The conjugation of reactive drug metabolites to GSH is considered an important detoxification mechanism that can be spontaneous and/or mediated by glutathione S-transferases (GSTs). In case GSTs play an important role in GSH conjugation, genetically determined deficiencies in GSTs may be a risk factor for adverse drug reactions (ADRs) resulting from reactive drug metabolites. So far, the role of GSTs in the detoxification of reactive intermediates of clozapine, a drug-causing idiosyncratic drug reactions (IDRs), has not been studied. In the present study, we studied the ability of four recombinant human GSTs (hGST A1-1, hGST M1-1, hGST P1-1, and hGST T1-1) to catalyze the GSH conjugation of reactive metabolites of clozapine, formed in vitro by human and rat liver microsomes and drug-metabolizing P450 BM3 mutant, P450 102A1M11H. Consistent with previous studies, in the absence of GSTs, three GSH conjugates were identified derived from the nitrenium ion of clozapine. In the presence of three of the GSTs, hGST P1-1, hGST M1-1, and hGST A1-1, total GSH conjugation was strongly increased in all bioactivation systems tested. The highest activity was observed with hGST P1-1, whereas hGST M1-1 and hGST A1-1 showed slightly lower activity. Polymorphic hGST T1-1 did not show any activity in catalyzing GSH conjugation of reactive clozapine metabolites. Interestingly, the addition of hGSTs resulted in major changes in the regioselectivity of GSH conjugation of the reactive clozapine metabolite, possibly due to the different active site geometries of hGSTs. Two GSH conjugates found were completely dependent on the presence of hGSTs. Chlorine substitution of the clozapine nitrenium ion, which so far was only observed in in vivo studies, appeared to be the major pathway of hGST P1-1-catalyzed GSH conjugation, whereas hGST A1-1 and hGST M1-1 also showed significant activity. The second GSH conjugate, previously also only found in in vivo studies, was also formed by hGST P1-1 and to a small extent by hGST A1-1. These results demonstrate that human GSTs may play a significant role in the inactivation of reactive intermediates of clozapine. Therefore, further studies are required to investigate whether genetic polymorphisms of hGST P1-1 and hGST M1-1 contribute to the interindividual differences in susceptibility to clozapine-induced adverse drug reactions.

Drug-induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by diclofenac and the proinflammatory cytokine tumor necrosis factor a (TNF-a). HepG2 cells were treated with diclofenac followed by TNF-a challenge and subsequent evaluation of necrosis and apoptosis. Diclofenac caused a mild apoptosis of HepG2 cells, which was strongly potentiated by TNF-a. A focused apoptosis machinery short interference RNA (siRNA) library screen identified that this TNF-a-mediated enhancement involved activation of caspase-3 through a caspase-8/Bid/APAF1 pathway. Diclofenac itself induced sustained activation of c-Jun N-terminal kinase (JNK) and inhibition of JNK decreased both diclofenac and diclofenac/TNF-a-induced apoptosis. Live cell imaging of GFPp65/RelA showed that diclofenac dampened the TNF-a-mediated nuclear factor kappaB (NF-jB) translocation oscillation in association with reduced NF-jB transcriptional activity. This was associated with inhibition by diclofenac of the TNF-ainduced phosphorylation of the inhibitor of NF-jB alpha (IjBa). Finally, inhibition of IjB kinase b (IKKb) with BMS-345541 as well as stable lentiviral short hairpin RNA (shRNA)-based knockdown of p65/RelA sensitized hepatocytes towards diclofenac/TNFa-induced cytotoxicity. Conclusion: Together, our data suggest a model whereby diclofenac-mediated stress signaling suppresses TNF-a-induced survival signaling routes and sensitizes cells to apoptosis. (HEPATOLOGY 2011;53:2027-2041 A dverse drug reactions are an important cause of morbidity and mortality in humans and druginduced liver injuries (DILIs) are the leading cause of acute liver failure. 1 In addition, DILI accounts for most of the drug attritions 2 and more than 10% of the occurring liver failures happen due to idiosyncratic DILIs. 1 We propose that the crosstalk between drug reactive metabolite-mediated stress responses and cytokine-mediated pro-and antiapoptotic signaling is an important component in the pathophysiology of DILI.Abbreviations: AIF, apoptosis inducing factor; AnxV, annexin V; APAF1, apoptotic protease activating factor 1; c-FLIP, cellular FLICE-like inhibitory protein; DILI, drug-induced liver injury; GFP, green fluorescent protein; IjBa, inhibitor of NF-jB a; IKKb, IjB kinase b; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; MAPK, mitogen activated protein kinase; NF-jB, nuclear factor-jB; NSAID, nonsteroidal antiinflammatory drug; PUMA, p53 up-regulated modulator of apoptosis; ROS, reactive oxygen species; RAIDD, RIP-associated protein with a death domain, siRNA, short interfering RNA; TNFR-1, TNF receptor-1; TNF-a, tumor necrosis factor a.From the

N-acetyl-meta-aminophenol (AMAP) is generally considered as a non-toxic regioisomer of the well-known hepatotoxicant acetaminophen (APAP). However, so far, AMAP has only been shown to be non-toxic in mice and hamsters. To investigate whether AMAP could also be used as non-toxic analog of APAP in rat and human, the toxicity of APAP and AMAP was tested ex vivo in precision-cut liver slices (PCLS) of mouse, rat and human. Based on ATP content and histomorphology, APAP was more toxic in mouse than in rat and human PCLS. Surprisingly, although AMAP showed a much lower toxicity than APAP in mouse PCLS, AMAP was equally toxic as or even more toxic than APAP at all concentrations tested in both rat and human PCLS. The profile of proteins released into the medium of AMAP-treated rat PCLS was similar to that of APAP, whereas in the medium of mouse PCLS, it was similar to the control. Metabolite profiling indicated that mouse PCLS produced the highest amount of glutathione conjugate of APAP, while no glutathione conjugate of AMAP was detected in all three species. Mouse also produced ten times more hydroquinone metabolites of AMAP, the assumed proximate reactive metabolites, than rat or human. In conclusion, AMAP is toxic in rat and human liver and cannot be used as non-toxic isomer of APAP. The marked species differences in APAP and AMAP toxicity and metabolism underline the importance of using human tissues for better prediction of toxicity in man.

Clozapine is known to cause hepatotoxicity in a small percentage of patients. Oxidative bioactivation to reactive intermediates by hepatic cytochrome P450s (P450s) has be proposed as a possible mechanism. However, in contrast to their role in formation of N-desmethylclozapine and clozapine N-oxide, the involvement of individual P450s in the bioactivation to reactive intermediates is much less well characterized. The results of the present study show that 7 of 14 recombinant human P450s were able to bioactivate clozapine to a glutathione-reactive nitrenium ion. CYP3A4 and CYP2D6 showed the highest specific activity. Enzyme kinetical characterization of these P450s showed comparable intrinsic clearance of bioactivation, implicating that CYP3A4 would be more important because of its higher hepatic expression, compared with CYP2D6. Inhibition experiments using pooled human liver microsomes confirmed the major role of CYP3A4 in hepatic bioactivation of clozapine. By studying bioactivation of clozapine in human liver microsomes from 100 different individuals, an 8-fold variability in bioactivation activity was observed. In two individuals bioactivation activity exceeded N-demethylation and Noxidation activity. Quinidine did not show significant inhibition of bioactivation in any of these liver fractions, suggesting that CYP2D6 polymorphism is not an important factor in determining susceptibility to hepatotoxicity of clozapine. Therefore, interindividual differences and drug-drug interactions at the level of CYP3A4 might be factors determining exposure of hepatic tissue to reactive clozapine metabolites.

Pistacia lentiscus L. is a Mediterranean shrub known for its health promoting effects attributed to a large extent to polyphenols accumulated in all parts of the plant. Microwave-assisted extraction is a green extraction technique enabling fast and effective isolation of plant polyphenols. Therefore, the aim of this research was to optimize the microwave-assisted extraction of polyphenols from Pistacia lentiscus L. leaves and fruit in terms of temperature, extraction time and microwave power and to evaluate their polyphenolic profile by UPLC/ESI-MS2 and antioxidant capacity by ORAC assay. Optimal extraction conditions for leaf polyphenols were 69 °C, 512 W and 12 min, while for fruit were slightly more intensive—75 °C, 602 W and 15 min. Obtained total phenolic content in leaves and fruit was similar to that obtained after 30 min of the heat-reflux method. The polyphenolic profile of extracts included 34 compounds, with myricetin glycosides being the most abundant compounds among flavonoids in Pistacia lentiscus L. leaves and fruit and gallic acid and its derivates among the phenolic acids. ORAC assay showed higher antioxidant capacity for Pistacia lentiscus L. leaves extract than for fruit, which is in correlation with their respective phenolic content.