The effects of carbon tetrachloride and some antioxidants on the hydroxylation of tetralin by rat liver homogenate

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The effects of carbon tetrachloride and some antioxidant$ on the hydroxylation of tetralin by rat liver homogenate Halogenated hydrocarbons, particularly carbon tetrachloride, have been known to cause fatty liver and other liver damage 1, 2. It has also been shown a 6 that some antioxidants are effective in protecting liver from such toxic effects. Evidence v is available to indicate that carbon tetrachloride stimulates lipid peroxidation in rat liver homogenate. It has been theorized T M that the primary cause of carbon tetrachloride hepatotoxicity is due to homolytic cleavage of its carbon-chloride bond to free radicals. We have shown previously 11 that the enzymatic hydroxylation of tetralin to tetralol can proceed by way of tetralin hydroperoxide. This is similar to nonenzymatic autoxidation of tetralin in which the formation of tetralyl radical is the initiating step ~2. Since carbon tetrachloride m a y initiate, whereas antioxidants protect against, the lipid peroxidation in hepatotoxicity, it is of interest to study the effect of carbon tetrachloride and antioxidants on the enzymatic hydroxylation of tetralin in rat liver homogenate. The perfused rat liver homogenate IOOOO × g supernatant fraction was used as the enzyme source. Incubations were carried out in a Warburg vessel, shaken at 37 °. The medium contained the liver enzyme, NADP ÷, glucose 6-phosphate, glucose6-phosphate dehydrogenase, and tetralin in 2/~1 acetone. Carbon tetrachloride and/or antioxidants were used where indicated. Sufficient o.I M phosphate buffer (pH 7.4) was used to make a total volume of 5 ml. After incubation, each mixture was extracted directly with 3o ml diethyl ether 3 times. The ether extract was dried over anhydrous Na2SO 4 and distilled to a small volume, which was then concentrated to about 5o/~1 under nitrogen. The amount of tetralin and its enzymatic products were determined by gas-liquid chromatography either directly or, in case of tetralol, as acetate. Acetylation of tetralol was carried out with I ,/~l of acetic anhydride and o.i #1 of pyridine overnight at room temperature. The gas-liquid chromatography instrument was equipped with a flame ionization detector as described previously n. The enzymatic conversion of tetralin to tetralol in rat liver IOOOO × g supernatant fraction was found to be affected by the addition of carbon tetrachloride. As shown in Fig. I, at low concentrations, carbon tetrachloride stimulated the conversion of tetralin to tetralol. With a concentration of 1.4 mg/ml, carbon tetrachloride stimulated the activity of the hydroxylation of tetralin 2-3-fold. However, with concentrations higher than 1.4 mg/ml, the degree of stimulation on tetralin hydroxylation decreased. Fig. 2 shows the effect of time of preincubation with different concentrations of carbon tetrachloride on the conversion of tetralin to tetralol. High concentration of carbon tetrachloride and long preincubation time lower the enzyme activity. This seems to indicate that carbon tetrachloride, per se, or lipid hydroperoxides induced by it m a y damage the hydroxylation enzyme in the liver microsomes. It has Biochim. Biophys. Acta, 192 (1969) 133-135

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been shown previously n that tetralin hydroperoxide, the intermediate in tetralin hydroxylation, could be accumulated by the removal of N A D P H generating system. With the omission of N A D P H generating system in both control and carbon tetrachloride-treated homogenate, the addition of carbon tetrachloride (1. 4 mg/ml) increased the amount of accumulated tetralin hydroperoxide by 50 %, as compared to control. The increase in hydroperoxide accumulation was small. However, repeated experiments indicated that it was, nevertheless, real. As there was always some endogenous N A D P H in the enzyme system, and as the reduction step was more than 30 times faster than the oxygenation step 11, higher yield of tetralin hydroperoxide cannot be expected. 20C \ 15C

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0 10 20 Prelncubat[on t}me with carbon tet r a c h l o r i d e ( rndn )

Fig. i. Conversion of t e t r a l i n to tetralol w i t h different c o n c e n t r a t i o n s of carbon tetrachloride. T e t r a l i n (0. 3 mM) was i n c u b a t e d w i t h different c o n c e n t r a t i o n s of c a r b o n tetrachloride, 0.8 m g N A D P +, 6 nlg Glc-6-P, i K o r n b e r g u n i t Glc-6-P d e h y d r o g e n a s e , a n d 17 m g p r o t e i n of e u z y m e p r e p a r a t i o n for 30 m i n in 5 ml p h o s p h a t e buffer (pH 7.4) a t 37 °. T h e a m o u n t of tetralol f o r m e d in t h e control e x p e r i m e n t was t a k e n as ioo. Fig. 2. T h e effect of t i m e s of p r e i n c u b a t i o n w i t h different c o n c e n t r a t i o n s of carbon tetrachloride on t h e c o n v e r s i o n of t e t r a l i n to tetralol. Different c o n c e n t r a t i o n s of carbon tetrachloride were i n c u b a t e d w i t h 0.8 m g N A D P +, 6 m g Glc-6-P, i K o r n b e r g u n i t Glc-6-P d e h y d r o g e n a s e , a n d 17 m g p r o t e i n of e n z y m e p r e p a r a t i o n for o, 5, io, or 20, m i n in 5 ml p h o s p h a t e buffer (pH 7-4) a t 37 °. T e t r a l i n (0. 3 mM) was t h e n a d d e d a n d i n c u b a t e d for a n o t h e r 3 ° m i n . T h e a m o u n t of tetralol formed w i t h o u t p r e i n c u b a t i o n a n d w i t h o u t a d d e d c a r b o n tetrachloride w a s t a k e n as IOO.

TABLE l EFFECTS

OF

CARBON

TETRACHLORIDE

AND

ANTIOXIDANTS

ON TETRALIN

HYDROXYLATION

T e t r a l i n (0. 3 mM) w a s i n c u b a t e d w i t h 17 m g p r o t e i n of e n z y m e p r e p a r a t i o n , 0.8 m g N A D P +, 6 m g Glc-6-P, I K o r n b e r g u n i t Glc-6-P d e h y d r o g e n a s e a n d w i t h or w i t h o u t c a r b o n tetrachloride, a n t i o x i d a n t for 3 ° m i n in 5 ml p h o s p h a t e buffer (pH 7.4) a t 37 %

Substance and concentration

Relative amount of tetralol formed

None

i oo

C a r b o n t e t r a c h l o r i d e (1.27 mg/ml)

2o6 33 31 38

N,N'-Diphenyl-p-phenylenediamine ( i

mM)

S o d i u m selenite (I mM) ~-Tocopherol a c e t a t e (I mM) C a r b o n t e t r a c h l o r i d e (1.27 m g / m l )

+ N,N'-diphenyl-p-phenylenediamine (i raM) C a r b o n t e t r a c h l o r i d e (1.27 m g / m l ) + s o d i u m selenite (i raM) C a r b o n t e t r a c h l o r i d e (1.27 m g / m l ) + a-tocopherol a c e t a t e (i raM)

Biochim. Biophys. Acta, 192 (1969) 133-135

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135

At the concentration studied, ~-tocopherol acetate, sodium selenite, and N,N'diphenyl-p-phenylenediamine all inhibited the enzymatic hydroxylation ( > 6 0 % ) (Table I). It has been established 13 that the function of antioxidants resides in their ability to trap radicals. If the stimulation of tetralin hydroxytation in rat liver homogenate by carbon tetrachloride involved a free radical, this stimulation effect should be counteracted by the addition of an antioxidant. ~-Tocopherol acetate and sodium selenite have been found to reduce the degree of stimulation by carbon tetrachloride to 50 %, whereas N,N'-diphenyl-p-phenylenediamine completely protects against carbon tetrachloride stimulation (Table I). This is in agreement with the results of GALLAGHER4 and DILUZlO14 that N,N'-diphenyl-p-phenylenediamine is the most efficient antioxidant in protecting against carbon tetrachloride induced fatty liver. Lipid hydroperoxidation can be radical in nature is. Since carbon tetrachloride in low concentrations stimulated both lipid hydroperoxidation and tetralin hydroxylation, while antioxidants inhibited both of these two reactions, it is reasonable to suggest that tetralin hydroxylation may also proceed via a radical mechanism similar to that of lipid peroxidation. These findings tend to support our previous proposed pathway that tetralin hydroperoxide is an intermediate in the hydroxylation of tetralin to tetralol in rat liver. This work was supported by National Institutes of Health Grant No. AM 09685 to C.C.

Department of Biochemistry, Northwestern University Medical School, Chicago, Ill. 6o6H (U.S.A.) I 2 3 4 5 6 7 8 9 IO II 12 13 14 15

CHIN-CHUNG L I N * CHIADAO CHEN

D. STETTEN, JR. AND J. SALCEDO, J. Biol. Chem., 156 (1944) 27. B. L. HARDIN, Ind. Med. Surg., 23 (1954) 93. E. L. HOVE, Arch. Biochem., 17 (1948) 467 . C. H. GALLAGHER,Australian J. Exptl. Biol. Med. Sci., 4 ° (1962) 241. N. R. DILUZI0 AND F. COSTALE, Exptl. Mol. Pathol., 4 (1965) 141. H. ZALKIN, A. L. TAPPEL AND J. P. JORDAN, Arch. Biochem. Biophys., 91 (196o) 117. M. COMPORTI, C. SACCOCCI AND M. U. DIAZANI, Enzymologia, 29 (1965) 185. R. O. RECKNAGEL AND A. K. GHOSHAL, Federation Proc., 24 (1965) 299. T. F. SLATER, Nature, 2o9 (1966) 36. R. O. RECKNAGEL AND A. K. GHOSHAL, Lab. Invest., 15 (1966) 132. C. CHEN AND C. C. LIN, Biochim. Biophys. Aeta, 17o (1968) 366. A. ROBERTSON AND W. A. WATERS, Trans. Faraday Soc., 42 (1946) 2Ol. J. L. BOLLAND AND H. TEN, Trans. Faraday Soc., 43 (1947) 2Ol. N. R. DILuZlO, Physiologist, 6 (1963) 169. K. LUNDBERG, in H. W. SCHULTZ, E. A. DAY AND R. O. SINNHUBER,Syrup. on Food; Lipids and Their Oxidation, A v i P u b l i s h i n g Co, W e s t p o r t , Conn. U.S.A., 1962.

Received July Ioth, 1969 * P r e s e n t a d d r e s s : D i v i s i o n of Biological Research, Schering Corporation, Bloomfield, N.J., U.S.A.

Biochim. Biophys. Aeta, 192 (I969) 133-135