首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
Cryopreserved human hepatocytes were extensively characterized in our laboratory. The post-thaw viability, measured via dye exclusion, ranged from 55 to 83%, for hepatocytes cryopreserved from 17 donors. Post-thaw viability and yield (viable cells per vial) were found to be stable up to the longest storage duration evaluated of 120 days. Drug-metabolizing enzyme activities of the cryopreserved hepatocytes (mean of ten donors) as percentages of the freshly isolated cells were: 97%, for cytochrome P450 isoform (CYP) 1A2, 78% for CYP2A6, 96% for CYP2C9. 86% for CYP2Cl9, 90% for CYP2D6, 164% for CYP3A4, 76% for UDP-glucuronidase, and 88% for umbelliferone sulfotransferase. Known species-differences in 7-ethoxycoumarin (7-EC) metabolism were reproduced by cryopreserved hepatocytes from human, rat, rabbit, dog, and monkey, illustrating the utility of cryopreserved hepatocytes from multiple animal species in the evaluation of species-differences in drug metabolism. Higher throughput screening (HTS) assays were developed using cryopreserved human hepatocytes for hepatotoxicity, metabolic stability, and inhibitory drug-drug interactions. Dose-dependent cytotoxicity, measured using MTT metabolism as an endpoint, was observed for the known hepatotoxic chemicals tamoxifen, clozapine, cadmium chloride, diclofenac, amiodarone, tranylcypromine, precocene II, but not for 2-thiouracil. Cell density- and time-dependent metabolism of 7-EC and dextromethorphan were observed in the HTS assay for metabolic stability. Known CYP isoform-specific inhibitors were evaluated in the HTS assay for inhibitory drug-drug interactions. Furafylline, sulfaphenazole, quinidine, and ketoconazole were found to be specific inhibitors of CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. Tranylcypromine and diethyldithiocarbamate were found to be less specific, with inhibitory effects towards several CYP isoforms, including CYP2A6, CYP2C9, CYP2C19, and CYP2E1. These results suggest that cryopreserved human hepatocytes represent a useful experimental tool for the evaluation of drug metabolism, toxicity, and inhibitory drug-drug interaction potential.  相似文献   

2.
Arsenic is a cancer chemotherapeutic but hepatotoxicity can be a limiting side effect. O2-vinyl 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO) is a nitric oxide (NO) donor prodrug and metabolized by liver cytochromes P450 (CYP450) to release NO. The effects of V-PROLI/NO pretreatment on the toxicity of arsenic (as NaAsO2) were studied in a rat liver cell line (TRL 1215). The cells acted upon the prodrug to release NO, as assessed by nitrite levels, in a time-dependent fashion to maximal levels of 8-fold above basal levels. Pretreatment with V-PROLI/NO markedly reduced arsenic cytolethality which was directly related to the level of NO produced by V-PROLI/NO treatment. Cyp1a1 expression was directly related to the level of NO production and to reduced arsenic cytotoxicity. V-PROLI/NO pretreatment markedly reduced arsenic-induced apoptosis and suppressed phosphorylation of JNK1/2. V-PROLI/NO pretreatment facilitated additional increases in arsenic-induced metallothionein, a metal-binding protein important in arsenic tolerance. Thus, V-PROLI/NO protects against arsenic toxicity in rat liver cells, reducing cytolethality, apoptosis and dysregulation of MAPKs, through generation of NO formed after metabolism by liver cell enzymes, possibly including Cyp1a1. CYP450 required for NO production from V-PROLI/NO treatment in the rat and human appears to differ as we have previously studied the ability of V-PROLI/NO to prevent arsenic toxicity in human liver cells where it reduced toxicity apparently through a CYP2E1-mediated metabolic mechanism. None-the-less, it appears that both rat and human liver cells act upon V-PROLI/NO via a CYP450-related mechanism to produce NO and subsequently reduce arsenic toxicity.  相似文献   

3.
The in vitro metabolism of precocene I by liver microsomes from control and treated rats and the effects of precocene I on the function and histology of the rat liver were examined. The major metabolites (80-90% of total metabolites) from all microsomal preparations were the cis and trans 3,4-diols of precocene I produced with a cis/trans isomer ratio of 1:2. These diols appear to arise mainly by spontaneous hydrolysis of precocene I 3,4-oxide. (+)-(3R,4R)-cis- and (-)-(3R,4S)-trans-precocene I 3,4-diols were the predominant enantiomers of the 3,4-diol formed. The enantiomeric excess of these diols (2-50%) is dependent on the microsomal preparation, with microsomes from control rats exhibiting the highest stereoselectivity and microsomes from phenobarbital-treated rats the least. 6-Hydroxyprecocene I was the next major metabolite and was formed to the extent of 5% (control), 10% and 17% (phenobarbital and 3-methylcholanthrene treatment, respectively) of total metabolites. Treatment of rats with a single i.p. dose of precocene I (300 mg/kg) resulted in extensive hepatic damage as evidenced by a marked increase of plasma glutamic pyruvic transaminase levels and histologic observation in liver sections of severe centrolobular necrosis. Although phenobarbital treatment of rats increased the rate of liver microsomal metabolism of precocene I by approximately 50% (nmol products/nmol cytochrome P-450/min) compared to liver microsomes from control rats, hepatic damage caused by precocene I was not significantly affected. Depletion of glutathione levels in the rats with diethyl maleate prior to precocene I treatment dramatically increased the severity of hepatic insult, whereas treatment of the rats with the mixed function oxidase inhibitor piperonyl butoxide prior to treatment with precocene I blocked hepatic damage. Treatment of rats with cysteamine prior to treatment with precocene I protected the animals against the toxic effects. Neither cis nor trans precocene I 3,4-diol nor 3,4-dihydroprecocene I elicited impaired liver function or cellular damage. The above results are consistent with the view that precocene I 3,4-oxide is the metabolite responsible for the hepatotoxic effects observed when precocene I is injected into rats.  相似文献   

4.
Reactive oxygen species (ROS), possibly produced during the metabolic conversion of benzo(a)pyrene (B[a]P), could be involved in B[a]P-induced genotoxicity and, eventually, carcinogenicity. Therefore, ROS formation by rat lung and liver microsomes was studied in vitro by electron spin resonance (ESR/EPR) spectrometry. B[a]P-mediated generation of ROS was detected in incubations with rat lung, but not with liver microsomes. Inhibition of cytochrome P450 (CYP450) by the non isoform-specific inhibitor SKF-525A resulted in a complete inhibition of B[a]P-dependent ROS formation, whereas ROS formation was not affected by inhibition of prostaglandin H synthase by indomethacin. Subsequently, bulky DNA adduct formation and 8-oxo-dG levels after a single oral dose of B[a]P were examined in vivo in rat lung and liver, in combination with urinary excretion of 8-oxodG. B[a]P exposure resulted in increased urinary 8-oxo-dG levels. On the contrary, 8-oxo-dG levels decreased in liver and lung after B[a]P exposure. Bulky DNA adducts reached higher levels and were more persistent in rat lung than in liver. These results indicate that ROS are generated during the CYP450 dependent metabolism of B[a]P, particularly in the rat lung, but this does not necessarily result in increased levels of oxidative DNA damage in vivo, possibly by induction of DNA repair mechanisms.  相似文献   

5.
The intermediate metabolites and redox status imbalance were supported as the two major points for N,N‐dimethylformamide (DMF)‐induced hepatotoxicity. However, the potential mechanism has not yet been concerned. By applying two inhibitors, this study tried to seek the major role in DMF‐induced toxicity on HL7702 cell. We observed that DMF induced cell apoptosis through mitochondrial‐dependent and p53 pathway. Inhibition reactive oxygen species by catalase remarkably attenuated the mitochondrial transmembrane potential (MMP), apoptotic proteins, and apoptosis. On the contrary, it reduced the biodegradation rate of DMF by coincubation with CYP2E1 antagonist (DDC) partially reduced late apoptosis. However, the change in MMP, the ratio of Bax to Bcl‐xl, and cleaved‐caspase 9 was not attenuated by DDC. The pathway in DDC coincubation groups was related to the p53 rather than the mitochondrial pathway. Restoring the redox balance during biodegradation is much more effective than attenuating the metabolite rate of DMF. This study may provide a suitable prevention method to occupational workers.  相似文献   

6.
7.
Precocene II (6,7-dimethoxy-2,2-dimethyl-2H-benzo[b]pyran), an insect growth regulator that is structurally related to several naturally occurring carcinogenic and non-carcinogenic alkenylbenzenes, is genotoxic and produces hepatic centrolobular necrosis in rats. This investigation was conducted to evaluate the effects of modulation of hepatic glutathione levels on the toxicity of precocene II. Administration of a toxic dose of precocene II (175 mg/kg) to male Sprague-Dawley rats rapidly depleted hepatic GSH, produced histopathological changes in the liver, and induced increases in serum aminotransferase activity. Concurrent administration of the cysteine pro-drug L-2-oxothiazolidine-4-carboxylic acid (OTC) prevented these toxic effects of precocene II. In contrast, pretreatment of rats with DL-buthionine-SR-sulfoximine (BSO), an inhibitor of glutathione synthesis, potentiated the toxicity of an otherwise non-toxic dose of precocene II (100 mg/kg). These results indicate that glutathione is important for protection from precocene II-induced hepatotoxicity.  相似文献   

8.
Drug-induced organ injury is a multifaceted process, involving numerous cell types and mediators, and remains a significant safety issue in pharmaceutical development and clinical therapy. Organotypic in vitro models, including precision-cut tissue slices, possess the multi-cellular, structural and functional features of in vivo tissue to facilitate the elucidation of mechanisms of drug-induced organ injury and to characterize species susceptibilities. This study reviews diclofenac-induced hepatotoxicity and presents data comparing the metabolism, specific binding of diclofenac products to cellular proteins and the effects on liver function in rat, monkey and human liver slices. Concentration- and time-dependent increases in specific protein binding demonstrate the progressive nature of the toxicity. Altered liver function correlated with the species differences in the extent of diclofenac metabolism (rat > monkey or human). Liver injury was not detectable within 24 h, unlike specific protein binding, yet it developed by 48 h and lower concentrations of diclofenac exhibited effects by 72 h, demonstrating that continued metabolism and the accumulation of specific protein binding could lead to altered cell function. The decline of liver slice ATP levels at concentrations not affecting GSH levels implicates mitochondrial dysfunction as a primary indicator of hepatotoxicity, of which oxidative stress may be a contributing cause. Diclofenac affected monkey liver slices function at similar concentrations as rat liver slices, while human liver slices exhibited less extensive specific protein binding and required higher diclofenac concentrations to alter cell function.  相似文献   

9.
Precocene II (6,7-dimethoxy-2,2-dimethylchromene), the most active anti-juvenile hormone isolated from Ageratum houstonianum, has been shown to be hepatotoxic in male Sprague-Dawley rats. A single 300-mg/kg dose of precocene II administered via i.p. injection caused extensive necrosis of parenchymal cells in the hepatic centrolobular areas. Liver functions were markedly affected as shown by the significant increases of glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase in the serum. By means of reversed-phase high pressure liquid chromatography (HPLC), [3H]precocene II was found to be rapidly metabolized in vitro by rat liver microsomes in an NADPH-generating system. Approximately 5% (3.4 nmol/mg protein) of the radioactivity from the [3H]precocene II substrate was covalently bound to the macromolecular pellet at the end of a 15-min incubation period when phenobarbital (PB)-induced microsomes were used. Results obtained from experiments using different incubation systems indicated the involvement of the cytochrome P-450-dependent monooxygenases in the metabolism of precocene II and the concurrent covalent binding. The most predominent metabolite was isolated and accounted for >90% of the radioactivity associated with the ethylacetate-extractable metabolites. Further analysis by mass spectrometry and proton nuclear magnetic resonance (NMR) spectroscopy identified it as a 37 : 63 stereoisomeric mixture of the cis and trans 3,4-dihydrodiols of precocene II. A highly reactive (3,4-epoxy-6,7-dimethyl-2,2-dimethylchromane (precocene-3,4-epoxide) was thus suggested as a crucial metabolic intermediate which may be responsible for the histopathological changes seen in rat liver.  相似文献   

10.
The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The Vmax for 6α-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent Km was 90 μM. Cytochrome b5 was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6α-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r2=0.97) and testosterone 6β-hydroxylation (r2=0.9). There was also a strong correlation between 6α-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6β-hydroxylation (r2=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6α-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 μM concentration. Other inhibitors, such as α-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent Km for 6α-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 μM). This might give an explanation for the limited formation of 6α-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.  相似文献   

11.
Five synthetic compounds analogous to pyrrolizidine alkaloids have been tested for toxicity in rats. These were the bis-N-ethylcarbamate esters of synthanecines A, B, C and D (Compounds I–IV) and the bis-diethylphosphate ester (V) of synthanecine A. The amino alcohol moiety in each of these had a single 5-membered heterocyclic ring in place of the pyrrolizidine amino alcohol (necine) moiety of natural pyrrolizidine alkaloids.The toxicity of these compounds differed considerably. The synthanecine A carbamate (I) was the most toxic, male and female rats being similarly susceptible. Like many hepatotoxic pyrrolizidine alkaloids, a single dose of compound I caused acute centrilobular necrosis of the liver, chronic hepatotoxicity involving the development of persistent giant hepatocytes, and chronic lung injury. Compound III had similar actions but was less toxic. The synthanecine D carbamate (IV) caused acute liver necrosis but no chronic hepatotoxicity, whereas the synthanecine A phosphate (V) had the opposite effect, with only chronic hepatotoxicity.The different toxic effects were related to the structure and metabolism of the compounds. Doses of compounds I, III and IV associated with a similar degree of acute hepatotoxicity led to similar levels of pyrrolic metabolites in the liver. Compound II, which was not hepatotoxic, gave very little liver pyrrole. The liver level of pyrrolic metabolite from the phosphate ester (V) decreased more rapidly than that from (I), and was not associated with acute toxicity.Antimitotic activity, indicated by the appearance of bizarre giant cells, was shown by compounds capable of forming pyrrolic metabolites which were bifunctional alkylating agents, but not by compound IV, which could only form a monofunctional alkylating agent. Pretreatment with phenobarbitone lowered the susceptibility of rats to compound I and greatly increased the liver level of pyrrolic metabolites associated with acute hepatotoxicity. Some rats given compounds I and III had kidney lesions primarily involving the glomerulus. The results confirm that toxic effects characteristic of many natural pyrrolizidine alkaloids can be reproduced using simplified synthetic analogues, and that such toxicity is associated with pyrrolic metabolites.  相似文献   

12.
Amitriptyline, the most widely used tricyclic antidepressant, has been associated with very rare but severe incidences of hepatotoxicity in patients. While the mechanism of idiosyncratic hepatotoxicity remains unknown, it is proposed that metabolic activation of amitriptyline and subsequent covalently binding of reactive metabolites to cellular proteins play a causative role. Studies were initiated to determine whether amitriptyline undergoes cytochrome P450 (P450)-mediated bioactivation in human liver microsomes to electrophilic intermediates. LC/MS/MS analysis of incubations containing amitriptyline and NADPH-supplemented microsomes in the presence of glutathione (GSH) revealed the formation of GSH conjugates derived from the addition of the sulfydryl nucleophile to hydrated metabolites of amitriptyline and nortriptyline, the major N-dealkylated metabolite of amitriptyline. Formation of GSH conjugates was primarily catalyzed by heterologously expressed recombinant CYP2D6, CYP3A4, CYP3A5, and to a less extent, CYP1A2. Corresponding dihydrodiol metabolites of amitriptyline and nortriptyline were also detected by tandem mass spectrometry. These findings are consistent with a bioactivation sequence involving initial P450-catalyzed oxidation of the aromatic nucleus in amitriptyline to an electrophilic arene oxide intermediate, which is subsequently attacked by glutathione and water yielding the sulfydryl conjugate and the dihydrodiol metabolite, respectively. The results from the current investigation constitute the first report on the cytochrome P450-catalyzed bioactivation of the antidepressants amitriptyline and nortriptyline. It is proposed that the arene oxide intermediate(s) may represent a rate-limiting step in the initiation of amitriptyline and nortriptyline-mediated hepatotoxicity.  相似文献   

13.
14.
Humanin vitro liver preparations — i.e., slices, hepatocyte suspensions, primary hepatocyte cultures and microsomes — are increasingly used in the drug development process. The main applications are prediction of drug metabolite profiles, drug-drug interactions and toxicity. The use of thesein vitro models is limited, however, because of their erratic availability, the absence of validated protocols and the difficulties of extrapolation ofin vitro data to thein vivo situation.  相似文献   

15.
Studies initiated to investigate the expression of cytochrome P450 2E1 (CYP2E1) in rat brain demonstrated low but detectable protein and mRNA expression in control rat brain. Though mRNA and protein expression of CYP2E1 in brain was several fold lower as compared to liver, relatively high activity of N-nitrosodimethylamine demethylase (NDMA-d) was observed in control rat brain microsomes. Like liver, pretreatment with CYP2E1 inducers such as ethanol or pyrazole or acetone significantly increased the activity of brain microsomal NDMA-d. Kinetic studies also showed an increase in the Vmax and affinity (Km) of the substrate towards the brain enzyme due to increased expression of CYP2E1 in microsomes of brain isolated from ethanol pretreated rats. In vitrostudies using organic inhibitors, specific for CYP2E1 and anti-CYP2E1 significantly inhibited the brain NDMA-d activity indicating that like liver, NDMA-d activity in rat brain is catalyzed by CYP2E1. Olfactory lobes exhibited the highest CYP2E1 expression and catalytic activity in control rats. Furthermore, several fold increase in the mRNA expression and activity of CYP2E1 in cerebellum and hippocampus while a relatively small increase in the olfactory lobes and no significant change in other brain regions following ethanol pretreatment have indicated that CYP2E1 induction maybe involved in selective sensitivity of these brain areas to ethanol induced free radical damage and neuronal degeneration.  相似文献   

16.
17.
A method for the primary culture of rat liver cells on collagen-coated dextran microcarriers is described. Ethoxycoumarin deethylase (EOD) activity 24 hr after inoculation was comparable for liver cells cultured on microcarriers and on collagen-coated dishes. Cells were cultured on microcarriers for up to 48 hr and retained 25% of the initial EOD-activity that was seen in freshly isolated liver cells. Microcarrier-attached hepatocytes were cocultured with BALB/c 3T3 cells to study the metabolism-mediated cytotoxicity of cyclophosphamide (CPA). In the absence of hepatocytes, growth of 3T3 cells was not affected by CPA at concentrations up to 3600 M. In coculture with hepatocytes, cytotoxicity of CPA was expressed in a time- and concentration-dependent manner. At high concentrations, CPA slightly depressed the EOD-activity of hepatocytes. Our results indicate that cocultivation of microcarrier-attached rat liver cells with target cells represents a valuable approach to the study of the metabolism-mediated toxicity of xenobiotics in vitro.Abbreviations CPA cyclophosphamide - EOD Ethoxycoumarin deethylase - FBS fetal bovine serum - HBSS Hanks' balanced salt solution - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - PASA Na-pyruvate, asparagine, serine, alanine - 7-HC 7-hydroxycoumarin This work was presented in part at the VIIIth Scandinavian Workshop on In Vitro Toxicology, Kongsvoll, Norway (1990).  相似文献   

18.
19.

Background

In recent years, there has been a growing interest to explore the association between liver injury and diabetes. Advanced glycated end product (AGE) formation which characterizes diabetic complications is formed through hyperglycemia mediated oxidative stress and is itself a source for ROS. Further, in VL-17A cells over-expressing ADH and CYP2E1, greatly increased oxidative stress and decreased viability have been observed with high glucose exposure.

Methods

In VL-17A cells treated with high glucose and pretreated with the different inhibitors of ADH and CYP2E1, the changes in cell viability, oxidative stress parameters and formation of AGE, were studied.

Results

Inhibition of CYP2E1 with 10 μM diallyl sulfide most effectively led to decreases in the oxidative stress and toxicity as compared with ADH inhibition with 2 mM pyrazole or the combined inhibition of ADH and CYP2E1 with 5 mM 4-methyl pyrazole. AGE formation was decreased in VL-17A cells when compared with HepG2 cells devoid of the enzymes. Further, AGE formation was decreased to the greatest extent with the inhibitor for CYP2E1 suggesting that high glucose inducible CYP2E1 and the consequent ROS aid AGE formation.

Conclusions

Thus, CYP2E1 plays a pivotal role in the high glucose induced oxidative stress and toxicity in liver cells as observed through direct evidences obtained utilizing the different inhibitors for ADH and CYP2E1.

General significance

The study demonstrates the role of CYP2E1 mediated oxidative stress in aggravating hyperglycemic insult and suggests that CYP2E1 may be a vital component of hyperglycemia mediated oxidative injury in liver.  相似文献   

20.
Aconitine, a major Aconitum alkaloid, is well known for its high toxicity that induces severe arrhythmias leading to death. The current study investigated the metabolism of aconitine and the effects of selective cytochrome P450 (CYP) inhibitors on the metabolism of aconitine in rat liver microsomes. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MS(n)) and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. Various selective inhibitors of CYP were used to identify the isoforms of CYP, that involved in the metabolism of aconitine. A total of at least six metabolites were found and characterized in rat liver microsomal incubations. Result showed that the inhibitor of CYP 3A had an inhibitory effect on aconitine metabolism in a concentration-dependant manner, the inhibitor of CYP1A1/2 had a modest inhibitory effect, whereas inhibitors of CYP2B1/2, 2D and 2E1 had no obvious inhibitory effects on aconitine metabolism. Aconitine might be metabolized by CYP 3A and CYP1A1/2 isoforms in rat liver microsome.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号