The effect of phenobarbitone on adult rat liver cells in primary culture

Phenobarbitone (PB) was toxic to rat liver fibroblasts within the concentration range 0.1–10 mM but was toxic to rat hepatocytes only at concentrations greater than 2 mM. This increase in cytotoxicity coincided with a decline in the PB-mediated inducibility of the microsomal monooxygenase (MMO) system. Benzanthracene (BA) was not cytotoxic to either cell type at maximal inducing concentration. Exposure of the cells to a mixture of PB + BA at maximal inducing concentrations produced synergistic toxicity to the fibroblasts, due possibly to PB-mediated induction of BA reactive metabolite formation.     

The effect of Aroclor 1254 pretreatment on the phase I and phase II metabolism of 7-ethoxycoumarin in isolated viable rat kidney cells

The metabolism of 7-ethoxy-and 7-hydroxy-coumarin was studied in viable kidney cortex cells isolated from control and Aroclor 1254-pretreated rats. Such pretreatment led to induction of the microsomal mono-oxygenase-mediated Phase I system but did not produce induction of Phase II glucuronidation or sulphation activity. Induction of the microsomal mono-oxygenase system led to an increase in the amount of unconjugated Phase I metabolite present during sequential Phase I and Phase II metabolism. It is suggested that this increase is due to some form of ‘uncoupling’ of the microsomal mono-oxygenase and glucuronidation systems.     

两种制剂BMP-7的骨诱导活性比较

目的通过试验评价选用不同载体的骨形成蛋白-7(BMP-7)的骨诱导活性。方法选择成年雄性昆明小鼠随机分为三组,其中实验组选用的BMP-7载体为PLA(聚乳酸)和PLA/明胶和对照组为只采用PLA。通过异位成骨实验,对不同制剂BMP-7的骨诱导活性进行分析评价。结果及结论从试验小鼠骨骼Ca含量测定结果可以看出,BMP-7/PLA/明胶组可较早的出现骨诱导活性,且BMP-7/PLA/明胶组比BMP-7/PLA组效价高。     

Relationship between inductions of monooxygenase activity and γ-glutamyltranspeptidase in rat hepatocyte primary cultures

The proposition that changes in activity of γ-glutamyltranspeptidase (GGT) in serum may provide a useful index of the extent of induction of liver drug-metabolizing enzymes by various drugs was examined by comparing control of GGT and monooxygenase activities in cultured hepatocytes. In rat hepatocyte monolayers maintained for up to 5 days the effects of xenobiotics and other factors on cellular GGT activity were compared with effects on a relatively broad measure of drug metabolism, the 7-ethoxycoumarin O-deethylase (ECD) activity of intact cells. A diverse group of drugs including phonobarbital and other barbiturates, diphenylhydantoin, glutethimide, aminopyrine and griseofulvin and the steroids dexamethasone and pregnenolone 16α-carbonitrile were shown to induce both GGT and ECD under comparable culture conditions. Inductions of both activities were potentiated by glucocorticoids and depressed (where tested) by dibutyryl cyclic AMP. Some other hormones or nutrients modulated the activities differently. The magnitude of GGT induction by different drugs did not correlate with relative ECD induction and for several drugs the concentration-dependence of the two effects was different. Interpretation is complicated by the possible contribution of multiple forms of cytochrome P-450 to ECD activity but it seems unlikely that drugs which induce both GGT and drug metabolism do so via a common regulatory mechanism. For such drugs changes in serum GGT could provide only a crude guide to likely changes in drug metabolism. Some compounds including polycyclic hydrocarbons and warfarin induced ECD but had no associated effect on GGT in hepatocytes.     

Relationship of serum triglyceride lowering to changes in hepatic composition induced by different classes of drugs

Dose-related serum trigylceride lowering has been demonstrated in the rat with the microsomal enzyme inducers, phenobarbital, 5,5′-diphenyl-2-thiohydantoin and chlorcyclizine and with the clinically tested anti-hyperlipidemic agents, clofibrate, 1-methyl-4-piperidyl bis(p-chlorophenoxy) acetate (SaH42-348) and 2-methyl-2-[p-(1,2,3,4-tetrahydro-1-naphthyl)-phenxoy] propionic acid (Su-13437). With both classes of drugs, serum lipid lowering was accompanied by hepatomegaly due to an increase in liver protein and phospholipid. Hepatomegaly was reversible upon cessation of drug treatment. The anti-hyperlipidemic drugs caused an increase in microsomal N-demethylase activity but this was slight when compared with the induction produced by known microsomal enzyme inducers. Examination of the subcellular compartments indicated that, even though both mitochondrial protein and phospholipid was increased with all the agents studied, the dominant change in the case of the microsomal enzyme inducers was in the microsomal compartment, whereas the anti-hyperlipidemic agents induced a larger increase in the mitochondrial fraction. The total accumulation of new membrane material, however, was similar for both classes of drugs. These data demonstrate that two classes of drugs which produce hepatomegaly through a proliferation of intracellular membranes also lower serum trigylceride levels.     

Correlation between Changes in Enzymatic Activities and Induction of Different Forms of Rat Liver Microsomal Cytochrome P-450 after Phenobarbital-, 3-Methylcholanthrene- and 16α-Cyanopregnenolone Treatment

Abstract Multiple forms of liver microsomal cytochrome P-450 in rats were identified on SDS-polyacrylamide gels stained for protein and peroxidase activity after induction with phenobarbital, 3-methylcholanthrene, and 16α-cyanopregnenolone. The induced forms were correlated to the in vitro metabolism of biphenyl, benzo(a)pyrene and the steroids 4-androstene-3,17-dione and 5α-androstane-3α,17β-diol. Induction of two forms with apparent molecular weights of 54,000 (RLvMc P-450₅₄) and 50,000 (RLvMc P-45 0₅₀) was obtained with phenobarbital, induction of RLvMc P-450₅₅ and RLvMc P-450₅₈ with 3-methylcholanthrene and induction of RLvMc P-450₅₄ with 16α-cyanopregnenolone. The RLvMc P-450₅₀ was mainly associated with the formation of benzo(a)pyrene-4,5-dihydrodiol, and 7α-hydroxy-4-androstene-3,17-dione. The RLvMc P-450₅₅ and/or the RLvMc P-450₅₈ was mainly associated with the formation of 2- and 3-hydroxybiphenyl and benzo(a)pyrene-7,8-dihydrodiol and RLvMc P-450₅₄ was to some extent associated with the formation of benzo(a)pyrene-4,5-dihydrodiol and several metabolites of 5α-androstane-3α, 17β-diol. It is suggested that SDS-polyacrylamide gel electrophoresis may be a valuable complement to enzyme assays in evaluating effects of drugs and environmental chemicals on the liver microsomal hydroxylase system.     

Emerging therapeutic strategies in myocardial preservation: focus on ATP-sensitive K channels

The development of cholesterol-lowering drugs, including a statins, bile acid sequestrants and cholesterol absorption inhibitors has expanded the options for cardiovascular prevention. Recent treatment guidelines emphasise that individuals at substantial risk for atherosclerotic coronary heart disease should meet defined lipid targets. Combination therapy with drugs that have different and complementary mechanisms of action is often needed to achieve these goals. Existing approaches to the treatment of hypercholesterolaemia are still ineffective in halting the progression of coronary artery disease in some patients despite combination therapies. Other patients are resistant to, or intolerant of, conventional pharmacotherapy and remain at high-risk of atherosclerotic cardiovascular disease, so that alternative approaches are needed. New agents, including inhibitors of microsomal triglyceride transfer protein (MTP), may play a future role, either alone or in combination, in the treatment of hyperlipidaemias. This review focuses on novel approaches to treat dyslipidaemias via the inhibition of MTP. Patients most suitable for use of MTP inhibitors include those with hepatic hypersecretion of apoB, including the metabolic syndrome, Type 2 diabetes mellitus and familial combined hyperlipidaemia, as well as homozygous and heterozygous familial hypercholesterolaemia. However, certain safety issues with these agents need resolving, particularly fatty liver disease.     

INTERACTION BETWEEN PHENYTOIN AND THEOPHYLLINE IN HEALTHY VOLUNTEERS

1. The effect of phenytoin, 100 mg thrice daily for 3 weeks, on theophylline disposition was studied in eight healthy volunteers. 2. The anticonvulsant significantly reduced the half-life of theophylline and this was associated with an increase in the rate of theophylline clearance. The volume of distribution was not significantly altered. 3. There was no correlation between initial theophylline clearance and its percentage increase after phenytoin pretreatment. 4. Coefficients of variation in theophylline clearance before and after phenytoin pretreatment were similar. 5. It is suggested that phenytoin induces theophylline metabolism, that initial theophylline metabolism is not a determinant of the extent of such induction, and that there is a similar potential for induction among healthy volunteers.     

Unique behaviour of benzene mono-oxygenase: activation by detergent and different properties of benzene- and phenobarbital-induced mono-oxygenase activities.

The benzene mono-oxygenase present in liver microsomes from untreated rats was activated 3.5-fold by the detergent Renex 690. This is the first report of an activation of a microsomal mono-oxygenase by detergent. Metyrapone also activated benzene mono-oxygenase, albeit not as strongly (1.5-fold). Treatment of the rats with benzene or phenobarbital induced the benzene mono-oxygenase activity about 3-fold. The benzene-induced form was also activated by the detergent and by metyrapone. However, the phenobarbital-induced activity was inhibited by both modulators.When the mono-oxygenase activity was measured with 7-ethoxycoumarin as a model substrate, which is accepted by many microsomal mono-oxygenases, the phenobarbital-induced activity was inhibited strongly (～ 80 per cent) by metyrapone or Renex 690; however, both the benzene-induced and the control activity were inhibited only weakly (～ 30 per cent).The results provide evidence for substantially different properties of the phenobarbital-induced mono-oxygenase(s) compared to control mono-oxygenase(s). Moreover, benzene appears to be a type of inducer different from the known types and of special interest since, whether assayed with benzene or 7-ethoxycoumarin as substrates, and Renex 690 or metyrapone as modulators, the benzene-induced mono-oxygenase activity possesses characteristics resembling the controls rather than that induced by phenobarbital.     

Decreased hepatic microsomal cytochrome P450 due to indomethacin: Protective roles of 16,16-dimethylprostaglandin F2α and inducing agents

Indomethacin administration to rats caused a dose-dependent decrease in hepatic microsomal cytochrome P450, aminopyrine N-demethylase, ethoxyresorufin O-de-ethylase and benzyloxyresorufin O-debenzylase, accompanied by selective alterations in microsomal sodium dodecylsulphate polyacrylamide gel electrophoretograms. High doses (?8.5 mg/kg) caused the disappearance of certain of the SDS-PAGE proteins tentatively identified as being different forms of cyt. P450, together with either increases, decreases or no change in some of the non-cyt. P450 proteins in the electrophoretogram. Concomitant administration of 16,16-dimethylprostaglandin F_2α gave dose-dependent protection against the deleterious effects of indomethacin on the enzymic and electrophoretic parameters of cyt. P450, but did not prevent the changes due to indomethacin in the non-cyt. P450 proteins on the electrophoretogram. In contrast, prior phenobarbitone or 3-methylcholanthrene induction prevented the effects of indomethacin on both cyt. P450 and the other microsomal proteins. Concomitant administration of SKF-525A exacerbated the effects of indomethacin on cyt. P450 and the other proteins. Indomethacin coadministration with 3-methylcholanthrene resulted in the major 3MC-induced putative cyt. P450 apoprotein having a lower mol. wt than usual. Conversely, indomethacin did not prevent the induction by SKF-525A of a different putative cyt. P450 apoprotein, despite causing decreases in cyt. P450 as determined spectrophotometrically and enzymologically. The results indicate that indomethacin rather than one of its metabolites is responsible for the decrease in cyt. P450 and that the mechanisms of protection by prostaglandin and inducing agents are, respectively, different.     

Differences between small and large intestine and liver in the inducibility of microsomal enzymes in response to stimulation by phenobarbitone and betanaphthoflavone in the diet

Rats were fed either sodium phenobarbitone (PB) or betanaphthoflavone (BNF) for seven days. Deethylation of 7-ethoxyresorufin ( 7ERR ) and 7-ethoxycoumarin ( 7EC ) was measured in small and large intestine and liver, and cytochrome P-450 in liver. Our semi-purified diet was shown to produce minimal levels of intestinal deethylation activity. BNF was added to the semi purified diet and fed at levels from 0.1 to 100 mg BNF/kg of diet. Significant (P less than 0.05) induction of deethylation in small intestine was seen at all dose levels, ranging from 2-fold at 0.1 mg/kg diet to greater than 100-fold at 100 mg/kg diet. A 3-fold increase was also seen in the large intestine at 50 mg/kg. A significant increase in hepatic deethylation was only seen at 100 mg/kg. PB was administered in drinking water at 50, 100 and 1000 mg PB/l. Significant (P less than 0.05) induction of hepatic deethylation was seen at all dose levels, ranging from 2-fold at 50 mg/l to 5-fold at 1000 mg/l. Hepatic cytochrome P450 was also increased. No significant increase in intestinal deethylation was seen at any of the doses used.     

Nicotine metabolism in isolated perfused lung and liver of phenobarbital- and benzoflavone-treated rats

The kinetics of nicotine elimination was investigated in isolated perfused lung and liver of phenobarbital (PB)- and 5,6-benzoflavone (BF)-pretreated rats. The estimated kinetic parameters demonstrated a high nicotine elimination rate in rat lung approaching the capacity of liver when both organs were in an uninduced state. The concentration-time profiles of cotinine as the main metabolite were almost identical for isolated lung and liver. In both organs the cotinine plasma concentrations reached a plateau level after 60 min of perfusion. Pretreatment of rats with 5,6-benzoflavone did not affect the rate of nicotine elimination and cotinine formation either in the lung or in the liver. Phenobarbital treatment, however, induced nicotine clearance in lung approximately 2-fold. This effect is quantitatively lower than the PB-related 8-fold induction of hepatic nicotine elimination observed in a previous study. The present results also indicate that the turnover of cotinine is markedly enhanced after PB induction. The elimination half-lives and clearance values for cotinine as the substrate were approximately 10-fold increased in rat liver after PB pretreatment. Thus, an important contribution of extrahepatic tissues to nicotine metabolism in rats has to be assumed. Moreover, since cotinine elimination is significantly increased after PB induction it is questionable whether cotinine plasma concentrations can further be used as suitable parameter for nicotine consumption.This study was supported by a grant from the German Council on Smoking and Health (Forschungsrat Rauchen und Gesundheit), Hamburg, FRG     

Review: Exploring anticarcinogenic agents in a rat hepatocarcinogenesis model--focus on selenium and statins

In this review, we describe a rat model for chemically induced hepatocarcinogenesis that can be used for studying the anticarcinogenic effects of different agents. In this model the process of carcinogenesis can be followed through the different stages of initiation, promotion and progression. Mechanistic studies of anticarcinogenic agents can be carried out and two examples are given by studies on selenium and statins as anticarcinogenic agents. These compounds suppress cancer via different mechanisms. In the case of selenium the induction of glutathione peroxidase 4 and inhibition of lipid peroxidation might be a part of the anticarcinogenic effect. In the case of statins, the inhibition of ubiquinone synthesis, as well as of the selenium-containing enzyme thioredoxin reductase 1 (TrxR1) might explain their anticarcinogenic properties. Interestingly, also in the case of selenium the inhibited carcinogenesis was associated with reduced TrxR activity, indicating an important role for this enzyme in carcinogenesis.     

Lack of evidence of carcinogenicity of technical-grade piperonyl butoxide in F344 rats: selective induction of ileocaecal ulcers

The carcinogenicity of technical-grade piperonyl butoxide was studied in F344/DuCrj rats fed a dietary level of 0.5 or 1% for 2 yr. Various tumours were detected in all groups, including the untreated control group, but no significant dose-related increase in the incidence of any tumour was found. Thus, it is concluded that under these experimental conditions piperonyl butoxide was not carcinogenic in F344 rats. Unexpectedly, however, ileocaecal ulcers were found in animals of both sexes in both experimental groups and the incidence was dose related. Further studies are required to establish the mechanism of induction of ileocaecal ulcers by piperonyl butoxide.     

Application and interpretation of hPXR screening data: Validation of reporter signal requirements for prediction of clinically relevant CYP3A4 inducers

A human pregnane X receptor (PXR) reporter-gene assay was established and validated using 19 therapeutic agents known to be clinical CYP3A4 inducers, 5 clinical non-inducers, and 6 known inducers in human hepatocytes. The extent of CYP3A4 induction (measured as RIF ratio in comparison to rifampicin) and EC50 was obtained from the dose-response curve. All of the clinical inducers (19/19) and human hepatocyte inducers (6/6) showed positive responses in the PXR assay. One out of five clinical non-inducers, pioglitazone, also showed a positive response. An additional series of 18 commonly used drugs with no reports of clinical induction was also evaluated as putative negative controls. Sixteen of these were negative (89%), whereas two of these, flutamide and haloperidol showed 16-fold (RIF ratio 0.79) and 10-fold (RIF ratio 0.48) maximal induction, respectively in the reporter-gene system. Flutamide and haloperidol were further demonstrated to cause CYP3A4 induction in human cryopreserved hepatocytes based on testosterone 6beta-hydroxylation activity. The induction potential index calculated based on the maximum RIF ratio, EC50, and in vivo maximum plasma concentration was used to predict the likelihood of CYP3A4 induction in humans. When the induction potential index is greater than 0.08, the compound is likely to cause induction in humans. A high-throughput screening strategy was developed based on the validation results at 1microM and 10microM for the same set of drugs. A RIF ratio of 0.4 was set as more practical screening cut-off to minimize the possibility of generating false positives. Thus, a tiered approach was implemented to use the human PXR reporter-gene assay from early lead optimization to late lead characterization in drug discovery.     

Induction of hepatic microsomal drug-metabolizing enzymes by inhibitors of 5-lipoxygenase (5-LO): studies in rats and 5-LO knockout mice.

The effect of 5-lipoxygenase (5-LO) inhibitors on the hepatic microsomal mixed-function oxidase (MFO) system of rodents was investigated. After establishing the relative in vitro and in vivo potencies of the 3 test compounds, male Crl:CD (SD) BR rats received CJ-11,802 (0, 10, 50, or 200 mg/kg/day), zileuton (0, 10, 60, or 300 mg/kg/day) or ZD2138 (0 or 200 mg/kg/day) once daily by oral gavage for 14 (zileuton and ZD2138) or 30 (CJ-11,802) consecutive days. Controls were given an equivalent volume of 0.5% methylcellulose vehicle. At necropsy, all livers were weighed, and sections from representative animals (control and highest dose for each compound) were utilized to prepare hepatic microsomal fractions, which were assayed for cytochrome P-450 (CYP) content and the activities of cytochrome c reductase (CRed), para-nitroanisole O-demethylase (p-NOD), ethoxyresorufin O-deethylase (EROD), and pentoxyresorufin O-dealkylase (PROD). A dose-related increase in liver weight occurred in rats given CJ-11,802 and zileuton, while animals administered ZD2138 were unaffected. Rats given CJ-11,802 (200 mg/kg/day) and zileuton (300 mg/kg/day) had increases in CYP, EROD, PROD, CRed and p-NOD compared to corresponding controls, while only the latter two activities were elevated in animals administered ZD2138. To determine if induction of the hepatic microsomal MFO system was related to 5-LO inhibition, male DBA wild-type and 5-LO knockout mice were administered either CJ-11,802 (200 mg/kg/day) or vehicle by oral gavage for 14 consecutive days. At necropsy, liver weight, CYP content, and CRed activity were measured and all were increased similarly in the treated wild-type and knockout mice compared to corresponding controls, indicating that induction was not related to inhibiting 5-LO.     

Bik subcellular localization in response to oxidative stress induced by chemotherapy,in Two different breast cancer cell lines and a Non‐tumorigenic epithelial cell line

Cancer chemotherapy remains one of the preferred therapeutic modalities against malignancies despite its damaging side effects. An expected outcome while utilizing chemotherapy is apoptosis induction. This is mainly regulated by a group of proteins known as the Bcl‐2 family, usually found within the endoplasmic reticulum or the mitochondria. Recently, these proteins have been located in other sites and non‐canonic functions have been unraveled. Bik is a pro‐apoptotic protein, which becomes deregulated in cancer, and as apoptosis is associated with oxidative stress generation, our objective was to determine the subcellular localization of Bik either after a direct oxidative insult due to H₂O₂, or indirectly by cisplatin, an antineoplastic agent. Experiments were performed in two human transformed mammary gland cell lines MDA‐MB‐231 and MCF‐7, and one non‐tumorigenic epithelial cell line MCF‐10A. Our results showed that in MCF‐7, Bik is localized within the cytosol and that after oxidative stress treatment it translocates into the nucleus. However, in MDA‐MB‐231, Bik localizes in the nucleus and translocates to the cytosol. In MCF10A Bik did not change its cellular site after either treatment. Interestingly, MCF10A were more resistant to cisplatin than transformed cell lines. This is the first report showing that Bik is located in different cellular compartments depending on the cancer stage, and it has the ability to change its subcellular localization in response to oxidative stress. This is associated with increased sensitivity when exposed to toxic agents, thus rendering novel opportunities to study new therapeutic targets allowing the development of more active and less harmful agents. Copyright © 2015 John Wiley & Sons, Ltd.