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
Selenium was found to be a novel regulator of ABSTRACT cellular heme metabolism in that the element induced both the mitochondrial enzyme 6-aminolevulinate synthase [succinylCoA:glycine C-succinyltransferase (decarboxylating); EC 2.3.1.37] and the microsomal enzyme heme oxygenase [heme, hydrogen-donor:oxygen oxidoreductase(a-methene-oxidizing, hydroxylating); EC 1.14.99.31 in liver. The effect of selenium on these enzyme activities was prompt, reaching a maximum within 2 hr after a single injection. Other changes in parameters of he atic heme metabolism occurred after administration of the element. Thirty minutes after injection the cellular content of heme was significantly increased; however, this value slightly decreased below control values within 2 hr, coinciding with the period of rapid induction of heme oxygenase. At later periods heme content returned to normal values. Selenium treatment caused only a slight decrease in microsomal cytochrome P450 content. However, drug-metabolizing activity was severely inhibited by higher doses of the element. Unlike other inducers of 5-aminolevulinate synthase, which as a rule are also porphyrinogenic agents, selenium induction of this enzyme was not accompanied by an increase in the cellular content of porphyrins. Wen rats were pretreated with selenium 90 min before administration of heme, a potent inhibitor of 6aminolevulinate synthase production, the inhibitory effect of heme on formation of this mitochondrial enzyme was completely blocked. Selenium, at high concentrations in vitro, was inhibitory to 6-aminolevulinate synthase activity. It is postulated that selenium may not be a direct inducer of heme oxygenase as is the case with trace metals such as cobalt, but may mediate an increase in heme oxygenase through increased production and cellular availability of "free" heme, which results from the increased heme synthetic activity of hepatocytes. Subsequently, the increased heme oxygenase activity is in turn responsible for the lack of increase in the microsomal heme content, thus maintaining heme levels at normal values despite the highly increased activities of both heme oxygenase and b-aminolevulinate synthase. It is further suggested that the increase in 6-aminolevulinate synthase activity is not due to a decreased rate of enzyme degradation or an activation of preformed enzyme, but to increased rate of synthesis of enzyme protein. Although selenium in trace amounts has been postulated to be involved in microsomal electron transfer process, the data from this study indicate that excess selenium can substantially inhibit microsomal drug metabolism.
effective inducer of both 6-aminolevulinate (AmLev) synthase [succinyl-CoA:glycine C-succinyl-transferase (decarboxylating); EC 2.3.1.37] and heme oxygenase [heme, hydrogen-donor: oxygen oxidoreductase (a-methene-oxidizing, hydroxylating); EC 1.14.99.3] activties. AmLev synthase is the rate-limiting enzyme in the heme synthetic pathway (8), and heme oxygenase is the microsomal enzyme that degrades heme to bile pigment. The effects of selenium on cellular heme metabolism were unusual in several respects, including its rather prompt induction of AmLev synthase together with a concomitant induction of heme oxygenase without major changes in cellular heme content. These effects are unlike those produced by trace metal inducers of AmLev synthase such as cobalt which elicit a clear biphasic response consisting of a substantial early inhibition of the enzyme that, after several hours, is followed by derepression of this enzyme synthesis (9, 10). A steady and substantial decrease in cellular heme content accompanies these changes in AmLev synthase activity (9). Moreover, in contrast to other chemical inducers of AmLev synthase such as allylisopropylacetamide (11) and dicarbethoxydehydrocollidine (12), which invariably produce a porphyrinogenic response in liver, selenium was shown not to be a porphyrinogenic agent despite its ability to induce the enzyme. The combination of these selenium actions on cellular heme metabolism is unique and may account for some of the biological functions of this element. MATERIALS AND METHODS Male Sprague-Dawley rats (150-180 g) were treated (subcutaneously) for various lengths of time with sodium selenite (Na2SeO3) solutions at pH 7.4. The dose routinely used was 5 ,umol/100 g; other doses were used in some experiments as indicated. Animals were also treated with a combination of sodium selenite and heme (hematin). In these experiments, the animals were first treated with Na2SeO3 90 min before the injection of heme (4 ,umol/100 g, intraperitoneally). The solution of heme was prepared as described elsewhere (13). After the last injection food was removed and animals were killed at different intervals as indicated. Livers were perfused in situ (0.9% NaCl) and homogenized in Tris-HCl (0.1 M, pH 7.4) containing 0.25 M sucrose. Aliquots were removed for assay of
Selenium has recently been identified as an essential element in mammalian nutrition (1). It is believed, with vitamin E, to function as an anti-oxidant in protection of biological membranes against lipid peroxidation (2) as well as maintaining normal levels of capillary permeability (3). Selenium deficiency causes a diminution in the induction response of the hepatic microsomal cytochrome P-450 system to barbiturates (4, 5), and it has been suggested that the element may participate in electron transfer reactions of some bacterial enzymes (6, 7). In the present study, we report a newly defined action of selenium on liver cell metabolism in which selenium was found to be an
AmLev synthase, 6-aminolevulinate dehydratase [porphobilinogen synthase; 5-aminolevulinatehydro-lyase (adding 5aminolevulinate and cyclizing); EC 4.2.1.24], and total porphyrin. The remainder was used for the preparation of microsomal fractions as described earlier (14). AmLev synthase activity was determined by the method of Marver et al. (15); AmLev dehydratase activity was determined as described by
Mauzerall and Granick (16). The total porphyrin content was determined by the method of Granick et al. (17). Microsomal heme and hemoprotein contents were measured by the methods of Paul et al. (18) and Omura and Sato (19), respectively. Oxi-
Abbreviation: AmLev, 6-aminolevulinate.
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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
Na2SeO3 (gmol/lOO g)
synthase
dehydratase
porphyrin
(pmol/mg-hr)
(nmol/mg-hr)
(pmol/mg)
Control 1 5 10
125.8 259.9 207.3 209.4
5.38 4.79 6.21 3.64
Microsomal heme (nmol/mg)
Heme oxygenase
(nmol/mg-hr)
3.57 1.50 1.85 1.38 3.30 3.09 3.32 1.47 5.55 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 expressed as the nmol of bilirubin formed per mg of protein per hr.
dative drug metabolizing activity was monitored with ethylmorphine as substrate in the system described earlier (13) measuring the formaldehyde produced (20). Microsomal heme oxygenase activity was determined by the method described earlier (9). The activity.of NADPH-cytochrome c reductase (ADPH:ferricytochrome oxidoreductase; EC 1.6.2.4) was measured as described by Williams and Kamin (21). Protein content was measured by the method of Lowry et al. (22). The data were analyzed by the "t" test; values of P < 0.05 were regarded as denoting significance.
RESULTS Selenium significantly modified cellular heme metabolism. Table 1 shows the responses of activities of various heme pathway enzymes and related parameters to sodium selenite administration; as shown, the effects on AmLev synthase and cellular porphyrin and heme contents were not dose-dependent, at least within the range of 1.0-10.0 ,mol/100 g of body weight. However, the activity of AmLev dehydratase was somewhat decreased by a high dose of selenium and that of heme oxygenase showed a dose-dependent increase after its injection. Fig. shows the time course of effects of selenium on AmLev synthase and AmLev dehydratase activities and on total hepatocyte porphyrin content. As shown, 30 min after injection there was a moderate inhibition (30%) of AmLev synthase which was followed by a rapid increase in the enzyme activity within 2 hr
that was sustained thereafter. The early inhibition of AmLev synthase may be due to the direct inactivation of the enzyme by selenium. As Table 2 shows, at high concentrations, selenium in vitro can markedly inhibit AmLev synthase activity. Since immediately after injection the cellular concentration of the compound is highly elevated, it is possible that the inhibition of AmLev synthase observed shortly after treatment is analogous to the situation in vitro. Despite the increase in AmLev synthase activity, no significant changes in hepatic porphyrin content were produced by selenium; neither was AmLev dehydratase activity altered. Selenium dioxide (SeO2) produced comparable changes in hepatic cell metabolism of heme in comparable doses (data not shown); at higher doses (>10 ,umol/100 g) this selenium compound was toxic to the animals. Fig. 2 shows the effect of selenium on microsomal content of heme and on heme oxygenase activity. Thirty minutes after injection the microsomal heme content was slightly. but significantly increased (22%). Four hours after injection there was a slight decrease observed in microsomal heme content, which coincided with the substantial increase of heme oxidation activity that had occurred. Despite this increase in heme oxygenase activity, no further depletion of cellular heme content was noted. The induction of heme oxygenase caused by selenium that was evident within 2 hr after treatment was not significantly altered during the subsequent 14 hr. A transient decrease 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, it isapparent that the inductions Table 2. Effect of sodium selenite in vitro on AmLev synthase and heme oxygenase activities
OlI
0.5
2
8
16
Time (hr) FIG. 1. Time course of selenium effects on activities of AmLev synthase and AmLev dehydratase and total porphyrin content in hepatocytes. Rats were treated with a solution of sodium selenite (subcutaneously,55gmol/100 g). At different intervals the animals were killed. Livers were perfused and then homogenized in Tris.HCl buffer (pH 7.4) containing 0.25 M sucrose. The assays were done as described in Materials and Methods. (-) AmLev synthase activity (pmol/mg-hr) X 102; (----) AmLev dehydratase activity (nmol/mg-hr) X 2; (- --) porphyrin content (pmol/mg).
nM Na2SeO3
AmLev synthase (pmol/mg-hr)
(nmol/mg-hr)
0 50 100 500 1000
83.70 88.26 86.74 16.13 12.17
1.55 1.70 1.40 1.25 1.70
Heme oxygenase
Whole homogenates were prepared from the perfused livers of starved rats and were used for the assay of AmLev synthase activity in the presence of indicated concentrations of sodium selenite. The heme oxygenase activity was determined using the microsomal fraction of the livers in the presence of above concentrations of
selenium.
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Biochemistry: Maines and Kappas
Proc. Natl. Acad. Sci. USA 73 (1976) Table 4. Effect of pretreatment with selenium on heme inhibition of AmLev synthase and heme oxygenase activities
5.0
4.0
Treatment 3.0
Control Selenium Heme Selenium + heme
AmLev synthase (pmol/ mg-hr)
Heme CytoTotal oxygenase chrome porphyrin (nmol/ P-450 (pmol/mg) mg-hr) (nmol/mg)
82.3 263.9 40.5
3.34 3.66 3.29
2.1 7.2 10.7
0.76 0.65 0.72
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
0.5
2
8
16
Time (hr) FIG. 2. Time course of selenium effects on hepatic microsomal 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 were measured as described in Materials and Methods. (-) Heme oxygenase activity (nmol/mg-hr); ---) microsomal heme content (nmol/mg).
by selenium of heme synthetic and heme degradation activities were biologically mediated effects rather than being chemical activation phenomena. On the basis of the findings from other laboratories (4-7) it was presumed that selenium might facilitate drug-metabolizing activity by enzymes of the endoplasmic reticulum. However, an unexpected finding in this study was that selenium inhibited microsomal drug-metabolizing activity by about 15-50% depending on dose (Table 3) despite the presence of an ample amount of microsomal cytochrome P-450 and the fact that NADPH-cytochrome c reductase activity was unchanged. It was noted above that one reported property of selenium is its ability to exert a "protective" effect on biological membranes (2, 3). Therefore, the possibility that increased activities of AmLev synthase and heme oxygenase after selenium might be due to a decrease in the rate of turnover of the enzymes rather than to increased synthesis was examined. It is known that exogenous heme is a potent inhibitor of the synthesis of AmLev synthase and is an inducer of heme oxygenase. AcTable 3. Effect of selenium treatment on microsomal enzyme activities and cytochrome P-450 content Dose of Na2SeO3 (/2mol/ 100 g)
Ethylmorphine N-demethylase (nmol/mg-hr)
NADPH-cytochrome c reductase (nmol/mg-hr)
Cytochrome P-450 (nmol/mg)
0 5 10
157.6 132.3 79.2
99.8 99.0 92.1
0.92 0.83 0.75
Rats were treated as described in the legend of Table 1. The hepatic microsomal fractions were prepared and assayed for ethylmorphine N-demethylase and NADPH-cytochrome c reductase activities; cytochrome P-450 content was measured. The procedure used for these assays Methods.
were
those described in Materials and
livers were perfused and homogenized, and aliquots were removed for the assay of AmLev synthase and determination of porphyrin content. Microsomal fractions were prepared and used for the assay of heme oxygenase and measurement of cytochrome P-450 and heme contents.
cordingly, in the present study the influence of selenium pretreatment on heme effects on AmLev synthase and heme oxygenase activities was studied. Table 4 shows that pretreatment with selenium (90 min) blocked heme inhibition of AmLev synthase formation. Moreover, heme administered to animals pretreated with selenium augmented induction of hepatic heme oxygenase activity. Microsomal contents of cytochrome P-450 were not significantly different in the animals treated with selenium alone or selenium plus heme. DISCUSSION
Selenium is an essential nutrient, and a number of functions ranging from serving as a biological anti-oxidant (2) to being a constituent of enzymes (6, 7) and possibly of the microsomal electron transport chain (4, 5) have been assigned to it. In the present study new actions of this element on heme metabolism were identified. The ability of selenium to induce AmLev synthase, the rate-limiting enzyme in heme synthesis (8), may be one of its most significant actions since hemoproteins constitute a major component of certain biological membranes and are directly involved in cellular oxidation reactions. Moreover, selenium is a unique compound in the effects it exerts on cellular heme metabolism. Unlike other inorganic elements, such as cobalt, which induce AmLev synthase after an initial prolonged and significant inhibition of this enzyme activity (10), or organic compounds, such as barbiturate analogues, in which an induction of AmLev synthase activity occurs concurrently with an increase in cellular content of porphyrins (10-12), no increase in porphyrins is observed with selenium induction of AmLev synthase. The fact that selenium does not increase cellular porphyrin content while inducing AmLev synthase indicates that the total amount of porphyrin synthesized is converted to heme which in turn is used for the formation of hemoproteins. This would explain the finding that in the presence of the substantially elevated heme oxidation activity produced by selenium no decrease in cellular heme content was observed. Conversely, the elevation of heme oxygenase activity elicited by the element was apparently responsible for the absence of an increase in cellular heme content despite the induction of AmLev synthase. Thus, a steady state was established after selenium administration in which the excess heme syn-
Biochemistry:
Maines and Kappas
thesized was degraded at an accelerated rate, maintaining the cellular content of heme at normal levels. Heme oxidation activity is induced by a number of compounds (9, 23-27), among which the most potent are certain transition elements and heavy metals (9, 22-26). Experimental evidence indicates that the induction effect elicited by such metals is most likely a direct one rather than an action mediated through alterations in intermediary cell components (28). It is possible that the selenium induction of heme oxygenase, however, does reflect a cellular response to an intermediary compound-specifically heme, the concentration of which is directly altered by the element. Selenium, by inducing AmLev synthase without causing concomitant increases in cell content of porphyrin or hemoproteins (i.e., cytochrome P-450), increases transiently the cellular pool of "free" heme (in 30 min, Fig. 2), and this heme fraction then may cause the induction of heme oxygenase. This effect of selenium is rather unusual since other compounds such as the barbiturates, which induce AmLev synthase and increase cellular heme content, do not induce heme oxygenase. Such compounds, however, concomitantly enhance hemoprotein formation, and it can be considered that the heme synthesized as a cellular response to agents such as the barbiturates is "committed" heme rather than "free" heme in that synthesis of a binding protein (apo-cytochrome) is simultaneously induced by the agent. The heme of the formed cytochromes does not induce heme oxygenase, presumably because of the higher affinity of heme for the apo-cytochrome moiety
(29).
One of the physiological functions attributed to selenium is the "protection" of biological membranes and their protein constituents. Therefore, it could be speculated that the increase in the rate of heme synthesis produced by selenium is due to suppression of the turnover rate of AmLev synthase protein in mitochondria, particularly since the enzyme has an exceedingly short half-life (30). However, the finding that pretreatment with selenium totally blocked the inhibition of AmLev synthase formation by heme indicates that selenium directly increases the rate of enzyme synthesis rather than decreasing its rate of degradation. Furthermore, the fact that the activity of AmLev synthase was the same in rats treated with selenium alone or with selenium plus heme suggests a direct action of the element in the regulation of this enzyme synthesis in contrast to its suggested effect on heme oxygenase synthesis. Finally, the fact that selenium treatment did not facilitate the activity of the microsomal electron transport chain, as indicated by a decreased rate of drug-metabolizing activity after its administration, may indicate that selenium does not play a limiting role in the electron transfer processes of microsomal enzymes except possibly in a marked deficiency state of the element. This research was supported by USPHS Grant ES-01055 and by individual grants from Exxon and Mobil Corporations and the Scaife
Proc. Natl. Acad. Sci. USA 73 (1976)
4431
Family Trust. We thank Mrs. Ilona Scher and Mr. Francis A. Farraye for their able and devoted technical assistance and Miss Ann Marie Quatela for typing of the manuscript. 1. Schwarz, K. & Foltz, C. M. (1958) J. Biol. Chem. 233, 245251. 2. Hoekstra, W. G. (1975) Fed. Proc. 34,,2083-2089. 3. Combs, G. F., Jr., Noguchi, T. & Scott, M. L. (1975) Fed. Proc. 34,2090-2095. 4. Burk, R. F., MacKinnon, A. M. & Simon, F. R. (1974) Biochem. Biophys. Res. Commun. 56,431-435. 5. Burk, R. F. & Masters, B. S. S. (1975) Arch. Biochem. Biophys. 170, 124-131. 6. Shum, A. C. & Murphy, J. C. (1972) J. Bacteriol. 110, 447452. 7. Turner, D. C. & Stadtman, T. C. (1973) Arch. Biochem. Biophys. 154,366-370. 8. Granick, S. (1966) J. Biol. Chem. 241, 1359-1375. 9. Maines, M. D. & Kappas, A. (1975) J. Biol. Chem. 250, 41714178. 10. Maines, M. D., Janousek, V., Tomio, J. M. & Kappas, A. (1976) Proc. Natl. Acad. Sci. USA 71, 4293-4297. 11. Schwartz, S. & Ikeda, K. (1955) in Porphyrin Biosynthesis and Metabolism, Ciba Foundation Symposium, eds. Wolstenholme, 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. 100,583-591. 13. Maines, M. D. & Kappas, A. (1975) J. Biol. Chem. 250, 23632369. 14. Maines, M. D. & Anders, M. W. (1973) Mol. Pharmacol. 9, 219-228. 15. Marver, H. S., Tschudy, D. P., Perlroth, M. G. & Collins, A. (1966) J. Biol. Chem. 241, 2803-2814. 16. Mauzerall, D. & Granick, S. (1958) J. Biol. Chem. 219, 435439. 17. Granick, S., Sinclair, P., Sassa, S. & Grieninger, G. (1975) J. Biol. Chem. 250, 9215-9225. 18. Paul, K. G., Theorell, H. & Akeson, A. (1953) Acta Chem. Scand. 7, 1284-1287. 19. Omura, T. & Sato, R. (1964) J. Biol. Chem. 239,2379-2385. 20. Nash, T. (1953) Biochem. J. 55, 416-421. 21. Williams, C. H. & Kamin, H. (1962) J. Biol. Chem. 237, 587595. 22. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 23. Maines, M. D. & Kappas, A. (1976) Biochem. J. 154, 125-131. 24. Kappas, A. & Maines, M. D. (1976) Science 192, 60-62. 25. Maines, M. D. & Kappas, A. (1974) Proc. Natl. Acad. Sci. USA 71,4295-4297. 26. Maines, M. D. & Kappas, A. (1976) Ann. Clin. Res. 8,39-46. 27. Pimstone, N. R., Engel, R., Tenhunen, R., Seitz, P. T., Marver, H. S. & Schmid, R. (1971) J. Clin.; Invest. 50, 2042-2051. 28. Maines, M. D. & Sinclair, P. (1976) J. Biol Chem. 251, in press. 29. Maines, M. D. (1976) Proceedings of the Third International Symposium on Microsomes and Drug Oxidation, Berlin, in press. 30. Whiting, M. & Granick, S. (1976) J. Biol. Chem. 251, 13401346.
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
Selenium was found to be a novel regulator of ABSTRACT cellular heme metabolism in that the element induced both the mitochondrial enzyme 6-aminolevulinate synthase [succinylCoA:glycine C-succinyltransferase (decarboxylating); EC 2.3.1.37] and the microsomal enzyme heme oxygenase [heme, hydrogen-donor:oxygen oxidoreductase(a-methene-oxidizing, hydroxylating); EC 1.14.99.31 in liver. The effect of selenium on these enzyme activities was prompt, reaching a maximum within 2 hr after a single injection. Other changes in parameters of he atic heme metabolism occurred after administration of the element. Thirty minutes after injection the cellular content of heme was significantly increased; however, this value slightly decreased below control values within 2 hr, coinciding with the period of rapid induction of heme oxygenase. At later periods heme content returned to normal values. Selenium treatment caused only a slight decrease in microsomal cytochrome P450 content. However, drug-metabolizing activity was severely inhibited by higher doses of the element. Unlike other inducers of 5-aminolevulinate synthase, which as a rule are also porphyrinogenic agents, selenium induction of this enzyme was not accompanied by an increase in the cellular content of porphyrins. Wen rats were pretreated with selenium 90 min before administration of heme, a potent inhibitor of 6aminolevulinate synthase production, the inhibitory effect of heme on formation of this mitochondrial enzyme was completely blocked. Selenium, at high concentrations in vitro, was inhibitory to 6-aminolevulinate synthase activity. It is postulated that selenium may not be a direct inducer of heme oxygenase as is the case with trace metals such as cobalt, but may mediate an increase in heme oxygenase through increased production and cellular availability of "free" heme, which results from the increased heme synthetic activity of hepatocytes. Subsequently, the increased heme oxygenase activity is in turn responsible for the lack of increase in the microsomal heme content, thus maintaining heme levels at normal values despite the highly increased activities of both heme oxygenase and b-aminolevulinate synthase. It is further suggested that the increase in 6-aminolevulinate synthase activity is not due to a decreased rate of enzyme degradation or an activation of preformed enzyme, but to increased rate of synthesis of enzyme protein. Although selenium in trace amounts has been postulated to be involved in microsomal electron transfer process, the data from this study indicate that excess selenium can substantially inhibit microsomal drug metabolism.
effective inducer of both 6-aminolevulinate (AmLev) synthase [succinyl-CoA:glycine C-succinyl-transferase (decarboxylating); EC 2.3.1.37] and heme oxygenase [heme, hydrogen-donor: oxygen oxidoreductase (a-methene-oxidizing, hydroxylating); EC 1.14.99.3] activties. AmLev synthase is the rate-limiting enzyme in the heme synthetic pathway (8), and heme oxygenase is the microsomal enzyme that degrades heme to bile pigment. The effects of selenium on cellular heme metabolism were unusual in several respects, including its rather prompt induction of AmLev synthase together with a concomitant induction of heme oxygenase without major changes in cellular heme content. These effects are unlike those produced by trace metal inducers of AmLev synthase such as cobalt which elicit a clear biphasic response consisting of a substantial early inhibition of the enzyme that, after several hours, is followed by derepression of this enzyme synthesis (9, 10). A steady and substantial decrease in cellular heme content accompanies these changes in AmLev synthase activity (9). Moreover, in contrast to other chemical inducers of AmLev synthase such as allylisopropylacetamide (11) and dicarbethoxydehydrocollidine (12), which invariably produce a porphyrinogenic response in liver, selenium was shown not to be a porphyrinogenic agent despite its ability to induce the enzyme. The combination of these selenium actions on cellular heme metabolism is unique and may account for some of the biological functions of this element. MATERIALS AND METHODS Male Sprague-Dawley rats (150-180 g) were treated (subcutaneously) for various lengths of time with sodium selenite (Na2SeO3) solutions at pH 7.4. The dose routinely used was 5 ,umol/100 g; other doses were used in some experiments as indicated. Animals were also treated with a combination of sodium selenite and heme (hematin). In these experiments, the animals were first treated with Na2SeO3 90 min before the injection of heme (4 ,umol/100 g, intraperitoneally). The solution of heme was prepared as described elsewhere (13). After the last injection food was removed and animals were killed at different intervals as indicated. Livers were perfused in situ (0.9% NaCl) and homogenized in Tris-HCl (0.1 M, pH 7.4) containing 0.25 M sucrose. Aliquots were removed for assay of
Selenium has recently been identified as an essential element in mammalian nutrition (1). It is believed, with vitamin E, to function as an anti-oxidant in protection of biological membranes against lipid peroxidation (2) as well as maintaining normal levels of capillary permeability (3). Selenium deficiency causes a diminution in the induction response of the hepatic microsomal cytochrome P-450 system to barbiturates (4, 5), and it has been suggested that the element may participate in electron transfer reactions of some bacterial enzymes (6, 7). In the present study, we report a newly defined action of selenium on liver cell metabolism in which selenium was found to be an
AmLev synthase, 6-aminolevulinate dehydratase [porphobilinogen synthase; 5-aminolevulinatehydro-lyase (adding 5aminolevulinate and cyclizing); EC 4.2.1.24], and total porphyrin. The remainder was used for the preparation of microsomal fractions as described earlier (14). AmLev synthase activity was determined by the method of Marver et al. (15); AmLev dehydratase activity was determined as described by
Mauzerall and Granick (16). The total porphyrin content was determined by the method of Granick et al. (17). Microsomal heme and hemoprotein contents were measured by the methods of Paul et al. (18) and Omura and Sato (19), respectively. Oxi-
Abbreviation: AmLev, 6-aminolevulinate.
4428
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
Na2SeO3 (gmol/lOO g)
synthase
dehydratase
porphyrin
(pmol/mg-hr)
(nmol/mg-hr)
(pmol/mg)
Control 1 5 10
125.8 259.9 207.3 209.4
5.38 4.79 6.21 3.64
Microsomal heme (nmol/mg)
Heme oxygenase
(nmol/mg-hr)
3.57 1.50 1.85 1.38 3.30 3.09 3.32 1.47 5.55 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 expressed as the nmol of bilirubin formed per mg of protein per hr.
dative drug metabolizing activity was monitored with ethylmorphine as substrate in the system described earlier (13) measuring the formaldehyde produced (20). Microsomal heme oxygenase activity was determined by the method described earlier (9). The activity.of NADPH-cytochrome c reductase (ADPH:ferricytochrome oxidoreductase; EC 1.6.2.4) was measured as described by Williams and Kamin (21). Protein content was measured by the method of Lowry et al. (22). The data were analyzed by the "t" test; values of P < 0.05 were regarded as denoting significance.
RESULTS Selenium significantly modified cellular heme metabolism. Table 1 shows the responses of activities of various heme pathway enzymes and related parameters to sodium selenite administration; as shown, the effects on AmLev synthase and cellular porphyrin and heme contents were not dose-dependent, at least within the range of 1.0-10.0 ,mol/100 g of body weight. However, the activity of AmLev dehydratase was somewhat decreased by a high dose of selenium and that of heme oxygenase showed a dose-dependent increase after its injection. Fig. shows the time course of effects of selenium on AmLev synthase and AmLev dehydratase activities and on total hepatocyte porphyrin content. As shown, 30 min after injection there was a moderate inhibition (30%) of AmLev synthase which was followed by a rapid increase in the enzyme activity within 2 hr
that was sustained thereafter. The early inhibition of AmLev synthase may be due to the direct inactivation of the enzyme by selenium. As Table 2 shows, at high concentrations, selenium in vitro can markedly inhibit AmLev synthase activity. Since immediately after injection the cellular concentration of the compound is highly elevated, it is possible that the inhibition of AmLev synthase observed shortly after treatment is analogous to the situation in vitro. Despite the increase in AmLev synthase activity, no significant changes in hepatic porphyrin content were produced by selenium; neither was AmLev dehydratase activity altered. Selenium dioxide (SeO2) produced comparable changes in hepatic cell metabolism of heme in comparable doses (data not shown); at higher doses (>10 ,umol/100 g) this selenium compound was toxic to the animals. Fig. 2 shows the effect of selenium on microsomal content of heme and on heme oxygenase activity. Thirty minutes after injection the microsomal heme content was slightly. but significantly increased (22%). Four hours after injection there was a slight decrease observed in microsomal heme content, which coincided with the substantial increase of heme oxidation activity that had occurred. Despite this increase in heme oxygenase activity, no further depletion of cellular heme content was noted. The induction of heme oxygenase caused by selenium that was evident within 2 hr after treatment was not significantly altered during the subsequent 14 hr. A transient decrease 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, it isapparent that the inductions Table 2. Effect of sodium selenite in vitro on AmLev synthase and heme oxygenase activities
OlI
0.5
2
8
16
Time (hr) FIG. 1. Time course of selenium effects on activities of AmLev synthase and AmLev dehydratase and total porphyrin content in hepatocytes. Rats were treated with a solution of sodium selenite (subcutaneously,55gmol/100 g). At different intervals the animals were killed. Livers were perfused and then homogenized in Tris.HCl buffer (pH 7.4) containing 0.25 M sucrose. The assays were done as described in Materials and Methods. (-) AmLev synthase activity (pmol/mg-hr) X 102; (----) AmLev dehydratase activity (nmol/mg-hr) X 2; (- --) porphyrin content (pmol/mg).
nM Na2SeO3
AmLev synthase (pmol/mg-hr)
(nmol/mg-hr)
0 50 100 500 1000
83.70 88.26 86.74 16.13 12.17
1.55 1.70 1.40 1.25 1.70
Heme oxygenase
Whole homogenates were prepared from the perfused livers of starved rats and were used for the assay of AmLev synthase activity in the presence of indicated concentrations of sodium selenite. The heme oxygenase activity was determined using the microsomal fraction of the livers in the presence of above concentrations of
selenium.
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 and heme oxygenase activities
5.0
4.0
Treatment 3.0
Control Selenium Heme Selenium + heme
AmLev synthase (pmol/ mg-hr)
Heme CytoTotal oxygenase chrome porphyrin (nmol/ P-450 (pmol/mg) mg-hr) (nmol/mg)
82.3 263.9 40.5
3.34 3.66 3.29
2.1 7.2 10.7
0.76 0.65 0.72
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
0.5
2
8
16
Time (hr) FIG. 2. Time course of selenium effects on hepatic microsomal 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 were measured as described in Materials and Methods. (-) Heme oxygenase activity (nmol/mg-hr); ---) microsomal heme content (nmol/mg).
by selenium of heme synthetic and heme degradation activities were biologically mediated effects rather than being chemical activation phenomena. On the basis of the findings from other laboratories (4-7) it was presumed that selenium might facilitate drug-metabolizing activity by enzymes of the endoplasmic reticulum. However, an unexpected finding in this study was that selenium inhibited microsomal drug-metabolizing activity by about 15-50% depending on dose (Table 3) despite the presence of an ample amount of microsomal cytochrome P-450 and the fact that NADPH-cytochrome c reductase activity was unchanged. It was noted above that one reported property of selenium is its ability to exert a "protective" effect on biological membranes (2, 3). Therefore, the possibility that increased activities of AmLev synthase and heme oxygenase after selenium might be due to a decrease in the rate of turnover of the enzymes rather than to increased synthesis was examined. It is known that exogenous heme is a potent inhibitor of the synthesis of AmLev synthase and is an inducer of heme oxygenase. AcTable 3. Effect of selenium treatment on microsomal enzyme activities and cytochrome P-450 content Dose of Na2SeO3 (/2mol/ 100 g)
Ethylmorphine N-demethylase (nmol/mg-hr)
NADPH-cytochrome c reductase (nmol/mg-hr)
Cytochrome P-450 (nmol/mg)
0 5 10
157.6 132.3 79.2
99.8 99.0 92.1
0.92 0.83 0.75
Rats were treated as described in the legend of Table 1. The hepatic microsomal fractions were prepared and assayed for ethylmorphine N-demethylase and NADPH-cytochrome c reductase activities; cytochrome P-450 content was measured. The procedure used for these assays Methods.
were
those described in Materials and
livers were perfused and homogenized, and aliquots were removed for the assay of AmLev synthase and determination of porphyrin content. Microsomal fractions were prepared and used for the assay of heme oxygenase and measurement of cytochrome P-450 and heme contents.
cordingly, in the present study the influence of selenium pretreatment on heme effects on AmLev synthase and heme oxygenase activities was studied. Table 4 shows that pretreatment with selenium (90 min) blocked heme inhibition of AmLev synthase formation. Moreover, heme administered to animals pretreated with selenium augmented induction of hepatic heme oxygenase activity. Microsomal contents of cytochrome P-450 were not significantly different in the animals treated with selenium alone or selenium plus heme. DISCUSSION
Selenium is an essential nutrient, and a number of functions ranging from serving as a biological anti-oxidant (2) to being a constituent of enzymes (6, 7) and possibly of the microsomal electron transport chain (4, 5) have been assigned to it. In the present study new actions of this element on heme metabolism were identified. The ability of selenium to induce AmLev synthase, the rate-limiting enzyme in heme synthesis (8), may be one of its most significant actions since hemoproteins constitute a major component of certain biological membranes and are directly involved in cellular oxidation reactions. Moreover, selenium is a unique compound in the effects it exerts on cellular heme metabolism. Unlike other inorganic elements, such as cobalt, which induce AmLev synthase after an initial prolonged and significant inhibition of this enzyme activity (10), or organic compounds, such as barbiturate analogues, in which an induction of AmLev synthase activity occurs concurrently with an increase in cellular content of porphyrins (10-12), no increase in porphyrins is observed with selenium induction of AmLev synthase. The fact that selenium does not increase cellular porphyrin content while inducing AmLev synthase indicates that the total amount of porphyrin synthesized is converted to heme which in turn is used for the formation of hemoproteins. This would explain the finding that in the presence of the substantially elevated heme oxidation activity produced by selenium no decrease in cellular heme content was observed. Conversely, the elevation of heme oxygenase activity elicited by the element was apparently responsible for the absence of an increase in cellular heme content despite the induction of AmLev synthase. Thus, a steady state was established after selenium administration in which the excess heme syn-
Biochemistry:
Maines and Kappas
thesized was degraded at an accelerated rate, maintaining the cellular content of heme at normal levels. Heme oxidation activity is induced by a number of compounds (9, 23-27), among which the most potent are certain transition elements and heavy metals (9, 22-26). Experimental evidence indicates that the induction effect elicited by such metals is most likely a direct one rather than an action mediated through alterations in intermediary cell components (28). It is possible that the selenium induction of heme oxygenase, however, does reflect a cellular response to an intermediary compound-specifically heme, the concentration of which is directly altered by the element. Selenium, by inducing AmLev synthase without causing concomitant increases in cell content of porphyrin or hemoproteins (i.e., cytochrome P-450), increases transiently the cellular pool of "free" heme (in 30 min, Fig. 2), and this heme fraction then may cause the induction of heme oxygenase. This effect of selenium is rather unusual since other compounds such as the barbiturates, which induce AmLev synthase and increase cellular heme content, do not induce heme oxygenase. Such compounds, however, concomitantly enhance hemoprotein formation, and it can be considered that the heme synthesized as a cellular response to agents such as the barbiturates is "committed" heme rather than "free" heme in that synthesis of a binding protein (apo-cytochrome) is simultaneously induced by the agent. The heme of the formed cytochromes does not induce heme oxygenase, presumably because of the higher affinity of heme for the apo-cytochrome moiety
(29).
One of the physiological functions attributed to selenium is the "protection" of biological membranes and their protein constituents. Therefore, it could be speculated that the increase in the rate of heme synthesis produced by selenium is due to suppression of the turnover rate of AmLev synthase protein in mitochondria, particularly since the enzyme has an exceedingly short half-life (30). However, the finding that pretreatment with selenium totally blocked the inhibition of AmLev synthase formation by heme indicates that selenium directly increases the rate of enzyme synthesis rather than decreasing its rate of degradation. Furthermore, the fact that the activity of AmLev synthase was the same in rats treated with selenium alone or with selenium plus heme suggests a direct action of the element in the regulation of this enzyme synthesis in contrast to its suggested effect on heme oxygenase synthesis. Finally, the fact that selenium treatment did not facilitate the activity of the microsomal electron transport chain, as indicated by a decreased rate of drug-metabolizing activity after its administration, may indicate that selenium does not play a limiting role in the electron transfer processes of microsomal enzymes except possibly in a marked deficiency state of the element. This research was supported by USPHS Grant ES-01055 and by individual grants from Exxon and Mobil Corporations and the Scaife
Proc. Natl. Acad. Sci. USA 73 (1976)
4431
Family Trust. We thank Mrs. Ilona Scher and Mr. Francis A. Farraye for their able and devoted technical assistance and Miss Ann Marie Quatela for typing of the manuscript. 1. Schwarz, K. & Foltz, C. M. (1958) J. Biol. Chem. 233, 245251. 2. Hoekstra, W. G. (1975) Fed. Proc. 34,,2083-2089. 3. Combs, G. F., Jr., Noguchi, T. & Scott, M. L. (1975) Fed. Proc. 34,2090-2095. 4. Burk, R. F., MacKinnon, A. M. & Simon, F. R. (1974) Biochem. Biophys. Res. Commun. 56,431-435. 5. Burk, R. F. & Masters, B. S. S. (1975) Arch. Biochem. Biophys. 170, 124-131. 6. Shum, A. C. & Murphy, J. C. (1972) J. Bacteriol. 110, 447452. 7. Turner, D. C. & Stadtman, T. C. (1973) Arch. Biochem. Biophys. 154,366-370. 8. Granick, S. (1966) J. Biol. Chem. 241, 1359-1375. 9. Maines, M. D. & Kappas, A. (1975) J. Biol. Chem. 250, 41714178. 10. Maines, M. D., Janousek, V., Tomio, J. M. & Kappas, A. (1976) Proc. Natl. Acad. Sci. USA 71, 4293-4297. 11. Schwartz, S. & Ikeda, K. (1955) in Porphyrin Biosynthesis and Metabolism, Ciba Foundation Symposium, eds. Wolstenholme, 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. 100,583-591. 13. Maines, M. D. & Kappas, A. (1975) J. Biol. Chem. 250, 23632369. 14. Maines, M. D. & Anders, M. W. (1973) Mol. Pharmacol. 9, 219-228. 15. Marver, H. S., Tschudy, D. P., Perlroth, M. G. & Collins, A. (1966) J. Biol. Chem. 241, 2803-2814. 16. Mauzerall, D. & Granick, S. (1958) J. Biol. Chem. 219, 435439. 17. Granick, S., Sinclair, P., Sassa, S. & Grieninger, G. (1975) J. Biol. Chem. 250, 9215-9225. 18. Paul, K. G., Theorell, H. & Akeson, A. (1953) Acta Chem. Scand. 7, 1284-1287. 19. Omura, T. & Sato, R. (1964) J. Biol. Chem. 239,2379-2385. 20. Nash, T. (1953) Biochem. J. 55, 416-421. 21. Williams, C. H. & Kamin, H. (1962) J. Biol. Chem. 237, 587595. 22. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 23. Maines, M. D. & Kappas, A. (1976) Biochem. J. 154, 125-131. 24. Kappas, A. & Maines, M. D. (1976) Science 192, 60-62. 25. Maines, M. D. & Kappas, A. (1974) Proc. Natl. Acad. Sci. USA 71,4295-4297. 26. Maines, M. D. & Kappas, A. (1976) Ann. Clin. Res. 8,39-46. 27. Pimstone, N. R., Engel, R., Tenhunen, R., Seitz, P. T., Marver, H. S. & Schmid, R. (1971) J. Clin.; Invest. 50, 2042-2051. 28. Maines, M. D. & Sinclair, P. (1976) J. Biol Chem. 251, in press. 29. Maines, M. D. (1976) Proceedings of the Third International Symposium on Microsomes and Drug Oxidation, Berlin, in press. 30. Whiting, M. & Granick, S. (1976) J. Biol. Chem. 251, 13401346.
