The role of hypoxia-inducible factor-1α in acetaminophen hepatotoxicity.

Hypoxia-inducible factor-1α (HIF-1α) is a critical transcription factor that controls oxygen homeostasis in response to hypoxia, inflammation, and oxidative stress. HIF has been implicated in the pathogenesis of liver injury in which these events play a role, including acetaminophen (APAP) overdose, which is the leading cause of acute liver failure in the United States. APAP overdose has been reported to activate HIF-1α in mouse livers and isolated hepatocytes downstream of oxidative stress. HIF-1α signaling controls many factors that contribute to APAP hepatotoxicity, including mitochondrial cell death, inflammation, and hemostasis. Therefore, we tested the hypothesis that HIF-1α contributes to APAP hepatotoxicity. Conditional HIF-1α deletion was generated in mice using an inducible Cre-lox system. Control (HIF-1α-sufficient) mice developed severe liver injury 6 and 24 h after APAP overdose (400 mg/kg). HIF-1α-deficient mice were protected from APAP hepatotoxicity at 6 h, but developed severe liver injury by 24 h, suggesting that HIF-1α is involved in the early stage of APAP toxicity. In further studies, HIF-1α-deficient mice had attenuated thrombin generation and reduced plasminogen activator inhibitor-1 production compared with control mice, indicating that HIF-1α signaling contributes to hemostasis in APAP hepatotoxicity. Finally, HIF-1α-deficient animals had decreased hepatic neutrophil accumulation and plasma concentrations of interleukin-6, keratinocyte chemoattractant, and regulated upon activation normal T cell expressed and secreted compared with control mice, suggesting an altered inflammatory response. HIF-1α contributes to hemostasis, sterile inflammation, and early hepatocellular necrosis during the pathogenesis of APAP toxicity.

Comparison of TNFα to lipopolysaccharide as an inflammagen to characterize the idiosyncratic hepatotoxicity potential of drugs: Trovafloxacin as an example.

Idiosyncratic drug reactions (IDRs) are poorly understood, unpredictable, and not detected in preclinical studies. Although the cause of these reactions is likely multi-factorial, one hypothesis is that an underlying inflammatory state lowers the tolerance to a xenobiotic. Previously used in an inflammation IDR model, bacterial lipopolysaccharide (LPS) is heterogeneous in nature, making development of standardized testing protocols difficult. Here, the use of rat tumor necrosis factor-α (TNFα) to replace LPS as an inflammatory stimulus was investigated. Sprague-Dawley rats were treated with separate preparations of LPS or TNFα, and hepatic transcriptomic effects were compared. TNFα showed enhanced consistency at the transcriptomic level compared to LPS. TNFα and LPS regulated similar biochemical pathways, although LPS was associated with more robust inflammatory signaling than TNFα. Rats were then codosed with TNFα and trovafloxacin (TVX), an IDR-associated drug, and evaluated by liver histopathology, clinical chemistry, and gene expression analysis. TNFα/TVX induced unique gene expression changes that clustered separately from TNFα/levofloxacin, a drug not associated with IDRs. TNFα/TVX cotreatment led to autoinduction of TNFα resulting in potentiation of underlying gene expression stress signals. Comparison of TNFα/TVX and LPS/TVX gene expression profiles revealed similarities in the regulation of biochemical pathways. In conclusion, TNFα could be used in lieu of LPS as an inflammatory stimulus in this model of IDRs.

Acute exposure to ozone exacerbates acetaminophen-induced liver injury in mice.

Ozone (O(3)), an oxidant air pollutant in photochemical smog, principally targets epithelial cells lining the respiratory tract. However, changes in gene expression have also been reported in livers of O(3)-exposed mice. The principal aim of the present study was to determine if acute exposure to environmentally relevant concentrations of O(3) could cause exacerbation of drug-induced liver injury in mice. Overdose with acetaminophen (APAP) is the most common cause of drug-induced liver injury in developed countries. In the present study, we examined the hepatic effects of acute O(3) exposure in mice pretreated with a hepatotoxic dose of APAP. C57BL/6 male mice were fasted overnight and then given APAP (300 mg/kg ip) or saline vehicle (0 mg/kg APAP). Two hours later, mice were exposed to 0, 0.25, or 0.5 ppm O(3) for 6 h and then sacrificed 9 or 32 h after APAP administration (1 or 24 h after O(3) exposure, respectively). Animals euthanized at 32 h were given 5-bromo-2-deoxyuridine 2 h before sacrifice to identify hepatocytes undergoing reparative DNA synthesis. Saline-treated mice exposed to either air or O(3) had no liver injury. All APAP-treated mice developed marked centrilobular hepatocellular necrosis that increased in severity with time after APAP exposure. O(3) exposure increased the severity of APAP-induced liver injury as indicated by an increase in necrotic hepatic tissue and plasma alanine aminotransferase activity. O(3) also caused an increase in neutrophil accumulation in livers of APAP-treated animals. APAP induced a 10-fold increase in the number of bromodeoxyuridine-labeled hepatocytes that was markedly attenuated by O(3) exposure. Gene expression analysis 9 h after APAP revealed differential expression of genes involved in inflammation, oxidative stress, and cellular regeneration in mice treated with APAP and O(3) compared to APAP or O(3) alone, providing some indications of the mechanisms behind the APAP and O(3) potentiation. These results suggest that acute exposure to near ambient concentrations of this oxidant air pollutant may exacerbate drug-induced liver injury by delaying hepatic repair.

Bacterial- and viral-induced inflammation increases sensitivity to acetaminophen hepatotoxicity.

Acetaminophen (APAP)-induced hepatotoxicity accounts for nearly half of acute liver failure cases in the United States. The doses that produce hepatotoxicity vary considerably and many risk factors have been proposed, including liver inflammation from viral hepatitis. Interestingly, inflammatory stress from another stimulus, bacterial endotoxin (lipopolysaccharide, LPS), renders the liver more sensitive to hepatotoxicity from numerous xenobiotic agents. The purpose of these studies was to test the hypothesis that inflammation induced by LPS or infection with reovirus increases sensitivity to APAP-induced liver injury. For LPS-induced inflammation, C57BL/6J mice were treated with either saline or LPS (44 x 10(6) EU/kg, ip) 2 h before treatment with APAP (100-400 mg/kg, ip) or saline. No elevation in serum alanine aminotransferase (ALT) activity was observed in mice that received vehicle or LPS alone. LPS co-treatment produced a leftward shift of the dose-response curve for APAP-induced hepatotoxicity and led to significantly greater tumor necrosis factor-alpha (TNF) production than APAP alone. Reovirus serotype 1 (10(8) PFU, iv) induced inflammation in Balb/c mice as evidenced by increases in hepatic mRNAs for macrophage inhibitory protein-2, interleukin-6, and TNF. Co-administration of reovirus and APAP at doses of 450 and 700 mg/kg (2 h after reovirus) led to increases in serum ALT activity, whereas neither reovirus nor APAP alone produced liver injury. Consistent with the increases in serum ALT activity, histopathologic examination revealed centrilobular necrosis with marked neutrophilic accumulation only in livers of mice treated with LPS/APAP or with reovirus/APAP. The results suggest that normally noninjurious doses of APAP are rendered hepatotoxic by modest inflammation, whether bacterial or viral in origin.

Role of the coagulation system in acetaminophen-induced hepatotoxicity in mice.

UNLABELLED: Acetaminophen (N-acetyl-p-aminophenol [APAP]) is one of the leading causes of acute liver failure, and APAP hepatotoxicity is associated with coagulopathy in humans. We tested the hypothesis that activation of the coagulation system and downstream protease-activated receptor (PAR)-1 signaling contribute to APAP-induced liver injury. Fasted C57BL/J6 mice were treated with either saline or APAP (400 mg/kg intraperitoneally) and were euthanized 0.5-24 hours later. Hepatotoxicity and coagulation system activation occurred by 2 hours after administration of APAP. Treatment with APAP also caused a rapid and transient increase in liver procoagulant activity. In addition, significant deposition of fibrin was observed in the liver by 2 hours, and the concentration of plasminogen activator inhibitor-1 in plasma increased between 2 and 6 hours. Pretreatment with heparin attenuated the APAP-induced activation of the coagulation system and hepatocellular injury and diminished hepatic fibrin deposition at 6 hours. Loss of hepatocellular glutathione was similar in APAP-treated mice pretreated with saline or heparin, suggesting that heparin did not diminish bioactivation of APAP. In mice deficient in tissue factor, the principal cellular activator of coagulation, APAP-induced liver injury, activation of coagulation, and hepatic fibrin deposition were reduced at 6 hours. Formation of the tissue factor-factor VIIa complex leads to the generation of thrombin that can activate cells through cleavage of PAR-1. Mice lacking PAR-1 developed less injury and hepatic fibrin deposits at 6 hours in response to APAP than control mice. CONCLUSION: Activation of the coagulation system and PAR-1 signaling contribute significantly to APAP-induced liver injury.

Modest inflammation enhances diclofenac hepatotoxicity in rats: role of neutrophils and bacterial translocation.

Idiosyncratic adverse drug reactions (IADRs) represent an important human health problem, yet animal models for preclinical prediction of these reactions are lacking. Recent evidence in animals suggests that some IADRs arise from drug interaction with an inflammatory episode that renders the liver sensitive to injury. Diclofenac (DCLF) is one of those drugs for which the clinical use is limited by idiosyncratic liver injury. We tested the hypothesis that modest inflammation triggered in rats by a small dose of lipopolysaccharide (LPS) renders a nonhepatotoxic dose of DCLF injurious to liver. Cotreatment of rats with nonhepatotoxic doses of LPS and DCLF resulted in elevated serum alanine aminotransferase activity and liver histopathologic changes 6 h after DCLF administration. Neither LPS nor DCLF alone had such an effect. Gene array analysis of livers revealed a unique gene expression pattern in the LPS/DCLF-cotreated group compared with groups given either agent alone. Antiserum-induced neutrophil (PMN) depletion in LPS/DCLF-cotreated rats protected against liver injury, demonstrating a role for PMNs in the pathogenesis of this LPS/DCLF interaction. Gut sterilization of LPS/DCLF-treated rats did not protect against liver injury. In contrast, gut sterilization did attenuate liver injury caused by a large, hepatotoxic dose of DCLF, suggesting that hepatotoxicity induced by large doses of DCLF is caused in part by its ability to increase intestinal permeability to endotoxin or other bacterial products. These results demonstrate that inflammation-DCLF interaction precipitates hepatotoxicity in rats and raise the possibility of creating animal models that predict human IADRs.

Modeling inflammation-drug interactions in vitro: a rat Kupffer cell-hepatocyte coculture system.

Xenobiotic-inflammation interactions lead to hepatotoxicity in vivo. Selected xenobiotic agents (acetaminophen, APAP; chlorpromazine, CPZ; allyl alcohol, AlOH; monocrotaline, MCT) for which this occurs were evaluated for ability to elicit the release of Kupffer cell (KC)-derived inflammatory mediators and to modulate lipopolysaccharide (LPS)-stimulated release of these mediators. Using KCs and hepatocytes (HPCs) isolated from rat, KC/HPC cocultures were treated with either LPS, xenobiotic, vehicle or a combination. Six hours later, the release of inflammatory mediators was assessed. LPS alone caused a concentration-dependent increase in TNF-alpha release but had no significant effect on the release of PGE(2). APAP by itself did not alter release of TNF-alpha, PGE(2), IL-10, Gro/KC or IFN-gamma; however, in the presence of LPS, APAP enhanced LPS-induced TNF-alpha and Gro/KC release. APAP also attenuated LPS-induced increases in IL-10 and MCP-1. CPZ alone caused a concentration-dependent increase in TNF-alpha release, which was approximately additive in the presence of LPS. AlOH alone did not affect TNF-alpha release, but decreased TNF-alpha production in the presence of LPS. AlOH increased PGE(2) production, and this effect was potentiated in the presence of LPS. MCT by itself did not affect release of TNF-alpha but increased the response to LPS. Neither MCT, LPS, nor the combination affected production of PGE(2). These results demonstrate that KC/HPC cocultures can be used to evaluate interactions of xenobiotics with LPS. Furthermore, data from these studies qualitatively mirror reported data from whole animal studies, suggesting that this model could be useful for predicting aspects of xenobiotic-inflammation interactions in vivo.

Coagulation-dependent gene expression and liver injury in rats given lipopolysaccharide with ranitidine but not with famotidine.

In an animal model of drug idiosyncrasy, rats cotreated with nonhepatotoxic doses of lipopolysaccharide (LPS) and ranitidine (RAN) develop hepatocellular injury, whereas rats treated with LPS and famotidine (FAM) do not. The coagulation system and neutrophils (PMNs) are requisite mediators of LPS/RAN-induced liver injury. We tested the hypothesis that unique gene expression in LPS/RAN-treated rats requires coagulation system activation and that these changes are absent in rats given LPS and FAM. Rats were treated with a nonhepatotoxic dose of LPS (44.4 x 10(6) endotoxin units/kg i.v.) or its vehicle, and then 1 h later, they were treated with heparin (3000 U/kg) or its vehicle. One hour thereafter, they were given RAN (30 mg/kg), FAM (6 mg/kg, a pharmacologically equiefficacious dose, or 28.8 mg/kg, an equimolar dose), or vehicle (i.v.). They were killed 2 or 6 h after drug treatment for evaluation of hepatotoxicity, coagulation system activation, and liver gene expression (2 h only). Statistical filtering of gene array results and real-time polymerase chain reaction identified groups of genes expressed in LPS/RAN-treated rats but not LPS/FAM-treated rats that were either changed or unchanged by heparin administration. For example, LPS/RAN-induced mRNA expression of the inflammatory mediators interleukin-6, cyclooxygenase-2, and macrophage inflammatory protein-2 (MIP-2) was reduced by anticoagulation. Enhancement of serum MIP-2 and plasminogen activator inhibitor-1 concentrations in LPS/RAN-treated rats was prevented by anticoagulation. The results suggest cross-talk between hemostasis-induced gene expression and inflammation (e.g., PMN function) in the genesis of hepatocellular injury in LPS/RAN-treated rats. In contrast, neither the expression of such genes nor hepatocellular necrosis occurred in rats treated with LPS/FAM.

Unique gene expression and hepatocellular injury in the lipopolysaccharide-ranitidine drug idiosyncrasy rat model: comparison with famotidine.

Rats cotreated with lipopolysaccharide (LPS) and ranitidine (RAN) but not LPS and famotidine (FAM) develop hepatocellular injury in an animal model of idiosyncratic drug reactions. Evaluation of liver gene expression in rats given LPS and/or RAN led to confirmation that the hemostatic system, hypoxia, and neutrophils (PMNs) are critical mediators in LPS/RAN-induced liver injury. We tested the hypothesis that unique gene expression changes distinguish LPS/RAN-treated rats from rats given LPS or RAN alone and from those cotreated with LPS/FAM. Rats were treated with a nonhepatotoxic dose of LPS (44.4 x 10(6) endotoxin units/kg, iv) or its vehicle. Two hours thereafter they were given RAN (30 mg/kg, iv), FAM (either 6 mg/kg, a pharmacologically equi-efficacious dose, or 28.8 mg/kg, an equimolar dose, iv), or vehicle. They were killed 2 or 6 h after drug treatment for evaluation of hepatotoxicity (2 and 6 h) and liver gene expression (2 h only). At a time before the onset of hepatocellular injury, hierarchical clustering distinguished rats treated with LPS/RAN from those given LPS alone. 205 probesets were expressed differentially to a greater or lesser degree only in LPS/RAN-treated rats compared to LPS/FAM or LPS alone, which did not develop liver injury. These included VEGF, EGLN3, MAPKAPK-2, BNIP3, MIP-2, COX-2, EGR-1, PAI-1, IFN-gamma, and IL-6. Expression of these genes was confirmed by real-time PCR. Serum concentrations of MIP-2, PAI-1, IFN-gamma, and IL-6 correlated with their respective gene expression patterns. Overall, the expression of several gene products capable of controlling requisite mediators of injury (i.e., hemostasis, hypoxia, PMNs) in this model were enhanced in livers of LPS/RAN-treated rats. Furthermore, enhanced expression of MAPKAPK-2 in RAN-treated rats and its target genes in LPS/RAN-treated rats suggests that p38/MAPKAPK-2 signaling is a regulation point for enhancement of LPS-induced gene expression by RAN.

Metabonomic evaluation of idiosyncrasy-like liver injury in rats cotreated with ranitidine and lipopolysaccharide.

Idiosyncratic liver injury occurs in a small fraction of people on certain drug regimens. The cause of idiosyncratic hepatotoxicity is not known; however, it has been proposed that environmental factors such as concurrent inflammation initiated by bacterial lipopolysaccharide (LPS) increase an individual's susceptibility to drug toxicity. Ranitidine (RAN), a histamine-2 receptor antagonist, causes idiosyncratic liver injury in humans. In a previous report, idiosyncrasy-like liver toxicity was created in rats by cotreating them with LPS and RAN. In the present study, the ability of metabonomic techniques to distinguish animals cotreated with LPS and RAN from those treated with each agent individually was investigated. Rats were treated with LPS or its vehicle and with RAN or its vehicle, and urine was collected for nuclear magnetic resonance (NMR)- and mass spectroscopy-based metabonomic analyses. Blood and liver samples were also collected to compare metabonomic results with clinical chemistry and histopathology. NMR metabonomic analysis indicated changes in the pattern of metabolites consistent with liver damage that occurred only in the LPS/RAN cotreated group. Principal component analysis of urine spectra by either NMR or mass spectroscopy produced a clear separation of the rats treated with LPS/RAN from the other three groups. Clinical chemistry (serum alanine aminotransferase and aspartate aminotransferase activities) and histopathology corroborated these results. These findings support the potential use of a noninvasive metabonomic approach to identify drug candidates with potential to cause idiosyncratic liver toxicity with inflammagen coexposure.

Microarray analysis of lipopolysaccharide potentiation of trovafloxacin-induced liver injury in rats suggests a role for proinflammatory chemokines and neutrophils.

Idiosyncratic drug toxicity refers to toxic reactions occurring in a small subset of patients and usually cannot be predicted during preclinical or early phases of clinical trials. One hypothesis for the pathogenesis of hepatic idiosyncratic drug reactions is that, in certain individuals, underlying inflammation results in sensitization of the liver, such that injury occurs from an agent that typically would not cause hepatotoxicity at a therapeutic dose. We explored this possibility by cotreating rats with nonhepatotoxic doses of bacterial lipopolysaccharide (LPS) and trovafloxacin (TVX), a drug that caused idiosyncratic hepatotoxicity in humans. The combination of LPS and TVX resulted in hepatotoxicity in rats, as determined by increases in serum alanine aminotransferase activity and hepatocellular necrosis, which were not observed with either agent alone. In contrast, treatment with LPS and levofloxacin, a fluoroquinolone without human idiosyncratic liability, did not result in these changes. Liver gene expression analysis identified unique changes induced by the combination of TVX and LPS, including enhanced expression of chemokines, suggestive of liver neutrophil (PMN) accumulation and activation. Consistent with a role for PMN in the hepatotoxicity induced by LPS/TVX, prior depletion of PMN attenuated the liver injury. The results suggest that gene expression profiles predictive of idiosyncratic liability can be generated in rats cotreated with LPS and drug. Furthermore, they identify gene expression changes that could be explored as biomarkers for idiosyncratic toxicity and lead to enhanced understanding of the mechanism(s) underlying hepatotoxicity induced by TVX.

Coagulation-mediated hypoxia and neutrophil-dependent hepatic injury in rats given lipopolysaccharide and ranitidine.

Idiosyncrasy-like liver injury occurs in rats cotreated with nonhepatotoxic doses of ranitidine (RAN) and bacterial lipopolysaccharide (LPS). Hepatocellular oncotic necrosis is accompanied by neutrophil (PMN) accumulation and fibrin deposition in LPS/RAN-treated rats, but the contribution of PMNs to injury has not been shown. We tested the hypothesis that PMNs are critical mediators of LPS/RAN-induced liver injury and explored the potential for interaction between PMNs and hemostasis-induced hypoxia. Rats were given either LPS (44.4 x 10(6) endotoxin units/kg) or its vehicle and then RAN (30 mg/kg) or its vehicle 2 h later. They were killed 3 or 6 h after RAN treatment, and hepatocellular injury was estimated from serum alanine aminotransferase activity and liver histopathology. Plasma PMN chemokine concentration and the number of PMNs in liver increased after LPS treatment at 3 h and were not markedly altered by RAN cotreatment. Depletion of circulating PMNs attenuated hepatic PMN accumulation and liver injury and had no effect on coagulation system activation. Anticoagulation with heparin attenuated liver fibrin deposition and injury in LPS/RAN-treated rats; however, heparin had little effect on liver PMN accumulation or plasma chemokine concentration. Liver hypoxia occurred in LPS/RAN-cotreated rats and was significantly reduced by heparin. In vitro, hypoxia enhanced the killing of rat hepatocytes by PMN elastase and shortened its onset, indicating a synergistic interaction between PMNs and hypoxia. The results suggest that PMNs are involved in the hepatocellular injury caused by LPS/RAN-cotreatment and that hemostasis increases sensitivity to PMN-induced hepatocellular injury by causing liver hypoxia.

Role of hepatic fibrin in idiosyncrasy-like liver injury from lipopolysaccharide-ranitidine coexposure in rats.

Coadministration of nonhepatotoxic doses of the histamine 2-receptor antagonist ranitidine (RAN) and bacterial lipopolysaccharide (LPS) results in hepatocellular injury in rats, the onset of which occurs in 3 to 6 hours. This reaction resembles RAN idiosyncratic hepatotoxicity in humans. Early fibrin deposition occurs in livers of rats cotreated with LPS/RAN. Accordingly, we tested the hypothesis that the hemostatic system contributes to liver injury in LPS/RAN-treated rats. Rats were given either LPS (44.4 x 10(6) EU/kg) or its vehicle, then RAN (30 mg/kg) or its vehicle 2 hours later. They were killed 2, 3, 6, 12, or 24 hours after RAN treatment, and liver injury was estimated from serum alanine aminotransferase activity. A modest elevation in serum hyaluronic acid, which was most pronounced in LPS/RAN-cotreated rats, suggested altered sinusoidal endothelial cell function. A decrease in plasma fibrinogen and increases in thrombin-antithrombin dimers and in serum concentration of plasminogen activator inhibitor-1 occurred before the onset of liver injury. Hepatic fibrin deposition was observed in livers from LPS/RAN-cotreated rats 3 and 6 hours after RAN. Liver injury was abolished by the anticoagulant heparin and was significantly attenuated by the fibrinolytic agent streptokinase. Hypoxia, one potential consequence of sinusoidal fibrin deposition, was observed in livers of LPS/RAN-treated rats. In conclusion, the results suggest that the hemostatic system is activated after LPS/RAN cotreatment and that fibrin deposition in liver is important for the genesis of hepatic parenchymal cell injury in this model.

Adverse hepatic drug reactions: inflammatory episodes as consequence and contributor.

Susceptibility to drug toxicity is influenced by a variety of factors, both genetic and environmental. The focus of this article is the evidence addressing the hypothesis that inflammation is both a result of and a susceptibility factor for drug toxicity, with an emphasis on liver as a target organ. Results of studies suggesting a role for inflammatory mediators in the hepatotoxicity caused by acetaminophen or ethanol are discussed. For several drugs, the evidence from animal models that concurrent inflammation increases injury is presented. In addition, the occurrence of adverse drug reactions in people with preexisting inflammatory diseases is considered. The special case of idiosyncratic drug reactions is discussed and the potential raised for development of animal models for this type of drug toxicity. The conclusion is that inflammatory factors should be considered as determinants of sensitivity to adverse drug reactions.

Gene expression analysis points to hemostasis in livers of rats cotreated with lipopolysaccharide and ranitidine.

Studies in rats have demonstrated that modest underlying inflammation can precipitate idiosyncratic-like liver injury from the histamine 2-receptor antagonist, ranitidine (RAN). Coadministration to rats of nonhepatotoxic doses of RAN and the inflammagen, bacterial lipopolysaccharide (LPS), results in hepatocellular injury. We tested the hypothesis that hepatic gene expression changes could be distinguished among vehicle-, LPS-, RAN- and LPS/RAN-treated rats before the onset of significant liver injury in the LPS/RAN-treated rats (i.e., 3 h post-treatment). Rats were treated with LPS (44 x 10(6) EU/kg, i.v.) or its vehicle, then two hours later with RAN (30 mg/kg, i.v.) or its vehicle. They were killed 3 h after RAN treatment, and liver samples were taken for evaluation of liver injury and RNA isolation. Hepatic parenchymal cell injury, as estimated by increases in serum alanine aminotransferase (ALT) activity, was not significant at this time. Hierarchal clustering of gene expression data from Affymetrix U34A rat genome array grouped animals according to treatment. Relative to treatment with vehicle alone, treatment with RAN and/or LPS altered hepatic expression of numerous genes, including ones encoding products involved in inflammation, hypoxia, and cell death. Some were enhanced synergistically by LPS/RAN cotreatment. Real-time PCR confirmed robust changes in expression of B-cell translocation gene 2, early growth response-1, and plasminogen-activator inhibitor-1 (PAI-1) in cotreated rats. The increase in PAI-1 mRNA was reflected in an increase in serum PAI-1 protein concentration in LPS/RAN-treated rats. Consistent with the antifibrinolytic activity of PAI-1, significant fibrin deposition occurred only in livers of LPS/RAN-treated rats. The results suggest the possibility that expression of PAI-1 promotes fibrin deposition in liver sinusoids of LPS/RAN-treated rats and are consistent with the development of local ischemia and consequent tissue hypoxia.

Application of high-throughput Fourier-transform infrared spectroscopy in toxicology studies: contribution to a study on the development of an animal model for idiosyncratic toxicity.

An evaluation of high-throughput Fourier-transform infrared spectroscopy (FT-IR) as a technology that could support a "metabonomics" component in toxicological studies of drug candidates is presented. The hypothesis tested in this study was that FT-IR had sufficient resolving power to discriminate between urine collected from control rat populations and rats subjected to treatment with a potent inflammatory agent, bacterial lipopolysaccharide (LPS). It was also hypothesized that co-administration of LPS with ranitidine, a drug associated with reports of idiosyncratic susceptibility, would induce hepatotoxicity in rats and that this could be detected non-invasively by an FT-IR-based metabonomics approach. The co-administration of LPS with "idiosyncratic" drugs represents an attempt to develop a predictive model of idiosyncratic toxicity and FT-IR is used herein to support characterization of this model. FT-IR spectra are high dimensional and the use of genetic programming to identify spectral sub-regions that most contribute to discrimination is demonstrated. FT-IR is rapid, reagentless, highly reproducible and inexpensive. Results from this pilot study indicate it could be extended to routine applications in toxicology and to supporting characterization of a new animal model for idiosyncratic susceptibility.

15-deoxy prostaglandin J2 enhances allyl alcohol-induced toxicity in rat hepatocytes.

Allyl alcohol causes hepatotoxicity that is potentiated by small doses of bacterial lipopolysaccharide (LPS) through a cyclooxygenase-2 (COX-2)-dependent mechanism. The COX-2 product prostaglandin D(2) (PGD(2)) increases hepatocyte killing by allyl alcohol in vitro. In the present study the ability of the nonenzymatic product of PGD(2), 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)), to increase the cytotoxicity of allyl alcohol was evaluated. In a concentration-dependent manner, 15d-PGJ(2) significantly augmented cell death caused by allyl alcohol in isolated rat hepatocytes. 15d-PGJ(2) also increased the cytotoxicity of acrolein, the active metabolite of allyl alcohol. An agonist for the PGD(2) receptor neither reproduced the increase in allyl alcohol-mediated cytotoxicity nor altered the response to 15d-PGJ(2). Similarly, these responses were not affected by either an agonist or an antagonist for the peroxisome proliferator-activated receptor-gamma. The enhancement by 15d-PGJ(2) of allyl alcohol-mediated cell killing was unaffected by augmentation or inhibition of cAMP. Protein synthesis was markedly decreased by 15d-PGJ(2), but inhibition of protein synthesis alone with cycloheximide did not increase allyl alcohol-mediated cell killing. Allyl alcohol at subtoxic concentrations increased translocation of nuclear factor kappa B (NF-kappaB), whereas at cytotoxic concentrations no translocation occurred. 15d-PGJ(2) inhibited translocation of NF-kappaB from the cytosol to the nucleus both in the presence and absence of allyl alcohol. Like 15d-PGJ(2), MG132, an inhibitor of NF-kappaB activation, enhanced allyl alcohol-induced hepatocyte death. Together these results indicate that 15d-PGJ(2) augments hepatocyte killing by allyl alcohol, and the mechanism may be related to the inhibition of NF-kappaB activation.

Modes of cell death in rat liver after monocrotaline exposure.

Monocrotaline (MCT) is a pyrrolizidine alkaloid (PA) plant toxin that produces sinusoidal endothelial cell (SEC) injury, hemorrhage, fibrin deposition, and coagulative hepatic parenchymal cell (HPC) oncosis in centrilobular regions of rat livers. Cells with apoptotic morphology have been observed in the livers of animals exposed to other PAs. Whether apoptosis occurs in the livers of MCT-treated animals and whether it is required for full manifestation of pathological changes is not known. To determine this, rats were treated with 300 mg MCT/kg, and apoptosis was detected by transmission electron microscopy and the TUNEL (TdT-mediated dUTP nick end labeling) assay. MCT produced significant apoptosis in the liver by 4 h after treatment. To determine if MCT kills cultured HPCs by apoptosis, HPCs were isolated from the livers of rats and exposed to MCT. MCT caused a concentration-dependent release of alanine aminotransferase (ALT), a marker of HPC injury. Furthermore, caspase 3 was activated and TUNEL staining increased in MCT-treated HPCs. MCT-induced TUNEL staining and release of ALT into the medium were completely prevented by the pancaspase inhibitors z-VAD.fmk and IDN-7314, suggesting that MCT kills cultured HPCs by apoptosis. To determine if caspase inhibition prevents MCT-induced apoptosis in the liver, rats were cotreated with MCT and IDN-7314. IDN-7314 reduced MCT-induced TUNEL staining in the liver and release of ALT into the plasma. Morphometric analysis confirmed that IDN-7314 reduced HPC oncosis in the liver by approximately 50%. Inasmuch as HPC hypoxia occurred in the livers of MCT-treated animals, upregulation of the hypoxia-regulated cell-death factor, BNIP3 (Bcl2/adenovirus EIB 19kD-interacting protein 3), was examined. BNIP3 was increased in the livers of mice treated 24 h earlier with MCT. Results from these studies show that MCT kills cultured HPCs by apoptosis but causes both oncosis and apoptosis in the liver in vivo. Furthermore, caspase inhibition reduces both apoptosis and HPC oncosis in the liver after MCT exposure.

Inflammation and drug idiosyncrasy--is there a connection?

"Drug idiosyncrasy" refers to untoward reactions to drugs that occur in a small fraction of patients and have no obvious relationship to dose or duration of therapy. The liver is a frequent target for toxicity. Much of the conventional thinking about mechanisms of drug idiosyncrasy has centered on hypotheses that the reactions have a metabolic basis involving drug metabolism polymorphisms or that they arise from a specific immune response to the drug or its metabolite(s). For very few drugs does convincing evidence exist for either of these mechanisms, however. The erratic temporal and dose relationships that characterize idiosyncratic drug responses suggest the possibility that some event during the course of therapy renders tissues peculiarly susceptible to toxic effects of the drug. For example, episodes of inflammation are commonplace in people, and results of numerous studies in animals indicate that a modest inflammatory response can enhance tissue sensitivity to a variety of toxic chemicals. These observations have led to the hypothesis that an episode of inflammation during drug therapy could decrease the threshold for drug toxicity and thereby render an individual susceptible to a toxic reaction that would not otherwise occur (i.e., an "idiosyncratic" response). This hypothesis can explain the features of drug idiosyncrasy using fundamental pharmacologic principles, and results of recent animal studies are supportive of this. Knowledge gaps that need to be filled before the hypothesis should be widely accepted are discussed.

Ranitidine treatment during a modest inflammatory response precipitates idiosyncrasy-like liver injury in rats.

Drug idiosyncrasy is an adverse event of unknown etiology that occurs in a small fraction of people taking a drug. Some idiosyncratic drug reactions may occur from episodic decreases in the threshold for drug hepatotoxicity. Previous studies in rats have shown that modest underlying inflammation triggered by bacterial lipopolysaccharide (LPS) can decrease the threshold for xenobiotic hepatotoxicity. The histamine-2 (H2)-receptor antagonist ranitidine (RAN) causes idiosyncratic reactions in people, with liver as a usual target. We tested the hypothesis that RAN could be rendered hepatotoxic in animals undergoing a modest inflammatory response. Male rats were treated with a nonhepatotoxic dose of LPS (44 x 10(6) endotoxin units/kg i.v.) or its vehicle and then 2 h later with a nonhepatotoxic dose of RAN (30 mg/kg i.v.) or its vehicle. Liver injury was evident only in animals treated with both RAN and LPS as estimated by increases in serum alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyl transferase activities within 6 h after RAN administration. LPS/RAN cotreatment resulted in midzonal liver lesions characterized by acute necrosuppurative hepatitis. Famotidine (FAM) is an H2-antagonist for which the propensity for idiosyncratic reactions is far less than RAN. Rats given LPS and FAM at a dose pharmacologically equipotent to that of RAN did not develop liver injury. In vitro, RAN sensitized hepatocytes to killing by cytotoxic products from activated neutrophils, whereas FAM lacked this ability. The results indicate that a response resembling human RAN idiosyncrasy can be reproduced in animals by RAN exposure during modest inflammation.