Isolation of two cytochrome P450 forms, CYP2A19 and CYP1A, from pig liver microsomes

Cytochromes P450 comprise a superfamily of proteins that play a crucial role in the biotransformation of numerous chemicals. Purified CYPs can be used e.g. in studies on structure or determining the drug metabolism pathways. In this work, purification of the porcine CYP1A and CYP2A19 to electrophoretic homogeneity from the pig hepatic microsomes using octylamino Sepharose and hydroxylapatite column chromatography is reported. The proteins have been clearly recognized by commercial antibodies against rat and human CYP1A2 (porcine CYP1A) and human CYP2A6 (CYP2A19) respectively, using Western blot. Activities of both enzymes were determined by specific substrates, 7-ethoxyresorufin, 7-methoxyresorufin (CYP1A) and coumarin (CYP2A19). The isolated enzymes show kinetic parameters similar to human counterparts. Taken together, pig cytochromes can be used for the testing of veterinary drug metabolism, useful for the determination of drug residues in meat of pigs. The results obtained show that the pigs may be a suitable model for biotransformation of xenobiotics in humans.     

Skatole Metabolism in the Pigs with Reduced Testicular Oestrogen Synthesis

The objectives of the study were to investigate the involvement of oestrogens in the regulation of skatole levels in pigs. In total, 44 intact male pigs, siblings from 10 litters, were included in the study. Pigs were orally treated weekly with either 0.1 mg letrozole/kg body weight to reduce endogenous oestrogens or the canola oil vehicle. Fat and liver samples were collected at slaughter at 16, 20 and 40 weeks of age. Skatole and androstenone levels in fat and activities of hepatic cytochrome P4501A1, CYP1A2, CYP2A19 and CYP2E1 were analysed. Letrozole treatment did not significantly change either the levels of skatole or activities of skatole‐metabolising enzymes, suggesting that oestrogens are not responsible for gender‐related differences in skatole concentrations in porcine tissues.     

In vitro and In vivo Association of Porcine Hepatic Cytochrome P450 3A and 2C Activities with Testicular Steroids

The aim of this study was to screen the inhibitory potential of several testicular steroids on cytochrome P450 3A (CYP3A) and 2C (CYP2C) activities in porcine liver microsomes. The microsomes used in this study were obtained from pubertal male pigs of two breeds, Landrace and Duroc. For the in vitro inhibition study, porcine microsomes were incubated in the presence of 17β‐estradiol, 17α‐estradiol, androstenone, dehydroepiandrosterone and dihydrotestosterone. Both reversible and mechanism‐based inhibitions were examined. 7‐benzyloxyresorufin (BR) and 7‐benzyloxy‐4‐trifluoromethylcoumarin (BFC) were used as substrates for CYP3A, and diclofenac and tolbutamide (TB) as substrates for CYP2C. 7‐benzyloxyresorufin O‐dealkylase (BROD) activity was inhibited by all tested steroids in the microsomes from Landrace pigs via mechanism‐based mode, but in the microsomes from Duroc pigs, BROD activities were inhibited only in the presence of 17β‐oestradiol. Mechanism‐based inhibition of BFC metabolism by the tested steroids was observed in the microsomes from both breeds, but this inhibition was weak and did not exceed 20%. TB hydroxylase (TBOH) activity in the microsomes from Duroc pigs was inhibited by 17α‐oestradiol through the mechanism‐based mode of inhibition. None of the investigated steroids inhibited TBOH activity in Landrace pigs. For the in vivo study, male pigs were injected with a single dose of human chorionic gonadotropin (hCG) to stimulate testicular steroid production by the Leydig cells. In vivo stimulation with hGC did not alter BROD activity either in Landrace or in Duroc pigs. BFC metabolism was significantly induced by hCG stimulation in both breeds and TBOH activity only in Duroc pigs. Activity of diclofenac hydroxylase was not detected in either Landrace or Duroc pigs. Breed significantly affected BROD and TBOH activity with BROD being higher in Landrace and TBOH in Duroc pigs. This study improved our understanding of the role of testicular steroids in the regulation of porcine CYP450 activity.     

Age‐related change of hepatic uridine diphosphate glucuronosyltransferase and sulfotransferase activities in male chickens and pigs

The hepatic activities of uridine diphosphate glucuronosyltransferase (UGT) and sulfotransferase (SULT) of male Ross 708 broiler chickens at the age of 1, 7, 14, 28, and 56 days and male Camborough‐29 pigs at the age of 1 day and 2, 5, 10, and 20 weeks were investigated. Glucuronidation and sulfation of 4‐nitrophenol were used to evaluate the activities. Porcine hepatic UGT and SULT activities were low at birth, peaked at around 5–10 weeks, and then declined. Both hepatic UGT and SULT activities of chickens were high at hatch and declined. Chicken hepatic UGT activity had a peak at the age of 28 days. Affinity of hepatic SULT to 4‐nitrophenol is similar in chickens and pigs, but the affinity of hepatic UGT in pigs was about 10 times higher than that in chickens. 4‐nitrophenol was predominantly conjugated by SULT instead of UGT in chicken livers from hatch to day 56. Conversely, hepatic UGT contributed predominantly in 4‐nitrophenol conjugation than the SULT in pigs from birth to 20 weeks. Therefore, age has significant impact on hepatic activities of UGT and SULT, and the importance of UGT and SULT on conjugation is different in chickens and pigs.     

Characterization of cytochrome P450-mediated drug metabolism in cats

In this study we examined activities of cytochrome P450 (CYP)1A, 2C, 2D and 3A using hepatic microsomes from five male and five female cats. CYP1A, 2C, 2D and 3A activities were referred by ethoxyresorufin O-deethylation (EROD), tolbutamide hydroxylation (TBH), bufuralol 1'-hydroxylation (BLH) and midazolam 1'- and 4-hydroxylation respectively. The anti-rat CYP1A2 and CYP3A2 serum significantly inhibited EROD and midazolam 1'- and 4-hydroxylation, suggesting that EROD and midazolam 1'- and 4-hydroxylation were catalysed by CYP1A and 3A in cats respectively. Quinidine inhibited BLH in cats microsomes at quite low concentrations, suggesting that BLH was catalysed by CYP2D in cats. Tolbutamide hydroxylation activities were negligible in hepatic microsomes from both male and female cats, suggesting CYP2C activities of cats are extremely low. This suggests that CYP2C substrates should be carefully administered to cats. Although there is no sexual difference in CYP1A activities, there are differences in CYP2D and 3A activities of cats. CYP2D activities were higher (3-fold), but CYP3A activities were lower (one-fifth) in female cats. These results might suggest that CYP2D and 3A substrates should be prescribed for male and female cats using different dosage regimen.     

In Vitro Cytochrome P450 2E1 and 2A Activities in the Presence of Testicular Steroids

Cytochrome P450 2E1 (CYP2E1) and 2A (CYP2A) are the main enzymes involved in the metabolism of skatole in pigs. In this study, physiological concentrations of androstenone, 17β‐oestradiol and testosterone were tested for their ability to regulate CYP2E1 and CYP2A activity in liver microsomes isolated from entire male and female pigs as well as in microsomes from Saccharomyces cerevisiae expressing either human recombinant CYP2E1 or CYP2A6. We found that physiological concentrations of androstenone and oestradiol had the ability to inhibit CYP2E1 activity. The magnitude of this inhibition (approximately 30%) was similar in recombinant human CYP2E1 and microsomes from entire male pigs. This inhibition was only seen when adding the steroid to the assay 15 min before the substrate. Interestingly, CYP2E1 activity in the microsomes from female pigs was not affected. None of the investigated steroids modified the activity of recombinant human CYP2A6. However, CYP2A activity was slightly increased in the microsomes from female pigs in the presence of oestradiol, but the magnitude of this increase was very low (below 10%) and probably irrelevant. Overall, these results indicate that physiological concentrations of androstenone and oestradiol have a potential to inhibit CYP2E1 activities in vitro, and that this inhibition is gender‐specific. Further studies are needed to investigate the biochemical mechanisms underlying those differences between the genders.     

Tiamulin selectively inhibits oxidative hepatic steroid and drug metabolism in vitro in the pig

The simultaneous use of the antibiotic tiamulin with certain ionophoric antibiotics (monensin, salinomycin) may give rise to a toxic interaction in pigs and poultry. In the present study, effects of tiamulin on hepatic cytochrome P450 activities in vitro were studied using pig liver microsomes. When tiamulin was added to the incubation medium the N-demethylation rate of ethylmorphine and the hydroxylation of testosterone at the 6β- and 1 lα- positions was sirongly inhibited. Tiamulin inhibited these activities more than SKF525A or cimetidine, but less than ketoconazole. The microsomal N-demethylation rate of erythromycin and the hydroxylation of testosterone at the 28- position were inhibited to a lesser degree, whereas the ethoxyresorufin-O-deethylation, aniline hydroxylation and testosterone hydroxylations at the 15α- and 15β- positions were not affected by tiamulin. No in vitro complexation by tiamulin of cytochrome P450 resulting in a loss of CO-binding capacity could be demonstrated. Results from the present study suggest a selective inhibition of cytochrome P450 enzymes in pigs, probably belonging to the P4503A subfamily. The mechanism of this interaction is still unclear. However, interactions between tiamulin and those veterinary drugs or endogenous compounds which undergo oxidative metabolism by P450 enzymes must be considered. More research is needed to reveal which of the P450 enzymes are affected by tiamulin in order to improve the understanding and probably the predictability of this interaction.     

Reaction phenotyping of vinblastine metabolism in dogs

Vinblastine is a vinca alkaloid used either as a single agent or in combination therapy for the treatment of canine mast cell tumours and lymphomas. The objective of this study was to determine which isoform of cytochrome P450 enzyme is responsible for the majority of vinblastine metabolism in dogs. A panel of eight recombinant canine cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP3A12, CYP3A26, CYP2B11, CYP2C41, CYP2C21 and CYP2D15) were incubated in vitro with vinblastine. Findings were confirmed by the use of canine polyclonal antibodies of cytochrome P450 enzymes (CYP1A1, CYP3A12, CYP2B11 and CYP2C21) that were pre‐incubated with individual and pooled hepatic microsomes that were purified from canine liver. Substrate depletion was observed in the presence of recombinant CYP3A12, whereas depletion did not substantially occur when microsomes were pre‐incubated with polyclonal antibodies against CYP3A12. These findings confirmed that CYP3A12 is the major cytochrome P450 isoform responsible for the metabolism of vinblastine in dogs.     

Comparative expression of liver cytochrome P450-dependent monooxygenases in the horse and in other agricultural and laboratory species

The apoprotein expression and the catalytic activities of cytochrome P450s involved in the biotransformation of xenobiotics were investigated in horse liver microsomes and compared with those of food producing (cattle, pigs, broiler chicks, and rabbits) and laboratory species (rats). Western blot analysis revealed the presence of proteins immunorelated to rat CYP 1A, CYP 2B, CYP 2E, and CYP 3A subfamilies in hepatic microsomes from horses and from any other examined species. With the exception of the N-demethylation of N-nitrosodimethylamine in broiler chicks, all the recorded interspecies differences were quantitative in nature. Equine preparations proved the most active in the biotransformation of the CYP 1A substrates ethoxy- and methoxyresorufin and the least active in the metabolism of aminopyrine and ethoxycoumarin. On a comparative basis, large differences were observed in the rate of the in vitro metabolism of model substrates between "minor" (rabbits, horses) and "major" food producing species. Taken in due consideration the limitations of the in vitro approach, results from this study reinforce the conclusion that studies on drug efficacy and residue depletion should be performed in each target species.     

Persistence of porcine reproductive and respiratory syndrome virus in intensive farrow-to-finish pig herds.

An epidemiological study of porcine reproductive and respiratory syndrome (PRRS) within pig herds was conducted in 8 intensive farrow-to-finish pig farms. Persistence of PRRS virus (PRRSV) in pig herds was demonstrated by regular postmortem examination on 2 farms for a period of 2 y. Virus isolation and serum neutralization (SN) tests were performed on the sera collected from 9 groups of pigs (10 pigs/group) of various ages on 8 pig farms. Except for 1 farm, isolation rates of PRRSV reached the highest level of 70 to 100% of pigs 6 to 8 wk of age, which coincided with the lowest levels of maternal immunity. In 1 pig herd, sows (39 in total) with SN titers of < or = 1:2, 1:4-1:8, and > or = 1:16 were designated as groups 1, 2, and 3, respectively. Sera were obtained from their progeny (3 pigs randomly selected from each litter) at various ages from 0 to 22 weeks. A positive correlation (r = 0.377, P < 0.001) between the SN titers of sows and those of their progeny (1-week-old piglets) was observed. Pigs at the age of 6 wk, only 7.9% of group 1 pigs compared to 72.4% of group 3 pigs were seropositive. A significant difference (P < 0.01) in the percentage of pigs with PRRSV viremia among the 3 groups was observed, with the lowest level found in group 3 pigs. The isolation rates of PRRSV from serum reached the maximum at the age of 9 wk for all 3 groups. The results indicated that passively acquired serum antibodies conferred a protective effect for piglets; however, loss of passive immunity at various ages of pigs produced susceptible pigs that resulted in PRRSV persistence in the pig herds. Pigs 6 to 9 weeks old were the major reservoir for PRRSV in farrow-to-finish pig herds.     

Effect of sex steroids on expression of sulfotransferase 2B1 immunoreactive protein in primary cultured porcine hepatocytes

An excessive accumulation of androstenone (5α-androst-16-en-3-one) in pig adipose tissue is one of the two major contributors to the phenomenon of boar taint. High levels of adipose tissue androstenone have been related to a low rate of hepatic androstenone metabolism, which includes two stages: oxidative and conjugative. Sulfotransferases (SULTs), alongside with other specific enzymes, play the key role in the conjugative stage of androstenone metabolism. The present study investigated the mechanism regulating expression of sulfotransferase 2B1 (SULT2B1) immunoreactive protein using primary cultured pig hepatocytes as a model system. A specific objective was to determine whether the expression of pig hepatic SULT2B1 is regulated by the sex steroids; androstenone, testosterone and estrone sulphate. The study was performed on entire male pigs of a Large White (40%) × Landrace (40%) × Duroc (20%) cross-breed, average carcass weight 72.2 kg. The study shows that SULT2B1 immunoreactive protein expression can be induced by testosterone (final concentrations, 10 and 500 nM) and repressed by estrone sulphate (final concentration, 100 nM). Androstenone had no significant effect on SULT2B1 immunoreactive protein expression in the range of concentration, 10 nM to 1 μM. Time-courses (0 to 48 h) of steroid effects were investigated. The maximum effects of testosterone and estrone sulphate were observed in 24 h after the steroid treatments. This study provides direct evidence for involvement of sex steroids in the regulation of porcine hepatic SULTs.     

Skatole metabolism in the intact pre-pubescent male pig: The relationship between hepatic enzyme activity and skatole concentrations in plasma and fat

The objective of this study was to evaluate, in the pre-pubescent intact male pig, the relationship between skatole levels and the activity of hepatic cytochrome P4502E1 (CYP2E1), cytochrome P4502A (CYP2A), aldehyde oxidase (AO), and phenol sulfotransferase 1A1 (SULT1A1). The activity of these enzymes has been positively associated with skatole clearance in mature boars. Twenty-four intact male pigs were weaned at 28 days of age and slaughtered 2 weeks postweaning, at which time caecal contents, blood, fat, and liver samples were collected. Caecal contents and fat were analyzed for skatole concentrations, and plasma was analyzed for skatole and steroid hormone (testosterone (T), dehydroepiandrosterone (DHEAS), estrone sulphate (E₁S)) concentrations. CYP2A, CYP2E1, and AO, as well as SULT1A1 activities were evaluated in liver samples. Stepwise regression was utilized considering plasma or fat skatole concentration as the dependent variables and hormone concentrations and enzyme activities as independent variables. The activities of the enzymes CYP2A, CYP2E1, and AO and concentrations of the hormones T, DHEAS, or E₁S were not correlated with concentrations of skatole in plasma or fat. However, SULT1A1 activity was negatively correlated with plasma (r = − 0.70, P < 0.05) and backfat (r = − 0.41, P < 0.05) skatole concentrations. Furthermore, this correlation was improved in plasma (r = − 0.88, P < 0.05) and fat (r = − 0.63, P < 0.05) when the concentrations of skatole in caecal contents was included as an independent variable in the multiple regression analysis, demonstrating the importance of measuring skatole production in these studies. T, DHEAS, and E₁S concentrations in plasma were not correlated with the activity of any of the enzymes evaluated. This study suggests that SULT1A1 is important in the metabolism of skatole in pre-pubescent pigs and the overall metabolism of skatole in the pre-pubescent pig differs from that in the mature boar.     

Feed–drug interaction of orally applied butyrate and phenobarbital on hepatic cytochrome P450 activity in chickens

The expression of hepatic drug‐metabolizing cytochrome P450 (CYP) enzymes may be affected by several nutrition‐derived compounds, such as by the commonly applied feed additive butyrate, possibly leading to feed–drug interactions. The aim of this study was to provide some evidence if butyrate can alter the activity of hepatic CYPs in chickens exposed to CYP‐inducing xenobiotics, monitoring for the first time the possibility of such interaction. Ross 308 chickens in the grower phase were treated with daily intracoelomal phenobarbital (PB) injection (80 mg/kg BW), applied as a non‐specific CYP‐inducer, simultaneously with two different doses of intra‐ingluvial sodium butyrate boluses (0.25 and 1.25 g/kg BW) for 5 days. Activity of CYP2H and CYP3A subfamilies was assessed by specific enzyme assays from isolated liver microsomes. According to our results, the lower dose of orally administered butyrate significantly attenuated the PB‐triggered elevation of both hepatic CYP2H and CYP3A activities, which might be in association with the partly common signalling pathways of butyrate and CYP‐inducing drugs, such as that of PB. Based on these data, butyrate may take part in pharmacoepigenetic interactions with simultaneously applied drugs or other CYP‐inducing xenobiotics, with possible consequences for food safety and pharmacotherapy. Butyrate was found to be capable to maintain physiological CYP activity by attenuating CYP induction, underlining the safety of butyrate application in poultry nutrition.     

Hepatic Ethoxy‐, Methoxy‐ and Pentoxyresorufin O‐Dealkylase Activities in Landrace and Duroc Pigs Stimulated with hCG

The effect of human chorionic gonadotropin (hCG) stimulation on the activities of ethoxyresorufin O‐deethylase (EROD), methoxyresorufin O‐demethylase (MROD) and pentoxyresorufin O‐depentylase (PROD) was studied in intact male pigs of purebred Landrace and Duroc breeds. Pigs were divided into four groups: two control groups of each breed, without hCG stimulation (n = 20 for each breed), and two experimental groups (n = 18 for each breed), with hCG stimulation (Pregnyl^®; N.V. Organon, Oss, The Netherlands, 30 IU/kg live weight). Pigs were slaughtered 3 days after hCG stimulation and enzyme activities were measured in hepatic microsomes using two approaches. First, only one substrate concentration was used for the analysis of each enzyme activity. We found that EROD activity was suppressed by hCG‐stimulation in Landrace (p = 0.004), but not Duroc pigs (p > 0.05). Generally, EROD activity was higher in Duroc pigs compared with Landrace (p = 0.017). Methoxyresorufin O‐demethylase and PROD activities did not differ between groups (p > 0.05). To further characterize EROD, MROD and PROD, enzyme kinetic studies were performed. V_max values for EROD and MROD in both breeds were lower after hCG stimuation (p < 0.001 for Landrace and p < 0.05 for Duroc). Additionally, V_max values for EROD significantly differed between Landrace and Duroc pigs being higher in Duroc pigs (p < 0.05). We concluded that both hCG stimulation and breed differences may be important in the regulation of EROD and MROD activities. This study provides the first data on the effect of hCG stimulation and thus high testicular steroids, on EROD, MROD and PROD activities. Further studies are needed to investigate individual CYP450 enzymes and their regulation in porcine tissues.     

Expressions of cytochrome P450 aromatase and anti-Müllerian hormone in testes of fattening pigs by the timing of the first vaccination for immunocastration

In practice, two injections of gonadotropin-releasing hormone (GnRH) vaccine are recommended for pig immunocastration for effective outcomes. The present study aimed to investigate the expressions of cytochrome P450 aromatase (P450_arom) and anti-Müllerian hormone (AMH) in testes, testicular length and testicular histomorphometry of the fattening pigs receiving the first injection of GnRH vaccine 6 weeks earlier than the standard protocol. Based on vaccination protocol, 24 pigs were equally divided into three groups: T1 was vaccinated at 15 and 19 weeks of age, T2 received vaccine at 9 and 19 weeks of age and C remained intact. P450_arom and AMH expressions were analysed using immunohistochemistry and Western blot. The results revealed that testicular length was highest in C pigs, but not different between T1 and T2 groups (6.5 ± 0.2 versus 6.9 ± 0.3 cm, p = .538). Histomorphometry demonstrated that the height of spermatogenic epithelia, the diameter of seminiferous tubules and the number of seminiferous tubules between T1 and T2 groups were not different (p > .05). For P450_arom, immunohistochemistry revealed that H-score of C group was significantly higher than that of both T1 and T2 groups. Western blot analysis showed that C group possessed the densest protein band. Moreover, H-score between T1 and T2 groups was not significantly different. Protein band intensity between both groups was not apparently different. As for AMH, C pigs had significantly lower H-score than both T1 and T2 pigs. Furthermore, T2 pigs possessed significantly higher H-score than T1 pigs. Western blot analysis showed that the most intense protein band was found in T2 group. In summary, GnRH vaccine affected testicular development and functions. The first injection could be performed either at 9 or 15 weeks of age since both protocols contributed to comparable results in aspect of testicular length, histomorphometry and expressions of P450_arom and AMH.     

Long-lasting enhancement of CYP activity after discontinuation of repeated administration of phenobarbital in dogs

We investigated how long in vivo hepatic cytochrome P450 (CYP) activity is enhanced even after discontinuation of repeated oral administration of phenobarbital (PB) in dogs using antipyrine clearance, which reflects hepatic CYP activity. A single antipyrine (5 mg/kg) was administered intravenously before and 34 days after the repeated oral administration of PB (5 mg/kg, bid) and 2, 4, 6, and 8 weeks after the discontinuation of PB in 5 dogs. Antipyrine clearance was increased by the repeated administration of PB, and remained increased 2 and 4, but not 6 and 8 weeks after the discontinuation of PB. The result suggests that hepatic CYP activity was enhanced by the repeated administration of PB, and this enhancement may last for at least 4 weeks even after its discontinuation.     

Modulation of porcine biotransformation enzymes by anthelmintic therapy with fenbendazole and flubendazole

Fenbendazole (FEN) and flubendazole (FLU) are benzimidazole anthelmintics often used in pig management for the control of nematodoses. The in vivo study presented here was designed to test the influence of FLU and FEN on cytochrome P4501A and other cytochrome P450 (CYP) isoforms, UDP-glucuronosyl transferase and several carbonyl reducing enzymes. The results indicated that FEN (in a single therapeutic dose as well as in repeated therapeutic doses) caused significant induction of pig CYP1A, while FLU did not show an inductive effect towards this isoform. Some of the other hepatic and intestinal biotransformation enzymes that were assayed were moderately influenced by FEN or FLU. Strong CYP1A induction following FEN therapy in pigs may negatively affect the efficacy and pharmacokinetics of FEN itself or other simultaneously or consecutively administered drugs. From the perspective of biotransformation enzyme modulation, FLU would appear to be a more convenient anthelmintic therapy of pigs than FEN.     

Gender-related differences in cytochrome P450 in porcine liver--implication for activity, expression and inhibition by testicular steroids

In pigs, the hepatic cytochrome P450 (CYP) 1A2, 2A and 2E1 activity is important in the regulation of skatole accumulation in adipose tissue. This study investigated gender-related differences in CYP1A2, 2A and 2E1 dependent activity, protein and mRNA expression. This study also investigated the gonadal steroid dependent inhibition of CYP activity in relation to gender and dietary composition. Microsomes were prepared from the liver of female and entire male pigs (Landrace × Yorkshire sire and Duroc boars) reared under similar conditions and slaughtered at an age of 164 days. A group of entire male pigs fed dried chicory root for 16 days prior to slaughter were included in the study. CYP activities were assessed by the use of probe substrates, whilst mRNA and protein expression were analysed by RT-PCR and Western blotting. Furthermore inhibition of CYP dependent activity by gonadal steroids was assessed in vitro. Microsomes from female pigs had greater CYP1A2 and 2A activity, as well as mRNA expression compared to entire male pigs. The antibodies used did not detected differences in protein expression. In vitro inhibition by 17β-oestradiol, oestrone, androstenone and 3β-OH androstenol of CYP2E1 activity in microsomes from entire male pigs as well as inhibition of CYP1A activity in chicory fed entire male pigs was observed. Apart from that no effect of steroids was shown. In conclusion, female pigs show greater CYP activity and mRNA expression. Including chicory in the diet for 16 days changed the gonadal steroid dependent inhibition of CYP activity in entire male pigs.     

Hepatic and pulmonary enzyme activities in horses

OBJECTIVE: To determine hepatic and pulmonary phase-I and phase-II enzyme activities in horses. SAMPLE POPULATION: Pulmonary and hepatic tissues from 22 horses that were 4 months to 32 years old. PROCEDURE: Pulmonary and hepatic tissues from horses were used to prepare cytosolic (glutathione S-transferase and soluble epoxide hydrolase) and microsomal (cytochrome P450 monooxygenases) enzymes. Rates of microsomal metabolism of ethoxyresorufin, pentoxyresorufin, and naphthalene were determined by high-performance liquid chromatography. Activities of glutathione S-transferase and soluble epoxide hydrolase were determined spectrophotometrically. Cytochrome P450 content was determined by carbon monoxide bound-difference spectrum of dithionite-reduced microsomes. Activity was expressed relative to total protein concentration. RESULTS: Microsomal protein and cytochromeP450 contents were detectable in all horses and did not vary with age. Hepatic ethoxyresorufin metabolism was detected in all horses; by comparison, pulmonary metabolism of ethoxyresorufin and hepatic and pulmonary metabolism of pentoxyresorufin were detected at lower rates. Rate of hepatic naphthalene metabolism remained constant with increasing age, whereas rate of pulmonary naphthalene metabolism was significantly lower in weanlings (ie, horses 4 to 6 months old), compared with adult horses. Hepatic glutathione S-transferase activity (cytosol) increased with age; however, these changes were not significant. Pulmonary glutathione S-transferase activity (cytosol) was significantly lower in weanlings than adult horses. Hepatic and pulmonary soluble epoxide hydrolase did not vary with age of horses. CONCLUSIONS AND CLINICAL RELEVANCE: Activity of cytochrome P450 isoforms that metabolize naphthalene and glutathione S-transferases in lungs are significantly lower in weanlings than adult horses, which suggests reduced ability of young horses to metabolize xenobiotics by this organ.     

Comparison of hepatic in vitro metabolism of the pyrrolizidine alkaloid senecionine in sheep and cattle

OBJECTIVE: To compare hepatic metabolism of pyrrolizidine alkaloids (PAs) between sheep and cattle and elucidate the protective mechanism of sheep. SAMPLE POPULATION: Liver microsomes and cytosol from 8 sheep and 8 cattle. PROCEDURE: The PA senecionine, senecionine N-oxide (nontoxic metabolite) and 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP; toxic metabolite) were measured in microsomal incubations. The kcat (turnover number) was determined for DHP and N-oxide formation. Chemical and immunochemical inhibitors were used to assess the role of cytochrome P450s, flavin-containing monooxygenases (FMOs), and carboxylesterases in senecionine metabolism. The CYP3A, CYP2B, and FMO concentrations and activities were determined, in addition to the role of glutathione (GSH) in senecionine metabolism. RESULTS: DHP concentration did not differ between species. Sheep formed more N-oxide, had higher N-oxide kcat, and metabolized senecionine faster than cattle. The P450 concentrations and isoforms had a large influence on DHP formation, whereas FMOs had a large influence on N-oxide formation. In cattle, CYP3A played a larger role in DHP formation than in sheep. FMO activity was greater in sheep than in cattle. Addition of GSH to in vitro microsomal incubations decreased DHP formation; addition of cytosol decreased N-oxide formation. CONCLUSIONS AND CLINICAL RELEVANCE: Hepatic metabolism differences alone do not account for the variation in susceptibility seen between these species. Rather, increased ruminal metabolism in sheep appears to be an important protective mechanism, with hepatic enzymes providing a secondary means to degrade any PAs that are absorbed from the rumen.