TOXICOLOGY AND APPLIED PHARMACOLOGY 71, 54-58 (1983)

Enhanced Inhibition of Hepatic Microsomal Calcium Pump Activity by CC& Treatment of Isopropanol-Pretreated Rats

LEONMOORE' ANDPRABHATIRAY

Department of Pharmacology, Unijbrmed Services University of the Health Sciences, &the&, Maryhd 20814

Received January 14, 1983; accepted May 31, 1983

Enhanced Inhibition of Hepatic Microsomal Calcium Pump Activity by CC& Treatment of Isopropanol-Pretreated Rats. MOORE, L., AND RAY, P. (1983). Toxicol. Appl. Pharmacol. 71, 54-58. Pretreatment of rats with isopropanol enhanced both hepatotoxicity and calcium pump inhibition after Ccl, exposure in vivo or in vitro. Animals were given isopropanol (I .25 ml/kg) 18 hr before Ccl, (0.01 to 1.0 ml/kg). Ccl, hepatotoxicity, judged as increased appearance of glutamic-pyruvate transaminase in serum, was enhanced by isopropanol pretreatment. Pretreatment of rats with isopropanol made CC4 as much as 20- to 30-fold more potent as an inhibitor of the calcium pump. Inhibition of another endoplasmic reticulum enzyme, glucose Gphosphatase. was also enhanced by isopropanol pretreatment. In contrast to the effect of CC4 in control animals, in isopropanol-pretreated rats given CC&, depletion of liver glutathione was observed. Altered CC4 metabolism in isopropanol-pretreated animals may result in production of increased amounts of phosgene (or other metabolites) responsible for inhibition of the liver microsome calcium pump and glutathione depletion.

Ccl4 is metabolized to a hepatotoxin, and pre- treatment of rats with selected compounds that enhance drug metabolism by the hepatic, cy- tochrome P-450, mixed function oxidase sys- tem (MFOS) enhances hepatotoxicity of CCL+. Likewise inhibition of the MFOS decreases CC& hepatotoxicity. Recently it has been sug- gested that metabolism of CCL, was associated with loss of calcium pump activity in a liver microsomal fraction (Lowery et al., 1981a). One would expect pretreatments that modify the MFOS also should modify the effect of CC4 on microsomal calcium pump activity. With certain pretreatments, the expected effect has been observed (Moore, 1980, 1982a,b). Isopropanol enhances Ccl4 hepatotoxicity (Traiger and Plaa, 197 1) and alters Ccl4 me- tabolism (Harris and Anders, 198 1; Reynolds et al., 1982). However, another report sug-

’ To whom correspondence should he addressed.

gested that even when isopropanol pretreat- ment enhanced CC&-induced hepatotoxicity it did not enhance CC&-induced loss of mi- crosomal calcium pump activity (Lowery et al., 198 1 b). The interaction between isopro- panol and Ccl4 has been reinvestigated, and this report suggests that the interaction can potentiate CCkinduced inhibition of micro- somal calcium pump activity.

METHODS

Animals and Ccl, administration. Male Sprague-Daw- ley rats (175 to 225 g, Taconic Farms) were maintained on a IZhr-light and dark schedule. Animals were allowed free access to feed (Agway, Northborough, Mass.) and water throughout the experiment. Isopropanol was diluted in water (25% isopropanol) and administered po as a single dose (I .25 ml isopropanol/kg) 18 hr before ip adminis- tration of corn oil or CC& diluted in corn oil (0.01 to 1 ml CC&/kg). Animals were killed 1 min to 24 hr later.

Assays. As previously described (Moore 1980, 1982a) calcium pump activity was determined in the microsomal fraction. The ability of microsomes to actively sequester

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54

CCl4, ISOPROPANOL, AND CALCIUM PUMP 55

45Ca’+ was used to measure pump activity. The isolation procedure was modified when microsomes were to be incubated in vitro with CC& and a NADPH generating system previously described (Ray and Moore, 1982).

Activity of glutamate-pyruvate transaminase in serum (SGPT) was determined with a reagent kit (Sigma Chem- ical Co., Inc., St. Louis, MO.). Activity of glutamate-py- ruvate transaminase in liver (GPT) was determined by substituting a diluted liver cytosol (supematant fraction from 105,OOOg for 60-min centrifugation) for serum in the SGPT assay. Glucose 6-phosphatase (G6Pase) activity was determined in microsomal preparations as described by Aronson and Touster (1974). Glutathione (GSH) levels were determined as non-protein sulfhydryls with 5,5’-di- thiobis-(2-nitrobenzoic acid) as described by Jaeger ef al. ( 1974). GSH levels were determined at periods from 1 min to I hr after Ccl, administration.

Two means were considered different when compared with the Student f test (p r 0.05. two tailed).

RESULTS

SGPT activity, 24 hr after CCL, adminis- tration, was used to evaluate CQ-induced hepatotoxicity in control and isopropanol- pretreated rats. SGPT activity was unaltered by isopropanol pretreatment (control 22 f 4.2 IU/liter, isopropanol 20 + 3.0 W/liter). Liver GPT activity was not altered by isopropanol pretreatment (control, 10260 t 1032 IU/g liver: isopropanol, 10260 ? 786 W/g liver). In control animals SGPT activity was signif- icantly increased only at 0.3 and 1 ml CClJ kg. In the control group, CC& (1 ml/kg) ele- vated SGPT activity ninefold. After isopro- panol pretreatment, CC& administration sig- nificantly increased SGPT activity at all Ccl4 doses tested. Activity increased 5-fold at 0.0 1 ml Ccl, and 55fold at 1 ml CC&/kg.

Pretreatment of rats with a single dose of isopropanol did not alter the amount of mi- crosomal protein/g liver. G6Pase activity, or calcium pump activity in microsomal prep- arations (data not shown). Compared to prep- arations from normal rats given CC14, signif- icantly less calcium pump activity was found in microsomal preparations isolated from iso- propanol-pretreated rats (Fig. 1). Displace- ment of the dose-response curve along the X- axis would suggest an approximately 20-fold

I t I 1 1

0.01 0.03 0.1 0.3 1.0

Ccl, (ml/kg)

FIG. I. Effect of various CC& doses on liver microsomal calcium pump activity in a normal (0) and isopropanol- pretreated (0) animals. Treatment of the animals, mi- crosome preparation, and determination of calcium pump activity are described in Methods. Microsomes were pre- pared from animals killed 1 hr after Ccl., administration. Data represent ,? t SE for the determination in groups of five to seven animals. Calcium pump activity was 247 + 47.2 nmol Ca/mg protein/30 min in control preparations and 278 * 56.3 in the isopropanol-pretreated control group.

increase in CCI, potency (evaluated at 0.01 ml/kg in the isopropanol-pretreated animals). If the effect of isopropanol was evaluated as increased inhibition at a specific dosage, dis- placement along the Y-axis demonstrates that inhibition increased fourfold at 0.0 1 ml Ccl,,/ kg and twofold at 0.03 ml CCL,/kg. Another enzyme activity localized in endoplasmic re- ticulum also undergoes significantly greater losses in isopropanol-pretreated rats. G6Pase activity was inhibited 23 f 6% 1 hr after Ccl, (0.01 ml/kg) in a group of control rats and 54 + 5% in a group of isopropanol-pretreated rats. Similar results were observed when cal- cium pump activity and G6Pase activity were determined in microsomal preparations iso- lated from rat liver 24 hr after CC& admin- istration.

The time course of loss of microsomal cal- cium pump activity was investigated. In these experiments equitoxic doses of CC& were ad- ministered to control and isopropanol-pre-

56 MOORE AND RAY

treated rats. These doses produced near max- imal inhibition of the pump within 1 hr. With both groups of animals loss of calcium pump activity occurred at approximately the same rate (Fig. 2). G6Pase activity was slightly de- creased 30 min after these doses of CC14. GSH levels did not change or were slightly elevated during the first 30 min after CC4 adminis- tration. However, by 1 hr after CC4 admin- istration a significant decrease of GSH had occurred only in the isopropanol-pretreated Ccl,-treated group (Fig. 2).

To further characterize this effect of CC1,

MINUTES

FIG. 2. Time course of effects of CC& on calcium pump activity. Animals received equitoxic doses of CCL, 3.0 ml CC&/kg in control (filled symbols), and 0.3 ml CC&/ kg in isopropanol-pretreated (open symbols) animals. Ex- perimental details for determination of microsomal cal- cium pump activity (circles), G6Pase activity (triangles), and GSH levels (squares) are provided in Methods. Data represent x + SE for samples from groups of three animals. Calcium pump activity of liver microsomes from control animals was 220 f 30.1 in normal and 24 1 f 43.5 nmol Ca/mg protein in isopropanol-pretreated groups. G6Pase activity of liver microsomes from control animals was 5.2 & 0.8 in normal and 5.2 + 1.3 pmol PO,/mg protein/20 min in isopropanol-pretreated animals. GSH content of livers of control animals was 1.43 + 0.17 in normal and 1.27 f 0.18 mg GSH/g liver in isopropanol-pretreated groups.

administration in the isopropanol-pretreated group, the dose-response relationship between CC4 administration and GSH level was ex- amined 1 hr after CC4 administration. There was no effect of isopropanol pretreatment on GSH levels (data not shown). At the lowest CC4 dose tested, GSH levels were unchanged or slightly elevated in both groups of animals when compared to the respective control. However at all higher doses of CC&, GSH levels in the two groups of rats differed. In animals that received no pretreatment, all doses of CC4 produced little effect on GSH content in the liver. But, 0.03 ml CC&/kg and higher doses reduced liver content of GSH to between 60 and 70% of control in animals pretreated with isopropanol (Fig. 3).

CCL,-induced loss of liver microsomal cal- cium pump activity in vitro is NADPH de- pendent and presumably this dependence re- flects activation of CCL by the MFOS (Lowery et al., 198 la). Microsomes from control and isopropanol-pretreated rats were incubated with CC& and an NADPH generating system in vitro. CC4 produced significantly more in- hibition of the calcium pump when incubated with microsomal preparations isolated from isopropanol-pretreated rats (Table 1).

DISCUSSION

Reexamination of the possible interaction between isopropanol and CC& on calcium pump activity demonstrated that isopropanol did increase Ccl,-induced loss of calcium pump activity after in vivo or in vitro exposure. The data suggested that the interaction be- tween isopropanol and CC& results in a greater amount of damage to the calcium pump sys- tem and not simply an increased rate of pro- duction of damage. Loss of calcium pump activity occurred before loss of an ER marker enzyme, G6Pase. Thus different enzymatic activities in the ER were lost at different rates. This difference may suggest a selective tem- poral or topological attack of certain activities in the ER. The data are compatible with ob- servations that suggest that isopropanol alters

CC&, ISOPROPANOL, AND CALCIUM PUMP 37

4 Wkn)

FIG. 3. Effect of CC4 on liver GSH levels in normal (0) and isopropanol-pretreated (0) rats. Experimental de- tails are provided in Methods. Isopropanol pretreatment did not significantly (p > 0.05) alter liver GSH levels (2.0 + 0.42 mg/g liver, control vs. 3.1 + 0.77 mg/g liver, iso- propanol pretreated). Data represent x + SE for the de- termination in groups of four to six animals.

the route of CCL metabolism both in vivo and in vitro (Harris and Anders, 198 1: Reynolds et al., 1982).

There are several differences between this study and the study reported by Lowery et al. ( 198 1 b) that may explain the differing effect of CC& on calcium pump activity in liver mi- crosomes isolated from isopropanol-pretreated

rats. Perhaps the most significant was the dif- ferent feeding schedules employed before and after isopropanol administration. Lowery et al. ( 198 1 b) fasted animals for 14 hr before and 18 hr after isopropanol. Fasting markedly in- creases CCL, hepatotoxicity (Krishnan and Stenger, 1966; Jaeger et al.. 1975: Nakajima and Sato, 1979). In the present study. animals were allowed free access to feed and water throughout the experimental procedure. It is possible that CC&-induced inhibition of the liver ER calcium pump was maximally stim- ulated by fasting in the study reported by Lowery et al., and thus no effect of isopropanol was observed. Comparison of Fig. 1 (this pa per) and the data of Lowery et al. (198 1 b, Table 2, saline pretreatment) at 0.1 ml CC&/ kg for 60 min suggests that fasting dramatically increased Ccl,-induced calcium pump inhi- bition. Other differences of animal preparation include the isopropanol dose employed and the route of CCL administration.

Interestingly, isopropanol pretreatment al- tered the response of liver GSH levels to Cc’lJ . Administration of CC& to phenobarbital-in- duced rats did not lower liver GSH levels (Docks and Krishna. 1976). A similar result was confirmed for normal rats in the present study. However, administration of CC& in doses of 0.03 to 1 ml/kg to isopropanol-pre- treated rats resulted in a 40% reduction of liver GSH (data not shown). This reduction

TABLE 1

INHIBITION OF LIVER MICROSOME CALCIUM FTJMP BY Ccl., IN C’ITRO

Isopropanol

Microsome calcium Pump activity

Control

195 * 17

cc14

117 f 11 (39 + 5)

Control

172 ‘- 17

CC-I, ---

27.0 i 6.9 (85 k 4)

Note. Microsomes were isolated from control or isopropanol-pretreated (I .25 ml/kg I8 hr) rats as described in Methods. Microsomes at a protein concentration of 2.0 to 2.5 mg protein/ml were incubated with a NADPH generating system. CCL, dissolved in DMSO, was added to the incubation medium (37 f I “C) for 5 min. The maximum volume of DMSO added was 5 &ml. This concentration of DMSO did not affect calcium pump activity (data not shown). The final concentration of CC& was 0.01 &ml. Data are the mean of pump activity (nmol Ca”/mg protein/30 min) t SE for the determination on preparations from six or seven animals.

58 MOORE AND RAY

occurred between 30 and 60 min after CCb administration. Phosgene is thought to be the CHCIJ metabolite responsible for GSH de- pletion after CHC13 administration (Pohl et al., 1980). Isopropanol pretreatment selec- tively induces production of phosgene from Ccl4 (Harris and Anders, 198 1). It is possible that enhanced phosgene production from CC4 was responsible for liver GSH depletion ob- served in isopropanol-pretreated rats. It is not clear if the effect on liver GSH contributed to or modified CC&-induced hepatotoxicity.

At least part of the effect of isopropanol responsible for inhibition of calcium pump activity appears localized in the microsomal fraction. Enhanced inhibition of calcium pump activity was observed when microsomes isolated from isopropanol-pretreated rats were incubated with CC& and an NADPH gener- ating system in vitro. Enhanced in vitro cal- cium pump inhibition in microsomes isolated from isopropanol-pretreated rats would sug- gest that more of a reactive CC4 metabolite has been produced in vitro, presumably by induction of a specific cytochrome P-450 (Sipes et al., 1973; Harris and Anders, 1981).

ACKNOWLEDGMENTS

The authors thank Michael Fraley and Beth Leary for excellent technical assistance. This research was supported by National Institutes of Health Grant ES 0269 I.

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