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A high-resolution screening (HRS) technology is described, which couples 2 parallel enzyme affinity detection (EAD) systems for substrates and inhibitors of rat cytosolic glutathione-S-transferases (cGSTs) and purified human GST P1 [...]

A high-resolution screening platform, coupling online affinity detection for mammalian cytochrome P450s (Cyt P450s) to gradient reversed-phase high-performance liquid chromatography (HPLC), is described. To this end, the online Cyt P450 [...]

The estrogenic mycotoxin zearalenone (ZEN) can undergo hepatic reductive metabolism to form the estrogenic α and β isomers of zearalenol. ZEN also undergoes cytochrome P450 monooxygenase (P450)-mediated oxidative metabolism to form monohydroxylated products, but until now nothing is known about the estrogenic potency of these metabolites. This study aimed at investigating the metabolism of ZEN by different P450 isoforms and to determine the estrogen receptor α (ERα) affinities of the in vitro P450-generated ZEN metabolites in an online high-resolution screening (HRS) setup. Human liver microsomes (HLM), recombinant P450s, and mutants of the bacterial P450 BM3 were used to investigate the oxidative metabolism of ZEN. It was shown that mutants of the bacterial P450 BM3 could be used to produce the human relevant 13- and 15-OH-ZEN catechol metabolites at such levels that their ERα affinity could be determined in an HRS setup, which was not possible with HLM. It was demonstrated that P450-mediated hydroxylation at the 13 and 15 positions of ZEN resulted in a loss of ERα affinity. The approach presented here can be used for the elucidation of the metabolism of other endocrine disrupting compounds and xenobiotics to get clear pictures of the total effects of these compounds and their metabolites. [ABSTRACT FROM AUTHOR]

Abstract: Inhibition of cytochrome P450s (CYPs) is a major cause of adverse drug-drug interactions. Alternatively, inhibition of glutathione S-transferases (GSTs) may increase harmful effects of electrophilic compounds or metabolites. In the present study, aqueous extracts of seven Ghanaian medicinal plants were investigated for their inhibitory potential towards recombinant human CYP1A2, CYP2C9, CYP2D6 and CYP3A4, heterologously expressed in Escherichia coli. Effects of these extracts on recombinant human GSTA1-1, GSTM1-1, GSTP1-1, human and rat cytosolic GSTs were also investigated. Seven extracts, including Phyllanthus amarus whole plant, leaf, stem and root, Cassia siamea and Momordica charantia, inhibited CYP1A2 and CYP2C9 with IC50 values ranging between 28.3–134.3μg/ml and between 63.4–425.9μg/ml, respectively. Similarly, both CYP2D6 and CYP3A4 were inhibited by five extracts including Phyllanthus amarus whole plant, leaf, stem and root and Cassia alata, with IC50 values ranging between 45.8–182.0μg/ml and between 79.2–158.8μg/ml respectively. Human and rat liver cytosolic GSTs were inhibited with IC50 values ranging between 25.2–95.5μg/ml and between 8.5–139.4μg/ml, respectively. GSTM1-1 was most susceptible to the inhibition by the extracts, with IC50 values ranging between 3.6–50.0μg/ml, whilst IC50 values of 8.9–159.0μg/ml and 68.6–157.0μg/ml were obtained for GSTA1-1 and GSTP1-1, respectively. These findings show a significant potential both for CYP- and GST-mediated herb–drug interactions of the Ghanaian medicinal plants investigated. [Copyright &y& Elsevier]

Abstract: Curcumin (diferuloylmethane) is a major yellow pigment and dietary component derived from Curcuma longa. It has potent anti-inflammatory, anticarcinogenic, antioxidant and chemoprotective activities among others. We studied the interactions of curcumin, a mixture of its decomposition products, and four of its individually identified decomposition products (vanillin, vanillic acid, ferulic aldehyde and ferulic acid) on five major human drug-metabolizing cytochrome P450s (CYPs). Curcumin inhibited CYP1A2 (IC50, 40.0μM), CYP3A4 (IC50, 16.3μM), CYP2D6 (IC50, 50.3μM), CYP2C9 (IC50, 4.3μM) and CYP2B6 (IC50, 24.5μM). Curcumin showed a competitive type of inhibition towards CYP1A2, CYP3A4 and CYP2B6, whereas a non-competitive type of inhibition was observed with respect to CYP2D6 and CYP2C9. The inhibitory activity towards CYP3A4, shown by curcumin may have implications for drug–drug interactions in the intestines, in case of high exposure of the intestines to curcumin upon oral administration. In spite of the significant inhibitory activities shown towards the major CYPs in vitro, it remains to be established, whether curcumin will cause significant drug–drug interactions in the liver, given the reported low systemic exposure of the liver to curcumin. The decomposition products of curcumin showed no significant inhibitory activities towards the CYPs investigated, and therefore, are not likely to cause drug–drug interactions at the level of CYPs. [Copyright &y& Elsevier]

In order to more rationally design thiourea-containing drugs and drug candidates, specifically thiourea-containing histamine H3 receptor antagonists, it is necessary to develop structure–toxicity relationships (STRs). For this purpose, the cytotoxicity of a series of thiourea-containing compounds was tested in precision-cut rat liver slices. A concentration of 1000 μM of N-p-bromophenyl, N′-(4-imidazole-ethyl)thiourea (8) or N-p-nitrophenyl, N′-(4-imidazole-ethyl)thiourea (9) was found to cause cytotoxicity, evidenced as LDH leakage, resulting in more than 95% LDH leakage after 6 h. N-p-Methoxyphenyl, N′-(4-imidazole-ethyl)thiourea (6) caused 40.6±19.7% LDH leakage after 6 h. Control levels of cell death (1% methanol as control vehicle) were below 20% in 6 h. After 6 h of exposure, N-p-chlorophenyl, N′-(4-imidazole-ethyl)thiourea (7), 8, and 9 were already found to cause significant cytotoxicity at a concentration of 100 μM. At 200 μM, 9 was found to cause significantly more cytotoxicity than 7 and 8. N-Naphthyl, N′-(4-imidazole-ethyl)thiourea (12) was found to cause significant cytotoxicity towards precision-cut rat liver slices after 6 h of exposure to a concentration of 500 μM. All other N-substituted, N′-(4-imidazole-ethyl)thiourea tested in this study were not found to be cytotoxic towards precision-cut rat liver slices within the 6 h of exposure up to a concentration of 1000 μM. Intracellular glutathione (GSH) content and mitochondrial MTT reduction activity were also examined after exposure of slices to N-substituted, N′-(4-imidazole-ethyl)thiourea. Both of these markers, however, were not found to provide additional information regarding the possible mechanisms of cytotoxicity, i.e. GSH depletion or reduced mitochondrial activity since these markers did not clearly precede LDH leakage.A correlation was found between cytotoxicity towards precision-cut rat liver slices and Vmax/Km values for the formation of sulfenic acids from N-substituted N′-(4-imidazole-ethyl)thiourea by hepatic rat flavin-containing monooxygenases (FMO). The compound with the highest Vmax/Km value for the formation of sulfenic acids, 9, was also the most cytotoxic. Compounds with a significantly lower Vmax/Km value, 7, 8, and 12, were less cytotoxic than 9. Compounds with a Vmax/Km value for the formation of sulfenic acids lower than 0.0788 ml/(min mg) were found not to be cytotoxic towards precision-cut rat liver slices for concentrations up to 1000 μM at an exposure time of 6 h.It is concluded, from this study, that N-phenyl substituted N′-(4-imidazole-ethyl)thiourea-containing electron-withdrawing p-substituents are cytotoxic towards precision-cut rat liver slices. Cytotoxicity is increased with increasing electron-withdrawing capacity of the p-substituent. A correlation was found to exist between Vmax/Km value for the formation of sulfenic acids by rat liver FMO enzymes and cytotoxicity. [Copyright &y& Elsevier]

Fotemustine is a clinically used DNA-alkylating 2-chloro-ethyl-substituted N-nitrosourea, which sometimes shows signs of haematotoxicity and reversible liver and renal toxicity as toxic side-effects. Mechanistic data on these side-effects are scarce and incomplete. In this study, firstly the cytotoxicity of fotemustine in freshly isolated rat hepato-cytes was investigated and secondly the metabolism of fotemustine and possible mechanisms involved in the observed cytotoxicity. Fotemustine caused concentration-and time-dependent cytotoxic effects in rat hepatocytes. Extensive GSH-depletion and formation of GSSG were first observed, followed by lipid peroxidation and finally by cell death measured as LDH-leakage. 2-Chloroethyl analogues of fotemustine, which in contrast to fotemustine have no carbamoylating potency, were not toxic to rat hepatocytes. The data suggest that the cytotoxicity of fotemustine is resulting from its reactive decompostition product, DEP-isocyanate. GSH-conjugation of DEP-iso-cyanate was shown to protect against the cytotoxicity of fotemustine, however, onlytemporary and not completely. Synthetical DEP-SG, the GSH-conjugate of DEP-iso-cyanate, was also toxic to rat hepatocytes,albeit to a significantly lesser extent than fotemustine. In rat liver microsomes, no fotemustine-induced LPO was observed, suggesting that reactive decomposition products of fotemustine do not directly cause peroxidation of cellular membranes.Fotemustine did not affect the antioxidant enzymes superoxide dismutase, catalase, GSH-peroxidase, GSSG-reductase and GSH S-transferases. Thus, direct effects on these antioxidant enzymes are not likely to explain the cytotoxic effects of fotemustine in hepatocytes. In conclusion, it is proposed that the cytotoxicity of fote mustine in rat hepatocytes is caused byrapid and extensive depletion of GSH by DEP-isocyanate, a reactive decomposition product of fotemustine, consequently hampering the endogenous protection against its own toxicity. Knowledge of molecular mechanisms of the cytotoxicity of fotemustine may contribute to a more rational design of selective protection against toxic side-effects which occur upon theraphyof patients with fotemustine. [ABSTRACT FROM PUBLISHER]

Modification of cellular macromolecules by reactive drug metabolites is considered to play an important role in the initiation of tissue injury by many drugs. Detection and identification of reactive intermediates is often performed by analyzing the conjugates formed after trapping by glutathione (GSH). Although sensitivity of modern mass spectrometrical methods is extremely high, absolute quantification of GSH-conjugates is critically dependent on the availability of authentic references. Although 1 H NMR is currently the method of choice for quantification of metabolites formed biosynthetically, its intrinsically low sensitivity can be a limiting factor in quantification of GSH-conjugates which generally are formed at low levels. In the present study, a simple but sensitive and generic method for absolute quantification of GSH-conjugates is presented. The method is based on quantitative alkaline hydrolysis of GSH-conjugates and subsequent quantification of glutamic acid and glycine by HPLC after precolumn derivatization with o-phthaldialdehyde/N-acetylcysteine (OPA/NAC). Because of the lower stability of the glycine OPA/NAC-derivate, quantification of the glutamic acid OPA/NAC-derivate appeared most suitable for quantification of GSH-conjugates. The novel method was used to quantify the concentrations of GSH-conjugates of diclofenac, clozapine and acetaminophen and quantification was consistent with 1 H NMR, but with a more than 100-fold lower detection limit for absolute quantification. [ABSTRACT FROM AUTHOR]