Enhancement of the predicted drug hepatotoxicity in gel entrapped hepatocytes within polysulfone-g-poly (ethylene glycol) modified hollow fiber

Collagen gel-based 3D cultures of hepatocytes have been proposed for evaluation of drug hepatotoxicity because of their more reliability than traditional monolayer culture. The collagen gel entrapment of hepatocytes in hollow fibers has been proven to well reflect the drug hepatotoxicity in vivo but was limited by adsorption of hydrophobic drugs onto hollow fibers. This study aimed to investigate the impact of hollow fibers on hepatocyte performance and drug hepatotoxicity. Polysulfone-g-poly (ethylene glycol) (PSf-g-PEG) hollow fiber was fabricated and applied for the first time to suppress the drug adsorption. Then, the impact of hollow fibers was evaluated by detecting the hepatotoxicity of eight selected drugs to gel entrapped hepatocytes within PSf and PSf-g-PEG hollow fibers, or without hollow fibers. The hepatocytes in PSf-g-PEG hollow fiber showed the highest sensitivity to drug hepatotoxicity, while those in PSf hollow fiber and cylindrical gel without hollow fiber underestimated the hepatotoxicity due to either drug adsorption or low hepatic functions. Therefore, the 3D culture of gel entrapped hepatocytes within PSf-g-PEG hollow fiber would be a promising tool for investigation of drug hepatotoxicity in vitro.     

Evaluation of the use of imaging parameters for the detection of compound-induced hepatotoxicity in 384-well cultures of HepG2 cells and cryopreserved primary human hepatocytes

Drug-induced liver injury (DILI) is a major cause of failed drug development, withdrawal and restricted usage. Therefore screening assays which aid selection of candidate drugs with reduced propensity to cause DILI are required. We have investigated the toxicity of 144 drugs, 108 of which caused DILI, using assays identified in the literature as having some predictivity for hepatotoxicity. The validated assays utilised either HepG2 cells, HepG2 cells in the presence of rat S9 fraction or isolated human hepatocytes. All parameters were quantified by multiplexed and automated high content fluorescence microscopy, at appropriate time points after compound administration (4, 24 or 48 h). The individual endpoint which identified drugs that caused DILI with greatest precision was maximal fold induction in CM-H2DFFDA staining in hepatocytes after 24 h (41% sensitivity, 86% specificity). However, hierarchical clustering analysis of all endpoints provided the most sensitive identification of drugs which caused DILI (58% sensitivity, 75% specificity). We conclude that multi-parametric high content cell toxicity assays can enable in vitro detection of drugs that have high propensity to cause DILI in vivo but that many DILI compounds exhibit few in vitro signals when evaluated using these assays.     

Role of CYP3A in regulating hepatic clearance and hepatotoxicity of triptolide in rat liver microsomes and sandwich-cultured hepatocytes

Triptolide (TP) is an active component of Tripterygium wilfordii Hook. F and widely used to treat autoimmune and inflammatory diseases. It has been demonstrated that cytochrome P450 (CYP) are involved in the metabolism of TP. However, the underlying mechanisms of TP-induced toxicity mediated by hepatic CYP have not been well delineated. In this study, rat liver microsomes (RLM) and sandwich-cultured rat hepatocytes (SCRH) were used to identify the mechanism involving the CYP3A inhibition by TP and to evaluate TP-induced liver damage after CYP3A modulation by the known inhibitor, ketoconazole, and the known inducer, dexamethasone. The results showed that TP itself had a time- and concentration-dependent inhibitory effect on CYP3A. When the CYP3A inhibitor and inducer were added, the enzyme activity and hepatotoxicity changed significantly. The enzyme inducer increased CYP3A activity and decreased the metabolic half life (t_1/2) of TP when compared to the control group, while the enzyme inhibitor had an opposite effect. Our findings reveal that TP is a weak CYP3A inhibitor involving the time-dependent inhibition mechanism. The induction or inhibition of CYP3A played an important role in TP-induced hepatotoxicity. Clinicians should be aware of the metabolic characteristics of TP to maximize therapeutic efficacy and reduce TP-induced toxicity.     

Molecular mechanism of trichloroethylene-induced hepatotoxicity mediated by CYP2E1

Cytochrome P450 (CYP) 2E1 was suggested to be the major enzyme involved in trichloroethylene (TRI) metabolism and TRI-induced hepatotoxicity, although the latter molecular mechanism is not fully understood. The involvement of CYP2E1 in TRI-induced hepatotoxicity and its underlying molecular mechanism were studied by comparing hepatotoxicity in cyp2e1^+/+ and cyp2e1^−/− mice. The mice were exposed by inhalation to 0 (control), 1000, or 2000 ppm of TRI for 8 h a day, for 7 days, and TRI-hepatotoxicity was assessed by measuring plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and histopathology. Urinary metabolites of trichloroethanol and trichloroacetic acid (TCA) were considerably greater in cyp2e1^+/+ compared to cyp2e1^−/− mice, suggesting that CYP2E1 is the major P450 involved in the formation of these metabolites. Consistent with elevated plasma ALT and AST activities, cyp2e1^+/+ mice in the 2000 ppm group showed histopathological inflammation. TRI significantly upregulated PPARα, which might function to inhibit NFκB p50 and p65 signalling. In addition, TRI-induced NFκB p52 mRNA, and significantly positive correlation between NFκB p52 mRNA expression and plasma ALT activity levels were observed, suggesting the involvement of p52 in liver inflammation. Taken together, the current study directly demonstrates that CYP2E1 was the major P450 involved in the first step of the TRI metabolism, and the metabolites produced may have two opposing roles: one inducing hepatotoxicity and the other protecting against the toxicity. Intermediate metabolite(s) from TRI to chloral hydrate produced by CYP2E1-mediated oxidation may be involved in the former, and TCA in the latter.     

Protective effect of caffeic acid phenethyl ester against carbon tetrachloride-induced hepatotoxicity in mice

This study was designed to investigate the protective effects of the phenethyl ester of caffeic acid (CAPE) against carbon tetrachoride (CCl₄)-induced hepatotoxicities in mice. Pretreatment with CAPE prior to administration of CCl₄ significantly prevented the increases in serum alanine, aspartate aminotransferase and alkaline phosphatase activities, hepatic lipid peroxidation formation, and depletion of glutathione content. In addition, CAPE prevented CCl₄-induced apoptosis and necrosis, as indicated by liver histopathology and DNA laddering studies. To determine whether the Fas/Fas ligand (FasL) pathway is involved in CCl₄-induced acute liver injury, Fas and FasL proteins and caspase-3 and -8 activities were tested by western blotting and ELISA. CAPE markedly decreased CCl₄-induced Fas/FasL protein expression levels and, in turn, attenuated CCl₄-induced caspase-3 and -8 activities in mouse liver. Moreover, the effect of CAPE on CYP2E1, the major isozyme involved in CCl₄ bioactivation, was investigated. Treatment with CAPE significantly decreased the CYP2E1-dependent hydroxylation of aniline. In addition, CAPE attenuated the CCl₄-mediated depletion of antioxidant enzyme (catalase, superoxide dismutase and glutathione-S-transferase) activities. These findings suggest that the protective effects of CAPE against CCl₄-induced acute liver injury may involve its ability to block CYP2El-mediated CCl₄ bioactivation and to protect against Fas/FasL-mediated apoptosis.     

Evidence for a potential protective effect of carnitine-pantothenic acid co-treatment on valproic acid-induced hepatotoxicity

Valproic acid is approved for treatment of seizures and manic episodes of bipolar disorder, and continues to be one of the most commonly prescribed antiepileptic drugs in the world. Hepatotoxicity is a rare but serious side effect resulting from its use, particularly in young patients. This adverse effect does not display normal dose–response curves and can be lethal in children. A review of the purported mechanisms of action suggest hepatotoxicity results from increased oxidative stress, caused by a reduction in beta-oxidation and an increase in activation of certain metabolizing enzymes. There is also evidence that both carnitine and pantothenic acid are involved in the regulation of valproic acid-induced hepatotoxic processes, and clinical evidence has shown that treatment with either compound shows protective effects against hepatotoxicity. These results suggest a potential increase in protective effects with cotreatment of carnitine and pantothenic acid.     

Bromuconazole-induced hepatotoxicity is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1 gene expression

Despite widespread use of bromuconazole as a pesticide for food crops and fruits, limited studies have been done to evaluate its toxic effects. Here, we evaluated the hepatotoxic effect of bromuconazole using classical toxicological (biochemical analysis and histopathological examination) and gene-based molecular methods. Male rats were treated either orally or topically with bromuconazole at doses equal to no observed adverse effect level (NOAEL) and 1/10 LD50 for 90?d. Bromuconazole increased activities of liver enzymes (ALT, AST, ALP, and ACP), and levels of bilirubin. It also induced hepatic oxidative stress as evidenced by significant decrease in the activities of superoxide dismutase (SOD), and significant increase in levels of malondialdehyde (MDA) in liver. In addition, bromuconazole caused an increase in liver weights and necrobiotic changes (vacuolation and hepatocellular hypertrophy). It also strongly induced the expression of PXR and its downstream target CYP3A1 gene as well as the activity of CYP3A1. However, it inhibited the expression of CAR and its downstream target CYP2B1 gene without significant changing in CYP2B1 activity. Overall, the oral route showed higher hepatotoxic effect and molecular changes than the dermal route and all changes were dose dependent. This is the first investigation to report that bromuconazole-induced liver oxidative damage is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1.     

Species-specific toxicity of troglitazone on rats and human by gel entrapped hepatocytes

Troglitazone, despite passing preclinical trials on animals, was shortly withdrawn from market due to its severe hepatotoxicity in clinic. As rat hepatocyte monolayer consistently showed sensitive troglitazone toxicity as human hepatocyte monolayer in contrast to the species-specific toxicity in vivo, this paper utilized both hepatocytes in three-dimensional culture of gel entrapment to reflect the species difference on hepatotoxicity. Rat hepatocytes in gel entrapment did not show obvious cellular damage even under a long-term exposure for 21 days while gel entrapped human hepatocytes significantly displayed oxidative stress, steatosis, mitochondrial damage and cell death at a short exposure for 4 days. As a result, the detected species-specific toxicity of troglitazone between gel entrapped rat and human hepatocytes consisted well with the situation in vivo but was in a sharp contrast to the performance of two hepatocytes by monolayer culture. Such contradictory toxicity of rat hepatocytes between monolayer and gel entrapment culture could be explained by the fact that troglitazone was cleared more rapidly in gel entrapment than in monolayer culture. Similarly, the differential clearance of troglitazone in rat and human might also explain its species-specific toxicity. Therefore, gel entrapment of hepatocytes might serve as a platform for evaluation of drug toxicity at early stage of drug development by reducing costs, increasing the likelihood of clinical success and limiting human exposure to unsafe drugs.     

Investigation of the metabolism of 14C/13C-practolol in rat using directly coupled radio-HPLC-NMR-MS

1. The metabolic fate of ¹⁴C ¹³C-practolol was investigated using on-line HPLCNMR-MS following oral administration to rat. The major route of elimination for the radiolabel was via the urine with the principal biotransformation products confirmed as the 2-hydroxy- and 2-hydroxyglucronide metabolites. 2. In addition, futile deacetylation, determined by the replacement of ¹³C-labelled acetyl groups with endogenous ¹²C-acetyls accounted for ~7-10% of the urinary metabolites, corresponding to ~5% of the dose undergoing N-deacetylation. 3. Evidence for chiral metabolism was sought via NMR of isolated metabolites using beta cyclodextrin as a chiral shift agent. Practolol was excreted as a racemate. However, some enantioselective metabolism excretion had occurred as the hydroxy- and hydroxyglucuronide were not excreted as racemic mixtures. 4. Directly coupled radio-HPLC-NMR-MS is extremely effective for the identification of the metabolites of radiolabelled xenobiotics in urine samples.     

Caveats of using acetaminophen hepatotoxicity models for natural product testing

In evaluating the potential of natural products and other chemicals to protect against acetaminophen-induced hepatotoxicity, it is critical to use clinically relevant experimental models and evaluate the initial metabolic activation and protein adduct formation. If these basic principles are not considered, the clinical relevance of the hepatoprotection by a natural product is questionable and conclusions regarding potential mechanisms of protection may be unreliable. Therefore, we feel it is necessary to express our concerns regarding a recent publication by Zhao et al. (2012).     

三七总皂苷对CYP450的影响及药物相互作用预测

目的：研究三七总皂苷（血塞通）对肝微粒体CYP450酶不同亚型的影响，进一步了解其在肝脏的代谢特点并据此对该药与其他药物相互作用的安全性进行预测，为临床合理应用提供依据。方法：检测肝药酶CYP1A2和CYP3A4的专属探针药物咖啡因、氨苯砜的原型浓度，采用原型药物减少初始速率测定法考察其体外代谢变化情况，从而评价三七总皂苷对CYPIA2、CYP3A4的诱导或抑制作用。结果：三七总皂苷对肝药酶CYPIA2有诱导作用，而对CYP3A4无影响。结论：三七总皂苷对CYP450。不同亚型酶的影响不同。在临床应用时，尤其是与CYPIA2代谢有关的药物合用时，应充分考虑到这种影响，以避免潜在的毒性或不良反应。     

中药止咳橘红颗粒对CYP3A4和CYP1A2抑制作用的研究

目的：在人体内研究止咳橘红对CYP3A4和CYP1A2的抑制作用，以预测止咳橘红与常用临床药物的相互作用。方法：咪哒唑仑和咖啡因分别作为CYP3A4和A2的探针药物，采取交叉设计，10名受试者在服用3d止咳橘红的前后均服用7.5mg咪哒唑仑和100mg咖啡因，服药后采血测定两者及代谢产物的代谢动力学参数，探讨针药物及代谢物的浓度用HPLC-MS法测定，Cmax,tmax从药时曲线中直接读出，AUC用梯形法计算，Ke用3P87程序进行拟合计算，分析服药前后CYP3A4和CYP1A2被抑制的情况，结果 服用止咳橘红后，咪哒唑仑的代谢受到了轻微的抑制，它的血药浓度，达峰时间和药时曲线下面积都有了升高趋势，但无显著差异。而咖啡因的代谢未受到影响。结论 止咳橘红对CYP3A4的活性有较弱的抑制作用，能够导致CYP3A4底物咪哒唑仑代谢的轻微抑制，而对CYP1A2的活性没有影响。止咳橘红长期使用或超过治疗剂量使用时是否会对CYP3A4产生显著性影响。尚需进一步的研究证明。     

DHA down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes by attenuating CAR translocation

The constitutive androstane receptor (CAR) plays an important role in regulating the expression of detoxifying enzymes, including cytochrome P450 2B (CYP 2B). Phenobarbital (PB) induction of human CYP 2B6 and mouse CYP 2b10 has been shown to be mediated by CAR. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA); however, the mechanism for this down-regulation by DHA was previously unknown. The objective of the present study was to determine whether change in CAR translocation is involved in the down-regulation by n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression in rat primary hepatocytes. We used 100 microM arachidonic acid, linoleic acid, eicosapentaenoic acid, and DHA to test this hypothesis. PB triggered the translocation of CAR from the cytosol into the nucleus in a dose-dependent and time-dependent manner in our hepatocyte system, and the CAR distribution in rat primary hepatocytes was significantly affected by DHA. DHA treatment decreased PB-inducible accumulation of CAR in the nuclear fraction and increased it in the cytosolic fraction in a dose-dependent manner. The down-regulation of CYP 2B1 expression by DHA occurred in a dose-dependent manner, and a similar pattern was found for the nuclear accumulation of CAR. The results of immunoprecipitation showed a CAR/RXR heterodimer bound to nuclear receptor binding site 1 (NR-1) of the PB-responsive enhancer module (PBREM) of the CYP 2B1gene. The EMSA results showed that PB-induced CAR binding to NR-1 was attenuated by DHA. Taken together, these results suggest that attenuation of CAR translocation and decreased subsequent binding to NR-1 are involved in DHA's down-regulation of PB-induced CYP 2B1 expression.     

Protective effect of saponins derived from the roots of Platycodon grandiflorum against carbon tetrachloride induced hepatotoxicity in mice

The purpose of this study was to investigate the protective effects of the saponins isolated from the root of Platycodi Radix (Changkil saponins: CKS) on carbon tetrachloride (CCl(4))-induced hepatotoxicities in mice. Pretreatment with CKS prior to the administration of CCl(4) significantly prevented the increase in serum alanine aminotransferase and aspartate aminotransferase activities and hepatic lipid peroxidation formation. In addition, CKS prevented CCl(4)-induced apoptosis and necrosis, as indicated by a liver histopathologic study and DNA laddering. To determine whether Fas/Fas ligand (FasL) pathway involved in CCl(4)-induced acute liver injury, Fas and FasL proteins and caspase-3, -8 activities were tested by western blotting and ELISA. CKS markedly decreased CCl(4)-induced Fas/FasL protein expression levels and in turn attenuated CCl(4)-induced caspase-3, -8 activities in mouse livers. Additionally, CKS protected the CCl(4)-induced depletion of hepatic glutathione levels. The effect of CKS on CYP2E1, the major isozyme involved in CCl(4) bioactivation, was investigated. Treatment with CKS resulted in a significant decrease in the CYP2E1-dependent hydroxylation of aniline. In addition, CKS exhibited antioxidant effects on FeCl(2)-ascorbate induced lipid peroxidation in liver homogenates, and on superoxide radical scavenging activity. These findings suggest that the protective effects of CKS against CCl(4)-induced acute liver injury possibly involve mechanisms related to its ability to block CYP2El-mediated CCl(4) bioactivation and its free radical scavenging effects, and that is also protects against Fas/FasL pathway mediated apoptosis.     

Effects of Guanxinning injection on rat cytochrome P450 isoforms activities in vivo and in vitro

Abstract

1. We aimed to investigate the regulatory effects of Guanxinning injection (GXNI) on activities of cytochrome P1A2 (CYP1A2), CYP2C11, CYP2D1 and CYP3A1/2 by probe drugs in rats in vivo and in vitro.

2. GXNI-treated and blank control groups were administered GXNI and physiological saline by caudal vein for 14 days consecutively, then they were given the probe drugs of caffeine (10?mg/kg), tolbutamide (10?mg/kg), metoprolol (20?mg/kg) and dapsone (10?mg/kg) by intraperitoneal injection. The blood samples were collected at different times for ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. Changes of the pharmacokinetics parameters between the GXNI-treated and the blank control groups were used to evaluate the effects of GXNI on the four CYP450 isoforms in rats in vivo. After blood collection, the livers of rats were taken and made microsomes for in vitro tests. The relevant metabolites of phenacetin, tolbutamide, dextromethorphan and testosterone were analyzed quantitatively by high-performance liquid chromatography (HPLC) after microsome incubation. The statistical differences between the two groups were observed to detect the effects of GXNI on the four CYP450 isoforms in rats in vitro.

3. The in vivo and in vitro results demonstrated that GXNI could induce CYP1A2 activity in rats, but had no significant effects on CYP2C11, CYP2D1 and CYP3A1/2.     

Identification and analysis of the reactive metabolites related to the hepatotoxicity of safrole

1.?Safrole is the main component of the volatile oil in Xixin, which has a strong antifungal effect. However, safrole has been shown to be associated with the development of hepatocellular carcinoma. Methylenedioxyphenyl and allyl-benzene substructures of safrole may cause a mechanism-based inhibition (MBI) of CYP450 enzymes (CYPs) and produce reactive metabolites (RMs), resulting in inhibition of enzyme activity and toxic effects.

2.?Based on the experiments of CYPs cocktail screening, glutathione (GSH) capture and the IC₅₀ data, we found that safrole had an inhibitory effect on CYP1A2. The test of enzyme activity recovery when adding GSH may help to verify the MBI of safrole.

3.?Two metabolites, 1,2-dihydroxy-4-allylbenzene (M1) and 1′-hydroxy safrole (M2) could be captured by GSH. The ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) method was used to identify the RMs through a detailed characterization of the safrole cleavage processes and the GSH-M1 adduct. The RMs identified are quinone and its tautomer. Thus, preliminary conclusion can be obtained that safrole is a mechanism-based inhibitor of CYP1A2.

4.?The cleavage process of the GSH-M1/M2 adduct was analyzed in further detail. We believe the safrole hepatotoxicity mechanism is related to the RMs mediated by CYP1A2. This work provides important information on predicting in vivo drug induced liver injury.     

慢性间断性低氧对大鼠肝脏CYP3A_2和CYP2E_1的影响研究

目的研究慢性间断性低氧对大鼠肝脏CYP3A2和CYP2E1的影响。方法将大鼠随机分为对照组、低氧3d组、低氧7d组、低氧14d组、低氧28d组,低氧处理结束24h后,常规腹腔注射麻醉,摘取眼球血液2ml制备血清,并测定丙氨酸氨基转移酶(ALT)、天门冬酸氨基转移酶(AST)、红霉素N-脱甲基酶(ERD)、苯胺羟化酶(ANH)活性;取新鲜肝组织以制备微粒体和提取核糖核酸(RNA),并以RT-PCR进行基因片段扩增以检测大鼠肝脏细胞色素CYP3A2、CYP2E1的mRNA表达水平。结果慢性间断性低氧对血清ALT、AST活性无明显影响;低氧7d后,大鼠肝脏ERD、ANH活性明显升高,28d时诱导率分别为155.5%、42.2%;同时,CYP3A2、CYP2E1mRNA的表达水平也分别增加了220.5%、102.8%。结论慢性间断性低氧能显著增加大鼠肝脏ERD、ANH活性,其机制可能与其在转录水平上提高肝脏CYP3A2和CYP2E1的基因表达水平有关。     

细胞色素P450(CYP450)遗传多态性研究进展

近年来对CYP450基因型和表型相关性的研究越来越受到重视,从临床合理用药方面来说,人们希望利用基因型分析来了解个体中药物代谢酶的活性,期望既能提高药物治疗水平同时又降低不良反应的发生;从新药研发角度来说,研究药物的代谢酶CYP450的功能能够指导新药的设计、筛选及优化。该文通过查阅国内外相关文献综述了近年来关于CYP450遗传多态性研究的进展,分别介绍了CYP2C19、CYP2C9、CYP3A4、CYP2D6、CYP1A2和CYP2E1这6种主要的药物代谢酶。研究CYP450对新药设计、筛选、评价及优化有重要意义。     

毛冬青胶囊对大鼠体内细胞色素P450 代谢活性的影响*

目的：采用Cocktail探针药物法研究毛冬青胶囊对大鼠CYP1A2、CYP3A2、CYP2C6、CYP2D1、CYP2D2和CYP2E1体内代谢活性的影响。方法：分别以茶碱、咪达唑仑、甲苯磺丁脲、奥美拉唑、右美沙芬和氯唑沙宗作为探针底物,将大鼠随机分为3组：空白对照组、毛冬青的低剂量组和高剂量组。低、高剂量组每日分别灌胃给予毛冬青胶囊1.8、3.6 g·kg-1,空白对照组每日给予与低剂量组等体积的生理盐水,各组均为1次/天,连续14 d。各组分别于第15天给予Cocktail探针药物,于给药前、后不同时间点取血,用LC-MS/MS检测各探针药物的血药浓度,计算药代动力学参数。结果：茶碱低剂量组cmax、AUC0-t有极显著差异（P≤0.01）;甲苯磺丁脲高剂量组和氯唑沙宗低剂量组AUC0-t有显著差异（P≤0.05）;其他无统计学差异。结论：毛冬青胶囊对大鼠体内CYP2C6和CYP1A2有强诱导作用,对CYP2E1有中强诱导作用,其他亚型基本无影响。