Relationship between metabolism of androstenone and skatole in intact male pigs.
The relationship between the metabolism of androsterone and skatole, the major compounds responsible for boar taint, was investigated in F4 Swedish Yorkshire x European Wild Pig intact males. The metabolism of androstenone and skatole were studied in liver microsomes, and the testicular steroid production was measured in testes microsomes. Including androstenone in the assays of skatole metabolism reduced the formation of 6-hydroxyskatole (pro-MII), and three other skatole metabolites (P<.05). The formation of three additional metabolites was not affected. Liver microsomal incubations of androstenone produced two metabolites, I and II. The rate of the formation of metabolite I and the rate of androstenone metabolism were correlated with the rate of skatole metabolism. Liver metabolism of androstenone was not related to levels of androstenone in fat. Testicular synthesis of 16-androstene steroids was correlated with combined synthesis of estrogens and androgens, plasma levels of androstenone, levels of skatole in fat, and skatole metabolism in the liver (P<.05). Plasma levels of estrone sulfate were correlated with levels of skatole in fat and with androstenone levels in fat and plasma and were negatively correlated with synthesis of skatole metabolite F-1 and pro-MII sulfation. These results indicate that the liver metabolism of androstenone and skatole are related. However, it is likely that the relationship between levels of androstenone and skatole in fat is due more to a link between the testicular synthesis of androstenone rather than to the metabolism of androstenone and skatole in the liver. Sex steroids may affect this relationship because of their biosynthesis along with androstenone and possible inhibition of skatole metabolism in the liver. (+info)
Specific dehydrogenation of 3-methylindole and epoxidation of naphthalene by recombinant human CYP2F1 expressed in lymphoblastoid cells.
3-Methylindole (3MI) is a naturally occurring pulmonary toxin that requires metabolic activation. Previous studies have shown that 3MI-induced pneumotoxicity resulted from cytochrome P-450-catalyzed dehydrogenation of 3MI to an electrophilic methylene imine (3-methyleneindolenine), which covalently bound to cellular macromolecules. Multiple cytochrome P-450s are capable of metabolizing 3MI to several different metabolites, including oxygenated products. In the present study, the role of human CYP2F1 in the metabolism of 3MI was examined to determine whether it catalyzes dehydrogenation rather than hydroxylation or ring oxidation. Metabolism was examined using microsomal fractions from human lymphoblastoid cells that expressed the recombinant human CYP2F1 P-450 enzyme. Expression of CYP2F1 in the lymphoblastoid cells proved to be an appropriate expression system for this enzyme. Products were analyzed using HPLC and the mercapturate, 3-[(N-acetylcystein-S-yl)methyl]indole, of the reactive intermediate was identified and quantified. Product analysis showed that human CYP2F1 efficiently catalyzed the dehydrogenation of 3MI to the methylene imine without detectable formation of indole-3-carbinol or 3-methyloxindole. High substrate concentrations of 3MI strongly inhibited production of the dehydrogenated product, a result that may indicate the existence of mechanism-based inhibition of CYP2F1 by 3MI. Recombinant CYP2F1 demonstrated remarkable selectivity for the bioactivation of 3MI to the putative dehydrogenated reactive electrophile. Bioactivation of naphthalene to its pneumotoxic epoxide by CYP2F1 was also demonstrated. (+info)
Identification of phase I metabolites of 3-methylindole produced by pig liver microsomes.
A study was conducted to investigate qualitative and quantitative aspects of the phase I metabolism of 3-methylindole (3MI) by porcine liver microsomes. Microsomal suspensions were prepared from the liver of 30 intact (uncastrated) male pigs. Metabolites produced in microsomal incubations were identified and quantitated with HPLC-UV, HPLC-fluorescence, and UV-spectral analysis; liquid chromatography-mass spectrometry (LC-MS) and NMR were used for the identification of a metabolite for which a reference compound was not available. The results showed that seven major metabolites of 3MI are produced by porcine microsomes, three of which had already been identified in pigs (3-OH-3-methyloxindole, 5-OH-3-methylindole, and 6-OH-3-methylindole). The other four major 3MI metabolites identified were 3-OH-3-methylindolenine, 3-methyloxindole, indole-3-carbinol, and 2-aminoacetophenone. On average, the metabolite that was produced in larger amounts was 3-OH-3-methylindolenine (45.1%), followed by the two oxindoles 3-methyloxindole (27.9%) and 3-OH-3-methyloxindole (18.5%). Average percentage of production of 6-OH-3-methylindole was 4.9%, whereas indole-3-carbinol accounted for 2.7% of all metabolites produced; 2-amino-acetophenone and 5-OH-3-methylindole were the metabolites produced in lesser amounts (0.5 and 0.3%, respectively). Large interindividual differences in the rate of production of all metabolites were observed. This variation could be attributed to differences in the activity and/or level of expression of phase I biotransformation enzymes and this issue should be further investigated. (+info)
Social effects and boar taint: significance for production of slaughter boars (Sus scrofa).
A study was conducted to elucidate the effects of social factors on the concentrations of boar taint substances, androstenone and skatole, in boars. The factors included dominance (social rank) and the effects of strongly tainted animals on other members of the group. Four successive replicates of 100 pigs (50 boars + 50 gilts) with an average live weight of 24 kg were randomly allocated to 10 pens of 10. Data for this study were collected during the period of 67 to 114 kg of live weight and included the repetitive recording of agonistic behavior during competitive feeding; blood sampling for determination of plasma androstenone, skatole and testosterone in boars; feces sampling for determination of skatole content; and collection of bulbourethral glands in boars, and uteri plus ovaries in gilts at slaughter, for the assessment of sexual maturity. Results show an influence of social rank on plasma concentrations of androstenone (P = .0001) and testosterone (P = .0001), the weight of the bulbourethral glands (P = .0001), and plasma skatole (P = .02). Pens were classified according to the pig with the highest concentration of androstenone in the pen into high, medium, and low maximum pens. In pens with high maximum concentrations of androstenone, the second-highest androstenone concentration (P = .0001), and the average concentration (P = .0003) in the pen were higher than those in pens with medium or low maximum concentrations of androstenone. Mean aggression level was also higher (P = .02), but pens with high maximum aggression level did not have higher mean androstenone concentration. Rank effect on androstenone was more important than aggression effect. Neither maximum androstenone concentration nor maximum aggression level in a pen was related to the pen mean stage of sexual maturity in either sex. No influences of rank, aggression, or aggression received were found on the feces skatole level, and no pheromonal communicative function was demonstrated for skatole. High androstenone concentrations did not have a suppressive effect on androstenone concentrations in other males of the group; on the contrary, the levels were increased. This may be due to a stimulating effect of androstenone and, possibly, mating activity. Consequently, in the production of boars for slaughter, strongly tainted animals should be avoided or removed and mating activity minimized. This could be facilitated by, for instance, slaughtering before sexual maturity or separate rearing of the sexes. (+info)
Metabolism of 3-methylindole by porcine liver microsomes: responsible cytochrome P450 enzymes.
The role of different cytochrome P450 enzymes on the metabolism of 3-methylindole (3MI) was investigated using selective chemical inhibitors. Eight chemical inhibitors of P450 enzymes were screened for their inhibitory specificity towards 3MI metabolism in porcine microsomes: alpha-naphthoflavone (CYP1A1/2), 8-methoxypsoralen (CYP2A6), menthofuran (CYP2A6), diethyldithiocarbamate (CYP2A6), 4-methylpyrazole (CYP2E1), sulphaphenazole (CYP2C9), quinidine (CYP2D6), and troleandomycin (CYP3A4). The production of 3MI metabolites was only affected by the presence of inhibitors of CYP2A6 and CYP2E1 in the microsomal incubations. In a second experiment, a set of porcine microsomes (n = 30) was analyzed for CYP2A6 content by protein immunoblot analysis and for their coumarin 7-hydroxylation activity (CYP2A6 activity). Both CYP2A6 content and enzymatic activity were found to be highly and negatively correlated with 3MI fat content. The results of the present study indicate that the CYP2A6 porcine ortholog plays an important role in the metabolism of 3MI and that measurement of CYP2A6 levels and/or activity could be a useful marker for 3MI-induced boar taint. (+info)
Characterization of acute interstitial pneumonia in cattle in southern Alberta feedyards.
Field data were collected over 2 consecutive years to characterize acute interstitial pneumonia (AIP) in feedyard cattle. Thirty-eight cattle with clinical symptoms of AIP were examined following emergency slaughter; 31 (all heifers) were confirmed to have AIP on the basis of gross and histological lung pathology. The 7 without AIP, plus 17 asymptomatic penmates, were used as contemporary controls. Plasma concentrations of 3-methylindole (3MI) metabolites were higher (P < 0.001) in heifers afflicted with AIP than in the control animals, and concentrations of 3MI mercapturates in the urine were lower (P < 0.007) in affected heifers. Concentrations of 3MI adducts in lung tissue and in microsomal protein did not differ (P > 0.05) between the 2 groups, and 3MI was not detected in ruminal fluid from either group. Total ruminal bacterial numbers and populations of lactobacilli and protozoa were similar (P > 0.05) between the AIP-positive and unafflicted groups, but fewer (P < 0.05) cellulolytic bacteria were present in the positive group. Bovine respiratory syncytial virus antigen was not found in lung tissue from any of the heifers confirmed to have AIP. To our knowledge, this study is the first to implicate 3MI metabolites as having a role in feedyard AIP. Further research is required to determine the factors responsible for the elevation in 3MI adducts in plasma and urine of feedyard cattle afflicted with AIP. (+info)
Selective dehydrogenation/oxygenation of 3-methylindole by cytochrome p450 enzymes.
3-Methylindole (3 MI) is a selective pulmonary toxicant, and cytochrome P450 (P450) bioactivation of 3 MI, through hydroxylation, epoxidation, or dehydrogenation pathways, is a prerequisite for toxicity. CYP2F1 and CYP2F3 exclusively catalyze the dehydrogenation of 3 MI to 3-methyleneindolenine, without detectable formation of the hydroxylation or epoxidation products. It was not known whether 3 MI is simply an excellent dehydrogenation substrate for all P450 enzymes, or whether certain cytochrome P450s responsible for 3 MI bioactivation have unique active sites that only catalyze the dehydrogenation of the molecule, while other P450s would catalyze only the oxygenation of 3 MI. Therefore, the kinetics of product formation by the CYP2F1 and CYP2F3 enzymes were compared with other cytochrome P450 enzymes. The enzymes tested were CYP1A1, CYP1A2, CYP1B1, and CYP2E1. The CYP1A1 and CYP1A2 enzymes produced all three 3 MI metabolites: the dehydrogenation product, 3-methyleneindolenine (V(max)/K(m) = 4 and 22, respectively); the hydroxylation product, indole-3-carbinol (V(max)/K(m) = 42 and 100, respectively); and the epoxidation product, 3-methyloxindole (V(max)/K(m) = 4 and 72, respectively). These CYP1A enzymes catalyzed oxygenation of 3 MI at much faster rates than dehydrogenation. CYP1B1 produced indole-3-carbinol (V(max)/K(m) = 85) and 3-methyloxindole (V(max)/K(m) = 7), and CYP2E1 only produced 3-methyloxindole (V(max)/K(m) = 98), but neither enzyme catalyzed the formation of the dehydrogenated product. Six additional P450 enzymes that were tested formed none of the dehydrogenation product. The ability of the various CYP1 family enzymes to catalyze the formation of all three major 3 MI metabolites, along with the specific oxygenation by CYP2E1, illustrates that dehydrogenation of 3 MI is not a substrate-directed process, but that the members of the CYP2F family possess unique active sites that specifically catalyze only the dehydrogenation mechanism. (+info)
Vaccination of boars with a GnRH vaccine (Improvac) eliminates boar taint and increases growth performance.
Peri- and postpubertal boars accumulate substances (e.g., androstenone and skatole) in their fatty tissue that are responsible for boar taint in pork. The objective of this study was to assess the efficacy of a GnRH vaccine, Improvac, in eliminating boar taint. Three hundred male (200 intact boars, 100 barrows) crossbred (Large White x Landrace) pigs were used in a 2 x 3 factorially arranged experiment. The respective factors were sex group (barrows, boars treated with placebo, or boars treated with Improvac) and slaughter age (23 or 26 wk). Vaccines were administered 8 and 4 wk before slaughter. All Improvac-treated pigs exhibited anti-GnRH titers. Testes and bulbo-urethral gland weights in treated pigs were reduced by approximately 50% (P < 0.001) and serum testosterone levels were below 2 ng/mL in the majority of treated boars (94 and 92% across both age groups at 2 and 4 wk, respectively). Boar taint, as assessed by the concentration of androstenone and skatole in s.c. fat, was suppressed to low or undetectable levels in 100% of Improvac-treated boars. No Improvac-treated pigs had significant concentrations of either androstenone (> 1.0 microg/g) or skatole (> 0.20 microg/g). In contrast, 49.5% of placebo-treated controls had significant androstenone and 10.8% had significant skatole levels, resulting in 10% of the control boars with high concentrations of both compounds. The mean concentrations of taint compounds in the Improvac-treated pigs were not significantly different from those in barrows. Improvac-treated boars grew more rapidly (P = 0.051 and < 0.001 for pigs slaughtered at 23 and 26 wk of age, respectively) than control boars over the 4 wk after the secondary vaccination, possibly because of reduced sexual and aggressive activities. Compared with barrows, Improvac-treated boars were leaner and had superior feed conversion efficiency. The vaccine was well tolerated by the pigs, and no observable site reactions could be detected at the time of slaughter. Vaccination of boars with Improvac allows production of heavy boars with improved meat quality through prevention and control of boar taint. (+info)