A study of the expression of the xenobiotic-metabolising cytochrome P450 proteins and of testosterone metabolism in bovine liver.

The expression of xenobiotic-metabolising cytochrome P450 proteins in the liver of cattle was determined using substrate probes and immunologically by Western blot analysis. Compared to the rat, cattle displayed much higher coumarin 7-hydroxylase (CYP2A) and ethoxyresorufin O-deethylase (CYP1) activity but, in contrast, it exhibited much lower debrisoquine 4-hydroxylase (CYP2D) and lauric acid hydroxylase activities (CYP4A). The ethoxyresorufin O-deethylase activity was markedly inhibited by furafylline and a-naphthoflavone, and coumarin 7-hydroxylase by 8-methoxypsoralen. Immunoblot analysis employing antibodies to rat CYP1A1 recognised two immunorelated proteins in bovine liver whose expression appeared to be higher compared with rat. Kinetic studies indicated that a single enzyme is likely to be responsible for the O-deethylation of 7-ethoxyresorufin in bovine liver. When bovine microsomes were probed with antibodies to rat CYP2A2, a single protein was detected in cattle liver. Kinetic analysis followed by construction of Eadie-Hofstee plots indicated that more than one enzyme contributes to the 7-hydroxylation of coumarin. Immunoblot analysis employing antibodies to human CYP2D6 and rat CYP4A1 revealed in both cases a single, poorly expressed immunoreacting band in bovine microsomes. Similar immunoblot studies detected proteins in cattle liver immunorelated to the CYP2B, CYP2C, CYP2E, and CYP3A subfamilies. Bovine microsomes metabolised testosterone but, in contrast to the rat, failed to produce 2alpha- and 16alpha-hydroxytestosterone. On the other hand, bovine microsomes produced levels of another hydroxylated metabolite, possibly 12-hydroxytestosterone. In conclusion, results emanating from this study indicate the presence of proteins in the cattle liver belonging to all the xenobiotic-metabolising families of cytochrome P450.

Evaluation of a recombinant yeast cell estrogen screening assay.

A wide range of chemicals with diverse structures derived from plant and environmental origins are reported to have hormonal activity. The potential for appreciable exposure of humans to such substances prompts the need to develop sensitive screening methods to quantitate and evaluate the risk to the public. Yeast cells transformed with plasmids encoding the human estrogen receptor and an estrogen responsive promoter linked to a reporter gene were evaluated for screening compounds for estrogenic activity. Relative sensitivity to estrogens was evaluated by reference to 17 beta-estradiol (E2) calibration curves derived using the recombinant yeast cells, MCF-7 human breast cancer cells, and a prepubertal mouse uterotrophic bioassay. The recombinant yeast cell bioassay (RCBA) was approximately two and five orders of magnitude more sensitive to E2 than MCF-7 cells and the uterotrophic assay, respectively. The estrogenic potency of 53 chemicals, including steroid hormones, synthetic estrogens, environmental pollutants, and phytoestrogens, was measured using the RCBA. Potency values produced with the RCBA relative to E2 (100) included estrone (9.6), diethylstilbestrol (74.3), tamoxifen (0.0047), alpha-zearalanol (1.3), equol (0.085), 4-nonylphenol (0.005), and butylbenzyl phathalate (0.0004), which were similar to literature values but generally higher than those produced by the uterotrophic assay. Exquisite sensitivity, absence of test compound biotransformation, ease of use, and the possibility of measuring antiestrogenic activity are important attributes that argue for the suitability of the RCBA in screening for potential xenoestrogens to evaluate risk to humans, wildlife, and the environment.

Functional integrity of precision-cut liver slices from deer and cattle.

Precision-cut bovine and cervine liver slices were incubated in RPMI 1640 media for up to 72 h, and cellular integrity was assessed utilizing the leakage of lactate dehydrogenase (LDH) and the formation of the formazan metabolite of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT). Leakage of LDH (80%) from the bovine and cervine slices was noted only following 10 h of culture, and similarly, the generation of MTT-formazan declined. Metabolic viability was determined using 7-ethoxycoumarin as the model substrate, which was metabolized by slices from both animal species in a time-dependent manner for at least 6 h to generate 7-hydroxycoumarin, which was secreted into the media primarily as glucuronide and sulphate conjugates. With both cervine and bovine slices metabolic activity decreased markedly after a 4-h preincubation as assessed following a further 2-h incubation in the presence of 7-ethoxycoumarin. Subsequently, ethoxycoumarin metabolism by bovine slices did not decrease further until 24 h and was still detectable at 72 h. In the case of cervine slices, activity declined gradually after 8 h, with no activity being detectable at 72 h. It may be concluded that cervine and bovine slices may be maintained metabolically active for 8-10 h, which should prove sufficient for xenobiotic metabolism studies to be performed.

Imidocarb depletion from cattle liver and mechanism of retention in isolated bovine hepatocytes.

Imizol injection (imidocarb) is used for the prevention and treatment of babesiosis in cattle. Studies in sheep indicate that imidocarb is retained in edible tissues (Aliu et al.). In the present study we have set up and validated a high-performance liquid chromatography based method to investigate the retention of imidocarb in cattle liver. Imidocarb was still detectable 224 d after a single therapeutic dose with a half-life of 42.7 d. The mechanism of imidocarb retention by bovine liver was modelled using isolated bovine hepatocytes. Incubations with isolated hepatocytes indicated that [14C]imidocarb binding was dependent on hepatocyte number and showed signs of saturation. Bound [14C]imidocarb could be eluted from hepatocytes with buffer and extracted with solvents. Equilibrium dialysis under denaturing conditions (Sun and Dent) indicated that 3% of the [14C]imidocarb was covalently bound to macromolecules. Although the hepatocyte preparations demonstrated the capacity for phase I and II 7-ethoxycoumarin metabolism no metabolites of [14C]imidocarb were found. Further in vitro binding studies involving sub-cellular fractionation indicated that [14C]imidocarb is partitioned largely in the nuclear fraction of bovine liver homogenates and that it binds to deoxyribonucleic acid.

Biotransformation, tissue distribution, and persistence of 4-nonylphenol residues in juvenile rainbow trout (Oncorhynchus mykiss).

Alkylphenols are weak oestrogenic environmental contaminants and have been implicated in the disruption of endocrine function in wildlife. The influence of biotransformation, tissue distribution, and elimination on biological activity was investigated in juvenile rainbow trout following a single iv dose of [3H]4-nonylphenol. Distribution and elimination of [3H]4-nonylphenol residues in tissues sampled 1, 2, 4, 24, 48, 72, and 144 hr after dosing was determined by sample combustion and liquid scintillation counting (LSC). Total 3H-labeled residue concentrations in trout 144 hr after dosing were in order: bile >> faeces >> liver > pyloric caecae > kidney > brain, gill, gonad, heart, plasma, skeletal muscle, and skin. The depletion kinetics of [3H]residues from tissues and plasma was biphasic with prolonged beta-phase half-lives in muscle and liver of 99 hr. Radio-HPLC analysis of metabolites in bile, liver, pyloric caecae, and faeces samples demonstrated similar profiles and contrasted with muscle where only parent compound was found. The predominant metabolite in bile was a glucuronide conjugate of 4-nonylphenol. Other metabolites included glucuronide conjugates of ring or side chain hydroxylated 4-nonylphenol. Liver contained a low level (1.7%) of covalently bound residues. Metabolism studies using isolated trout hepatocytes produced a similar range of metabolites and a sulfate conjugate of hydroxylated 4-nonylphenol. Despite rapid metabolism and excretion, a substantial depot of parent compound remained in muscle which will have implications for the maintenance of 4-nonylphenol residues and associated biological activity.

Imidocarb residues in edible bovine tissues and in vitro assessment of imidocarb metabolism and cytotoxicity.

Imidocarb residues in liver and muscle were measured by HPLC after a single therapeutic dose to cattle (3 mg imidocarb dipropionate kg-1). Imidocarb and 7-ethoxycoumarin metabolism were compared in three different in vitro systems prepared from bovine liver: cultures of hepatocyte monolayers, precision-cut liver slices, and microsomes. The potential hepatotoxicity of imidocarb residues was tested on hepatocyte monolayers and assessed using the neutral red and lactate dehydrogenase leakage assays. The concentration of imidocarb (mean +/- SD) decreased between days 14 and 224 after treatment from 5.40 +/- 0.61 to 0.12 +/- 0.01 and from 1.05 +/- 0.31 to 0.06 +/- 0.02 microgram g-1 in liver and muscle, respectively. The depletion kinetics of imidocarb fitted a two-compartment model with alpha- and beta-phase half-lives of 31.7 and 48.5 days in liver and 34.9 and 120.7 days in muscle, respectively. Imidocarb metabolites were not detected in any in vitro system. 7-Ethoxycoumarin metabolism was found in all in vitro systems; the predominant metabolite produced by hepatocyte and liver slice cultures was umbelliferone glucuronide. Cytotoxicity of imidocarb (100 microM) to hepatocyte monolayers was maximal after 72 hr treatment and dose-dependent above 10 microM imidocarb. It is most likely that the hepatotoxicity of imidocarb is caused by the parent compound, because no evidence for imidocarb metabolism was found.