Phase 1 and 2 Metabolism in Freshly Isolated Hepatocytes and Subcellular Fractions from Rat,Mouse, Chicken and Ox Livers

In toxicological studies hepatocytes offer an excellent alternative to whole-animal experiments, provided their metabolic competence has been established. We have compared Phase 1 and 2 metabolism in rat, mouse, chicken and ox liver microsomes and cytosol with freshly isolated hepatocytes. The relative amounts of total cytochrome P450 in microsomes and hepatocytes were equivalent. Rat liver had the highest P450 content while chicken liver had the lowest content (148·2(±75·7) and 20·6(±11·5) pmol mg^-1 hepatocellular protein, respectively). The metabolism of testosterone was assessed to determine selective cytochrome P450 isoenzyme activities. Only two metabolite products were common to all four species, namely 6β-hydroxytestosterone (6β-OHT) and androstenedione (ASD), which co-eluted with 6-dehydrotestosterone (6DHT). 16α-OHT was present in all incubations except for ox microsomes. The rate of metabolism of testosterone was generally lower in microsomes than hepatocytes, with the exception of the ox, but the pattern and quantity of metabolite formation was similar. The quantity of total products formed was 15- to 27-fold higher in rat and mouse livers than in chicken or ox. The major product formed in freshly isolated hepatocytes from mice and chickens was ASD/6DHT which accounted for 60% and 76% of the total metabolites, respectively. ASD/6DHT formation accounted for only 33% and 17% of the total metabolites formed by rat and ox hepatocytes, respectively. 2α-OHT production occurred in rat and mouse hepatocytes (14% of the total metabolites in rat and 7% in mouse hepatocytes) but was lacking in chicken or ox cells. The stability of P450 isoforms in culture was species-dependent. Rat and mouse hepatocyte cultures lost 54% and 31% of their initial P450 content after 72 h, while there was no loss in chicken hepatocytes over the same period. There was a good correlation between the relative glutathione S-transferase (GST) activities in cytosol and freshly isolated hepatocytes. Mouse liver exhibited highest GST activity (664·2(±203·5)) compared with rat, chicken or ox (320·4(±64·0), 341·5(±13·9) and 256·3(±109·9) nmol min^-1 mg^-1 cytosolic protein, respectively). © 1997 SCI.     

Stereoselective metabolism of fipronil in water hyacinth (Eichhornia crassipes)

The enantioselective metabolism of racemic fipronil in water hyacinth (Eichhornia crassipes) had been investigated. In this study, the degradation data and the enantiomer fraction (EF) were determined by chiral high-performance liquid chromatography (HPLC) with a column cellulose-tri-(3, 5-dimethylphe-nylcarbamate)-based chiral stationary phase (CDMPC-CSP). During the uptake phase, the EF value of plant sample increased from 0.50 at 1st day to 0.72 at 63rd day, while it was almost unchanged in water. For the depuration phase, the S- and R-enantiomer of fipronil in water hyacinth plants were degraded 92.22% and 82.07% after 17 days, respectively. The process of the degradation of the two enantiomers was followed first-order kinetics (R² ? 0.94). Stereoselective behavior was observed in both accumulation and degradation process. In this study, fipronil-sulfone and fipronil-sulfide, the metabolites of fipronil, were detected by GC-MS to show the main metabolic pathway of fipronil in water hyacinth.     

Studies on the chiral isomers of fonofos and fonofos oxon: III. In vivo metabolism

The metabolism of the chiral isomers of ³⁵S-labeled fonofos was examined in the house fly and white mouse. Metabolism of the chiral isomers of fonofos oxon also was investigated in the mouse. Little difference in either the rate of penetration or pattern of metabolism was observed between house flies treated with the (R)_P and (S)_P enantiomers of fonofos. At the higher dosage of 8 mg/kg the less toxic (S)_P enantiomer was degraded in mice and eliminated in significantly larger amounts than (R)_P-fonofos. However, at the sublethal dosage of 4 mg/kg little difference in degradation and total elimination was observed between the two isomers although the excretion rate appeared to be faster initially with the less toxic enantiomer. Overall, metabolism and excretion of the chiral isomers of both fonofos and fonofos oxon took place more rapidly and to a greater extent with the less toxic enantiomer.     

Stereoselective degradation of ethofumesate in turfgrass and soil

The stereoselective degradation of ethofumesate in turfgrasses and several agricultural soils was investigated to provide details of the fate of this chiral herbicide. Racemic ethofumesate was either foliar applied to two species of turfgrass or fortified into four types of agricultural soils. (+)- and (−)-Enantiomers were extracted and analyzed by a validated chiral HPLC method which involved extraction of samples with organic solvent followed by separation on cellulose-Tris-(3,5-dimethylphenylcarbamate)-based chiral column and quantification by UV absorbance at 230 nm. Mean recoveries of each enantiomer fortified at 0.5, 5, and 10 μg g⁻¹ ranged from 82.3 ± 5.84 to 92.5 ± 2.87% in turfgrasses and from 86.0 ± 5.09 to 98.1 ± 2.51% in soil. As a measure of this composition, the enantiomeric ratio (ER) was used, defined as the concentration ratio of (+)/(−)-enantiomer. Similarly, preferential degradation of the (−)-enantiomer was observed in both grass species with the largest ER of about 3 and in one of the test soil with ER = 1.65, resulting in residues enriched with (+)-enantiomer. This stereoselective degradation in this soil led to significant difference on half-lives between the two enantiomers. No stereoselective degradation was observed in other soils.     

Hepatoprotective activity of angiotensin-converting enzyme (ACE) inhibitors, captopril and enalapril, against paraquat toxicity

Paraquat is a highly toxic herbicide that is used in most countries without restriction. The cytotoxic action of paraquat is mediated by reactive radicals that are products of its metabolic reduction in cells. It has already been hypothesized that some angiotensin-converting enzyme inhibitors (e.g., captopril and enalapril) could show antioxidant and radical scavenging activity through their structural thiol groups, increasing antioxidant enzymes production or nitric oxide synthesis. In this study the hepatoprotective effect of captopril and enalapril against paraquat induced oxidative stress cytotoxicity was evaluated in isolated rat hepatocyte. Subtoxic concentrations of captopril (0.2 mM) and enalapril (0.2 mM) significantly (p < 0.05) protected the hepatocytes against paraquat (2 mM) induced oxidative stress cytotoxicity markers including: cell lysis, reactive oxygen species (ROS) generation, lipid peroxidation, glutathione depletion, mitochondrial membrane potential decrease, lysosomal membrane oxidative damage and cellular proteolysis. Moreover, we showed that non-thiol enalapril acts as well as thiol containing captopril at inhibiting oxidative stress cytotoxicity markers. Finally, our results support the hypothesis that it is the increase in nitric oxide synthesis and not the presence of the thiol group that accounts for the antioxidant activity of ACE inhibitors.     

Studies on the chiral isomers of fonofos and fonofos oxon: I. Toxicity and antiesterase activities

The toxicity of the (R)_P and (S)_P chiral isomers and racemates of fonofos and fonofos oxon to insects and white mice were determined. (R)_P-Fonofos and (S)_P-fonofos oxon were 2- to 12-fold more toxic to house flies, mosquito larvae, and mice than were the corresponding enantiomers. The racemates were intermediate in toxicity. Stereoselectivity also was observed in the in vitro inhibition of house fly-head and bovine erythrocyte acetylcholinesterase, horse serum cholinesterase, chymotrypsin, trypsin, and a variety of esterases. In all cases the (S)_P-oxon was a more potent inhibitor than the (R)_P-oxon with k₁ ratios of $\text{(S)}{}_{\mathrm{<mtext>P</mtext>}}\text{(R)}{}_{\mathrm{<mtext>P</mtext>}}$ ranging from 4- to 60-fold. Further, differences in levels of house fly-head, mouse brain, and blood cholinesterase obtained from house flies and mice treated with the enantiomers and racemates of fonofos and fonofos oxon were observed. Differences in toxicity of the enantiomers and racemates to house flies and mice were more closely related to in vivo than to in vitro cholinesterase inhibition.     

Studies on the chiral isomers of fonofos and fonofos oxon: II. In vitro metabolism

The in vitro metabolism of the chiral isomers of fonofos and fonofos oxon in the presence of mouse liver mixed-function oxidase and serum esterase was investigated. The metabolism of ³⁵S-labeled phenyl-(S)_P-fonofos mediated by mixed-function oxidase took place stereoselectively, resulting predominantly in (R)_P-fonofos oxon. Similarly, (R)_P-fonofos was converted to (S)_P-oxon. In each case, however, a significant amount of racemization occurred. Other products were diphenyl disulfide and diphenyl disulfide oxide. In addition to stereospecificity, the oxidative metabolism of (R)_P-fonofos proceeded at a rate faster than that of (S)_P-fonofos. Stereoselective rate differences also were observed in mouse or rat serum-catalzyed degradation of the fonofos oxon enantiomers, the (S)_P isomer being degraded about twofold faster than its enantiomer. The differences in toxicities of the isomers of fonofos and fonofos oxon were consistent with the in vitro metabolism data.     

Enantioselective degradation kinetics of metalaxyl in rabbits

Metalaxyl [methyl-N-(2′-methoxyacetyl)-N-(2,6-dimethylphenyl)-d,l- alaninate] is a potent phenylamide fungicide. The (−)-(R)-isomer accounts for most of the fungicidal activity. A possible stereo and/or enantioselective kinetics of metalaxyl in rabbits was investigated by intravenous injection. The concentrations of (−)-(R)- and (+)-(S)-metalaxyl in plasma, liver, and kidney tissue were determined by HPLC with a cellulose-Tris-(3,5-dimethylphenylcarbamate)-based chiral stationary phase and gas chromatography-mass spectroscopy. After intravenous administration of racemic metalaxyl (40 mg/kg), the (+)-(S)-enantiomer levels in plasma, liver, and kidney decreased more rapidly than the (−)-(R)-isomer. The area ratio of the (−)-(R)-/(+)-(S)-enantiomer under the concentration-time curve (AUC_0 → ∞) in plasma after drug application was 1.62. The total plasma clearance value of the (+)-(S)-enantiomer was 1.53 and higher than that of the (−)-(R)-enantiomer. The [R]/[S] ratio in plasma was >1 for standard rac-metalaxyl at each time point. The other pharmacokinetic parameters of the enantiomers were also different. The results indicate substantial stereoselectivity in the degradation of metalaxyl enantiomers in rabbits.     

Perfusion analysis of periportal and centrilobular metabolism of paraoxon in the rat liver

Paraoxon infused into the rat liver during perfusion in situ with Waymouth's medium underwent chromatographic translobular migration with an apparent hepatic transit time of 3 min. Intralobular heterogeneity of paraoxon metabolism was examined by analyzing metabolites produced under conditions minimizing the chromatographic translobular migration of paraoxon. Periportal and centrilobular activities were estimated following forward and retrograde infusion of paraoxon, respectively. Centrilobular hepatocytes exhibited nearly twice the metabolic rate of the periportal cells. Pretreatment of the rat with DDE resulted in about a threefold increase in the ratio of oxidative deethylation to hydrolytic dearylation in the centrilobular region. The differentials observed by these analyses were less pronounced than expected from enzyme analyses in vitro, possibly reflecting secondary metabolism or intracellular heterogeneity of metabolic activities.     

The enantiomeric composition of triadimenol produced during metabolism of triadimefon by fungi: III. Relationship with sensitivity to triadimefon

Fifteen species of fungi, grown in shake culture in a liquid nutrient medium containing a 50:50 mixture of the 1R and 1S enantiomers of triadimefon, reduced triadimefon to triadimenol, the extent of reduction varying from <1% to >95% according to species. Each produced its own characteristic pattern of two or more of the four possible enantiomers of triadimenol and the stereoselectivity and stereospecificity of the reduction is discussed. The sensitivity of each fungus to individual enantiomers and a mixture of enantiomers of triadimefon and of triadimenol was assessed. Three broadly defined groups of fungi were found: (i) sensitive to both triadimefon and tridimenol, (ii) insensitive to both, and (iii) insensitive to triadimefon but moderately sensitive to triadimenol. In many cases it was possible to predict, at least semiquantitatively, the sensitivity of an organism to triadimefon from a knowledge of its characteristic metabolism to triadimenol and its sensitivity to the individual triadimenol enantiomers. No evidence for synergism or antagonism was found.     

Affinity and phosphonylation constants for the inhibition of cholinesterases by the optical isomers of O-2-butyl S-2-(dimethylammonium)ethyl ethylphosphonothioate hydrogen oxalate

The synthesis of the four optical isomers of known absolute configuration of O-2-butyl S-2-(dimethylammonium)ethyl ethylphosphonothioate hydrogen oxalate is described. Values for the affinity constant (K_a), phosphonylation constant (k_p), and bimolecular inhibition rate constant (k_i) for the inhibition of bovine erythrocyte acetylcholinesterase, housefly-head acetylcholinesterase, and horse serum cholinesterase by the chiral isomers and the racemic mixture are reported. Using a relatively simple spectrophotometric technique, inhibition times as low as 0.5 sec were used. The phosphorus isomers of S_p configuration were more potent inhibitors than their R_p enantiomers by 1630-fold against the bovine enzyme, 9120-fold against the fly-head enzyme, and 40-fold against the horse serum enzyme. The differences in anticholinesterase activity were attributable to differences in the affinity constant, K_a, and the phosphonylation constant, k_p. Small but consistent inhibition rate differences were attributable to asymmetry at carbon. Against horse serum cholinesterase, the S_C isomers indicated the presence of three kinetic forms in this enzyme preparation.     

Paraoxon-induced release of β-glucuronidase from rat hepatocytes in vitro

A suspension culture of isolated rat hepatocytes was used to reproduce in vitro the paraoxon-induced release of hepatic β-glucuronidase observed in vivo. After a short latent period, exposure of hepatocytes to paraoxon at 10^?7 to 10^?4M resulted in a typical dose-dependent response, with highest release occurring at 10^?4M paraoxon. With 10^?3M paraoxon, however, response was anomalous with a much-decreased enzyme release. As expected from earlier results in vivo, SV₁-oxon exhibited less effect than paraoxon.     

Endocrine disruption and metabolic changes following exposure of Cyprinus carpio to diethyl phthalate

Diethyl phthalate (DEP) enter into aquatic environment from industries manufacturing cosmetics, plastic and many commercial products and can pose potential fish and human health hazard. This experiment evaluated effects of DEP in adult male (89 g) common carp (Cyprinus carpio) by exposing them to fractions of LC₅₀ (1/500-1/2.5) doses with every change of water for 28 days. Vitellogenin induction metabolic enzymes, somatic indices and bioaccumulation were studied on 7th, 14th, 21st and 28th day. The 96th hour LC₅₀ of DEP in fingerlings was found to be 48 mg/L. Compared to control, except increase (P < 0.01) in alkaline phosphatase activity (EC 3.1.3.1) and liver size, there was decrease (P < 0.01) in activity of acid phosphatase (EC 3.1.3.2), aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2) and testiculosomatic index following exposure to 1, 5 and 20 ppm DEP. Significant (P < 0.01) dose dependant vitellogenin induction was observed with exposure of fish to 0.1, 1 and 5 ppm DEP. The bioaccumulation of DEP in testis, liver, brain, gills and more importantly in muscle tissues of fish increased significantly (P < 0.01) with increase of dose from 1 to 5 ppm. Significant interaction (P < 0.01) of dose and duration of exposure indicated that exposure period of a week to two was sufficient to bring about changes in quantifiable parameters studied. Fish exposed to 20 ppm DEP became lethargic and discolored during onset of the 4th week. This is the first report describing metabolic changes and vitellogenin induction following exposure of C. carpio to DEP dose that is as low as 1/500th fraction of LC₅₀.     

Metabolic and histological parameters of silver catfish (Rhamdia quelen) exposed to commercial formulation of 2,4-dichlorophenoxiacetic acid (2,4-D) herbicide

The objective of this study was to investigate the effects of commercial formulation of herbicide 2,4-D on metabolic parameters, acetylcholinesterase (AChE) activity and liver histological evaluation of silver catfish (Rhamdia quelen) exposed for 96 h. AChE activity increased in brain (600 and 700 mg L⁻¹) and decreased in all concentrations tested in muscle tissue. Hepatic glycogen was reduced after 2,4-D exposure ranging from 47.67% (400 mg L⁻¹) until 59.3% (700 mg L⁻¹). Hepatic tissue showed lactate reduction at all 2,4-D concentrations tested and glucose was reduced only at 700 mg L⁻¹. In the highest concentration tested hepatic glycogen and glucose reduced instead plasma glucose levels increased. White muscle tissue showed glycogen reduction in fingerlings exposed to all herbicide concentrations and glucose reduction at 700 mg L⁻¹. Muscle lactate levels increase at all 2,4-D concentrations tested. Vacuolation of hepatocytes and changes in its arrangement cords were observed by histologic analysis in group treated with 700 mg/L of 2,4-D. These results suggest that silver catfish exposed to concentrations of 2,4-D near of CL₅₀ showed metabolic and histological response to compensate some stress caused by herbicide exposure. Taken together parameters measured can be used as biomarkers to monitor herbicide contaminated water.     

Metabolism of the 1,2,4-triazolylmethane fungicides,triadimefon, triadimenol,and diclobutrazol,by Aspergillus niger (van Tiegh.)

Metabolism of the triazolylmethane fungicides triadimefon, triadimenol, and diclobutrazol by Aspergillus niger was studied using a replacement culture technique and ¹⁴C substrates. Components of metabolite mixtures were characterized by TLC, GLC, radio-GC, and GC-MS analyses of the free materials and their trifluoroacetate and trimethylsilyl ether derivatives. The three compounds underwent a common metabolic change involving oxidation of C(CH₃)₃ to C(CH₃)₂CH₂OH. In this work the isopropyl analog of triadimefon, previously reported as a metabolite, was an artifact and resulted from nonbiological oxidation of the corresponding primary alcohol. The fungus also reduced triadimefon to triadimenol, giving a mixture of 1R2S, 1S2R and 1R2R, 1S2S diastereoisomers. The less fungitoxic 1R2S, 1S2R triadimenol predominated, so that this conversion may be directly associated with the relative insensitivity of A. niger to triadimefon. Implications of oxidative and reductive metabolism of these fungicides are suggested with particular reference to the differing fungitoxicities of diastereoisomers and enantiomers.     

Cytochrome P-450 content and aldrin epoxidation to dieldrin in isolated rat hepatocytes

A rat hepatocyte suspension effectively epoxidized aldrin to dieldrin with a V_max of 7.19 mol/mol P-450/min and a K_m of 9.27 μM. Viability and metabolic activity were stable for 6 hr after isolation when cells were maintained at room temperature (20°C) with the gentle introduction of $\text{O}{}_2\text{CO}{}_2$ onto the surface of the suspension. The cytochrome P-450 content of the suspension was 303 pmol/10⁶ cells. Primary maintenance culture of the cells also epoxidized aldrin. During culture for 3 days, metabolic activity decreased slowly day by day. Metabolic activity of microsomal fraction from rat liver was also examined. Microsomes epoxidized aldrin with a V_max of 5.11 mol/mol P-450/min and a K_m of 1.64 μM. Significant loss of some subspecies of cytochrome P-450 during fractionation of liver homogenate was indicated.     

Species differences in the in vitro inhibition of brain acetylcholinesterase and carboxylesterase by mipafox,paraoxon, and soman

Acute intraperitoneal toxicity of mipafox, paraoxon, and soman was highest in chicken, followed by rat, and lowest in frog. Species differences in organophosphorus toxicity were not related to differences in the specific activities of either acetylcholinesterase or carboxylesterase in brain. The sensitivity to inhibition of brain acetylcholinesterase in vitro by the organophosphorus compounds was closely related to the susceptibility of the species to acute organophosphorus poisoning. Both the acute toxicity and the sensitivity of brain acetylcholinesterase to inhibition in vitro by organophosphorus compounds in all the species were in the following order of increasing activity: mipafox < paraoxon < soman. The sensitivity of brain carboxylesterase to inhibition by the organophosphorus compounds were less than that of acetylcholinesterase and it could not be related to species susceptibility to acute organophosphorus toxicity. Paraoxon-insensitive phenyl valerate hydrolase in chicken brain was more sensitive to inhibition by mipafox and soman compared to rat; in chicken the sensitivity of paraoxon-insensitive phenyl valerate hydrolase to inhibition by soman was 9000 times more than that by mipafox, while in rat it was 100 times more. Frog brain had no paraoxon-insensitive phenyl valerate hydrolase activity. No evidence of age dependence was noticed in the specific activities of brain acetylcholinesterase, carboxylesterase, neurotoxic esterase, and paraoxon-insensitive phenyl valerate hydrolase or in the sensitivity of these enzymes to in vitro inhibition by organophosphorus compounds in both rats and chickens.     

手性农药己唑醇和腈菌唑对黄酒酿造微生物酶活性的影响

在食品发酵过程中,残留的农药会对酿造微生物及产品组分产生一定影响,而酶在此过程中发挥着关键作用。作者在对映异构体水平上研究了手性农药己唑醇和腈菌唑对黄酒酵母和米曲霉中典型酶的酶活性和蛋白质含量的影响。结果表明:不同农药对映异构体对微生物酶活性的影响存在显著差异,其中:（+）-己唑醇对黄酒酵母超氧化物歧化酶（SOD酶）活性具有较明显的抑制作用,而（-）-己唑醇则对其有激活作用;低浓度的（+）-腈菌唑对黄酒酵母脂肪酶活性的抑制作用小于（-）-腈菌唑,而高浓度（+）-腈菌唑对脂肪酶活性的抑制作用却大于（-）-腈菌唑;（+）-己唑醇对米曲霉中的脂肪酶活性有明显的激活作用,而（-）-己唑醇对其则有抑制作用。本研究结果证明,手性农药对映异构体对发酵微生物酶活性的影响存在差异,并可能影响黄酒的组分和品质,使黄酒安全性存在较大风险。     

Species and sex differences in the reductive dechlorination of DDT by supplemented liver preparations

p,p′-DDT was converted to DDD, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane, by unheated 12,000g supernate supplemented with NADPH, made from liver of rat, mouse, hamster, quail, chicken and pigeon. The additional presence of exogenous riboflavin did not augment reduction. If, however, riboflavin was added to unheated microsomes supplemented with NADPH or NADH the rate of reduction was more than doubled.Heated 12,000g supernate, unsupplemented or containing only exogenous riboflavin, did not reduce DDT. When NAD(P)H was present, big species differences in activity occurred, the pigeon and cockerel scarcely reducing any DDT in 2 hr, the Wistar rat supernate being most active. Addition to heated 12,000g supernate of 30 μg riboflavin as well as NAD(P)H resulted in increased reductive activity for all six species. The species difference in effect of exogenous riboflavin between the pigeon and rat was also observed using four DDT analogs as substrates. A sex-related difference in activity of preheated, supplemented 12,000g supernate from livers of the chicken and Norwegian hooded rat was not found when unheated preparations were used. In contrast to the activity possessed by preheated 12,000g supernate of rat liver supplemented with NAD(P)H, similarly supplemented preheated microsomes did not reduce DDT. On adding riboflavin as well as NAD(P)H, however, preheated hepatic microsomes of both pigeon and rat produced DDD, and this activity was further increased by addition of unheated microsomal (105,000g) supernate. The biocatalytic system functioning in preheated preparations appears to need at least one component of heated microsomes, NAD(P)H and one or more water-soluble components.