Vol.
167,
March
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
16, 1990
PROTECTION
BY ACIDOTIC
TO RAT HEPATOCYTES
Anna-Liisa
pH AND FRUCTOSE
AGAINST
LETHAL
FROM MITOCHONDRIAL INHIBITORS, AND OXIDANT CHEMICALS1 Nieminen, Thomas L. Dawson,
Toru Kawanishi3,
600-606
INJURY
IONOPHORES
Gregory J. Gores2,
Brian Herman and John J. Lemasters4
Laboratories for Cell Biology, Department of Cell Biology & Anatom , School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, N E 27599-7090 Received
January
11,
1990
The importance of mitochondrial ATP formation and extracellular acidosiswas evaluated in hepatocyte suspensionsafter different toxic treatments. Acidotic pH was protective against cell killing from all toxic treatments examined except for pronase, a toxic protease. Fructose, a substrate for glycolytic ATP formation, provided good protection against toxicity from cyanide, oli omycin, t-butyl hydro eroxide, menadione and cystamine. Protection by fructose against ECCP, gramicidin and E!r-A23187 required oligomycin. This indicated that these ionophores were causing cytotoxicity by uncoupling oxidative phosphorylation. Fructose provided little protection against pronase and HgC12,the latter compound being a potent inhibitor of glycolysis. In conclusion, disruption of mitochondrial ATP formation was a common event contributing to the toxicity of chemical oxidants and ionophores. Acidotic pH was generally protective under these conditions of impaired ATP generation. 0 1990 Academic Press.Inc.
Depletion of ATP is a typical feature of hypoxic and toxic injury (e.g., ref l-3). In hypoxia, ATP depletion is causedby inhibition of aerobic mitochondrial ATP formation, an effect which can be mimicked by the respiratory inhibitor, cyanide. Mitochondria may also be a target of injury by toxic chemicals (2-4). For example, oxidative injury by the strong oxidant, HgC12,appears to causeATP depletion by depolarizing the mitochondrial membrane lThis work was supported by Grants AGO7218 and DK30874 from the National Institutes of Health and Grant J-1433 from the Office of Naval Research. %he present addressof Dr. Gores is GI Research Unit, Mayo Clinic, Rochester, MN 55905. %‘he resent addressof Dr. Kawanishi is Biological Safety Research Center, National Institute of R ygienic Sciences,Tokyo 158, Japan. ?To whom correspondence should be addressed. Abbreviations used are: BSA, bovine serum albumin; t-BuOOH, tert-butyl hydroperoxide; CCCP, carbonyl cyanide m-chlorophenylh drazone; IAA, iodoacetic acid; KRH, Krebs-Ringers-Hepes buffer containing 115 mM KaC1, 5 mM KCl, 2 mM CaC12, 1 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHEPES buffer, pH 7.4; MPP+, l-methyl-C phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3@etrahydropyridine. 0006-291X/90 Copyright All rights
$1.50 0 1990 by Academic Press, of reproduction in any form
Inc. reserved.
600
Vol.
167,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(3). However, it is not clear to what extent ATP depletion by itself is the principal factor leading to cell killing in different models of acute toxic injury. Previously, fructose was shown to protect against lethal cell injury from anoxia and cyanide in isolated rat livers (5,6) and hepatocyte suspensions (1,7). Fructose also protected hepatocytes against MPTP and its toxic metabolite, MPP+ (8). Fructose protection in all these circumstances was mediated by glycolytic formation of ATP. Other sugars which are not readily metabolized by liver such as glucose did not protect. Extracellular acidosis also protects against hypoxic cell death (1,9), an effect mediated by intracellular acidification (10,ll). Little is known, however, concerning protection by acidosis and fructose against toxic cell injury. Thus, the aim of this study was to evaluate fructose and1 acidosis as protective agents in models of acute toxic injury to hepatocyte suspensions. The results indicate that mitochondrial dysfunction and consequent disruption of ATP formation are common events leading to cell death caused by mitochondrial inhibitors, ionophores, and chemical oxidants. Acidosis substantially delays cell killing by these toxic treatments and may be generally protective after cellular ATP depletion. MATERIALS
AND METHODS
Hepatocyte kolation - Hepatocytes were isolated from 18-20 hour-fasted male Sprague-Dawley rats (200-250g) by collagenase perfusion as previously described (1). Viability of the cells was ~90% determined by trypan blue exclusion. Qtotoxicity and enzyme assays - Cell viability in hepatocyte suspensions was monitored using propidium iodide fluorescence (1). Freshly isolated hepatocytes were incubated in KRH buffer containing 1 PM propidium iodide and In experiments where monensin was employed, cells were incubated in 10 mM Na+, 105 mM choline-substituted KRH buffer, pH 7.4. Propidium iodide fluorescence was measured every 30 main with a Sequoia-Turner Model 450 filter fluorometer (Mountain View, CA) using 520 run excitation and 605 nm emission filters. Under these conditions, fluorescence is linearly proportional to loss of cell viability as measured by lactate dehydro enase release and nuclear staining with trypan blue (1). At the end of each experiment, 37 s PM digitonin was added to permeabilize all cells and obtain a maximal fluorescence reading corresponding to 100% cell death. Glyceraldehyde-3-phosphate dehydrogenase was assayed spectroph~a;;;~lly by NADH oxidation in the presence of 1,3-bisphosphoglycerate (12). - Bovine glyceraldehyde-3-phosphate dehydrogenase was obtained from Sigma (St. Louis, MO). BrA23187, the non-fluorescent derivative of the calcium ionophore, A23187, was obtained from Calbiochem (San Diego, CA). All other reagents were of analytical grade and obtained from the usual commercial sources. RESULTS Protection by jkctose against lethal cell injury - In isolated hepatocytes and intact perfused livers, glycolytic ATP formation utilizing endogenous glycogen or exogenous fructose protects against cell killing in models of hypoxic injury (l&7). In order to evaluate whether protection by fructose is a general phenomenon in hepatocellular injury, cell viability was measured during different toxic treatments in the presence and absence of 20 mM fructose (Fig. 1). Fructose provided substantial protection against cytotoxicity by the mitochondrial inhibitors, cyanide (2.5 mM) and oligomycin (10 &ml), but little protection against the uncoupler, CCCP (10 FM). Iodoacetic acid (0.5 mM), which inhibits the glycolytic enzyme 601
Vol.
167,
No.
2, 1990
BIOCHEMICAL
0
AND
7.4
BIOPHYSICAL
I
Fructose
Gramicidin
Br-A23187
D
COMMUNICATIONS
eZa 6.5
CCCP
Oligomycin
RESEARCH
KCNIIAA
Pronase
100
50
0 Menadione
t-BuOOH
Cystamine
Hg’X
Figure 1. Effect of acidosis andfnrctose on hepatocyte viability during treatments with various toxtc chemicals - Fresh1 isolatedhe atocyteswere suspended in unmodifiedKRH, KRH adjustedto pH 6.5 with ACl, or KR r-f supplemented with 20 mM fructose. After 30 min preincubation,toxic chemicalswere added,andviability wasassessed after variousperiodsof time asdescribedin MATERIALS AND METHODS. Concentrationsand timesof
aspercent of the viability of control cellsat pH cate determinationsfrom 2 or morecell isolations. glyceraldehyde-3-phosphate dehydrogenase, abolished protection by fructose against cyanide toxicity. Fructose alone provided little protection against the toxicity of the ionophores, gramicidin D (250 nM) and Br-A23187 (10 PM), and the protease, pronase (0.5 mg/ml). However, fructose provided substantial protection against toxicity by the oxidant chemicals, menadione (50 PM), t-BuOOH (50 PM) and cystamine (2.5 mM). Fructose did not protect against HgCl2 (50 PM) which at this concentration was a potent inhibitor of bovine glyceraldehyde-3-phosphate dehydrogenase(data not shown). Fructose provided good protection against cyanide and oligomycin toxicity but little protection against CCCP. An explanation may be that CCCP, but not cyanide or 602
Vol.
167,
No.
BIOCHEMICAL
2, 1990
100
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
A
e i50e
AND
50 0
f
--
P
6 t &
s
o-
C t 50-
O-
.-==Ys 0
30
Time
60
90
120
(min)
Fi re 2. Effect of fructoseand oligomycinon the cytotoxicityof CCCP,Br-A23187and gramzc m D - Freshly isolatedhepatocyteswere incubatedin KRH in the resenceor absenceof 20 mM fructose.After 30min, CCCP(10 PM, panelA B), and gramicidinD (250 nM, panel C) were added with or without 10 &ml ohgomycin. Cell viabihty was assessed asdescribedin Additions are: none, open circles;toxic chemical(CCCP, Br-A23187 or gramicidin D), closedcircles;toxic chemicalplusfructose,closedtrian es;toxic chemicalplusfructoseand oligomydn,closedsquares.Data representmeansf d.E. of triplicate determinationsfrom 3 or more cellisolations.
7i.F
oligomycin, stimulates ATP hydrolysis by the mitochondrial FIFO-ATPase. Such uncoupled ATP consumption would counteract the benefit of glycolytic ATP generation from fructose. To test this hypothesis, we examined whether oligomycin, a specific inhibitor of mitochondrial ATPase, might confer upon fructose the property of protecting against CCCP toxicity (Fig. 2). In confirmation, oligomycin (10 pg/ml) plus fructose conferred complete protection against CCCP toxicity. This occurred despite the fact that oligomycin by itself was toxic, causing 50% cell killing in 90 min (Fig. 1) and 90% cell killing in 120 min. Oligomycin plus fructose also1provided complete protection against toxicity by BrA23187 and substantial protection against gramicidin D toxicity (Fig. 2). Thus, cytotoxicity by BrA23187 and gramicidin D appeared to be mediated by uncoupling of oxidative phosphorylation and intracellular A’TP hydrolysis. Protection againsttoxic cell killing by acidoticpH - Previously, acidotic pH was shown to be protective in models of hypoxic cell injury (1,9-11). To determine whether acidosiswas generally protective in hepatocellular injury, cell killing at pH 7.4 and pH 6.5 was compared after exposure of hepatocytes to various toxic chemicals (Fig. 1). Acidotic pH protected against lethal cell injury from exposure to all toxic chemicalsexamined except pronase. Role of intracellular acidosisin protection by fnrctose - To determine if protection by fructose was due to lactic acid formation and subsequent intracellular acidosis,hepatocytes 603
Vol.
167,
No.
2, 1990
BIOCHEMICAL
0'
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
I 0
30
60
90
120
Time (min)
Figure 3. Effect of fructoseon cell viabilig in monensin-treated hepatocytes- Freshly isolated heuatocvtes were incubated in modified KRH at DH 7.4. Fructose (20 mMj wasincluded where i
167,
March
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
16, 1990
PROTECTION
BY ACIDOTIC
TO RAT HEPATOCYTES
Anna-Liisa
pH AND FRUCTOSE
AGAINST
LETHAL
FROM MITOCHONDRIAL INHIBITORS, AND OXIDANT CHEMICALS1 Nieminen, Thomas L. Dawson,
Toru Kawanishi3,
600-606
INJURY
IONOPHORES
Gregory J. Gores2,
Brian Herman and John J. Lemasters4
Laboratories for Cell Biology, Department of Cell Biology & Anatom , School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, N E 27599-7090 Received
January
11,
1990
The importance of mitochondrial ATP formation and extracellular acidosiswas evaluated in hepatocyte suspensionsafter different toxic treatments. Acidotic pH was protective against cell killing from all toxic treatments examined except for pronase, a toxic protease. Fructose, a substrate for glycolytic ATP formation, provided good protection against toxicity from cyanide, oli omycin, t-butyl hydro eroxide, menadione and cystamine. Protection by fructose against ECCP, gramicidin and E!r-A23187 required oligomycin. This indicated that these ionophores were causing cytotoxicity by uncoupling oxidative phosphorylation. Fructose provided little protection against pronase and HgC12,the latter compound being a potent inhibitor of glycolysis. In conclusion, disruption of mitochondrial ATP formation was a common event contributing to the toxicity of chemical oxidants and ionophores. Acidotic pH was generally protective under these conditions of impaired ATP generation. 0 1990 Academic Press.Inc.
Depletion of ATP is a typical feature of hypoxic and toxic injury (e.g., ref l-3). In hypoxia, ATP depletion is causedby inhibition of aerobic mitochondrial ATP formation, an effect which can be mimicked by the respiratory inhibitor, cyanide. Mitochondria may also be a target of injury by toxic chemicals (2-4). For example, oxidative injury by the strong oxidant, HgC12,appears to causeATP depletion by depolarizing the mitochondrial membrane lThis work was supported by Grants AGO7218 and DK30874 from the National Institutes of Health and Grant J-1433 from the Office of Naval Research. %he present addressof Dr. Gores is GI Research Unit, Mayo Clinic, Rochester, MN 55905. %‘he resent addressof Dr. Kawanishi is Biological Safety Research Center, National Institute of R ygienic Sciences,Tokyo 158, Japan. ?To whom correspondence should be addressed. Abbreviations used are: BSA, bovine serum albumin; t-BuOOH, tert-butyl hydroperoxide; CCCP, carbonyl cyanide m-chlorophenylh drazone; IAA, iodoacetic acid; KRH, Krebs-Ringers-Hepes buffer containing 115 mM KaC1, 5 mM KCl, 2 mM CaC12, 1 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHEPES buffer, pH 7.4; MPP+, l-methyl-C phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3@etrahydropyridine. 0006-291X/90 Copyright All rights
$1.50 0 1990 by Academic Press, of reproduction in any form
Inc. reserved.
600
Vol.
167,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(3). However, it is not clear to what extent ATP depletion by itself is the principal factor leading to cell killing in different models of acute toxic injury. Previously, fructose was shown to protect against lethal cell injury from anoxia and cyanide in isolated rat livers (5,6) and hepatocyte suspensions (1,7). Fructose also protected hepatocytes against MPTP and its toxic metabolite, MPP+ (8). Fructose protection in all these circumstances was mediated by glycolytic formation of ATP. Other sugars which are not readily metabolized by liver such as glucose did not protect. Extracellular acidosis also protects against hypoxic cell death (1,9), an effect mediated by intracellular acidification (10,ll). Little is known, however, concerning protection by acidosis and fructose against toxic cell injury. Thus, the aim of this study was to evaluate fructose and1 acidosis as protective agents in models of acute toxic injury to hepatocyte suspensions. The results indicate that mitochondrial dysfunction and consequent disruption of ATP formation are common events leading to cell death caused by mitochondrial inhibitors, ionophores, and chemical oxidants. Acidosis substantially delays cell killing by these toxic treatments and may be generally protective after cellular ATP depletion. MATERIALS
AND METHODS
Hepatocyte kolation - Hepatocytes were isolated from 18-20 hour-fasted male Sprague-Dawley rats (200-250g) by collagenase perfusion as previously described (1). Viability of the cells was ~90% determined by trypan blue exclusion. Qtotoxicity and enzyme assays - Cell viability in hepatocyte suspensions was monitored using propidium iodide fluorescence (1). Freshly isolated hepatocytes were incubated in KRH buffer containing 1 PM propidium iodide and In experiments where monensin was employed, cells were incubated in 10 mM Na+, 105 mM choline-substituted KRH buffer, pH 7.4. Propidium iodide fluorescence was measured every 30 main with a Sequoia-Turner Model 450 filter fluorometer (Mountain View, CA) using 520 run excitation and 605 nm emission filters. Under these conditions, fluorescence is linearly proportional to loss of cell viability as measured by lactate dehydro enase release and nuclear staining with trypan blue (1). At the end of each experiment, 37 s PM digitonin was added to permeabilize all cells and obtain a maximal fluorescence reading corresponding to 100% cell death. Glyceraldehyde-3-phosphate dehydrogenase was assayed spectroph~a;;;~lly by NADH oxidation in the presence of 1,3-bisphosphoglycerate (12). - Bovine glyceraldehyde-3-phosphate dehydrogenase was obtained from Sigma (St. Louis, MO). BrA23187, the non-fluorescent derivative of the calcium ionophore, A23187, was obtained from Calbiochem (San Diego, CA). All other reagents were of analytical grade and obtained from the usual commercial sources. RESULTS Protection by jkctose against lethal cell injury - In isolated hepatocytes and intact perfused livers, glycolytic ATP formation utilizing endogenous glycogen or exogenous fructose protects against cell killing in models of hypoxic injury (l&7). In order to evaluate whether protection by fructose is a general phenomenon in hepatocellular injury, cell viability was measured during different toxic treatments in the presence and absence of 20 mM fructose (Fig. 1). Fructose provided substantial protection against cytotoxicity by the mitochondrial inhibitors, cyanide (2.5 mM) and oligomycin (10 &ml), but little protection against the uncoupler, CCCP (10 FM). Iodoacetic acid (0.5 mM), which inhibits the glycolytic enzyme 601
Vol.
167,
No.
2, 1990
BIOCHEMICAL
0
AND
7.4
BIOPHYSICAL
I
Fructose
Gramicidin
Br-A23187
D
COMMUNICATIONS
eZa 6.5
CCCP
Oligomycin
RESEARCH
KCNIIAA
Pronase
100
50
0 Menadione
t-BuOOH
Cystamine
Hg’X
Figure 1. Effect of acidosis andfnrctose on hepatocyte viability during treatments with various toxtc chemicals - Fresh1 isolatedhe atocyteswere suspended in unmodifiedKRH, KRH adjustedto pH 6.5 with ACl, or KR r-f supplemented with 20 mM fructose. After 30 min preincubation,toxic chemicalswere added,andviability wasassessed after variousperiodsof time asdescribedin MATERIALS AND METHODS. Concentrationsand timesof
aspercent of the viability of control cellsat pH cate determinationsfrom 2 or morecell isolations. glyceraldehyde-3-phosphate dehydrogenase, abolished protection by fructose against cyanide toxicity. Fructose alone provided little protection against the toxicity of the ionophores, gramicidin D (250 nM) and Br-A23187 (10 PM), and the protease, pronase (0.5 mg/ml). However, fructose provided substantial protection against toxicity by the oxidant chemicals, menadione (50 PM), t-BuOOH (50 PM) and cystamine (2.5 mM). Fructose did not protect against HgCl2 (50 PM) which at this concentration was a potent inhibitor of bovine glyceraldehyde-3-phosphate dehydrogenase(data not shown). Fructose provided good protection against cyanide and oligomycin toxicity but little protection against CCCP. An explanation may be that CCCP, but not cyanide or 602
Vol.
167,
No.
BIOCHEMICAL
2, 1990
100
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
A
e i50e
AND
50 0
f
--
P
6 t &
s
o-
C t 50-
O-
.-==Ys 0
30
Time
60
90
120
(min)
Fi re 2. Effect of fructoseand oligomycinon the cytotoxicityof CCCP,Br-A23187and gramzc m D - Freshly isolatedhepatocyteswere incubatedin KRH in the resenceor absenceof 20 mM fructose.After 30min, CCCP(10 PM, panelA B), and gramicidinD (250 nM, panel C) were added with or without 10 &ml ohgomycin. Cell viabihty was assessed asdescribedin Additions are: none, open circles;toxic chemical(CCCP, Br-A23187 or gramicidin D), closedcircles;toxic chemicalplusfructose,closedtrian es;toxic chemicalplusfructoseand oligomydn,closedsquares.Data representmeansf d.E. of triplicate determinationsfrom 3 or more cellisolations.
7i.F
oligomycin, stimulates ATP hydrolysis by the mitochondrial FIFO-ATPase. Such uncoupled ATP consumption would counteract the benefit of glycolytic ATP generation from fructose. To test this hypothesis, we examined whether oligomycin, a specific inhibitor of mitochondrial ATPase, might confer upon fructose the property of protecting against CCCP toxicity (Fig. 2). In confirmation, oligomycin (10 pg/ml) plus fructose conferred complete protection against CCCP toxicity. This occurred despite the fact that oligomycin by itself was toxic, causing 50% cell killing in 90 min (Fig. 1) and 90% cell killing in 120 min. Oligomycin plus fructose also1provided complete protection against toxicity by BrA23187 and substantial protection against gramicidin D toxicity (Fig. 2). Thus, cytotoxicity by BrA23187 and gramicidin D appeared to be mediated by uncoupling of oxidative phosphorylation and intracellular A’TP hydrolysis. Protection againsttoxic cell killing by acidoticpH - Previously, acidotic pH was shown to be protective in models of hypoxic cell injury (1,9-11). To determine whether acidosiswas generally protective in hepatocellular injury, cell killing at pH 7.4 and pH 6.5 was compared after exposure of hepatocytes to various toxic chemicals (Fig. 1). Acidotic pH protected against lethal cell injury from exposure to all toxic chemicalsexamined except pronase. Role of intracellular acidosisin protection by fnrctose - To determine if protection by fructose was due to lactic acid formation and subsequent intracellular acidosis,hepatocytes 603
Vol.
167,
No.
2, 1990
BIOCHEMICAL
0'
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
I 0
30
60
90
120
Time (min)
Figure 3. Effect of fructoseon cell viabilig in monensin-treated hepatocytes- Freshly isolated heuatocvtes were incubated in modified KRH at DH 7.4. Fructose (20 mMj wasincluded where i
