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Previously we have shown that liver cells of mice intoxicated with high dose of acetaminophen (Paracetamol, APAP) synthesize increased quantities of prostaglandin E2 (PGE2), prostaglandin I2 (PGI2) and thromboxane (TXA2). The aim of study was to determine which of these prostanoids contribute to defensive reaction of host to toxic effect of APAP (i.e. is hepatoprotective) and those which increase the toxic effect of APAP (i.e. is pathogenic). The toxicity of APAP was determined by observing the survival of mice given a lethal dose of APAP by gastric lavage and concentration of serum aminotransferases (AST and ALT) in mice given a sublethal dose of APAP. The concentration of particular prostanoid in body fluid of mice was modulated either by giving exogenously its stabile analog or giving specific anti-PG antibodies or inhibitors of specific receptors. In previous investigations we have shown that 16-16-dimethyl-PGE2 (a stable analogue of PGE2) had a strong protective effect if given to mice before or after APAP ; in contrast, application of specific anti- PGE2 antibodies increased toxic effect of APAP. Here we have shown that anti-TXB2 antibodies have opposite effect than anti-PGE2 antibodies, i.e., when given to APAP intoxicated mice, they decreased significantly serum AST and ALT levels. Recombinant TXB2 (0, 2 mg/kg) had no significant effect on AST and ALT levels in same mice. Presently we are testing the effect of various prostanoids agonists and antagonist on survival of intoxicated mice. The effect protective PG-s on synthesis of hepatotrophic cytokine, IL-6 and induction of Stat3 protein will also be tested.

INTRODUCTION. Previously we have shown that inflammatory cytokines (IL-1α /β and IL-6) have hepatoprotective effect if given to mice before administration of acetaminophen (APAP) or D-Galactozamine + LPS and that this effect is partially mediated by PGE2. Since many inflammatory cytokines stimulate the synthesis of cAMP as well as PGE2, we investigated the effect of agonists, antagonists and inhibitors of cAMP degradation on APAP toxicity. MATHERIALS AND METHODS. APAP (300 mg/kg) was administered by gastric lavage to mice which were given i.p., 1-2 hours before or up to 3 hours after a stable agonist (dibutyryl-cAMP – Db-cAMP) or antagonist (2, 3-dideoxyadenosine – DDA) of cAMP or inhibitor of phoshodiesterase IV (Rolipram). The survival of mice was followed for 72 hours and level of serum aminotransferases (AST and ALT) were determined 18-24 hours after administration of APAP. RESULTS. Db-cAMP (25 mg/kg) as well as Rolipram (8 mg/kg) significantly increased the survival of mice and reduced AST and ALT serum concentration if given 1-2 hours before or up to 2 hours after AAP administration. On the contrary, DDA (160 μ g/kg) decreased the survival of mice and increased serum aminotransferase concentration if given 2 hours before or ½ ; ; hours after APAP, but only effect on aminotransferase level was statistically significant. The treatment of animals with APAP greatly decreased the level of cAMP (20 or more times) in liver in vivo. IL-1β , which was previously shown to have protective effect on APAP toxicity, if given before APAP, greatly increased the synthesis of cAMP in liver in vivo in comparison to saline control (to approximately 2/3 of level in normal mice), and this effect could be blocked almost completely with DDA, given after IL-1α but before APAP. Similar results with Db-cAMP were obtained in mice intoxicated with D-galactosamine + lipopolysaccharide. Presently, we are investigating signaling ways of this protective effect, using more specific inhibitors of cAMP or its downstream intracellular mediators. Specially, we are investigating the role of NF-κ B and protein kinase A (PKA) in protective action of cAMP. CONCLUSIONS. cAMP has a significant protection on liver toxicity induced by xenobiotics.

It is known that proinflammatory cytokines influence the effect of hepatotoxic chemicals on liver (1). Previously we have shown that IL-1alpha and IL-6 have hepatoprotective effect if given to mice before administration of acetaminophen (APAP) and that this effect is partially mediated by PGE2 (1, 2). Since many inflammatory cytokines stimulate the synthesis of cAMP as well as PGE2, we investigated the effect of agonists, antagonists and inhibitors of cAMP degradation on APAP toxicity. APP (300 mg/kg) was administered intragastrically to mice which were given one to two hours before or up to 3 hours after a stable agonist of cAMP (dibutyryl-cAMP – Db-cAMP), antagonist (2, 3-dideoxyadenosine – DDA) or inhibitor of phoshodiesterase IV (Rolipram). All agents were given i.p. The survival of mice was followed for 72 hours and level of serum aminotransferases (AST and ALT) were determined 18-24 hours after administration of APAP. Db-cAMP (25 mg/kg) as well as Rolipram (8 mg/kg) significantly increased the survival of mice and reduced AST and ALT serum concentration if given 1-2 hours before or up to 2 hours after AAP administration. Db-cAMP had the same effect if given 3 hours after AAP, but effect was not statistically significant. On the contrary, DDA (160 microg/kg) decreased the survival of mice and increased serum aminotransferase concentration if given 2 hours before or ½ ; hours after AAP, but only effect on aminotransferase level was statistically significant. The treatment of animals with APAP greatly decreased the level of cAMP (20 or more times) in liver in vivo (the degradation of cAMP was blocked by rolipram). IL-1alpha, which was previously shown to have protective effect on APAP toxicity, if given before APAP, greatly increased the synthesis of cAMP in liver in vivo in comparison to saline control (to approximately 2/3 level in normal mice), and this effect could be blocked almost completely with DDA, given after IL-1α but before AAP. Presently, we are investigating signaling ways of this protective effect, using more specific inhibitors of cAMP or its downstream intracellular mediators.

It is known that Kupffer and other non-parenchymal liver cells are activated in hepatic injury, even if this is induced acutely by administration of high doses of hepatotoxic drugs. These cells secrete prostaglandins, proinflammatory cytokines and other products, which may contribute to pathogenic or repair processes. In these experiments we investigated the role of prostaglandin E2 (PGE2), prostaglandin I2 (PGI2) and thromboxane (TXA2) in acute liver toxicity induced in mice by Paracetamol (acetaminophen, AAP). Mice were given a lethal dose of AAP by gastric lavage and production of PGs was determined in supernatants of short term culture of livers slices, taken from mice at various times (2-24 hours) after AAP administration. Several agents which are known to have hepatoprotective effect and/or modulate PG synthesis (IL-1 , ketoconazole and dipyiridamole) were given to mice 1-2 hours before AAP. The synthesis of all prostanoids was greatly increased in liver slices of mice given AAP in comparison to mice given saline. Pretreatment of mice with IL-1 before administration of AAP further increased the synthesis of PGE2 and PGI2 and reduced the synthesis of TXA2 in liver slices. These changes in level of PG synthesis were significant at most time observed time intervals after AAP administration. The effect of ketoconazole and dipyridamole on PG synthesis was measured only at 6 hour interval after AAP administration. Both agents had similar effect as IL-1 , i.e., they increased the synthesis of PGE2 or PGI2 and reduced the synthesis of TXA2. All agents had a significant protective effect in vivo when given before AAP, i.e., they reduced the mortality of mice and level of serum aminotransferases in survived mice (ALT and AST). 16-16-dimethyl-PGE 2 (a stable analogue of (PGE 2 ) had a strong protective effect if given to mice before or up to two hours after AAP. These data suggest that PGE2 and PGI2 might have a protective and TXA2 noxious effect in acute liver injury.

Background and Purpose. We have shown previously that IL-la is highly protective in mice with acute hepatototoxicity induced by AAP In these experiments we investigated whether PCE2 and IL-6 are involved in IL-la-induced hepatoprotection. In addition, we investigated the effect of spontaneously produced IL-6 on AAP-induced hepatotoxicity, and the ef&not ; fect of IL-la on heptotoxicity induced by LPS in D-galactosamine sensi&not ; tized mice. Materials and Methods. The mice received toxic dose of AAP by gas&not ; tric route. Recombinant human IL-lα or recombinant mouse IL-6 were given to mice i.p. 2 hours before AAP polycolnal anti-PGE2 and mono&not ; clonal anti-IL-6 antibodies were given 3 hours before IL-lα , or, when given alone, 5 hours before AAP The mortality of mice was followed, and serum aminotransferase levels (AST and ALT) were determined 24 hours after the AAP administration. Results. IL-lα significantly reduced and the serum ALT and AST levels in the AAP-treated mice. It also decreased the mortality of mice which re&not ; ceived LPS after D-galactosamine. Pretreatment of mice with anti-PGE2 or anti-IL-6 antibodies before IL-lα administration decreased its protective effect in those with AAP-induced hepatotoxicity. Administration of anti-IL-6 antibodies alone before AAP increased the mortality of mice and the se&not ; rum aminotransferase levels, while, in contrast, administration of recombinant mouse IL-6 before AAP had the opposite, i.e. beneficial effect. Conclusion. Both PGE2 and IL-6 mediate, perhaps in mutually interde&not ; pendent way, the hepatoprotective effect of IL-lα .

Acetaminophen (AAP) administered to mice via gastric tube in a dose of 300-350 mg/kg induced heavy liver injury and killed 55%- 80% mice within 48 hours. Dimethyl-16-16prostaglandin E2 (PGE2), given intraperitoneally (0.025-0.2 mg/kg) 15 to 30 minutes before AAP, ameliorated the liver injury and reduced the mortality in mice to 20%-30%. PGE2 also significantly reduced the increase in plasma transamina.se (ALT, AST) levels induced by AAP. In the same doses, PGE1 had a slight but not significantly protective effect. When given 2 hours after AAP administration, PGE2 delayed the death of mice but had no significant effect on total mortality. Pretreatment of mice with indomethacin (4 mg/kg i.p.) had no effect on AAP toxicity, while pretreatment of mice with specific anti-PGE2 antibodies increased this toxicity. Thus, PGE2 was concluded to have a protective role in AAP-induced hepatotoxicity.

Acetaminophen (AAP) administered to mice via gastric tube in a dose of 300-350 mg/kg induced heavy liver injury and killed 55% - 80% mice within 48 hours. Dimethyl-16-16 prostaglandin E2 (PGE2), given intraperitoneally (0.025-0.2 mg/kg) 15 to 30 minutes before AAP, ameliorated the liver injury and reduced the mortality in mice to 20%-30%. PGE2 also significantly reduced the increase in plasma transaminase (ALT, AST) levels induced by AAP. In the same doses, PGE1 had a slight but not significantly protective effect. When given 2 hours after AAP administration, PGE2 delayed the death of mice but had no significant effect on total mortality. Pretreatment of mice with indomethacin (4 mg/kg i.p.) had no effect on AAP toxicity, while pretreatment of mice with specific anti-PGE2 antibodies increased this toxicity. Thus, PGE2 was concluded to have a protective role in AAP-induced hepatotoxicity.