinity of heme for the apo-cytochrome moiety 219-228. (29). 15. Marver, H. S., Tschudy, D. P., Perlroth, M. G. & Collins, A. (1966) One of the physiological functions attributed to selenium is J. Biol. Chem. 241, 2803-2814. the "protection" of biological membranes and their protein 16. Mauzerall, D. & Granick, S. (1958) J. Biol. Chem. 219, 435- constituents. Therefore, it could be speculated that the increase 439. in the rate of heme synthesis produced by selenium is due to 17. Granick, S., Sinclair, P., Sassa, S. & Grieninger, G. (1975) J. Biol. Chem. 250, 9215-9225. suppression of the turnover rate of AmLev synthase protein in 18. Paul, K. G., Theorell, H. & Akeson, A. (1953) Acta Chem. Scand. mitochondria, particularly since the enzyme has an exceedingly 7, 1284-1287. short half-life (30). However, the finding that pretreatment with 19. Omura, T. & Sato, R. (1964) J. Biol. Chem. 239,2379-2385. selenium totally blocked the inhibition of AmLev synthase 20. Nash, T. (1953) Biochem. J. 55, 416-421. formation by heme indicates that selenium directly increases 21. Williams, C. H. & Kamin, H. (1962) J. Biol. Chem. 237, 587- the rate of enzyme synthesis rather than decreasing its rate of 595. degradation. Furthermore, the fact that the activity of AmLev 22. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. synthase was the same in rats treated with selenium alone or (1951) J. Biol. Chem. 193,265-275. with selenium plus heme suggests a direct action of the element 23. Maines, M. D. & Kappas, A. (1976) Biochem. J. 154, 125-131. in the regulation of this enzyme synthesis in contrast to its 24. Kappas, A. & Maines, M. D. (1976) Science 192, 60-62. 25. Maines, M. D. & Kappas, A. (1974) Proc. Natl. Acad. Sci. USA suggested effect on heme oxygenase synthesis. 71,4295-4297. Finally, the fact that selenium treatment did not facilitate 26. Maines, M. D. & Kappas, A. (1976) Ann. Clin. Res. 8,39-46. the activity of the microsomal electron transport chain, as in- 27. Pimstone, N. R., Engel, R., Tenhunen, R., Seitz, P. T., Marver, dicated by a decreased rate of drug-metabolizing activity after H. S. & Schmid, R. (1971) J. Clin.;Invest. 50, 2042-2051. its administration, may indicate that selenium does not play a 28. Maines, M. D. & Sinclair, P. (1976) J. Biol Chem. 251, in limiting role in the electron transfer processes of microsomal press. enzymes except possibly in a marked deficiency state of the 29. Maines, M. D. (1976) Proceedings of the Third International element. Symposium on Microsomes and Drug Oxidation, Berlin, in press. This research was supported by USPHS Grant ES-01055 and by 30. Whiting, M. & Granick, S. (1976) J. Biol. Chem. 251, 1340- individual grants from Exxon and Mobil Corporations and the Scaife 1346. Downloaded by guest on September 24, 2021