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Mechanisms of dideoxynucleoside induced hepatitis: Effect on mitochondria DNA replication?
HEPATOLOGY Vol. 22, No. 4, Pt. 2, 1995 1345
AASLD ABSTRACTS
LIFE-THREATENING FOOD ALLERGY IN A CHILD TREATED WITH FKb06. F. Lacaille, J. Laurent, J. Bousquet, J. Schmitz. Department of Psediatrics, Enfants Malades Hospital, Paris, and Department of Respiratory Diseases, Montpellier~ France. A 7,month-old girl was transplanted for biliary atresia. Her mother had had asthma in childhood. Prior to liver transplantation (LT ~) she had been on a normal diet. After LT she wae given a protein hydrolysate. Immunosuppression was FKb06, prednisone and azathioprine. She was well until 5 months after LT, when she had 3 episodes of severe angioedema with generalized urticaria, responsive to high dose steroids. She was then hospitalized in ICU for a severe attack of asthma, responsive to steroids and epinephrine. Giant urticaria appeared after contact with latex. Each episode followed an accidental ingestion of cow's milk. Total IgE were elevated (190 kIU/l), so were specific IgE (PAST) against casein, egg yolk~ peanut, and latex. After the last episode she was given a mixture of aminoacids (Neocate*~ SHS), and other nutrients were reintroduced one by one on hospital supervision. Latex was excluded from the environment. She was though readmitted several times for asthma, and once in ICU where she needed mechanical ventilation. She also developed eczema. At sge 17 months, she is treated with oral steroids, ketotifen, sodium eromoglycate, and nebulizations of salbutamol and steroids. The liver tests have always been normal. FKb06 blood level has been maintained between 5 and 7 ng/ml. Severe allergic reactions to cow's milk are not rare in ehildren~ but they usually do not occur after such a long period of tolerance (pre-LT). It is also uncommon to see so many allergic manifestations. We are testing this in vitro in lymphoeytes from normal and allergic subjects by studying proliferation and cytokine production (particularly IL4) with and without FK506. With the results, we may switch the child to cyelosporine.
1347 MULTIPLE PATHWAYS FOR ESTRONE SULFATE UPTAKE IN
1346
443A
MECHANISMS OF DIDEOXYNUCLEOSlDE INDUCED HEPATITIS: EFFECT ON MITOCHONDRIA DNA REPLICATION? (3. Lake-Bakaar and Kate Dickman. Department of Medicine,VAMC Northportand SUNY Health ScienceCenter at Stony Brook, NY. INTRODUCTION: Dideoxynucleosides(ddNs) like dideoxyinosineddl, which are used in the treatment of HIV disease, are potent inhibitors of HIV-1 replication and are readily and preferentiallyincorporetedinto elongating HIV DNA by viral reverse transcdptase, causing chain termination. Although nuclear DNA poiymereses alpha and delta do not readily utilize ddNs, DNA polymerase gamma which catalyzes mitochondfial DNA replication, is readily inhibited by ddNs. The mitochondrial genome contains genes which encode protein components for several enzymes involved in terminal electron transport/oxidativephosphorylation (oxphos), a major pathway for intrecellular ATP generation. The unpredictable delayed hepatitis associatedwith ddN treatmentof HIV disease may be relatedto delayedinhibition of mitochonddal replication. METHODS: Hep 2G cellswere grown followinga 1 to 6 split for 9 days in culture media containing ddl at concentrationsof 20 uM or 50 uM, Lactate content was measured spectrephotometricallyusing an NAD-Iinked assay. Basal and uncoupled respiration (in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, FCCP) was measured polarigraphicallywith a Clark-type oxygen electrode and an oxymeter.All measurementswere normalizedfor total protein.
Control ddl 20 uM ddlS0uM
Lactate protein 25.6 28.3 28.4
mM/mg Basal QO2 FCCP QO2 mmollminlmg mmollmin/mg 5.8 19.1 6.3 11.8 5.6 14.3
SUMMARY: Lactateproduction by Hep-2Gcoils under the conditions of the present experiment is high and is little affected by exposure to ddl. Basal oxygen consumption is also unaffected by ddl. By contrast, ddl at both 20 uM and 50 uM reduced FCCPstimulated respiration,suggesting possible mitochondriaEdysfunction in cells exposedto ddl for prolonged periods. CONCLUSION: Prolongedexposureof Hep-2G cells to the dideoxynucleoside ddl, is associated with reduced mitochonddal respiratory function and suggests a possible mechanism for the delayed idiosyncratichepatocallulardamage associated with dideoxynucleosidetreatment.
1348
ISOLATED RAT HEPATOCYTES. D Lam', A Hassenu, and KS Pan_~. Faculty of Pharmacy* and Department of Pharmacology*, University of Toronto, Canada.
INTERACTION OF HEPATOCYTE GROWTH FACTOR WITH CHANGES PRODUCED IN THE LIVER AFTER PARTIAL HEPATECTOMY. L Lambotte. JP Germ. A Salllz. B U~ Laboratory of Expadmentsi Stage;y, Univem~ty of LotNain Medical School, Brussels, Belgium.
The uptake of estrone sulfate, EIS (1 to 400 /zM, traced by [3H]E~S) was studied by rapid filtration in isolated rat bepatocytes (viability > 90%). The rate of accumulation of E~S in liver ceils was constant up to 1 rain, and the plot exhibited a positive yintercept suggestive of nonspeeifie binding. Regression of the rate of E1S accumulation in cells vs. concentration displayed uptake curves which were subsequently segregated into saturable (Kin of 155:7 /zM and Vm~ of 0.785:0.62 umol/min/g) and nonsaturable (linear, clearance of 0.0028+0.0025 ral/mirgl@ cells; n=6) components• by curve-fitting techniquesl In the presence of 1 mM ouabain and replacement of sodium with choline, E~S uptake was significantly suppressed (43% and 38% of control, respectively). The rate of E1S was also diminished in the presence of temperature (at 27°C, 48%) and metabolic inhibitors: 15 pg/ml antimycin A, 22%; 30/~M rotenone, 20%; 2 mM KCN, 47%. In the presence of 4,4'-diisothiocyanostilbene-2-2'-disulfonicacid (DIDS, 2 raM), an inhibitor of the anion transporter, a reduction in E~S uptake (34%) was further found. The sinusoidal transfer of E1S was also reduced (53%) by harmol sulfate (HS, 600 #M), which has been shown to enter the liver cell by saturable and nonsaturable systems (Km and Vm~ 123/~M and 0.48 nmol/rain/106 cells, respectively, and the linear transmembrane clearance was 0.001 ml/mirg 106 cells) that are DIDS-sensitive and energy but not sodium dependent. The data strongly suggest that the hepatocellular uptake of E~S in the rat is via multiple pathways that require sodium, energy, and are shared, at least in part, with other anions such as DIDS and HS.
In o~ler to play a role in the liver regeneration, HGF, a potent mitogen with a great vadaty of sites of Ixoductton and action, has most likely to combine with more Uver-spaclfic factors. An increased sensitivity of hepatocytes (Hx) to HGF after a 1/3 partial hepatectomy {PH) has been repaltod by others. In the present expadment, IHGF (100 pg) was administered in mall Wistar rats, immediately after (time zero) and ten houm after such a 1/3PH but also after two protocols susceptible of indudng a "state of Iximing" of Hx : a sham operation (SH) and a temparepj 70% PH (TPH) in which the liver lobes are devescullrized for 3 hr with the protection of local hypothem~ and then reconnected. A stimulatoW effect of HGF, evatu~ed by the incorporation of [3H]4hymldlne in the DNA, compared to non-treated similarly operated rats, is not detected 24 hr after a 81-I ( 1 2 . 6 , 3 . 9 versus 12.0 ± 2.1 DPM/pg DNA, M + SL=) but Is obsorved after a TPH (44.6 + 8.7 v 11.3:1: 1.3; p < 0.01), which by itself does not induce DNA Slmthe~s and is even more important after a 1/3PH (148.7:1: 27:1 v40.4:1: 2.6; p < 0.001). The dlffemnee between SH and TPH, both of which induce an acceleration of the DNA synthesis after a subsequent 2/3PH and thus some form of palming, could he related tothe fact that TPH but not SH induces an increased exlxeSsien of o-myc which may thus he required to ntnln an effect of HGF. In a second sedes of e x ~ m e n t s , a singlldoesofl00pgd'lGFwesgiveneltherzemor 10hr aftera 1/3PH. A two-way Anova ~ e s that the effects of edministmtkm at zero (82.1 * 1.2) or at 10 hr (115 ± 10) are signWcantly different flora the control (40.4 ± 2.6), equivalent and, wtNm companxl to the double edminislmtlln, additive without interaction. Until now. the effect of HGF on Hx proliferation /n v/vo has only been repoded Mter continuous or repeated adminlatretlon. Due to its shod hell-life, it seerns unlikely that the response elicited at time zero tom,spends to en increased sensitlvtiy to HGF bnt it can be suggested that the sequence of events HGF produces has to be combined with other changes produced by the PH and that the chronology of this interadion is not cdtinel.
AASLD ABSTRACTS
LIFE-THREATENING FOOD ALLERGY IN A CHILD TREATED WITH FKb06. F. Lacaille, J. Laurent, J. Bousquet, J. Schmitz. Department of Psediatrics, Enfants Malades Hospital, Paris, and Department of Respiratory Diseases, Montpellier~ France. A 7,month-old girl was transplanted for biliary atresia. Her mother had had asthma in childhood. Prior to liver transplantation (LT ~) she had been on a normal diet. After LT she wae given a protein hydrolysate. Immunosuppression was FKb06, prednisone and azathioprine. She was well until 5 months after LT, when she had 3 episodes of severe angioedema with generalized urticaria, responsive to high dose steroids. She was then hospitalized in ICU for a severe attack of asthma, responsive to steroids and epinephrine. Giant urticaria appeared after contact with latex. Each episode followed an accidental ingestion of cow's milk. Total IgE were elevated (190 kIU/l), so were specific IgE (PAST) against casein, egg yolk~ peanut, and latex. After the last episode she was given a mixture of aminoacids (Neocate*~ SHS), and other nutrients were reintroduced one by one on hospital supervision. Latex was excluded from the environment. She was though readmitted several times for asthma, and once in ICU where she needed mechanical ventilation. She also developed eczema. At sge 17 months, she is treated with oral steroids, ketotifen, sodium eromoglycate, and nebulizations of salbutamol and steroids. The liver tests have always been normal. FKb06 blood level has been maintained between 5 and 7 ng/ml. Severe allergic reactions to cow's milk are not rare in ehildren~ but they usually do not occur after such a long period of tolerance (pre-LT). It is also uncommon to see so many allergic manifestations. We are testing this in vitro in lymphoeytes from normal and allergic subjects by studying proliferation and cytokine production (particularly IL4) with and without FK506. With the results, we may switch the child to cyelosporine.
1347 MULTIPLE PATHWAYS FOR ESTRONE SULFATE UPTAKE IN
1346
443A
MECHANISMS OF DIDEOXYNUCLEOSlDE INDUCED HEPATITIS: EFFECT ON MITOCHONDRIA DNA REPLICATION? (3. Lake-Bakaar and Kate Dickman. Department of Medicine,VAMC Northportand SUNY Health ScienceCenter at Stony Brook, NY. INTRODUCTION: Dideoxynucleosides(ddNs) like dideoxyinosineddl, which are used in the treatment of HIV disease, are potent inhibitors of HIV-1 replication and are readily and preferentiallyincorporetedinto elongating HIV DNA by viral reverse transcdptase, causing chain termination. Although nuclear DNA poiymereses alpha and delta do not readily utilize ddNs, DNA polymerase gamma which catalyzes mitochondfial DNA replication, is readily inhibited by ddNs. The mitochondrial genome contains genes which encode protein components for several enzymes involved in terminal electron transport/oxidativephosphorylation (oxphos), a major pathway for intrecellular ATP generation. The unpredictable delayed hepatitis associatedwith ddN treatmentof HIV disease may be relatedto delayedinhibition of mitochonddal replication. METHODS: Hep 2G cellswere grown followinga 1 to 6 split for 9 days in culture media containing ddl at concentrationsof 20 uM or 50 uM, Lactate content was measured spectrephotometricallyusing an NAD-Iinked assay. Basal and uncoupled respiration (in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, FCCP) was measured polarigraphicallywith a Clark-type oxygen electrode and an oxymeter.All measurementswere normalizedfor total protein.
Control ddl 20 uM ddlS0uM
Lactate protein 25.6 28.3 28.4
mM/mg Basal QO2 FCCP QO2 mmollminlmg mmollmin/mg 5.8 19.1 6.3 11.8 5.6 14.3
SUMMARY: Lactateproduction by Hep-2Gcoils under the conditions of the present experiment is high and is little affected by exposure to ddl. Basal oxygen consumption is also unaffected by ddl. By contrast, ddl at both 20 uM and 50 uM reduced FCCPstimulated respiration,suggesting possible mitochondriaEdysfunction in cells exposedto ddl for prolonged periods. CONCLUSION: Prolongedexposureof Hep-2G cells to the dideoxynucleoside ddl, is associated with reduced mitochonddal respiratory function and suggests a possible mechanism for the delayed idiosyncratichepatocallulardamage associated with dideoxynucleosidetreatment.
1348
ISOLATED RAT HEPATOCYTES. D Lam', A Hassenu, and KS Pan_~. Faculty of Pharmacy* and Department of Pharmacology*, University of Toronto, Canada.
INTERACTION OF HEPATOCYTE GROWTH FACTOR WITH CHANGES PRODUCED IN THE LIVER AFTER PARTIAL HEPATECTOMY. L Lambotte. JP Germ. A Salllz. B U~ Laboratory of Expadmentsi Stage;y, Univem~ty of LotNain Medical School, Brussels, Belgium.
The uptake of estrone sulfate, EIS (1 to 400 /zM, traced by [3H]E~S) was studied by rapid filtration in isolated rat bepatocytes (viability > 90%). The rate of accumulation of E~S in liver ceils was constant up to 1 rain, and the plot exhibited a positive yintercept suggestive of nonspeeifie binding. Regression of the rate of E1S accumulation in cells vs. concentration displayed uptake curves which were subsequently segregated into saturable (Kin of 155:7 /zM and Vm~ of 0.785:0.62 umol/min/g) and nonsaturable (linear, clearance of 0.0028+0.0025 ral/mirgl@ cells; n=6) components• by curve-fitting techniquesl In the presence of 1 mM ouabain and replacement of sodium with choline, E~S uptake was significantly suppressed (43% and 38% of control, respectively). The rate of E1S was also diminished in the presence of temperature (at 27°C, 48%) and metabolic inhibitors: 15 pg/ml antimycin A, 22%; 30/~M rotenone, 20%; 2 mM KCN, 47%. In the presence of 4,4'-diisothiocyanostilbene-2-2'-disulfonicacid (DIDS, 2 raM), an inhibitor of the anion transporter, a reduction in E~S uptake (34%) was further found. The sinusoidal transfer of E1S was also reduced (53%) by harmol sulfate (HS, 600 #M), which has been shown to enter the liver cell by saturable and nonsaturable systems (Km and Vm~ 123/~M and 0.48 nmol/rain/106 cells, respectively, and the linear transmembrane clearance was 0.001 ml/mirg 106 cells) that are DIDS-sensitive and energy but not sodium dependent. The data strongly suggest that the hepatocellular uptake of E~S in the rat is via multiple pathways that require sodium, energy, and are shared, at least in part, with other anions such as DIDS and HS.
In o~ler to play a role in the liver regeneration, HGF, a potent mitogen with a great vadaty of sites of Ixoductton and action, has most likely to combine with more Uver-spaclfic factors. An increased sensitivity of hepatocytes (Hx) to HGF after a 1/3 partial hepatectomy {PH) has been repaltod by others. In the present expadment, IHGF (100 pg) was administered in mall Wistar rats, immediately after (time zero) and ten houm after such a 1/3PH but also after two protocols susceptible of indudng a "state of Iximing" of Hx : a sham operation (SH) and a temparepj 70% PH (TPH) in which the liver lobes are devescullrized for 3 hr with the protection of local hypothem~ and then reconnected. A stimulatoW effect of HGF, evatu~ed by the incorporation of [3H]4hymldlne in the DNA, compared to non-treated similarly operated rats, is not detected 24 hr after a 81-I ( 1 2 . 6 , 3 . 9 versus 12.0 ± 2.1 DPM/pg DNA, M + SL=) but Is obsorved after a TPH (44.6 + 8.7 v 11.3:1: 1.3; p < 0.01), which by itself does not induce DNA Slmthe~s and is even more important after a 1/3PH (148.7:1: 27:1 v40.4:1: 2.6; p < 0.001). The dlffemnee between SH and TPH, both of which induce an acceleration of the DNA synthesis after a subsequent 2/3PH and thus some form of palming, could he related tothe fact that TPH but not SH induces an increased exlxeSsien of o-myc which may thus he required to ntnln an effect of HGF. In a second sedes of e x ~ m e n t s , a singlldoesofl00pgd'lGFwesgiveneltherzemor 10hr aftera 1/3PH. A two-way Anova ~ e s that the effects of edministmtkm at zero (82.1 * 1.2) or at 10 hr (115 ± 10) are signWcantly different flora the control (40.4 ± 2.6), equivalent and, wtNm companxl to the double edminislmtlln, additive without interaction. Until now. the effect of HGF on Hx proliferation /n v/vo has only been repoded Mter continuous or repeated adminlatretlon. Due to its shod hell-life, it seerns unlikely that the response elicited at time zero tom,spends to en increased sensitlvtiy to HGF bnt it can be suggested that the sequence of events HGF produces has to be combined with other changes produced by the PH and that the chronology of this interadion is not cdtinel.