Protective Effect of Chromium( III) on Acute Lethal Toxicity of Carbon Tetrachloride in Rats and Mice Masakatsu Tezuka, Keiko Momiyama, Toshiyuki Edano, and Shoji Okada Department of Radiobiwhemistry, Shizuoka, Shizuoka-shi, Japan

School of Pharmaceutical Sciences, University of

ABSTRACT Trivalent chromium (Cr(III)) preadministered intraperitoneally (5 mg Cr/kg body weight) to rats and mice protected these animals from acute lethal toxicity of carbon tetracbloride (Ccl,). Some other metals, Cr(Vl), Cu(ll), and Zn(II), had no effect on Ccl, lethal toxicity. DL-ar-tocopherol, one of the antioxidative agents, showed similar protective effects to Cr(III). Activities of serum COT and GPT in mice were increased sharply by the administration of Ccl,, but these elevations were depressed by Cr(III) preadministration. Serum glucose levels of mice increased transiently after Ccl, administration and then in the control group fell to hypoglycemic levels after 6 hr, whereas the Cr(JIl)-pretreated group kept to homeostatic levels. Lipid peroxidation of microsomes in mice 24 hr after Cr(IlI) administration was lower than that of the control. These results suggest that Cr(IlI) preadministered to mice might act as a radical scavenger to Ccl, to form trichloromethyl radicals which are a major initial product of Ccl, in liver cells.

INTRODUCTION Chromium has been known to be au essential trace element for the maintenance of normal glucose tolerance and normal lipid metabolism [ 1, 21. Besides this action, it was recently reported by us that the administration of trivalent chromium (Cr(III)) caused a marked enhancement of nucleolar RNA synthesis in regenerating rat liver at the early stage after partial hepatectomy [3-91. This observation suggests that Cr(IlI) plays some role in the process of gene expression on cell proliferation and also that it may be informative to perform an experiment to see the effect of Cr(IlJJ on the repairing process of liver after the treatment with hepatotoxic agents. In the course

Address reprint requests and correspondence to: Professor Shoji Okada, Department of Radiobiochemistry, School of Pharmaceutical Sciences, University of Shixuoka, 395 Yada, Shixuoka-shi, 422 Japan. 1 Journal of Inorganic Biochemistry, 42, 1-8 (1991) 0 1991 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/91/$3.50

2

M. Tezuka et al.

of this experiment using rats dosed with carbon tetrachloride (Ccl,), we unexpectedly found that the administration of Cr(II1) protected animals from the acute lethal toxicity induced by high doses of Ccl,. The present paper demonstrates the protective effect of Cr(III) on the lethal toxicity of Ccl, in rats and mice together with the change of biochemical parameters in serum and liver of mice after the administration of Cr(II1) and Ccl,.

MATERIALS

AND METHODS

Animals Wistar male rats (100-150 g body weight) and ddY male mice (25-30 g body weight) were purchased from Japan SLC Inc. (Hamamatsu, Japan). They were kept at 25 + 1 “C and fed with laboratory chow (MF, Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum until used for the experiment. ChemicaIs Chromic chloride (CrCl,) and sodium chromate (Na,CrG,) were obtained from Nacalai Tesque Inc. (Kyoto, Japan). Ccl, and DL-cw-tocopherol were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan) and Sigma Chemical Co. (St. Louis, U.S.), respectively. Other compounds used were of reagent grade. Administration

of Metal Compounds

and DL-cY-tocopherol

Male rats were injected intraperitoneally with CrCl, at a dose of 5 mg Cr/kg body weight and then fasted for 24 hr. They were administered with a mixture of Ccl, and olive oil (1 : 1, v/v) at doses of 1.39-2.4 ml Ccl, /kg. Mice were intrapcritoneally administered with CrCl,, Na,CrG,, ZnCl,, CuCI,, or DL-cr-tocopherol. The dose of these compounds was 5 mg metals/kg body weight and of DL-cr-tocopherol was 41 mg/kg. After fasting for 24 hr, the mice were administered with Ccl, at a dose of 3.0 ml/kg. Biochemical

Determinations

Mice injected with Ccl, were anesthetized with ether and blood was collected from the inferior vena cava. The serum was obtained by centrifuging the blood after coagulation. The activities of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) in the serum were determined by using the commercial test kits, GOT-UV Test Wako and GPT-UV Test Wako (Wako Pure Chemical Industries), respectively. Serum glucose contents were measured with Glucose Test Wako according to the o-toluidine-borate reaction. Contents of ammonia and total bile acids in serum were determined with Ammonia Test Wako and Total Bile Acids Test Wako, respectively. Determinations of Cytochrome Activity in Microsomes

P-450,

Lipid

Peroxidation,

and

Glucose&phosphatase

The livers isolated after perfusion with ice-cold 0.25 M sucrose solution were homogenized in two volumes of the same solution with a Potter-Elvehjem glass-Teflon homogenizer. Microsomes were prepared according to the method of Aronson and Touster [lo]. The microsomal pellets obtained after centrifugation at 105,000 g for 60 min were suspended in ice-cold 0.25 M sucrose. In case of the determination of

Cr(lB) PROTECTS PROM Ccl,

TABLEl.

EffcctofCr(IlI)PmdminismiononLethal Toxicity of CCl,

in Rats

WW

(mg Cr/kg, i.p.) 1.39 1.67 2.0 2.4

TOXICITY

0 5 0 5 0 5 0 5

wc13 was injected 24 hr bcfolt. ccl,

SurvivalTime (h) (?dcaaf S.D.) 21.5 29.4 14.6 31.3 6.0 31.5 5.1 32.5

f 15.0 f 5.1 f 5.3 f 4.0c f 0.0 f 3.9c f 1.0 f 4.0’

ILSb (96) 37 114 425 537

admsmtion.

bPcrccnt increase in life-span over control. cSi@kant

di%remx from c4mt.d (p < 0.05, II = 4).

cytochrome P-450, the pellets were suspended in 100 n&l potassium phosphate buffer @H 7.4). The protein content in microsomes was determined by using the Bio-Rad Protein Assay. Activity of glucose-6-phosphatase was measured according to the method of Aronson and Touster [lo] by determining the rate of release of inorganic phosphate from glucose 6-phosphate. Cytochrome P450 content of microsomal suspension in the potassium phosphate buffer was e&mated according to the method of Omura and Sato [ 111. Lipid peroxidation in microsomes was determined by the method of Lee et al. [12].

RESULTS Protedve

Effect of C!.r@l) on Acute Lethal Toxicity of Ccl,

In order to investigate the effect of Cr(III) on the repairing process after liver injury induced by the administration of Ccl, to rats, RNA synthesis of the liver was determined. By the pmadministration of Cr(BI) at a dose of 5 mg/kg body weight, rat liver RNA synthesis 4 hr after Ccl, (2 ml/kg) admin&ation was increased to 1.5 fold of control CCl,dosed rats (to be published elsewhere). In the course of these experiments, we found that Cr(IB) protected rats from the acute lethal toxicity induced by Ccl, administmtion at high dose (Table 1). Survival time of rats injected with Ccl, intraperitoneally at doses of 1.67-2.4 ml/kg was significantly elongated by Cr(llI) pmadminktration at 24 hr prior to Ccl, dosing. The protective effect of Cr(IB) was also observed in mice (Table 2). Then, subsequent experiments were carried out with mice. To see if the protective action was Cr(IB)-specific, the effect of some other inorganic metal compounds, Cr(VI), Cu(II), and Zn(II), on the lethal toxicity of Ccl, was examined. As shown in Table 3, no elongation of survival time was observed with these metals administered 24 hr before Ccl, (3 ml/kg), suggesting that the prokction was not metallothionein-induced or nonspecitic but Cr(IB)-specific. On the other hand, DL-a-tocopherol, one of the antioxidative agents, showed the protective effect on Ccl, lethal toxicity similar to Cr(lB).

4 M. Tezuka et al. TABLE 2. Effect of ccl, ml/kg, i.p.

CrGW mg Cr/kg, i.p.

SUWiV~b

016 016 0 0.5

316’ 016 O/6

016 5 0 0.5 2 5

4

416’

016 016 116 016

‘CrCl, was injected 24 hr before CCl, administration. bSurvival/total munbcr of mice 2 wk after CCl, administratioo. csigni6callt diercxc from control @ c 0.05).

Changea

Ccl,

AdministratIon

Mice

As shown in Figure the levels GOT elevated remarkably at 0.5-6 hr after Ccl, (3 ml/kg) administration in control animals. were significantly depressed by Cr(III) preadministration, not by Cr(VI), during the assay period hr. Serum glucose was also varied by administration Ccl, (Fig. 2). the value increased ca. mg/dl at hr after Ccl, administration and decreased ca. 50 mg/dl, than normal, hr. In both the and Cr(V1) the initial values were significantly than control and, 0.5 CCl, administration,

of Pmdmhistration of Some or DL-a-Tocopherol on Lethal of CCl, in Mice F%eadmistration~

atedoscof5mgn~tal/kgbodywc&l1torDGcr-tocopherol of 41 tug/kg was inJf,ctcdintrapcritoncally24 hr before ccl, (3 ml/kg, i.p.) adminddon. bSurvivd/tutal number of mice 2 wk after Ccl, administration. “SigniticantdiEfxence from control @ < 0.05).

Cr(RI) P R O T E C T S F R O M CC14 L E T H A L T O X I C I T Y

4i

I.I

o..

I.I

o

ILl

$

9"

1

,~e

i

i

0 15

i

i

Hours a f t e r CCI4(Sml/kg) a d m i n i s t r a t i o n F I G U R E 1.

Effect o f CI~III) on serum GOT alKI GPT in mic¢ 8fear CCI 4 8dmlnlgU'ation.

Mice were preadministered i.p. with CrCI 3 (5 ms Cr/k8, A), Na2CrO 4 (5 mg Cr/ks, II), or H20 (Q) 24 hr before CC14 athninistration. Each point and bar Icpresents ~ + S.D. (n = 6-8). Stars indicate a significant difference (p < 0.05) from each control.

FIGURE 2. Effect of c K m ) on serum glucose in mice after C C I 4 administration. Mice were _ p r f . ~ 4 m l n i ~ i.p. with CrCI 3 (5 mg Cr/kg, A), NazCrO 4 (5 mg Cr/kg, II), or H 2 0 (e) 24 hr before CCI 4 8dminimzation. Each point and bar retnsenm mean+S.D. ( n : 6-8). Stars indicate a significant difference (p < 0.05) from each coatrol.

U

G

01o s

i

Hours a f t e r CCIs(Sml/kg) a d m i n i s t r a t i o n

6 iU. Tauka et al. TABLE4. ChangeaofBio&emkalPammctersinSenunaadMirrosomes . . AdlWU&ab ‘on of c%(m) aIMI ccl, CrO’ 041 Crlkg, i.p.)

Hour8d?cr Ccl, (3 d/kg, 0

of Mice after i.p.) admidrdon

0.5

2

6

-

398*48 395 f 38 41 f 18 29s~

318 24%*47 26zt.6 28* 7

InSeNm 0

ab

bile tidsb

178k

7

18f

4 4

0

53 f 23 16

Inmicrosomas cytocllrome

252 f 52 5

f 87

5

93 f 25

383i46 18k

Yrcl,wasiajected24brbefofecxl,rdminirtntioa. b(zontaaof

15

6 10

14 f 13 27 f 13

Cr(III) PROTECTS FROM Ccl, LETHAL TGXICITY

7

FIGURE 3. Effect of Cr(IU) on lipid peroxidation of mice microsomcs after ccl, sldmsx&on. Mice were preadministered i.p. with CrCl, (5 rug Cr/kg, A) or H,O (0) 24 hr before Ccl,

. . lldmmmioa.Eachpointandbarnpro seas meanfS.D. (x1=4-8). Star indiHourr aftor CCk(i)ml/kg)

ldminlotr~tlon

cates a signilicaat difkence @< 0.05) from control.

Chromitmt(IJI)did not protect mice against lethal toxicity of some other hepatotoxins, D-( + )-galactosamine, diethylnitrosamine, and cumene hydroperoxide (data not shown). With regard to the hepatotoxic effect of these compounds, Reppler et al. [14] reported that D-(+)-galactosamine markedly reduced the hepatic contents of uridine phosphates and then inhibited RNA synthesis of rat liver, and Stoner [15] described that diethyl&osamk inhibited RNA and DNA syntheses of rat Liver. Jewell et al. [16] pointed out that tert-butylhydroperoxide caused the depletion of intracelhtlar ghuathione and the extensive lipid peroxidation in isolated rat hepatocytes. Theeffect of cumene hydroperoxide to rat heputocytes may be homologous to that of tert-butylhydroperoxide. Differing to these compounds, CCl, is well known to cause hepatic lipid peroxidntion in rats and mice. glater [ 171 described that a major initial product of CCl, for lipid peroxidation was trichloromethyl radical produced by cytochrome PASO and/or NADPH-cytochrome C reductase in microsomes and liver injury was induced. Therefore, the differential protective effect of Cr(RI) on the lethal toxicity between Ccl, and the other compounds may be due to the difference of their hepatotoxic actions. DL-cr-tocopherol, one of the antioxidative factors in animals, showed the protective effect similar to Cr(III) on Ccl, lethal toxicity. The present finding that lipid peroxidation in microsomes was depressed by Cr(III) pmadministration suggests that Cr(IIl) may have an antioxidative activity on metabolic activation of CCl, to form free radicals in liver microsomes in analogy with DL-ar-tocopherol. Furthermore, we measured some biochemical parameters in serum and liver after Ccl, administration. The rapid increase of serum GOT and GPT activities by Ccl, administration was significantly depressed in Cr(III)-pretreated mice, indicating that Cr(lII) might reduce CCl,-induced liver damage. Serum glucose content of Cr(llI)-pmadministered mice was lower than the control level and increased transiently by Ccl, administration, and then kept at homeostak

8

iu. Tczuka et al. level for at least 6 hr, while in control mice it fell to hypoglycemic levels. This action seems to be advantageous to protect mice from Ccl, lethal toxicity. Here, we found a new biological fimction of Cr(IE), i.e., the pro&dive effect on acute lethal toxicity of Ccl,, similar to DL-docopherol. Cr(III) might act as an antioxidative agent, probably as a radical scavenger at an early period of Ccl, activation. Our current investigations axe revealing that Cr(III) acts as a radical scavenger in the formation of trichloromethyl radical from CCI, in liver cells.

RJSERENCES 1. K. Schwarz and W. Mertz, Arch. Biochem. Biophp. 8!$ 292-295 (1959). 2. W. Mertz, scicncc 213, 1332-1338 (1981). 3. S. Okada, M. !%mki, H. Tsukada, and H. Ohba, Chem. Pharm. Bull. 30,3439-3441 (1982). 4. S. Ok&a, M. !&n&i, and H. Ohba, J. Znorg. Biochem. 19,95- 103 (1983). 5. S. Okada, H. Tsukada, ard H. Ohba, J. Znorg. Biochem. 21, 113-124 (1984). 6. S. Okda and H. Tsukada, in 7kace h&tents in Mm and Animals, C. F. Mills, I. Bmmner, and J. K. chtot#s, E!ds., C-A-B., Slough, 1985, pp. 133-139. 7. S. Okada, H. Tsukada, T. Kiyohara, and M. Tezuka, in Toxicoio&~of Met&-Clinical and ~rimental Resuzrch, S. S. Brown and Y. Kodama, Eds., Hal&d Press, New York, 1987, pp. 371-372. 8. H. Tsukada ad S. OkxIa, in Toxkdooay of Meta&Clinicol and Eqwrimental Rc mh, S. S. Brown and Y. Kodama, EIds., Hakd Press, New York, 1987, pp. 372-373. 9. S. Okada, H. Tsukada, and M. Tezuka, Biof. Tracx Element Res. 21, 35-39 (1989). 10. N. N. Aronstm Jr. and 0. Touter, hfethodk in Enqymol. 31.90-l&? (1974). 11. T. Omura and R. Seto, J. Biol. Chem. 239, 2370-2385 (1964). 12. P. Y. Lee, P. B. I&Cay, and K. R. Hombmok, Biochem. Pharmad. 31,405~409 (1982). 13. S. Z. Cagen and C. D. Kkassen, Tokcol. A&. Pharmucol. 51, 107-116 (1979). 14. D. Kep@r, J. Paw& ad K. Dadrcr, J. Biol. Chem. 249,211-216 (1974). 15. H. B. Staosr, Brit. J. Exp. Pathd. 37, 176-198 (1956). 16. S. A. Jewell, D. D. Monte, P. Richdmi. G. Bellomo, and S. Orrenius, J. Biochem. Toxid 1, 13-22 (1986). 17. T. F. Slater, Biochem. J. 222, 1-15 (1984). Received June 20,lIXX.l; aaxpted August 14,19&W