Effects of Sublethal Exposure to a Glyphosate-Based Herbicide Formulation on Metabolic Activities of Different Xenobiotic-Metabolizing Enzymes in Rats.

The activities of different xenobiotic-metabolizing enzymes in liver subcellular fractions from Wistar rats exposed to a glyphosate (GLP)-based herbicide (Roundup full II) were evaluated in this work. Exposure to the herbicide triggered protective mechanisms against oxidative stress (increased glutathione peroxidase activity and total glutathione levels). Liver microsomes from both male and female rats exposed to the herbicide had lower (45%-54%, P < 0.01) hepatic cytochrome P450 (CYP) levels compared to their respective control animals. In female rats, the hepatic 7-ethoxycoumarin O-deethylase (a general CYP-dependent enzyme activity) was 57% higher (P < 0.05) in herbicide-exposed compared to control animals. Conversely, this enzyme activity was 58% lower (P < 0.05) in male rats receiving the herbicide. Lower (P < 0.05) 7-ethoxyresorufin O-deethlyase (EROD, CYP1A1/2 dependent) and oleandomycin triacetate (TAO) N-demethylase (CYP3A dependent) enzyme activities were observed in liver microsomes from exposed male rats. Conversely, in females receiving the herbicide, EROD increased (123%-168%, P < 0.05), whereas TAO N-demethylase did not change. A higher (158%-179%, P < 0.01) benzyloxyresorufin O-debenzylase (a CYP2B-dependent enzyme activity) activity was only observed in herbicide-exposed female rats. In herbicide-exposed rats, the hepatic S-oxidation of methimazole (flavin monooxygenase dependent) was 49% to 62% lower (P < 0.001), whereas the carbonyl reduction of menadione (a cytosolic carbonyl reductase-dependent activity) was higher (P < 0.05). Exposure to the herbicide had no effects on enzymatic activities dependent on carboxylesterases, glutathione transferases, and uridinediphospho-glucuronosyltransferases. This research demonstrated certain biochemical modifications after exposure to a GLP-based herbicide. Such modifications may affect the metabolic fate of different endobiotic and xenobiotic substances. The pharmacotoxicological significance of these findings remains to be clarified.

Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes. Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate oxidation.

7-Ethoxy (OEt) coumarin has been used as a model substrate in many cytochrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin O-deethylation in human liver microsomes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuterium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reactions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for O-demethylation, 6.1 for O-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for O-demethylation, 6.1 for O-deethylation) was attenuated in the noncompetitive intermolecular experiments. High noncompetitive intermolecular kinetic isotope effects were seen for 7-OEt coumarin O-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3-8.1 for O-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limiting step applies to all of the systems studied with this model P450 1A2 reaction including human liver microsomes, the most physiologically relevant.

Kinetic analysis of oxidation of coumarins by human cytochrome P450 2A6.

Human cytochrome P450 (P450) 2A6 catalyzes 7-hydroxylation of coumarin, and the reaction rate is enhanced by cytochrome b5 (b5). 7-Alkoxycoumarins were O-dealkylated and also hydroxylated at the 3-position. Binding of coumarin and 7-hydroxycoumarin to ferric and ferrous P450 2A6 are fast reactions (k(on) approximately 10(6) m(-1) s(-1)), and the k(off) rates range from 5.7 to 36 s(-1) (at 23 degrees C). Reduction of ferric P450 2A6 is rapid (7.5 s(-1)) but only in the presence of coumarin. The reaction of the ferrous P450 2A6 substrate complex with O2 is rapid (k > or = 10(6) m(-1) s(-1)), and the putative Fe2+.O2 complex decayed at a rate of approximately 0.3 s(-1) at 23 degrees C. Some 7-hydroxycoumarin was formed during the oxidation of the ferrous enzyme under these conditions, and the yield was enhanced by b5. Kinetic analyses showed that approximately 1/3 of the reduced b5 was rapidly oxidized in the presence of the Fe2+.O2 complex, implying some electron transfer. High intrinsic and competitive and non-competitive intermolecular kinetic deuterium isotope effects (values 6-10) were measured for O-dealkylation of 7-alkoxycoumarins, indicating the effect of C-H bond strength on rates of product formation. These results support a scheme with many rapid reaction steps, including electron transfers, substrate binding and release at multiple stages, and rapid product release even though the substrate is tightly bound in a small active site. The inherent difficulty of chemistry of substrate oxidation and the lack of proclivity toward a linear pathway leading to product formation explain the inefficiency of the enzyme relative to highly efficient bacterial P450s.

Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices.

Hepatocytes are anchorage-dependent cells sensitive to microenvironment; the control of the physicochemical properties of the extra-cellular matrices may be useful to the maintenance of hepatocyte functions in vitro for various applications. In a microcapsule-based 3-D hepatocyte culture microenvironment, we could control the physical properties of the collagen nano-fibres by fine-tuning the complex-coacervation reaction between methylated collagen and terpolymer of hydroxylethyl methacrylate-methyl methacrylate-methylacrylic acid. The physical properties of the nano-fibres were quantitatively characterized using back-scattering confocal microscopy to help optimize the physical support for hepatocyte functions. We further enhanced the chemical properties of the collagen nano-fibres by incorporating galactose onto collagen, which can specifically interact with the asialoglycoprotein receptor on hepatocytes. By correlating a range of collagen nano-fibres of different physicochemical properties with hepatocyte functions, we have identified a specific combination of methylated and galactosylated collagen nano-fibres optimal for maintaining hepatocyte functions in vitro. A model of how the physical and chemical supports interplay to maintain hepatocyte functions is discussed.

Mechanistic studies of 9-ethynylphenanthrene-inactivated cytochrome P450 2B1.

The mechanism of inactivation of the major phenobarbital-inducible cytochrome P450 of rat liver, P450 2B1, by 9-ethynylphenanthrene (9EPh) has been investigated. Matrix-assisted laser desorption ionization-mass spectrometry analysis of the cyanogen bromide-generated peptides from 9EPh-inactivated P450 2B1 confirmed the addition of a phenanthrylacetyl group to the peptide corresponding to residues 290 to 314. When this peptide was further digested with pepsin, the site of attachment could be assigned to one of the amino acids in the peptide Phe297 to Leu307 [Roberts, E. S., Hopkins, N. E., Zaluzec, E. J., Gage, D. A., Alworth, W. L., and Hollenberg, P. F. (1995) Arch. Biochem. Biophys. 323, 000-000]. The inactivation by 9EPh resulted in a 90-95% loss in the NADPH-supported deethylation of 7-ethoxy-4-trifluoromethylcoumarin (EFC), but had no effect on the iodosobenzene- or cumene hydroperoxide-supported metabolism of EFC. The loss of NADPH-supported activity was not affected by the addition of cytochrome b5 or the presence of excess levels of reductase. The magnitude of the Type 1 spectral change upon the addition of benzphetamine was decreased with the 9EPh-modified protein. There was no decrease in the ability of modified 2B1 to form the steady-state level of the CO-reduced complex either enzymatically with NADPH and reductase or chemically with sodium dithionite, but the rate of reduction by reductase under anaerobic conditions was 57% that of native protein in the absence of substrate and 35% that of native protein in the presence of substrate. The 9EPh-modified 2B1 had an overall slower rate of NADPH oxidation, H2O2 formation, and formaldehyde formation during metabolism of benzphetamine compared to native 2B1. The ratio of H2O2 to HCHO was 1.0:1.0 for the native and 1.6:1.0 for the modified protein. The ability of the modified protein to form the steady-state level of the oxygen-iron complex in the presence of cyclohexane was decreased. These results are consistent with the idea that the covalent modification of one of the residues in the peptide Phe297 to Leu307 by the phenanthrylacetyl group impairs the reduction of P450 2B1 by reductase and also causes the uncoupling of NADPH utilization and oxygen consumption from product formation.

Identification of active-site peptides from 3H-labeled 2-ethynylnaphthalene-inactivated P450 2B1 and 2B4 using amino acid sequencing and mass spectrometry.

2-Ethynylnaphthalene (2EN) is a mechanism-based inactivator of rat cytochrome P450 (P450) 2B1 with 1.3 mol of adduct bound per mole of P450 inactivated [Roberts, E.S., Hopkins, N.E., Alworth, W.L., & Hollenberg, P.F. (1993) Chem. Res. Toxicol. 6, 470-479]. Further studies have shown that 2EN is also an efficient mechanism-based inactivator of the 7-ethoxycoumarin O-deethylase activity of rabbit P450 2B4 with 0.83 mol of adduct bound per mole of P450. Cleavage of [3H]2EN-inactivated 2B1 with cyanogen bromide, separation of the peptides by HPLC, and further purification of the radiolabeled fraction by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) led to the identification by autoradiography of a radiolabeled peptide (M(r) approximately 3000). Amino acid sequence analysis of the first 12 N-terminal residues revealed the sequence ISLLSLFFAGTE corresponding to positions 290-301 in the protein. When the radiolabeled fraction from the HPLC separation was analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), peaks at m/z 2722.5 and 2890.6 were detected. The lower mass peak corresponds to the molecular ion (average mass) of the cyanogen bromide peptide Ile290 to Met314 (theoretical 2722.2), while the higher mass peak corresponds to the same peptide with a bound 2-naphthylacetyl group (theoretical 2890.4). When [3H]2EN-inactivated 2B4 was treated with cyanogen bromide, the peptides were separated by HPLC, and the fractions were analyzed by Tricine-SDS-PAGE, two radiolabeled peptides (M(r) = 5000 and 8000) were identified by autoradiography. Amino acid sequence analysis of the first 11 residues revealed identical N-termini with the sequence EKDKSDPSSEF corresponding to positions 273-283.(ABSTRACT TRUNCATED AT 250 WORDS)