Effect of sodium selenite upon bromobenzene toxicity in rats

TOXICOLOGY

ANDAPPLIEDPHARMACOLOGY

83,271-278(1986)

Effect of Sodium Selenite upon Bromobenzene

Toxicity in Rats

I. Hepatotoxicity

B. ALEXMERRICK,*

MARCH.DAVIES,* ROYHEIHASEGAWA,~ SAMUELM.COHEN,~-ANDR.CRAIGSCHNELL*~'

MARGARET K.

ST.JOHN,~

*Depurtment of Pharmacodynamics and To.uicolog.v, College of’ Pharmacy, University of Nebraska Medical Center. 42nd & Dewev Avenue, Omaha, Nebraska 68105: and tDepartment of Pathology and Laborutory Medicine, College of Medicine. University of Nebraska Medical Center. 42nd & Dewey Avenue, Omaha, Nebraska 68105

Received

October

15. 1985: accepted

November

5. 1985

Effect of Sodium Selenite upon Bromobenzene Toxicity in Rats. 1. Hepatotoxicity. MERRICK, B. A.. DAVIES, M. H., HASEGAWA. R., ST.JOHN, M. K.,COHEN, S. M.. ANDSCHNELL, R. C. (1986). Toxicol. Appl. Pharmacol. 83,271-278. The effects of sodium selenite on bromobenzene hepatotoxicity were examined in male rats. Rats pretreated with sodium selenite ( 12.5 or 30 pmol/ kg, ip) 72 hr prior to injection of bromobenzene (7.5 mmol/kg, ip) showed a marked reduction in bromobenzene-induced liver injury as evidenced by decreased plasma alanine and aspartate transaminase values, sorbitol dehydrogenase activity, and reduced histologic damage. Administration of bromobenzene did not affect the selenium content of blood or liver. At 72 hr after treatment with selenite, hepatic reduced (GSH) and oxidized (GSSG) glutathione values or GSH synthetic and degradation enzyme activities were not altered. However, from 3 to 12 hr following bromobenzene administration, hepatic GSH and cysteine amounts declined less rapidly in selenitetreated rats compared to control. Thus, acute selenite treatment ameliorated bromobenzene hepatotoxicity in a manner suggesting facilitation of hepatic GSH production by sefenite for use in bromobenzene detoxication. 0 1986 Academic Press, Inc.

Selenium is found asan essentialtrace element with GSH, the amounts of GSH and the acin mammals as an integral component of the tivities of GSH synthetic enzymes may be inenzyme, glutathione peroxidase(Schwartz and creased(Eaton et al., 1980; Chung and Maines, Foltz, 1957; Rotruck et al., 1973). However, 1981; LeBeouf and Hoekstra, 1983; Merrick excess selenium exposure can exert many et al., 1984). Furthermore, a direct interaction pharmacological and toxicological effects upon of selenium or its thiolated metabolites can cellular processesin addition to nutritional alter the disposition and toxicity of heavy functions. When selenium is administered in metals (Ridlington and Whanger, 1981; Early the form of sodium seleniteat quantities which and Schnell, 1981; Berry et al., 1984) in a exceed nutritional requirements, selenite is manner which may be relevant for necrotoxic processed for pulmonary and urinary elimi- xenobiotics (Moxon et al., 1940). nation by interaction with the tripeptide, gluRecently, it wasreported that acute selenite tathione (GSH) (Ganther and Hsieh, 1974). treatment ameliorated bromobenzene hepaAs a consequence of the reaction of selenite totoxicity in male rats in which alterations in GSH peroxidase and GSH S-transferaseactivities did not appear involved (Merrick et al., ’ Burroughs-Wellcome Scholar in Toxicology, 1983. To 1984). Bromobenzene is metabolized by cywhom correspondence should be addressed: Dean of tochrome P-450 enzymes forming reactive Graduate Studies and Research, North Dakota State University, Fargo, N.D. 58105. epoxide intermediates which are primarily de271

0041-008X/86

$3.00

Copyright Q 1986 by Academx Press, Inc. All rights of reproduction in any form resewed

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toxified by enzymatic conjugation with GSH or which form hydroxylated metabolites (Brodie et al., 197 1; Reid et al., 197 1; Jollow et al., 1974). If cellular amounts of GSH are depleted, the epoxides are hypothesized to bind covalently to tissue macromolecules leading to cytotoxicity (Reid et al., 197 1; Jollow et al., 1974; Monks et al.. 1982). Alterations in hepatic production of GSH amounts or in selenium disposition were investigated as possible factors by which selenite attenuates bromobenzene hepatotoxicity. METHODS Animals. Male, Sprague-Dawley rats (Sasco, Inc., Omaha, Nebr.) weighing 230-300 g were allowed free access to feed (Purina Lab Chow No. 50 12, Ralston Purina Co., St. Louis, MO.) and tap water. The animals maintained on a 12-hr light/dark cycle (L:O600-I 800 hr) and were housed in community stainless-steel cages for at least 1 week prior to use. Seienite and bromobenzene trealments. All injections of sodium selenite or bromobenzene were made intraperitoneally (ip) between 1030 and 1200 hr. Selenite, dissolved in deionized distilled water, was administered either in a dose of 12.5 pmol Se/kg which has no effect on hepatic microsomal drug metabolism (Early and Schnell, 1981) or 30 prnol Se/kg which can inhibit drug metabolism (Schnell and Early. 198 la,b). Control rats received saline (2 ml/kg). Rats were pretreated with selenite 72 hr prior to injection with bromobenzene. This treatment regimen substantially reduces hepatotoxicity (Met-rick et al., 1984). Bromobenzene was dissolved in sesame oil (50% solution) and was administered in a dose of 7.5 mmol/kg which results in reproducible hepatic damage. Control rats received an equivalent volume of sesame oil. Assessment of hepatotoxicitv. Hepatic damage was assessed by measuring plasma enzyme activities and liver histopathology. Rats were anesthetized with ether immediately prior to termination. Alanine aminotransferase (ALT), aspartate aminotransferase (AST). and sorbitol dehydrogenase (SDH) activities in plasma were determined with commercial kits (Nos. 505-OP and 50 UV. Sigma Chemical Co., St. Louis, MO.). The activities of these enzymes were expressed in Sigma-Frankel units (SF U). Blood was collected in a heparinized syringe from the inferior vena cava and plasma was separated by centrifuging whole blood at 2500 rpm for 15 min. Sections of the right and left lobes of liver were analyzed for necrosis after fixation in neutral 10% buffered Formalin solution. Paraffin-embedded tissue was stained with hematoxylin-eosin. Slides were labeled by a coded sequence and evaluated by the pathologist without knowledge of the treatment. Histologic damage was graded by slightly modifying the criteria of Brodie el al. (197 I) with a point

ET

AL

scale of 0 to 4 in order of increasing damage: O-no observed hepatic necrosis; l-a few degenerating parenchymal cells and trace necrosis: 2-minimal necrosis; 3extensive necrosis in which central veins were surrounded by several layers of dead or degenerating cells; and 4massive necrosis of extensive liver areas. Bio&emical analyses. Hepatic reduced glutathione (GSH) was measured by the procedure of Sedlak and Lindsay ( 1968). Analysis of hepatic oxidized glutathione (GSSG) in the same acid soluble supematant fractions was performed according to the method of Davies ef al. ( 1984). Hepatic and plasma quantities of cysteine were measured by the method of Gaitonde ( 1967). Hepatic cysteine content was determined from supematant fractions derived from acid-precipitated liver fractions as described for GSH analysis. Plasma derived from heparinized whole blood was deproteinized by adding 1 vol of TCA (5%) containing I mM EDTA. Selenium contents of liver or whole blood were determined by the method of Watkinson (1966). Liver was analyzed after preparing a tissue homogenate (20% w/v) of 0. I M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.5). Measurement of hepatic y-glutamylcysteine synthetase and GSSG reductase activities were determined by the methods of Sekura and Meister (I 977) and Racker (1955). respectively. These enzyme activities were measured from the cytosolic fraction obtained by centrifugation of liver homogenate ( I g in 4 vol of 0.1 M Tris buffer, pH 7.5) at 97,6OOg,, at 4°C for 60 min in a Beckman Ultracentrifuge (Model L5-5OB). Hepatic y-glutamyltransferase activity was determined by the procedures of Tate and Meister (198 1). Cytosolic protein was solubilized with 1% (w/v) of 7-deoxycholic acid prior to ultracentrifugation as described.

Protein analysis was performed according to the method of Lowry ut al. (I 95 I) with crystalline bovine serum albumin as a standard. Staiisfical an&s& Data were analyzed by analysis of variance (ANOVA). Student’s t test, or linear regression on a Hewlett-Packard calculator (Model 985 I A) with a cassette program package (No. 098 15-l 5014). Post-hoc tests were performed when appropriate by Duncan’s New Multiple Range Test for multiple comparisons among means (Bruning and Kintz. 1977). The level set for statistical significance wasp < 0.05.

RESULTS

Efect oj‘Selenite Treatment upon Bromobenxene-Induced Increases in Plasma Enzymes und Histologic Damage Preceding studiesindicated that, of the time periods examined (4 to 72 hr), treatment with selenite for 72 hr prior to bromobenzene afforded maximal protection from bromoben-

EFFECT

OF

SELENITE

ON

BROMOBENZENE

zene-induced hepatotoxicity exhibited by increases in plasma ALT and AST activities (Merrick et al., 1984). Experiments were designed to determine if amelioration of bromobenzene-induced increases in plasma enzyme activities by selenite would also extend to plasma sorbitol dehydrogenase (SDH), an enzyme primarily found in liver, and histologic damage. Control rats treated with saline and sesame oil showed low circulating values of plasma ALT, AST, and SDH activities (Table 1). Selenite treatment decreased bromobenzene-induced increases in plasma enzyme activities as compared to saline and bromobenzenetreated rats in a dose-related manner (Table 1). Treatment with 12.5 pmol Se/kg lowered bromobenzene-induced increases in plasma ALT, AST, and SDH activities to 37,46, and 39%, respectively, versus saline- and bromobenzene-treated rats. Reduction by 30 pmol Se/kg of increases in ALT, AST, and SDH activities produced by bromobenzene were 22, 3 1, and 26%, respectively, compared to salineand bromobenzene-treated rats. Results of histologic studies (same groups as above) show that as the dose of selenite increased, the severity of hepatic necrosis produced by bromobenzene was lessened compared to rats receiving only bromobenzene (Table 2). Selenite treatment alone was not associated with any detectable histologic damage (data not shown). Thus, pretreatment

with selenite before bromobenzene intoxication was associated with diminished hepatic necrosis and lowered enzyme activities compared to control.

Efect of Bromobenzene Treatment upon Selenium Content in Liver and Whole Blood from Selenite- Treated Rats Studies by Moxon et al. (1940) have suggested that a biochemical interaction may occur between selenium and bromobenzene, inferring that bromobenzene treatment may alter the selenium content of tissues from selenium-treated animals. Since accumulation of the metalloid occurs in liver and blood, the effect of bromobenzene on the concentration of selenium in these two tissues was investigated. Rats pretreated 72 hr prior with either saline or selenite received sesame oil or bromobenzene and then were killed 24 hr later. Preliminary data from our laboratory and other reports (Bopp et al., 1982) show that tissue amounts of selenium after parenteral injection stabilize at 72 hr after injection of selenite into rats. Data in Table 3 show a dose-related increase in selenium in liver and blood of rats treated 72 hr prior with selenite in the presence or absence of bromobenzene. Comparison between the two sets of rats receiving selenite. or selenite and bromobenzene. reveal that the selenium contents of blood and liver were al-

TABLE EFFECT OFSELENITE

273

TOXICITY

1

PRETREATMENTONBROMOBENZENE-INDUCEDINCREASEINPLASMA ALT, AST. AND SDH ACTIVITIES

Treatment” Saline (-) bromobenzene Saline (+) bromobenzene 12.5 rmol Se/kg (+) bromobenzene 30 pmol Se/kg (+) bromobenzene

ALT 10 f 54 -c 20 k 12+

AST 2b 4c 2d lb

43 f 4b 188 + IO’ 81 f 5d 59* 4’

SDH 80* 13* 4070 + 393’ 1570 * 188d 1058 f 58’

’ Male rats received either saline or selenite (ip) 72 hr prior to administration of sesame oil (- bromobenzene) or bromobenzene (7.5 mmol/kg, ip). Rats were killed 24 hr after injection with bromobenzene, and plasma ALT, AST. and SDH activities were analyzed as described. Each value represents the X ? SE for nine rats except in the saline and oil group which contained 6 rats. Results are expressed in sigma units/ml. b-e Within columns, values not sharing common letters are significantly different (ANOVA, Duncan’s New Multiple Range Test; p < 0.05).

274

MERRICK TABLE

ET

AL.

2

EFFECTOF~ELENITE TREATMENTUPONHISTOLOGIC DAMAGE PRODUCEDBYBROMOBENZENE Hepatic Treatment”

I

0

Saline (-) bromobenzene Saline (+) bromobenzene 12.5 Krnol Se/kg (+) bromobenzene 30 hmol Se/kg (+) bromobenzene

necrosis 2

-

6 -

-

4

5 4 1

3

I 3 4

2 4

3

’ Rats were pretreated with selenite or saline for 72 hr prior to receiving sesame oil (- bromobenzene) or bromobenzene (7.5 mmol/kg, ip). Twenty-four hours after bromobenzene, rats were killed and sections were removed for histologic analysis. The criteria for hepatic necrosis were: 0 = no necrosis; 1 = trace: 2 = minimal: 3 = extensive; and 4 = massive. Each value represents the number of animals in each category. Corresponding plasma enzyme activities for each group are shown in Table I.

most identical at each dose. These data demonstrate that bromobenzene treatment did not alter selenium content of blood and liver in selenite-treated animals.

Eflect qfSelenite 011Heputic GSH, GSSG, und GSH Metabolism E~~qvmes Acute selenium treatment has been shown to elevate rat liver GSH up to 48 hr after parenteral injection (Eaton et al., 1980; Chung and Maines, 1981). After 72 hr, liver GSH content subsidesto control values (Merrick et ul., 1984). However, selenite may produce a sustained effect upon GSH metabolism enzymes. Thus, the effect of selenite treatment upon hepatic GSH and GSSG and GSH meTABLE

tabolism enzymes was investigated (Table 4). Selenite significantly enhanced GSSG reductase activity to 30 and 49% above control for 12.5 pmol Se/kg and 30 pmol Se/kg, respectively. Liver GSH and GSSG contents were unchanged in selenite-treated animals compared to saline controls. Activity of the ratelimiting enzyme in GSH synthesis, y-glutamylcysteine synthetase, did not differ from control values at 72 hr after selenite injection. y-Glutamyl transferase activity, which is involved in GSH degradation. was not significantly lowered by selenitetreatment. Thus, the metabolic capabilities for hepatic GSH production in rats treated for 72 hr with selenite appeared similar to control animals. The ca3

EFFECTOFBROMOBENZENE TREATMENTUPON~ELENWM CONTENTOFLIVERANDBLOOD FROMMALERATSPRETREATEDWITHSELENITE~ Saline Liver (-) (+) Blood (-) (+)

12.5 pmol

Se/kg

30 rmol

Se/kg

Bromobenzene Bromobenzene

40.6 + 1.0” 40.8 t 1.2*

54.5 k 2.8’ 55.1 * 1.7’

83.1 + 3.2d 77.5 k 3.6d

Bromobenzene Bromobenzene

2 I .4 + 0.9h 20.8 f 0.4h

33.1 t 1.6’ 35.8 t 1.0

85.5 * 5. I d 84.3 k 4.4d

’ Male rats were treated with saline or selenite (ip), followed 72 hr later by injection with sesame oil (- bromobenzene) or bromobenzene (7.5 mmol/kg, ip). Rats were killed after 24 hr. Selenium contents of whole blood (pg/dl) and liver (fig/100 g) were determined as described. Each value represents the X + SE for 5-6 rats per group. b-d Across rows and within columns for liver or blood, values not sharing a common letter are significantly different (ANOVA. Duncan’s New Multiple Range Test: p < 0.05).

EFFECT OF SELENITE

ON BROMOBENZENE

275

TOXICITY

TABLE 4 EF’FECTOF SELENITE TREATMENT ON GLUTATHIONE AND GLUTATHIONE SYNTHETIC AND DEGRADATIVE ENZYME ACTIVITIES’ Parameter Glutathione (GSH) Glutathione (GSSG) y-Glutamylcysteine synthetase y-Glutamyl transferase Glutathione (GSSG) reductase

Saline 49.7 3.5 46.6 0.8 84

+ I.Ob i 0.5h k 2.6h f 0.2h &4b

12.5 rmol Se/kg 52.9 3.3 45.1 0.5 109

f + f * f

0.6’ 0.6’ 1.7b O.lb 4’

30 pmol Se/kg 51.9 + I.Ob 3.5 ?z 0.4” 45.1 f 2.4’ 0.6? O.lb 125 ?I 12’

a Male rats were treated with saline or selenite (ip). At 72 hr. rats were killed and the above parameters were measured as described in text according to the following units: nmol GSH/mg liver protein, nmol GSSG/mg liver protein, nmol P, released/min/mg cytosolic protein for -/-GCS, nmol pnitroaniline formed/min/mg liver protein for y-GT. and nmol NADPH oxidized/min/mg protein for GSSG reductase. b-cAcross rows, values not sharing a common letter are significantly different. (ANOVA. Duncan’s New Multiple Range Test; p < 0.05).

pacity to maintain the reduced status of glutathione. via GSSG reductase, may be enhanced by selenite.

of 0 hr GSH value) at 12 hr after bromobenzene injection. The declinesin hepatic cysteine concentrations after bromobenzene exposure in selenite-treated rats were lessthan controls Selenite EJk*ts on Hepatic GSH and Cysteine as were observed for GSH decline. Twelve Decline qfier Bromobenzene Administration hours after bromobenzene treatment, saline Studies were designed to examine if treat- and 12.5 pmol Se/kg treated rats had comment with selenite could alter the decline of parable hepatic cysteine contents (35 and 29% hepatic GSH and cysteine content over time of 0 hr, respectively). Rats treated with 30 after administration of bromobenzene. Plasma pmol Se/kg had significantly elevated liver ALT activity was measuredas an index of he- cysteine content (67% of 0 hr level) compared patic damage. Plasma cysteine content was to controls at 12 hr after bromobenzene indetermined asa possiblesource of extrahepatic jection. Plasma concentrations of cysteine in cysteine available to liver for GSH synthesis. saline or selenite-treated rats remained relaResults in Fig. 1 show the effect of bro- tively unaffected by bromobenzene treatment mobenzene treatment from 3 to 12 hr after at the time intervals examined. Plasma ALT injection upon hepatic GSH and cysteine activity was substantially increased from 3 to amounts in rats treated 72 hr prior with saline 12 hr after administration of bromobenzene or selenite. Immediately prior to bromobenin salinecontrols. However, selenitetreatment zene exposure. at time 0 hr, the amounts of ameliorated the rise in plasma ALT activity GSH and cysteine were not different between produced by bromobenzene (Figure 1). selenite and saline-treated control rats. FolDISCUSSION lowing injection of bromobenzene, the decline in hepatic GSH content proceeded more rapThe correlation between reduced plasma idly in control animals compared to selenite- enzyme activities and histologic damagedemtreated rats from 3 to 9 hr. At 12 hr after bro- onstrates that acute selenite treatment diminmobenzene injection, similar GSH values were ished bromobenzene hepatotoxicity. In studyobserved in 12.5 pmol Se/kg treated rats and ing the protective effect of selenite, alterations saline controls. However, liver GSH concen- in hepatic production of GSH by selenite or tration in rats treated with 30 pmol Se/kg (43% changes in selenium disposition by bromoof 0 hr GSH value) remained significantly el- benzene were examined. evated above that in saline-treated rats (13% The possibility that seleniteand bromoben-

276

MERRICK

HEPATIC

ET

AL

HEPATIC

GSH

CYSTEINE

250

150

PLASMA

ALT b

PLASMA CYSTEINE n 30 umole Setkg A

12 5 urn&

l

Salme

100

; !i a E

50

5 1 01

250

P G ; c 2

Se/kg

hours

FIG. I. Decline of hepatic GSH and cysteine content in rats treated with selenite. Rats were treated with saline or selenite 72 hr prior to receiving bromobenzene (7.5 mmol/kg, ip). (a) Hepatic cysteine and GSH concentrations, plasma cysteine concentrations, and plasma ALT activity were measured at 0 to 12 hr after bromobenzene as described in text. At each time point of each parameter, values having no letters (b. c) in common are significantly different (ANOVA. Duncan’s New Multiple Range Test, p < 0.05). No significant differences were noted for plasma cysteine concentrations between treatment groups.

zene may undergo a direct interaction has been previously considered when it was shown that bromobenzene increased the rate of urinary excretion of selenium in selenium-intoxicated cattle and dogs (Moxon et al., 1940) and in humans (Lemley, 1940). Moxon et al. (1940) speculated that selenoamino acids combined with bromobenzene, resulting in the excretion of a selenomercapturic acid. Westfall and Smith (1941) were unable to confirm an increasein urinary excretion of selenium when bromobenzene was administered to seleniumtoxic, male rabbits. In the presentexperiments, bromobenzene did not alter the disposition of selenite in blood or liver. This suggeststhat

direct interaction between selenite and bromobenzene was of little consequencein rats. Several studieshave shown that bromobenzene hepatotoxicity is diminished under conditions which facilitate GSH biosynthesis (Jollow et al., 1974; Thor et al., 1978a,b, 1979). Selenite has been shown to increase hepatic GSH contents in rats up to 48 hr after injection (Eaton ezal., 1980; Chung and Maines, 1981: LeBoeuf and Hoekstra, 1983; Merrick et al., 1984). In addition, enhanced synthesisof GSH may be related to heightened activities of yglutamylcysteine synthetase and GSSG reductase which temporally parallel the selenite mediated increase in GSH up to 24 hr after

EFFECT OF SELENITE

ON BROMOBENZENE

selenite injection (Chung and Maines, 1981). It is interesting that at 72 hr after selenite injection, liver GSH contents were comparable to control values but such animals remained protected from bromobenzene toxicity in the present study. Furthermore, the activities of hepatic synthetic and degradation enzymes, y-glutamylcysteine synthetase, y-glutamyl transferase, were unaltered by treatment with selenite. Since the metabolism of bromobenzene does not produce GSSG, the enhancement of GSSG reductase activity does not appear to be directly related to the protective effect of selenite. However, it has been suggested that in viva production of GSH is substratelimited in that hepatic cysteine concentrations are five times less than the Km for y-glutamylcysteine synthetase, while glycine and glutamine are in relative abundance (Tateishi et al., 1977, 198I). This infers that increasesin GSH synthetic enzyme activity may not necessarily invoke an increase in GSH concentration without a concomitant increase in cysteine amounts. Since prior studiesshowed that depletion of liver GSH at 24 hr after bromobenzene exposure did not differ in controls and rats pretreated 72 hr prior with selenite (Merrick er al., 1984) it was possible that selenite might influence the initial decline of liver GSH after bromobenzene, eventually becoming equally depressedto control values 24 hr later. Experiments designed to measure hepatic GSH contents, hepatic and plasma cysteine concentrations, and plasma ALT activities from 0 to 12 hr after bromobenzene in rats treated 72 hr prior with saline or selenite showedthese parameters were at similar values immediately prior to bromobenzene administration in each group. Following injection of bromobenzene, the decline in hepatic GSH and cysteine content proceeded more rapidly in control animals compared to selenite-treated rats. The diminished decline in hepatic GSH and cysteine contents in rats treated with selenite and bromobenzene was accompanied by reduced plasmaALT activities which indicates reduced hepatotoxicity. Rats treated with 30 pmol Se/ kg appeared to have a greater capacity to

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277

maintain liver GSH and cysteine contents after bromobenzene than rats treated with 12.5 pmol Se/kg. Since plasma cysteine concentrations did not vary in selenite or saline-treated rats injected with bromobenzene, this suggests that extrahepatic cysteine was not supplied to the liver during bromobenzene toxicity. These results suggestthat hepatic capacity required to synthesize GSH might be enhanced in selenite-treated rats which, in turn, may be dependent upon an increased hepatic ability to generate cysteine. The possibility that reduced hepatotoxicity correlates with increased mercapturic acid formation in selenium-treated rats is considered in an accompanying paper (Merrick et al., 1986) which examines the effect of selenium upon bromobenzene metabolism. ACKNOWLEDGMENTS Support for this work was provided by NIEHS Research Grant ES-02425, a Blanch Widaman Fellowship (B.A.M.), and a Burroughs-Wellcome Toxicology Scholar Award (R.C.S.).

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