Reactive Oxygen Species during Ischemia-Reflow Injury in Isolated Perfused Rat Liver

Transcription

1 Reative Oxygen Speies during shemiareflo njury in solated Perfused Rat Liver Hartmut Jaeshke, harles V. Smith, and Jerry R. Mithell Setion on Hypertension/linial Pharmaology, Department ofmediine, and enterfor xperimental Therapeutis, Baylor ollege ofmediine, Houston, Texas 773 Abstrat The hypothesis that intraellular generation of reative oxygen speies in hepatoytes or retiuloendothelial ells may ause ishemiareperfusion injury as tested in isolated perfused livers of male Fisher rats. GSSG as measured in perfusate, bile, and tissue as a sensitive index of oxidative stress. After a preperfusion phase of 3 min, the perfusion as stopped (global ishemia) for various times (3, 12 min) and the liver as reperfused for another 6 min. The bile flo (1.48±.17,il/min X gram liver eight), the biliary efflux of total glutathione (6.54±.94 nmol GSH eq/min X g), and GSSG (1.59±.23 nmol GSH eq/min X g) reovered to 6986% after shortterm ishemia and to 3672% after 2 h of ishemia hen ompared ith values obtained from ontrol livers perfused for the same period of time. During reperfusion, the sinusoidal efflux of total glutathione (16.4±2.1 nmol GSH eq/min X g) and GSSG (.13±.5 nmol GSH eq/min X g) did not hange exept for an initial 13s inrease during reperfusion ashout. No inreased GSSG seretion into bile as detetable at any time during reperfusion. The liver ontent of total glutathione (32.5±3.5 nmol GSH eq/mg protein) and GSSG (.27±.9 nmol GSH eq/mg protein) did not hange signifiantly during any period of ishemia or reperfusion. We onlude, therefore, that at most only a minor amount of reative oxygen speies ere generated during reperfusion. Thus, reative oxygen speies are unlikely to ause ishemia/reperfusion injury in rat liver by lipid peroxidation or tissue thiol oxidation. ntrodution shemia/reperfusionindued tissue injury is a major pathophysiologial proess responsible for severe organ damage in important human health problems, suh as myoardial infartion, stroke, and organ transplantation (1). There is evidene that reative oxygen speies may play a role in ishemia/reperfusion injury in various organs, i.e., liver (26), heart (7, 8), intestine (9), kidney (1), and brain (1 1). The mehanism of reative oxygen generation through superoxide formation as proposed by Mord (12) involves the enhaned degradation of adenosine to hypoxanthine during ishemia as ell as the onversion ofthe ytosoli enzyme xanthine dehydrogenase to xanthine oxidase (13), hih uses oxygen instead of NAD+ as Address orrespondene to Dr. Jaeshke, Baylor ollege of Mediine, Room 826, One Baylor Plaza, Houston, TX 773. Reeivedfor publiation 1 June 1987 and in revisedform 28 September J. lin. nvest. The Amerian Soiety for linial nvestigation, n /88/4/124/7 $2. Volume 81, April 1988, eletron aeptor. Sine the oxygen onentration in tissue is extremely lo during the ishemi period, the metabolism of hypoxanthine by xanthine oxidase is inhibited due to the lak ofthe seond substrate ofthis reation. Upon reflo, oxygen is available and formation of reative oxygen speies ould take plae, ausing reperfusion injury. Hoever, hepatoytes and endothelial ells (14) as ell as other ell types ontain very effetive protetion mehanisms against reative oxygen speies, i.e., superoxide dismutase and glutathione peroxidase. The biliary and sinusoidal export of GSSG is a ellestablished and sensitive indiator of oxidative stress in vivo (15, 16) and in the isolated perfused rat liver (17, 18). f relevant amounts of reative oxygen speies ere formed at any time during the reperfusion period, an inreased formation of GSSG in the liver ould be expeted. We therefore have measured the effets of ishemia and reperfusion on the formation and effilux of GSSG in the isolated perfused rat liver to test diretly the hypothesis that reflo to previously ishemi tissue auses prodution of suffiient quantities of reative oxygen speies to produe strutural ell damage. Although leukoyte ativation may be an important fator in reflo injury, e hose a leukoytefree system for the present study to test the hypothesis that reflo to ishemi liver auses the hepati generation of reative oxygen speies and that these reative intermediates are produed in suffiient quantities to kill hepatoytes. The results reported here indiate a minimal, if any, enhanement ofthe formation of GSSG by the liver. Thus, no evidene as found for intraellular generation of amounts of reative oxygen speies by hepatoytes or endothelial ells during global ishemia and reperfusion of the liver likely to be suffiient quantitatively to exeed ellular protetive defenses against reative oxygen injury. Methods solated perfused rat liver. Male Fisher 344 rats (1823 g) ere purhased from Harlan SpragueDaley n. (Houston, TX) and alloed free aess to food (Wayne rodent ho) and tap ater. After anesthetizing the animals ith pentobarbital (5 mg/kg i.p.) the livers ere perfused as desribed previously (19, 2) ith hemoglobin and albuminfree KrebsHenseleit biarbonate buffer (ph 7.4, 37 ) gassed ith arbogen (95% 2, 5% O2). The perfusate as pumped through the livers ith a peristalti pump at a onstant flo rate of ml/min X g liver eight in a single pass mode. The bile dut as annulated ith P 1tubing and bile as olleted in preeighed test tubes ontaining 4% sulfosaliyli aid in 1min intervals. After annulating the portal vein (t, min), the livers ere perfused for 3 min and then the perfusion as stopped. After an ishemi period (global ishemia) of 3 or 12 min at room temperature, the livers ere perfused for another 6 min. n additional ishemia experiments, solutions of diquat (a generous gift from Dr. an Wyatt of mperial hemial ndustries, Ltd., Malesfield, ngland) or tbutyl hydroperoxide (Sigma hemial o., St. Louis, MO) ere infused into the 124 H. Jaeshke,. V. Smith, and J. R. Mithell

2 portal vein annula during the reperfusion period. The duration of the experiments had been 12 min (3 min of ishemia) or 21 min (12 min of ishemia) ith a total perfusion period of 9 min. Therefore, "ontrol" experiments (ithout ishemia) ere performed ith a perfusion time of 9 min. The validity of the method and viability riteria ofperfused rat liver have been desribed and disussed in detail (222). The viability of the livers as heked by measurement of the latate dehydrogenase (LDH)' efflux (< 2 mu/min X g liver eight at 3 min), the latatetopyruvate ratio (five to six at 3 min), and the bile flo. Methods. Total soluble glutathione (GSH and GSSG) as measured in bile, perfusate, and in aidi homogenate from freezelamped livers by the method of Tietze (23). For the determination of GSSG a volume of bile, perfusate, or liver homogenate as mixed immediately after olletion or preparation ith 1 mm Nethylmaleimide (NM) in 1 mm potassium phosphate buffer (ph 6.5) as desribed in detail (15). To separate GSSG from NM and NMGSH adduts, an aliquot of the solution as passed through a 18SepPak artridge (Waters Asso., Div. of Millipore orp., Milford, MA) folloed by 1 ml of buffer (15). GSSG as determined in the ombined eluates by the methods of Tietze (23). Hepati ATP in the aidi homogenate of freezelamped liver, LDH, latate, and pyruvate in the perfusate ere determined aording to standard tests (24). Statistis. All data are expressed as the mean±sd. omparison of data sets ere performed ith the unpaired Student's t test. Results When livers from male Fisher rats ere perfused ith Krebs Henseleit biarbonate buffer in a single pass mode, a bile flo of 1.48±.17,ul/min X g liver eight, a total biliary glutathione efflux of 6.54±.94 nmol GSH eq/min X g, and a GSSG efflux of 1.59±.23 nmol GSH eq/min X g (n = 5) ere measured 3 min after annulating the portal vein. The sinusoidal efflux of total glutathione at that time as 16.4±2.1 nmol GSH eq/min X g and the GSSG efflux as.13±.5 nmol GSH eq/min X g. Although the efflux rates of glutathione into the perfusate remained onstant over a total perfusion period of 9 min, the biliary parameters delined gradually and ere determined as 1.1±.1 Al/min X g (bile flo), 3.59±.47 nmol GSH eq/min X g (total glutathione efflux), and ±.25 nmol GSH eq/min X g (GSSG efflux) at the end of the experiment (n = 5). When livers ere reperfused after 3 min of global ishemia (Fig. 1), bile flo reovered to 95% and total glutathione efflux and GSSG efflux into bile reovered to values of 7% (P <.5) ithin 3 min hen ompared ith ontrol livers perfused for the same period of time. No inreased GSSG efflux into bile as observed. The sinusoidal efflux rates ofgsh and GSSG ere inreased only during a short ashout period and returned to ontrol values after 3 s ithout any further hange (Fig. 1). When the ishemi period as extended to 12 min, a tremendous selling of the liver, i.e., a 4% inrease in liver eight, ourred during the first 6 s, and ontinued during the first fe minutes of reperfusion. Aordingly, no bile flo as detetable (Fig. 2). After 5 to 1 min, hen the liver had returned to normal size, i.e., preishemi eight, bile flo reovered to 72% (P <.5), the biliary glutathione efflux to 55% (P <.5), and GSSG efflux to 36% (P <.5) of ontrol values ithin 1 h of reperfusion. All values are ompared statistially ith ontrol livers perfused for 9 min. As after shortterm ishemia, no inreased biliary GSSG 1. Abbreviations used in this paper: LDH, latate dehydrogenase; NM, Nethylmaleimide a x ) 3 o.s.2 1OF Bile 1.5 Perfusate T l 1 o f O 1. :.5 L m GSH.Gsa Av... GS5G......N. 2 t t t (min) Figure 1. Bile flo and hepati glutathione efflux after 3 min ishemia. ah liver as preperfused for 3 min. After a global ishemia period of 3 min, the livers ere reperfused for another 6 min. The total glutathione efflux (given as GSH equivalents) and the GSSG efflux (given as GSH equivalents) into bile (top) and perfusate (bottom) is shon (mean±sd, n = 4). P <.5 ompared ith ontrols. efflux as observed (Fig. 2). The sinusoidal efflux rates ofgsh and GSSG ere enhaned during the ashout period (3 s after starting reperfusion). Although the GSSG efflux delined rapidly and returned to ontrol values as after 3min ishemia, the GSH efflux stayed inreased for several minutes and then gradually returned to ontrol efflux rates (Fig. 2). The LDH release as similar to the GSH efflux (Fig. 3) in that LDH release after shortterm ishemia as inreased during the ashout phase (3 s) and returned to ontrol values ithin 5 min. No signifiant liver ell damage (evaluated as enhaned LDH release) as observed during the 6 min of reperfusion. Longterm ishemia, hoever, aused a greater release of hepati LDH that delined but did not return to basal values and again rose rapidly ithin 3 min of reperfusion, indiating severe ell damage at this time (Fig. 3). The lose orrelation beteen sinusoidal GSH efflux and LDH release in the early phases of reperfusion after long and shortterm ishemia is shon in Fig. 4 in a higher resolution presentation of the initial reperfusion period. The peak efflux rates of LDH and GSH (2 h ishemia) ourred at the time of the maximum liver eight gain (4% higher than preishemi liver eight), indiating an enhaned leakiness of ell membranes possibly due to mehanial stress. n ontrast, GSSG efflux from the liver did not orrelate ith LDH efflux and liver eight gain (Fig. 4). The reason for the onsiderable uptake of ater into liver ells might be due to a hyperosmolar state Oxidant Stress and shemiareflo njury 1241

3 Or 4 2 'i % T T Bile T_;1 T r:,,l 41 _ 3 3: > i.5 D 1 O m 2 x 1 sehmla 1 12 min.2 4 Perfusate O a The objetive of the present study as to investigate the hypothesis that intraellular generation of reative oxygen spe i A...T 2 3 SO 1T T T l H+88 GSHSG m D t (min) Figure 2. Bile flo and hepati glutathione efflux after 12 min ishemia. ah liver as preperfused for 3 min. After a global ishemia period of 12 min, the livers ere reperfused for another 6 min. The total glutathione efflux (given as GSH equivalents) and the GSSG efflux (given as GSH equivalents) into bile (top) and perfusate (bottom) is shon (mean±sd, n = 5). P <.5 ompared ith ontrols. seondary to the aumulation of metabolites, e.g., gluose and latate. ndeed, during the first 1 min of reperfusion, the ontrol efflux rates of gluose (1.1.5,mol/min X g liver eight) and latate (1.1.2,umol/min X g) inreased dramatially. After longterm ishemia, peak values of 68,umol/ min X g (gluose) and 112 umol/min X g (latate) ere measured after 3 s ith a gradual deline to ontrol efflux rates afterards. There is evidene that the seretion ofgssg out of various ell types is a arriermediated transport that requires ATP (2529). Beause the hepati ATP levels are signifiantly redued during ishemia and do not reover totally upon reperfusion (5), the GSSG seretion of the liver might be impaired due to the lak ofatp. Therefore, e investigated the degradation and resynthesis of ATP during various ishemia/reperfusion periods. As shon in Fig. 5, the hepati ATP ontent dropped exponentially to 17% of ontrols after 3 min of ishemia and to 1% of ontrols after 12 min. Hoever, after starting reperfusion the ATP ontent reovered rapidly and reahed a ne equilibrium ithin 15 min, although these ne ATP levels ere signifiantly loer than the values measured before ishemia for the same livers. Tissue GSH and GSSG onentrations did not hange signifiantly during any O L t_& "s, T r1 ~", T 3 min 41 so 1 12 (mi) Figure 3. Hepati LDH efflux after ishemia. LDH effilux rates ere shon before the ishemi period (23 min) and during a 6min reperfusion period after 3 min ishemia (612 min) or after 12 min ishemia (152 1 min). P <.5 ompared ith ontrols. period of ishemia and reperfusion hen ompared ith ontrols perfused for the same period of time (Fig. 6). Beause no inrease in hepati GSSG efflux into bile nor aumulation of GSSG in the liver as detetable upon reoxygenation, a solution of a superoxidegenerating ompound as infused to ensure that the protetive mehanisms against reative oxygen speies ere still operative. After 3 min of ishemia, the infusion of diquat into the liver to provide an intraellular oxidative stress resulted in a massive efflux of GSSG into bile and perfusate (Fig. 7). The average total effilux of GSSG from the liver during reperfusion as 18.2±3.1 nmol GSH eq/min X g liver eight. The time ourse of the LDH release, hoever, as idential to the 3min ishemia experiments shon in Fig. 3. After 1 h of ontinuous oxidative stress in livers subjeted to ishemia and reflo, LDH efflux rates of 21.3±1.8 mu/min X g ere measured. Diquat infusion also aused a signifiant inrease ofthe tissue GSSG ontent as ell as a signifiant derease of the hepati GSH levels (Fig. 6). Shortterm infusion of tbutyl hydroperoxide (2,uM for 1 min) during the reperfusion period after 3 min or after 12 min of ishemia resulted in total hepati GSSG efflux rates of 33±2 nmol GSH eq/min X g, also ithout affeting the LDH release signifiantly (data not shon). ontrol and previously ishemi livers responded quantitatively for 3 min ith the same GSSG efflux rates to oxidative stress indued by diquat (Jaeshke, H.,. V. Smith, and J. R. Mithell, unpublished observation). Disussion 1242 H. Jaeshke,. V. Smith, and J. R. Mithell

4 m 8 T x 11_ ) 'f A x L) o o 4 5s o11 4 a ' 1 2 Figure 4. Sinusoidal LDH and glutathione efflux during the initial reperfusion period. The hepati LDH, total glutathione (given as GSH equivalents), and GSSG (given as GSH equivalents) are shon during the initial reperfusion (5 min) after 3 min (A) or 12 min (v) l ATP 3 6 ill give rise to an inreased GSSG formation and effilux from Figure 5. Hepati ATP ATP levels ere measure( dnteint duing ishemia anriod reflo. the liver. Hoever, e observed very little, if any, inrease in and during reperfusion (m)after 3 min of ishemia or after 12 min the seretion of GSSG into bile or into perfusate. Likeise, no of ishemia. The ATP orntent is given as perent of the value mea hange ourred in the intraellular GSSG ontent. sured before starting ishemia (1%, 3.9±.7 Amol ATP/g liver Nevertheless, hen the hepati glutathione defense system t; n 1). = Data are given as mean±sd of n equals four to ten. P as hallenged by infusion of a peroxide or a superoxidegeneishemi liver ATP ontent. erating ompound, marked inrease in GSSG efflux rates <.5 ompared ith pr( o s 3 5 : 12, 3 A t (min) ishemia. Given are the mean of n equals four to five perfusions and the standard deviation, if the standard deviation is bigger than the symbol. P <.5 ompared ith ontrols. ies may be responsible for ishemiareflo injury in the liver (26). The major support for this theory ame from experiments that shoed protetive effets of extraellular superoxide dismutase and atalase (3), as ell as alpha toopherol (5). The protetive effet of allopurinol, a xanthine oxidase inhibitor, suggested this enzyme as the likely soure of superoxide (24). Our approah as based on the rapid dismutation of superoxide to oxygen and hydrogen peroxide by superoxide T dismutase, an enzyme that is found in high onentrations in the ytosol and in mitohondria (3). One hydrogen peroxide is formed, glutathione peroxidase, loalized in the same ellular ompartment, ill redue the peroxide to ater hile oxi dizing glutathione (GSH) to its disulfide (GSSG) (31). GSSG is redued 1' by glutathione redutase at the expense of NADPH. n a situation of enhaned formation of GSSG, ells atively / serete GSSG (25, 28, 29), and this efflux has been used as a sensitive indiator of intraellular oxidative stress in vivo ( 15, 16) and in isolated perfused organs, i.e., liver (17, 18), heart (27, 32), and lungs (33). Therefore, if at any time during ish ' ' ' emia and reperfusion a signifiant formation of superoxide t (min) takes plae, the enzymati defense systems ill detoxify it and Oxidant Stress and shemiareflo njury 1243

5 t F 1o \ a in ' UU 1 3 go 15 r 1i.s t (min) x 1. 1m.5 i UJ..75.SO 4.25 D O o D Do T / "' tt T p i ikt h r t (min) Figure 6. Hepati GSH and GSSG ontent during ishemia and reflo. Hepati levels of total glutathione (top) and GSSG (bottom) ere measured during an ishemi period of 12 min (A) and during reperfusion (i) after 3 or 12 min of ishemia as ell as after reflo ith 2 AM diquat in the perfusate (e) (mean±sd, n equals four to ten). P <.5 (reflo ith vs. ithout diquat after 3 min ishemia). urred, demonstrating a funtional glutathione peroxidase and GSSG exretion system during the reperfusion period after global ishemia. Taken together, these results provide no evidene for a massive or even a moderate generation of reative oxygen during ishemia and reperfusion of the liver. The initial 13s inrease in GSSG efflux from the liver after starting reperfusion an be interpreted as ashout of extraellular aumulated GSH, hih oxidized spontaneously in the sinusoidal spae. This vie is supported by the folloing observations: (a) The highest GSSG onentrations in the perfusate ere measured during the first fe seonds, then GSSG dropped sharply and returned to ontrol values after 2 to 3 s (Fig. 4); (b) Metabolites like gluose and latate, hih aumulated only to a lesser part extraellularly, shoed a different efflux pattern. Latate and gluose efflux inreased during the first 2 s of reperfusion, had a maximum beteen 2 and 3 s, and delined gradually during the first 1 min. Thus, it is unlikely that the initial GSSG efflux represents intraellular GSSG. On the other hand an up to tenfold higher GSH efflux into the perfusate as observed during the initial period of o 3 4 o2 (D Perfusate T _. teiii_._ 1 ssa T A.r T. T o t (min) Figure 7. Bile flo and hepati glutathione efflux after 3 min ishemia and diquat infusion. The livers ere preperfused for 3 min. After a global ishemi period of 3 min, the livers ere reperfused ith KrebsHenseleit biarbonate buffer ontaining 2 MM of diquat for another 6 min. The total glutathione efflux (given as GSH equivalents) and the GSSG efflux (given as GSH equivalents) into bile (top) and perfusate (bottom) are shon (mean±sd, n = 3). P <.5 ompared ith livers ithout diquat after 3 min ishemia (Fig. 1). reperfusion after longterm ishemia. The enhaned GSH efflux paralleled an inreased LDH effilux and the selling of the liver, indiating a nonspeifi leakage of hepatoytes, perhaps due to the mehanial stress. Sine reperfusion is sloer and thus liver selling and GSH efflux is muh more extended after hepati ishemia in vivo, these findings may explain the elevated onentrations ofgsh and GSSG observed in vivo in plasma but not in bile (34). Ativation ofleukoytes also ould explain the observed inrease in GSSG in the vasular ompartment, but not in bile in vivo. The toxiity of reative oxygen speies is ell knon (35), and leads to damage of various biomoleules (36). Lipid peroxidation in partiular has been disussed as a possible mehanism of hemialindued aute hepatotoxiity in vivo (3742). Sine reative intermediates an alter biomoleules through multiple mehanisms, the ontribution of reative oxygen speies, thiol oxidation, and lipid peroxidation to ell nerosis is diffiult to evaluate. Hoever, studies ith the superoxideproduing agents diquat (4143) and paraquat (44) and infusion of various peroxides into the isolated perfused rat 1244 H. Jaeshke,. V. Smith, and J. R. Mithell

6 liver ( 17, 45, 46), demonstrate learly that the rat liver is able to ithstand a large amount of oxidative stress ithout signifiant damage (evaluated as enhaned LDH release, redued bile flo, redued seretion of glutathione, and inreased onentration of lipid peroxidation produts). The present study onfirms and extends these observations. A simulation of the oxygen radial hypothesis, i.e., the massive intraellular generation of superoxide during reperfusion of a previously ishemi liver, revealed no additional ell damage (LDH release) or impaired organ funtion (bile flo, GSSG seretion). An average hepati GSSG release of 1 nmol GSSG/min X g during diquat infusion is the onsequene of 36 nmol GSSG/min X g formed ithin the hepatoytes. These alulations are based on the tbutyl hydroperoxide infusion experiments: an infusion rate of 6 nmol peroxide/min X g resulted in an efflux rate of 17 nmol GSSG/min X g (=2.8%), assuming total uptake ofthe peroxide into the liver (46). Thus, during the 1h infusion period of diquat, 72 nmol X 6 min = 43,mol superoxide per gram liver as generated and detoxified ithout damage to the liver. f one assumes that the short ashout of GSSG at the beginning of the reperfusion period (Figs. 1, 2, and 4) does not represent extraellularly oxidized GSH, but atually indiates an intraellular oxidative stress, the average efflux rate of nmol GSSG/min X g during 3 s ould then be the result of.36,umol superoxide per gram liver formed inside the ells. Therefore, it seems very unlikely that this small oxidative stress ould produe strutural damage of the liver, hen a more than three orders of magnitude higher reative oxygen formation after diquat administration (or after tbutyl hydroperoxide) does not affet the viability of the reperfused liver. The signifiane of the present observations is not simply the lak of oxidative stress responses during ishemiareflo injury in the isolated perfused rat liver, but the additional evidene that the methods used readily detet subtoxi oxidant stress responses. The data presented here make it highly unlikely that suffiient quantities of reative oxygen speies are generated to overhelm endogenous defense mehanisms. Thus, the hypothesis that xanthine oxidase, loated in parenhymal and in endothelial ells, auses ishemiareflo injury in the liver by massive generation of superoxide (26, 12) is not onsistent ith our data. The ell damage and impaired organ funtion after ishemiareflo seen in the leukoytefree isolated perfused rat liver is less severe than after the same hepati ishemi period in vivo, here massive ell nerosis as observed (34, 47). This impliates an important additional unknon proess in the pathogenesis of ishemiareflo injury, perhaps involving ativation of leukoytes or retiuloendothelial ells as potential soures of superoxide prodution. n summary, the present study shoed that global ishemia and reperfusion ause impaired organ funtion and ell damage (evaluated as enhaned LDH release) in rat liver ithout signifiant GSSG generation. We onlude, therefore, that at most a minor amount of reative oxygen speies may be generated during reperfusion. Thus, oxygen radials are unlikely to ause ishemia/reperfusion injury in rat liver by diret strutural damage through lipid peroxidation or tissue thiol oxidation. Aknoledgments The expert tehnial assistane of Ms. Marilyn L. Samuel Wyllie is gratefully appreiated. Oens and Mr. This ork as supported by National nstitutes of Health grant GM3412. Referenes 1. Bulkley, G. B The role of oxygen free radials in human disease proesses. Surgery. 94: Siems, W., B. Mielke, M. Mueller,. Heumann, L. Raeder, and G. Gerber Status of glutathione in the rat liver. nhaned formation of oxygen radials at lo oxygen tension. Biomed. Biohim. Ata. 42: Granger, D. N., D. Adkison, M.. Hollarth, J. N. Benoit, D. A. Parks, J. M. Mord, and P. R. Kvietys Role of oxygen free radials in ishemiareperfusion injury in the liver. Gastroenterology. 88:1662. (Abstr.) 4. Nordstroem, G., T. Seeman, and P.O. Hasselgren Benefiial effet of allopurinol in liver ishemia. Surgery. 97: Marubayashi, S., K. Dohi, K. Ohi, and T. Kaasaki Role of free radials in ishemi rat liver ell injury: prevention of damage by alphatoopherol administration. Surgery. 99: Takekaa, S., H. shii, H. Takahashi, D. to, T. Takagi, and M. Tsuhiya Role of free radials in experimental ishemi hepati injury assessed by leuoyte hemiluminesene. Hepatology. 6:1131. (Abstr.) 7. Gardner, T. J., J. R. Steart, A. S. asale, J. M. Doney, and D.. hambers Redution of myoardial ishemi injury ith oxygenderived free radial savengers. Surgery. 94: hambers, D.., D. A. Parks, G. Patterson, R. Roy, J. M. Mord, S. Yoshida, L. F. Parmley, and J. M. Doney Xanthine oxidase as a soure of free radial damage in myoardial ishemia. J. Mol. ell ardiol. 17: Parks, D. A., and D. N. Granger Oxygenderived radials and ishemiaindued tissue injury. n Oxy Radials and Their Savenger Systems.. ellular and Medial Aspets. R. A. Greenald and G. ohen, editors. lsevier Siene/NorthHolland, Ne York Paller, M. S., and R. P. Hebbel thane prodution as a measure of lipid peroxidation after renal ishemia. Am. J. Physiol. 251 :F839F toh, T., M. Kaakami, Y. Yamauhi, S. Shimizu, and M. Nakamura ffet of allopurinol on ishemia and reperfusionindued erebral injury in spontaneously hypertensive rats. Stroke. 17: Mord, J. M Oxygenderived free radials in postishemi tissue injury. N. ngl. J. Med. 312: Roy, R. S., and J. M. Mord Superoxide and ishemia: onversion of xanthine dehydrogenase to xanthine oxidase. n Oxy Radials and Their Savenger Systems.. ellular and Medial Aspets. R. A. Greenald and G. ohen, editors. lsevier Siene/ NorthHolland, Ne York Suttorp, N., W. Toepfer, and L. Roka Antioxidant defense mehanisms of endothelial ells: glutathione redox yle versus atalase. Am. J. Physiol. 251: Adams, J. D., B. H. Lauterburg, and J. R. Mithell Plasma glutathione and glutathione disulfide in the rat: regulation and response to oxidative stress. J. Pharmaol. xp. Ther. 227: Lauterburg, B. H.,. V. Smith, H. Hughes, and J. R. Mithell Biliary exretion of glutathione and glutathione disulfide in the rat. J. lin. nvest. 73: Sies, H.,. Gersteneker, H. Menzel, and L. Flohe Oxidation in the NADP system and release of GSSG from hemoglobinfree perfused rat liver during peroxidati oxidation of glutathione by hydroperoxides. FBS (Fed. ur. Biohem. So.) Lett. 27: Akerboom, T., M. Bilzer, and H. Sies The relationship of biliary glutathione disulfide efflux and intraellular glutathione disulfide ontent in perfused rat liver. J. Bio. hem. 257: Sies, H The use of perfusion of liver and other organs for the study of mirosomal eletrontransport and ytohrome P45 systems. Methods nzymol. 52:4859. Oxidant Stress and shemiareflo njury 1245

7 2. Krell, H., H. Jaeshke, H. Hoeke, and. Pfaff Bile seretion in hemoglobinfree perfused rat liver. HoppeSeyler's Z. Physiol. hem. 365: Jaeshke, H., H. Krell, and. Pfaff No inrease ofbiliary permeability in ethinylestradioltreated rats. Gastroenterology. 85: Jaeshke, H., H. Krell, and. Pfaff Quantitative estimation of transellular and paraellular pathays of biliary surose in isolated perfused rat liver. Biohem. J. 241: Tietze, F nzymati method for quantitative determination of nanogram amounts of total and oxidized glutathione. Anal. Biohem. 27: Bergmeyer, H. U., editor Methods of nzymati Analysis. Aademi Press, n., Ne York , , , Beutler, Ative transport ofglutathione disulfide from erythroytes. n Funtions ofglutathione: Biohemial, Physiologial, Toxiologial and linial Aspets. A. Larsson, S. Orrenius, A. Holmgren, and B. Mannervik, editors. Raven Press, Ne York Akerboom, T., M. noue, H. Sies, R. Kinne, and. M. Arias Biliary transport ofglutathione disulfide studied ith isolated rat liver analiularmembrane vesiles. ur. J. Biohem. 141: shikaa, T., and H. Sies ardia transport of glutathione disulfide and Sonjugate. J. Biol. hem. 259: Niotera, P., M. Moore, G. Bellomo, F. Mirabelli, and S. Orrenius Demonstration and partial haraterization of glutathione disulfidestimulated ATPase ativity in the plasma membrane fration from rat hepatoytes. J. Biol. hem. 26: shikaa, T., M. Zimmer, and H. Sies nergylinked ardia transport system for glutathione disulfide. FBS (Fed. ur. Biohem. So) Lett. 2: Fridovih, Superoxide radial and superoxide dismutases. n Oxygen and Living Proesses. D. L. Gilbert, editor. Springer Verlag, Heidelberg, FRG Flohe, L Glutathione peroxidase brought into fous. n Free Radials in Biology. Vol. V. W. A. Pryor, editor. Aademi Press, n., Ne York Xia, Y., K.. Hill, and R. F. Burk ffet of selenium defiieny on hydroperoxideindued glutathione release from the isolated rat heart. J. Nutr. 115: Jenkinson, S. G., T. H. Spene, R. A. Larene, K.. Hill,. A. Dunan, and K. H. Johnson Rat lung glutathione release: response to oxidative stress and selenium defiieny. J. Appl. Physiol. 62: Jaeshke, H.,. V. Smith, H. Hughes, and J. R. Mithell No evidene for oxidative hepati damage during ishemia/reflo injury. The Pharmaologist. 29:425. (Abstr.) 35. Halliell, B., and J. M.. Gutteridge Oxygen toxiity, oxygen radials, transition metals and disease. Biohem. J. 219: Sies, H Biohemistry of oxidative stress. Ange. hem. nt. d. ngl. 25: Rao, K. S., and R.. Reknagel arly onset of lipoperoxidation in rat liver after arbon tetrahloride administration. xp. Mol. Pathol. 9: Wendel, A., S. Feuerstein, and K. H. Konz Aute paraetamol intoxiation of starved mie leads to lipid peroxidation in vivo. Biohem. Pharmaol. 28: Burk, R. F., R. A. Larene, and J. M. Lane Liver nerosis and lipid peroxidation in the rat as the result of paraquat and diquat administration. J. lin. nvest. 65: Jaeshke, H.,. Kleinaehter, and A. Wendel The role of allyl aloholindued lipid peroxidation and liver ell damage in mie. Biohem. Pharmaol. 36: Smith,. V ffet of BNU pretreatment on diquatindued oxidant stress and hepatotoxiity. Biohem. Biophys. Res. ommun. 144: Smith,. V videne for the partiipation of lipid peroxidation and iron in diquatindued hepati nerosis in vivo. MoL Pharmaol. 32: Smith,. V., H. Hughes, B. H. Lauterburg, and J. R. Mithell Oxidant stress and hepati nerosis in rats treated ith diquat. J. Pharmaol. xp. Ther. 235: Brigelius, R., and M. S. Aner nreased biliary GSSGseretion and loss of hepati glutathione in isolated perfused rat liver after paraquat treatment. Res. ommun. hem. Pathol. Pharmaol. 31: Bartoli, G. M., and H. Sies Redued and oxidized glutathione efflux from liver. FBS (Fed. ur. Biohem. So.) Lett. 86: Sies, H., and K. H. Summer Hydroperoxidemetabolizing systems in rat liver. ur. J. Biohem. 57: Mithell, J. R.,. V. Smith, H. Hughes, M. Lenz, H. Jaeshke, L. Mihael, and M. L. ntman No evidene for reative oxygen damage in ishemiareflo injury. lin. Res. 35:642A. (Abstr.) 1246 H. Jaeshke,. V. Smith, and J. R. Mithell