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Bifunctional effect of tumor necrosis factor alpha (TNF-α) on the oxidative stress response and NF-κB activation of cultured rat hepatocytes and HEPG2 cells
HEPATOLOGY Vol. 22, No. 4, P t . 2, 1995
AASLD
ABSTRACTS
237A
521 ~ o~ DEFm~ ~ Mra~HCr,~aAL ~ C~ C~H EIHANOL-FI~ (EIOH) RA'IS:~ ~ COM~Sr[I(:~ AND EFF~2S OF S-ADt~(~'L-L-MEIIqIONINE (SAM). A Cole~C. Garda-Ruiz, A IV~rales,I. Redes. N. Kapbwitz* and !.C. F e m a n d e z ~ Liver Ur~ Hosl:/~ C]k~ici Provk~al, B~dona, Spa~ and *GI andLiver Dmas~,USC SchoolofMedk~,LK, CK Chronic ethanol intake depletes the mitochondrial pool of GSH by impairing the transport of GSH from cytosol into mitochondria. It has been recently shown that mitochondria from ETOH fed rats supplemented with SAM displayed a preserved capacity to transport GSH from cytosol into mitochondria (Hepatology 21: 207-214, 1995). Since chronic ethanol exposure and SAM affect biological membrane properties, the purpose of our study was to determine the lipid composition of mitochondria in relation to the kinetics of GSH transport in isolated mitochondria from rats fed ethanol for 1 month with or without SAM administration (02 mmol/Kg/day) in the liquid diet. The initial rate of 35SGSH (0.1-50mM) transport into isolated mitochondria followed Michaelis-Menten kinetics revealing two saturable transport components that were affected by chronic ETOH feeding: 2 fold increase in Km of the high affinity component (0.095~+0.008 vs. 0.047-+0.002 mM) and a 50% decrease in Vmax (0.76+0.08 vs. 1.41_+0.2 n m o l / 1 5 s e c / m g prot) of the low affinity component. ETOH- SAM supplemented mitochondria transported GSH with kinetic properties nearly identical to pair-fed controls. L i p i d s from ETOH mitochondria revealed a significant increase in total cholesterol/phospholipid molar ratio compared to pair-fed control (0.102+0.043 vs. 0.045_+0.009) due to increased cholesterol content. SAM supplementation to ETOH rats prevented the increase in cho]est~l/ph~ho~ici ratio. Choles~o!-eurki~ control mitochondria to levels similar to those found in ETOH mitochondria displayed a 50% decrease in the initial rate of 35S-GSH uptake (at I and 10mM). Conclusions: Isolated mitochondria from ETOH fed rats exhibit a defect in GSH transport kinetics. Mitochondria contained increased cholesterol after ethanol feeding. Control mitochondria loaded with cholesterol exhibit impaired GSH uptake. SAM prevents the lipid compositional and GSH transport changes. These results suggest that ethanol-induced changes in lipid composition impair the function of the GSH carrier in mitochondria.
522 GLUTATHIONE SUPPRESSION OF ENDOTOXIN-INDUCED NF-rB
523 BIFUNCTIONAL EFFECTOFTUIvIORNB:~CSEFACTORALPHA(rNF-cz)ON THE OXIDATIVE STRESS R E S ~ S E AND NF-rd3 ACTIVATION (XJLTURt~RAT HEPATC~q~ES ANDI--IEIK~Ch'Il ~.A. Morales.C Carda-Ruiz, A. Colell.A. Ballesta*.and I. C F e r n ~ z - C h e ~ Uver Unit, *Servido Bio~mica, HospitalClinici Provindal, Unive~daddeBarm_lona, Barcelona,Slmin. TNF~, a pleiotropiccytokine is capableof indudng strikingly c~fferentcellular readion~ Among other mechanisms reactive ox3om ~x~des (RC6) have been propo~i as an imporiant factor in mediaiing the cy~toxic effects of TNF-(z. The purlx~ of our studywasto ~ the r~]afion of helmticGSH of TNF~ in relation to o~4dafiveeffeclsand activation of NF-cB in mt oal0Lredhepatocytes and human HepG2 m_llline. Rat exltured hepa~y0es and Hep(X! cells were incubated with recomlinantTNF-a(fh'omega,2.7.107 UAng)at250,500,2O00and10.000U/ml for 24 hr, meamringRCS and lipidlCm~mddaliontry 2'-7 -did'domfluorem~ ([X~ and dsparinaric, re~edively and GSH levels by HPLC TNF-a increased GSH levels compared to conirol hepa~-ytes (27_+6nmol/106 ~ vs 44~ nrnol/l(36 mils) with no increase in DCF or lore of ds-l~naric, indurating atmnce of oxidative stress at conamWaiion up to 2300 U/ml. This indudive effect refted~d an ina'eased GSH synthetic capadty, sino~ cdl-fi~e ex~ac~ of TNF-~-treated hepa~3/~s d~lay~d a geafDer synthetic GSH rate from ~ e del~xmined by monochlorol~nane (028_+0.05va 0375:0~8nmolAnin/mgproL) which was alreadyot:mrved at 4 hr of incubation. The TNF-~ indudive effectwas atto~led by preincubafion of mils with adinomT~Sn D, indicating the requirement for de novo RNA synthesi~ Nudear extractsfrom TNF-(z(up to ~300U//nl) lzeatedhepat(xy~esdispla3~l adivafion of N F/¢.]3in a do~,-dependent marme~ (2-4fold) compared to basal h ~ At higher TNF-aclmemtrations ~10.000 U/ml) or by the adchliortof a cy~kine oxkiail,IL-I~, IFNff, LPS and TNF~ (5U, 100U, 11~ and 500 U per mL req~vely), GSH w ~ severely&~leted (40-50%)resultinginan in~rea~ein DCF (3 fold), loss of ds-Fal~aric add (40-60%) and supefindudion of NF-rJ3 (10-12 fold). Condu~ons TNF-a di~ayed a dual effecton fire rag~ation of hepatic GSH, indudng the synthetic GSH enzymes at moderate conoantrations with no produdion of ROS and activation of NF-r,B.At highe*conomtrations, TNF-a depleted hepaiic GSH resultingin oxidative stressleadingtoa g~eaterinduction of NF-KB.
5 2 4 ALTERATION OF GSH HOMEOSTASIS
ACTIVATION AND TUMOR NECROSIS FACTOR RELEASE FROM ISOLATED KUPFFER CELLS. BA Neuschwandar-Tetri, JM Bellezzo~ RS Britton and ES Fox. Saint Louis Univ. School of Medicine, St. Louis, MO. Hepatic Kupffer cells may play an important role in mediating liver injury. Activation of macrophages causes the release of potent cytokines and is regulated by the intmcellular activation and nuclear translocation of the transcription factor NF-*zB. Because this signallingpathway can be suppressed pharmacologicallyby N-acetylcysteine, these experiments were undertaken to determine if glutathione (GSH), the most abundant physiologic intraceUular thiol, can have a similar regulatory effect in Kupffer cells. Methods. Rat Kupffer ceils were isolated by collagenase perfusion and centrifugal elutriati0n without endotoxin contamination. Cells were cultured overnight and treated with glutathione monoethyl ester (GSHEE) beginning 1 hr before exposure to 10 ng/ml lipopolysaccharide (LPS). Nuclear NF-~B was measured by electrophoretic gel-shift assay 1 hr after addition' of LPS; cellular total GSH was measured by HPLC and TNF release was measured by ELISA 5 hr after LPS. Results. GSH (l~mol/mgDNA) NF-~cB TNF (n~g DNA) No LPS 105+33 + < 0.03 + l m M GSHEE 224_+28 < 0.03 + 7.5 mM GSHEE 366_+77' < 0.03 + 15 mM GSHEE 393-+24" + < 0.03 LPS 121-+16 ++++ 49±6 + t mM GSHEE 197+_20 64_+7 + 7.5 mM GSHEE 292_+63' 10±8' + 15 mM GSHEE 400+_36" + < 0.03' Values are means ±SEM; n=4 cell preparations. Asterisks denote significant differences from respective control cells not treated with GSHEE. Kupffer cell GSH at 15, 30, and 60 rain after LPS did not differ from control. Conefusions. Augmenting Kupffer cell GSH content with GSHEE suppressed LPS-induced NF-~B activation and TNF release. Although LPS alone did not induce Kupffar cell GSH depletion either acutely (during the time frame of NF-~B activation) or over 5 hr (the time frame of TNF release), elevating the intraceUular GSH content above normal effectively inhibited LPS-stimulated cytokine release. This observation raises the possibility that therapeutic interventions aimed at manipulating hepatic glutathione could diminish liver injury by down-regulating Kupffer cell cytokine release, Supported by NIH grants DK44305 and DK41816,
IN EHBR RATS: MECHANISMS AND ORGAN SPECIFICITY. $. C. Lu. J. Cai. W. Sun. H. Takikawa, S. Horie and N. KaDIowitz. Division of GI and Liver Diseases, USC School of Medicine, LA, CA, Eisai Co., Ltd., and Teikyo University, Japan. EHBR rats are mutant Sprague-Dawley rats (similar to the TR-, mutant Wistar rats) which exhibit impaired biliary organic anion and GSH secretion. In addition, liver GSH levels in both types of mutant rats are twice that of age-matched controls. The exact mechanism for the increase in cell GSH is unknown. The aims of our current study were to elucidate the mechanisms of increased hepatic GSH level and to examine organ specificity of altered GSH homeostasis in EHBR rats. FI.ESULTS: GSH levels in liver, kidney, duodenal, jejunal and ileal mucosa of EHBR rats were 200-300% of age-matched control rats (n=5 each). Parameters that affect the intracellular steady state GSH level, namely the availability of cysteine and the activity of y-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis, were measured. Cysteine level was significantly increased (169% of controls) in livers of EHBR rats compared to controls. GCS activity as measured by previously described methods (J. Clin. Invest. 90:524-532, 1992) in both liver and kidney cytosol of EHBR rats was also significantly increased (161-200% of controls) compared to controls. GSH synthetase activity in liver cytosol of EHBR rats was not different from that of controls. We excluded the possibility that the changes in GSH synthesis are due to impaired biliary GSH secretion since these changes are absent in diabetic rats which also exhibit impaired biliary GSH secretion. In summary, steady state GSH level is increased in multiple organs of the EHBR rat and is most likely explained by both increased availability o f cysteine and GCS activity. The mechanism by which a primary genetic defect in the ATP-driven organic anion transporter leads indirectly to alterations in GSH homeostasis remains to be elucidated.
AASLD
ABSTRACTS
237A
521 ~ o~ DEFm~ ~ Mra~HCr,~aAL ~ C~ C~H EIHANOL-FI~ (EIOH) RA'IS:~ ~ COM~Sr[I(:~ AND EFF~2S OF S-ADt~(~'L-L-MEIIqIONINE (SAM). A Cole~C. Garda-Ruiz, A IV~rales,I. Redes. N. Kapbwitz* and !.C. F e m a n d e z ~ Liver Ur~ Hosl:/~ C]k~ici Provk~al, B~dona, Spa~ and *GI andLiver Dmas~,USC SchoolofMedk~,LK, CK Chronic ethanol intake depletes the mitochondrial pool of GSH by impairing the transport of GSH from cytosol into mitochondria. It has been recently shown that mitochondria from ETOH fed rats supplemented with SAM displayed a preserved capacity to transport GSH from cytosol into mitochondria (Hepatology 21: 207-214, 1995). Since chronic ethanol exposure and SAM affect biological membrane properties, the purpose of our study was to determine the lipid composition of mitochondria in relation to the kinetics of GSH transport in isolated mitochondria from rats fed ethanol for 1 month with or without SAM administration (02 mmol/Kg/day) in the liquid diet. The initial rate of 35SGSH (0.1-50mM) transport into isolated mitochondria followed Michaelis-Menten kinetics revealing two saturable transport components that were affected by chronic ETOH feeding: 2 fold increase in Km of the high affinity component (0.095~+0.008 vs. 0.047-+0.002 mM) and a 50% decrease in Vmax (0.76+0.08 vs. 1.41_+0.2 n m o l / 1 5 s e c / m g prot) of the low affinity component. ETOH- SAM supplemented mitochondria transported GSH with kinetic properties nearly identical to pair-fed controls. L i p i d s from ETOH mitochondria revealed a significant increase in total cholesterol/phospholipid molar ratio compared to pair-fed control (0.102+0.043 vs. 0.045_+0.009) due to increased cholesterol content. SAM supplementation to ETOH rats prevented the increase in cho]est~l/ph~ho~ici ratio. Choles~o!-eurki~ control mitochondria to levels similar to those found in ETOH mitochondria displayed a 50% decrease in the initial rate of 35S-GSH uptake (at I and 10mM). Conclusions: Isolated mitochondria from ETOH fed rats exhibit a defect in GSH transport kinetics. Mitochondria contained increased cholesterol after ethanol feeding. Control mitochondria loaded with cholesterol exhibit impaired GSH uptake. SAM prevents the lipid compositional and GSH transport changes. These results suggest that ethanol-induced changes in lipid composition impair the function of the GSH carrier in mitochondria.
522 GLUTATHIONE SUPPRESSION OF ENDOTOXIN-INDUCED NF-rB
523 BIFUNCTIONAL EFFECTOFTUIvIORNB:~CSEFACTORALPHA(rNF-cz)ON THE OXIDATIVE STRESS R E S ~ S E AND NF-rd3 ACTIVATION (XJLTURt~RAT HEPATC~q~ES ANDI--IEIK~Ch'Il ~.A. Morales.C Carda-Ruiz, A. Colell.A. Ballesta*.and I. C F e r n ~ z - C h e ~ Uver Unit, *Servido Bio~mica, HospitalClinici Provindal, Unive~daddeBarm_lona, Barcelona,Slmin. TNF~, a pleiotropiccytokine is capableof indudng strikingly c~fferentcellular readion~ Among other mechanisms reactive ox3om ~x~des (RC6) have been propo~i as an imporiant factor in mediaiing the cy~toxic effects of TNF-(z. The purlx~ of our studywasto ~ the r~]afion of helmticGSH of TNF~ in relation to o~4dafiveeffeclsand activation of NF-cB in mt oal0Lredhepatocytes and human HepG2 m_llline. Rat exltured hepa~y0es and Hep(X! cells were incubated with recomlinantTNF-a(fh'omega,2.7.107 UAng)at250,500,2O00and10.000U/ml for 24 hr, meamringRCS and lipidlCm~mddaliontry 2'-7 -did'domfluorem~ ([X~ and dsparinaric, re~edively and GSH levels by HPLC TNF-a increased GSH levels compared to conirol hepa~-ytes (27_+6nmol/106 ~ vs 44~ nrnol/l(36 mils) with no increase in DCF or lore of ds-l~naric, indurating atmnce of oxidative stress at conamWaiion up to 2300 U/ml. This indudive effect refted~d an ina'eased GSH synthetic capadty, sino~ cdl-fi~e ex~ac~ of TNF-~-treated hepa~3/~s d~lay~d a geafDer synthetic GSH rate from ~ e del~xmined by monochlorol~nane (028_+0.05va 0375:0~8nmolAnin/mgproL) which was alreadyot:mrved at 4 hr of incubation. The TNF-~ indudive effectwas atto~led by preincubafion of mils with adinomT~Sn D, indicating the requirement for de novo RNA synthesi~ Nudear extractsfrom TNF-(z(up to ~300U//nl) lzeatedhepat(xy~esdispla3~l adivafion of N F/¢.]3in a do~,-dependent marme~ (2-4fold) compared to basal h ~ At higher TNF-aclmemtrations ~10.000 U/ml) or by the adchliortof a cy~kine oxkiail,IL-I~, IFNff, LPS and TNF~ (5U, 100U, 11~ and 500 U per mL req~vely), GSH w ~ severely&~leted (40-50%)resultinginan in~rea~ein DCF (3 fold), loss of ds-Fal~aric add (40-60%) and supefindudion of NF-rJ3 (10-12 fold). Condu~ons TNF-a di~ayed a dual effecton fire rag~ation of hepatic GSH, indudng the synthetic GSH enzymes at moderate conoantrations with no produdion of ROS and activation of NF-r,B.At highe*conomtrations, TNF-a depleted hepaiic GSH resultingin oxidative stressleadingtoa g~eaterinduction of NF-KB.
5 2 4 ALTERATION OF GSH HOMEOSTASIS
ACTIVATION AND TUMOR NECROSIS FACTOR RELEASE FROM ISOLATED KUPFFER CELLS. BA Neuschwandar-Tetri, JM Bellezzo~ RS Britton and ES Fox. Saint Louis Univ. School of Medicine, St. Louis, MO. Hepatic Kupffer cells may play an important role in mediating liver injury. Activation of macrophages causes the release of potent cytokines and is regulated by the intmcellular activation and nuclear translocation of the transcription factor NF-*zB. Because this signallingpathway can be suppressed pharmacologicallyby N-acetylcysteine, these experiments were undertaken to determine if glutathione (GSH), the most abundant physiologic intraceUular thiol, can have a similar regulatory effect in Kupffer cells. Methods. Rat Kupffer ceils were isolated by collagenase perfusion and centrifugal elutriati0n without endotoxin contamination. Cells were cultured overnight and treated with glutathione monoethyl ester (GSHEE) beginning 1 hr before exposure to 10 ng/ml lipopolysaccharide (LPS). Nuclear NF-~B was measured by electrophoretic gel-shift assay 1 hr after addition' of LPS; cellular total GSH was measured by HPLC and TNF release was measured by ELISA 5 hr after LPS. Results. GSH (l~mol/mgDNA) NF-~cB TNF (n~g DNA) No LPS 105+33 + < 0.03 + l m M GSHEE 224_+28 < 0.03 + 7.5 mM GSHEE 366_+77' < 0.03 + 15 mM GSHEE 393-+24" + < 0.03 LPS 121-+16 ++++ 49±6 + t mM GSHEE 197+_20 64_+7 + 7.5 mM GSHEE 292_+63' 10±8' + 15 mM GSHEE 400+_36" + < 0.03' Values are means ±SEM; n=4 cell preparations. Asterisks denote significant differences from respective control cells not treated with GSHEE. Kupffer cell GSH at 15, 30, and 60 rain after LPS did not differ from control. Conefusions. Augmenting Kupffer cell GSH content with GSHEE suppressed LPS-induced NF-~B activation and TNF release. Although LPS alone did not induce Kupffar cell GSH depletion either acutely (during the time frame of NF-~B activation) or over 5 hr (the time frame of TNF release), elevating the intraceUular GSH content above normal effectively inhibited LPS-stimulated cytokine release. This observation raises the possibility that therapeutic interventions aimed at manipulating hepatic glutathione could diminish liver injury by down-regulating Kupffer cell cytokine release, Supported by NIH grants DK44305 and DK41816,
IN EHBR RATS: MECHANISMS AND ORGAN SPECIFICITY. $. C. Lu. J. Cai. W. Sun. H. Takikawa, S. Horie and N. KaDIowitz. Division of GI and Liver Diseases, USC School of Medicine, LA, CA, Eisai Co., Ltd., and Teikyo University, Japan. EHBR rats are mutant Sprague-Dawley rats (similar to the TR-, mutant Wistar rats) which exhibit impaired biliary organic anion and GSH secretion. In addition, liver GSH levels in both types of mutant rats are twice that of age-matched controls. The exact mechanism for the increase in cell GSH is unknown. The aims of our current study were to elucidate the mechanisms of increased hepatic GSH level and to examine organ specificity of altered GSH homeostasis in EHBR rats. FI.ESULTS: GSH levels in liver, kidney, duodenal, jejunal and ileal mucosa of EHBR rats were 200-300% of age-matched control rats (n=5 each). Parameters that affect the intracellular steady state GSH level, namely the availability of cysteine and the activity of y-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis, were measured. Cysteine level was significantly increased (169% of controls) in livers of EHBR rats compared to controls. GCS activity as measured by previously described methods (J. Clin. Invest. 90:524-532, 1992) in both liver and kidney cytosol of EHBR rats was also significantly increased (161-200% of controls) compared to controls. GSH synthetase activity in liver cytosol of EHBR rats was not different from that of controls. We excluded the possibility that the changes in GSH synthesis are due to impaired biliary GSH secretion since these changes are absent in diabetic rats which also exhibit impaired biliary GSH secretion. In summary, steady state GSH level is increased in multiple organs of the EHBR rat and is most likely explained by both increased availability o f cysteine and GCS activity. The mechanism by which a primary genetic defect in the ATP-driven organic anion transporter leads indirectly to alterations in GSH homeostasis remains to be elucidated.