Selenium antagonism of cadmium-induced inhibition of hepatic drug metabolism in the male rat

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

58,

57-66 (1981)

Selenium Antagonism of Cadmium-Induced Inhibition Drug Metabolism in the Male Rat JOHNNIE Department

of Pharmacology Purdue

Received

L. EARLY,

JR.’ AND R. CRAIG SCHNELL~

and Toxicology, School of Pharmacy and Pharmacal University, West Lafayette, Indiana 47907

August

of Hepatic

29, 1980;

accepted

November

Sciences,

10, 1980

Selenium Antagonism of Cadmium-Induced Inhibition of Hepatic Drug Metabolism in the Male Rat. EARLY, J. L., JR., AND SCHNELL, R. C. (1981). Toxicol. Appl. Pharmacol. S&57-66. Experiments were undertaken to examine the effect of selenium, an essential trace element, on cadmium-induced inhibition of drug metabolism in male, Sprague-Dawley derived rats. Prior administration of sodium selenite (1.6 mg Se/kg, ip) blocked the cadmiuminduced (0.84 mg Cd/kg, ip) prolongation of hexobarbital-induced hypnosis and inhibition of hepatic microsomal biotransformation of ethylmorphine or aniline. Selenium also blocked cadmium-induced reduction in microsomal cytochrome P-450 content and the microsomal binding of both ethylmorphine and aniline. However, pretreatment of rats with selenium did not prevent the inhibitory effect of cadmium (10-O to 10e3 M) added in vitro on either ethylmorphine or aniline biotransformation. In addition, the reduction in biotransformation of both substrates following in vivo cadmium administration was not reversed following the in vitro administration of selenium but, in fact, selenium produced further concentrationdependent decreases in drug metabolism. In additional in vitro experiments it was found that the inhibition in drug metabolism induced by in vitro additions of cadmium is not affected by similar additions of selenium when added to the incubation vessel either before or after the cadmium. Thus, for selenium to prevent the cadmium-induced inhibition of hepatic drug metabolism requires in vivo administration of selenium.

Cadmium has been shown to induce a wide variety of toxic manifestations (reviewed by Friberg et al., 1974). Many of these toxic effects have been elicited in laboratory animals following parenteral or oral administration of this heavy metal. Recently, considerable interest has been generated in assessing the toxicity not of a single toxic entity but that of various combinations of toxicants. Also, of interest has been the study of alterations in heavy metal toxicity

by concomitant administration of other essential trace elements. For example, cadmium toxicity has been increased by dietary deficiencies of essential minerals such as calcium, copper, iron, or zinc (Fox, 1974). There is also considerable evidence that the essential trace element, selenium, interacts with a number of toxic heavy metals such as lead, mercury, methylmercury, silver, thallium, arsenic, and cadmium, and renders these substances less toxic (Nordberg, 1976). With respect to cadmium toxicity, selenium has been shown to protect against lethality in rats (Mason et al., 1964) and in mice (Gunn et al., 1%8), against testicular necrosis (Mason et al., 1964; Gunn et al., 1968), against pancreatic changes and alterations in gluconeogenic

1 Present address: Department of Pharmacology, School of Pharmacy, Florida A&M University, Tall% hassee, Florida 32307. * Send correspondence to: Department of Pharmacodynamics and Toxicology, College of Pharmacy, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebraska 68105. 57

0041-008X/81/040057-10$02.00/0 Copyright All rights

6 1981 by Academic Press, Inc. of reproduction in any form reserved.

58

EARLY AND SCHNELL

enzymes (Merali and Singhal, 1973, and against cadmium-induced reductions in plasma and testicular glutathione peroxidase activity in the rat (Omaye and Tappel, 1975; Prohaska et al., 1977). Previous reports from our laboratory have demonstrated that cadmium treatment can potentiate drug response and inhibit hepatic microsomal drug metabolism in male rats (Hadley et al., 1974; Johnston et al., 1975; Pence et al., 1977; Schnell, 1978). This inhibitory effect of cadmium on this monooxygenase system is primarily mediated by a decrease in the concentration of hepatic microsomal cytochrome P-450 (Krasny and Holbrook, 1977; Means et al., 1979; Means and Schnell, 1979). Studies by Caygill et al. (1971) and Burk et al. (1974) have attempted to elucidate the role of selenium on this hepatic monooxygenase system. Burk and Masters (1975) reported that selenium deficiences did not alter the activity of the monooxygenase enzymes of the levels of cytochromes P-450 orb, or NADPH-cytochrome reductase activity. However, these investigators did report that the ability of phenobarbital to induce the activity of this enzyme system was impaired in selenium-deficient rats. The objective of this study was to determine if the inhibition of hepatic drug metabolism by cadmium could be prevented by selenium. To accomplish this, experiments were conducted to examine the interaction of both elements when administered in vivo as well as to examine this interaction when both elements were added in vitro to microsomal fractions. METHODS Animals. Male Sprague-Dawley derived rats (Laboratory Supply Co., Indianapolis, IN) weighing 225-265 g were used throughout these studies. The animals were housed in community stainless-steel cages for at least 1 week prior to use and allowed free access to food (Wayne Lab Blox, Allied Mills, Chicago, IL) and tap water. Chemicals. Cadmium acetate (Fisher Scientific Company, Fairlawn, NJ) or sodium selenite (Alpha Products, Danver, MA) were dissolved in double

distilled water prior to intraperitoneal injection. Unless otherwise noted, selenium was administered in the lower right quadrant immediately prior to the administration of cadmium in the opposite quadrant. Control animals received normal saline. Pharmacological response. The duration of hypnosis induced by hexobarbital(lO0 mg/kg, ip) was measured as the time elapsing from the loss of the righting reflex until the animal could successfully right itself from a supine position twice within 30 sec. Microsomal drug metabolism. Microsomes were prepared by homogenizing a liver mince in 3 volumes of cold (0-4°C) 0.1 M Tris-KC1 (PH 7.5) buffer using a Potter-Elvehjem homogenizer with a Teflon pestle. The homogenate was centrifuged at 10,000 rpm (11,73Og,,,; type 30 rotor) for 20 min at 4°C in a Beckman Model L5-65 ultracentrifuge. The pellet was discarded and the supematant was centrifuged at 34,ooO rpm ( 105,000g,,,aX; type 40 rotor) for 60 min at 4°C. The supematant was discarded and the microsomal pellet was resuspended in the original volume of Tris-KC1 buffer and recentrifuged as before for 60 min. The resulting pellet was resuspended in Tris-HCl buffer (pH 7.5) such that 1.O ml of microsomes contained the equivalent of 250 mg of whole liver. Ethylmorphine (20 pmol) or aniline (5 pmol) was incubated in 25-ml Erlenmeyer flasks with 1.0 ml of microsomal suspension in mixtures containing 4 pm01 of NADP, 30 pmol of glucose-6-phosphate, 50 pmol of magnesium chloride, and 2 IU of glucose-dphosphate dehydrogenase and 45 pmol of semicarbazide (ethylmorphine-N-demethylase) in Tris-HCI buffer to give a total volume of 5.0 ml (4.0 ml for aniline hydroxylase). Incubations were carried out at 37°C under oxygencarbon dioxide (95%:5%) with shaking (120 oscillations/min) in a Dubnoff metabolic shaking incubator for periods of 15 (ethylmorphine) or 20 (aniline) min. Ethylmorphine-N-demethylase was determined by measuring the formation of formaldehyde following the procedure of Nash (1953) modified by Anders and Mannering (1966). Aniline hydroxylase was determined by measuring the formation of p-aminophenol according to the procedure of Imai et al. (1966). Cytochrome P-4.50. For the measurement of cytochrome P-450, livers were perfused in situ with 1.15% KC1 and homogenized initially in 1.875 volumes of buffer prior to the described centrifugation procedure. The content of cytochrome P-450 was determined from the carbon monoxide difference spectrum of dithionitetreated microsomes assuming a value of 91 mM-’ cm-’ for the molar extinction coefficient between 450 and 4% nM (Omura and Sato, 1964). Substrate-induced binding spectra. The binding of ethylmorphine or aniline to microsomes was determined from difference spectra described by Remmer et al. (1966). Protein determination. The protein content of microsomes was determined by the method of Lowry ef al.

SELENIUM

PROTECTION

AGAINST

59

CADMIUM

C

-I-

I

”

Normal

Saline

Se 1.6 ma/kg

Cd 0.04

Se 1.6 mg/kg” Cd 0.64 mg/kg

mg/kg

cd 0.6;4mg/kgY

Se ltn;g/kg

Se 1.6 mg/kg

Cd 0.04

-%

mg/kg

FIG. 1. Effect of selenium on cadmium-induced prolongation of hexoharhital-induced hypnosis in male rats. Groups of male rats (225-265 g) received normal saline, sodium selenite (1.6 mg Se/kg, ip), and/or cadmium acetate (0.84 mg Cd/kg, ip). Seventy-two hours later animals were challenged with hexobarbital-Na (100 mg/kg, ip) and the duration of hypnosis was determined. Values represent mean t SEM for four to seven rats. Data were analyzed statistically by one-way ANOVA followed by application of Dunnett’s test. “Selenium was administered in the lower right quadrant immediately preceding cadmium in the opposite quadrant. “Cadmium and selenium were administered in the lower right quadrant at intervals of 7 hr. cSignificantly different from control (p < 0.05). (1951) using crystalline the standard. Statistical

analysis.

bovine serum albumin

as

The data were analyzed staTABLE

tistically by use of analysis of variance (ANOVA) followed by the application of Dunnett’s test for multiple comparisons with a control (Dunnett, 1955) 1

EFFECT ON SELENIUM ON CADMIUM-INDUCED INHIBITION OF HEPATIC MICROSOMAL N-DEMETHYLASE AND ANILINE HYDROXYLASE ACTIVITY” Cadmium (0.84 mg/kg)

Se 1.6 mg/kg Cd 0.84 mg/kg

236 2 27 (77)

98 ?z 18” (32)

213 2 296 (70)

35 + 2 (76)

18 ” 3h (39)

36 k 5

Selenium Measurement

Saline

Ethylmorphine N-demethylase (nmol formaldehyde formed/mg microsomal protein05 min)

306 k 43 (100)

Aniline hydroxylase (nmol p-aminophenol formed/mg microsomal protein/20 min)

45 A 3 (1W

ETHYLMORPHINE

(1.6m&d

(81)

n Male albino rats (225-265 g) received normal saline, sodium selenite (1.6 mg Se/kg, ip) and/or cadmium acetate (0.84 mg Cd/kg). Seventy-two hours later animals were killed and hepatic microsomal metabolism quantitated. Values represent mean * SEM for six animals. Values in parentheses represent percentage control. * Significantly different from respective control (p < 0.05).

60

EARLY

TABLE

AND

2

EFFECT OF SELENIUM ADMINISTRATION ON CADMIUM-INDUCED ALTERATIONS IN HEPATIC MICROSOME-SUBSTRATE BINDING SPECTRA IN MALE RATS”

Treatment

Cytochrome P-450 content 0miol cytochrome P-45Oimg microsomal protein)

Magnitude of spectral binding COD,-, A Abs x 1091 mg microsomal protein) Ethylmorphine

Aniline

Normal saline

0.87 + 0.04 1100)

13 + 2 (N-w

23 -+ 1 (W

Se 1.6 mg/kg

0.87 f 0.09 ( 100)

12 f I (92)

20 + 2 (87)

Cd 0.84 mdkg

0.65 2 OM” (75)

6 f Lo (46)

13 -r 1” (57)

Se 1.6 mglkg Cd 0.84 m&g

0.95 t 0.04 (109)

11 2 2 (85)

20 t 2 (87)

a Microsomes were derived from male albino rats (225-265 g) treated as indicated. Animals were killed 72 hr following treatment and microsomal cytochrome P-450 content and the magnitude of spectral binding ofethyhnorphine and aniline determined. Data represent mean ? SEM for five animals. Values in parentheses represent percentage control. b Significantly different from control (P < 0.05).

or Duncan’s new multiple range test for determining significant differences between means (Steel and Torrie, 1960). The acceptable level of significance was established at p < 0.05.

RESULTS Effect of Selenium on Cadmium-Induced Prolongation of Hexobarbital Hypnosis in Male Rats

In the first series of experiments, the effect of cadmium and selenium on the duration of hexobarbital induced hypnosis was studied. Selenium (1.6 mg/kg, ip) was administered immediately prior to cadmium (0.84 mg/kg, ip) in the opposite quadrant or in the same quadrant either 7 hr before or after cadmium administration. The data are presented in Fig. 1. As expected, cadmium treatment alone significantly (p < 0.05) increased the duration of hexobarbital-induced hypnosis while selenium administered alone did not alter the hypnotic response to hexobarbital. When selenium was administered immediately prior to, or 7 hr before or after the cad-

SCHNELL

mium, prolongation longer observed.

of hypnosis

was no

Effect of Selenium on Cadmium-Induced Inhibition of Hepatic Microsomal Drug Metabolism in Male Rats

The in vitro metabolism of both ethylmorphine and aniline was determined 72 hr following treatment with either selenium (1.6 mg/kg), cadmium (0.84 mg/kg), or a combination in which selenium was administered just prior to cadmium. The results are presented in Table 1. Cadmium treatment significantly reduced the rate of metabolism of both substrates. Selenium alone did not alter the rate of ethylmorphine metabolism but did produce a slight but significant reduction in aniline metabolism. Simultaneous administration of both elements blocked the inhibitory effect of cadmium on the microsomal metabolism of both substrates. Effect of Selenium on Cadmium-Induced Reduction of Hepatic Microsomal Cytochrome P-450 Content and Hepatic Microsomal Substrate Binding in Male Rats

Using a similar treatment regimen, the effect of the selenium-cadmium interaction on microsomal cytochrome P-450 levels and substrate binding was examined. As can be seen from the data in Table 2, cadmium alone decreased both the microsomal content of cytochrome P-450 and the microsomal binding of both substrates. Selenium alone did not alter any of these parameters. Pretreatment with selenium prevented the cadmium-induced reductions in cytochrome P-450 content and microsomal binding of either ethylmorphine or aniline. Thus, these data are consistent and supportive of the previous data concerned with the effects of the cadmium-selenium interaction on the hepatic microsomal drug-metabolizing enzyme activity.

SELENIUM 400

r

AGAINST

CADMIUM

.; 300 2

61

ANILINE

ETHYLMORPHINE

ti ti

T

-2

E E 5 loo '"E H{ 2000 I_ $E 400

PROTECTION

00

I

lxlo- IxlC

2 B y 300 1

I I E = 200 100 0

CADMIUM

CONCENTRATION

(MI 0 Normal

69%

l6mg/kg

FIG. 2. Effect of cadmium added in vitro on ethylmorphine N-demethylase and aniline hydroxylase activity in microsomes derived from selenium-treated male rats. Male rats (225-265 g) received normal saline or 1.6 mg Se/kg. Seventy-two hours following treatment animals were killed and in vitro ethylmorphine N-demethylase and aniline hydroxylase activity were determined. Each value represents the mean f SEM five to six animals. *Significantly different from control value (p < 0.05).

Effect of Cadmium Added in Vitro on Drug Metabolizing Activity in Microsomes Derived from Selenium-Treated Male Rats Concentration-dependent inhibition of the metabolism of various substrates has been reported following the in vitro addition of cadmium to hepatic microsomes derived from untreated male rats (Hadley et al., 1974; Means et al., 1979). The purpose of this experiment was to determine if selenium pretreatment could render the microsomes nonresponsive to cadmium inhibition. As shown in Fig. 2, concentration-dependent decreases in the rate of ethylmorphine metabolism were recorded with significant reductions occurring at cadmium concentrations of 10e4 and 10e3 M in microsomes derived from both control and selenium-treated rats. Concentration-

dependent decreases were also observed in the metabolism of aniline in cadmium concentrations of 10e5 M and greater. Thus, these results indicate that the microsomal enzymes derived from selenium (in viva)-treated rats remain susceptible to the inhibitory action of cadmium added in vitro. Effect of Selenium Added in Vitro on Drug Metabolism by Microsomes from Cadmium-Treated Male Rats Since selenium (1.6 n&kg) administration did not alter the inhibitory action of cadmium added in vitro, it was of interest to determine if selenium added in vitro would alter the inhibitory effect of in vivo cadmium (0.84 mg/kg) treatment. Microsomes from control and cadmium-treated

62

EARLY AND SCHNELL ETHYLMORPHINE

l-7

ANILINE

50

v a

a

a

l-k * lxld

lxld

a

0.0 lxl@ IXld 1x164 SELENIUM CONCENTRATION

lxl@ (MI

SELENIUM Saline

61 Cd 0.84

CONCENTRATION

(MI

mg/kg

FIG. 3. Effect of selenium added in vitro on ethylmorphine N-demethylase and aniline hydroxylase activity in microsomes derived from cadmium-treated male rats. Male rats (225-265 g) received normal saline or cadmium (0.84 mg Cd/kg). Seventy-two hours following treatment, animals were killed and in vitro ethylmorphine N-demethylase and aniline hydroxylase activity were determined. Each value represents the mean 2 SEM for six animals. “Significantly different from the respective control value (p < 0.05).

rats were exposed to varying concentrations of selenium (10d6 to lop3 M) and the effect on drug metabolism was quantitated. A significant (p < 0.05) reduction in both ethylmorphine and aniline metabolism was observed in microsomes derived from cadmium-treated rats in the absence of selenium (Fig. 3). The in vitro addition of selenium resulted in further concentrationdependent decreases in the rate of ethylmorphine and aniline metabolism by microsomes from both control and cadmiumtreated rats. Significant (p < 0.05) reductions in drug metabolism were observed at selenium concentrations of 10m4and 10e3 M (control), and at 10e4 M (cadmium-treated). It is of interest to note that although the rate of ethylmorphine metabolism is reduced 49% by cadmium treatment, the mag-

nitude of the reductions in drug metabolism by in vitro selenium is on the same order in microsomes derived from control and cadmium-treated rats. Similarly, concentrationdependent decreases in aniline metabolism were comparable in microsomes derived from control and cadmium-treated rats. Thus, in vitro selenium does not affect cadmium-induced inhibition of drug metabolism, and conversely, cadmium administration does not alter the inhibitory effect of in vitro selenium. Effect of Selenium and Cadmium Added in Vitro on Hepatic Microsomal Drug Metabolism in Male Rats Since selenium alter the inhibitory

administration did not effect of in vitro cad-

SELENIUM

PROTECTION

mium, and conversely, in vitro selenium did not alter the inhibitory effect of cadmium administration on drug metabolism, experiments were conducted to examine the interaction of these elements in a more direct manner. In these experiments both elements were added in vitro to microsomal incubates. The first element was added to the incubation medium followed (as indicated by the order of listing) by swirling and a period of 5 min elapsed prior to the introduction of the second element. The enzymatic reactions were then initiated by addition of the appropriate substrate. Selenium was added in concentrations of 10V6 to 10m3M, and cadmium in a concentration Of

1o-4

M.

Table 3 summarizes the data in which selenium was added to the incubation vessels before cadmium. Cadmium ( lop4 M) significantly (p < 0.05) reduced the rate of ethylmorphine and aniline metabolism. Sig-

TABLE EFFECT

OF

in Vitro ON YLASE .AND MICROSOMES RATS”

SELENIUM

HEPATK ANILINE

Selenium or cadmium concentration (M) 0.0 (Control) Cd lo-’ Se 10-O Se 10-j Se 10m4 Se 10m3 Se IO-Wd lo-’ Se lo-Wd 10m4 Se 10-“/Cd lo-* Se IO-YCd lo-*

AND

FROM

CADMIUM

+ + t ? k 5 + + + +

43 18” 37 34 28’ 21” 196 16b 14b 14*

ADDED

N-DEMETHACTIVITY

UNTREATED

Ethylmorphine (nmol HCHO formed/mg microsomal protein05 min) 277 109 250 227 180 141 105 108 100 92

63

CADMIUM

TABLE EFFECT OF CADMIUM AND ON HEPATIC ETHYLMORPHINE ANILINE

HYDROXYLASE

DERIVED

FROM

(M)

0.0 Cd Se Se Se Se Cd Cd Cd Cd

SELENIUM ADDEDin N-DEMETHYLASE ACTIVITY

UNTREATED

Cadmium or selenium concentration (Control) 1O-4 10e6 10m5 1O-4 10e3 lo-We lo-” lO%Se 10m5 IO-We 10m4 lo-We 10m3

4

MALE

Ethylmorphine (nmol HCHO formed/mg microsomal protein/15 min) 277 109 250 227 180 141 83 84 83 79

2 t r + k 2 k t t k

43 18 31 34 28b 21* ll* 13* 11* 12*

IN

Vitro AND

MICROSOMES

RATS”

Aniline (nmol p-aminophenol formed/ mg microsomal protein/20 min) 44 19 43 43 35 30 18 18 19 21

2 1 2 1 2 2 2 1 f 10 + lb t lb zk 1” k I* k lb

n Ethylmorphine N-demethylase and aniline hydroxylase activities were determined in microsomes derived from untreated male rats (225-265 g). Values represent mean ? SEM for four animals. * Significantly different from control value (p < 0.05).

3

ETHYLMORPHINE HYDROXYLASE

DERIVED

AGAINST

IN

MALE

Aniline (nmol p-aminophenol formed/ mg microsomal protein/20 min) 43 + 12 + 43 2 41 2 32 + 2.5 k 13 t 13 + 14 k 1.5 k

2 lb 2 2 1” 1” lb lb 10 1”

’ Ethylmorphine N-demethylase and aniline hydroxylase activities were determined in microsomes derived from untreated male rats (225-265 g). Values represent mean k SEM for four animals. ’ Significantly different from control value (p < 0.05).

nificant (p < 0.05) reductions were observed at lop4 and lop3 M selenium. Subsequent to the introduction of cadmium, however, significant (p < 0.05) reductions in drug metabolism were observed at each concentration of selenium (lOA6 to 10m3 M). Table 4 summarizes those experiments in which cadmium was added to the incubation medium first. Cadmium (10m4 M) significantly (p < 0.05) reduced the rate of ethylmorphine and aniline metabolism. Selenium (lo+ to 1O-3 M) produced concentration-dependent decreases in drug metabolism and significantly (p < 0.05) reduced drug metabolism at lop4 and 10e3 M. Cadmium (10e4 M) added to the incubation vessels prior to selenium (1O-6 to low3 M) significantly (p < 0.05) reduced drug metabolism regardless of selenium concentration. These experiments suggest that cadmium and selenium do not antagonize the inhibitory actions of either element when addedin vitro to microsomes.

64

EARLY

AND

DISCUSSION The results of this investigation have demonstrated that selenium can prevent cadmium-induced hepatotoxicity which is manifested as a decrease in the activity of the microsomal monooxygenase enzyme system. Interestingly, this protective effect was observed only after the in viva administration by both elements. Moreover, this enzyme system, isolated from seleniumtreated rats, was susceptible to inhibition by cadmium added in vitro. Furthermore, the simultaneous addition of both elements in vitro to the isolated enzyme system actually produced an enhancement in the inhibition produced by each element when added alone. Early studies by Gunn et al. (1968) showed that selenium treatment blocked cadmium-induced testicular toxicity. These investigators also found that selenium treatment increased the testicular levels of cadmium which led them to propose that the selenium formed a chemical complex with the cadmium, thus preventing free cadmium from reacting with sensitive tissue sites and producing necrosis. The results of our investigation would certainly argue against the formation of a nontoxic Se:Cd complex. The in vitro studies rather clearly showed that both cadmium and selenium in sufficient concentrations could inhibit this monooxygenase enzyme system. Thus, if a chemical complexation of the two elements were the mechanism of detoxification, the inhibitory effect of one element should be decreased in the presence of the other. In fact, we found that the inhibitory effect of each element was enhanced in the presence of the other. However, it is possible that the form of selenium that binds cadmium may not be the selenite. The protective effect of selenium against cadmium toxicity may involve the role of selenium as an integral and necessary component of glutathione peroxidase (Rotruck et al., 1972; Hoekstra, 1975), a cytosolic enzyme which is important in destroying

SCHNELL

noxious hydroperoxides (Hoekstra, 1975). This action against lipid hydroperoxides is particularly important for maintaining the integrity of cellular and subcellular membranes from oxidative damage (Hoekstra, 1975). Other studies have shown that selenium deficiency results in a decrease in glutathione peroxidase activity (Rotruck et al., 1972) and, in turn, an increase in tissue susceptibility to peroxidation (Van Vleet and Ferrans, 1976). Within the hepatic microsomal fractions both polyunsaturated lipids and hemoproteins (cytochrome P-450) are strong catalysts of lipid peroxidation (Combs et al., 1975). A preliminary report by Stacey et al. (1979) has suggested that cadmium may induce its toxicity by lipid peroxidation. Consistent with this viewpoint, Omaye and Tappel (1975) have reported a significant decrease in testicular glutathione peroxidase activity and a significant increase in lipid peroxidation following cadmium treatment in rats. All of these were prevented by prior in vivo treatment with selenium. Although no experiments were conducted to test this hypothesis, our data do not preclude this possible mechanism. Another possible explanation for this protective effect of selenium involves the ability of selenium to alter the tissue distribution of cadmium. The studies by Chen et al. (1975) are typical. Following selenium pretreatment cadmium levels are increased in the blood (22-fold) and testes (3-fold), while those in the liver (48%) and kidney (12%) are decreased. Using similar experimental designs Piotrowski et al., (1977), Gunn et al., (1968), and Magos and Webb (1976) have also reported decreases in liver cadmium levels in selenium-pretreated animals. Apparently, a considerable proportion of the cadmium in these seleniumpretreated rats is diverted to the blood compartment (Chen et al., 1975; Gasiewicz and Smith, 1978). In addition, Stowe (1976) has shown that selenium pretreatment increases the rate of biliary excretion of cadmium. However, the ability of selenium to antagonize cadmium toxicity cannot simply be

SELENIUM

PROTECTION

explained by the phenomenon of organ redistribution of cadmium. As previously mentioned, Gunn et al. (1968) found that selenium protected against cadmium-induced testicular toxicity as the testicular concentration of the heavy metal increased. While the complexation hypothesis proposed by Gunn et al. (1968) has been discounted, further studies by Chen et al. (1975) and Prohaska et al. (1977) have shown that increased cadmium levels in testicular cytosolic fraction have been redistributed from lowmolecular-weight proteins (15,000 to 34,000) to higher-molecular-weight proteins (110,000 to 115,000). Chen et al. (1975) also reported similar findings with respect to distribution of cadmium in the hepatocyte with the metal being redistributed to higher-molecularweight proteins (115,000) from lower-molecular-weight proteins (ca. 10,000) in the selenium-treated rats. While our data are consistent with the latter two hypotheses for explaining the ability of selenium to prevent cadmium toxicity, the exact molecular mechanisms remain to be elucidated. ACKNOWLEDGMENTS This research was supported by NIEHS Environmental Toxicology Training Grant No. ES-00071, a Purdue University Doctoral Fellowship for Black Students and Ethnic Minorities, and NIEHS Research grant ES-02425.

REFERENCES ANDERS, M. W., AND MANNERING, G. J. (1966). Inhibition of drug metabolism. I. Kinetics of the inhibition of the N-demethylation of ethylmorphine by 2-diethylaminoethyl 2,2-diphenylvalerate HCl (SKF 525A) and related compounds. Mol. Pharmacol. 2, 319-327. BURK, R. F., MACKINNON, A. M., AND SIMON, F. R. (1974). Selenium and hepatic microsomal hemoproteins Biochem. Biophys. Res. Commun. 50, 431-436. BURK, R. F., AND MASTERS, B. S. S. (1975). Some effects of selenium deficiency on the hepatic microsomal cytochrome P-450 system in the rat. Arch. Biochem. Biophys. 170, 124-131. CAYGILL, D. P. J., LUCY, J. A., AND DIPLOCK, A. T. (1971). The effect of vitamin E on the intracellular

AGAINST

CADMIUM

65

distribution of the different oxidation states of selenium in rat liver. Biochem. J. 125, 407-416. CHEN, R. W., WHANGER, P. D., AND Weswig, P. H. (1975). Selenium-induced redistribution of cadmium binding to tissue proteins: A possible mechanism of protection against cadmium toxicity. Bioinorg. Chem. 4, 125-133. COMBS, G. F., NOGUCHI, T., AND SCOTT, M. L. (1975). Mechanism of action of selenium and vitamin E in protection of biological membranes. Fed. Proc. 34,2090-2095. DUNNETT, C. W. (1955). A multiple comparison procedure for comparing several treatments with a control. Amer. Stat. Assoc. 50, 1096- 1121. Fox, M. R. S. (1974). Effect of essential minerals on cadmium toxicity. A review. J. Food Sci. 39, 321-335. FRIBERG, L., PISCATOR, M., NORDBERG, G. F., AND KJELLSTROM, T. (1974). Cadmium in the Environment, 2nd ed., pp. 93-202. CRC Press, Cleveland. GASIEWICZ, T. A., AND SMITH, J. C. (1978). Properties of the cadmium and selenium complex formed in rat plasma in vivo and in vitro. Chem.-Biol. Znferact 23, 171-183. GuNN,S. A., GOULD,T. C., AND ANDERSON, W. A. D. (1%8). Selectivity of organ response to cadmium injury and various protective measures. J. Pathol. Bacterial. %, 89-96. HADLEY, W. M., MIYA, T. S., AND BOUSQUET, W. F. (1974). Cadmium inhibition of hepatic drug metabolism in the rat. Toxicol. Appl. Pharmacol. 28, 284291. HOEKSTRA, W. G. (1975). Biochemical function of selenium and its relation to vitamin E. Fed. Proc. 34, 2083-2089. IMAI, Y., ITO, A., AND SATO, K. (1966). Evidence for biochemically different types of vesicles in the hepatic microsomal fraction. J. Biochem. 60, 417428. JOHNSTON, R. E., MIYA, T. S., AND SCHNELL, R. C. (1975). Cadmium potentiation of drug-response role of the liver. Biochem. Pharmacol. 24,877-881. KRASNY, H. C., AND HOLBROOK, D. J. (1977). Effects of cadmium on microsomal hemoproteins and heme oxygenase in rat liver. Mol. Pharmacol. 13,759-765. LOWRY, 0. H., ROSEBROUGH,N. J.,FARR, A. L., AND RANDALL, R. J. (1951). Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265-275. MAGOS, L., AND WEBB, M. (1976). Differences in distribution and excretion of selenium and cadmium or mercury after their simultaneous administration subcutaneously in equimolar doses. Arch. Toxicol. 36, 63-69. MASON, K. E., YOUNG, J. O., AND BROWN, J. A. (1964). Effectiveness of selenium and zinc in protecting against cadmium-induced injury in the rat testis. Anra. Rec. 148, 309. MEANS, J. R., CARLSON, G. P., AND SCHNELL, R. C.

66

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