An in vitro drug-induced hepatotoxicity screening system using CYP3A4-expressing and γ-glutamylcysteine synthetase knockdown cells

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ABSTRACT:The double null mutation of glutathione transferase, GSTM1 and GSTT1, is reported to influence troglitazone-associated abnormal increases of alanine aminotransferase and aspartate aminotransferase. However, no nonclinical data with a bearing on the clinical outcomes and underlying mechanisms have hitherto been reported. To investigate whether deficiency in GSTM1 and/or GSTT1 is related to troglitazone hepatotoxicity in vitro, the covalent binding level (CBL) (an index of reactive metabolite formation) and cytotoxicity of troglitazone and rosiglitazone, another thiazolidinedione but with low hepatotoxicity, were examined using human liver samples phenotyped for cytochrome P450s and genotyped for GSTM1 and GSTT1. Despite addition of GSH, CBLs of troglitazone and rosiglitazone in human liver microsomes were correlated with CYP3A (or CYP2C8) and CYP2C8 activities, respectively. With addition of recombinant GSTM1, the microsomal CBLs of troglitazone and rosiglitazone decreased. However, the CBLs of troglitazone in GSTM1/GSTT1 wild-type hepatocytes were unexpectedly higher than those in null hepatocytes. Although this discrepancy has not been fully explained, the GSTM1 and GSTT1 null mutations increased the cytotoxicity of troglitazone, independent of CYP3A or CYP2C8 activities. Furthermore, a GSH adduct of troglitazone, M2, limited to GSTM1 wild-type hepatocytes was detected. Of clear interest, GSTM1 and/or GSTT1 null mutation-dependent cytotoxicity and higher exposure to the reactive metabolite trapped as M2 as for troglitazone were not observed for rosiglitazone. This result might at least partly explain the findings related to clinical hepatotoxicity, suggesting that measurement of GSH adducts or cytotoxicity using GSTM1-and GSTT1-genotyped hepatocytes might offer an important in vitro system to assist in better prediction of idiosyncratic hepatotoxicity.

Section: Gstm1/gstt1 Defects and Troglitazone-induced Liver Injurysupporting

confidence: 73%

In Vitro Investigation of the Glutathione Transferase M1 and T1 Null Genotypes as Risk Factors for Troglitazone-Induced Liver Injury

Usui

Hashizume²,

Katsumata³

et al. 2011

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“…Recombinant adenoviruses expressing CYP3A4 (AdCYP3A4), CYP2C9 (AdCYP2C9), and green fluorescent protein (AdGFP) were constructed as in our previous studies (Hosomi et al, 2010;Iwamura et al, 2011). Recombinant adenoviruses expressing PGRMC1 (AdPGRMC1) and CYP2E1 (AdCYP2E1) were created as follows.…”

Section: Methodsmentioning

confidence: 99%

Progesterone Receptor Membrane Component 1 Modulates Human Cytochrome P450 Activities in an Isoform-Dependent Manner

Oda

Nakajima²,

Toyoda³

et al. 2011

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ABSTRACT:Cytochromes P450 (P450s) catalyze the metabolism of a wide spectrum of compounds. Recently, progesterone receptor membrane component 1 (PGRMC1), which shares a key structural motif with cytochrome b 5 , has been reported to bind to sterol-or steroidsynthesizing P450s, enhancing their activities. In this study, we investigated whether PGRMC1 affects human drug-metabolizing P450 activities. Using coexpression systems for PGRMC1 and P450s (CYP3A4, CYP2C9, or CYP2E1) in HepG2 cells, we found that PGRMC1 decreased the V max values and increased the K m values of the CYP3A4 activities, and it decreased the V max values but did not affect the K m values of the CYP2C9 activities. In contrast, PGRMC1 hardly affected the CYP2E1 activities. These results suggest that PGRMC1 negatively modulates the drug-metabolizing activities of P450, although it was isoform but not substrate dependent. It is worth noting that coimmunoprecipitation analysis using coexpression systems for FLAG-PGRMC1 and Myc-P450s in human embryonic kidney 293 cells revealed that PGRMC1 interacts with all three P450s, although the affinity seemed to vary. In 29 human liver microsomes (HLMs), there was a 5-fold variability in the PGRMC1 protein levels. By the correlation analyses using the P450 activities and the PGRMC1 levels, we could neither observe the contribution of PGRMC1 to the P450 activities in HLMs nor that of the NADPH-cytochrome P450 reductase or cytochrome b 5 . In conclusion, in contrast to sterol-or steroid-synthesizing P450s, we found that PGRMC1 negatively modulates the human drug-metabolizing activities of P450 through direct interaction. Further studies are needed to determine the clinical significance of PGRMC1 in the pharmacokinetics of drugs.

“…It was reported that CYP3A4 is involved in the metabolism of losartan (Stearns et al, 1995). However, no cytotoxicity of losartan was induced when the cells were infected with AdCYP3A4 constructed previously (Hosomi et al, 2010) instead of AdCYP2C9 (data not shown). Therefore, to detect adducts specifically generated by CYP2C9, losartan was incubated with CYP2C9 or CYP3A4 (negative control) Supersomes.…”

Section: Detection Of Semicarbazide Adducts Of Losartanmentioning

confidence: 83%

“…Tienilic acid is metabolized to reactive intermediates, the thiophene sulfoxide or the thiophene epoxide, by CYP2C9 (Koenigs et al, 1999). In recent studies, we developed useful in vitro cell-based assays using adenovirus to sensitively evaluate the involvement of CYP3A4 and superoxide dismutase 2 in drug-induced cytotoxicity (Yoshikawa et al, 2009;Hosomi et al, 2010). In the present study, a highly sensitive cytotoxicity assay system for CYP2C9-mediated metabolic activation was established in a similar way, and the druginduced cytotoxicity was evaluated with the established assay system.…”

Section: Introductionmentioning

confidence: 99%

CYP2C9-Mediated Metabolic Activation of Losartan Detected by a Highly Sensitive Cell-Based Screening Assay

Iwamura¹,

Fukami²,

Hosomi³

et al. 2011

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ABSTRACT:Drug-induced hepatotoxicity is a major problem in drug development, and reactive metabolites generated by cytochrome P450s are suggested to be one of the causes. CYP2C9 is one of the major enzymes in hepatic drug metabolism. In the present study, we developed a highly sensitive cell-based screening system for CYP2C9-mediated metabolic activation using an adenovirus vector expressing CYP2C9 (AdCYP2C9). Human hepatocarcinoma HepG2 cells infected with our constructed AdCYP2C9 for 2 days at multiplicity of infection 10 showed significantly higher diclofenac 4-hydroxylase activity than human hepatocytes. AdCYP2C9-infected cells were treated with several hepatotoxic drugs, resulting in a significant increase in cytotoxicity by treatment with losartan, benzbromarone, and tienilic acid. Metabolic activation of losartan by CYP2C9 has never been reported, although the metabolic activations of benzbromarone and tienilic acid have been reported. To identify the reactive metabolites of losartan, the semicarbazide adducts of losartan were investigated by liquid chromatographytandem mass spectrometry. Two CYP2C9-specific semicarbazide adducts of losartan (S1 and S2) were detected. S2 adduct formation suggested that a reactive metabolite was produced from the aldehyde metabolite E3179, but a possible metabolite from S1 adduct formation was not produced via E3179. In conclusion, a highly sensitive cell-based assay to evaluate CYP2C9-mediated metabolic activation was established, and we found for the first time that CYP2C9 is involved in the metabolic activation of losartan. This cell-based assay system would be useful for evaluating drug-induced cytotoxicity caused by human CYP2C9.