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Glutathione Depletion with L-Buthionine-(S,R)-Sulfoximine Demonstrates Deleterious Effects in Acute Pancreatitis of the Rat G. ALSFASSER, MD,* M. GOCK, MD,* L. HERZOG, MD,* M.M. GEBHARD, MD,† C. HERFARTH, MD,* E. KLAR, MD,* and J. SCHMIDT, MD* A common pathway in the pathogenesis of acute pancreatitis is the generation of free oxygen radicals. The most important defense mechanisms are free radical scavengers, especially glutathione. This study evaluates the influence of the inhibition of glutathione synthesis with L-buthionine-(S,R)-sulfoximine (BSO) on the course of experimentally induced acute pan- creatitis in rats and the effects on isolated pancreatic acini and their secretion pattern. Thus acute necrotizing pancreatitis was induced with intraductal infusion of low-dose glycodeoxy- cholic acid and subsequent hyperstimulation with cerulein with and without pretreatment with BSO. In vitro pancreatic acini were isolated and stimulated with different concentrations of cerulein with and without BSO. The BSO-treated group showed a significantly reduced survival, more necrosis, and a decreased secretion of amylase in vivo. No effect on secretion pattern in either groups was seen in vitro and BSO did not exert toxic effects. Based on the data presented, this study demonstrates deleterious effects of scavenger depletion on the course of experimental pancreatitis. This is due to the systemic effects of free oxygen radicals rather than to local effects. KEY WORDS: pancreatitis; BSO; acini; radicals; scavenger. Acute pancreatitis is defined as an acute inflamma- tory process of the pancreas with involvement of adjacent or distant organ systems (1). The pathologic features extend from microscopic interstitial edema formation and fat necrosis of the peripancreatic tissue to macroscopic pancreatic necrosis and hemorrhage. The etiology of acute pancreatitis is varied in and its pathogenesis is complex. It ranges from occlusion of a common channel with subsequent biliopancreatic re- flux (2, 3), to intrapancreatic activation of pancreatic enzymes (4, 5), and to microcirculatory disturbances (6, 7) with expression of various adhesion molecules and consequent leukocyte infiltration (8, 9). A com- mon pathway is the generation of oxygen-derived free radicals. Many studies emphasize the importance of these radicals (10, 11). They influence leukocyte activation and production of cytokines, produce endothelial damage, influence microcirculation and the severity of the disease, and play a central role in all forms of acute pancreatitis (12). These multiple effects are due to the oxidizing property of the oxygen molecule, which binds electrons, and is used by the cellular cytochrome system, where the complete (tetravalent) Manuscript received September 27, 2001; accepted March 3, 2002. From the Departments of *Surgery and †Experimental Surgery, University of Heidelberg, Heidelberg, Germany. Address for reprint requests: Jan Schmidt, MD, Department of Surgery, Kirschnerstr. 1, 69120 Heidelberg, Germany. Digestive Diseases and Sciences, Vol. 47, No. 8 (August 2002), pp. 1793–1799 (© 2002) 1793 Digestive Diseases and Sciences, Vol. 47, No. 8 (August 2002) 0163-2116/02/0800-1793/0 © 2002 Plenum Publishing Corporation

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Page 1: Glutathione Depletion with l-Buthionine-(S,R)-Sulfoximine Demonstrates Deleterious Effects in Acute Pancreatitis of the Rat

Glutathione Depletion withL-Buthionine-(S,R)-Sulfoximine

Demonstrates Deleterious Effects inAcute Pancreatitis of the Rat

G. ALSFASSER, MD,* M. GOCK, MD,* L. HERZOG, MD,* M.M. GEBHARD, MD,†C. HERFARTH, MD,* E. KLAR, MD,* and J. SCHMIDT, MD*

A common pathway in the pathogenesis of acute pancreatitis is the generation of free oxygenradicals. The most important defense mechanisms are free radical scavengers, especiallyglutathione. This study evaluates the influence of the inhibition of glutathione synthesis withL-buthionine-(S,R)-sulfoximine (BSO) on the course of experimentally induced acute pan-creatitis in rats and the effects on isolated pancreatic acini and their secretion pattern. Thusacute necrotizing pancreatitis was induced with intraductal infusion of low-dose glycodeoxy-cholic acid and subsequent hyperstimulation with cerulein with and without pretreatmentwith BSO. In vitro pancreatic acini were isolated and stimulated with different concentrationsof cerulein with and without BSO. The BSO-treated group showed a significantly reducedsurvival, more necrosis, and a decreased secretion of amylase in vivo. No effect on secretionpattern in either groups was seen in vitro and BSO did not exert toxic effects. Based on thedata presented, this study demonstrates deleterious effects of scavenger depletion on thecourse of experimental pancreatitis. This is due to the systemic effects of free oxygen radicalsrather than to local effects.

KEY WORDS: pancreatitis; BSO; acini; radicals; scavenger.

Acute pancreatitis is defined as an acute inflamma-tory process of the pancreas with involvement ofadjacent or distant organ systems (1). The pathologicfeatures extend from microscopic interstitial edemaformation and fat necrosis of the peripancreatic tissueto macroscopic pancreatic necrosis and hemorrhage.The etiology of acute pancreatitis is varied in and itspathogenesis is complex. It ranges from occlusion of acommon channel with subsequent biliopancreatic re-

flux (2, 3), to intrapancreatic activation of pancreaticenzymes (4, 5), and to microcirculatory disturbances(6, 7) with expression of various adhesion moleculesand consequent leukocyte infiltration (8, 9). A com-mon pathway is the generation of oxygen-derived freeradicals.

Many studies emphasize the importance of theseradicals (10, 11). They influence leukocyte activationand production of cytokines, produce endothelialdamage, influence microcirculation and the severityof the disease, and play a central role in all forms ofacute pancreatitis (12). These multiple effects are dueto the oxidizing property of the oxygen molecule,which binds electrons, and is used by the cellularcytochrome system, where the complete (tetravalent)

Manuscript received September 27, 2001; accepted March 3,2002.

From the Departments of *Surgery and †Experimental Surgery,University of Heidelberg, Heidelberg, Germany.

Address for reprint requests: Jan Schmidt, MD, Department ofSurgery, Kirschnerstr. 1, 69120 Heidelberg, Germany.

Digestive Diseases and Sciences, Vol. 47, No. 8 (August 2002), pp. 1793–1799 (© 2002)

1793Digestive Diseases and Sciences, Vol. 47, No. 8 (August 2002)0163-2116/02/0800-1793/0 © 2002 Plenum Publishing Corporation

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reduction of oxygen to water is utilized for the pro-duction of adenosine triphosphate. During this reac-tion the superoxide anion (O2

�) and the hydroxylradical (�OH) develop, which are able to degradehyaluronic acid and collagen. They can also directlydamage the cell membranes through peroxidation ofstructurally important polyunsaturated fatty acids withinthe phospholipid structure of the membrane itself.

One of the characteristics of free radical reactivityis the tendency to generate a chain reaction of radicalspecies production, which results in amplification ofthe ultimate destructive effect. Membrane damagemay also affect lysosomes, whose destruction resultsin release of lysosomal enzymes, which can also po-tentiate free-radical induced cellular damage. De-struction of acini induces release of pancreatic en-zymes. Oxygen-derived free radicals probably play amajor role in any disease process that involves hyper-oxygenation, ischemia (particularly ischemia followedby reperfusion), or tissue inflammation. Natural de-fense mechanisms against radicals and their toxicpotential are scavengers, such as glutathione and vi-tamins C and E.

Glutathione is a tripeptide, consisting of �-glutamicacid, cysteine, and glycine, and it is a very effectivebiologic redox system. Glutathione is synthesized in-tracellulary, and 75% is stored in mitochondrias and25% within the cytosol (13). Another important fea-ture is restoring the pools of vitamins C and E. Thereare many possibilities to induce a glutathione deple-tion, which, however, are not specific (14). Blockageof glutathione synthetase leads to metabolic acidosis(15). The most efficient substance is L-buthionine-(S,R)-sulfoximine (BSO), which very specifically in-hibits �-glutamylcysteine synthetase (13) and does notexert toxic effects (14).

This study consists of two experiments. First, weexamined the effects of systemic glutathione deple-tion in an in vivo rat model. Second, we studied theeffects of inhibition of glutathione synthesis on cellu-lar level in vitro. For this purpose we investigatedglutathione depletion in isolated pancreatic acini, be-cause it is the smallest functional unit of the exocrinepancreas.

MATERIALS AND METHODS

Male Wistar rats (weighing 300 � 50 g) were used for theexperiments. Care was provided in accordance to the pro-cedures outlined in the Guide for Care and Use of Labora-tory Animals [DHHS Publication No. (NIH) 85-23 revised1985, Office of Science and Health Reports, Bethesda,Maryland 20892] and §7 and §4, Deutsches Tierschutzge-

setz. The study was approved by the committee on animalresearch at the Regierungsprasidium Karlsruhe, Germany.Rats were housed individually in hanging cages at standardconditions (12-hr light–dark cycle and 21° � 1°C) and fedregular rat chow. The animals were fasted overnight beforethe experiment but allowed free access to water.

All reagents were obtained from Sigma (St. Louis, Mis-souri) unless otherwise specified.

Anesthesia and Catheter Placement. Surgical anesthesiawas induced by intraperitoneal pentobarbital (Narcoren 60mg/kg; Fa. Merial GmbH, Hallbergmoos, Germany) andintramuscular ketamine BM (Ketanest 100 mg/kg; Parke-Davis, Freiburg, Germany). The right internal jugular veinwas cannulated using a soft polyethylene catheter (0.5 mmID, 0.9 mm OD; Braun, Melsungen, Germany) for infusion.A second catheter of the same size was placed in the leftcarotid artery for blood sampling and hemodynamic mon-itoring. All catheters were then tunneled subcutaneously tothe subscapular area and brought out via a flow-throughtether, which permitted free movement.

Induction of Pancreatitis. Experimental severe necrotiz-ing pancreatitis was induced as previously described (16).Briefly, the biliopancreatic duct was cannulated with a24-gauge angiocath (Becton-Dickinson, Sandy, Utah), andbile and pancreatic juice were drained by gravity in a 60°reverse Trendelenburg position for 5 min. During the last 2min of this procedure, the main duct was clamped below theliver to allow complete emptying of the biliary and pancre-atic ductal system and to facilitate the subsequent intraduc-tal infusion. Freshly prepared glycodeoxycholic acid(GDOC) in glycylglycine–NaOH-buffered solution (pH 8.0at room temperature) at a low concentration of 10 mmol/liter then was infused in a pressure- (30 mm Hg) andvolume- (0.15 ml/100 g) controlled manner over 10 min.After completion of the intraductal infusion, all animalsreceived continuous secretory hyperstimulation for 6 hrwith intravenous cerulein (5 �g/kg/h) (Takus 40, Farmitalia,Carlo Erba GmbH, Freiburg, Germany) at 8 ml/kg/hr asbaseline hydration. Sodium bicarbonate (0.2 ml/100 g) andketamine (0.2 ml) were added to this infusion.

Experimental Design. Two groups were employed: ani-mals with no pretreatment (group I, N � 6) and animalspretreated with BSO (8 mmol/liter) (group II, N � 8). Bothgroups received either 1 ml of NS (Fresenius, Homburg,Germany) (group I) or 1 ml BSO (group II) after placementof catheters. All animals were observed for 24 hr and timeof death was noted. Blood samples were taken 6 hr and 24hr after placement of catheters, for measuring amylase,lipase, LDH, hematocrit, and arterial blood gas analysis.After 24 hr the animals were killed with an overdose ofpentobarbital. At time of death the pancreas was removedand fixed with buffered formalin (Merck, Darmstadt, Ger-many) for evaluating the extent of pancreatic necrosis.

Morphometric Analysis of Histology. The specimenswere rinsed with tap water, dehydrated with increasingconcentrations of ethanol and chloroform (JT Baker, De-venter, The Netherlands) and embedded in paraffin(Merck).

Sections were made from the head and tail of eachpancreatic specimen. The slices then were stained withhematoxylin–eosin. The entire section was meticulouslyevaluated for the amount of necrotic acini versus intact

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ones using morphometric methods for exact quantification.We focused on the difference between necrotic versus in-tact, because this is the only hard criteria of evaluation offunctional integrity of pancreatic acini. With the help of agrid (Carl Zeis, Jena, Germany) within the ocular view, 100acini were counted per field with 170-fold magnification. Inthis way, 1500–2500 acini were evaluated per slice.

Isolation of Pancreatic Acini. After decapitation of therat (N � 8), the pancreas was taken out without removingfat tissue and put in 6 ml of cold (0°C) buffer-solutioncontaining 200 units of collagenase. Subsequently the pan-creas was cut in small pieces and incubated in a warm waterbath for 10 min at 37°C with carbogen gas. Finally, theentire content was placed in a 15-ml centrifuge tube andcarefully shaken by hand, afterwards the cells were pouredthrough a 200-�m mesh and washed three times with buffersolution at 600 rpm.

Stimulation of Pancreatic Acini. Before starting the stim-ulation experiment, acini were evaluated by light micros-copy. Signs of good quality of the acini were three-dimensional integrity and centroacinar granulation. At firstbaseline values of amylase and lipase were determinedusing the method described above. After 15 min of prein-cubation in a warm water bath at 37°C, the stimulation withcerulein concentrations described below was started. Inorder to measure enzymes of the different suspensions afterexactly 30 min, the stimulations were started 2 min apart.Enzymes were measured before preincubation (0), beforestarting cerulein stimulation (T0), and 10 min (T10) and 30min (T30) after starting stimulation. The suspensions withdifferent cerulein concentrations were run in duplicate, onewith 1 mM BSO and the other without (serving as control).

The isolated acini were stimulated with six different con-centrations of cerulein (10�12–10�7 M) with a maximumstimulation at 10�10 M. At each time point, amylase andlipase were measured. To do so the acini were put in tubesfilled with a mixture of phthalic acid dibutylester–phthalicacid diisonylester and centrifuged at 13,000 rpm. In this waythe acini were separated from the supernatant and couldnot influence the enzyme concentration.

In order to prepare the acini, collagenase (Worthington,Freehold, New Jersey) was dissolved in twice-distilled waterto a concentration of 1 unit/�l. KRH solution served as abuffer containing HEPES (Roth, Karlsruhe, Germany),NaCl, KCl, KH2PO4 and MgSO4, 90 mg glucose, 20 mgtrypsin inhibitor (Merck), 2 ml nonessential amino acids, 4ml essential amino acids (Gibco Life Technology, Eggen-stein, Germany), 400 mg BSA (bovine albumin fraction V,pH 7.0; Serva, Heidelberg, Germany) and completed withtwice-distilled water. The pH was adjusted to 7.4 with 5 MNaOH. Finally buffer solution was incubated with carbogengas for 20 minutes in a waterbath at 37°C.

Measurement of Amylase and Lipase. Amylase andlipase were measured on an automated analyzer BM/Hitachi 717 (Boehringer, Mannheim, Germany).

RESULTS

In Vivo Experiments. Blood gas analysis was per-formed at all time points, but there were no patho-logical changes in either group.

Survival. All control animals survived 24 hr. Of theBSO group only one animal survived 24 hr. Controlanimals lived an average of 24 hr, whereas BSO-treated animals survived only 11 hr. This is a signifi-cant decrease in mean survival in the BSO group (P �0.005, Mann-Whitney U test). Comparing survivalcurves with the log-rank test reveals a significantdifference (P � 0.01; Figure 1).

Enzyme Kinetics. Enzymes were measured beforeinduction of acute pancreatitis, after 6 and 24 hr. Acomparison after 24 hr was not made, because onlyone BSO animal survived to 24 hr.

Before induction of pancreatitis there were no dif-ferences in plasma levels of amylase between groups.Amylase concentration in the control group was1656 � 80.32 units/liter and in the BSO treated group2023 � 247.5 units/liter. After 6 hr, enzyme levels inthe control group were significantly higher at42,131 � 5073.1 units/liter compared to 12,008 �4349.1 units/liter in the BSO group (P � 0.002,Student’s t test, Figure 2).

Lipase and LDH concentrations were not differentat any time between the two groups (Figures 3 and 4).

Morphometric Analysis of Histology. Histologicalanalysis of necrosis versus nonecrosis was comparedbetween the two groups. Per animal two representa-tive slices of the head and tail of the pancreas wereanalyzed together (1500–2500 acini per animal). In

Fig 1. Survival of both groups, *Significant difference betweengroups (P � 0.01, log-rank test).

Fig 2. Amylase in both groups over time (*P � 0.002).

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the control animals an average of 7.7 � 0.72% of aciniwere necrotic compared to 18.7 � 3.37% in BSO-treated animals. This is a statistically significant dif-ference (P � 0.025, Student’s t test, Figure 5).

In Vitro Stimulation of Isolated Pancreatic Acini.Before comparing both groups, the stimulation pat-tern of isolated pancreatic acini had to be evaluated.After 15 min of preincubation, the cells were stimu-lated with cerulein for another 30 min. A specific andreproducible pattern of enzyme secretion after stim-ulation with different concentrations of cerulein wasnoted.

After placement into a water bath, at 37°C, theconcentration of amylase and lipase in the superna-tants increased after 45 min even without stimulation.Under stimulation with cerulein, enzyme concentra-tions were notably higher. Enzyme secretion rosecontinuously from 10�12 M to 10�10 M and reached amaximum at 10�10 M. With rising concentration ofcerulein from 10�9 M to 10�7 M, a decrease inenzyme secretion was noted, whereas there was nodifference between 10�8 M and 10�7 M. These resultsare in line with the literature and demonstrate anintact preparation.

In Vitro Amylase and Lipase Secretion of IsolatedPancreatic Acini With and Without BSO. When eval-uating the functionality of isolated pancreatic acini,absolute amounts of enzymes are not important. De-pending on the size of the pancreas, differentamounts of isolated acini are generated. That is thereason why the absolute concentration of amylase and

lipase differ between each cell suspension. Therefore,we compared the relative secretion of amylase andlipase. Each concentration was compared to the con-centration of enzymes before preincubation, definedas baseline value. This value represents 0% secretionof amylase or lipase. Concentrations after 45 min withand without cerulein stimulation were expressed asthe percentage of this baseline value. Comparing bothgroups, there were no differences in the secretionpattern between BSO-treated acini and controls (Fig-ures 6 and 7).

The relative secretion of lipase in controls rangedfrom 265 � 44% without cerulein up to 900 � 186%with stimulation and in the BSO group 268 � 41% upto 914 � 169%, respectively.

Statistics. The stimulation experiments were ana-lyzed with a multifactorial univariate analysis of vari-ance. There was no statistical difference in amylasenor lipase secretion (P � 0.866) between those acinistimulated with or without BSO.

DISCUSSION

This study evaluates the effect of glutathione de-pletion at two different levels. First, we examined the

Fig 4. LDH in both groups over time (NS)

Fig 5. Percentage of acinar necrosis of head and tail. *P � 0.025.morphometric analysis of 1500–2000 acini per pancreas.

Fig 6. Relative secretion of enzymes as percent of baseline value. 0and �12 to �7 are different concentrations of cerulein from 0 M to10�7 M and demonstrate relative secretion of amylase after 30 minof stimulation.

Fig 3. Lipase in both groups over time (NS).

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effect on isolated pancreatic acini, because they rep-resent the smallest functional unit of the exocrinepancreas. We also studied the systemic effect of scav-enger depletion in experimentally induced acute pan-creatitis in rats. Our results can be summarized asfollows: First, survival after induction of an acutepancreatitis was significantly reduced in BSO-treatedanimals. Second, in the BSO group there was signif-icantly more necrosis of the pancreas. Third, secre-tion of amylase was decreased in the BSO group.These results demonstrate a deleterious effect of scav-enger depletion with BSO on the course of acutepancreatitis.

Before evaluating the effect of BSO on isolatedpancreatic acini, we had to analyze the secretionpattern of enzymes with stimulation with differentconcentrations of cerulein to determine the validity ofthe test. Each stimulation experiment showed an in-crease in enzyme concentration, and the peak secre-tion was reached at 10�10 M. Higher concentrationsof cerulein resulted in a decrease of secretion. Stim-ulation at 10�10 M is defined as maximal stimulation,and at lower or higher concentrations it is defined assubmaximal and supramaximal, respectively. This se-cretion kinetic confirmed the results of Wank et al(17).

Special elements of the cytoskeleton, microtubulesand microfilaments, play an important role in trans-port and exocytosis of enzymes. Jungermann et alshowed that a supramaximal stimulation of pancreaticacini with cerulein led to a degradation of structuralprotein such actin and �-tubulin (18). This is anexplanation for the decrease of enzyme concentrationin the stimulation experiments. If we compare thoseacini stimulated under the influence of BSO withcontrol acini, there is no difference with any concen-tration of cerulein. This stimulation model is com-pletely isolated from physiological control circuits,and therefore solely dependent on the ability of the

isolated acini in secreting enzymes. Obviously inhibi-tion of glutathione synthesis plays a minor role in thissetting. Additionally, we could demonstrate that BSOdid not exert toxic effects and did not influence theintegrity and functionality of isolated pancreatic acini.This observation is confirmed by the studies of Mar-tensson et al (14). In our experiment depletion ofglutathione on the level of acinar cells was not able todeliver an answer about the role of oxygen-derivedfree radicals in acute pancreatitis. Obviously interac-tion with a variety of cellular biochemical cascadesand organ systems is required. Therefore an in vivoexperiment was necessary.

In our in vivo experiments we compared enzymekinetics, length of survival, and histological changes.We could demonstrate three major differences be-tween BSO treated animal and controls.

First, after 6 hr the concentration of amylase incontrols was significantly increased and the same ten-dency was noted for lipase concentration. Serum ac-tivity of amylase and lipase was measured beforeinduction of pancreatitis and after 6 and 24 hr. In thecontrol group there was a peak of both enzymes after6 hr and an obvious tendency to normalization after24 hr.

A similar kinetic of enzyme secretion was noted ina study of Tani et al (21), who evaluated biochemicalalterations in experimentally induced acute pancre-atitis in a cerulein hyperstimulation model. Peak con-centration of amylase and lipase were seen after 6 hrand normalization occurred after 18 hr. Concentra-tion of enzymes only increased 10-fold, but Tani et alused less cerulein and a model with induction of amilder form of pancreatitis without intraductal infu-sion of GDOC (19). The only study comparing amy-lase and lipase levels in acute pancreatitis under theinfluence of BSO was performed by Fu et al (20) inmice, and thus is not comparable to our study. Theauthors could not demonstrate any influence of BSOon hyperamylasemia (20). In our study, the higherhyperamylasemia in controls can be explained by thepresence of more vital pancreatic acini, which wereable to secrete more enzymes, because the rate ofnecrosis was significantly lower compared to the BSOgroup.

Nagelkerke et al (21) performed a study on isolatedhepatocytes of rats and demonstrated that an inhibi-tion of glutathione synthesis inhibits secretion ofLDL. The authors found using immunofluorescentstaining that microtubuli within hepatocytes weredamaged and hypothesized a modification of specialthiole groups, which in turn inhibits secretion (21).

Fig 7. Relative secretion of enzymes as percent of baseline value. 0and �12 to �7 are different concentrations of cerulein from 0 M to10�7 M and demonstrate relative secretion of lipase after 30 min ofstimulation.

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Therefore, a depletion of glutathione and the subse-quent load of oxygen free radicals could damagemicrotubuli, which are responsible for exocytosis,with less enzymes that could be secreted. Addition-ally, Reinheckel et al (22) demonstrated an earlymodification of intracellular proteins mediated byoxygen derived free radicals in acute pancreatitis.This cellular damage occurs before lipid peroxidationof cell membranes (22).

Second, histologic analysis revealed a significantlyhigher degree of necrosis in BSO-treated animals. Inorder to get a complete evaluation of the functionalintegrity of pancreatic acini, we examined all acini fornecrosis versus nonnecrosis. In the BSO group therewas significantly more necrosis. There are no furtherstudies evaluating the degree of necrosis under theinfluence of BSO in experimentally induced acutepancreatitis in rats. However, Schoenberg et al (23)demonstrated a significant reduction in the amount ofnecrosis after treatment with scavengers. Similar ob-servations are reported by Rau et al (24), who eval-uated tissue destruction at various time points withapplication of different radical scavengers. Luthen etal (25) could not demonstrate any effect on histologicappearance after administration of BSO alone inmice without induction of acute pancreatitis (25).Thus the best explanation for the higher rate ofnecrosis is that of increased lipid peroxidation.

Third, survival in BSO-treated animals was signifi-cantly reduced. Comparing length of survival withinthe first 24 hr after induction of acute pancreatitis,there was a significant decrease in survival in theBSO-treated animals. Survival in the BSO group wasdecreased 55% compared to controls.

As mentioned earlier, Martensson et al (14) dem-onstrated that BSO did not exert toxic effects, whichwas also shown by our stimulation experiments.Therefore BSO per se is not responsible for the re-duced survival. BSO specifically inhibits �-glutamyl-cysteine synthetase (13) and therefore induces scav-enger depletion. Subsequently, the concentration ofoxygen-derived free radicals increases. These radicalsnot only act on the pancreas, but damage distantorgan systems as well, ie, the lungs (26). Coursin andCihla demonstrated that BSO administration alonedoes not induce damage to the lungs (27). On theother hand, Chardavoyne et al (28) showed a cardio-circulatory breakdown 1 hr after induction of acutenecrotizing pancreatitis in dogs. Administration ofradical scavengers improved the clinical situation(28). Other studies also demonstrated beneficial ef-fects of prophylactic administration of free radical

scavengers (10, 11). Additionally, Herzog et al (29)demonstrated an increased survival in the samemodel after administration of glutathione monoeth-ylester in the treatment group. These studies supportour findings that glutathione depletion, through inhi-bition of �-glutamylcysteine synthetase with L-buthionine-(S,R)-sulfoximine, exerts a deleterious ef-fect on the course of acute pancreatitis.

In summary, the inhibition of the endogenous syn-thesis of glutathione with L-buthionine-(S,R)-sulfoximine has the following effects in experimen-tally induced acute pancreatitis in rats and in isolatedpancreatic acini: (1) survival was significantly de-creased in the BSO-treated animals, (2) BSO-treatedanimals had significantly more pancreatic necrosis,(3) secretion of amylase was decreased with BSOtreatment, (4) the secretion pattern of isolated pan-creatic acini under cerulein stimulation is unchanged,and (5) BSO has no effect on excretion of amylaseand lipase in vitro and does not exert direct toxiceffects. These results, together with the finding thatglutathione monoethylester represents an effectivetherapy of acute pancreatitis, should be considered inthe planning of future studies of human acute pan-creatitis.

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