Sheep Liver Microsomes Research Articles

Uterotropic and enzyme induction effects of 2,2?,5-trichlorobiphenyl

Polychlorinated biphenyls (PCBs) are among the ubiquitously persistent chemicals possessing endocrine-disrupting effects. Most of the environmentally abundant CB congeners have one or more ortho chlorines in the biphenyl ring. Because ortho-chlorinated CB congeners do not avidly bind to the aryl hydrocarbon (Ah) receptor, this clearly suggests that the estrogenicity of PCBs is not related to potency as Ah receptor agonists. It is important to investigate the estrogenicity of those ortho-chlorinated CB congeners in order to more accurately predict the hazards from PCB residues in the environment. The hydroxylated metabolites are presumably more reactive with the estrogen receptor, but induction of phase 2 enzymes at the higher doses could sequester the metabolites resulting in the lower serum residues of parent compound and attenuated estrogenic activity observed at higher doses. It was of interest to determine if a di-ortho CB congener which is more readily metabolized and an ineffective PROD inducer exhibited similar nonlinear dose:response relationships for estrogenicity and serum residues as observed for CB 153. CB 18 (2,2{prime}, 5-trichlorobiphenyl) is very rapidly metabolized to more polar products by sheep liver microsomes, rats and house flies. The detection of greater amounts of mono-hydroxylated metabolites from slower metabolized congeners than from CB 18more » reflects further secondary metabolism in these extended (up to 1 hour) microsomal incubations. Such secondary metabolism should be relatively constant up to very high doses in vivo, since CB 18 should be a very poor P450 2B1 inducer. Even though CB 18 commonly occurs in environmental samples, there is little information regarding its biological effects. In this study, we used immature Sprague-Dawley female rats as an animal model to examine the uterotropic response, thyrotoxic effect and enzyme induction potency of CB 18. 17 refs., 3 tab.« less

In vitro fenoprofenyl-coenzyme A thioester formation: interspecies variations.

In vitro coenzyme A thioester formation from (-)-(R)-fenoprofen (FPF) and palmitic acid has been studied using liver microsomes from rat, guinea pig, sheep, and dog. In every species with both palmitic acid or (-)-(R)-fenoprofen, the Lineweaver-Burk plot was linear in the substrate concentration range used and as a consequence agrees with the involvement of only one isoenzyme (or different isoenzymes of similar Km values). The Vmax values for the thioesterification of (-)-(R)-fenoprofen present large species variations from 2.1 +/- 1.0 with sheep liver microsomes to 60.6 +/- 11 nmol/min/mg with dog liver microsomes. These values statistically significantly correlate (r = 0.94) to the Vmax values observed when palmitic acid was used as a substrate. Furthermore palmitic acid inhibited (-)-(R)-fenoprofen-CoA formation in the same extent in all animal species. The stereoselectivity of the thioesterification was also species dependent.

Participation of a cytochrome P450 enzyme from the 2C subfamily in progesterone 21-hydroxylation in sheep liver

Progesterone 21-hydroxylation in hepatic microsomes from adult male sheep is a quantitatively important metabolic pathway (0.27±0.08 nmol deoxycorticosterone formed/min/mg protein; representing 13–25% of total progesterone conversion). This study was undertaken to determine whether the ovine hepatic progesterone 21-hydroxylase may be another member of the P450 2C subfamily, normally associated with progesterone 21-hydroxylation in rodent liver. An IgG preparation raised in rabbits against purified rat liver microsomal cytochrome P450 2C6 was found to recognize a single antigen (M w 52 kDa) in sheep liver microsomes. This protein was present in sheep liver (apparent concentration 16±4 ng/ μg microsomal protein) representing approx. 28% of the corresponding content of P450 2C6 in untreated rat liver. Preincubation of the anti- P450 2C6 IgG with hepatic microsomes was found to decrease the rate of progesterone 21-hydroxylation to 50–80% of uninhibited control. Taken together, from these findings it is apparent that a P450 enzyme, most likely from the 2C subfamily, catalyses deoxycorticosterone formation from progesterone in sheep liver and that this is a quantitatively important pathway of progesterone hydroxylation in these fractions.

Functional expression of an ovine growth hormone receptor in transfected Chinese hamster ovary cells

Structural heterogeneity has been demonstrated for growth hormone (GH) receptors from a number of species, and both high and low affinity art receptors have been characterised by ligand binding studies. In the present study, we have transfected Chinese hamster ovary (CHO-K1) cells with a cDNA clone encoding a full-length transmembrane ovine (o) GH receptor, under the regulatory control of the human metallothionein IIA promoter. A stably transfected cell line was established (GHR9.5) which expresses on the cell surface a single class of receptor which binds 220,000 [ 125I]oGH molecules at high affinity ( K d = 0.30 nM) which is comparable to the affinity established for endogenous oGH receptors in postnatal sheep liver microsomes ( K d = 0.27 nM, Freemark et al. (1987) Endocrinology 120, 1865–1872). The expressed receptor also binds ovine placental lactogen (oPL, 205, 000 binding sites per cell) with high affinity ( K d = 0.76 nM). The presence of two species of oGH receptor was detected in GHR9.5 cells using affinity cross-linking analysis ( M r 148,000 and M r 73,000) and given that the oGH receptor cDNA codes for a non-glycosylated receptor of M r 69,914, it is likely that these cross-linked species correspond to homodimeric and monomeric forms of the oGH receptor, each binding to a single molecule of GH. Parallel cross-linking studies with sheep liver microsomes also demonstrated two oGH receptor species ( M r 133,000 and M r 58,000), the difference in relative molecular weights between the transfected and endogenous receptors presumably resulting from tissue-specific post-translational modifications. In the presence of oGH, the GHR9.5 cells respond by increasing total cellular protein synthesis by 27% relative to non-GH-exposed GHR9.5 cells, indicating the functionality of the expressed receptor. We also demonstrate unequivocally that oPL, through a specific interaction with the transfected oGH receptor, is able to mediate a similar cellular response (38% protein synthesis induction). Responsiveness to oGH and oPL in the GHR9.5 cells is dependent on serum starvation prior to oGH exposure and occurs only with prolonged exposure (greater than 2 h) to oGH. This cellular stimulation occurs independently of c- fos transcription which has previously been shown to be one of the earliest events associated with GH action in tissues expressing endogenous GH receptors (Doglio et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1148–1152; Slootweg et al. (1990) J. Mol. Endocrinol. 4, 265–274).

Ontogenic development of liver progesterone metabolism in female sheep. Contribution of cytochrome P4502B and P4503A subfamilies

Age-related changes in progesterone hepatic metabolism were measured in Lacaune ewes in the foetal, neonatal (1 and 4 weeks), growing (7 months), pregnant (11 months) and adult (6 years) stages. 6β-Hydroxylation and 20α-reduction were found to be the most efficient metabolic process in ovine microsomes. These activities were detected in 3-month-old foetuses and they increased rapidly during the first month of life, in a similar manner to the developmental expression of the cytochrome P4503A subfamily. 16α- and 21-hydroxylation of progesterone were characterized by low, constant turn over in sheep liver microsomes during development. The hepatic ovine P450B isozyme was purified to electrophoretic homogeneity by means of successive DEAE cellulose, hydroxylapatite and CM cellulose chromatographic separations. This hemoprotein had an apparent molecular weight of 51 kDa and was characterized by spectral data, NH 2-terminal amino-acid sequence, immunological and catalytic properties. The relative contribution of this form and of the previously purified ovine P4503A subfamily was investigated in liver progesterone metabolism by immunoinhibition studies using polyclonal antibodies raised in rabbits and form the existence of induction and of significant correlations between microsomal activity and specific P450 content. In sheep liver microsomes, it would appear that cytochrome P450B is involved in progesterone 21-hydroxylation whereas P4503A participates in the 6β- and 16α-hydroxylation and possibly in the reductive conversion of progesterone in its 20α-hydroxy derivative.

Glutathione-dependent inhibition of lipid peroxidation in sheep liver microsomes

Considerable evidence supports the role of oxidative stress in the pathogenesis of Alzheimer's disease (AD). Previous studies suggest that the central nervous system (CNS) administration of β-amyloid peptide, the major constituent of senile plaque in AD, induces oxidative stress in rodents which may contribute to the learning and memory deficits verified in the β-amyloid model of AD. In the present study, we compared the effects of a single intracerebroventricular (i.c.v.) injection of aggregated β-amyloid peptide-(1–40) (Aβ1–40) (400 pmol/mouse) on spatial learning and memory performance, synaptic density and the glutathione (GSH)-dependent antioxidant status in adult male C57BL/6 and Swiss albino mice. Seven days after Aβ1–40 administration, C57BL/6 and Swiss mice presented similar spatial learning and memory impairments, as evaluated in the water maze task, although these impairments were not found in Aβ40–1-treated mice. Moreover, a similar decline of synaptophysin levels was observed in the hippocampus (HC) and prefrontal cortex (PFC) of both Swiss and C57BL/6 mice treated with Aβ1–40, which suggests synaptic loss. C57BL/6 mice presented lower levels of glutathione-related antioxidant defences (total glutathione (GSH-t) levels, glutathione peroxidase (GPx) and glutathione reductase (GR) activity) in the HC and PFC in comparison to Swiss mice. Despite the reduced basal GSH-dependent antioxidant defences observed in C57BL/6 mice, Aβ1–40 administration induced significant alterations in the brain antioxidant parameters only in Swiss mice, decreasing GSH-t levels and increasing GPx and GR activity in the HC and PFC 24 h after treatment. These results indicate strain differences in the susceptibility to Aβ1–40-induced changes in the GSH-dependent antioxidant defences in mice, which should be taken into account in further studies using the Aβ model of AD in mice. In addition, the present findings suggest that the spatial learning and memory deficits induced by β-amyloid peptides in rodents may not be entirely related to glutathione-dependent antioxidant response.

Distinct pulmonary and hepatic forms of flavin-containing monooxygenase in sheep

1. 1. Flavin-containing monooxygenase (FMO) in pulmonary and hepatic microsomes from sheep was analyzed by western blotting by probing with antibodies raised against FMO purified from rabbit lung and pig liver. 2. 2. Pulmonary microsomes from sheep contain a single major protein which cross-reacts with the antibody to rabbit lung FMO, but no band can be observed when probed with the antibody to the pig liver enzyme. Likewise, sheep liver microsomes contain a protein which cross-reacts with the antibody to pig liver FMO, but no significant staining is observed following incubation with antibody to the lung enzymes. 3. 3. Sheep pulmonary and hepatic microsomal FMO also display a difference in activity toward chlorpromazine and n-dodecylamine. 4. 4. Preliminary evidence suggests that sheep FMO may be induced (liver) or repressed (lung) during pregnancy. 5. 5. Sheep are similar to rodents (rat, mouse, guinea pig, hamster and rabbit) in having distinct forms of pulmonary and hepatic FMO. The immunochemical and catalytic difference between sheep liver and lung FMO is similar to that of rabbit.

Demethylation and conjugation of formononetin and daidzein in sheep and cow liver microsomes

Demethylation and conjugation of formononetin and daidzein in sheep and cow liver microsomes

Characterization of sheep liver and lung microsomal ethylmorphine N-demethylase

1. 1. Ethylmorphine N-demethylase activity of the sheep liver and lung microsomes was reconstituted in the presence of solubilized microsomal cytochrome P-450, NADPH-cytochrome c reductase and synthetic lipid, phosphatidylcholine dilauroyl. 2. 2. The K m of the lung microsomal ethylmorphine N-demethylase was calculated to be 4.84 mM ethylmorphine from its Lineweaver-Burk graph and lung enzyme was inhibited by its substrate, ethylmorphine, when its concn was 25 mM and above, reaching to 67% inhibition at 50 mM concn. 3. 3. The Lineweaver-Burk and Eadie-Hofstee plots of the liver enzyme were found to be curvilinear. From these graphs, two different K m values were calculated for the liver enzyme as 4.17 mM and 0.40 mM ethylmorphine. 4. 4. Ethylmorphine N-demethylase activities of both liver and lung microsomes were inhibited by NiCl 2, CdCl 2 and ZnSO 4. Ethylalcohol inhibited N-demethylation of ethylmorphine in lung and liver microsomes. 5. 5. Acetone (5%) slightly enhanced the N-demethylase activity of the liver enzyme, whereas 5% acetone completely inhibited the lung enzyme. 6. 6. Phenylmethylsulfonyl fluoride at 0.10 mM and 0.25 mM concn had no effect on liver enzyme activity, while at these concns, it inhibited the activity of the lung enzyme by about 35%.

Comparative studies of sheep liver and lung microsomal aniline 4-hydroxylase

1. The specific activity of the aniline 4-hydroxylase which catalyses hydroxylation of aniline to p-aminophenol was found to be 0.65 (N = 10) and 0.15 (N = 13) nmol p-aminophenol formed/mg protein, min. in sheep liver and lung microsomes, respectively. 2. The effects of aniline concentration. pH. cofactors. amount of enzyme and incubation period, on enzyme activity were studied, and the optimum conditions for maximum activity of liver and lung microsomes were determined. 3. Liver and lung microsomal aniline 4-hydroxylase activity was found to be completely dependent on the presence of cofactor NADPH. 4. The Lineweaver-Burk and Eadie-Hofstee plots of the liver enzyme were found to be curvilinear, suggesting that the enzyme did not follow the Michaelis-Menten kinetics. From these graphs, two different K m values were calculated for the liver enzyme as 3.21 and 0.072 mM aniline. K m of the lung enzyme was calculated to be 1.43 mM aniline from its Lineweaver-Burk graph. 5. The effects of magnesium, nickel and cadmium ions on the liver and lung aniline 4-hydroxylase activity were examined. Magnesium ion was found to have stimulatory effect, whereas nickel and cadmium ions inhibited the activity of the both liver and lung enzyme.

Purification by affinity chromatography and some properties of microsomal galactosyltransferase from pig thyroid

Membrane-bound 4-β-galactosyltransferase (lactose synthase; UDP galactose: d-glucose 4-β-galactosyltransferase, EC 2.4.1.22) was purified 1500-fold to near homogeneity from pig thyroid microsomes with about 30% yield. The purified enzyme behaved as a lipophilic protein, rapidly losing activity and aggregating if not supplemented with either Triton X-100 or serum albumin (both of these were equally effective for long-term stabilization). The enzyme preparation showed an absolute requirement for Mn 2+, which could not be replaced by other cations. Catalytic properties were very similar to those reported for soluble forms of the enzyme in biological fluids. The purified galactosyltransferase showed a major protein band of approx. 74 000 daltons on sodium dodecyl sulfate gel electrophoresis. On gel filtration, enzyme activity was eluted at approx. 70 000 daltons. It is concluded that the membrane-bound thyroid galactosyltransferase is a monomeric protein significantly larger than the soluble forms of this enzyme described earlier; but it resembles recently reported galactosyltransferases from sheep mammary Golgi membranes and liver microsomes.

Tissue distribution of PCB components in swine and sheep fed three different rations containing Aroclors 1242 and 1254

Growing swine and sheep were fed three dietary variations containing 20 ppm of Aroclors 1242 or 1254 for 13 to 15 weeks. Generally, Aroclor 1254 residues were higher; higher 1242 residues in swine blood, spleen, and ovary are attributed to persistent major components of this Aroclors. Some minor differences in total PCB were observed with varying diets, but peak composition did not vary. Some lower chlorinated components maintained higher levels in the blood relative to other components; other components were selectively accumulated in tissues such as fat and muscle. Blood and fat residues in sheep declined during the last weeks of feeding. Microsomal oxidase levels were elevated in response to PCB and diet in both species, but the response was greater in sheep. Sheep liver microsomes were capable of metabolizing pure analogs and components of Aroclor 1242. Major differences in Aroclor profiles can be demonstrated between swine and sheep residues and total residues can be estimated by measuring selected peaks in blood and backfat. The peaks which provide the most reliable estimate of total PCB residue vary with species and Aroclor.

Electron transport in sheep ( Ovis aries) liver microsomes. Characteristics, effect of lipid peroxidation and aqueous acetone extraction

1. 1. Activities and contents of the electron transport components of sheep ( Ovis aries) liver microsomes are given. Enzymes or enzyme systems assayed are NADH-cytochrome c reductase, NADH-ferrieyanide reductase, NADH-dichlorophenol-indophenol reductase, NADH- and NADPH-neotetrazolium reductase, cytochrome b 5, cytochrome P-450 and the cyanide binding protein. 2. 2. Prior lipid peroxidation of sheep liver microsomes did not markedly alter NADH- and NADPH-cytochrome c reductase or NADH-ferricyanide reductase activities but decreased NADPH-dependent aniline hydroxylation activity. Intermediate amounts of prior lipid peroxidation enhanced the activity of NADPH-dependent lipid peroxidation. 3. 3. NADH-cytochrome c reductase activity of sheep liver microsomes was decreased 39–56% when 60% of the microsomal organic phosphorus was removed by acetone:water 90:10 (v/v) extraction but was not markedly altered by the removal of 25 and 44% of the microsomal organic phosphorus by acetone:water 100:4 (v/v) and acetone:water 100:7 (v/v) extractions.

Effect of aqueous acetone extractions and vitamin E on nadph-dependent lipid peroxidation of sheep and rat liver microsomes

1. 1. Extraction of sheep ( Ovis aries) liver microsomes with acetone : water 100 :4 (v/v), acetone : water 100 : 7 (v/v) and acetone : water 90 :10 (v/v) removed 25, 44 and 60%, respectively, of the organic phosphorus. 2. 2. Extraction of rat ( Rattus norvegicus) liver microsomes with acetone : water 100 :4 (v/v) caused a 5 % increase in the organic phosphorus content. 3. 3. The removal of 25, 44 and 60% of the organic phosphorus from sheep liver microsomes caused losses of NADPH-dependent lipid peroxidation activity that were far larger than the removal of phospholipid substrate would cause. Increasing the organic phosphorus content of rat liver microsomes by 5 % caused a slight increase in NADPH-dependent lipid peroxidation activity. 4. 4. The phospholipid compositions of the aqueous acetone extracts of sheep liver microsomes were similar to that of the total microsomal lipid extract. 5. 5. The hypothesis is put forward that NADPH-dependent lipid peroxidation has a structural phospholipid requirement and that more than one protein is involved in this enzymic process. 6. 6. Liver microsomes from rats fed 600 mg α-tocopheryl acetate/kg diet possessed negligible NADPH-dependent lipid peroxidation activity. Extraction of these microsomes with acetone : water 100 :4 (v/v) reversed the inhibition of NADPH-dependent lipid peroxidation by dietary vitamin E.

Changes in kinetic characteristics of sheep liver microsomes following induction and dietary stress

The effects of dietary stress and polychlorinated biphenyls (PCB) on the kinetic characteristics of ovine liver microsomal oxidases were studied. Curvilinear Lineweaver-Burke kinetic plots were obtained for the O-deethylation of p-nitrophenetole. Increasing linearity of the plots resulted from microsomal induction due to both dietary stress and PCB exposure. This suggests two enzyme systems with different magnitudes of induction catalyzing the same reaction.

Alkylamidase of sheep liver

1. 1. An amidase from sheep liver microsomes which catalyzes the hydrolysis of N-monosubstituted and N,N-disubstituted amides was purified 59-fold by hydroxylapatite and benzyl-DEAE-cellulose column chromatography. 2. 2. Acrylamide gel electrophoresis of the final enzyme preparation indicated that it was a single protein. 3. 3. The enzyme had an optimum at approximately pH 9, was not stimulated by divalent cations and had a molecular weight, as determined by gel filtration, of 230 000 to 250 000. 4. 4. The enzyme was unable to hydrolyze the lower members of a series of N- methyl-substituted amides and only had a trace of activity to N- methyl butyramide. The activity reached a maximum with N- methyl caproamide and decreased with an increase in chain length. The enzyme hydrolyzed N- methyl, N- ethyl and N- propyl caproamide at approximately equal rates. Of all the compounds investigated as substrates. N- phenyl caproamide had the shortest enzymatic half-life. Of the N,N- dialkyl-substituted caproamides evaluated, only N,N- dimethyl caproamide was hydrolyzed.