Natural killer cells mediate severe liver injury in a murine model of halothane hepatitis.

Severe halothane (HAL)-induced hepatotoxicity occurs in one in 6000-30,000 patients by an unknown mechanism. Female sex is a risk factor in humans and rodents. We tested the hypothesis that a sex difference in natural killer (NK) cell activity contributes to HAL-induced liver injury. HAL (15 mmol/kg, ip) treatment resulted in severe liver injury by 12 h in female, wild-type BALB/cJ mice, and the magnitude of liver injury varied with stage of the estrous cycle. Ovariectomized (OVX) mice developed only mild liver injury. Plasma interferon-gamma (IFN-γ) was elevated 10-fold in HAL-treated females compared with similarly treated male mice or with OVX female mice. IFN-γ knockout mice were resistant to severe HAL-induced liver injury. The deactivation of NK cells with anti-asialo GM1 treatment attenuated liver injury and the increase in plasma IFN-γ compared with immunoglobulin G-treated control mice. Mice with a mutated form of perforin, a protein involved in granule-mediated cytotoxicity, were protected from severe liver injury. Furthermore, HAL increased the activity of NK cells in vivo, as indicated by increased surface expression of CD69, an early activation marker. In response to HAL, NK cell receptor ligands on the surface of hepatocytes were expressed in a manner that can activate NK cells. These results confirm the sexual dimorphic hepatotoxic response to HAL in mice and suggest that IFN-γ and NK cells have essential roles in the development of severe HAL-induced hepatotoxicity.

A mouse model of severe halothane hepatitis based on human risk factors.

Halothane (2-bromo-2-chloro-1,1,1-trifluoro-ethane) is an inhaled anesthetic that induces severe, idiosyncratic liver injury, i.e., "halothane hepatitis," in approximately 1 in 20,000 human patients. We used known human risk factors (female sex, adult age, and genetics) as well as probable risk factors (fasting and inflammatory stress) to develop a murine model with characteristics of human halothane hepatitis. Female and male BALB/cJ mice treated with halothane developed dose-dependent liver injury within 24 h; however, the liver injury was severe only in females. Livers had extensive centrilobular necrosis, inflammatory cell infiltrate, and steatosis. Fasting rendered mice more sensitive to halothane hepatotoxicity, and 8-week-old female mice were more sensitive than males of the same age or than younger (4-week-old) females. C57BL/6 mice were insensitive to halothane, suggesting a strong genetic predisposition. In halothane-treated females, plasma concentration of tumor necrosis factor-alpha was greater than in males, and neutrophils were recruited to liver more rapidly and to a greater extent. Anti-CD18 serum attenuated halothane-induced liver injury in female mice, suggesting that neutrophil migration, activation, or both are required for injury. Coexposure of halothane-treated male mice to lipopolysaccharide to induce modest inflammatory stress converted their mild hepatotoxic response to a pronounced, female-like response. This is the first animal model of an idiosyncratic adverse drug reaction that is based on human risk factors and produces reproducible, severe hepatitis from halothane exposure with lesions characteristic of human halothane hepatitis. Moreover, these results suggest that a more robust innate immune response underlies the predisposition of female mice to halothane hepatitis.

Trovafloxacin enhances TNF-induced inflammatory stress and cell death signaling and reduces TNF clearance in a murine model of idiosyncratic hepatotoxicity.

Therapy employing the fluoroquinolone antibiotic, trovafloxacin (TVX) was curtailed due to idiosyncratic hepatotoxicity. Previous studies in mice showed that a nonhepatotoxic inflammatory stress induced by tumor necrosis factor alpha (TNF) synergized with a nonhepatotoxic dose of TVX to cause liver injury. The purpose of this study was to explore mechanisms by which TVX interacts with TNF to cause liver injury. TVX pretreatment prolonged the peak of plasma TNF after its administration. This prolongation of TNF by TVX was critical to the development of hepatotoxicity. The prolongation of TNF concentration in plasma was primarily due to reduced clearance when compared with secondary biosynthesis. TNF is cleared from plasma by binding to soluble TNF receptors (TNFRs) which are eliminated by the kidney; however, the plasma concentrations of soluble TNFRs were not reduced, and biomarkers of renal dysfunction were not elevated in TVX/TNF-treated mice. Two injections of TNF mimicked the prolongation of the TNF peak by TVX and caused liver injury, but injury was less severe than after TVX/TNF coexposure. TVX enhanced the induction of proinflammatory cytokines by TNF. Additionally, TVX sensitized Hepa1c1c7 cells to TNF-induced killing in a concentration-dependent manner and increased both potency and efficacy of TNF to activate effector caspases that were critically involved in cell death from TVX/TNF coexposure. In summary, TVX reduced the clearance of TNF independent of either receptor shedding or kidney dysfunction. Additionally, TVX interacted with TNF to enhance inflammation and sensitize hepatocytes to TNF-induced cell death.

p38 mitogen-activated protein kinase stimulates estrogen-mediated transcription and proliferation through the phosphorylation and potentiation of the p160 coactivator glucocorticoid receptor-interacting protein 1.

Nuclear hormone receptors, such as the estrogen receptors (ERs), are regulated by specific kinase signaling pathways. Here, we demonstrate that the p38 MAPK stimulates both ERalpha- and ERbeta-mediated transcription in MCF-7 breast carcinoma, Ishikawa endometrial adenocarcinoma, and human embryonic kidney 293 cells. Inhibition of this potentiation using the p38 inhibitor, RWJ67657, blocked estrogen-mediated transcription and proliferation. Activated ERs promote gene expression in part through the recruitment of the p160 class of coactivators. Because no direct p38 phosphorylation sites have been determined on either ERalpha or beta, we hypothesized that p38 could target the p160 class of coactivators. We show for the first time using pharmacological and molecular techniques that the p160 coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) is phosphorylated and potentiated by the p38 MAPK signaling cascade in vitro and in vivo. S736 was identified as a necessary site for p38 induction of GRIP1 transcriptional activation. The C terminus of GRIP1 was also demonstrated to contain a p38-responsive region. Taken together, these results indicate that p38 stimulates ER-mediated transcription by targeting the GRIP1 coactivator.

Sensitization of apoptotically-resistant breast carcinoma cells to TNF and TRAIL by inhibition of p38 mitogen-activated protein kinase signaling.

The mitogen-activated protein kinase (MAPK) cascade is a critical component in the regulation of cell survival and proliferation decisions. In breast carcinoma cells, activation of the p38-MAPK member of this family occurs in response to pro-inflammatory cytokines and cellular stress. The involvement of p38-MAPK in the activation of the transcription factor, NF-kappaB, suggests a potential role and mechanism for regulation of cell survival and drug resistance. Generation of the resistant MCF-7 variant (MCF-7TN-R) was achieved by prolonged exposure of MCF-7N cells to increasing concentrations of TNF. Differences in MAPK activation and function in the MCF-7 cell variants were determined. The role of the p38-MAPK pathway in regulation of resistance was determined using pharmacological (SB 203580) or molecular [Dominant Inhibitory (DI)-p38] inhibition. The effect of p38 inhibition on NF-kappaB transcriptional activation was analyzed. As compared to the sensitive MCF-7N parent cell line, the MCF-7TN-R cell line displayed significant resistance to TNF- and TRAIL-induced cell death. Analysis of the expression and phosphorylation of members of the MAPK family revealed an increased basal activation of p38 in the MCF-7TN-R variant. The p38-mediated phosphorylation and transcriptional activity were suppressed by pharmacologic inhibition with SB 230580. Treatment of MCF-7TN-R cells with SB partially restored sensitivity to TNF-induced cell death. In addition, use of a DI-p38 construct with or without the addition to TNF induced cell death, thus restoring TNF-sensitivity to these cells. The ability of p38 inhibition to restore apoptotic sensitivity was correlated with suppression of the TNF-induced cell survival pathway, NF-kappaB. The increased activation of p38-MAPK in MCF-7TN-R cells demonstrates that this signaling pathway through activation of NF-kappaB is an important route for control of resistance to cell death in breast carcinoma. Molecular and pharmacological inhibition of p38-MAPK signaling may represent a mechanism for sensitizing cancer cells to chemotherapeutic regimens and restoration of apoptotic signaling.