Identification and quantitative determination of 3-chloro-2-hydroxypropylmercapturic acid and α-chlorohydrin in urine of rats treated with epichlorohydrin

Epichlorohydrin (ECH) is used in many industrial processes. Different toxic effects of ECH were found in rodents. The metabolism of ECH was investigated before in rats using [¹⁴C]ECH. The aim of this investigation was the development of non-radioactive quantitative analytical methods for measuring two urinary metabolites of ECH, namely 3-chloro-2-hydroxypropylmercapturic acid (CHPMA) and α-chlorohydrin (α-CH). The identity of CHPMA and α-CH excreted in urine of rats treated with 5 to 35 mg/kg ECH was confirmed by GC-MS. The quantitative analysis of CHPMA, involving ethyl acetate extraction from acidified urine and subsequent methylation and analysis by gas chromatography-flame photometric detection (GC-FPD), showed a method limit of detection of 2 μg/ml. The analysis of α-CH, based on ethyl acetate extraction and subsequent analysis by GC-ECD, showed a method limit of detection of 2 μg/ml. CHPMA and α-CH derivatives could be determined quantitatively down to concentrations of 0.5 and 0.4 μg/ml urine, respectively, by selected-ion monitoring GC-MS under EI conditions. Cumulative urinary excretion of CHPMA and α-CH by rats treated with ECH were found to be 31 ± 10 and 1.4 ± 0.6% (n = 13) of the ECH dose, respectively. For CHPMA, the dose-excretion relationship suggested partially saturated ECH metabolism. For α-CH, the dose-excretion relationship was linear. With fractionated urine collection it was found that approximately 74 and 84% of the total cumulative excretion of CHPMA and α-CH, respectively, took place within the first 6 h after administration of ECH. From these investigations it is concluded that the GC-FPD and GC-ECD based methods developed are sufficiently sensitive to measure urinary excretion of CHPMA and α-CH in urine from rats administered 5 to 35 mg/kg ECH. It is anticipated that the analysis of CHPMA and α-CH based on GC-MS may be sufficiently sensitive to investigate urinary excretion from humans occupationally exposed to ECH.     

Aerobic degradation of polychlorinated biphenyls by Alcaligenes sp. JB1: metabolites and enzymes

In contrast to the degradation of penta-and hexachlorobiphenyls in chemostat cultures, the metabolism of PCBs by Alcaligenes sp. JB1 was shown to be restricted to PCBs with up to four chlorine substituents in resting-cell assays. Among these, the PCB congeners containing ortho chlorine substituents on both phenyl rings were found to be least degraded. Monochloro-benzoates and dichlorobenzoates were detected as metabolites. Resting cell assays with chlorobenzoates showed that JB1 could metabolize all three monochlorobenzoates and dichlorobenzoates containing only meta and para chlorine substituents, but not dichlorobenzoates possessing an ortho chlorine substituent. In enzyme activity assays, meta cleaving 2,3-dihydroxybiphenyl 1,2-dioxygenase and catechol 2,3-dioxygenase activities were constitutive, whereas benzoate dioxygenase and ortho cleaving catechol 1,2-dioxygenase activities were induced by their substrates. No activity was found for pyrocatechase II, the enzyme that is specific for chlorocatechols. The data suggest that complete mineralization of PCBs with three or more chlorine substituents by Alcaligenes sp. JB1 is unlikely.Abbreviations PCB polychlorinated biphenyls - CBA chlorobenzoate - D di - Tr tri - Te tetra - Pe penta- - H hexa     

Characterization of biotransformation enzyme activities in primary rat proximal tubular cells

The proximal tubule is a frequent target for nephrotoxic compounds due to it's ability to transport and accumulate xenobiotics and their metabolites, as well as by the presence of an organ-selective set of biotransformation enzymes. The aim of the present study was to characterize the activities of different biotransformation enzymes during primary culturing of rat proximal tubular cells (PT cells). Specific marker substrates for determining cytochrome P450 (CYP450) activity of primary cultured PT cells include 7-ethoxyresorufin (CYP1A1), caffeine (CYP1A), testosterone (CY2B/C, CYP3A), tolbutamide (CYP2C) and dextromethorphan (CYP2D1). Activities of the CYP450 isoenzymes decreased considerably during culture with the greatest loss in activity within 24 h of culture. In addition, expression of CYP450 apoprotein, including CYP1A, CYP2C, CYP2D, CYP2E and CYP4A, was detected in microsomes from freshly isolated PT cells by immunoblotting using specific antibodies. CYP2B and CYP3A apoprotein could not be detected. Activity of the phase II biotransformation enzymes GST, GGT, beta-lyase and UGT was determined with 1-chloro-2,4-dinitrobenzene, L-glutamic acid gamma-(7-amido-4-methyl-coumarin), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine and 1-naphthol, respectively, as marker substrates. Activity of the phase II enzymes remained more stable and, in contrast to CYP450 activity, significant activity was still expressed after 1 week of PT cell culture. Thus, despite the obvious advantages of PT cells as an in-vitro model for studies of biotransformation mediated toxicity, the strong time dependency of especially phase I and, to a lesser extent, phase II biotransformation activities confers limitations to their application.     

Tissue distribution of cytosolic beta-elimination reactions of selenocysteine Se-conjugates in rat and human

Selenocysteine Se-conjugates (e.g. methylselenocysteine) have been shown to be potent chemopreventive and chemoprotective agents, and inducers of apoptosis. Although the mechanism of action remains to be elucidated, beta-elimination of these compounds by beta-lyase enzymes into corresponding selenols, pyruvate and ammonia is thought to be critical. This study describes in vitro beta-lyase activity in nine rat organs using three selenocysteine Se-conjugates and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine. For all substrates the highest beta-elimination rates were found in kidney, followed by liver, while brain, spleen, heart, large and small intestine, thyroid and lung were of minor importance. Since liver plays an important role in beta-elimination, hepatic beta-lyase activity was extensively studied using 23 selenocysteine Se-conjugates and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine and was compared with previously obtained renal beta-lyase data. The results showed that hepatic beta-lyase activities were 4-25-fold lower than the corresponding renal beta-lyase activities. Hepatic beta-elimination of the substrates appeared to be exclusively catalyzed by the pyridoxal 5'-phosphate-dependent beta-lyase enzyme kynureninase. Studies performed with human hepatic cytosols of three individuals showed that hepatic beta-lyase activity was 2-5-fold higher when compared with the previously obtained human renal activity. Significant correlation was obtained between human hepatic beta-lyase activities of three individuals. The relevance of this data for using SeCys-conjugates as chemopreventive and a chemoprotective agent is discussed. Based on the large differences in organ-selective beta-elimination and specific beta-lyase activity between rat and humans, the rat might not be a good model to investigate nephrotoxicity of cysteine S-conjugates, and chemoprevention and chemoprotection of SeCys-conjugates in man.     

Free energies of binding of <Emphasis Type="Italic">R</Emphasis>- and <Emphasis Type="Italic">S</Emphasis>-propranolol to wild-type and F483A mutant cytochrome P450 2D6 from molecular dynamics simulations

Detailed molecular dynamics (MD) simulations have been performed to reproduce and rationalize the experimental finding that the F483A mutant of CYP2D6 has lower affinity for R-propranolol than for S-propranolol. Wild-type (WT) CYP2D6 does not show this stereospecificity. Four different approaches to calculate the free energy differences have been investigated and were compared to the experimental binding data. From the differences between calculations based on forward and backward processes and the closure of thermodynamic cycles, it was clear that not all simulations converged sufficiently. The approach that calculates the free energies of exchanging R-propranolol with S-propranolol in the F483A mutant relative to the exchange free energy in WT CYP2D6 accurately reproduced the experimental binding data. Careful inspection of the end-points of the MD simulations involved in this approach, allowed for a molecular interpretation of the observed differences. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Combining substrate dynamics, binding statistics, and energy barriers to rationalize regioselective hydroxylation of octane and lauric acid by CYP102A1 and mutants

Hydroxylations of octane and lauric acid by Cytochrome P450-BM3 (CYP102A1) wild-type and three active site mutants--F87A, L188Q/A74G, and F87V/L188Q/A74G--were rationalized using a combination of substrate orientation from docking, substrate binding statistics from molecular dynamics simulations, and barrier energies for hydrogen atom abstraction from quantum mechanical calculations. Wild-type BM3 typically hydroxylates medium- to long-chain fatty acids on subterminal (omega-1, omega-2, omega-3) but not the terminal (omega) positions. The known carboxylic anchoring site Y51/R47 for lauric acid, and hydrophobic interactions and steric exclusion, mainly by F87, for octane as well as lauric acid, play a role in the binding modes of the substrates. Electrostatic interactions between the protein and the substrate strongly modulate the substrate's regiodependent activation barriers. A combination of the binding statistics and the activation barriers of hydrogen-atom abstraction in the substrates is proposed to determine the product formation. Trends observed in experimental product formation for octane and lauric acid by wild-type BM3 and the three active site mutants were qualitatively explained. It is concluded that the combination of substrate binding statistics and hydrogen-atom abstraction barrier energies is a valuable tool to rationalize substrate binding and product formation and constitutes an important step toward prediction of product ratios.     

Online biochemical detection of glutathione-S-transferase P1-specific inhibitors in complex mixtures

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 to gradient reversed-phase high-performance liquid chromatography (HPLC). The cGSTs and GST P1 EAD systems were optimized and validated first in flow injection analysis (FIA) mode, and optimized values were subsequently used for HPLC mode. The IC(50) values of 8 ligands thus obtained online agreed well with the IC(50) values obtained with microplate reader-based assays. For ethacrynic acid, an IC(50) value of 1.8 +/- 0.4 microM was obtained with the cGSTs EAD system in FIA mode and 0.8 +/- 0.6 microM in HPLC mode. For ethacrynic acid with the GST P1 EAD system, IC(50) values of 6.0 +/- 2.9 and 3.6 +/- 2.8 microM were obtained in FIA and HPLC modes, respectively. An HRS GST EAD system, consisting of both the cGSTs and the GST P1 EAD system in HPLC mode in parallel, was able to separate complex mixtures of compounds and to determine online their individual affinity for cGSTs and GST P1. Finally, a small library of GST inhibitors, synthesized by reaction of several electrophiles with glutathione (GSH), was successfully screened with the newly developed parallel HRS GST EAD system. It is concluded that the present online gradient HPLC-based HRS screening technology offers new perspectives for sensitive and simultaneous screening of general cGSTs and specific GST P1 inhibitors in mixtures.