Toxicology Letters, 52 (1990) 179-190 Elsevier
179
TOXLET 02352
Hepatic mitochondrial function and lysosomal enzyme activity in ethanol-potentiated aflatoxin B 1 hepatotoxicity
Chaivat Toskulkao and Thirayudh Glinsukon Department of Physiology, Faculty of Science, Mahidol University, Bangkok (Thailand)
(Received 8 May 1989) (Accepted 20 February 1990) Key words: Aflatoxin B,; Ethanol; Mitochondrial
enzymes; Lysosomal enzymes
SUMMARY The possible role of hepatic mitochondrial function and lysosomal enzyme activity in ethanol-enhanced aflatoxin B, (AFB,) hepatotoxicity was studied in male rats. Hepatic ATP content was significantly decreased in rats treated with ethanol (4.0 g/kg body wt.) and AFB, (2.0 mg/kg body wt.) compared with rats treated with AFB, alone at 12-72 h after AFB, administration. The decrease in hepatic ATP content was due to the decrease in the activity of NADH-cytochrome c reductase whereas cytochrome oxidase activity did not differ in rats treated with ethanol and AFB, when compared to AFB, alone. Total and free activities of hepatic lysosomal enzymes (glucuronidase, arylsulfatase and acid phosphatase) were significantly increased in rats treated with ethanol and AFB, at 24-36 h after AFB, administration when compared to AFB, alone. The increase in hepatic lysosomal enzyme activities correlated well with the increase in the lipid peroxide level of lysosomes in rats treated with ethanol and AFB,. These findings indicate that the decrease in hepatic mitochondrial respiratory enzyme activities and the increase in lipid peroxide level of lysosomes might lead to a decrease in hepatic ATP content, and that the increase in the activities of hepatic lysosomal enzymes, respectively, enhance the AFB, hepatotoxicity of ethanol.
INTRODUCTION
The intracellular ATP content is considered to be an important energy source in regulating the cations pump and cellular volume. It has been shown that tissue injury or necrosis is related to the degree of inhibition of hepatic mitochondrial respiratory
Address for correspondence: Dr. Chaivat Toskulkao, Department of Physiology, Faculty of Science, Mahi-
do1 University, Rama VI Road, Bangkok 10400, Thailand. 0378-4274/90/S 3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
180
functions and the total ATP content [1,2]. Studies have shown that aflatoxin Bi (AFBi) induced a higher percentage inhibition of guinea-fowl liver mitochondrial respiration which was about 100 times lower than that reported for the rat. It has been also suggested that the degree and sites.of inhibition by aflatoxin depend on the toxin concentration. We hereby report a possible potentiated action of ethanol on AFBt hepatotoxicity with respect to hepatic mitochondrial respiratory enzyme activity and total ATP content. It is well known that the peroxidation of membrane lipids leads to loss of integrity of membrane structure and function [3,4]. Many investigators have shown that the lysis of membranes or subcellular organelles can often be attributed to lipid peroxidation in their membranes, and that the peroxidative damage of membranes is prevented by inhibitors of lipid peroxidation [5,6]. Breakdown of lysosomes and the release of degradative lysosomal enzymes into the surrounding tissue are obviously implicated in the hepatic necrosis and hemorrhage consistently observed with acutely toxic doses of AFBi [7]. In vitro treatment of rat lysosomal preparations with AFBi also produced a dose-dependent release of the marker enzymes [8]. Therefore, it is our aim to present a possible potentiated action of ethanol on AFBi hepatotoxicity with respect to the correlation between lysosomal lipid peroxidation and lysosomal enzyme activity. MATERIALS AND METHODS
Male Wistar rats (18&200 g) were supplied by the National Laboratory Animal Production Center (Mahidol University, Thailand). All animals were housed in an air-conditioned room at 25 f 2°C with a relative humidity of about 65 % and were provided with commercial rat chow and water ad libitum. AFBi and ethanol were purchased from Makor Chemical Co. (Israel) and E. Merck Co. (F.R.G.), respectively. Thiobarbituric acid (TBA), 1,1,3,3-tetramethoxypropane (TMP) and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). This study was divided into two major experiments as follows: Experiment
I
The effect of ethanol on the potentiation of AFBi hepatotoxicity was studied with respect to hepatic mitochondrial enzyme activity and total hepatic ATP content at various time intervals after AFBt administration. The rats were divided into 6 groups as follows: Groups I and II rats were treated with 4 oral doses of 4.0 g/kg body wt. water and ethanol (40%, v/v) at 48,45, 24 and 21 h prior to sacrifice by decapitation, respectively. Groups III and IV rats were pretreated with 4 oral doses of 4.0 g/kg body wt. water and ethanol at 48,45,24 and 21 h prior to a single i.p. administration of DMSO (0.5 g/kg body wt.), respectively. Groups V and VI rats were pretreated with 4 oral doses of water and ethanol at 48, 45, 24 and 21 h prior to a single ip.
181
administration of AFBi (2.0 mg/kg body wt.), respectively. After 12, 24, 36, 48 and 72 h of DMSO or AFBr administration, rats were sacrificed by decapitation. The livers of the rats were perfused with cold saline solution and rapidly removed and homogenized. The mitochondria were prepared [9] for determination of mitochondrial protein content [lo], NADH-cytochrome c reductase activity [9,11] and cytochrome oxidase activity [9,12]. Total ATP content in whole homogenates was determined (Sigma Kit No. 366UV). Experiment II The effect of ethanol on the potentiation of AFBi hepatotoxicity was studied with respect to hepatic lysosomal lipoxidation and the activities of lysosomal enzymes in lysosome and cytosol at various time intervals after AFB, administration. The rats were divided into 6 groups and pretreated with water or ethanol and with DMSO or AFBi as already described in Experiment I. After 12,24,36,48 and 72 h of DMSO or AFBi administration, the rats were sacrificed by decapitation. The livers were perfused with cold saline solution and rapidly removed and homogenized. Total hepatic protein was determined [lo] and total activities of lysosomal enzymes were determined in the homogenates. The lysosomal and cytosol fractions were isolated [13] for determination of lysosomal enzyme activities in lysosomes and for free lysosomal enzyme activities in cytosol, lysosomal protein and cytosolic protein [lo] and lysosoma1 lipid peroxide level in the lysosomal fraction [14]. Glucuronidase activity [15], acid phosphatase activity [ 161and arylsulfatase activity [ 171were taken as parameters of lysosomal enzyme activities. RESULTS
Hepatic mitochondrial protein content significantly decreased from 16.5 + 0.3 to 14.5 50.5 mg/g liver (PcO.01) in rats treated with AFB, when compared to control rats at 48 h after AFBi administration. The mitochondrial protein content was not significantly different in rats pretreated with ethanol and treated with AFB, when compared to rats treated with AFBr alone at various time intervals after AFBi administration [ 181.After AFBi administration at 72 h, the hepatic ,mitochondrial protein content gradually returned to the control level. Figures 1 and 2 illustrate the activities of hepatic respiratory enzymes of rats pretreated with ethanol and treated with AFBl as indicated by the activities of cytochrome oxidase and NADH-cytochrome c reductase, respectively. Ethanol treatment slightly decreased the activities of cytochrome oxidase and NADH-cytochrome c reductase at 48 h after the first dose of ethanol and they gradually returned to the control level. At 48 h after AFBi administration in rats pretreated with water, cytochrome oxidase activities decreased maximally from 1.20 + 0.03 to 0.88 + O.lO~mol/mg protein/min (P-C 0.01) when compared to control rats. Cytochrome oxidase activity gradually returned to the control level later than 72 h after AFB, administration.
182
1
1
0
G-Q
WATER - DMSO
w O---O
ETHANOL - DMSO
H
ETHANOL - AFBl
WATER
-
I 12 Time
AFBl
I 24 after
AFBt
I
I
36
46
admlnistration
I
72
Ch
1
Fig. 1. Time course of changes in hepatic mitochondrial cytochrome oxidase activity following ethanol and AFB, administration in rats. Rats were pretreated with 4 oral doses of water or ethanol (4.0 g/kg body wt.) prior to i.p. administration of AFB, (2.0 mg/kg body wt.). Rats were sacrificed at the time indicated. Values are means * SE of 5 rats.
Ethanol further decreased the activity of cytochrome oxidase, without statistical significance, from 0.88 f 0.10 to 0.76 f0.08 pmol/mg protein/min after 48 h of AFBt administration when compared to rats treated with AFBt alone. The activity of cytochrome oxidase was still lower than that of the control at 72 h after AFBt administration. In rats treated with AFB,, NADH-cytochrome c reductase activity decreased maximally from 0.504+0.039 to 0.356+0.019 pmol/mg protein/min (P-C 0.01) at 36 h after AFBi administration when compared to control rats. The activity of NADHcytochrome c reductase gradually returned to the control level about 72 h after AFBt administration. NADH-cytochrome c reductase activity was highly significantly decreased in rats pretreated with ethanol at various time intervals after AFBi administration and decreased maximally at 36 h after AFBi administration from 0.356 + 0.19 to 0.296 + 0.013 pmol/mg protein/min (P < 0.05) when compared to rats treated with AFBi alone. In ethanol- and AFBi-treated rats, the activity of NADH-cytochrome c reductase was still lower than that of the control level.
183
DMSO
O_-a
WATER -
w
ETHANOL -
0-Q
WATER -
H
ETHANOL - AFill
DMSO
AFBl
I
I
I
I
I
0
12
24
36
48
Time
after
AFBt
Fig. 2. Time course of changes in hepatic mitochondrial ethanol and AFB, administration
edmlnlstratlon
NADH-cytochrome
I
72 t h 1
c reductase activity following
in rats. Rats were pretreated with 4 oral doses of water or ethanol (4.0
g/kg body wt.) prior to i.p. administration
of AFB, (2.0 mg/kg body wt.). Rats were sacrificed at the time indicated. Values are means f SE of 5 rats. *PC 0.05, **P< 0.01, significantly different from water-AFB, and ethanol-AFB,.
The hepatic ATP content in rats pretreated with ethanol and treated with AFBt is shown in Figure 3. Ethanol treatment caused a slight decrease in ATP content at 48 h after the first dose but it gradually returned to the control level. At 48 h after AFBt administration in rats pretreated with water, the ATP content decreased maximally from 0.992 f 0.030 to 0.591+ 0.061 pmol/g liver (P < 0.001) when compared to the control rats. The hepatic ATP content gradually returned to the control level and then slightly increased from 1.013 f 0.036 to 1.321 f 0.094 pmol/g liver (PC 0.05) in rats treated with AFBt alone at 72 h. In rats pretreated with ethanol and treated with AFBt, the ATP content was highly significantly decreased in rats treated with AFBt alone at various time intervals after AFBt administration and decreased maximally at 48 h after AFBl administration from 0.591 f0.061 to 0.347f 0.058 pmol/g liver (P-C 0.05) when compared to rats treated with AFBt alone. At 72 h after AFBt administration in rats pretreated with ethanol, the hepatic ATP content was lower than that in rats treated with water or ethanol or AFBt alone. Figure 4 illustrates the lysosomal lipid peroxide level of rats pretreated with ethanol and AFBt. Ethanol treatment produced a significant increase in the lipid peroxide level of lysosomes at 48 h after the first dose of ethanol and it gradually returned to
184 1.4
1.2
k > =
o---O
IdATER -
N
ETHANOL -
b-4
L’ATER
CI
ETHANOL -
-
DMSO DW.0
AFBl MB1
0.8 t
I
1
0
I 24
I
12 Time
after
AFBt
I
I
36
48
admlnstration
I
72 t II 1
Fig. 3. Time course of changes in hepatic ATP content following ethanol and AFB, administration in rats. Rats were pretreated with 4 oral doses of water or ethanol (4.0 g/kg body wt.) prior to i.p. administration of AFB, (2.0 mg/kg body wt.). Rats were sacrificed at the time indicated. Values are means f SE of 5 rats. *PC 0.05, significantly different from water-AFB, and ethanol-AFB,.
the control level. At 24 h after AFBi administration in rats pretreated with water, the lysosomal lipid peroxide level increased maximally from 180.6 f 19.8 to 886.5+ 120.8 nmol MDA/100 mg protein (PC 0.001) when compared to the control rats and gradually returned to the control level at 48 h after AFBi administration. In rats pretreated with ethanol and AFBi, the lysosomal lipid peroxide level was slightly significantly increased at 12, 24 and 48 h after AFBi administration and increased maximally at 24 h after AFBi administration from 886.5+ 120.8 to 1418.7 + 191.3 nmol MDA/100 mg protein (P < 0.05) when compared to rats treated with AFBi alone. In ethanol- and AFBi-treated rats, the lysosomal lipid peroxide concentration returned to the control level later than 72 h after AFBt administration. The activities of hepatic free lysosomal enzymes of rats pretreated with ethanol and treated with AFBi are shown in Figures 5-7. Ethanol treatment caused a slight increase in the free activity of acid phosphatase (Fig. 5) and significantly increased the free activity of arylsulfatase (Fig. 6) and glucuronidase (Fig. 7) at 48 h after the first
185
1600 LVSOSOME e-0 WATER-
2oo0
12
Time
24 at ter AFB,
36
H
ETHANOL
m--Q
WATER
w--a
ETHANOL
MS0 - DMSO - AFBl - A+
48
administration
72 (h 1
Fig. 4. Time course of changes in lipid peroxide level in hepatic lysosomal fraction following ethanol and AFB, administration in rats. Rats were pretreated with 4 oral doses of water or ethanol (4.0 g/kg body wt.) prior to i.p. administration of AFB, (2.0 mg/kg body wt.). Rats were sacrificed at the time indicated. Values are means & SE of 5 rats. bZ’
179
TOXLET 02352
Hepatic mitochondrial function and lysosomal enzyme activity in ethanol-potentiated aflatoxin B 1 hepatotoxicity
Chaivat Toskulkao and Thirayudh Glinsukon Department of Physiology, Faculty of Science, Mahidol University, Bangkok (Thailand)
(Received 8 May 1989) (Accepted 20 February 1990) Key words: Aflatoxin B,; Ethanol; Mitochondrial
enzymes; Lysosomal enzymes
SUMMARY The possible role of hepatic mitochondrial function and lysosomal enzyme activity in ethanol-enhanced aflatoxin B, (AFB,) hepatotoxicity was studied in male rats. Hepatic ATP content was significantly decreased in rats treated with ethanol (4.0 g/kg body wt.) and AFB, (2.0 mg/kg body wt.) compared with rats treated with AFB, alone at 12-72 h after AFB, administration. The decrease in hepatic ATP content was due to the decrease in the activity of NADH-cytochrome c reductase whereas cytochrome oxidase activity did not differ in rats treated with ethanol and AFB, when compared to AFB, alone. Total and free activities of hepatic lysosomal enzymes (glucuronidase, arylsulfatase and acid phosphatase) were significantly increased in rats treated with ethanol and AFB, at 24-36 h after AFB, administration when compared to AFB, alone. The increase in hepatic lysosomal enzyme activities correlated well with the increase in the lipid peroxide level of lysosomes in rats treated with ethanol and AFB,. These findings indicate that the decrease in hepatic mitochondrial respiratory enzyme activities and the increase in lipid peroxide level of lysosomes might lead to a decrease in hepatic ATP content, and that the increase in the activities of hepatic lysosomal enzymes, respectively, enhance the AFB, hepatotoxicity of ethanol.
INTRODUCTION
The intracellular ATP content is considered to be an important energy source in regulating the cations pump and cellular volume. It has been shown that tissue injury or necrosis is related to the degree of inhibition of hepatic mitochondrial respiratory
Address for correspondence: Dr. Chaivat Toskulkao, Department of Physiology, Faculty of Science, Mahi-
do1 University, Rama VI Road, Bangkok 10400, Thailand. 0378-4274/90/S 3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
180
functions and the total ATP content [1,2]. Studies have shown that aflatoxin Bi (AFBi) induced a higher percentage inhibition of guinea-fowl liver mitochondrial respiration which was about 100 times lower than that reported for the rat. It has been also suggested that the degree and sites.of inhibition by aflatoxin depend on the toxin concentration. We hereby report a possible potentiated action of ethanol on AFBt hepatotoxicity with respect to hepatic mitochondrial respiratory enzyme activity and total ATP content. It is well known that the peroxidation of membrane lipids leads to loss of integrity of membrane structure and function [3,4]. Many investigators have shown that the lysis of membranes or subcellular organelles can often be attributed to lipid peroxidation in their membranes, and that the peroxidative damage of membranes is prevented by inhibitors of lipid peroxidation [5,6]. Breakdown of lysosomes and the release of degradative lysosomal enzymes into the surrounding tissue are obviously implicated in the hepatic necrosis and hemorrhage consistently observed with acutely toxic doses of AFBi [7]. In vitro treatment of rat lysosomal preparations with AFBi also produced a dose-dependent release of the marker enzymes [8]. Therefore, it is our aim to present a possible potentiated action of ethanol on AFBi hepatotoxicity with respect to the correlation between lysosomal lipid peroxidation and lysosomal enzyme activity. MATERIALS AND METHODS
Male Wistar rats (18&200 g) were supplied by the National Laboratory Animal Production Center (Mahidol University, Thailand). All animals were housed in an air-conditioned room at 25 f 2°C with a relative humidity of about 65 % and were provided with commercial rat chow and water ad libitum. AFBi and ethanol were purchased from Makor Chemical Co. (Israel) and E. Merck Co. (F.R.G.), respectively. Thiobarbituric acid (TBA), 1,1,3,3-tetramethoxypropane (TMP) and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). This study was divided into two major experiments as follows: Experiment
I
The effect of ethanol on the potentiation of AFBi hepatotoxicity was studied with respect to hepatic mitochondrial enzyme activity and total hepatic ATP content at various time intervals after AFBt administration. The rats were divided into 6 groups as follows: Groups I and II rats were treated with 4 oral doses of 4.0 g/kg body wt. water and ethanol (40%, v/v) at 48,45, 24 and 21 h prior to sacrifice by decapitation, respectively. Groups III and IV rats were pretreated with 4 oral doses of 4.0 g/kg body wt. water and ethanol at 48,45,24 and 21 h prior to a single i.p. administration of DMSO (0.5 g/kg body wt.), respectively. Groups V and VI rats were pretreated with 4 oral doses of water and ethanol at 48, 45, 24 and 21 h prior to a single ip.
181
administration of AFBi (2.0 mg/kg body wt.), respectively. After 12, 24, 36, 48 and 72 h of DMSO or AFBr administration, rats were sacrificed by decapitation. The livers of the rats were perfused with cold saline solution and rapidly removed and homogenized. The mitochondria were prepared [9] for determination of mitochondrial protein content [lo], NADH-cytochrome c reductase activity [9,11] and cytochrome oxidase activity [9,12]. Total ATP content in whole homogenates was determined (Sigma Kit No. 366UV). Experiment II The effect of ethanol on the potentiation of AFBi hepatotoxicity was studied with respect to hepatic lysosomal lipoxidation and the activities of lysosomal enzymes in lysosome and cytosol at various time intervals after AFB, administration. The rats were divided into 6 groups and pretreated with water or ethanol and with DMSO or AFBi as already described in Experiment I. After 12,24,36,48 and 72 h of DMSO or AFBi administration, the rats were sacrificed by decapitation. The livers were perfused with cold saline solution and rapidly removed and homogenized. Total hepatic protein was determined [lo] and total activities of lysosomal enzymes were determined in the homogenates. The lysosomal and cytosol fractions were isolated [13] for determination of lysosomal enzyme activities in lysosomes and for free lysosomal enzyme activities in cytosol, lysosomal protein and cytosolic protein [lo] and lysosoma1 lipid peroxide level in the lysosomal fraction [14]. Glucuronidase activity [15], acid phosphatase activity [ 161and arylsulfatase activity [ 171were taken as parameters of lysosomal enzyme activities. RESULTS
Hepatic mitochondrial protein content significantly decreased from 16.5 + 0.3 to 14.5 50.5 mg/g liver (PcO.01) in rats treated with AFB, when compared to control rats at 48 h after AFBi administration. The mitochondrial protein content was not significantly different in rats pretreated with ethanol and treated with AFB, when compared to rats treated with AFBr alone at various time intervals after AFBi administration [ 181.After AFBi administration at 72 h, the hepatic ,mitochondrial protein content gradually returned to the control level. Figures 1 and 2 illustrate the activities of hepatic respiratory enzymes of rats pretreated with ethanol and treated with AFBl as indicated by the activities of cytochrome oxidase and NADH-cytochrome c reductase, respectively. Ethanol treatment slightly decreased the activities of cytochrome oxidase and NADH-cytochrome c reductase at 48 h after the first dose of ethanol and they gradually returned to the control level. At 48 h after AFBi administration in rats pretreated with water, cytochrome oxidase activities decreased maximally from 1.20 + 0.03 to 0.88 + O.lO~mol/mg protein/min (P-C 0.01) when compared to control rats. Cytochrome oxidase activity gradually returned to the control level later than 72 h after AFB, administration.
182
1
1
0
G-Q
WATER - DMSO
w O---O
ETHANOL - DMSO
H
ETHANOL - AFBl
WATER
-
I 12 Time
AFBl
I 24 after
AFBt
I
I
36
46
admlnistration
I
72
Ch
1
Fig. 1. Time course of changes in hepatic mitochondrial cytochrome oxidase activity following ethanol and AFB, administration in rats. Rats were pretreated with 4 oral doses of water or ethanol (4.0 g/kg body wt.) prior to i.p. administration of AFB, (2.0 mg/kg body wt.). Rats were sacrificed at the time indicated. Values are means * SE of 5 rats.
Ethanol further decreased the activity of cytochrome oxidase, without statistical significance, from 0.88 f 0.10 to 0.76 f0.08 pmol/mg protein/min after 48 h of AFBt administration when compared to rats treated with AFBt alone. The activity of cytochrome oxidase was still lower than that of the control at 72 h after AFBt administration. In rats treated with AFB,, NADH-cytochrome c reductase activity decreased maximally from 0.504+0.039 to 0.356+0.019 pmol/mg protein/min (P-C 0.01) at 36 h after AFBi administration when compared to control rats. The activity of NADHcytochrome c reductase gradually returned to the control level about 72 h after AFBt administration. NADH-cytochrome c reductase activity was highly significantly decreased in rats pretreated with ethanol at various time intervals after AFBi administration and decreased maximally at 36 h after AFBi administration from 0.356 + 0.19 to 0.296 + 0.013 pmol/mg protein/min (P < 0.05) when compared to rats treated with AFBi alone. In ethanol- and AFBi-treated rats, the activity of NADH-cytochrome c reductase was still lower than that of the control level.
183
DMSO
O_-a
WATER -
w
ETHANOL -
0-Q
WATER -
H
ETHANOL - AFill
DMSO
AFBl
I
I
I
I
I
0
12
24
36
48
Time
after
AFBt
Fig. 2. Time course of changes in hepatic mitochondrial ethanol and AFB, administration
edmlnlstratlon
NADH-cytochrome
I
72 t h 1
c reductase activity following
in rats. Rats were pretreated with 4 oral doses of water or ethanol (4.0
g/kg body wt.) prior to i.p. administration
of AFB, (2.0 mg/kg body wt.). Rats were sacrificed at the time indicated. Values are means f SE of 5 rats. *PC 0.05, **P< 0.01, significantly different from water-AFB, and ethanol-AFB,.
The hepatic ATP content in rats pretreated with ethanol and treated with AFBt is shown in Figure 3. Ethanol treatment caused a slight decrease in ATP content at 48 h after the first dose but it gradually returned to the control level. At 48 h after AFBt administration in rats pretreated with water, the ATP content decreased maximally from 0.992 f 0.030 to 0.591+ 0.061 pmol/g liver (P < 0.001) when compared to the control rats. The hepatic ATP content gradually returned to the control level and then slightly increased from 1.013 f 0.036 to 1.321 f 0.094 pmol/g liver (PC 0.05) in rats treated with AFBt alone at 72 h. In rats pretreated with ethanol and treated with AFBt, the ATP content was highly significantly decreased in rats treated with AFBt alone at various time intervals after AFBt administration and decreased maximally at 48 h after AFBl administration from 0.591 f0.061 to 0.347f 0.058 pmol/g liver (P-C 0.05) when compared to rats treated with AFBt alone. At 72 h after AFBt administration in rats pretreated with ethanol, the hepatic ATP content was lower than that in rats treated with water or ethanol or AFBt alone. Figure 4 illustrates the lysosomal lipid peroxide level of rats pretreated with ethanol and AFBt. Ethanol treatment produced a significant increase in the lipid peroxide level of lysosomes at 48 h after the first dose of ethanol and it gradually returned to
184 1.4
1.2
k > =
o---O
IdATER -
N
ETHANOL -
b-4
L’ATER
CI
ETHANOL -
-
DMSO DW.0
AFBl MB1
0.8 t
I
1
0
I 24
I
12 Time
after
AFBt
I
I
36
48
admlnstration
I
72 t II 1
Fig. 3. Time course of changes in hepatic ATP content following ethanol and AFB, administration in rats. Rats were pretreated with 4 oral doses of water or ethanol (4.0 g/kg body wt.) prior to i.p. administration of AFB, (2.0 mg/kg body wt.). Rats were sacrificed at the time indicated. Values are means f SE of 5 rats. *PC 0.05, significantly different from water-AFB, and ethanol-AFB,.
the control level. At 24 h after AFBi administration in rats pretreated with water, the lysosomal lipid peroxide level increased maximally from 180.6 f 19.8 to 886.5+ 120.8 nmol MDA/100 mg protein (PC 0.001) when compared to the control rats and gradually returned to the control level at 48 h after AFBi administration. In rats pretreated with ethanol and AFBi, the lysosomal lipid peroxide level was slightly significantly increased at 12, 24 and 48 h after AFBi administration and increased maximally at 24 h after AFBi administration from 886.5+ 120.8 to 1418.7 + 191.3 nmol MDA/100 mg protein (P < 0.05) when compared to rats treated with AFBi alone. In ethanol- and AFBi-treated rats, the lysosomal lipid peroxide concentration returned to the control level later than 72 h after AFBt administration. The activities of hepatic free lysosomal enzymes of rats pretreated with ethanol and treated with AFBi are shown in Figures 5-7. Ethanol treatment caused a slight increase in the free activity of acid phosphatase (Fig. 5) and significantly increased the free activity of arylsulfatase (Fig. 6) and glucuronidase (Fig. 7) at 48 h after the first
185
1600 LVSOSOME e-0 WATER-
2oo0
12
Time
24 at ter AFB,
36
H
ETHANOL
m--Q
WATER
w--a
ETHANOL
MS0 - DMSO - AFBl - A+
48
administration
72 (h 1
Fig. 4. Time course of changes in lipid peroxide level in hepatic lysosomal fraction following ethanol and AFB, administration in rats. Rats were pretreated with 4 oral doses of water or ethanol (4.0 g/kg body wt.) prior to i.p. administration of AFB, (2.0 mg/kg body wt.). Rats were sacrificed at the time indicated. Values are means & SE of 5 rats. bZ’
