Development of a highly sensitive cytotoxicity assay system for CYP3A4-mediated metabolic activation.

Drug-induced hepatotoxicity, which is a rare but serious adverse reaction to a large number of pharmaceutical drugs, is sometimes associated with reactive metabolites produced by drug-metabolizing enzymes. In the present study, we constructed a cell-based system to evaluate the cytotoxicity of reactive metabolites produced by CYP3A4 using human hepatoma cells infected with an adenovirus vector expressing human CYP3A4 (AdCYP3A4). When seven hepatoma cell lines (HepG2, Hep3B, HLE, HLF, Huh6, Huh7, and Fa2N4 cells) were infected with AdCYP3A4, HepG2 cells showed the highest CYP3A4 protein expression and testosterone 6ß-hydroxylase activity (670 pmol · min(-1) · mg(-1)). With the use of AdCYP3A4-infected HepG2 cells, the cytotoxicities of 23 drugs were evaluated by the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt assay, and the cell viability when treated with 11 drugs (amiodarone, desipramine, felbamate, isoniazid, labetalol, leflunomide, nefazodone, nitrofurantoin, tacrine, terbinafine, and tolcapone) was significantly decreased. Moreover, the transfection of siRNA for nuclear factor erythroid 2-related factor 2 (Nrf2) to decrease the cellular expression level of Nrf2 exacerbated the cytotoxicity of some drugs (troglitazone, flutamide, acetaminophen, clozapine, terbinafine, and desipramine), suggesting that the genes regulated by Nrf2 are associated with the detoxification of the cytotoxicities mediated by CYP3A4. We constructed a highly sensitive cell-based system to detect the drug-induced cytotoxicity mediated by CYP3A4. This system would be beneficial in preclinical screening in drug development and increase our understanding of the drug-induced cytotoxicity associated with CYP3A4.

CYP2C9-mediated metabolic activation of losartan detected by a highly sensitive cell-based screening assay.

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 chromatography-tandem 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.

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

Drug-induced hepatotoxicity is often caused by CYP-dependent activation of the drugs into reactive metabolites. CYP3A4 is one of the main isoforms involved in the metabolic activation of drugs. In this study, an adenovirus vector expressing CYP3A4 (AdCYP3A4) was constructed. After 3days infection of AdCYP3A4, the testosterone 6beta-hydroxylase activity reached to 325pmol/min/mg protein in H4IIE (rat hepatoma) cells. To knockdown the gamma-glutamylcysteine synthetase heavy chain subunit (GCSh) and decrease the intrinsic glutathione (GSH) level, we used an adenovirus vector with short hairpin RNA against rat GCSh (AdGCSh-shRNA). Three days infection of AdGCSh-shRNA and AdCYP3A4 simultaneously with H4IIE cells decreased the intracellular GSH level by 50-60% without affecting the expression level of CYP3A4. Using this cell-based system sensitive to the cytotoxicity of reactive metabolites, drugs known for their hepatotoxicity were evaluated. As a result, troglitazone, flutamide, and acetaminophen caused significant decreases of cell viability in AdCYP3A4/AdGCSh-shRNA group compared to the other groups (AdGFP, AdCYP3A4, AdGFP/AdGCSh-shRNA groups), indicating that reactive metabolite(s) produced by CYP3A4 and subsequently conjugated by GSH would be involved in the cytotoxicity. These results suggest that this cell-based assay system expressing CYP3A4 with GCSh knockdown would be useful for the prediction of CYP3A4-mediated cytotoxicity in preclinical drug development.

Knockdown of superoxide dismutase 2 enhances acetaminophen-induced hepatotoxicity in rat.

Drug-induced hepatotoxicity is a major problem in drug development, and oxidative stress is known as one of the causes. Superoxide dismutases (SODs) are important antioxidant enzymes against reactive oxygen species (ROS). Mitochondria are the major source of superoxide production, and SOD2 is mainly localized in mitochondria and, with other SODs, plays an important role in scavenging superoxide. Previously, we reported the establishment of an adenovirus vector with short hairpin RNA against rat SOD2 (AdSOD2-shRNA), and applied this to evaluate drug-induced cytotoxicity. In this study, infection of AdSOD2-shRNA to Fisher 344 rats resulted in a significant decrease of SOD2 mRNA, protein expression, and SOD2 enzyme activity to 28%, 35%, and 39%, respectively, 7 days after infection. Serum AST and ALT were significantly increased by single oral administration of acetaminophen (1000 mg/kg) to these SOD2-knockdown rats without fasting compared with the control adenovirus infected groups. Heme oxygenase-1 protein, known to be induced by oxidative stress, was detected in SOD2-knockdown rats administered acetaminophen. In addition, protein carbonyl and lipid peroxidation, also known to be induced by oxidative stress, were significantly increased in SOD2 knockdown rats. This is the first report of a SOD2-knockdown rat model that could be useful to evaluate the drug-induced hepatotoxicity with high sensitivity.

Drug-induced hepatotoxicity test using gamma-glutamylcysteine synthetase knockdown rat.

Idiosyncratic drug-induced liver injury (DILI) is a major clinical problem for drug development. It is generally known that DILI is mainly caused by hepatic glutathione (GSH) depletion. The glutathione S-transferase activity of rodent is higher than that of human, which could make the prediction of DILI more difficult. Recently, we reported that an experimental rat model of GSH-depletion displayed high susceptibility to acetaminophen-induced hepatotoxicity. To deplete GSH, we used an adenovirus vector with short hairpin RNA against gamma-glutamylcysteine synthetase heavy chain subunit (AdGCSh-shRNA). In this study, we further investigated the usefulness of this rat model for determining drug-induced sensitive acute and subacute toxicity. Rats were administered diclofenac and flutamide which have been reported as idiosyncratic hepatotoxic drugs. In the acute (6 or 24h) or subacute (7 days) toxicity tests, rats were administered the drugs once or once a day for a week, respectively. Plasma biochemical markers for hepatotoxicity were measured. The 6 and 24h toxicity test of diclofenac, and the 24h and 7 days toxicity tests of flutamide showed significant ALT elevations. Additionally, the 24h toxicity test of flutamide showed a slight bilirubin elevation, and histological hepatotoxicity. The 7 days toxicity test of flutamide also demonstrated histological hepatotoxicity. In conclusion, this rat model would contribute to evaluating acute and subacute DILI in preclinical drug development.

Establishment of knockdown of superoxide dismutase 2 and expression of CYP3A4 cell system to evaluate drug-induced cytotoxicity.

Drug-induced hepatotoxicity is a major problem in drug development, and oxidative stress is known as one of the causes. Superoxide dismutases (SODs) are important antioxidant enzymes against reactive oxygen species (ROS). Mitochondria are the major source of superoxide production, and SOD2 is mainly localized in mitochondria and, with other SODs, plays an important role in scavenging superoxide. In this study, we established SOD2-knockdown cells. An adenovirus vector with short hairpin RNA against rat SOD2 (AdSOD2-shRNA) was constructed, and infection of AdSOD2-shRNA to rat hepatic BRL3A cells resulted in significant decreases of SOD2 mRNA and protein by 60%, and SOD2 activity by 50% after 3 days infection. We previously constructed an adenovirus expressing cytochrome P450 3A4 (AdCYP3A4). Co-infection of AdSOD2-shRNA and AdCYP3A4 to BRL3A cells was carried out to evaluate the superoxide- and CYP3A4-mediated formation of active metabolites, and mitochondrial toxicity, ROS and superoxide radical production and lipid peroxidation were selected to assess the cell viability. Albendazole, carbamazepine, dapsone, flutamide, isoniazid, nifedipine, sulfamethoxazole, trazodone, troglitazone, and zidovudine demonstrated significant increases of SOD2- and CYP3A4-mediated cytotoxicity. In conclusion, we constructed a highly sensitive cell system to evaluate oxidative stress and CYP3A4 mediated cytotoxicity that could be useful in preclinical drug development.

Knock down of gamma-glutamylcysteine synthetase in rat causes acetaminophen-induced hepatotoxicity.

Drug-induced hepatotoxicity is mainly caused by hepatic glutathione (GSH) depletion. In general, the activity of rodent glutathione S-transferase is 10 to 20 times higher than that of humans, which could make the prediction of drug-induced hepatotoxicity in human more difficult. Gamma-glutamylcysteine synthetase (gamma-GCS) mainly regulates de novo synthesis of GSH in mammalian cells and plays a central role in the antioxidant capacity of cells. In this study, we constructed a GSH-depletion experimental rat model for the prediction of human hepatotoxicity. An adenovirus vector with short hairpin RNA against rat gamma-GCS heavy chain subunit (GCSh) (AdGCSh-shRNA) was constructed and used to knock down the GCSh. In in vitro study in H4IIE cells, a rat hepatoma cell line, GCSh mRNA and protein were significantly decreased by 80% and GSH was significantly decreased by 50% 3 days after AdGCSh-shRNA infection. In the in vivo study in rat, the hepatic GSH level was decreased by 80% 14 days after a single dose of AdGCSh-shRNA (2 x 10(11) pfu/ml/body), and this depletion continued for at least 2 weeks. Using this GSH knockdown rat model, acetaminophen-induced hepatotoxicity was shown to be significantly potentiated compared with normal rats. This is the first report of a GSH knockdown rat model, which could be useful for highly sensitive tests of acute and subacute toxicity for drug candidates in preclinical drug development.