Toxicity In Human Hepatocytes Research Articles

Integration of Metabolomics and Lipidomics Reveals Metabolic Mechanisms of Triclosan-Induced Toxicity in Human Hepatocytes.

Triclosan (TCS), an extensively used antimicrobial agent, has raised considerable concern due to its hepatocarcinogenic potential. However, previous hepatotoxicity studies primarily focused on the activation of specific intracellular receptors, the underlying mechanisms still warrant further investigation at the metabolic level. Herein, we applied metabolomics in combination with lipidomics to unveil TCS-related metabolic responses in human normal and cancerous hepatocytes. Endogenous and exogenous metabolites were analyzed for the identification of metabolic biomarkers and biotransformation products. In L02 normal cells, TCS exposure induced the up-regulation of purine metabolism and amino acid metabolism, caused lipid accumulation, and disturbed energy metabolism. These metabolic disorders in turn enhanced the overproduction of reactive oxygen species (ROS), leading to the alteration of antioxidant enzyme activities, down-regulation of endogenous antioxidants, and peroxidation of lipids. TCS-induced oxidative stress is thus considered to be one crucial factor for hepatotoxicity. However, in HepG2 cancer cells, TCS underwent fast detoxification through phase II metabolism, accompanied by the enhancement of energy metabolism and elevation of antioxidant defense system, which contributed to the potential effects of TCS on human hepatocellular carcinoma development. These different responses of metabolism between normal and cancerous hepatocytes provide novel and robust perspectives for revealing the mechanisms of TCS-triggered hepatotoxicity.

Synthesis of sulfamoylbenzamide derivatives as HBV capsid assembly effector

The synthesis of novel series of sulfamoylbenzamides as HBV capsid assembly effector is reported. The structure was divided into five parts which were independently modified as part of our lead optimization. All synthesized compounds were evaluated for their anti-HBV activity and toxicity in human hepatocytes, lymphocytes and other cells. Additionally, we assessed their effect on HBV cccDNA formation in an HBeAg reporter cell-based assay. Among the 27 compounds reported, several analogs exhibited submicromolar activities and significant reduction of HBeAg secretion. Selected compounds were studied under negative-stain electron microscopy for their ability to disrupt the HBV capsid formation. Structures were modeled into a binding site recently identified in the HBV capsid protein for similar molecules to rationalize the structure-activity relationships for this family of compounds.

CYP4F2 repression and a modified alpha-tocopherol (vitamin E) metabolism are two independent consequences of ethanol toxicity in human hepatocytes

The expression of CYP4F2, a form of cytochrome P-450 with proposed role in α-tocopherol and long-chain fatty acid metabolism, was explored in HepG2 and HepaRG human hepatocytes during ethanol toxicity.Cytotoxicity, ROS production, and JNK and ERK1/2 kinase signaling increased in a dose and time-dependent manner during ethanol treatments; CYP4F2 gene expression decreased, while other CYP4F forms, namely 4F11 and 12, increased along with 3A4 and 2E1 isoforms. α-Tocopherol antagonized the cytotoxicity and CYP4F2 gene repression effect of ethanol in HepG2 cells. Ethanol stimulated the tocopherol-ω-hydroxylase activity and the other steps of vitamin E metabolism, which points to a minor role of CYP4F2 in this metabolism of human hepatocytes. PPAR-γ and SREBP-1c followed the same expression pattern of CYP4F2 in response to ethanol and α-tocopherol treatments. Moreover, the pharmacological inhibition of PPAR-γ synergized with ethanol in decreasing CYP4F2 protein expression, which suggests a role of this nuclear receptor in CYP4F2 transcriptional regulation.In conclusion, ethanol toxicity modifies the CYP expression pattern of human hepatic cells impairing CYP4F2 transcription and protein expression. These changes were associated with a lowered expression of the fatty acid biosynthesis regulators PPAR-γ and SREBP-1c, and with an increased enzymatic catabolism of vitamin E. CYP4F2 gene repression and a sustained vitamin E metabolism appear to be independent effects of ethanol toxicity in human hepatocytes.

Cytokine-associated drug toxicity in human hepatocytes is associated with signaling network dysregulation.

Idiosyncratic drug hepatotoxicity is a major problem in pharmaceutical development due to poor prediction capability of standard preclinical toxicity assessments and limited knowledge of its underlying mechanisms. Findings in animal models have shown that adverse effects of numerous drugs with idiosyncratic hepatotoxicity in humans can be reproduced in the presence of coincident inflammatory cytokine signaling. Following these observations, we have recently developed an in vitro drug/inflammatory cytokine co-treatment approach that can reproduce clinical drug hepatotoxicity signatures-particularly for idiosyncratic drugs-in cultured primary human hepatocytes. These observations have suggested that drug-induced stresses may interact with cytokine signaling to induce hepatic cytotoxicity, but the hepatocyte signaling mechanisms governing these interactions are poorly understood. Here, we collect high-throughput phosphoprotein signaling and cytotoxicity measurements in cultured hepatocytes, from multiple human donors, treated with combinations of hepatotoxic drugs (e.g. trovafloxacin, clarithromycin) and cytokines (tumor necrosis factor-alpha, interferon-gamma, interleukin-1 alpha, and interleukin-6). We demonstrate, through orthogonal partial least-squares regression (OPLSR) modeling of these signal-response data, that drug/cytokine hepatic cytotoxicity is integratively controlled by four key signaling pathways: Akt, p70 S6 kinase, MEK-ERK, and p38-HSP27. This modeling predicted, and experimental studies confirmed, that the MEK-ERK and p38-HSP27 pathways contribute pro-death signaling influences in drug/cytokine hepatic cytotoxicity synergy. Further, our four-pathway OPLSR model produced successful prediction of drug/cytokine hepatic cytotoxicities across different human donors, even though signaling and cytotoxicity responses were both highly donor-specific. Our findings highlight the critical role of kinase signaling in drug/cytokine hepatic cytotoxicity synergies and reveal that hepatic cytotoxicity responses are governed by multi-pathway signaling network balance.

A multipathway phosphoproteomic signaling network model of idiosyncratic drug- and inflammatory cytokine-induced toxicity in human hepatocytes.

Idiosyncratic drug hepatotoxicity is a hepatotoxicity subset that occurs in a very small fraction of human patients, is poorly predicted by standard preclinical models and in clinical trials, and frequently leads to postapproval drug failure. Animal models utilizing bacterial LPS co-administration to induce an inflammatory background and hepatocyte cell culture models utilizing cytokine mix cotreatment have successfully reproduced idiosyncratic hepatotoxicity signatures for certain drugs, but the hepatocyte signaling mechanisms governing these drug-cytokine toxicity synergizes are largely unclear. Here, we summarize our efforts to computationally model the signaling mechanisms regulating inflammatory cytokine-associated idiosyncratic drug hepatotoxicity. We collected a "cue-signal-response" (CSR) data compendium in cultured primary human hepatocytes treated with many combinations of idiosyncratic hepatotoxic drugs and inflammatory cytokine mixes ("cues") and subjected this compendium to orthogonal partial-least squares regression (OPLSR) to computationally relate the measured intracellular phosphoprotein signals and hepatocellular death responses. This OPLSR model suggested that hepatocytes specify their cell death responses to toxic drug/cytokine conditions by integrating signals from four key pathways - Akt, p70 S6K, ERK, and p38. An OPLSR model focused on data from these four signaling pathways demonstrated accurate predictions of idiosyncratic drug- and cytokine-induced hepatotoxicities in a second human hepatocyte donor, suggesting that hepatocytes from different individuals have shared network control mechanisms governing toxicity responses to diverse combinations of idiosyncratic hepatotoxicants and inflammatory cytokines.

Does CYP2E1 play a major role in the aggravation of isoniazid toxicity by rifampicin in human hepatocytes?

Isoniazid and rifampicin are first-line anti-tubercular drugs. In a recent paper, Shen et al. provided interesting findings that rifampicin exacerbated isoniazid toxicity in human hepatocytes but not in rat hepatocytes. The main conclusion was that the difference in cytochrome P450 2E1 (CYP2E1) induction by rifampicin between rat and human hepatocytes accounted for the difference in exacerbation of isoniazid hepatotoxicity by rifampicin. 4-Nitrophenol hydroxylase (4-NP) activity was the only probe of CYP2E1 activity used in the paper. The authors presented data showing that rifampicin enhanced 4-NP activity and CYP2E1 mRNA expression in human hepatocytes, but not in rat hepatocytes. However, CYP3A also makes a significant contribution to 4-NP activity in humans and rats, which has been confirmed by both CYP3A-specific inducer and inhibitors. Rifampicin is a strong inducer of human CYP3A; thus, the increase in 4-NP activity in human hepatocytes could be due to the induction of CYP3A. Rifampicin did not increase 4-NP activities in rat hepatocytes, which could reflect a lack of the induction of rat CYP3A by rifampicin. Additionally, more experiments are needed to support the conclusion that rifampicin increased CYP2E1 mRNA expression in human hepatocytes because of the small sample size and the limitations of semi-quantitative RT-PCR. The study by Shen et al. suggests that another drug-metabolizing enzyme rather than CYP2E1 could be involved in the aggravation of isoniazid toxicity by rifampicin in human hepatocytes.

Toxicity caused by Chelidonium majus in primary human, canine, monkey, and rat hepatocytes

Herbal extracts of Chelidonium majus have been widely used in European countries and in Chinese herbal medicines for different applications, based on their anti‐viral, anti‐inflammatory and anti‐microbial properties and their use against various liver disorders including cancer. Recently, several cases of liver disease have been related to C. majus intake. Therefore, we have used primary hepatocyte cultures of human, rat, canine and monkey to elucidate the potential hepatotoxicity of C. majus.Water‐soluble extracts were prepared and incubated with primary hepatocytes. Cellular viability and functions were analyzed by means of lactate dehydrogenase (LDH) and urea release and via the MTT assay. The amounts of different alkaloids present in C. majus extracts were determined by HPLC analysis.The liquid extract showed a significant concentration‐dependent toxicity in human hepatocytes and at concentrations of 7.5 mg/ml for animal hepatocytes. In addition, C. majus lead to a decrease in urea release. The decline in urea production varied between the different species with regard to cellular sensitivity and to its maximal extent.The data suggest, that human hepatocytes seemed to be more sensitive to C. majus extracts than canine, rat and monkey hepatocytes, with rat hepatocytes being the most insensitive cells. In addition, these results demonstrate the necessity to use human hepatocytes in order to evaluate possible toxicological effects of complex plant extracts in humans.

Rifampicin exacerbates isoniazid‐induced toxicity in human but not in rat hepatocytes in tissue‐like cultures

Rifampicin has been extensively reported to exacerbate the hepatotoxicity of isoniazid in patients with tuberculosis. However, this was controversially claimed by previous reports using rat models. This study evaluated the effect of rifampicin on isoniazid-induced hepatocyte toxicity by using human and rat hepatocytes in tissue-like culture. Hepatocytes in tissue-like gel entrapment were used to examine isoniazid toxicity, as shown by cell viability, intracellular glutathione content and albumin secretion. For demonstration of the differential effects of rifampicin on human and rat hepatocytes, induction by rifampicin of cytochrome P450 (CYP) 2E1, a major enzyme associated with isoniazid hepatotoxicity, was detected by 4-nitrocatechol formation and RT-PCR analysis. Rifampicin (12 microM) enhanced isoniazid-induced toxicity in human hepatocytes but not in rat hepatocytes. Enhanced CYP 2E1 enzymic activity and mRNA expression were similarly detected in human hepatocytes but not in rat hepatocytes. Both rat and human hepatocytes in gel entrapment were more sensitive to isoniazid treatment compared with the corresponding hepatocytes in a monolayer culture. The difference in induction of CYP 2E1 by rifampicin between rat and human hepatocytes accounted for the difference in exacerbation of isoniazid hepatocyte toxicity by rifampicin, with more significant toxicity in gel entrapment than in monolayer cultures. Thus, human hepatocytes in tissue-like cultures (gel entrapment) could be an effective model for hepatotoxicity research in vitro, closer to the in vivo situation.

Kinetics of Acetaminophen Glucuronidation by UDP-Glucuronosyltransferases 1A1, 1A6, 1A9 and 2B15. Potential Implications in Acetaminophen−Induced Hepatotoxicity

The importance of uridine 5'-diphosphate-glucuronosyltranferases (UGT) 2B15 and other UGT enzymes (1A1, 1A6, and 1A9) in glucuronidating acetaminophen (APAP) is demonstrated. The kinetics and contributions of various UGTs in glucuronidating APAP are presented using clinically and toxicologically relevant concentrations of the substrate. UGT 1A9 and UGT 2B15 contribute significantly toward glucuronidating APAP when incubations were conducted in either phosphate or Tris-HCl buffers at 0.1 and 1.0 mM substrate concentrations. At 10 mM APAP, UGT 1A9 is a significant enzyme responsible for metabolizing APAP in either one of the buffers. UGT 1A1 is the next most important enzyme in glucuronidating APAP at this high substrate concentration. The contribution of UGT 1A6 at 10 mM APAP concentration became obscured by similar relative activities exhibited by UGTs 1A7, 1A8, and 2B7. These observations may reflect the differences in kinetic parameters for APAP glucuronidation by the individual UGTs. UGT 1A1 demonstrated Hill kinetics while UGT 1A9 displayed Michaelis-Menten kinetics. Substrate inhibition kinetics is observed with UGT 1A6, UGT 2B15, and human liver microsomes. The substrate inhibition is confirmed by employing stable isotope-labeled APAP as the substrate, while APAP glucuronide is used to test for inhibition of d4-APAP glucuronide. The in vitro hepatotoxicity caused by APAP in combination with phenobarbital or phenytoin is demonstrated in this study. The inhibition of APAP glucuronidation by phenobarbital leads to an increase in APAP-mediated toxicity in human hepatocytes. The toxicity to hepatocytes was further increased by coadministering APAP with phenytoin and phenobarbital. This synergistic increase in toxicity is postulated to be due to inhibition of UGTs (1A6, 1A9, and 2B15) responsible for detoxifying APAP through the glucuronidation pathway.

Inhibition of Hepatobiliary Transport as a Predictive Method for Clinical Hepatotoxicity of Nefazodone

Treatment with the antidepressant nefazodone has been associated with clinical idiosyncratic hepatotoxicty. Using membranes expressing human bile salt export pump (BSEP), human sandwich hepatocytes, and intact rats, we compared nefazodone and its marketed analogs, buspirone and trazodone. We found that nefazodone caused a strong inhibition of BSEP (IC(50) = 9 microM), inhibition of taurocholate efflux in human hepatocytes (IC(50) = 14 microM), and a transient increase in rat serum bile acids 1 h after oral drug administration. Buspirone or trazodone had no effect on biliary transport system. Nefazodone produced time- and concentration-dependent toxicity in human hepatocytes with IC(50) = 18 microM and 30 microM measured by inhibition of protein synthesis after 6 h and 24 h incubation, respectively. Toxicity was correlated with the amount of unmetabolized nefazodone. Partial recovery in toxicity by 24 h has been associated with metabolism of nefazodone to sulfate and glucuronide conjugates. The saturation of nefazodone metabolism resulted in sustained decrease in protein synthesis and cell death at 50 microM. The toxicity was not observed with buspirone or trazodone. Addition of 1-aminobenzotriazole (ABT), an inhibitor of CYP450, resulted in enhancement of nefazodone toxicity at 10 microM and was associated with accumulation of unmetabolized nefazodone. In human liver microsomes, ABT also prevented metabolism of nefazodone and formation of glutathione conjugates. We suggest that inhibition of bile acid transport by nefazodone is an indicator of potential hepatotoxicity. Our findings are consistent with the clinical experience and suggest that described methodology can be applied in the selection of nonhepatotoxic drug candidates.

Human hepatocytes are protected from ethanol-induced cytotoxicity by DADS via CYP2E1 inhibition

We investigated the protective effects of diallyl disulfide (DADS), a potent inhibitor of cytochrome P450 2E1 (CYP2E1), on ethanol-induced toxicity in human hepatocytes. We found a clear dose-dependent response between ethanol and CYP2E1 activity. The ethanol-dependent CYP2E1 enzyme activity and protein expression, lactate dehydrogenase and aspartate transaminase release, malondialdehyde formation and caspase-3 activity decreased dramatically in the presence of DADS. Furthermore, DADS increased the hepatocellular glutathione (GSH) content and prevented the ethanol-dependent cellular GSH depletion. Our data show that DADS reduces ethanol-induced toxicity in human hepatocytes by reducing CYP2E1 activity and/or stabilizing the cellular GSH content, which might be of therapeutic interest.