RESUMEN

Testicular steroids can alter the activity and expression of enzymes within the liver and may influence the metabolism of skatole and androstenone, which are responsible for boar taint. Plasma levels of estrone sulfate (E1S) are indicative of the steroidogenic capacity of the boar and are variable between animals of similar live weights at slaughter. This study aimed to characterize the relationship between steroidogenic capacity and the metabolism of boar taint compounds by relating plasma E1S levels at slaughter weight to the expression levels of genes regulating the metabolism of androstenone and skatole, along with their respective metabolite profiles. RT-qPCR was used to evaluate gene expression in the liver. Hepatocytes were also isolated and treated with androstenone or skatole, with metabolite levels in the incubation media quantified by high-performance liquid chromatography. Plasma E1S levels ranged from 2.2-108.5 ng/mL and were positively correlated with overall skatole metabolism (p = 0.038), the production of metabolites 3-methyloxindole (p = 0.026) and 3-hydroxy-3-methyloxindole (p = 0.036), and expression levels of key genes involved in skatole metabolism, specifically CYP2C33 (p = 0.0042), CYP2C49 (p = 0.022), and CYB5R1 (p = 0.017). There was no association between androstenone metabolism and plasma E1S concentrations; however, there was evidence of possible co-regulation amongst genes involved in the metabolism of androstenone, skatole, and estrogens. These findings indicate that steroidogenic capacity is related to the rate of skatole, but not androstenone metabolism, in slaughter-weight boars.

Asunto(s)

RESUMEN

Ketosis, evidenced by hyperketonemia with elevated blood ß-hydroxybutyrate (BHB) levels, is a significant metabolic disorder of dairy cattle, typically diagnosed within the first 6 weeks post-calving when high energy levels are essential to milk production. Our study aimed to identify genetic markers linked to hyperketonemia (HYK) patterns in Holstein cows during early lactation and compare these to HYK-negative cows. We screened 964 cows for HYK using a threshold of BHB ≥1.2 mmol/L during the first 2 weeks postpartum (screening period, SP). Cows that tested negative initially were retested the following week. Cows were deemed HYK-negative (CON group) if BHB levels were below 1.2 mmol/L in both tests, while those with BHB levels exceeding this threshold at any test were treated and classified as HYK-positive (HYK+). Post-treatment, HYK+ cows were monitored for two-week follow-up period (FP) and classified based on their recovery: cured (CUR; consistently low BHB), recurrent (REC; fluctuating BHB levels), severe (SEV; high initial BHB that decreased), or chronic (CHR; persistently high BHB). Using 489 cows that were genotyped, a GWAS was conducted using GCTA software, revealing significant associations of several SNPs across different HYK patterns when compared to the CON group. These SNPs were primarily linked to genes affecting milk traits and were enriched in biological pathways relevant to protein glycosylation, inflammatory response, glucose homeostasis, and fatty acid synthesis. Our findings highlight genomic regions, potential candidate genes, and biological pathways related to ketosis, underscoring potential targets for improving health management in dairy cattle. These insights could lead to better strategies for managing ketosis through genetic selection, ultimately enhancing dairy cattle welfare and productivity. Further research with a larger number of cows is recommended to validate these findings and help confirm the implicated SNPs and genes.

RESUMEN

Boar taint, an unfavorable odor in the meat of intact male pigs, is caused primarily by the accumulation of two compounds: androstenone and skatole. This multifactorial trait is regulated by numerous dietary, management and genetic factors. At the mechanistic level, there are many genes known to be involved in boar taint metabolism. Cytochrome P450 2E1 (CYP2E1) impacts boar taint through the phase I metabolism of skatole. The aim of this study was to identify single-nucleotide polymorphisms (SNPs) within the CYP2E1 gene promoter and explore their relationship with the expression of CYP2E1 mRNA and protein. Sequencing of the promoter region using pools of genomic DNA identified seven promoter region SNPs at -159, -586, -1693, -1806, -2322, -2369 and -2514 bp upstream of the ATG start site. Genomic DNA was obtained from 65 boars from the three major swine breeds: Duroc, Landrace and Yorkshire, and individual animals were genotyped for the identified SNPs. RNA was isolated from liver tissue and quantitative PCR was performed to measure CYP2E1 gene expression, while levels of CYP2E1 protein in liver were measured by Western blotting. Significant within-breed variation in CYP2E1 protein and mRNA expression was observed, indicating significant differences in gene expression among individuals. However, levels of CYP2E1 mRNA and protein were not significantly correlated. Two SNPs within the promoter were significantly associated with CYP2E1 mRNA expression, but not with protein expression. This study provides evidence of additional mutations affecting the gene expression of CYP2E1 and suggests that factors that affect the differences in translation of CYP2E1 mRNA may also be important in affecting skatole metabolism.

RESUMEN

Equine leaky gut syndrome is characterized by gastrointestinal hyperpermeability and may be associated with adverse health effects in horses. The purpose was to evaluate the effects of a prebiotic Aspergillus oryzae product (SUPP) on stress-induced gastrointestinal hyperpermeability. Eight horses received a diet containing SUPP (0.02 g/kg BW) or an unsupplemented diet (CO) (n = 4 per group) for 28 days. On Days 0 and 28, horses were intubated with an indigestible marker of gastrointestinal permeability (iohexol). Half the horses from each feeding group underwent 60 min of transport by trailer immediately followed by a moderate-intensity exercise bout of 30 min (EX), and the remaining horses stayed in stalls as controls (SED). Blood was sampled before iohexol, immediately after trailering, and at 0, 1, 2, 4, and 8 h post-exercise. At the end of the feeding period, horses were washed out for 28 days before being assigned to the opposite feeding group, and the study was replicated. Blood was analyzed for iohexol (HPLC), lipopolysaccharide (ELISA), and serum amyloid A (latex agglutination assay). Data were analyzed using three-way and two-way ANOVA. On Day 0, the combined challenge of trailer transport and exercise significantly increased plasma iohexol in both feeding groups; this increase was not seen in SED horses. On Day 28, EX increased plasma iohexol only in the CO feeding group; this increase was completely prevented by the provision of SUPP. It is concluded that combined transport and exercise induce gastrointestinal hyperpermeability. Dietary SUPP prevents this and therefore may be a useful prophylactic for pathologies associated with gastrointestinal hyperpermeability in horses.

RESUMEN

Creatine is a nitrogenous compound essential for cellular energy homeostasis found in animal protein; however, when heat-processed for pet food, creatine is degraded to creatinine, which is not metabolically active and excreted in urine. The objective of the present investigation was to define the postprandial plasma creatine and creatinine response in dogs fed a commercial diet (CON) formulated for adult dogs, top-dressed with a combination of creatine (9.6 g/kg dry matter, DM), carnitine (2.13 g/kg DM) and choline (0.24 g/kg DM; CCC), methionine (2.6 g/kg DM; MET), or taurine (0.7 g/kg DM; TAU). Eight adult Beagles were fed one of the four diets for 7 days in a Latin Square design with no washout period. On day 7, cephalic catheters were placed and blood samples were collected before being fed (fasted) and up to 6 h post-meal. Creatine and creatinine were analyzed using HPLC and data analyzed using PROC GLIMMIX in SAS. Plasma creatine concentrations were higher in dogs fed CCC (103 ± 10 µmol/L) compared to MET (72 ± 7 µmol/L) at fasted (P < 0.05) and higher compared to all other treatments from 15 to 360 min post-meal (P < 0.05). Plasma creatinine concentrations were higher in dogs fed CCC from 60 to 180 min compared to all other treatments. These data suggest that when creatine, carnitine and choline are top-dressed for 7 days, plasma creatine is rapidly absorbed and remains elevated up to 6 h post-meal. This may have implications for energy metabolism and should be considered when using creatinine as a diagnostic tool in dogs.

RESUMEN

The nuclear receptors PXR, CAR, and FXR are activated by various ligands and function as transcription factors to control the expression of genes that regulate the synthesis and metabolism of androstenone and skatole. These compounds are produced in entire male pigs and accumulate in the fat to cause the development of a meat quality issue known as boar taint. The extent of this accumulation is influenced by the synthesis and hepatic clearance of androstenone and skatole. For this reason, PXR, CAR, and FXR-mediated signaling pathways have garnered interest as potential targets for specialized treatments designed to reduce the development of boar taint. Recent research has also identified several metabolites produced by gut microbes that act as ligands for these nuclear receptors (e.g., tryptophan metabolites, short-chain fatty acids, bile acids); however, the connection between the gut microbiome and boar taint development is not clear. In this review, we describe the nuclear receptor signaling pathways that regulate the synthesis and metabolism of boar taint compounds and outline the genes involved. We also discuss several microbial-derived metabolites and dietary additives that are known or suspected nuclear receptor ligands and suggest how these compounds could be used to develop novel treatments for boar taint.

RESUMEN

Acute interstitial pneumonia (AIP) of cattle has been recognized for many decades. While the pathogenesis and risk factors for this condition in pastured cattle are relatively well characterized, there remains a poor understanding of the disease as it occurs in intensively fed cattle such as in beef feedlots. Specifically, in pastured cattle, AIP results from excessive ruminal production of the pneumotoxicant 3-methylindole (3-MI). In feedlot cattle, the evidence to substantiate the role of 3-MI is comparatively deficient and further investigations into the cause, pathogenesis, and control are sorely needed. This review highlights our current understanding of AIP with a focus on the disease as it occurs in feedlot cattle. Additionally, it illustrates the need for further work in understanding the specific animal factors (e.g. the ruminal microbiome, and the role of concurrent diseases), management factors (e.g. animal stocking and vaccination protocols), and dietary factors (e.g. dietary supplements) that may impact the development of AIP and which are relatively unique to the feedlot setting. All stakeholders in the beef industry stand to benefit from a greater understanding of what remains a pressing yet poorly understood issue in beef production.

Asunto(s)

RESUMEN

Boars express high testicular levels of sulfotransferase enzymes, and consequently, the boar taint causing compound androstenone predominantly circulates as a steroid sulfate. Androstenone sulfate is suspected to function as a steroid reservoir that can be deconjugated to provide a source of free androstenone for accumulation. Therefore, the purpose of this study was to characterize the uptake and deconjugation of androstenone sulfate in the adipose tissue of the boar. Real-time PCR was used to quantify the expression of steroid sulfatase (STS) and several organic anion transporting polypeptides (OATPs) in the adipose tissue. Additionally, [3H]-androstenone sulfate was incubated with adipocytes or supernatant from homogenized fat to assess steroid uptake and conversion, respectively. A positive correlation existed between OATP-B expression and androstenone sulfate uptake (r = 0.86, p = 0.03), as well as between STS expression and androstenone sulfate conversion (r = 0.76, p < 0.001). Moreover, fat androstenone concentrations were positively correlated (r = 0.85, p < 0.001) with androstenone sulfate conversion and tended to increase with STS expression in early maturing boars. This suggests that androstenone sulfate uptake and deconjugation are mediated by OATP-B and STS, respectively, which may influence the development of boar taint in early maturing animals.

RESUMEN

Androstenone circulates in the plasma bound to albumin before accumulating in the fat, resulting in the development of boar taint. Androstenone sulfate is more abundant in the circulation than free androstenone; however, it is unclear how androstenone sulfate is transported in the plasma and if steroid transport affects the development of boar taint. Therefore, the purpose of this study was to characterize the binding of androstenone sulfate in boar plasma and determine if variability in steroid binding affects the accumulation of androstenone in the fat. [3H]-androstenone sulfate was incubated with plasma and the steroid binding was quantified using gel filtration chromatography. Inter-animal variability was assessed by quantifying androstenone binding specificity in plasma obtained from boars that had high or low fat androstenone concentrations at slaughter. Androstenone sulfate bound minimally in the plasma and to isolated albumin, which suggests that it is transported primarily in solution. The specific binding of androstenone quantified in plasma and isolated albumin from low fat androstenone animals was significantly higher (p = 0.01) than in high fat androstenone boars. These results indicate that the binding of androstenone to albumin varies amongst individual animals and affects the transport of androstenone in the plasma and accumulation in the fat of the boar.

RESUMEN

Hepatic glucuronidation represents an important phase II biotransformation reaction in both mammals and fish. The kinetic characteristics of uridine 5'-diphosphate (UDP) glucuronosyltransferases (UDPGTs) in rainbow trout liver microsomes were examined using p-nitrophenol (p-NP) as an aglycone and UDP-glucuronic acid (UDPGA) as a glucuronyl donor according to an existing protocol. The kinetic data obtained with varying concentrations of p-NP best fit the Hill equation and UDPGT activity was successfully induced following an i.p. injection of ß-naphthoflavone (ß-NF). The assay was subsequently adapted to a microplate method for determination of UDPGT activity in microsomal samples obtained from rainbow trout as well as Nile tilapia. In contrast to rainbow trout, UDPGT activity of Nile tilapia was best described by Michaelis-Menten kinetics. Based on the linearity of p-NP glucuronide formation, a p-NP concentration of 0.60 mM and a UDPGA concentration of 6.89 mM were determined to be suitable for assaying UDPGT activity in samples from rainbow trout and Nile tilapia. The microplate method offers several advantages over the historical assay; most notably it enables the observation of successive kinetics which ensures that enzyme activity is calculated in the most linear (initial) rate of the reaction. It also provides practical advantages in terms of ease-of-use and efficiency. This may be relevant to researchers investigating exposure of wild or farmed fish to environmental or feed-borne contaminants which are substrates of UDPGTs.

Asunto(s)

RESUMEN

Dietary fiber affects canine physiology in many ways, such as increasing colonic absorption of water and improving gut health, both of which may positively impact exercise performance. The objectives of this study were to investigate the effects of increased dietary soluble fiber and incremental training on respiratory rate (RR), internal body temperature (BT), body composition, and fecal metabolites in mid-distance training sled dogs. Fourteen dogs (12 Siberian and 2 Alaskan Huskies) were blocked by age, sex, and body weight (BW) and then randomly allocated into one of two diet groups. Seven dogs were fed a dry extruded control diet (Ctl) with an insoluble:soluble fiber ratio of 4:1 (0.74% soluble fiber on a dry-matter basis), and seven dogs were fed a dry extruded treatment diet (Trt) with an insoluble:soluble fiber ratio of 3:1 (2.12% soluble fiber on a dry-matter basis). Fecal samples were taken once a week. All dogs underwent 9 weeks of incremental exercise conditioning where the running distance was designed to increase each week. Every 3 weeks, external telemetry equipment was used to non-invasively measure and record RR and internal BT at resting, working, and post-exercise recovery states. Body composition was measured on weeks -1 and 9 using quantitative magnetic resonance. Body composition, RR, BT, and fecal metabolites were analyzed using a mixed model with dog as a random effect and week and diet group as fixed effects. Dogs on Trt had lower working and post-exercise BT than Ctl (P < 0.05). In addition, Trt dogs had lower recovery BT at weeks 2 and 5 than Ctl dogs (P < 0.05). Treatment dogs had greater fecal short-chain fatty acid concentrations than Ctl (P < 0.05). Diet had no effect on RR or body composition (P > 0.10), but exercise resulted in an overall 7% increase in lean and 3.5% decrease in fat mass (P < 0.05). These data suggest that increasing dietary soluble fiber may positively influence BT and gut health; however, it has no effect on RR or body composition. Soluble fiber did not negatively impact any measures of overall health and performance and should be considered for use in performance dogs.

RESUMEN

Growth performance, liver and spleen weight, plasma, and ceca digesta metabolites and incidences of diarrhea were investigated in growing pigs fed spent biomass of Pichia kudriavzevii. Ninety six barrows (~25 kg, 4 pigs/pen) were fed 1 of 4 experimental diets (n = 6) for 7 weeks. The diets were control, corn-, and soybean meal-based diet or control plus 2.5%, 3.75%, or 5.0% P. kudriavzevii. Diets were formulated to be isocaloric and iso nitrogenous. Feed intake and body weight (BW) were recorded weekly for calculation of average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F). Fecal scores were taken 3 d/wk to assess incidence and severity of diarrhea. One pig/pen close to pen average was bled for plasma metabolites on days 7 and 49 and subsequently euthanized for spleen and liver weight, ileal and cecum digesta samples for concentration of short-chain fatty acids (SCFA). The concentration of crude protein, crude fat, and non-fiber carbohydrates in P. kudriavzevii biomass was 36.4%, 9.6%, and 50.8% DM, respectively. Inclusion of P. kudriavzevii tended (P = 0.06) to linearly reduce ADG from days 8 through 49 resulting in a trend (P = 0.06) for linear reduction in the final BW. The final BW was 79.0, 79.2, 76.8, and 75.5 kg for the 0%, 2.5%, 3.75%, and 5.0% P. kudriavzevii, respectively. Diets had no effect (P > 0.10) on ADFI, G:F, spleen, and liver weight throughout the entire experiment. On day 7, there was cubic (P = 0.03) decrease and quadratic (P = 0.02) increase in plasma concentration of creatinine and urea N, respectively. However, there were no (P > 0.10) diet effects on plasma metabolites on day 49. There was a tendency (P = 0.08) for linear increase in cecum digesta concentration of acetic acid. There were no diet effects (P > 0.10) on fecal score in the first 4 wk of feeding. In conclusion, feeding P. kudriavzevii yeast tended to depress growth and stimulate cecum fermentation at higher dose and had no detrimental effects on organ weights or plasma metabolites in growing pigs.

RESUMEN

Boar taint is caused by the accumulation of androstenone and skatole and other indoles in the fat; this is regulated by the balance between synthesis and degradation of these compounds and can be affected by a number of factors, including environment and management practices, sexual maturity, nutrition, and genetics. Boar taint can be controlled by immunocastration, but this practice has not been accepted in some countries. Genetics offers a long-term solution to the boar taint problem via selective breeding or genome editing. A number of short-term strategies to control boar taint have been proposed, but these can have inconsistent effects and there is too much variability between breeds and individuals to implement a blanket solution for boar taint. Therefore, we propose a precision livestock management approach to developing solutions for controlling taint. This involves determining the differences in metabolic processes and the genetic variations that cause boar taint in specific groups of pigs and using this information to design custom treatments based on the cause of boar taint. Genetic, proteomic or metabolomic profiling can then be used to identify and implement effective solutions for boar taint for specific populations of animals.

RESUMEN

Treating fibrous feed ingredients with exogenous feed enzymes may improve their utilization in monogastric animals. An in vitro study was conducted to determine the effects of steeping corn distillers dried grains with solubles (DDGS) or wheat middlings (WM) with exogenous feed enzymes. Four treatments were arranged as follows: 1) co-product steeped with water (CON), 2) CON plus 0.5-g fiber degrading enzymes (FDE), 3) CON plus 0.5-g protease (PRO), and 4) CON plus 0.5-g FDE and 0.5 g PRO (FDEPRO). The FDE contained about 62,000, 37,000, and 8,000 U/g of xylanase, cellulase, and ß-glucanase, respectively, whereas activities in PRO amounted to 2,500,000, 1,300,000, and 800,000 U/g of acid, alkaline, and neutral proteases, respectively. Briefly, 50 g of DDGS or WM samples (n = 8) were mixed with 500-mL water with or without enzymes and steeped for 0 to 72 h at 37 °C with continuous agitation. The pH, concentration of monosaccharides, and organic acids in the supernatant and apparent disappearance (AD) of fiber in solids were measured at 0, 12, 24, 48, and 72 h. There was treatment and time interaction (P < 0.005) on monosaccharides concentration. At 12 h, arabinose and glucose concentrations were similar (P > 0.05) between FDE and FDEPRO but higher (P = 0.002) than for CON in DDGS. For WM, FDE, and FDEPRO had higher (P < 0.001) xylose concentration than CON and PRO, whereas glucose concentration was higher (P < 0.001) for enzymes than CON at 12 h. However, FDEPRO had higher (P < 0.001) xylose concentration than CON, whereas xylose concentration for FDE and PRO was intermediate at 24 h. There was an interaction (P < 0.05) between treatment and time effect on lactic acid concentration in DDGS and WM (P < 0.005), and acetic acid concentration in WM (P < 0.001). In general, monosaccharide concentration was higher between 12 and 24 h and decreased after 48 h, whereas the pH decreased, and concentration of organic acids increased continuously over time (P < 0.05). The AD of NDF and ADF in DDGS was greater (P = 0.001) for FDE and FDEPRO than CON and PRO at 72 h. In WM, enzymes increased (P = 0.007) AD of NDF relative to CON at 72 h. Nonetheless, greater (P < 0.05) AD of fiber was observed between 48 and 72 h. In conclusion, although there were differences in responses among co-products, fiber degrading enzymes increased release of fermentable monosaccharides from co-products at 12 to 24 h of steeping and these effects were not extended with the addition of protease.

RESUMEN

Cytochrome P450 17A1 (CYP17A1) catalyses the 17α-hydroxylation and 17,20 lyase reactions to convert pregnenolone to 17α-hydroxypregnenolone (17OHP) and subsequently the androgen dehydroepiandrosterone (DHEA). In pigs and humans, CYP17A1 also catalyses the delta-16-synthase reaction to produce the 16-androstene steroid 5,16-androstadien-3ß-ol (16A), which is then further metabolised to the sex pheromone androstenone. Cytochrome b5A (CYB5A) stimulates the 17,20 lyase reaction and is required for the delta 16-synthase reaction. We have identified and mutated residues in porcine CYP17A1 and CYB5A that may alter the synthesis of DHEA and 16A. This included residues in the steroid binding pocket of CYP17A1 and residues on the surface of CYP17A1 and CYB5A that are involved in binding of CYP17A1 to CYB5A. We then expressed the various mutations of CYB5A and CYP17A1 along with porcine cytochrome P450 oxidoreductase (POR) and cytochrome b5 reductase (CYB5R3) in HEK293 cells and measured the formation of metabolites 16A, 17OHP and DHEA from radiolabelled pregnenolone by high performance liquid chromatography (HPLC). Mutations were identified in both CYP17A1 and CYB5A that affected the production of the different metabolites and also affected the overall production of metabolites. Several combinations of mutations decreased the production of both 16A and DHEA and increased production of 17OHP, while the N62S mutation of CYB5A with wild type CYP17A1 increased production of both 16A and DHEA. The best combination of mutations to reduce the production of 16A, while maintaining the production of DHEA and the overall conversion similar to wild type are the N21K, L28V, N21K/L28V and the R52 M/N62S mutations of CYB5A with the D103S mutation of CYP17A1.

Asunto(s)

RESUMEN

Genetic selection of farm animals plays an important role in genetic improvement programs. Regularized regression methods on single nucleotide polymorphism (SNP) data from a set of candidate genes can help to identify genes that are associated with the trait of interest. This complex task must also consider the relative effect sizes on the desired trait and account for the relationships among the candidate SNPs so that selection of a SNP does not promote other undesirable traits through breeding. We present the Doubly Sparse Regression Incorporating Graphical structure (DSRIG), a novel regularized method for genetic selection that exploits the relationships among candidate SNPs to improve prediction. DSRIG was applied in the prediction of skatole and androstenone levels, two compounds known to be associated with boar taint. DSRIG was shown to provide a predictive benefit when compared to ordinary least squares (OLS) and the least absolute shrinkage and selection operator (LASSO) in a cross-validation procedure. The relative sizes of the coefficient estimates over the cross-validation procedure were compared to determine which SNPs may have the greatest impact on expression of the boar taint compounds and a consensus graph was used to infer the relationships among SNPs.

Asunto(s)

RESUMEN

Increased public interest in the welfare of pigs reared for pork production has led to an enhanced effort in finding alternatives to castration for controlling the unpleasant odour and flavour from heated pork products known as boar taint. The purpose of this study was to investigate the testicular metabolism of androstenone, one of the major components of boar taint. Leydig cells were isolated from mature boars and incubated with radiolabeled androstenone for 10 min, 1 h, 4 h, 8 h, and 12 h. Steroid profiles were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS/MS). Sulfoconjugated, but not glucuronidated steroids were produced by Leydig cells. Approximately 85% of androstenone was converted into sulfoconjugated metabolites in Leydig cell incubations after 8 h. This sulfoconjugate fraction included androstenol-3-sulfate and two major sulfated forms of androstenone. Following removal of the sulfate group, these two sulfated forms of androstenone returned the parent compound androstenone, and not a hydroxylated metabolite. These findings provided direct evidence for the testicular production of sulfoconjugated forms of androstenone and androstenol in the boar. The high proportion of sulfoconjugates produced by the Leydig cells emphasizes the importance of steroid conjugation, which serves to regulate the amount of unconjugated steroid hormones available for accumulation in adipose tissue.

Asunto(s)

RESUMEN

Porcine sulfotransferase 2A1 (pSULT2A1) is a key enzyme involved in the testicular and hepatic sulfoconjugation of steroids such as dehydroepiandrosterone (DHEA) and potentially androstenone. This latter steroid is a major cause of boar taint, which is an unpleasant off-odour and off-flavour in pork from male pigs. Sulfotransferase 2B1 (pSULT2B1) may also be important, although no direct evidence exists for its involvement in sulfoconjugation of steroids. The purpose of this study was to investigate the sulfoconjugation activity of human and porcine sulfotransferases towards DHEA and androstenone. pcDNA 3.1 vectors expressing porcine (p) SULT2A1, pSULT2B1, human (h) SULT2A1, hSULT2B1a, and hSULT2B1b enzymes were transfected into human embryonic kidney cells. Transfected cells were then incubated with either androstenone or dehydroepiandrosterone (DHEA) in both time-course and enzyme kinetics studies. The production of sulfonates of androstenone metabolites and DHEA sulfonate increased over time for all enzymes with the exception of pSULT2B1. Enzyme kinetics analysis showed that androstenone and DHEA were poor substrates for the human orthologs, hSULT2B1a and hSULT2B1b. Human and porcine SULT2A1 showed substantially different substrate affinities for androstenone (Km 5.8 ±â€¯0.6 µM and 74.1 ±â€¯15.9 µM, respectively) and DHEA (Km 9.4 ±â€¯2.5 µM and 3.3 ±â€¯1.9 µM, respectively). However, these enzymes did show relatively similar sulfonation efficiencies for DHEA (Vmax/Km 50.5 and 72.9 for hSULT2A1 and pSULT2A1, respectively). These results highlight the species differences in sulfonation activity and provide direct evidence, for the first time, suggesting that pSULT2B1 is not involved in sulfonation of either androstenone metabolites or DHEA.

Asunto(s)

RESUMEN

1. Nuclear receptors CAR (NR1I3) and PXR (NR1I2) are major ligand-activated transcriptional regulators of xenobiotic metabolism and disposition and modulators of endobiotic metabolism. Differences in xenobiotic selectivity between the human and rodent receptors are well recognized but there is lack of such information on properties of CAR and PXR in important domestic animals. 2. The pig and bovine receptors were cloned and their ligand profiles were systematically compared to corresponding human and mouse forms utilizing a panel of xenobiotics and structural analysis. 3. Pig CAR and PXR resemble their human counterparts which can be rationalized by only modest amino acid changes between critical residues of the human ligand-binding pockets (H203Q for CAR, L210V and M243I for PXR). 4. In contrast, bovine CAR shows a blunted response to CAR agonists and inverse agonists. These changes are likely due to disruptive mutations at or near critical hydrogen bond-forming residues (N165I, Y326F). The unresponsiveness of bovine PXR to human- and mouse-selective agonists may be related to substitutions at important ligand-contacting residues R410Q and F305V, respectively. 5. Our findings have implications for regulation of drug-metabolizing enzymes and transporters and pharmacokinetics in cattle and pigs.

Asunto(s)

RESUMEN

INTRODUCTION: Parenteral nutrition (PN) in preterm infants leads to PN-associated liver disease (PNALD). PNALD has been linked to serum accumulation of phytosterols that are abundant in plant oil but absent in fish oil emulsions. HYPOTHESIS: Whether modifying the phytosterol and vitamin E composition of soy and fish oil lipid emulsions affects development of PNALD in preterm pigs. METHODS: We measured markers of PNALD in preterm pigs that received 14 days of PN that included 1 of the following: (1) Intralipid (IL, 100% soybean oil), (2) Intralipid + vitamin E (ILE, d-α-tocopherol), (3) Omegaven (OV, 100% fish oil), or (4) Omegaven + phytosterols (PS, ß-sitosterol, campesterol, and stigmasterol). RESULTS: Serum levels of direct bilirubin, gamma glutamyl transferase, serum triglyceride, low-density lipoprotein, and hepatic triglyceride content were significantly lower (P < .05) in the ILE, OV, and PS compared to IL. Hepatic cholesterol 7-hydroxylase and organic solute transporter-α expression was lower (P < .05) and portal plasma FGF19 higher in the ILE, OV, and PS vs IL. Hepatic expression of mitochondrial carnitine palmitoyltransferase 1A and microsomal cytochrome P450 2E1 fatty acid oxidation genes was higher in ILE, OV, and PS vs IL. In vivo (13)C-CDCA clearance and expression of pregnane X receptor target genes, cytochrome P450 3A29 and multidrug resistance-associated protein 2, were higher in ILE, OV, and PS vs IL. CONCLUSIONS: α-tocopherol in Omegaven and added to Intralipid prevented serum and liver increases in biliary and lipidemic markers of PNALD in preterm piglets. The addition of phytosterols to Omegaven did not produce evidence of PNALD.

Asunto(s)