Hepatic biotransformation pathways and ruminal metabolic stability of the novel anthelmintic monepantel in sheep and cattle

Monepantel (MNP) is a new amino‐acetonitrile derivative anthelmintic drug used for the treatment of gastrointestinal (GI) nematodes in sheep. The present work investigated the main enzymatic pathways involved in the hepatic biotransformation of MNP in sheep and cattle. The metabolic stability in ruminal fluid of both the parent drug and its main metabolite (monepantel sulphone, MNPSO₂) was characterized as well. Additionally, the relative distribution of both anthelmintic molecules between the fluid and particulate phases of the ruminal content was studied. Liver microsomal fractions from six (6) rams and five (5) steers were incubated with a 40 μm of MNP. Heat pretreatment (50 °C for 2 min) of liver microsomes was performed for inactivation of the flavin‐monooxygenase (FMO) system. Additionally, MNP was incubated in the presence of 4, 40, and 80 μm of methimazole (MTZ), a FMO inhibitor, or equimolar concentrations of piperonyl butoxide (PBx), a well‐known general cytochrome P450 (CYP) inhibitor. In both ruminant species, MNPSO₂ was the main metabolite detected after MNP incubation with liver microsomes. The conversion rate of MNP into MNPSO₂ was fivefold higher (P < 0.05) in sheep (0.15 ± 0.08 nmol/min·mg) compared to cattle. In sheep, the relative involvement of both FMO and CYP systems (FMO/CYP) was 36/64. Virtually, only the CYP system appeared to be involved in the production of MNPSO₂ in cattle liver. Methimazole significantly reduced (41 to 79%) the rate of MNPSO₂ production in sheep liver microsomes whereas it did not inhibit MNP oxidation in cattle liver microsomes. On the other hand, PBx inhibited the production of MNPSO₂ in liver microsomes of both sheep (58 to 98%, in a dose‐dependent manner) and cattle (almost 100%, independently of the PBx concentration added). The incubation of MNP and MNPSO₂ with ruminal contents of both species showed a high chemical stability without evident metabolism and/or degradation as well as an extensive degree of adsorption (83% to 90%) to the solid phase of the ruminal content. Overall, these results are a further contribution to the understanding of the metabolic fate of this anthelmintic drug in ruminants.     

Assessment of the main metabolism pathways for the flukicidal compound triclabendazole in sheep

Triclabendazole (TCBZ) is an halogenated benzimidazole (BZD) compound worldwide used to control immature and adult stages of the liver fluke Fasciola hepatica. The purpose of this investigation was to characterize in vitro the patterns of hepatic and ruminal biotransformation of TCBZ and its metabolites in sheep. TCBZ parent drug was metabolized into its sulphoxide (TCBZSO), sulphone (TCBZSO2) and hydroxy derivatives by sheep liver microsomes. The same microsomal fraction was also able to oxidize TCBZSO into TCBZSO2 and hydroxy-TCBZSO (HO-TCBZSO). TCBZ sulphoxidation was significantly (P < 0.001) inhibited after inactivation of the flavin-monooxygenase (FMO) system (77% inhibition) as well as in the presence of the FMO substrate methimazole (MTZ) (71% inhibition). TCBZ sulphoxidative metabolism was also reduced (24% inhibition, P < 0.05) by the cytochrome P450 inhibitor piperonyl butoxide (PB). The rate of TCBZSO conversion into TCBZSO2 was also significantly inhibited by PB (55% inhibition), MTZ (52% inhibition) and also following FMO inactivation (58% inhibition). The data reported here indicate that the FMO is the main enzymatic pathway involved in TCBZ sulphoxidation (ratio FMO/P450 = 3.83 +/- 1.63), although both enzymatic systems participate in a similar proportion in the sulphonation of TCBZSO to form the sulphone metabolite (ratio FMO/P450 = 1.31 +/- 0.23). Additionally, ketoconazole (KTZ) did not affect TCBZ sulphoxidation but decreased (66% inhibition, P < 0.05) the formation of TCBZSO2. Similarly, inhibition of TCBZSO2 production was observed after incubation of TCBZSO in the presence of KTZ and erythromycin (ETM). Conversely, thiabendazole (TBZ) and fenbendazole (FBZ) did not affect the oxidative metabolism of both incubated substrates. The sheep ruminal microflora was able to reduce the sulphoxide (TCBZSO) into the parent thioether (TCBZ). The ruminal sulphoreduction of the HO-TCBZSO derivative into HO-TCBZ was also demonstrated. The rate of sulphoreduction of HO-TCBZSO was significantly (P < 0.05) higher than that observed for TCBZSO. The metabolic approach tested here contributes to the identification of the different pathways involved in drug biotransformation in ruminant species. These findings on the pattern of hepatic and ruminal biotransformation of TCBZ and its main metabolites are a further contribution to the understanding of the pharmacological properties of widely used anthelmintics in ruminants. Comprehension of TCBZ metabolism is critical to optimize its flukicidal activity.     

Benzydamine as a useful substrate of hepatic flavin‐containing monooxygenase activity in veterinary species

Capolongo, F., Santi, A., Anfossi, P., Montesissa, C. Benzydamine as a useful substrate of hepatic flavin‐containing monooxygenase activity in veterinary species. J. vet. Pharmacol. Therap. doi: 10.1111/j.1365‐2885.2009.01145.x. Benzydamine (BZ), a weak base and an indazole derivative with analgesic and antipyretic properties used in human and veterinary medicine, is metabolized in human, rat, cattle and rabbit to a wide range of metabolites. One of the main metabolites, BZ N‐oxide (BZ‐NO), is produced in the liver and brain by flavin‐containing monooxigenases (FMOs), by liver and brain enzymes. To evaluate the suitability of BZ as an FMO probe in veterinary species, BZ metabolism was studied in vitro using liver microsomes from bovine, rabbit and swine. Kinetic parameters, K_m and V_max, of BZ‐NO production, were evaluated to corroborate the pivotal role of FMOs. Inhibition studies were carried out by heat inactivation and by specific FMO chemical inhibitors: trimethylamine and methimazole. The results confirmed the presence of FMO activity in the liver and the role of BZ as a suitable marker of FMO enzyme activities for the veterinary species considered.     

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.     

Stability of ivermectin in rumen fluids

To determine whether ivermectin is metabolized in the rumen, in vitro studies were conducted with the tritium-labelled H₂B_1a component of ivermectin in rumen fluid from sheep and cattle. No detectable metabolism occurred over 24 h in in vitro incubations at 38°. The viability of the microbes in the rumen fluids was demonstrated by the conversion of 17% and 11% of [¹⁴C]cellulose to ¹⁴CO₂ in 24 h in the incubations with sheep and steer rumen fluids respectively. The results indicate that ivermectin is not metabolized in the rumen. Based on the lack of in vitro metabolism of ivermectin in rumen fluid, the similarity of in vitro liver microsomal metabolism with in vivo metabolism of the avermectins and the physicochemical properties of the avermectins, any disappearance of ivermectin in vitro from rumen fluid is probably a result of binding to solids or surfaces. Apparent discrimination by dung beetles, where observed, between control faeces and faeces from cattle or sheep treated with ivermectin or abamectin therefore must be attributable to chance, to factors unrelated to treatment or to factors such as changes in amino acid composition rather than the production of volatile metabolites of ivermectin.     

In vitro Metabolism of 14C-Moxidectin by Hepatic Microsomes from Various Species

Moxidectin is an antiparasitic drug widely used in cattle, sheep and companion animals. No data were available on its metabolism in wild species or in monogastrics. The in vitro metabolism of ¹⁴C-moxidectin was studied using hepatic microsomes from several different species: cow (Bos taurus), sheep (Ovis ovis), goat (Capra hircus), deer (Cervus dama), rat (Rattus norvegicus), pig (Sus scrofa and rabbit (Oryctolagus cuniculus). After separation and quantification by HPLC, the extent of metabolism of ¹⁴C-moxidectin was greatest with microsomes from sheep (32.7%) as compared to those from cows (20.6%), deer (15.4%), goats (12.7%), rabbits (7.0%) or rats (3.0%). The least metabolism occurred with microsomes from pigs, with 0.8% of total detected metabolites. A C₂₉ monohydroxymethyl metabolite was detected in the greatest amounts, providing 0.4% out of the total detected radioactivity in pigs and 19.3% in sheep. In addition, the importance of P450 3A in the metabolism of ¹⁴C-moxidectin was confirmed by using in vivo induced P450 in combination with various P450 inhibitors.     

The oxidative metabolism of fenbendazole: a comparative study

The oxidative metabolism of fenbendazole (FBZ) was studied in hepatic fractions prepared from livers of cattle, sheep, goats, chickens, ducks, turkeys, rats, rabbits and catfish. All species produced the sulfoxide metabolite (oxfendazole; FBZ-SO), and p-hydroxyfenbendazole (FBZ-OH) was produced by all species except sheep. The product of demethoxycarbonylation, fenbendazole amine (FBZ-NH2), was not produced by liver preparations of any species. A fourth metabolite, resulting from the further oxidation of oxfendazole, fenbendazole sulfone (FBZ-SO2), was formed in all species but at highly varying rates. The chicken exhibited the highest overall rate of FBZ metabolism, followed by the duck, goat, sheep, steer, catfish, rat, rabbit, and turkey. Considerable variation was evident among avian species, the duck and turkey produced substantially less of the FBZ-OH and FBZ-SO2 metabolites than the chicken. Catfish liver preparations formed equivalent amounts of metabolite at 25 degrees C and 37 degrees C incubation temperatures. The formation of the sulfone metabolite (FBZ-SO2), however, was practically nonexistent in catfish.     

Influence of verapamil on the efflux and metabolism of 14C moxidectin in cultured rat hepatocytes

Moxidectin (MOX) is an antiparasitic drug widely used in cattle, sheep and companion animals. As a result of the implication of cytochrome P450 3 A in the metabolism of MOX and the role of competitor substrates of P-glycoprotein (Pgp) in modification of the bioavailability of endectocides, we studied the influence of verapamil (a multidrug-resistance reversing agent) on the metabolism of 14C moxidectin in cultured rat hepatocytes over 72 h. The metabolism of MOX remained low: 10.79 +/- 1.99% of the total 14C moxidectin for the main detected metabolite in verapamil-treated cells and 7.17 +/- 0.74% for the control cells after 24 h. The main detected metabolite in rat hepatocytes was the same as that detected in rat hepatic microsomes (the C29 monohydroxymethyl metabolite). Verapamil increased the quantity of MOX in the cells after 24, 48 and 72 h. Examination of the Area Under the concentration time Curve (AUC) of the main detected metabolite revealed a significant increase in the exposure of cells to MOX after verapamil treatment throughout the experiment. It is hypothesized that verapamil interfered with MOX as a substrate for Pgp during the initial incubation period. After this initial interaction, verapamil metabolites were able to interfere with Pgp. This experiment demonstrated the implication of Pgp in the transport of MOX and allowed prediction of the drug-drug interactions which might modify the bioavailability of endectocides.     

Phase 1 and phase 2 metabolic activities along the small intestine in adult male sheep

Maté, L., Virkel, G., Lifschitz, A., Sallovitz, J., Ballent, M., Lanusse, C. Phase 1 and phase 2 metabolic activities along the small intestine in adult male sheep. J. vet. Pharmacol. Therap. 33 , 537–545. Metabolic activities of several xenobiotic metabolizing enzymes were evaluated in both hepatic and enteric subcellular fractions obtained from Corriedale × Merino crossbreed rams by using a biochemical approach. Microsomes obtained from the different segments of sheep small intestinal mucosa displayed cytochrome P450 (CYP)‐dependent N‐demethylations but not O‐deethylase activities apparently occurred. CYP‐mediated N‐demethylations neither decreased nor increased along the small intestinal mucosa. Percentages of activity for erythromycin N‐demethylase in the small intestine were between 29% (duodenum) and 45% (ileum) from that measured in the liver, whereas those determined for triacetyl‐oleandomycin N‐demethylation ranged between 10% (duodenum) and 15% (jejunum) of the same hepatic activity. Conversely, metabolic rates for aminopyrine and chlorfeniramine N‐demethylations in the gut mucosa ranged between 3% and 7% compared to their respective hepatic enzyme activities. Sheep enteric mucosa also displayed metabolic reactions typically mediated by flavin‐containing monooxygenases (FMOs), carbonyl reductases (CBRs), carboxylesterases (CES), glutathione S‐transferases (GSTs) and uridine diphosphoglucuronyltransferases (UGTs). The FMO‐mediated sulfoxidation of methimazole was 2.6‐fold higher (P < 0.01) in the ileal compared to the duodenal mucosa. Percentages of activity for the microsomal CBR‐dependent biotransformation of menadione were between 12% (ileum) and 19% (duodenum–jejunum) of the total activity measured in the liver; metabolic rates measured in duodenum and jejunum were ～1.7‐fold higher (P < 0.05) than that observed in the ileum. The microsomal CES activity (using p‐nitrophenyl acetate as substrate) was around twofold higher in duodenum (P < 0.05) and jejunum (P < 0.01) in comparison to the ileum. Cytosolic GST‐dependent activities (toward 1‐chloro, 2,4‐dinitrobenzene) were similar in the mucosa of duodenum, jejunum and ileum. Microsomal UGT activities (toward 1‐naphthol) in duodenum and jejunum were three‐ and fourfold higher, respectively, compared to that measured in the ileum. The small intestinal mucosa may play a critical defensive role due to its involvement in the detoxification of toxic compounds prior to absorption. In addition, gut metabolic reactions may contribute to the presystemic metabolism of orally administered drugs. These results are a further contribution to the understanding of the relevance of the extra‐hepatic metabolism of xenobiotics in ruminant species.     

Species differences in hepatic biotransformation of the anthelmintic drug flubendazole

Flubendazole (FLBZ) is a broad‐spectrum benzimidazole anthelmintic used in pigs, poultry, and humans. It has been proposed as a candidate for development for use in elimination programmes for lymphatic filariasis and onchocerciasis in humans. Moreover, FLBZ has shown promise in cancer chemotherapy, particularly for neuroblastoma. This work investigated the hepatic carbonyl‐reducing pathway of FLBZ in different species, including humans. Microsomal and cytosolic fractions were obtained from sheep, cattle, pig, hen, rat, and human liver. Both subcellular fractions of each species converted FLBZ into a reduced metabolite (red‐FLBZ). The rate of microsomal red‐FLBZ production was highest in sheep (1.92 ± 0.13 nmol/min.mg) and lowest in pigs (0.04 ± 0.02 nmol/min.mg); cytosolic red‐FLBZ production ranged from 0.02 ± 0.01 (pig) to 1.86 ± 0.61 nmol/min.mg (sheep). Only subcellular fractions from sheep liver oxidized red‐FLBZ to FLBZ in a NADP⁺‐dependent oxidative reaction. Liver microsomes from both pigs and humans transformed FLBZ to red‐FLBZ and a hydrolyzed metabolite. Very significant differences in the pattern of FLBZ metabolism were observed among the tested species and humans. These results reinforce the need for caution in extrapolating data on metabolism, efficacy, and safety of drugs derived from studies performed in different species.     

Effect of breed and gender on bovine liver cytochrome P450 3A (CYP3A) expression and inter-species comparison with other domestic ruminants

The cytochrome P450 (P450) superfamily represents a group of relevant enzymes in the field of drug metabolism and several exogenous or constitutional factors contribute to regulate its expression. Cattle represent an important source of animal-derived food-products and studies concerning the P450 expression are needed for the extrapolation of pharmacotoxicological data from one species to another and for the evaluation of the consumer's risk associated with the consumption of harmful residues found in foodstuffs. In the present study, possible breed-, gender- and species-differences in P4503A (the P450 subfamily more expressed in the human liver) expression were studied in vitro in Piedmontese (PDM) and Limousin (LIM) meat cattle breeds of both sexes and in domestic Ruminants (cattle, sheep and goats). Cytochrome P450 and P4503A contents as well as CYP3A-dependent drug metabolising enzymes (DME) were measured in liver microsomes. Significant lower levels of P450 (P < 0.001) and P4503A (P < 0.05) contents were observed in PDM vs. LIM of both sexes; the P4503A-dependent DME activities were significantly (P values ranging from 0.05 up to 0.001) higher in PDM cattle, particularly in males. A gender-effect in DME activities was noticed (P < 0.05) only in PDM male cattle. With regards to the species, the expression of both P4503A apoprotein and some of the related DME activities were more pronounced in sheep (P < 0.01 vs. cattle) and in goats (P < 0.05 vs. sheep; P < 0.01 vs. cattle) than in cattle. The significant differences in P4503A expression observed in LIM and PDM cattle are consistent with previously published data on strain- and breed-differences pointed out in rats and men. As far as a possible sex-effect is concerned, no clear-cut evidence is likely to be drawn. Finally, P4503A expression was more relevant in small ruminants.     

Characterization of xenobiotic biotransformation in hepatic, renal and gut tissues of cattle and sheep

Microsomal and cytosolic preparations of hepatic, renal, ileal and ruminal tissues of cattle and sheep were used to measure oxidative, hydrolative and conjugative biotransformations of 11 xenobiotic substrates. Within species, enzyme activities were generally higher (P less than .05) in hepatic than non-hepatic tissue but, in both species, non-hepatic tissue exhibited considerable capacities for metabolizing certain substrates. Sheep rumen wall (with papillae) was notably high in cytochrome P-450 content (34% of hepatic value), in glutathione conjugation of ethacrynic acid (223% of hepatic activity; P less than .05), and UDP-glucuronidation of estrone (290% of hepatic activity; P less than .05). Sheep differed (P less than .05) from cattle, having lower cytochrome P-450 content in liver and ileum (but not kidney); lower N-demethylase activity in liver, but two- to threefold higher activity in kidney; lower sulfotransferase activity in liver and kidney; and higher glutathione S-transferase activity toward certain substrates. UDP-glucuronidation varied too widely among substrates to afford strong generalization in comparisons among tissues or between species. Non-hepatic tissues in ruminants exhibit considerable capacities for oxidative, hydrolative and conjugative metabolism of xenobiotics. Sheep and cattle differ widely in hepatic and non-hepatic capacities for biotransforming certain xenobiotics.     

In vitro complex formation and inhibition of hepatic cytochrome P450 activity by different macrolides and tiamulin in goats and cattle

In humans, clinically relevant drug-drug interactions occur with some macrolide antibiotics via the formation of stable metabolic intermediate (MI) complexes with enzymes of the cytochrome P4503A (CYP3A) subfamily. The formation of such complexes can result in a decreased biotransformation rate of simultaneously administered drugs. In previous studies it was shown that the veterinary antibiotic tiamulin was also able to form a stable MI complex in pigs and rats. In the present study the relative CYP3A inhibiting potency and MI complex formation of a series of macrolide antibiotics and tiamulin were studied in microsomal fractions of goat and cattle and in a cell-line expressing bovine CYP3A. Tiamulin and triacetyloleandomycin (TAO) were found to be effective inhibitors of CYP450 activity in all systems tested. Erythromycin and tilmicosin were found to be relatively less effective inhibitors of CYP450 activity in microsomes, and their activity in the bovine CYP3A4 expressing cell line was relatively weak. Tylosin was shown to be a weak inhibitor in microsomes and not in the cell line, whereas spiramycin had no effect at all. MI-complex formation measured by spectral analysis was seen with TAO, tiamulin, erythromycin and tylosin, but not with tilmicosin and spiramycin. Although additional factors play a role in vivo, these results may explain potential drug-drug interactions and differences between related compounds in this respect.     

Inhibition of aflatoxin M1 production by bovine hepatocytes after intervention with oltipraz

It is well known that cattle ingesting aflatoxin B1 contaminated feed commodities excrete aflatoxin M1 into their milk. As aflatoxin M1 originates from hepatic metabolism, measures to prevent aflatoxin M1 formation need to be directed to either the immobilization of aflatoxin B1 in the gastrointestinal tract or the modification of hepatic metabolism of aflatoxin B1. Here we studied the influence of oltipraz and a second dithiolthione, (1,2) dithiolo (4,3-c)-1,2-dithiole-3,6 dithione (DDD) on bovine hepatic aflatoxin B1 biotransformation. Oltipraz inhibited aflatoxin B1 metabolism as no aflatoxin M1 and no aflatoxin B1-dihydrodiol, the second metabolite found in bovine hepatocytes, was formed. DDD did not significantly inhibit aflatoxin B1 metabolism. It could be demonstrated that the inhibition of aflatoxin B1 metabolism was due to the inhibition of several cytochrome P450 enzyme activities by oltipraz. In contrast, DDD inhibited only ethoxyresorufin O-deethylation activity. These findings suggest a high efficacy of oltipraz in inhibiting aflatoxin M1 contamination of milk from dairy cows exposed to aflatoxin B1 contaminated feeds.     

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.     

Zearalenone is bioactivated in the river Buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>): hepatic biotransformation

Zearalenone (ZEA) as a mycoestrogen is found frequently in human foods and animal feeds. Its estrogenic effects depend on its biotransformation fate including both first- and second-phase reactions, which are predominantly governed by hydroxylation and glucuronidation, respectively. In this study, we investigate the hepatic biotransformation of ZEA in river buffalo. To evaluate the hepatic biotransformation of ZEA, both subcellular fractions of the liver were prepared. ZEA was incubated with intracellular subfractions in the presence of nicotinamide dinucleotide phosphate, and the products were determined by means of high-performance liquid chromatography. Moreover, in the same frame of experiment and in the presence of uridine diphosphate glucuronic acid, the rate of glucuronidation for substrate and products were estimated as well. We found that α-zearalenol (α-ZOL) is the major hydroxylated hepatic metabolite of ZEA produced by both studied subcellular fractions. The enzymatic kinetics analyses indicated that the α-ZOL and β-ZOL production by microsomal fraction were two- and three-fold higher than those by postmitochondrial fraction, respectively. The calculated data showed that α-ZOL is conjugated with glucuronic acid more than ZEA and β-ZOL, especially at the lower concentrations, which seems to be more applicable. Our data suggest that unlike other domestic ruminants including cattle and sheep, the hepatic biotransformation of ZEA in river buffalo results in bioactivation and formation of potent estrogenic metabolite. Moreover, at the relevant concentrations, the produced potent estrogenic metabolite is entirely conjugated with glucuronic acid and, consequently, may cause the prolongation of presence of the compound in the body due to enterohepatic cycle.     

Interlobular distribution of hepatic xenobiotic-metabolizing enzyme activities in cattle, goats and sheep

Microsomal and cytosolic enzymes that metabolize xenobiotics were measured in composite samples representing entire livers and in samples from three lobes, using livers of cattle, goats and sheep. Within individual species, concentrations of cytochrome P-450 and b5 and activities of NADPH cytochrome c reductase, aldrin epoxidase, aminopyrine N-demethylase, ethoxycoumarin O-deethylase, microsomal and cytosolic stilbene oxide (epoxide) hydrolase and glutathione S-transferase were not different (P greater than .05) among the various hepatic lobes. Among species, several activities differed (P less than .05), with cattle livers generally having lower values than sheep or goats.     

Intracellular distribution of copper and zinc in the liver of copper-exposed cattle from northwest Spain

The intracellular distribution of copper (Cu) and zinc (Zn) in the livers of normal and moderately Cu-exposed cattle was investigated with the aim of improving understanding of the pathophysiology of Cu accumulation in cattle. At total liver Cu concentrations within the generally accepted normal range (25-100 mg/kg fresh weight) the large-granule fraction was the main subcellular compartment for Cu accumulation, followed by the cytosol and the nucleus, whereas in the microsomal fraction Cu accumulation was very low. With increasing Cu exposure, the capacity of the large-granule fraction to accumulate Cu decreased, proceeding towards a plateau (estimated at about 80 mg/kg at a projected total liver Cu concentrations of 450 mg/kg), accompanied by progressively greater Cu accumulation in the nucleus and cytosol. Total liver Cu concentration had little influence on subcellular Zn distribution, with hepatic Cu concentrations being only moderately correlated with Zn concentration in the large-granule fraction. There was a strong association between the proportion of total Cu bound to metallothionein (MT) and the proportion of total Zn in the large-granule fraction, suggesting that Zn displaced from MT is taken up by the lysosomes for excretion. This pattern of Cu accumulation, as in sheep, may be due to the limited capacity for metallothionein binding of Cu and excretion in bile.     

Antibiotic effects on cytochromes P450 content and mixed-function oxygenase (MFO) activities in the American alligator,Alligator mississippiensis

There are no Food and Drug Administration (FDA)-approved antimicrobial agents for use in cultured American alligators (Alligator mississippiensis) destined for human consumption yet some producers administer antibiotics for prophylaxis. The cytochromes P450-dependent mixed-function oxygenases (MFO) catalyze the oxidation of xenobiotic compounds such as drugs, pesticides and polycyclic aromatic hydrocarbons. Herein, we describe the effects of oxytetracycline, ceftazidime and enrofloxacin on the MFO system of the American alligator, Alligator mississippiensis. Juvenile alligators (4 animals/treatment) were administered these antibiotics intraperitoneally in an effort to induce hepatic microsomal cytochromes P450. Alligators treated with enrofloxacin exhibited emesis and convulsive spasms within 5 min of the initial injection. Total hepatic cytochromes P450 contents were significantly decreased in oxytetracycline-and enrofloxacin-pretreated alligators. In vitro hepatic microsomal benzyloxyresorufin O-dealkylase (BROD) activity was significantly decreased by enrofloxacin pretreatment. Western blots of proteins from antibiotic-pretreated alligator hepatic microsomes incubated with several mammalian and fish cytochromes P450 (CYP) antibodies exhibited little or no induction of CYP1A1, 2B, 2C and 2E1. In vitro incubation with enrofloxacin and oxytetracycline caused a concentration-dependent decrease in alkyl-substituted phenoxazone dealkylase activities catalyzed by phenobarbital- and 3-methylcholanthrene-induced alligator hepatic microsomes.