Robert A. Roth

Neutrophil migration during endotoxemia

Endotoxemia is marked by a global activation of inflammatory responses, which can lead to shock, multiple organ failure, and the suppression of immune and wound healing processes. Neutrophils (PMNs) play a central role in some of these responses by accumulating in tissues and releasing reactive oxygen species and proteases that injure host structures. This review focuses on altered PMN migratory responses that occur during endotoxemia and their consequences in the development of pulmonary infection. The inflammatory mediators that might be responsible for these altered responses are discussed. The oxidant potential of PMNs is increased after exposure to endotoxin both in vitro and during clinical and experimental endotoxemia. However, other functions such as chemotaxis and phagocytosis are often depressed in these same cells. Endotoxin exposure renders PMNs hyperadhesive to endothelium. The sum of these effects produces activated inflammatory cells that are incapable of leaving the vasculature. As such, the endotoxic PMN is more likely to promote tissue injury from within microvascular beds than to clear pathogens from extravascular sites. Moreover, the functional characteristics of endotoxic PMNs are similar to those observed during trauma, burn injury, sepsis, surgery, and other inflammatory conditions. Accordingly, several clinical conditions might have a common effector in the activated, yet migratorially dysfunctional, PMN. Direct effects of endotoxin on PMNs as well as effects of endogenous mediators released during endotoxemia are discussed. Understanding PMN behavior during endotoxemia may provide basic and critical insights that can be applied to a number of inflammatory scenarios. J. Leukoc. Biol. 66: 10–24; 1999.

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.

Novel mechanisms in chemically induced hepatotoxicity.

This review focuses on cellular events that modulate hepatotoxicity subsequent to initial liver insult. Cellular events that determine the nature and extent of hepatotoxic injury and the ultimate outcome of that injury are also discussed. The roles of cell types other than hepatocytes, hepatocyte organelle‐spccific processes, and regeneration in progression or recovery from liver injury are emphasized. Leukocyte activities are key events in two distinct hepatotoxicities. Neutrophil‐mediated, periportal inflammation appears to play a primary role in progression of α‐naphthylisothiocyanate‐induced cholangiolitic hepatitis. However, a humorally mediated autoimmune response to protein adducts that occurs after anesthesia is critical in onset of halothane‐induced hepatitis. New insights into specific events at the hepatocyte level are also emerging. Although reducing gap junctional communication between hepatocytes can protect against progression of liver injury, down‐regulation of the subunit proteins (connexins) can isolate neoplastic cells from growth regulation. Acidic intracellular pH characteristic of hypoxia is protective against both hypoxic and toxicant‐induced cell injury In oxidative injury, a pH‐mediated mitochondrial permeability transition causes mitochondrial uncoupling and ATP loss and leads to cell death. The ultimate outcome of hepatotoxic injury depends on the extent of tissue repair. Stimulation of tissue repair after a sublethal dose of CCl4 appears to be the central mechanism in protection against death from a subsequent large dose. Taken together, these examples illustrate the importance of events subsequent to initial liver injury as determinants of extent of liver damage.—Mehendale, H. M., Roth, R. A., Gandolfi, A. J., Klaunig, J. E., Lemasters, J. J., Curtis, L. R. Novel mechanisms in chemically induced hepatotoxicity. FASEB J. 8, 1285‐1295 (1994)

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.

Inflammatory Stress and Idiosyncratic Hepatotoxicity: Hints from Animal Models

Adverse drug reactions (ADRs) present a serious human health problem. They are major contributors to hospitalization and mortality throughout the world (Lazarou et al., 1998; Pirmohamed et al., 2004). A small fraction (less than 5%) of ADRs can be classified as “idiosyncratic.” Idiosyncratic ADRs (IADRs) are caused by drugs with diverse pharmacological effects and occur at various times during drug therapy. Although IADRs affect a number of organs, liver toxicity occurs frequently and is the primary focus of this review. Because of the inconsistency of clinical data and the lack of experimental animal models, how IADRs arise is largely undefined. Generation of toxic drug metabolites and induction of specific immunity are frequently cited as causes of IADRs, but definitive evidence supporting either mechanism is lacking for most drugs. Among the more recent hypotheses for causation of IADRs is that inflammatory stress induced by exogenous or endogenous inflammagens is a susceptibility factor. In this review, we give a brief overview of idiosyncratic hepatotoxicity and the inflammatory response induced by bacterial lipopolysaccharide. We discuss the inflammatory stress hypothesis and use as examples two drugs that have caused IADRs in human patients: ranitidine and diclofenac. The review focuses on experimental animal models that support the inflammatory stress hypothesis and on the mechanisms of hepatotoxic response in these models. The need for design of epidemiological studies and the potential for implementation of inflammation interaction studies in preclinical toxicity screening are also discussed briefly.

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

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. (HEPATOLOGY 2007.)

Concurrent inflammation as a determinant of susceptibility to toxicity from xenobiotic agents

Sensitivity to the toxic effects of xenobiotic agents is influenced by a number of factors. Recent evidence derived from studies using experimental animals suggests that inflammation is one of these factors. For example, induction of inflammation by coexposure to bacterial endotoxin, vitamin A or Corynebacterium parvum increases injury in response to a number of xenobiotic agents that target liver. These agents are diverse in chemical nature and in mechanism of hepatotoxic action. Factors critical to the augmentation of liver injury by inflammation include Kupffer cells, neutrophils, cytokines such as tumor necrosis factor-alpha (TNF-alpha) and lipid mediators such as prostaglandins, but these may vary depending on the xenobiotic agent and the mechanisms by which it alters hepatocellular homeostasis. In addition, the timing of inflammagen exposure can qualitatively alter the toxic response to chemicals. Inflammation-induced increases in susceptibility to toxicity are not limited to liver. Concurrent inflammation also sensitizes animals to the toxic effects of agents that damage the respiratory tract, kidney and lymphoid tissue. It is concluded that inflammation should be considered as a determinant of susceptibility to intoxication by xenobiotic exposure.

Transurethral ultrasound-guided laser-induced prostatectomy (tulip procedure) : a canine prostate feasibility study

We describe the TULIP procedure, a new system to relieve bladder outlet obstruction caused by benign prostatic hyperplasia. This device is composed of a real-time 7.5 MHz ultrasound transducer coupled to a Nd:YAG laser with a 1.064 microns wavelength that fires through an intact intraprostatic balloon. A series of feasibility studies in 21 canine prostate glands was performed with a follow-up time to 3 months. Results indicate that the Nd:YAG laser in the 20 to 40 W range at a pull rate of approximately 1 mm. per second is an effective means of removing substantial amounts of canine benign prostatic hyperplasia. Transurethral ultrasonography was a reliable means of identifying essential landmarks and of controlling the laser. Prostatectomy by laser coagulation necrosis resulted in no bleeding or postoperative obstruction. Intraoperative irrigation fluids were not required, eliminating systemic volume related problems.

Intrinsic versus Idiosyncratic Drug-Induced Hepatotoxicity—Two Villains or One?

“Intrinsic” and “idiosyncratic” drug-induced liver injury reactions are commonly thought to arise by different modes of action. Intrinsic toxicity is reproducible in animals and occurs dose-dependently at sublethal doses. Environmental and genetic sensitivity factors can influence the toxicity of intrinsic hepatotoxicants. Among these is inflammatory stress. For example, exposure of mice to inflammatory bacterial lipopolysaccharide (LPS) causes a leftward shift in the dose-response relationship for acetaminophen hepatotoxicity; that is, acetaminophen toxicity is enhanced by LPS-induced inflammatory stress. Idiosyncratic reactions present themselves very differently than intrinsic ones; they happen in a minority of patients, with variable time of onset and no obvious relationship to drug dose, and they are not reproducible in usual animal tests. Although these characteristics seem to distinguish them from intrinsic reactions, consideration of fundamental principles of dose response can explain the differences. For a drug that causes idiosyncratic hepatotoxicity, the liver may not be a typical target for toxicity because the dose-response curve for hepatotoxicity lies to the right of the lethal dose. However, a sporadically occurring sensitivity factor, such as an inflammatory episode, could shift the dose-response curve for hepatotoxicity to the left, thereby bringing hepatotoxic doses into the therapeutic range. This hypothesis can account for the bizarre characteristics of idiosyncratic reactions and is supported by recent results showing that several drugs associated with human idiosyncratic reactions can be rendered hepatotoxic to rodents upon interaction with an inflammatory stimulus. In light of this view, intrinsic and idiosyncratic reactions may not be that different after all.

Identification of factors from rat neutrophils responsible for cytotoxicity to isolated hepatocytes

Activated polymorphonuclear neutrophils (PMNs) have been shown to be cytotoxic to rat hepatic parenchymal cells in vitro. This cytotoxicity could be observed without direct cell‐cell contact, since the conditioned medium from PMNs activated with formyl‐Met‐Leu‐Phe (fMLP) was effective in hepatocyte killing. To identify the toxic factor(s) released by PMNs, degranulation was induced by fMLP in PMNs pretreated with cytochalasin B. The contents released from the phagocytes were subjected to gel filtration on a Sephadex G‐100 column. Resulting fractions were tested for cytotoxicity to isolated hepatocytes by using release of alanine aminotransferase as a marker for hepatocyte injury. Cytotoxicity was associated with fractions containing cathepsin G and elastase and not with other fractions, including those containing myeloperoxidase. The former two enzymes were purified to homogeneity with a carboxymethyl cellulose column. Each of these enzymes demonstrated concentration‐dependent cytotoxicity to hepatocytes at concentrations >2 μg/mL. Moreover, they exhibited an additive cytotoxic effect. Effective concentrations for the combined cathepsin C and elastase in the incubation mixture were similar to the concentrations of these enzymes in PMN‐conditioned medium that produced cytotoxicity to hepatocytes. Cytotoxicity of either purified enzyme or of conditioned medium could be prevented by plasma α‐1‐antitrypsin or soybean trypsin‐chymotrypsin inhibitor, which were also potent inhibitors of enzymic activity of both cathepsin G and elastase. By contrast, the serine protease inhibitors, aprotinin and 4‐(2‐aminoethyl)‐benzenesulfonyl fluoride, were less effective in inhibiting cathepsin G and elastase activities as well as cytotoxicity caused by the purified proteases or PMN‐conditioned medium. These results support the hypothesis that cathepsin G and elastase are important mediators of hepatic parenchymal cell killing produced by activated PMNs in vitro.