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Enhanced inhibition of hepatic microsomal calcium pump activity by CCl4 treatment of isopropanol-pretreated rats
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
71, 54-58 (1983)
Enhanced Inhibition of Hepatic Microsomal Calcium Pump Activity by CC& Treatment of Isopropanol-Pretreated Rats LEONMOORE' Department
of Pharmacology,
Unijbrmed
Received
Services
January
ANDPRABHATIRAY University
of the Health
14, 1983; accepted
May
Sciences,
31,
&the&,
Maryhd
20814
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 pretreatment of rats with selectedcompounds that enhance drug metabolism by the hepatic, cytochrome P-450, mixed function oxidase system (MFOS) enhanceshepatotoxicity of CCL+. Likewise inhibition of the MFOS decreases CC& hepatotoxicity. Recently it hasbeen suggestedthat 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, 1971) and alters Ccl4 metabolism (Harris and Anders, 1981; Reynolds et al., 1982). However, another report sug-
’ To whom
0041-008X/83 Coprnght All
rights
ic
1983
of reprodurtmn
correspondence
should
$3.00 by Academx m any
he addressed.
54 Press.
Inc.
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reserved
gested that even when isopropanol pretreatment enhanced CC&-induced hepatotoxicity it did not enhance CC&-induced loss of microsomal calcium pump activity (Lowery et al., 1981b). The interaction between isopropanol and Ccl4 has been reinvestigated, and this report suggeststhat the interaction can potentiate CCkinduced inhibition of microsomal calcium pump activity. METHODS Animals and Ccl, administration. Male Sprague-Dawley 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 administration 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
CCl4,
ISOPROPANOL,
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 Chemical Co., Inc., St. Louis, MO.). Activity of glutamate-pyruvate 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’-dithiobis-(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, administration, was used to evaluate CQ-induced hepatotoxicity in control and isopropanolpretreated 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 significantly increased only at 0.3 and 1 ml CClJ kg. In the control group, CC& (1 ml/kg) elevated SGPT activity ninefold. After isopropanol pretreatment, CC& administration significantly 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 microsomal protein/g liver. G6Pase activity, or calcium pump activity in microsomal preparations (data not shown). Compared to preparations from normal rats given CC14, significantly less calcium pump activity was found in microsomal preparations isolated from isopropanol-pretreated rats (Fig. 1). Displacement of the dose-response curve along the Xaxis would suggest an approximately 20-fold
AND
CALCIUM
55
PUMP
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 isopropanolpretreated (0) animals. Treatment of the animals, microsome preparation, and determination of calcium pump activity are described in Methods. Microsomes were prepared 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, displacement 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 reticulum 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 calcium pump activity and G6Pase activity were determined in microsomal preparations isolated from rat liver 24 hr after CC& administration. The time course of loss of microsomal calcium pump activity was investigated. In these experiments equitoxic doses of CC& were administered to control and isopropanol-pre-
MOORE
56
treated rats. These doses produced near maximal 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 decreased 30 min after these doses of CC14. GSH levels did not change or were slightly elevated during the first 30 min after CC4 administration. However, by 1 hr after CC4 administration a significant decrease of GSH had occurred only in the isopropanol-pretreated Ccl,-treated group (Fig. 2). To further characterize this effect of CC1,
AND RAY
administration in the isopropanol-pretreated group, the dose-response relationship between CC4 administration and GSH level was examined 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 calcium pump activity in vitro is NADPH dependent and presumably this dependence reflects 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 inhibition of the calcium pump when incubated with microsomal preparations isolated from isopropanol-pretreated rats (Table 1). DISCUSSION
MINUTES
FIG. 2. Time course of effectsof 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. Experimental details for determination of microsomal calcium 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.
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 between isopropanol and CC& results in a greater amount of damage to the calcium pump system and not simply an increased rate of production 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 temporal or topological attack of certain activities in the ER. The data are compatible with observations that suggest that isopropanol alters
CC&,
4
ISOPROPANOL,
AND
FIG. 3. Effect of CC4 on liver GSH levels in normal (0) and isopropanol-pretreated (0) rats. Experimental details 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, isopropanol pretreated). Data represent x + SE for the determination 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 1b) that may explain the differing effect of CC& on calcium pump activity in liver microsomes isolated from isopropanol-pretreated
TABLE OF LIVER MICROSOME
37
PUMP
rats. Perhaps the most significant was the different feeding schedules employed before and after isopropanol administration. Lowery et al. ( 1981b) fasted animals for 14 hr before and 18 hr after isopropanol. Fasting markedly increases CCL, hepatotoxicity (Krishnan and Stenger, 1966; Jaeger et al.. 1975: Nakajima and Sato, 1979). In the present study. animals were allowed free accessto feed and water throughout the experimental procedure. It is possible that CC&-induced inhibition of the liver ER calcium pump was maximally stimulated 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 1b, Table 2, saline pretreatment) at 0.1 ml CC&/ kg for 60 min suggeststhat fasting dramatically increased Ccl,-induced calcium pump inhibition. Other differencesof animal preparation include the isopropanol dose employed and the route of CCL administration. Interestingly, isopropanol pretreatment altered the responseof liver GSH levels to Cc’lJ . Administration of CC& to phenobarbital-induced 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 dosesof 0.03 to 1 ml/kg to isopropanol-pretreated rats resulted in a 40% reduction of liver GSH (data not shown). This reduction
Wkn)
INHIBITION
CALCIUM
1
CALCIUM
FTJMP BY Ccl., IN
C’ITRO
Isopropanol Control Microsome calcium Pump activity
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
occurred between 30 and 60 min after CCb administration. Phosgene is thought to be the CHCIJ metabolite responsible for GSH depletion after CHC13 administration (Pohl et al., 1980). Isopropanol pretreatment selectively 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 observed 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 generating system in vitro. Enhanced in vitro calcium pump inhibition in microsomes isolated from isopropanol-pretreated rats would suggest 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.
REFERENCES ARONSON, N. N., AND TOUSTER, 0. (1974). Isolation of liver plasma membrane fragments in isotonic sucrose. Methods
in Enzymology
31,A,
93-94.
DOCKS, E. L., AND KRISHNA, G. (1976). The role of glutathione in chloroform induced hepatotoxicity. Exp. Mol.
Pathol.
24, 13-22.
HARRIS, R. N.. AND ANDERS, M. W. (1981). Phosgene: A possible role in the potentiation ofcarbon tetrachloride hepatotoxicity by 2-propanol. Life Sci. 29, 503-507.
AND RAY JAEGER, R. J., CONNOLLY, R. B., AND MURPHY, S. D. (1974). Effect of 18 hr fast and glutathione depletion on I,1 -dichloroethylene-induced hepatotoxicity and lethality in rats. Exp. Mol. Pathol. 20, 187-198. JAEGER, R. J., CONOLLY, R. B., AND MURPHY, S. D. (1975). Short-term inhalation toxicity of halogenated hydrocarbons. Effects on fasting rats. Arch. Environ. Health
30, 26-30.
KRISHNAN, N., AND STENGER, R. (1966). Effects of starvation on the hepatotoxicity of carbon tetrachloride. Amer.
J. Pathol.
49, 239-246.
LOWERY, K., GLENDE, E. A., JR., AND RECKNAGEL, R. 0. ( 198 la). Destruction of liver microsomal calcium pump activity by carbon tetrachloride and bromotrichloromethane. Biochem. Pharmacol. 30, 139- 140. LOWERY, K., GLENDE, E. A., JR., AND RECKNAGEL, R. 0. ( 198 1b). Failure of ethanol or isopropanol pretreatment to affect carbon tetrachloride-induced inhibition of hepatic microsomal calcium pump activity. Drug
Chem.
Toxicol.
4, 263-273.
MOORE, L. (1980). Inhibition of liver-microsome calcium pump by in vivo administration of Ccl+ CHC& and I, I -dichloroethylene (vinylidene chloride). Biochem. Pharmacol.
29,2505-25
11.
MOORE, L. (1982a). 1,I-Dichloroethylene inhibition of liver endoplasmic reticulum calcium pump function. Biochem. Pharmacol. 31, 1463-1465. MOORE, L. (1982b). Carbon disulfide hepatotoxicity and inhibition of liver microsome calcium pump. Biochem. Pharmacol.
31, 1466-1467.
NAKAJIMA, T., AND SATO, A. (1979). Enhanced activity of liver drug-metabolizing enzymes for aromatic and chlorinated hydrocarbons following food deprivation. Toxicol.
Appl. Pharmacol.
50, 549-556.
POHL, L. R., MARTIN, J. L., ANDGEROGE, J. W. (1980). Mechanism of metabolic activation of chloroform by rat liver microsomes. Biochem. Pharmacol. 29, 327 l3276.
RAY, P., AND MOORE, L. (1982). l,l-Dichloroethylene inhibition of liver microsomal calcium pump in vitro. Arch.
Biochem.
Biophys.
218, 26-30.
REYNOLDS, E. S., MOSLEN, M. T.. AND TREINEN, R. J. (1982). Isopropanol enhancement of carbon tetrachloride metabolism in vivo. Life Sci. 31, 661-669. SIPES, I. G., STRIPP, B., KRISHNA, G., MALING, H. M., AND GILLETTE, J. R. (1973). Enhanced hepatic microsomal activity by pretreatment of rats with acetone or isopropanol. Proc. Sot. Exp. Biol. Med. 142, 237-240. TRAIGER, G. J., AND PLAA, G. L. (197 1). Differences in the potentiation of carbon tetrachloride in rats by ethanol and isopropanol pretreatment. Toxicol. Appl. Pharmacol. 20, 105-l 12.
AND
APPLIED
PHARMACOLOGY
71, 54-58 (1983)
Enhanced Inhibition of Hepatic Microsomal Calcium Pump Activity by CC& Treatment of Isopropanol-Pretreated Rats LEONMOORE' Department
of Pharmacology,
Unijbrmed
Received
Services
January
ANDPRABHATIRAY University
of the Health
14, 1983; accepted
May
Sciences,
31,
&the&,
Maryhd
20814
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 pretreatment of rats with selectedcompounds that enhance drug metabolism by the hepatic, cytochrome P-450, mixed function oxidase system (MFOS) enhanceshepatotoxicity of CCL+. Likewise inhibition of the MFOS decreases CC& hepatotoxicity. Recently it hasbeen suggestedthat 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, 1971) and alters Ccl4 metabolism (Harris and Anders, 1981; Reynolds et al., 1982). However, another report sug-
’ To whom
0041-008X/83 Coprnght All
rights
ic
1983
of reprodurtmn
correspondence
should
$3.00 by Academx m any
he addressed.
54 Press.
Inc.
form
reserved
gested that even when isopropanol pretreatment enhanced CC&-induced hepatotoxicity it did not enhance CC&-induced loss of microsomal calcium pump activity (Lowery et al., 1981b). The interaction between isopropanol and Ccl4 has been reinvestigated, and this report suggeststhat the interaction can potentiate CCkinduced inhibition of microsomal calcium pump activity. METHODS Animals and Ccl, administration. Male Sprague-Dawley 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 administration 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
CCl4,
ISOPROPANOL,
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 Chemical Co., Inc., St. Louis, MO.). Activity of glutamate-pyruvate 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’-dithiobis-(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, administration, was used to evaluate CQ-induced hepatotoxicity in control and isopropanolpretreated 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 significantly increased only at 0.3 and 1 ml CClJ kg. In the control group, CC& (1 ml/kg) elevated SGPT activity ninefold. After isopropanol pretreatment, CC& administration significantly 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 microsomal protein/g liver. G6Pase activity, or calcium pump activity in microsomal preparations (data not shown). Compared to preparations from normal rats given CC14, significantly less calcium pump activity was found in microsomal preparations isolated from isopropanol-pretreated rats (Fig. 1). Displacement of the dose-response curve along the Xaxis would suggest an approximately 20-fold
AND
CALCIUM
55
PUMP
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 isopropanolpretreated (0) animals. Treatment of the animals, microsome preparation, and determination of calcium pump activity are described in Methods. Microsomes were prepared 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, displacement 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 reticulum 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 calcium pump activity and G6Pase activity were determined in microsomal preparations isolated from rat liver 24 hr after CC& administration. The time course of loss of microsomal calcium pump activity was investigated. In these experiments equitoxic doses of CC& were administered to control and isopropanol-pre-
MOORE
56
treated rats. These doses produced near maximal 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 decreased 30 min after these doses of CC14. GSH levels did not change or were slightly elevated during the first 30 min after CC4 administration. However, by 1 hr after CC4 administration a significant decrease of GSH had occurred only in the isopropanol-pretreated Ccl,-treated group (Fig. 2). To further characterize this effect of CC1,
AND RAY
administration in the isopropanol-pretreated group, the dose-response relationship between CC4 administration and GSH level was examined 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 calcium pump activity in vitro is NADPH dependent and presumably this dependence reflects 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 inhibition of the calcium pump when incubated with microsomal preparations isolated from isopropanol-pretreated rats (Table 1). DISCUSSION
MINUTES
FIG. 2. Time course of effectsof 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. Experimental details for determination of microsomal calcium 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.
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 between isopropanol and CC& results in a greater amount of damage to the calcium pump system and not simply an increased rate of production 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 temporal or topological attack of certain activities in the ER. The data are compatible with observations that suggest that isopropanol alters
CC&,
4
ISOPROPANOL,
AND
FIG. 3. Effect of CC4 on liver GSH levels in normal (0) and isopropanol-pretreated (0) rats. Experimental details 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, isopropanol pretreated). Data represent x + SE for the determination 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 1b) that may explain the differing effect of CC& on calcium pump activity in liver microsomes isolated from isopropanol-pretreated
TABLE OF LIVER MICROSOME
37
PUMP
rats. Perhaps the most significant was the different feeding schedules employed before and after isopropanol administration. Lowery et al. ( 1981b) fasted animals for 14 hr before and 18 hr after isopropanol. Fasting markedly increases CCL, hepatotoxicity (Krishnan and Stenger, 1966; Jaeger et al.. 1975: Nakajima and Sato, 1979). In the present study. animals were allowed free accessto feed and water throughout the experimental procedure. It is possible that CC&-induced inhibition of the liver ER calcium pump was maximally stimulated 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 1b, Table 2, saline pretreatment) at 0.1 ml CC&/ kg for 60 min suggeststhat fasting dramatically increased Ccl,-induced calcium pump inhibition. Other differencesof animal preparation include the isopropanol dose employed and the route of CCL administration. Interestingly, isopropanol pretreatment altered the responseof liver GSH levels to Cc’lJ . Administration of CC& to phenobarbital-induced 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 dosesof 0.03 to 1 ml/kg to isopropanol-pretreated rats resulted in a 40% reduction of liver GSH (data not shown). This reduction
Wkn)
INHIBITION
CALCIUM
1
CALCIUM
FTJMP BY Ccl., IN
C’ITRO
Isopropanol Control Microsome calcium Pump activity
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
occurred between 30 and 60 min after CCb administration. Phosgene is thought to be the CHCIJ metabolite responsible for GSH depletion after CHC13 administration (Pohl et al., 1980). Isopropanol pretreatment selectively 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 observed 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 generating system in vitro. Enhanced in vitro calcium pump inhibition in microsomes isolated from isopropanol-pretreated rats would suggest 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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