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2-Ethylhexanol uncouples oxidative phosphorylation in rat liver mitochondria
Toxicology Letters, 57 (1991) 113-120 @ 1991 Elsevier Science Publishers B.V. 03784274/91/$3.50 ADONIS037842749100071K
113
TOXLET 02574
2-Ethylhexanol uncouples oxidative phosphorylation in rat liver mitochondria
Barbara J. Keller, Decai Liang and Ronald G. Thurman Laboratory of Hepatobiology and Toxicology, Department of Pharmacology. University of North Carolina at Chapel Hill, Chapel Hill, NC (U.S.A.)
(Received 20 August 1990) (Accepted 18December 1990) Key words: Mitochondria,
uncoupler; Plasticizers; 2-Ethylhexanol; Liver
SUMMARY 2-Ethylhexanol (70 PM), a non-genotoxic carcinogen and peroxisome proliferator, stimulated oxygen uptake in the perfused rat liver by about 10%during the first 10 min of infusion. Perfusion with a higher, hepatotoxic dose of ethylhexanol(3 mM) led to a transient increase in oxygen uptake followed by a rapid inhibition of respiration of over 50% in 10 min. Lactate dehydrogenase (LDH) release, indicative of irreversible cell death, was detected in the effluent perfusate after 20 min. After 10 min of perfusion with ethylhexanol, livers were freeze-clamped, acid extracts were prepared and adenine nucleotides were measured by high-pressure liquid chromatography. Ethylhexanol decreased the ATP/ADP ratio from 2.5 to 0.9. Thus, marked decreases in hepatic energy state due to inhibition of respiration preceded cell death. To attempt to understand this phenomenon, the effect of ethylhexanol on isolated mitochondria was studied. Similar to classical uncoupling agents, ethylhexanol stimulated state-4 rates of respiration, diminished coupled rates of respiration, and decreased the P/O ratio in a dose-dependent manner in isolated mitochondria. Ethylhexanol also decreased uptake of radiolabeled 45CaC12by isolated mitochondria 4- to 5-fold. Therefore, we hypothesize that ethylhexanol initially uncouples oxidative phosphorylation leading to diminished ATP synthesis and collapse of ion gradients across the mitochondrial membrane.
INTRODUCTION
It is well known that the widely used plasticizer di(ethylhexyl)phthalate (DEHP) induces peroxisomes and is a non-genotoxic carcinogen in rodents by mechanisms which remain to be elucidated [ 11.DEHP is metabolized in the gut to mono(ethylhexAddress for correspondence: Dr. Ronald G. Thurman, Laboratory of Hepatobiology and Toxicology, Department of Pharmacology CB 7365, Faculty Laboratory Office Building, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365, U.S.A.
114
yl)phthalate and 2-ethylhexanol[2]. Recently, ethylhexanol was shown to inhibit oxygen uptake in the perfused rat liver by over 50% and cause cell death predominantly in periportal regions of the liver lobule [3]. Further, oxygen uptake was inhibited nearly exclusively in oxygen-rich periportal regions of the liver lobule [3]. These data suggested a direct action of ethylhexanol on the mitochondria. Therefore, the present work was designed to study the effect of ethylhexanol on respiration in perfused liver and isolated mitochondria in an attempt to understand the mechanism of acute hepatotoxicity. METHODS
AND MATERIALS
Liver perfusion Female Sprague-Dawley rats (100-180 g) were fasted 24 h prior to use. Rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.) and livers were perfused via the portal vein with Krebs-Henseleit bicarbonate buffer (pH 7.4, 37°C) saturated with 95% 0,/S% COZ in a non-recirculating system. Samples of effluent perfusate were collected and assayed for lactate dehydrogenase (LDH) using standard enzymatic techniques [4]. Oxygen concentration in the effluent perfusate was monitored continuously with a Teflon-shielded, Clark-type 02 electrode. Rates of lactate dehydrogenase release and oxygen uptake by the liver were calculated from the influenteffluent concentration differences, the flow rate, and the liver wet weight. Measurement of adenine nucleotides Livers were perfused with oxygen-saturated Krebs-Henseleit buffer for 10 min in the presence or absence of ethylhexanol, then freeze-clamped with aluminum tongs chilled in liquid nitrogen [5]. Samples of frozen liver weighing about 500 mg were powdered and extracted with 8% KC104 and neutralized with 2 M KHC03. Highpressure liquid chromatography was performed on tissue extracts using a Cts-Bondapak reverse-phase column. Adenine nucleotides were eluted with 0.15 M KHzPOa (pH 5.5) isocratically at a flow rate of 1 ml/min, determined at 254 nm and compared with authentic standards. Average retention times for ATP, ADP and AMP were 6.4, 7.9 and 16 min, respectively. Isolation of mitochondria Rat liver mitochondria were isolated by standard procedures of differential centrifugation [6]. Mitochondria were incubated at room temperature in a 2-ml volume of buffer (pH 7.2) containing 100 mM KCl, 50 mM sucrose, 20 mM Tris-HCl, 5 mM Tris-phosphate and 10 ,uM rotenone. Oxygen uptake was measured in a closed vessel with a Clark-type 02 electrode using succinate (1 pmol) as substrate. The Pi concentration was determined by the method of Sumner [7]. Preparations of mitochondria (1 mg/ml) incubated with 1.O,Ei 45CaC12and 450 ,uM CaC& for 5 min were passed through 1.2 pm Millipore filters and rinsed with 6 ml of ice-cold mannitol(225 mM)/
115
sucrose (75 mM) buffer (pH 7.0). Dried filters were placed in scintillation vials containing 7 ml of scintillation fluid and radioactivity was measured with a /?-counter. Protein concentration was determined calorimetrically [8]. Materials
2-Ethylhexanol was obtained from Aldrich Chemical Co. and 45CaC12was purchased from Amersham. All other chemicals and reagents were of the highest available purity from standard commercial sources. RESULTS
Basal rates of hepatic oxygen uptake ranged from 125-150 pmol/g/h prior to addition of ethylhexanol (Fig. lA,B). Upon infusion of a low dose of ethylhexanol (70 PM), oxygen uptake increased rapidly by about 10% (Fig. 1A) and maintained steady-state levels for 60 min. Oxygen uptake remained elevated even after infusion of ethylhexanol was terminated. Ethylhexanol is highly lipophilic and accumulates in the liver during perfusion [3], which may explain why oxygen uptake remained elevated after infusion was terminated. Perfusion with higher doses of ethylhexanol (3 mM; Fig. 1B) caused an initial small transient increase in oxygen uptake followed by a rapid decrease over 10 min to values around 90 pmol/g/h. Oxygen uptake then declined slowly for the next 30 min reaching values around 60 ,umol/g/h. When infusion of ethylhexanol was terminated, oxygen uptake returned slowly towards basal, reaching values around 100 pmol/g/h by 70 min of perfusion (Fig. 1B). Lactate dehydrogenase release, an indicator of irreversible cell damage, was negligible during perfusion with 70 PM ethylhexanol (Fig. 1C). However, LDH was detected in the effluent perfusate as early as 20 min after infusion of 3 mM ethylhexanol (Fig. 1D). Enzyme release increased sharply for the next 2@-30 min, reaching maximal values of 250 U/g/h, but diminished rapidly after ethylhexanol infusion was terminated. The effect of ethylhexanol on hepatic adenine nucleotide levels was assessed after oxygen uptake was inhibited but before cell death occurred (i.e., perfusion with ethylhexanol for 10 min). Ethylhexanol decreased the ATP/ADP ratio from 2.5 to 0.9 while concomitantly elevating hepatic AMP content from 239 to 1319 ,umol/kg liver (Table I). Since oxygen uptake by the perfused liver is due predominantly to mitochondria [9], the effect of ethylhexanol on respiration of isolated mitochondria was examined. In the absence of ADP, mitochondria took up oxygen at expected rates. Values increased over 3-fold when ADP was added, indicating that the mitochondria were well coupled (Fig. 2). In contrast, stimulation by ADP was minimal in the presence of ethylhexanol. Moreover, the percent coupling of oxidative phosphorylation was inhibited in a dose-dependent manner by ethylhexanol (i.e., state-3 minus state-4 in the absence of ethylhexanol = 100%; Fig. 3). Uncoupling was also clear since ethylhex-
116
anol stimulated state-4 rates of respiration in a dose-dependent manner (Fig. 3). It is well known that uncoupling agents diminish the P/O ratio of isolated mitochondria [lo]. In this study, ethylhexanol decreased the P/O ratio significantly (P< 0.001) from 1.54 _+0.2 to 0.54 + 0.12 with succinate as substrate (n = 6). One would predict from these data that energy-dependent ion uptake would be affected by uncoupling of oxidative phosphorylation due to ethylhexanol. Indeed, uptake of radiolabeled 45CaC12 by isolated mitochondria was decreased in a dose-dependent manner by ethylhexanol (half-maximal inhibition was observed at concentrations between 2 and 6 pmol; Fig. 4).
Minutes Fig. 1. Effect of ethylhexanol liver. Livers
from
female
buffer in a non-recirculating
on oxygen Sprague-Dawley
uptake
of Perfusion
and lactate dehydrogenase
rats were perfused
system. Ethylhexanol(70
(LDH)
release in perfused
with oxygen-saturated
PM or 3 mM) was infused during
rat
Krebs-Henseleit the time designat-
ed by the horizontal bar and vertical arrows. Oxygen uptake (A and B) and LDH release (C and D) were measured and calculated as described in ‘Methods’. Representative experiments for oxygen uptake; LDH data reflects mean f SEM (n = 8).
117 TABLE I EFFECT OF ETHYLHEXANOL LIVER
Control Ethylhexanol
ON ADENINE NUCLEOTIDE
ATP
ADP
1695+216
613 f 89
301 f 19**
LEVELS IN THE PERFUSED
AMP @mol/kg liver)
334f28*
239k38 1319*132**
ATP/ADP
2.5 0.9**
Livers from fasted rats were perfused in the presence or absence of ethylhexanol(3 mM) for 10 min. Tissue was then freeze-clamped, acid extracts were prepared, and adenine nucleotides were determined as described in ‘Methods’. Mean f SEM (n = 4). *PC 0.01 for comparison with control. **P
DISCUSSION
Ethylhexanol inhibits oxidativephosphorylation
At low doses, ethylhexanol stimulated oxygen uptake in the perfused liver in much the same manner as the classical uncoupler dinitrophenol [l 11. At high doses, however, ethylhexanol first stimulated transiently, then inhibited, oxygen uptake in the perfused liver. While these experiments suggest uncoupling, conclusive proof that ethylhexanol is an uncoupler of oxidative phosphorylation came from experiments with isolated mitochondria. Ethylhexanol increased state-4 rates of respiration and
50
-1
1 mln I*
Time Fig. 2. Effect of ethylhexanol (EH) on oxygen uptake by isolated mitochondria. Mitochondria were isolated from rat liver and incubated at room temperature in a 2-ml volume of buffer as described in ‘Methods’. Oxygen concentration was measured in a closed vessel with a Clark-type oxygen electrode after addition of succinate (1~01) and ADP (0.5 ~01) in the presence or absence of ethylhexanol.
118
24g
g -.3
Ethylhexanol
P
(pmols)
Fig, 3. Uncoupling of oxidative phosphorylatron by ethylhexanol m isolated mitochondria. Conditions as in Fig. 2. State-4 rates of respiration (A-A) were calculated from the change in oxygen concentration per unit time after addition of succinate (1 pmol). Percent uncoupling (O-O) was determined by dividing state-3 (succinate and ADP) minus state-4 (succinate only) rates of respiration in the presence of ethylhexanol by state-3 minus state-4 rates in the absence of ethylhexanol multiplied by 100.
01
0
2
l_M-
Ethylhexanol
6
10
(pmols)
Fig. 4. Effect of ethylhexanol on WaC12 uptake by isolated mitochondria. Mitochondria were isolated as described in ‘Methods’ and incubated (1 mg/ml) for 5 min in a sucrose/mannitol buffer containing 0.45 mM CaC& and 1 &i WaC12 in the presence or absence of ethylhexanol. Uptake of WaC12 was determined as described in ‘Methods’. Typical experiment.
119
decreased coupled rates of respiration as well as the P/O ratio in a dose-dependent manner (Fig. 3). Thus, based on the results of this study with the perfused liver and isolated mitochondria, it is clear that ethylhexanol is an uncoupler of oxidative phosphorylation. Mitochondria as a target of ethylhexanol hepatotoxicity
Is uncoupling of oxidative phosphorylation involved in the mechanism of hepatotoxicity due to ethylhexanol? Because of its lipophilicity, it is proposed that ethylhexanol accumulates in the cell, most likely in the mitochondrial membrane, since respiration did not decline when infusion of low concentrations of ethylhexanol was terminated (Fig. 1A). This leads to uncoupling of oxidative phosphorylation (Fig. 3). It is well known that chemicals which uncouple at low doses inhibit oxidative phosphorylation at higher concentrations. This could explain the observation that respiration was inhibited (Fig. 1B) concomitant with a decline in the ATP/ADP ratio in the intact cell after perfusion with ethylhexanol (Table I). Therefore, the energy state is disrupted, preventing ion pumping mechanisms from operating normally. This could lead to elevation of cytosolic free calcium due to the inability of mitochondria to sequester this toxic divalent ion. In support of this hypothesis, it was observed that ethylhexanol caused a dose-dependent decrease in mitochondrial calcium uptake (Fig. 4) which was accompanied by a decrease in the mitochondrial membrane potential [3]. Therefore, one early key event in the mechanism of hepatotoxicity due to ethylhexanol may be an elevation of intracellular free calcium as a result of uncoupling of mitochondrial oxidative phosphorylation. ACKNOWLEDGEMENT
This study was supported in part by a grant from NIEHS (No. E&)4325). REFERENCES 1 Reddy, J.K. and Lalwai, N.D. (1989) Carcinogenesis by hepatic peroxisome proliferators: evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. CRC Crit. Rev. Biochem. 7,305-310. 2 Albro, P. (1975) The metabolism of 2-ethylhexanol in rats. Xenobiotica 5,625636. 3 Keller, B.J., Yamanaka, H., Liang, D., Kauffman, F.C. and Thurman, R.G. (1990) Oxygen-dependent hepatotoxicity due to ethylhexanol in the perfused rat liver: mitochondria as a site of action. J. Pharmacol. Exp. Ther. 252, 1355-1360. 4 Bergmeyer, H.U. (1988) Methods of Enzymatic Analysis. Academic Press, New York. 5 Wollenberger, A., Ristau, 0. and Schoffa, G. (1960) A simple technique for extremely rapid freezing of large pieces of tissue. Pfliiger’s Arch. 270,399412. 6 Schneider, W. and Hogeboom, G.H. (1950) Intracellular distribution of enzymes. V. Further studies on the distribution of cytochrome c in rat liver. J. Biol. Chem. 183, 123-128. 7 Sumner, I.G., Freedman, R.B. and Lodola, A. (1983) Characterization of hepatocyte sub-populations generated by centrifugal elutriation. Eur. J. Biochem. 134, 539-545.
120 8 Lowry, O.H., Rosebrough, N.J., Farr, AL. and Randall, R.J. (1951) Protein measurement with the Folm phenol reagent. J. Biol. Chem. 193.265-275. 9 Thurman, R.G. and Scholz, R. (1969) Mixed function oxidation in perfused rat liver. Oxygen uptake following aminopyrine addition. Eur. J. Biochem. 10,459-467. IO Woods, T.A., Decker, G L. and Pedersen. P.L. (1977) Antih~erIIpidemic drugs - in vitro effect on the function and structure of rat liver mitochondria. J. Mol. Cell Cardiol. 9,807-822. 11 Edsall, G. (1934) Biological actions of dimtrophenol and related compounds: a review. N. Engl. J. Med. 211, 385-390.
113
TOXLET 02574
2-Ethylhexanol uncouples oxidative phosphorylation in rat liver mitochondria
Barbara J. Keller, Decai Liang and Ronald G. Thurman Laboratory of Hepatobiology and Toxicology, Department of Pharmacology. University of North Carolina at Chapel Hill, Chapel Hill, NC (U.S.A.)
(Received 20 August 1990) (Accepted 18December 1990) Key words: Mitochondria,
uncoupler; Plasticizers; 2-Ethylhexanol; Liver
SUMMARY 2-Ethylhexanol (70 PM), a non-genotoxic carcinogen and peroxisome proliferator, stimulated oxygen uptake in the perfused rat liver by about 10%during the first 10 min of infusion. Perfusion with a higher, hepatotoxic dose of ethylhexanol(3 mM) led to a transient increase in oxygen uptake followed by a rapid inhibition of respiration of over 50% in 10 min. Lactate dehydrogenase (LDH) release, indicative of irreversible cell death, was detected in the effluent perfusate after 20 min. After 10 min of perfusion with ethylhexanol, livers were freeze-clamped, acid extracts were prepared and adenine nucleotides were measured by high-pressure liquid chromatography. Ethylhexanol decreased the ATP/ADP ratio from 2.5 to 0.9. Thus, marked decreases in hepatic energy state due to inhibition of respiration preceded cell death. To attempt to understand this phenomenon, the effect of ethylhexanol on isolated mitochondria was studied. Similar to classical uncoupling agents, ethylhexanol stimulated state-4 rates of respiration, diminished coupled rates of respiration, and decreased the P/O ratio in a dose-dependent manner in isolated mitochondria. Ethylhexanol also decreased uptake of radiolabeled 45CaC12by isolated mitochondria 4- to 5-fold. Therefore, we hypothesize that ethylhexanol initially uncouples oxidative phosphorylation leading to diminished ATP synthesis and collapse of ion gradients across the mitochondrial membrane.
INTRODUCTION
It is well known that the widely used plasticizer di(ethylhexyl)phthalate (DEHP) induces peroxisomes and is a non-genotoxic carcinogen in rodents by mechanisms which remain to be elucidated [ 11.DEHP is metabolized in the gut to mono(ethylhexAddress for correspondence: Dr. Ronald G. Thurman, Laboratory of Hepatobiology and Toxicology, Department of Pharmacology CB 7365, Faculty Laboratory Office Building, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365, U.S.A.
114
yl)phthalate and 2-ethylhexanol[2]. Recently, ethylhexanol was shown to inhibit oxygen uptake in the perfused rat liver by over 50% and cause cell death predominantly in periportal regions of the liver lobule [3]. Further, oxygen uptake was inhibited nearly exclusively in oxygen-rich periportal regions of the liver lobule [3]. These data suggested a direct action of ethylhexanol on the mitochondria. Therefore, the present work was designed to study the effect of ethylhexanol on respiration in perfused liver and isolated mitochondria in an attempt to understand the mechanism of acute hepatotoxicity. METHODS
AND MATERIALS
Liver perfusion Female Sprague-Dawley rats (100-180 g) were fasted 24 h prior to use. Rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.) and livers were perfused via the portal vein with Krebs-Henseleit bicarbonate buffer (pH 7.4, 37°C) saturated with 95% 0,/S% COZ in a non-recirculating system. Samples of effluent perfusate were collected and assayed for lactate dehydrogenase (LDH) using standard enzymatic techniques [4]. Oxygen concentration in the effluent perfusate was monitored continuously with a Teflon-shielded, Clark-type 02 electrode. Rates of lactate dehydrogenase release and oxygen uptake by the liver were calculated from the influenteffluent concentration differences, the flow rate, and the liver wet weight. Measurement of adenine nucleotides Livers were perfused with oxygen-saturated Krebs-Henseleit buffer for 10 min in the presence or absence of ethylhexanol, then freeze-clamped with aluminum tongs chilled in liquid nitrogen [5]. Samples of frozen liver weighing about 500 mg were powdered and extracted with 8% KC104 and neutralized with 2 M KHC03. Highpressure liquid chromatography was performed on tissue extracts using a Cts-Bondapak reverse-phase column. Adenine nucleotides were eluted with 0.15 M KHzPOa (pH 5.5) isocratically at a flow rate of 1 ml/min, determined at 254 nm and compared with authentic standards. Average retention times for ATP, ADP and AMP were 6.4, 7.9 and 16 min, respectively. Isolation of mitochondria Rat liver mitochondria were isolated by standard procedures of differential centrifugation [6]. Mitochondria were incubated at room temperature in a 2-ml volume of buffer (pH 7.2) containing 100 mM KCl, 50 mM sucrose, 20 mM Tris-HCl, 5 mM Tris-phosphate and 10 ,uM rotenone. Oxygen uptake was measured in a closed vessel with a Clark-type 02 electrode using succinate (1 pmol) as substrate. The Pi concentration was determined by the method of Sumner [7]. Preparations of mitochondria (1 mg/ml) incubated with 1.O,Ei 45CaC12and 450 ,uM CaC& for 5 min were passed through 1.2 pm Millipore filters and rinsed with 6 ml of ice-cold mannitol(225 mM)/
115
sucrose (75 mM) buffer (pH 7.0). Dried filters were placed in scintillation vials containing 7 ml of scintillation fluid and radioactivity was measured with a /?-counter. Protein concentration was determined calorimetrically [8]. Materials
2-Ethylhexanol was obtained from Aldrich Chemical Co. and 45CaC12was purchased from Amersham. All other chemicals and reagents were of the highest available purity from standard commercial sources. RESULTS
Basal rates of hepatic oxygen uptake ranged from 125-150 pmol/g/h prior to addition of ethylhexanol (Fig. lA,B). Upon infusion of a low dose of ethylhexanol (70 PM), oxygen uptake increased rapidly by about 10% (Fig. 1A) and maintained steady-state levels for 60 min. Oxygen uptake remained elevated even after infusion of ethylhexanol was terminated. Ethylhexanol is highly lipophilic and accumulates in the liver during perfusion [3], which may explain why oxygen uptake remained elevated after infusion was terminated. Perfusion with higher doses of ethylhexanol (3 mM; Fig. 1B) caused an initial small transient increase in oxygen uptake followed by a rapid decrease over 10 min to values around 90 pmol/g/h. Oxygen uptake then declined slowly for the next 30 min reaching values around 60 ,umol/g/h. When infusion of ethylhexanol was terminated, oxygen uptake returned slowly towards basal, reaching values around 100 pmol/g/h by 70 min of perfusion (Fig. 1B). Lactate dehydrogenase release, an indicator of irreversible cell damage, was negligible during perfusion with 70 PM ethylhexanol (Fig. 1C). However, LDH was detected in the effluent perfusate as early as 20 min after infusion of 3 mM ethylhexanol (Fig. 1D). Enzyme release increased sharply for the next 2@-30 min, reaching maximal values of 250 U/g/h, but diminished rapidly after ethylhexanol infusion was terminated. The effect of ethylhexanol on hepatic adenine nucleotide levels was assessed after oxygen uptake was inhibited but before cell death occurred (i.e., perfusion with ethylhexanol for 10 min). Ethylhexanol decreased the ATP/ADP ratio from 2.5 to 0.9 while concomitantly elevating hepatic AMP content from 239 to 1319 ,umol/kg liver (Table I). Since oxygen uptake by the perfused liver is due predominantly to mitochondria [9], the effect of ethylhexanol on respiration of isolated mitochondria was examined. In the absence of ADP, mitochondria took up oxygen at expected rates. Values increased over 3-fold when ADP was added, indicating that the mitochondria were well coupled (Fig. 2). In contrast, stimulation by ADP was minimal in the presence of ethylhexanol. Moreover, the percent coupling of oxidative phosphorylation was inhibited in a dose-dependent manner by ethylhexanol (i.e., state-3 minus state-4 in the absence of ethylhexanol = 100%; Fig. 3). Uncoupling was also clear since ethylhex-
116
anol stimulated state-4 rates of respiration in a dose-dependent manner (Fig. 3). It is well known that uncoupling agents diminish the P/O ratio of isolated mitochondria [lo]. In this study, ethylhexanol decreased the P/O ratio significantly (P< 0.001) from 1.54 _+0.2 to 0.54 + 0.12 with succinate as substrate (n = 6). One would predict from these data that energy-dependent ion uptake would be affected by uncoupling of oxidative phosphorylation due to ethylhexanol. Indeed, uptake of radiolabeled 45CaC12 by isolated mitochondria was decreased in a dose-dependent manner by ethylhexanol (half-maximal inhibition was observed at concentrations between 2 and 6 pmol; Fig. 4).
Minutes Fig. 1. Effect of ethylhexanol liver. Livers
from
female
buffer in a non-recirculating
on oxygen Sprague-Dawley
uptake
of Perfusion
and lactate dehydrogenase
rats were perfused
system. Ethylhexanol(70
(LDH)
release in perfused
with oxygen-saturated
PM or 3 mM) was infused during
rat
Krebs-Henseleit the time designat-
ed by the horizontal bar and vertical arrows. Oxygen uptake (A and B) and LDH release (C and D) were measured and calculated as described in ‘Methods’. Representative experiments for oxygen uptake; LDH data reflects mean f SEM (n = 8).
117 TABLE I EFFECT OF ETHYLHEXANOL LIVER
Control Ethylhexanol
ON ADENINE NUCLEOTIDE
ATP
ADP
1695+216
613 f 89
301 f 19**
LEVELS IN THE PERFUSED
AMP @mol/kg liver)
334f28*
239k38 1319*132**
ATP/ADP
2.5 0.9**
Livers from fasted rats were perfused in the presence or absence of ethylhexanol(3 mM) for 10 min. Tissue was then freeze-clamped, acid extracts were prepared, and adenine nucleotides were determined as described in ‘Methods’. Mean f SEM (n = 4). *PC 0.01 for comparison with control. **P
DISCUSSION
Ethylhexanol inhibits oxidativephosphorylation
At low doses, ethylhexanol stimulated oxygen uptake in the perfused liver in much the same manner as the classical uncoupler dinitrophenol [l 11. At high doses, however, ethylhexanol first stimulated transiently, then inhibited, oxygen uptake in the perfused liver. While these experiments suggest uncoupling, conclusive proof that ethylhexanol is an uncoupler of oxidative phosphorylation came from experiments with isolated mitochondria. Ethylhexanol increased state-4 rates of respiration and
50
-1
1 mln I*
Time Fig. 2. Effect of ethylhexanol (EH) on oxygen uptake by isolated mitochondria. Mitochondria were isolated from rat liver and incubated at room temperature in a 2-ml volume of buffer as described in ‘Methods’. Oxygen concentration was measured in a closed vessel with a Clark-type oxygen electrode after addition of succinate (1~01) and ADP (0.5 ~01) in the presence or absence of ethylhexanol.
118
24g
g -.3
Ethylhexanol
P
(pmols)
Fig, 3. Uncoupling of oxidative phosphorylatron by ethylhexanol m isolated mitochondria. Conditions as in Fig. 2. State-4 rates of respiration (A-A) were calculated from the change in oxygen concentration per unit time after addition of succinate (1 pmol). Percent uncoupling (O-O) was determined by dividing state-3 (succinate and ADP) minus state-4 (succinate only) rates of respiration in the presence of ethylhexanol by state-3 minus state-4 rates in the absence of ethylhexanol multiplied by 100.
01
0
2
l_M-
Ethylhexanol
6
10
(pmols)
Fig. 4. Effect of ethylhexanol on WaC12 uptake by isolated mitochondria. Mitochondria were isolated as described in ‘Methods’ and incubated (1 mg/ml) for 5 min in a sucrose/mannitol buffer containing 0.45 mM CaC& and 1 &i WaC12 in the presence or absence of ethylhexanol. Uptake of WaC12 was determined as described in ‘Methods’. Typical experiment.
119
decreased coupled rates of respiration as well as the P/O ratio in a dose-dependent manner (Fig. 3). Thus, based on the results of this study with the perfused liver and isolated mitochondria, it is clear that ethylhexanol is an uncoupler of oxidative phosphorylation. Mitochondria as a target of ethylhexanol hepatotoxicity
Is uncoupling of oxidative phosphorylation involved in the mechanism of hepatotoxicity due to ethylhexanol? Because of its lipophilicity, it is proposed that ethylhexanol accumulates in the cell, most likely in the mitochondrial membrane, since respiration did not decline when infusion of low concentrations of ethylhexanol was terminated (Fig. 1A). This leads to uncoupling of oxidative phosphorylation (Fig. 3). It is well known that chemicals which uncouple at low doses inhibit oxidative phosphorylation at higher concentrations. This could explain the observation that respiration was inhibited (Fig. 1B) concomitant with a decline in the ATP/ADP ratio in the intact cell after perfusion with ethylhexanol (Table I). Therefore, the energy state is disrupted, preventing ion pumping mechanisms from operating normally. This could lead to elevation of cytosolic free calcium due to the inability of mitochondria to sequester this toxic divalent ion. In support of this hypothesis, it was observed that ethylhexanol caused a dose-dependent decrease in mitochondrial calcium uptake (Fig. 4) which was accompanied by a decrease in the mitochondrial membrane potential [3]. Therefore, one early key event in the mechanism of hepatotoxicity due to ethylhexanol may be an elevation of intracellular free calcium as a result of uncoupling of mitochondrial oxidative phosphorylation. ACKNOWLEDGEMENT
This study was supported in part by a grant from NIEHS (No. E&)4325). REFERENCES 1 Reddy, J.K. and Lalwai, N.D. (1989) Carcinogenesis by hepatic peroxisome proliferators: evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. CRC Crit. Rev. Biochem. 7,305-310. 2 Albro, P. (1975) The metabolism of 2-ethylhexanol in rats. Xenobiotica 5,625636. 3 Keller, B.J., Yamanaka, H., Liang, D., Kauffman, F.C. and Thurman, R.G. (1990) Oxygen-dependent hepatotoxicity due to ethylhexanol in the perfused rat liver: mitochondria as a site of action. J. Pharmacol. Exp. Ther. 252, 1355-1360. 4 Bergmeyer, H.U. (1988) Methods of Enzymatic Analysis. Academic Press, New York. 5 Wollenberger, A., Ristau, 0. and Schoffa, G. (1960) A simple technique for extremely rapid freezing of large pieces of tissue. Pfliiger’s Arch. 270,399412. 6 Schneider, W. and Hogeboom, G.H. (1950) Intracellular distribution of enzymes. V. Further studies on the distribution of cytochrome c in rat liver. J. Biol. Chem. 183, 123-128. 7 Sumner, I.G., Freedman, R.B. and Lodola, A. (1983) Characterization of hepatocyte sub-populations generated by centrifugal elutriation. Eur. J. Biochem. 134, 539-545.
120 8 Lowry, O.H., Rosebrough, N.J., Farr, AL. and Randall, R.J. (1951) Protein measurement with the Folm phenol reagent. J. Biol. Chem. 193.265-275. 9 Thurman, R.G. and Scholz, R. (1969) Mixed function oxidation in perfused rat liver. Oxygen uptake following aminopyrine addition. Eur. J. Biochem. 10,459-467. IO Woods, T.A., Decker, G L. and Pedersen. P.L. (1977) Antih~erIIpidemic drugs - in vitro effect on the function and structure of rat liver mitochondria. J. Mol. Cell Cardiol. 9,807-822. 11 Edsall, G. (1934) Biological actions of dimtrophenol and related compounds: a review. N. Engl. J. Med. 211, 385-390.