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Some effects of selenium deficiency on the hepatic microsomal cytochrome P-450 system in the rat
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
170,
124-131
Some Effects of Selenium Microsomal Cytochrome RAYMOND Liver Unit, Depatiment Health Science Center,
F. BURK2 of Internal Southwestern
Medicine Medical Received
Deficiency on the Hepatic P-450 System in the Rat1 BETTIE
AND
(1975)
SUE
SILER
MASTERS
and Department of Biochemistry, The School, 5323 Harry Hines Boulevard, February
University of Texas, Dallas, Texas 75.235
28, 1975
The hepatic microsomal cytochrome P-450 system was investigated in male rats fed a selenium-deficient Torula yeast diet for 3 mo after weaning and in control rats fed the same diet with 0.5 pg selenium as Na$!leOs added per gram of diet. Cytochrome P-450 and b, coqtents, NADPH-cytochrome c reductase, ethylmorphine demethylase, 2- and 4biphenyl hydroxylase activities, and pentobarbital sleeping time were measured and the effect of phenobarbital pretreatment on these parameters was determined. The effect of 3-methylcholanthrene pretreatment on all parameters except sleeping time was studied. Selenium deficiency caused no alterations in any of the above activities in untreated or 3-methylcholanthrene-treated rats. However, phenobarbital treatment in seleniumdeficient rats produced an increase in cytochrome P-450 content of only 70% as compared to 150% in the similarly treated controls. The induction of ethylmorphine demethylase activity by phenobarbital was also impaired in selenium-deficient rats. No such impairment in the induction of cytochrome b, content, NADPH-cytochrome c reductase activity, biphenyl hydroxylase activity, or pentobarbital sleeping time occurred. The percentage of the carbon monoxide-reactive cytochrome P-450 which bound metyrapone was the same in phenobarbital-treated selenium-deficient rats and in phenobarbital-treated controls. Also the cytochrome P-450 ethyl isocyanide binding spectra were similar in phenobarbital-treated selenium-deficient rats and in phenobarbital-treated control rats. These data indicate a specific effect of selenium in the hepatic microsomal cytochrome P450 system. The biochemical function of selenium giving rise to this effect is unknown.
Until the last few years the biochemical functions of selenium were unknown. Recently, in rapid succession, the element has been shown to be an essential constituent of several enzymes. In animals it is part of a prosthetic group in glutathione peroxidase (1) and it has been reported to be present in a soluble cytochrome of sheep muscle (2). Studies with bacteria show it to be part of formic dehydrogenase in Escherich& coli (3) and of protein A of the glytine reductase system in Clostridium stickZandii (4). It has been suggested that sele-
nium may participate in electron transfer in each of these proteins (5). Another electron transport system of great importance is the hepatic microsoma1 cytochrome P-450 system. A recent report indicated that the induction of cytochromes P-450 and bs by phenobarbital was impaired in selenium-deficient rats (6). The present study is a detailed examination of the constituents of that system and some of its functions in selenium deficiency. METHODS
’ This work was supported by U. S. Public Health Service Grants ES-01017, AM05490 (R.F.B.), GM16488 and HL13619 (B.S.S.M.). Presented in part at FASEB, April 17, 1975 and published in abstract form in Fed. Proc. 34, 924 (Abs.) 1975. * Research and Education Associate of the Veterans Administration.
Diets. Two diets were fed. The composition of the selenium-deficient diet is shown in Table I. The selenium-adequate or control diet differed only in that 0.67% of the sucrose was replaced with a Na.SeOl-sucrose mixture to provide a final selenium concentration of 0.5 *g/g of diet. Approxi124
Copyright
0 1975 by Academic
All
of reproduction
rights
in any
Press, Inc. form reserved.
SELENIUM TABLE
DEFICIENCY
AND
I
DIET COMPOSITION Torula yeast5 Sucrose Fat (stripped corn Salt mixture’ Vitamin mixtured
oil or lard)*
30.0 58.8 6.7 3.5 1.0
a Purchased from St. Regis, Rhinelander, WI. * Purchased from Nutritional Biochemicals Co., Cleveland, OH. c Williams-Briggs formula (J. N&r. 103, 536, 1973) mixed by the authors. d Same as in J. Nutr. 102,1049 (1972) except dl-otocopherol supplied at 100 III/kg diet. mately half of the experiments were carried out with lard stripped of vitamin E as the fat source; the other half used corn oil stripped of vitamin E. Vitamin E was supplied in the vitamin mixture. All experiments utilized internal controls and the significant results reported here were noted with both dietary fat sources. Tap water and experimental diet were provided ad lib. Glutathione peroxidase levels in liver supernatant fractions and in plasma from rats fed the selenium-deficient diet for 3 mo were approximately 25 and 3% respectively, of those from the control animals as measured by the method of Paglia and Valentine (7) with cumene hydroperoxide as substrate. Moreover, dietary liver necrosis was produced in weanling male rats fed this diet when o-tocopherol was omitted. Both of these findings demonstrate that the diet was selenium-deficient. Animals. Male weanling Holtzman rats were fed the experimental diet for 3 mo before they were used for experiments. Most animals weighed 200-300 g. All experiments except the induction time course included controls injected with vehicle only. Inducing agents (sodium phenobarbital, 75 mg/kg in 0.14 M NaCl; 3-methylcholanthrene, 18 mg/kg in corn oil) were administered by intraperitoneal injection for 4 days, and the animals were fasted for 24 h after the last injection. They were weighed before killing which was done between 4 and 8 AM. In earlier experiments, animals were killed by exsanguination from the aorta under ether anesthesia, but later experiments used decapitation. No difference in results was noted when this change was made. Microsomal preparation. Immediately after death, the liver was perfused with 12 ml of cold 0.14 M NaCl via the portal vein. It was then removed and weighed. Eight grams or the whole liver (if it weighed less than 8 g) was minced and washed with 50 ml of cold 0.14 M NaCl. It was then homogenized in 3 vol of 0.25 M sucrose by three passes of a Teflon pestle at 2000 rpm. The homogenate was centrifuged at 8700 g for 10 min and the supernatant was recen-
CYTOCHROME
P-450
SYSTEM
125
trifuged at 18,800 g for 10 min. The resulting supernatant was centrifuged at 105,000 g for 60 min and the pellet was resuspended in 0.15 M KC1 and centrifuged again at 105,000 g for 30 min. The final pellet was then suspended in 0.05 M Tris-HCl, 0.25 M sucrose, pH 7.4 buffer. Protein concentration was determined by the biuret method (81, and the microsomes were kept at 4 C until used. Assays. All assays were carried out within 30 h of the preparation of microsomes. There was no loss of cytochrome P-450 in this interval (see Results). Drug metabolism assays carried out within 4 h of microsomal preparation had slightly higher values than those stored overnight. All experiments included controls so that storage effect on conclusions should be eliminated. Cytochromes b, and P-450 in microsomal suspensions of 2 mg protein/ml were determined by the methods of Omura and Sato (9) using an Aminco-Chance DW-2 spectrophotometer. NADPH-cytochrome c reductase activity was determined by the method of Masters et al. (10). Ethylmorphine demethylase activity was measured by the determination of the rate of formaldehyde formation using the Nash reaction (11). Freshly prepared reaction mixture-50 mM Tris, 150 mM KCl, 10 mM MgCl,, 8 mM ethylmorphine, 8 mM isocitrate, 0.25 units isocitrate dehydrogenase -at pH 7.4 was incubated in a shaking water bath for 2 min at 25°C. Then microsomes were added to a tinal concentration of 2 mg protein per milliliter. After 1 min, NADPH was added to a concentration of 250 FM, and at 1-min intervals for 10 min, 1 ml of the mixture was removed and the reaction was stopped with 1 ml of 15% trichloroacetic acid. After centrifugation, 1.5 ml of the supernatant was added to 1.5 ml of Nash reagent and incubated 8 min at 58°C. After cooling, absorbance was determined at 412 nm. The 0 time sample was used as a reagent blank. Formaldehyde contents were read from a standard curve. The initial rate was estimated graphically. The biphenyl hydroxylase assay used was that of Creaven, Parke, and Williams (12). Pentobarbital sleeping times were carried out with rats fasted 24 h. The drug was administered intraperitoneally (45 mg/kg) and the time was noted when the rat lost its righting reflex. This was induction time. The rat was not disturbed further until it turned over. It was then replaced on its back twice and the time it turned over for the third time was noted. That period (from induction to righting) was recorded as the sleeping time. The unpaired t test was used to determine significant differences. Metyrapone-binding cytochrome P-450 was determined. Microsomes were suspended in 0.25 M sucrose, 0.05 M Tris, pH 7.4, at a concentration of 2 mg protein/ml and reduced with sodium dithionite. They were then put into two cuvettes and metyrapone was added to the sample cuvette. The metyrapone-binding cytochrome P-450 was calculated using
126
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AND
MASTERS
lation was unrelated to selenium status. A slight induction of cytochrome bs was obtained with phenobarbital treatment, but it was the same in selenium-deficient and control animals. Selenium status had a striking effect on the induction of cytochrome P-450 by phenobarbital (Table II). The microsomal content of cytochrome P-450 in untreated animals was unaffected by selenium status. Treatment with phenobarbital, however, caused a greater increase of cytochrome P450 in control animals than in seleniumdeficient ones. Treatment with phenobarbital resulted in a 2.5fold cytochrome P-450 content in control rats but only a 1.7-fold content in selenium-deficient ones. NADPH-cytochrome c reductase was not significantly affected by selenium status. Table III presents data illustrating the effect of 3-methylcholanthrene stimulation. Treatment with this agent caused a shift of the peak in the carbon monoxide difference spectrum of reduced microsomes from 450 nm to 448 nm. This is considered to be a form of cytochrome P-450 and is referred to here as cytochrome P-446. No effect attributable to selenium status was observed in cytochrome bs, cytochrome P448, or NADPH-cytochrome c reductase determinations. Thus, selenium deficiency results in decreased induction of cytochrome P-450 by phenobarbital with no effect on the content of cytochrome b5 or NADPH-cytochrome c reductase activity. Also, selenium de& ciency does not affect the response of cyto-
the absorbance differences at 446 and 500 nm and an arbitrary extinction coefficient of 91 mM-’ cm-‘. The carbon monoxide-reactive cytochrome P-450 was also determined and the percentage of it which bound metyrapone was calculated. Ethyl isocyanide binding spectra were obtained by the method of Sladek and Mannering (13). The suspending buffer was 0.2 M Tris, 150 mM KCl, 10 mM MgCl,, pH 7.4, and the scans were started exactly 30 s after the injection of ethyl isocyanide into the cuvette. The heights of the peaks at 455 and 430 nm were measured by comparison with the absorbance at 500 nm. MATERIALS Sodium phenobarbital was purchased from Mallinckrodt Chemical Works and 3-methylcholanthrene from Eastman. NADH and NADPH were products of P-L.Biochemicals. Cytochrome c, isocitrate dehydrogenase, trisodium isocitrate, and ethyl isocyanide were purchased from Sigma. Ethylmorphine hydrochloride was obtained from Merck, Sharp, and Dohme. Biphenyl, purchased from Eastman, and 2-OH and 4-OH biphenyls, purchased from K and K Laboratories, Inc., were recrystallized from ethanol. Pentobarbital was a product of Abbott Labs. Metyrapone was a gift of Dr. J. J. Chart of Ciba-Geigy Corp. RESULTS
The effect of phenobarbital stimulation on liver weight and on the contents of some hepatic microsomal constituents in selenium-deficient and control rats is shown in Table II. Liver weight per 100 g body wt was greater in selenium-deficient rats than controls whether treated with phenobarbital or not, but the relative weight increase with phenobarbital stimuTABLE EFFECT
Diet
OF PHENOBARBITAL
Treatment
ON LIVER
Liver
weight
Cytochrome
g/100 g body n 0 Se 0 Se 0.5 Se 0.5 Se
Saline Phenobarbital Saline Phenobarbital
n* b* c Values
having
12 13 10 10 same
wt
+ SD 2.9 4.4 2.5 3.7
superscript
II
WEIGHT AND MICROSOMAL AND CONTROL RATS
2 ++ 2
different
Cytochrome
nmles/mg protein n
0.2” 0.36 0.2” o.3b
b,
CONSTITUENTS
12 13 10 10
P-450
NADPHcytochrome reductase
nmoles/mg protein
+ SD 0.39 0.51 0.40 0.55
IN SELENIUM-DEFICIENT
k ‘+ ?I
0.04 0.05 0.05 0.05
c
nmoles/mg proteimmin
72
2 SD
n
12 13 10 10
0.83 k 0.12 1.4 ? 0.2’ 0.88 2 0.14 2.2 ? 0.3c
10 11 10 10
P < 0.001; only 0 Se and 0.5 Se compared.
‘- SD 72 160 83 179
* 2 I k
11 18 18 31
SELENIUM
DEFICIENCY
AND
CYTOCHROME
TABLE EFFECT
OF %METRYLCHOLANTHRENE
ON LIVER
Diet
Treatment
Liver
AND
weight
0 Se 0 Se 0.5 Se 0.5 Se n Values
Corn 3-MC Corn 3-MC having
oil oil
same
4 5 4 4
2.9 3.7 2.6 3.2
superscript
‘, + k +
AND
MICROSOMAL
CONTROL
Cytochrome
n
b,
IN
Cyt;t&me
nmoles/mg protein k SD
n
0.39 0.55 0.38 0.61
4 5 4 4
-c + 2 k
CONSTITUENTS
SELENIUM-
RATS
0.07 0.07 0.03 0.11
NADPHcytochrome c reductase n
nmoles/mg proii%
0.83 1.7 0.86 1.7
+
0.2 0.2” 0.1 0.3”
4 5 4 4
different
P < 0.01; only 0 Se and 0.5 Se compared.
chrome P-448, cytochrome bg, or NADPHcytochrome c reductase to 3-methylcholanthrene administration. The influence of selenium status on several cytochrome P-450 dependent drug metabolizing systems was studied. Table IV shows the effect of phenobarbital treatment. Selenium deficiency had no significant effect on ethylmorphine demethylase activity in untreated animals. However, with phenobarbital treatment, controls had 1.8 times the activity of selenium-deficient animals. This corresponds closely with the effect observed on cytochrome P450. Unlike ethylmorphine demethylase activity, the induction of 2- and 4-hydroxylation of biphenyl was unaffected by selenium status. Likewise, pentobarbital sleeping time was shortened to the same extent in selenium-deficient and control rats by the prior administration of phenobarbital. Treatment with 3-methylcholanthrene caused a decrease of ethylmorphine demethylase activity and an increase in biphenyl hydroxylase activity. These changes were unaffected by selenium status as is shown in Table V. In order to define further the impairment of cytochrome P-450 response to phenobarbital administration in selenium deliciency, a time course experiment was carried out. In Fig. 1A liver weight per 100 g body wt is seen to have increased steadily during the 4 days of phenobarbital treatment. Cytochrome P-450 increased less in livers of selenium-deficient rats than in livers of selenium-adequate control rats
127
SYSTEM
III
WEIGHT
DEFICIENT
P-450
f 0.22 IO.2 k 0.04 i- 0.2
4 5 4 4
nmoles/mg protein/ min k SD 58 66 79 69
of- 14 + 14 f 23 r 12
(Fig. 1B). Ethylmorphine demethylase (Fig. 1C) induction was also less in selenium-deficient livers. Since the induction of cytochrome P-450 by phenobarbital was impaired in selenium deficiency, somequalitative studies of the hemoprotein were done. Table VI shows that the percentage of the carbon monoxide-reactive cytochrome P-450 which bound metyrapone was the same in selenium deficiency and adequacy in both saline- and phenobarbital-injected animals. Also, the ratios of the 455 nm peak to the 430 nm peak of the ethyl isocyanide binding spectra were unaffected by selenium status. These studies failed to demonstrate that the cytochrome P-450 induced by phenobarbital in selenium-deficient rats is qualitatively different from that in similarly treated controls. It was considered that selenium could be protecting the phenobarbital-induced cytochrome P-450 from destruction, perhaps through its role in glutathione peroxidase. Therefore, cytochrome P-450 was measured 2 and 24 h following microsomal preparation from two selenium-deficient and two control rats which had received phenobarbital for 3 days. Microsomes were stored at 4°C under air. In each case the cytochrome P-450 content of the microsomes changed by less than 5% indicating that virtually no cytochrome P-450 was destroyed between 2 and 24 h under these conditions. In addition, experiments were done in which livers of selenium-deficient animals treated for 4 days with phenobar-
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AND
MASTERS
TABLE EFFECT
OF PHENOBARBITAL
Diet
Treatment
ON SOME
Ethylmorphine demethylase activity n
0 Se 0 Se 0.5 Se 0.5 Se
Saline Phenobarbital Saline Phenobarbital
a Different b Negligible
10 11 10 10
P < 0.001; activity.
only
Biphenyl
nmoles HCHO/mg proteinlmin + SD 2.5 6.6 3.2 11.7
2 r k +
n
Diet
0.6 1.6O 1.0 1.6”
3 6 3 5
’ Negligible
Pentobarbital sleeping time (min) n
AND
Sleeping time k SD
Induction time k SD
4-OH
-b 0.38 2 0.08 -b 0.37 -c 0.06
0.89 4.72 0.84 4.95
f r k +
0.11 0.62 0.08 1.45
10 10 8 9
3.6 5.1 3.1 4.8
k + f ”
0.9 1.2 0.5 0.8
139 15.0 111 14.5
? 2 -+ 2
28 4.3 18 2.4
compared.
V
OF %METHYLCHOLANTHRENE ON SOME PARAMETERS OF DRUG METABOLISM IN SELENIUM-DEFICIENT AND CONTROL RATS
Treatment
Corn 3-MC Corn 3-MC
IN SELENIUM-DEFICIENT
protein/ k SD
Z-OH
0 Se and 0.5 Se values
Ethylmorphine demethylase activity n
0 Se 0 Se 0.5 Se 0.5 Se
METABOLISM
hydroxylase activity
nmoles/mg min
TABLE EFFECT
IV
PARAMETERS OF DRUG CONTROL RATS
oil oil
4 5 4 4
Biphenyl
nmoles HCHO/mg protein/min k SD 2.3 1.7 3.1 2.4
k ? k ?
1.2 0.6 0.8 0.7
n
hydroxylase activity
nmoles/mg
protein/min 2 SD
2-OH 3 4 3 3
-a 1.2 + 0.3 -a 1.2 ? 0.1
4-OH 0.78 2.3 0.83 2.3
2 t + L
0.23 0.6 0.13 0.2
activity.
bital were split and one part was homogenized with 250 mg dl-a-tocopheryl acetate added per 8 g of liver. The other part was homogenized in the conventional manner. In one experiment, microsomal cytochrome P-450 content was 1.17 and 1.28 mnoles/mg protein with and without added vitamin E, respectively, and in another experiment the values were 1.56 and 1.45. These results do not support the hypothesis that selenium protects cytochrome P-450 from destruction. DISCUSSION
An effect of selenium status on the hepatic microsomal cytochrome P-450 system has been demonstrated. The effect-a depressed cytochrome P-450 content-seems to be specific since it was found in pheno-
barbital-treated rats but not in saline- or in 3-methylchdlanthrene-treated rats. No effect of selenium status on the specific contents of NADPH-cytochrome c reductase or cytochrome b, was observed under any conditions. This study confirms a preliminary report by Burk, Mackinnon, and Simon (6) of impairment of cytochrome P-450 induction by phenobarbital in selenium-deficiency but does not agree with the report that cytochrome b, induction was also impaired. Basal cytochrome b5 levels were higher in that study due to the different method of microsomal preparation and possibly to the somewhat different diet, but there is no readily apparent explanation for the different response to phenobarbital. Caygill et al. (14) found no impairment
SELENIUM
261
DEFICIENCY
AND
B
E
0.6
E
2 $
5 e e
E
p.
1 e-4
Se-defment
o--C,
Se-adequate
CYTOCHROME
P-450
nium levels are still appreciable after 2 wk of a selenium-deficient regimen (15). More severe depletion is probably necessary for the effect reported here. It was expected that parameters of cytochrome P-450-dependent drug metabolism would be affected by selenium deficiency. The induction of ethylmorphine demethylase activity was decreased in selenium deficiency; but surprisingly, biphenyl hydroxylase activity and pentobarbital sleeping time were induced by phenobarbital to the same extent in the selenium-deficient rats as in selenium-adequate rats in spite of decreased induction of cytochrome P-450 in the selenium-deficient rats. The explanation for these findings is not known. Siami et al. (16) found no impairment of phenobarbital induction of aminopyrine demethylase and hexobarbital oxidase activity in hepatic microsomes of selenium-deflcient female rats. These investigators did not report cytochrome P-450 levels or ethylmorphine demethylase activity. An important difference in protocol between their study and the present one was that they administered phenobarbital intermittently in the drinking water. The nature of the involvement of selenium with cytochrome P-450 is unknown. It has been reported that cytochrome P-450 TABLE
c
129
SYSTEM
VI
EFFECT OF PHENOBARBITAL ON METYRAP~NE AND ETHYL ISOCYANIDE BINDING SPECTRA OF CYTOCHROME P-450 IN SELENIUM-DEFICIENT AND CONTROL RATS OJ, 0
24 hours
48
of phenobarbital
72
t 96
Diet
Treatment
cytochrome P-450 measured with metyrapone
treatment
FIG. 1. Effect of selenium deficiency on phenobarbital induction of liver weight (A), microsomal cytochrome P-450 content (B), and ethylmorphine demethylase activity (0. Each point represents one rat. Rats fed the diet containing corn oil were used. They were injected with phenobarbital at 24-h intervals until 24 h before killing. Thus, the animals killed at 0 time never received phenobarbital, those killed at 24 h received one injection of it at 0 time, etc. All rats were fasted 24 h before killing.
of cytochrome P-450 induction by phenobarbital in rats fed selenium-deficient diets for 2 wk. That does not necessarily conflict with the present findings since tissue sele-
Metyranone c-ytochrome P-450/ carbon monoxide P-450
nmoles/ mg protein” 0 Se 0 Se 0.5 Se 0.5 Se
Saline Phenobarbital Saline Phenobarbital
(443505 x$ in ethyl isocyanide binding spectrumD (pH 7.4)
0.10 0.72
0.11 0.48
0.53 0.60
0.13 0.96
0.15 0.46
0.61 0.70
I
a Average
Ratio of veak heights
of two animals.
I
130
BURK
AND
heme is present in approximately 40-fold excess over selenium in rat liver microsomes (6), and this seems to preclude a role for selenium as part of cytochrome P450.Glutathione peroxidase activity is severely depressed in livers from seleniumdeficient rats (17), and the possibility exists that this enzyme might protect cytochrome P-450 from lipid peroxide-induced damage. This argument is made somewhat tenuous by the finding of unimpaired induction of cytochrome P-448 by 3-methylcholanthrene in selenium-deficient rats. It would have to be postulated that the glutathione peroxidase protects only a species of cytochrome P-450 induced by phenobarbital. Also, that no decrease of cytochrome P-450 occurs during storage between 2 and 24 h after microsomal preparation further weakens this hypothesis. Finally, the inclusion of the antioxidant a-tocopherol in the homogenizing solution failed to increase measurable cytochrome P-450. Thus, it seems unlikely that the effect of selenium in this system is mediated by glutathione peroxidase. The possibility that heme synthesis is depressed by selenium deficiency was not examined directly in this work. However, the normal induction of cytochrome bs and cytochrome P-448 reported here suggest that heme synthesis is not impaired in selenium deficiency. Nevertheless, direct investigation of this problem is needed. Several forms of cytochrome P-450 have been shown to be present in microsomes (18). Evidence was sought that selenium deficiency causes a selective decrease in some form of cytochrome P-450 by the qualitative studies of metyrapone binding and the ethyl isocyanide binding spectrum (Table V). Similar results were obtained in selenium-deficient and selenium-adequate phenobarbital-treated rats, failing to demonstrate any effect on a specific form of cytochrome P-450. However, the rather selective effect of selenium deficiency on a portion of the phenobarbital-induced cytochrome P-450 and the ethylmorphine demethylase activity but not biphenyl hydroxylase activity tend to support this hypothesis and this possibility must remain open. Diplock and his co-workers (19) have hy-
MASTERS
pothesized that selenium is part of a nonheme iron protein in the microsomes which is analogous to adrenodoxin in the adrenal mitochondrial cytochrome P-450 system. The existence of such a protein which might transfer electrons from NADPH-cytochrome c reductase to cytochrome P-450 has been postulated previously based on the mitochondrial system (20), but little evidence for a nonheme iron protein in the microsomes other than ferritin has been found (21-24). The presence of an analogous iron-selenium protein should have been easily detected in the microsomes by electron paramagnetic resonance studies (25) but no such signal has been found. However, part of Diplock’s hypothesis is that the iron-selenium protein is oxidant-labile and these studies did not use antioxidants to protect such a protein. Stronger evidence against the existence of this protein are recent reconstitution studies using highly purified NADPH-cytochrome c reductase and cytochrome P-450 which suggest that such a factor is not required for at least some drug metabolism to take place (26, 27). The present studies shed little light on this possible role of selenium, and it is difficult to understand how a decrease in such a nonheme iron-selenium-containing protein could lead to the findings reported here. The possibility remains that a specific protein induced by barbiturates in the liver endoplasmic reticulum requires the presence of selenium and is responsible for the specific induction of certain oxidative N-demethylation activities and the cytochrome(s) P450 required for them. ACKNOWLEDGMENTS The authors are grateful to Dr. Jurgen Werringloer for determining the metyrapone-binding cytochrome P-450 and the ethyl isocyanide binding spectra, to Dr. M. Danny Burke for the biphenyl hydroxylase determinations, to Mrs. Barbara Radighieri for the ethylmorphine demethylase determinations, and to Mrs. Elizabeth L. Isaacson for the NADPHcytochrome c reductase assays. REFERENCES 1. OH, S. H., GANTHER, H. E., AND HOEKSTRA, W. G. (1974) Biochemistry 13, 1825. 2. WHANGER, P. D., PEDEREIEN, N. D., AND WEsWIG, P. H. (1973) Biochem. Biophys. Res. Cornmun. 53, 1031.
SELENIUM
DEFICIENCY
AND
3. SHUM, A. C., AND MURPHY, J. C. (1972)5. Bacteria?. 110, 447. 4. TURNER, D. C., AND STADTMAN, T. C. (1973) Arch. Biochem. Biophys. 154, 366. 5. STADTMAN, T. C. (1974) Science 183, 915. 6. BURK, R. F., MACKINNON, A. M., AND SIMON, F. R. (1974) B&hem. Biophys. Res. Commun. 56,431. 7. PAGLIA, D. E., AND VALENTINE, W. N. (1967) J. Lab. Clin. Med. 70, 1.58. 8. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M. (1949) J. Biol. Chem. 177, 751. 9. OMURA, T., AND SATO, R. (1964) J. Biol. Chem. 239, 2370. 10. MASTERS, B. S. S., WILLIAMS, C. H., JR., AND KAMIN, H. (1967) in Methods in Enzymology (Estabrook, R. W. and Pullman, M., eds.), Vol. 10, p. 565, Academic Press, New York. 11. NASH, T. (1953) Biochem. J. 55,416. 12. CREAVEN, P. J., PARK, D. V., AND WILLIAMS, R. T. (1965) Biochem J. 96, 879. 13. SLADEK, N. E., AND MANNERING, G. J. (1966) Biochem. Biophys. Res. Common. 24, 668. 14. CAYGILL, C. P. J., DIPLOCK, A. T., AND JEFFERY, E. H. (1973) Biochem. J. 136, 851. 15. BURK, R. F., WITNEY, R., FRANK, H., ANDPEARSON, W. N. (1968) J. Nutr. 95, 420. 16. SIAMI, G., SCHULERT, A. R., AND NEAL, R. A. (1972) J. Nutr. 102, 857.
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17. HAFEMAN, D. G., SUNDE, R. A., AND HOEKSTRA, W. G. (1974) J. Nutr. 104, 580. 18. HILDEBRANDT, A., REMMER, H., AND ESTABROOK, H. W. (1969) Biochem. Biophys. Res. Commun. 30, 607. 19. DIPLOCK, A. T., AND LUCY, J. A. (1973) FEBS Lett. 29, 205. 20. ESTABROOK, R. W. (1971) in Handbook of Experimental Pharmacology, (Brodie, B. B. and Gillette, J. R., eds.), Vol. 28, No. 2, p. 264, Springer Verlag, Berlin. 21. MONTGOMERY, M. R., CLARK, C., AND HOLTZMAN, J. L. (1974) Arch. Biochem. Biophys. 160, 113. 22. MIYAKE, Y., MASON, H. S., AND LANDGRAF, W. (1967) J. Biol. Chem. 242, 393. 23. MASTERS, B. S. S., BARON, J., TAYLOR, W. E., ANDISAACSON, E. L. (1971) J. Biol. Chem. 246, 4143. 24. BARON, J., TAYLOR, W. E., AND MASTERS, B. S. S. (1972)Arch. Biochem. Biophys. 150,105. 25. TSIBRIS, J. C. M., NAMTVEDT, M. J., ANDGUNSALUS, I. C. (1968) Biochem. Biophys. Res. Commun. 30, 323. 26. VAN DER HOEVEN, T. A., HAUGEN, D. A., AND COON, M. J. (1974) Biochem. Biophys. Res. Commun. 60, 569. 27. VERMILION, J. L. AND COON, M. J. (1974) Biochem. Biophys. Res. Commun. 60,1315.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
170,
124-131
Some Effects of Selenium Microsomal Cytochrome RAYMOND Liver Unit, Depatiment Health Science Center,
F. BURK2 of Internal Southwestern
Medicine Medical Received
Deficiency on the Hepatic P-450 System in the Rat1 BETTIE
AND
(1975)
SUE
SILER
MASTERS
and Department of Biochemistry, The School, 5323 Harry Hines Boulevard, February
University of Texas, Dallas, Texas 75.235
28, 1975
The hepatic microsomal cytochrome P-450 system was investigated in male rats fed a selenium-deficient Torula yeast diet for 3 mo after weaning and in control rats fed the same diet with 0.5 pg selenium as Na$!leOs added per gram of diet. Cytochrome P-450 and b, coqtents, NADPH-cytochrome c reductase, ethylmorphine demethylase, 2- and 4biphenyl hydroxylase activities, and pentobarbital sleeping time were measured and the effect of phenobarbital pretreatment on these parameters was determined. The effect of 3-methylcholanthrene pretreatment on all parameters except sleeping time was studied. Selenium deficiency caused no alterations in any of the above activities in untreated or 3-methylcholanthrene-treated rats. However, phenobarbital treatment in seleniumdeficient rats produced an increase in cytochrome P-450 content of only 70% as compared to 150% in the similarly treated controls. The induction of ethylmorphine demethylase activity by phenobarbital was also impaired in selenium-deficient rats. No such impairment in the induction of cytochrome b, content, NADPH-cytochrome c reductase activity, biphenyl hydroxylase activity, or pentobarbital sleeping time occurred. The percentage of the carbon monoxide-reactive cytochrome P-450 which bound metyrapone was the same in phenobarbital-treated selenium-deficient rats and in phenobarbital-treated controls. Also the cytochrome P-450 ethyl isocyanide binding spectra were similar in phenobarbital-treated selenium-deficient rats and in phenobarbital-treated control rats. These data indicate a specific effect of selenium in the hepatic microsomal cytochrome P450 system. The biochemical function of selenium giving rise to this effect is unknown.
Until the last few years the biochemical functions of selenium were unknown. Recently, in rapid succession, the element has been shown to be an essential constituent of several enzymes. In animals it is part of a prosthetic group in glutathione peroxidase (1) and it has been reported to be present in a soluble cytochrome of sheep muscle (2). Studies with bacteria show it to be part of formic dehydrogenase in Escherich& coli (3) and of protein A of the glytine reductase system in Clostridium stickZandii (4). It has been suggested that sele-
nium may participate in electron transfer in each of these proteins (5). Another electron transport system of great importance is the hepatic microsoma1 cytochrome P-450 system. A recent report indicated that the induction of cytochromes P-450 and bs by phenobarbital was impaired in selenium-deficient rats (6). The present study is a detailed examination of the constituents of that system and some of its functions in selenium deficiency. METHODS
’ This work was supported by U. S. Public Health Service Grants ES-01017, AM05490 (R.F.B.), GM16488 and HL13619 (B.S.S.M.). Presented in part at FASEB, April 17, 1975 and published in abstract form in Fed. Proc. 34, 924 (Abs.) 1975. * Research and Education Associate of the Veterans Administration.
Diets. Two diets were fed. The composition of the selenium-deficient diet is shown in Table I. The selenium-adequate or control diet differed only in that 0.67% of the sucrose was replaced with a Na.SeOl-sucrose mixture to provide a final selenium concentration of 0.5 *g/g of diet. Approxi124
Copyright
0 1975 by Academic
All
of reproduction
rights
in any
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SELENIUM TABLE
DEFICIENCY
AND
I
DIET COMPOSITION Torula yeast5 Sucrose Fat (stripped corn Salt mixture’ Vitamin mixtured
oil or lard)*
30.0 58.8 6.7 3.5 1.0
a Purchased from St. Regis, Rhinelander, WI. * Purchased from Nutritional Biochemicals Co., Cleveland, OH. c Williams-Briggs formula (J. N&r. 103, 536, 1973) mixed by the authors. d Same as in J. Nutr. 102,1049 (1972) except dl-otocopherol supplied at 100 III/kg diet. mately half of the experiments were carried out with lard stripped of vitamin E as the fat source; the other half used corn oil stripped of vitamin E. Vitamin E was supplied in the vitamin mixture. All experiments utilized internal controls and the significant results reported here were noted with both dietary fat sources. Tap water and experimental diet were provided ad lib. Glutathione peroxidase levels in liver supernatant fractions and in plasma from rats fed the selenium-deficient diet for 3 mo were approximately 25 and 3% respectively, of those from the control animals as measured by the method of Paglia and Valentine (7) with cumene hydroperoxide as substrate. Moreover, dietary liver necrosis was produced in weanling male rats fed this diet when o-tocopherol was omitted. Both of these findings demonstrate that the diet was selenium-deficient. Animals. Male weanling Holtzman rats were fed the experimental diet for 3 mo before they were used for experiments. Most animals weighed 200-300 g. All experiments except the induction time course included controls injected with vehicle only. Inducing agents (sodium phenobarbital, 75 mg/kg in 0.14 M NaCl; 3-methylcholanthrene, 18 mg/kg in corn oil) were administered by intraperitoneal injection for 4 days, and the animals were fasted for 24 h after the last injection. They were weighed before killing which was done between 4 and 8 AM. In earlier experiments, animals were killed by exsanguination from the aorta under ether anesthesia, but later experiments used decapitation. No difference in results was noted when this change was made. Microsomal preparation. Immediately after death, the liver was perfused with 12 ml of cold 0.14 M NaCl via the portal vein. It was then removed and weighed. Eight grams or the whole liver (if it weighed less than 8 g) was minced and washed with 50 ml of cold 0.14 M NaCl. It was then homogenized in 3 vol of 0.25 M sucrose by three passes of a Teflon pestle at 2000 rpm. The homogenate was centrifuged at 8700 g for 10 min and the supernatant was recen-
CYTOCHROME
P-450
SYSTEM
125
trifuged at 18,800 g for 10 min. The resulting supernatant was centrifuged at 105,000 g for 60 min and the pellet was resuspended in 0.15 M KC1 and centrifuged again at 105,000 g for 30 min. The final pellet was then suspended in 0.05 M Tris-HCl, 0.25 M sucrose, pH 7.4 buffer. Protein concentration was determined by the biuret method (81, and the microsomes were kept at 4 C until used. Assays. All assays were carried out within 30 h of the preparation of microsomes. There was no loss of cytochrome P-450 in this interval (see Results). Drug metabolism assays carried out within 4 h of microsomal preparation had slightly higher values than those stored overnight. All experiments included controls so that storage effect on conclusions should be eliminated. Cytochromes b, and P-450 in microsomal suspensions of 2 mg protein/ml were determined by the methods of Omura and Sato (9) using an Aminco-Chance DW-2 spectrophotometer. NADPH-cytochrome c reductase activity was determined by the method of Masters et al. (10). Ethylmorphine demethylase activity was measured by the determination of the rate of formaldehyde formation using the Nash reaction (11). Freshly prepared reaction mixture-50 mM Tris, 150 mM KCl, 10 mM MgCl,, 8 mM ethylmorphine, 8 mM isocitrate, 0.25 units isocitrate dehydrogenase -at pH 7.4 was incubated in a shaking water bath for 2 min at 25°C. Then microsomes were added to a tinal concentration of 2 mg protein per milliliter. After 1 min, NADPH was added to a concentration of 250 FM, and at 1-min intervals for 10 min, 1 ml of the mixture was removed and the reaction was stopped with 1 ml of 15% trichloroacetic acid. After centrifugation, 1.5 ml of the supernatant was added to 1.5 ml of Nash reagent and incubated 8 min at 58°C. After cooling, absorbance was determined at 412 nm. The 0 time sample was used as a reagent blank. Formaldehyde contents were read from a standard curve. The initial rate was estimated graphically. The biphenyl hydroxylase assay used was that of Creaven, Parke, and Williams (12). Pentobarbital sleeping times were carried out with rats fasted 24 h. The drug was administered intraperitoneally (45 mg/kg) and the time was noted when the rat lost its righting reflex. This was induction time. The rat was not disturbed further until it turned over. It was then replaced on its back twice and the time it turned over for the third time was noted. That period (from induction to righting) was recorded as the sleeping time. The unpaired t test was used to determine significant differences. Metyrapone-binding cytochrome P-450 was determined. Microsomes were suspended in 0.25 M sucrose, 0.05 M Tris, pH 7.4, at a concentration of 2 mg protein/ml and reduced with sodium dithionite. They were then put into two cuvettes and metyrapone was added to the sample cuvette. The metyrapone-binding cytochrome P-450 was calculated using
126
BURK
AND
MASTERS
lation was unrelated to selenium status. A slight induction of cytochrome bs was obtained with phenobarbital treatment, but it was the same in selenium-deficient and control animals. Selenium status had a striking effect on the induction of cytochrome P-450 by phenobarbital (Table II). The microsomal content of cytochrome P-450 in untreated animals was unaffected by selenium status. Treatment with phenobarbital, however, caused a greater increase of cytochrome P450 in control animals than in seleniumdeficient ones. Treatment with phenobarbital resulted in a 2.5fold cytochrome P-450 content in control rats but only a 1.7-fold content in selenium-deficient ones. NADPH-cytochrome c reductase was not significantly affected by selenium status. Table III presents data illustrating the effect of 3-methylcholanthrene stimulation. Treatment with this agent caused a shift of the peak in the carbon monoxide difference spectrum of reduced microsomes from 450 nm to 448 nm. This is considered to be a form of cytochrome P-450 and is referred to here as cytochrome P-446. No effect attributable to selenium status was observed in cytochrome bs, cytochrome P448, or NADPH-cytochrome c reductase determinations. Thus, selenium deficiency results in decreased induction of cytochrome P-450 by phenobarbital with no effect on the content of cytochrome b5 or NADPH-cytochrome c reductase activity. Also, selenium de& ciency does not affect the response of cyto-
the absorbance differences at 446 and 500 nm and an arbitrary extinction coefficient of 91 mM-’ cm-‘. The carbon monoxide-reactive cytochrome P-450 was also determined and the percentage of it which bound metyrapone was calculated. Ethyl isocyanide binding spectra were obtained by the method of Sladek and Mannering (13). The suspending buffer was 0.2 M Tris, 150 mM KCl, 10 mM MgCl,, pH 7.4, and the scans were started exactly 30 s after the injection of ethyl isocyanide into the cuvette. The heights of the peaks at 455 and 430 nm were measured by comparison with the absorbance at 500 nm. MATERIALS Sodium phenobarbital was purchased from Mallinckrodt Chemical Works and 3-methylcholanthrene from Eastman. NADH and NADPH were products of P-L.Biochemicals. Cytochrome c, isocitrate dehydrogenase, trisodium isocitrate, and ethyl isocyanide were purchased from Sigma. Ethylmorphine hydrochloride was obtained from Merck, Sharp, and Dohme. Biphenyl, purchased from Eastman, and 2-OH and 4-OH biphenyls, purchased from K and K Laboratories, Inc., were recrystallized from ethanol. Pentobarbital was a product of Abbott Labs. Metyrapone was a gift of Dr. J. J. Chart of Ciba-Geigy Corp. RESULTS
The effect of phenobarbital stimulation on liver weight and on the contents of some hepatic microsomal constituents in selenium-deficient and control rats is shown in Table II. Liver weight per 100 g body wt was greater in selenium-deficient rats than controls whether treated with phenobarbital or not, but the relative weight increase with phenobarbital stimuTABLE EFFECT
Diet
OF PHENOBARBITAL
Treatment
ON LIVER
Liver
weight
Cytochrome
g/100 g body n 0 Se 0 Se 0.5 Se 0.5 Se
Saline Phenobarbital Saline Phenobarbital
n* b* c Values
having
12 13 10 10 same
wt
+ SD 2.9 4.4 2.5 3.7
superscript
II
WEIGHT AND MICROSOMAL AND CONTROL RATS
2 ++ 2
different
Cytochrome
nmles/mg protein n
0.2” 0.36 0.2” o.3b
b,
CONSTITUENTS
12 13 10 10
P-450
NADPHcytochrome reductase
nmoles/mg protein
+ SD 0.39 0.51 0.40 0.55
IN SELENIUM-DEFICIENT
k ‘+ ?I
0.04 0.05 0.05 0.05
c
nmoles/mg proteimmin
72
2 SD
n
12 13 10 10
0.83 k 0.12 1.4 ? 0.2’ 0.88 2 0.14 2.2 ? 0.3c
10 11 10 10
P < 0.001; only 0 Se and 0.5 Se compared.
‘- SD 72 160 83 179
* 2 I k
11 18 18 31
SELENIUM
DEFICIENCY
AND
CYTOCHROME
TABLE EFFECT
OF %METRYLCHOLANTHRENE
ON LIVER
Diet
Treatment
Liver
AND
weight
0 Se 0 Se 0.5 Se 0.5 Se n Values
Corn 3-MC Corn 3-MC having
oil oil
same
4 5 4 4
2.9 3.7 2.6 3.2
superscript
‘, + k +
AND
MICROSOMAL
CONTROL
Cytochrome
n
b,
IN
Cyt;t&me
nmoles/mg protein k SD
n
0.39 0.55 0.38 0.61
4 5 4 4
-c + 2 k
CONSTITUENTS
SELENIUM-
RATS
0.07 0.07 0.03 0.11
NADPHcytochrome c reductase n
nmoles/mg proii%
0.83 1.7 0.86 1.7
+
0.2 0.2” 0.1 0.3”
4 5 4 4
different
P < 0.01; only 0 Se and 0.5 Se compared.
chrome P-448, cytochrome bg, or NADPHcytochrome c reductase to 3-methylcholanthrene administration. The influence of selenium status on several cytochrome P-450 dependent drug metabolizing systems was studied. Table IV shows the effect of phenobarbital treatment. Selenium deficiency had no significant effect on ethylmorphine demethylase activity in untreated animals. However, with phenobarbital treatment, controls had 1.8 times the activity of selenium-deficient animals. This corresponds closely with the effect observed on cytochrome P450. Unlike ethylmorphine demethylase activity, the induction of 2- and 4-hydroxylation of biphenyl was unaffected by selenium status. Likewise, pentobarbital sleeping time was shortened to the same extent in selenium-deficient and control rats by the prior administration of phenobarbital. Treatment with 3-methylcholanthrene caused a decrease of ethylmorphine demethylase activity and an increase in biphenyl hydroxylase activity. These changes were unaffected by selenium status as is shown in Table V. In order to define further the impairment of cytochrome P-450 response to phenobarbital administration in selenium deliciency, a time course experiment was carried out. In Fig. 1A liver weight per 100 g body wt is seen to have increased steadily during the 4 days of phenobarbital treatment. Cytochrome P-450 increased less in livers of selenium-deficient rats than in livers of selenium-adequate control rats
127
SYSTEM
III
WEIGHT
DEFICIENT
P-450
f 0.22 IO.2 k 0.04 i- 0.2
4 5 4 4
nmoles/mg protein/ min k SD 58 66 79 69
of- 14 + 14 f 23 r 12
(Fig. 1B). Ethylmorphine demethylase (Fig. 1C) induction was also less in selenium-deficient livers. Since the induction of cytochrome P-450 by phenobarbital was impaired in selenium deficiency, somequalitative studies of the hemoprotein were done. Table VI shows that the percentage of the carbon monoxide-reactive cytochrome P-450 which bound metyrapone was the same in selenium deficiency and adequacy in both saline- and phenobarbital-injected animals. Also, the ratios of the 455 nm peak to the 430 nm peak of the ethyl isocyanide binding spectra were unaffected by selenium status. These studies failed to demonstrate that the cytochrome P-450 induced by phenobarbital in selenium-deficient rats is qualitatively different from that in similarly treated controls. It was considered that selenium could be protecting the phenobarbital-induced cytochrome P-450 from destruction, perhaps through its role in glutathione peroxidase. Therefore, cytochrome P-450 was measured 2 and 24 h following microsomal preparation from two selenium-deficient and two control rats which had received phenobarbital for 3 days. Microsomes were stored at 4°C under air. In each case the cytochrome P-450 content of the microsomes changed by less than 5% indicating that virtually no cytochrome P-450 was destroyed between 2 and 24 h under these conditions. In addition, experiments were done in which livers of selenium-deficient animals treated for 4 days with phenobar-
128
BURK
AND
MASTERS
TABLE EFFECT
OF PHENOBARBITAL
Diet
Treatment
ON SOME
Ethylmorphine demethylase activity n
0 Se 0 Se 0.5 Se 0.5 Se
Saline Phenobarbital Saline Phenobarbital
a Different b Negligible
10 11 10 10
P < 0.001; activity.
only
Biphenyl
nmoles HCHO/mg proteinlmin + SD 2.5 6.6 3.2 11.7
2 r k +
n
Diet
0.6 1.6O 1.0 1.6”
3 6 3 5
’ Negligible
Pentobarbital sleeping time (min) n
AND
Sleeping time k SD
Induction time k SD
4-OH
-b 0.38 2 0.08 -b 0.37 -c 0.06
0.89 4.72 0.84 4.95
f r k +
0.11 0.62 0.08 1.45
10 10 8 9
3.6 5.1 3.1 4.8
k + f ”
0.9 1.2 0.5 0.8
139 15.0 111 14.5
? 2 -+ 2
28 4.3 18 2.4
compared.
V
OF %METHYLCHOLANTHRENE ON SOME PARAMETERS OF DRUG METABOLISM IN SELENIUM-DEFICIENT AND CONTROL RATS
Treatment
Corn 3-MC Corn 3-MC
IN SELENIUM-DEFICIENT
protein/ k SD
Z-OH
0 Se and 0.5 Se values
Ethylmorphine demethylase activity n
0 Se 0 Se 0.5 Se 0.5 Se
METABOLISM
hydroxylase activity
nmoles/mg min
TABLE EFFECT
IV
PARAMETERS OF DRUG CONTROL RATS
oil oil
4 5 4 4
Biphenyl
nmoles HCHO/mg protein/min k SD 2.3 1.7 3.1 2.4
k ? k ?
1.2 0.6 0.8 0.7
n
hydroxylase activity
nmoles/mg
protein/min 2 SD
2-OH 3 4 3 3
-a 1.2 + 0.3 -a 1.2 ? 0.1
4-OH 0.78 2.3 0.83 2.3
2 t + L
0.23 0.6 0.13 0.2
activity.
bital were split and one part was homogenized with 250 mg dl-a-tocopheryl acetate added per 8 g of liver. The other part was homogenized in the conventional manner. In one experiment, microsomal cytochrome P-450 content was 1.17 and 1.28 mnoles/mg protein with and without added vitamin E, respectively, and in another experiment the values were 1.56 and 1.45. These results do not support the hypothesis that selenium protects cytochrome P-450 from destruction. DISCUSSION
An effect of selenium status on the hepatic microsomal cytochrome P-450 system has been demonstrated. The effect-a depressed cytochrome P-450 content-seems to be specific since it was found in pheno-
barbital-treated rats but not in saline- or in 3-methylchdlanthrene-treated rats. No effect of selenium status on the specific contents of NADPH-cytochrome c reductase or cytochrome b, was observed under any conditions. This study confirms a preliminary report by Burk, Mackinnon, and Simon (6) of impairment of cytochrome P-450 induction by phenobarbital in selenium-deficiency but does not agree with the report that cytochrome b, induction was also impaired. Basal cytochrome b5 levels were higher in that study due to the different method of microsomal preparation and possibly to the somewhat different diet, but there is no readily apparent explanation for the different response to phenobarbital. Caygill et al. (14) found no impairment
SELENIUM
261
DEFICIENCY
AND
B
E
0.6
E
2 $
5 e e
E
p.
1 e-4
Se-defment
o--C,
Se-adequate
CYTOCHROME
P-450
nium levels are still appreciable after 2 wk of a selenium-deficient regimen (15). More severe depletion is probably necessary for the effect reported here. It was expected that parameters of cytochrome P-450-dependent drug metabolism would be affected by selenium deficiency. The induction of ethylmorphine demethylase activity was decreased in selenium deficiency; but surprisingly, biphenyl hydroxylase activity and pentobarbital sleeping time were induced by phenobarbital to the same extent in the selenium-deficient rats as in selenium-adequate rats in spite of decreased induction of cytochrome P-450 in the selenium-deficient rats. The explanation for these findings is not known. Siami et al. (16) found no impairment of phenobarbital induction of aminopyrine demethylase and hexobarbital oxidase activity in hepatic microsomes of selenium-deflcient female rats. These investigators did not report cytochrome P-450 levels or ethylmorphine demethylase activity. An important difference in protocol between their study and the present one was that they administered phenobarbital intermittently in the drinking water. The nature of the involvement of selenium with cytochrome P-450 is unknown. It has been reported that cytochrome P-450 TABLE
c
129
SYSTEM
VI
EFFECT OF PHENOBARBITAL ON METYRAP~NE AND ETHYL ISOCYANIDE BINDING SPECTRA OF CYTOCHROME P-450 IN SELENIUM-DEFICIENT AND CONTROL RATS OJ, 0
24 hours
48
of phenobarbital
72
t 96
Diet
Treatment
cytochrome P-450 measured with metyrapone
treatment
FIG. 1. Effect of selenium deficiency on phenobarbital induction of liver weight (A), microsomal cytochrome P-450 content (B), and ethylmorphine demethylase activity (0. Each point represents one rat. Rats fed the diet containing corn oil were used. They were injected with phenobarbital at 24-h intervals until 24 h before killing. Thus, the animals killed at 0 time never received phenobarbital, those killed at 24 h received one injection of it at 0 time, etc. All rats were fasted 24 h before killing.
of cytochrome P-450 induction by phenobarbital in rats fed selenium-deficient diets for 2 wk. That does not necessarily conflict with the present findings since tissue sele-
Metyranone c-ytochrome P-450/ carbon monoxide P-450
nmoles/ mg protein” 0 Se 0 Se 0.5 Se 0.5 Se
Saline Phenobarbital Saline Phenobarbital
(443505 x$ in ethyl isocyanide binding spectrumD (pH 7.4)
0.10 0.72
0.11 0.48
0.53 0.60
0.13 0.96
0.15 0.46
0.61 0.70
I
a Average
Ratio of veak heights
of two animals.
I
130
BURK
AND
heme is present in approximately 40-fold excess over selenium in rat liver microsomes (6), and this seems to preclude a role for selenium as part of cytochrome P450.Glutathione peroxidase activity is severely depressed in livers from seleniumdeficient rats (17), and the possibility exists that this enzyme might protect cytochrome P-450 from lipid peroxide-induced damage. This argument is made somewhat tenuous by the finding of unimpaired induction of cytochrome P-448 by 3-methylcholanthrene in selenium-deficient rats. It would have to be postulated that the glutathione peroxidase protects only a species of cytochrome P-450 induced by phenobarbital. Also, that no decrease of cytochrome P-450 occurs during storage between 2 and 24 h after microsomal preparation further weakens this hypothesis. Finally, the inclusion of the antioxidant a-tocopherol in the homogenizing solution failed to increase measurable cytochrome P-450. Thus, it seems unlikely that the effect of selenium in this system is mediated by glutathione peroxidase. The possibility that heme synthesis is depressed by selenium deficiency was not examined directly in this work. However, the normal induction of cytochrome bs and cytochrome P-448 reported here suggest that heme synthesis is not impaired in selenium deficiency. Nevertheless, direct investigation of this problem is needed. Several forms of cytochrome P-450 have been shown to be present in microsomes (18). Evidence was sought that selenium deficiency causes a selective decrease in some form of cytochrome P-450 by the qualitative studies of metyrapone binding and the ethyl isocyanide binding spectrum (Table V). Similar results were obtained in selenium-deficient and selenium-adequate phenobarbital-treated rats, failing to demonstrate any effect on a specific form of cytochrome P-450. However, the rather selective effect of selenium deficiency on a portion of the phenobarbital-induced cytochrome P-450 and the ethylmorphine demethylase activity but not biphenyl hydroxylase activity tend to support this hypothesis and this possibility must remain open. Diplock and his co-workers (19) have hy-
MASTERS
pothesized that selenium is part of a nonheme iron protein in the microsomes which is analogous to adrenodoxin in the adrenal mitochondrial cytochrome P-450 system. The existence of such a protein which might transfer electrons from NADPH-cytochrome c reductase to cytochrome P-450 has been postulated previously based on the mitochondrial system (20), but little evidence for a nonheme iron protein in the microsomes other than ferritin has been found (21-24). The presence of an analogous iron-selenium protein should have been easily detected in the microsomes by electron paramagnetic resonance studies (25) but no such signal has been found. However, part of Diplock’s hypothesis is that the iron-selenium protein is oxidant-labile and these studies did not use antioxidants to protect such a protein. Stronger evidence against the existence of this protein are recent reconstitution studies using highly purified NADPH-cytochrome c reductase and cytochrome P-450 which suggest that such a factor is not required for at least some drug metabolism to take place (26, 27). The present studies shed little light on this possible role of selenium, and it is difficult to understand how a decrease in such a nonheme iron-selenium-containing protein could lead to the findings reported here. The possibility remains that a specific protein induced by barbiturates in the liver endoplasmic reticulum requires the presence of selenium and is responsible for the specific induction of certain oxidative N-demethylation activities and the cytochrome(s) P450 required for them. ACKNOWLEDGMENTS The authors are grateful to Dr. Jurgen Werringloer for determining the metyrapone-binding cytochrome P-450 and the ethyl isocyanide binding spectra, to Dr. M. Danny Burke for the biphenyl hydroxylase determinations, to Mrs. Barbara Radighieri for the ethylmorphine demethylase determinations, and to Mrs. Elizabeth L. Isaacson for the NADPHcytochrome c reductase assays. REFERENCES 1. OH, S. H., GANTHER, H. E., AND HOEKSTRA, W. G. (1974) Biochemistry 13, 1825. 2. WHANGER, P. D., PEDEREIEN, N. D., AND WEsWIG, P. H. (1973) Biochem. Biophys. Res. Cornmun. 53, 1031.
SELENIUM
DEFICIENCY
AND
3. SHUM, A. C., AND MURPHY, J. C. (1972)5. Bacteria?. 110, 447. 4. TURNER, D. C., AND STADTMAN, T. C. (1973) Arch. Biochem. Biophys. 154, 366. 5. STADTMAN, T. C. (1974) Science 183, 915. 6. BURK, R. F., MACKINNON, A. M., AND SIMON, F. R. (1974) B&hem. Biophys. Res. Commun. 56,431. 7. PAGLIA, D. E., AND VALENTINE, W. N. (1967) J. Lab. Clin. Med. 70, 1.58. 8. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M. (1949) J. Biol. Chem. 177, 751. 9. OMURA, T., AND SATO, R. (1964) J. Biol. Chem. 239, 2370. 10. MASTERS, B. S. S., WILLIAMS, C. H., JR., AND KAMIN, H. (1967) in Methods in Enzymology (Estabrook, R. W. and Pullman, M., eds.), Vol. 10, p. 565, Academic Press, New York. 11. NASH, T. (1953) Biochem. J. 55,416. 12. CREAVEN, P. J., PARK, D. V., AND WILLIAMS, R. T. (1965) Biochem J. 96, 879. 13. SLADEK, N. E., AND MANNERING, G. J. (1966) Biochem. Biophys. Res. Common. 24, 668. 14. CAYGILL, C. P. J., DIPLOCK, A. T., AND JEFFERY, E. H. (1973) Biochem. J. 136, 851. 15. BURK, R. F., WITNEY, R., FRANK, H., ANDPEARSON, W. N. (1968) J. Nutr. 95, 420. 16. SIAMI, G., SCHULERT, A. R., AND NEAL, R. A. (1972) J. Nutr. 102, 857.
CYTOCHROME
P-450
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17. HAFEMAN, D. G., SUNDE, R. A., AND HOEKSTRA, W. G. (1974) J. Nutr. 104, 580. 18. HILDEBRANDT, A., REMMER, H., AND ESTABROOK, H. W. (1969) Biochem. Biophys. Res. Commun. 30, 607. 19. DIPLOCK, A. T., AND LUCY, J. A. (1973) FEBS Lett. 29, 205. 20. ESTABROOK, R. W. (1971) in Handbook of Experimental Pharmacology, (Brodie, B. B. and Gillette, J. R., eds.), Vol. 28, No. 2, p. 264, Springer Verlag, Berlin. 21. MONTGOMERY, M. R., CLARK, C., AND HOLTZMAN, J. L. (1974) Arch. Biochem. Biophys. 160, 113. 22. MIYAKE, Y., MASON, H. S., AND LANDGRAF, W. (1967) J. Biol. Chem. 242, 393. 23. MASTERS, B. S. S., BARON, J., TAYLOR, W. E., ANDISAACSON, E. L. (1971) J. Biol. Chem. 246, 4143. 24. BARON, J., TAYLOR, W. E., AND MASTERS, B. S. S. (1972)Arch. Biochem. Biophys. 150,105. 25. TSIBRIS, J. C. M., NAMTVEDT, M. J., ANDGUNSALUS, I. C. (1968) Biochem. Biophys. Res. Commun. 30, 323. 26. VAN DER HOEVEN, T. A., HAUGEN, D. A., AND COON, M. J. (1974) Biochem. Biophys. Res. Commun. 60, 569. 27. VERMILION, J. L. AND COON, M. J. (1974) Biochem. Biophys. Res. Commun. 60,1315.