Dietary Fermentation Product of Aspergillus Oryzae Prevents Increases in Gastrointestinal Permeability ('Leaky Gut') in Horses Undergoing Combined Transport and Exercise.

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.

The combination of trailer transport and exercise increases gastrointestinal permeability and markers of systemic inflammation in horses.

BACKGROUND: Leaky gut syndrome (LGS) is an idiopathic disorder characterised by alterations in intestinal permeability and low-grade systemic inflammation. Factors contributing to development of LGS are not well-understood but physiological stressors such as exercise and transport may play a role which may be of pathophysiological relevance in horses. OBJECTIVES: To characterise the combined effect of transport stress and exercise on gastrointestinal permeability, and to determine whether these effects are associated with increased inflammatory biomarkers in plasma. STUDY DESIGN: Controlled, randomised and cross-over study. METHODS: Horses (n = 8 per group) were given a gastrointestinal permeability tracer (iohexol; 5.6% solution; 1 ml/kg bwt) via nasogastric entubation prior to being assigned to a stressed (EX; 1 h of trailer transport immediately followed by 30 min moderate intensity exercise; n = 4) or sedentary control (CON; n = 4) group. Plasma samples were obtained prior to iohexol administration (P1), after transport (P2), at exercise cessation (P3), and at 1 (P4), 2 (P5), 4 (P6) and 8 (P7) hours after cessation of exercise and were analysed for iohexol, inflammatory biomarkers (SAA, LPS, IFABP and LBP) and tight junction proteins (zonulin). Faecal samples were collected at times corresponding to before and after stress from both groups and analysed for zonulin. Data were analysed using a 2-way RM ANOVA. RESULTS: In EX horses, a significant increase in iohexol was observed at P2 (1.5 ± 0.24 µg/ml; p = 0.03), P3 (2.1 ± 0.29 µg/ml; p < 0.001), P4 (2.1 ± 0.17 µg/ml; p < 0.001) compared with P1 (0.7 ± 0.21 µg/ml); iohexol was significantly higher in EX than CON horses at P3 (p < 0.001), P4 (p < 0.001) and P5 (p = 0.003). LPS and SAA were significantly higher in EX than CON at P4 (p < 0.001) and P6 (p = 0.04), respectively. MAIN LIMITATIONS: Data from our small sample size may not be generalisable to the larger equine population. CONCLUSIONS: Combined transport and exercise increases gastrointestinal permeability and systemic SAA and LPS. The model described herein may be useful in further studies on the role of alterations in gastrointestinal permeability in equine disease.

Development of a microplate method for the determination of hepatic UDP-glucuronosyltransferase activity in rainbow trout (Oncorhynchus mykiss) and Nile tilapia (Oreochromis niloticus).

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.

Effect of mutations in porcine CYB5A and CYP17A1 on the metabolism of pregnenolone.

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.

The characterization of androstenone transport in boar plasma.

The transport of steroids by plasma proteins influences the amount of steroid available for uptake by the target tissue. In the boar, androstenone is transported to the adipose tissue where it accumulates to cause an off-odour or off-flavour in pork, known as boar taint. The mechanism of the transport of androstenone in the boar remains unclear, and the plasma protein responsible for binding androstenone has yet to be identified. Therefore, the purpose of the present study was to characterize the binding of androstenone to plasma proteins in the boar. The binding specificity of androstenone to plasma proteins was first investigated using a HPLC gel filtration method. [3H]-androstenone was incubated with plasma in the presence or absence of unlabeled competitors and the displacement of androstenone from plasma proteins was measured. In the presence of excess unlabeled competitors, [3H]-androstenone was only partially displaced from plasma proteins, indicating it binds to a low affinity high capacity plasma protein. Binding kinetics studies were also conducted to characterize the binding of androstenone and dehydroepiandrosterone (DHEA) to plasma proteins. The Bmax of androstenone and DHEA was approximately the same (89.1% and 92.3%, respectively). However, the binding affinity (K) of androstenone was 6.5 fold greater than DHEA (0.39 nmol/ml and 0.06 nmol/ml, respectively). Affinity chromatography was used to remove albumin from the plasma proteins. Following incubations with androstenone and DHEA, the binding observed in the albumin free protein fraction was reduced 2.6 and 2.1 fold, respectively relative to the binding in the albumin protein fractions. These results provide direct evidence that androstenone is transported non-specifically by albumin in the plasma of the boar.

Sulfiredoxin involved in the protection of peroxiredoxins against hyperoxidation in the early hyperglycaemia.

As a direct consequence of hyperglycaemia, the excessive generation of ROS is central to the pathogenesis of diabetic cardiomyopathy. We hypothesize that stimulation of high glucose (HG) results in an increased sulfiredoxin (Srx) expression, which regulates ROS signaling through reducing the hyperoxidized peroxiredoxins (Prxs). We show that hyperoxidized Prxs were initially reduced in the preliminary stage but then dramatically increased in advanced stage and these changes corresponded to a significant increase of Srx expression in the heart of diabetic rats. These time-dependent changes were also confirmed in neonatal cardiomyocytes and H9c2 cells treated with HG. Moreover, the reduction rate of hyperoxidized Prxs was greatly improved in the HG 24h group, which had an elevated expression of Srx. Our data also show that HG-induced AP1 activation and Srx expression were almost abolished by JNK inhibitor and N-acetylcysteine (NAC). In addition, siRNA-Srx suppressed HG-induced ANP and ß-MHC gene expression. These observations suggest that activation of AP1 induced by HG is important for the expression of Srx and the reduction of hyperoxidized Prxs in cardiomyocytes. This Srx induction maybe is the pivotal compensatory protection mechanism against oxidative stress in diabetes or hyperglycaemia. Most interestingly, hyperoxidized Prxs/Srx pathway may be involved in the cardiac hypertrophy signaling of diabetes.

Functional polymorphism in porcine CYP2E1 gene: Its association with skatole levels.

Raising intact male pigs would have a significant economic impact on the pork industry. However, the presence of skatole (a major cause of boar taint) in meat from intact male pigs could be highly objectionable to consumer. The excessive accumulation of skatole in fat is a major cause of boar taint, and is associated with defective expression of cytochrome P4502E1 (CYP2E1). In pigs, it has been found that CYP2E1 is negatively correlated with accumulation of skatole. The searching for polymorphism of CYP2E1 and the relevant functional analysis would help develop a genetic marker for the selection of pigs with low skatole levels in fat. The aim of this study was to measure the expression pattern of CYP2E1 mRNA in various tissues of the pig, to identify genetic polymorphisms, and to evaluate the functional relevance of polymorphic sites with respect to the skatole level in fat. We show herein that a substitution of G --> A at base 1423 of the CYP2E1 gene in the liver causes a significant decrease in the expressed CYP2E1 level. Our data suggest that the G --> A substitute might be at least partially responsible for a high level of skatole in pigs. We believe that this is an important step toward the selection of genetic markers for boar taint by lowering fat levels of skatole in fat.

The role of CYP2A and CYP2E1 in the metabolism of 3-methylindole in primary cultured porcine hepatocytes.

The accumulation of 3-methylindole (3MI) in uncastrated male pigs (boars) is a major cause of boar taint, which negatively affects the quality of meat from the animal. Previously, CYP2E1 and CYP2A have been identified as cytochrome P450 (P450) isoforms involved in the metabolism of 3MI using porcine liver microsomes. This study further examines the role of these isoforms in the metabolism of 3MI using a primary porcine hepatocyte model by examining metabolic profiles of 3MI after incubation with P450 inhibitors. Incubation of hepatocytes with 4-methylpyrazole resulted in a selective inhibition of CYP2E1 activity as determined by p-nitrophenol hydroxylase activity and an associated significant decrease in the production of the 3MI metabolites 3-hydroxy-3-methyloxindole and 3-methyloxindole. Furthermore, inhibition of CYP2A, as assayed by coumarin 7-hydroxylase activity, using 8-methoxypsoralen and diethyldithiocarbamate was not associated with any further significant inhibition of the production of 3MI metabolites. Treatment with general P450 inhibitors resulted in further decreases in CYP2E1 activity and a more dramatic decrease in the production of 3MI metabolites, suggesting that additional P450s may be involved in the phase 1 metabolism of 3-methylindole. In conclusion, CYP2E1 activity levels are more important than CYP2A activity levels for the metabolism of 3-methylindole in isolated pig hepatocytes.

Using human microarrays to identify differentially expressed genes associated with increased steroidogenesis in boars.

Human microarrays are readily available, and it would be advantageous if they could be used to study gene expression in other species, such as pigs. The objectives of this research were to validate the use of human microarrays in the analysis of porcine gene expression, to assess the variability of the data generated, and to compare gene expression in boars with different levels of steroidogenesis. Cytochrome b5 (CYB5) expression was used to assess array detection sensitivity. Samples having high or low CYB5 RNA levels were hybridized to microarrays to determine if the known expression difference could be detected. Six hybridizations were conducted using human microarrays containing 3840 total spots representing 1718 characterized human ESTs. To analyze gene expression in boars with different levels of steroidogenesis, testis RNA from four boars with high levels of plasma estrone sulphate was hybridized to testis RNA from four boars with lower levels. Eight microarray hybridizations were conducted including fluor-flips. Self-self hybridizations were also conducted to assess the variability of array experiments. The Cy5 and Cy3 intensity values for each array were normalized using a locally weighted linear regression (LOESS). Statistical significance was assessed using a Student's t-test followed by the Benjamini and Hochberg multiple testing correction procedure. Quantitative real-time PCR (Q-RT-PCR) was used to verify select gene expression differences. The results show that CYB5 was significantly overexpressed in the high CYB5 sample by 1.8 fold (P < 0.05), verifying the known expression difference. The average log2 ratio of the majority of genes (1643) falls within one standard deviation of the mean, indicating the data were reproducible. In the high versus low steroidogenesis experiment, seven genes were significantly overexpressed in the high group (P < 0.05). Quantitative real-time PCR was used to validate five genes with the highest fold change, and the results corroborated those found by the microarray experiments. The results of the self-self hybridizations showed that no genes were significantly differentially expressed following the application of the Benjamini and Hochberg multiple testing correction procedure. The results presented in this report show that human arrays can be used for gene expression analysis in pigs.

Identification of a single nucleotide polymorphism in porcine testis cytochrome P450-c17 (CYP17) and its effect on steroidogenesis.

Raising uncastrated male pigs could have significant economic benefits for pig production. Uncastrated male pigs can accumulate high levels of 16-androstene steroids, however, resulting in boar taint, which is highly objectionable to consumers. Cytochrome P450-c17 (CYP17) interacts with cytochrome b5 in the biosynthesis of the 16-androstene steroids and the sex steroids from pregnenolone. Amino acid substitutions in CYP17 could therefore affect the ability of this enzyme to catalyze the reactions leading to the production of androstenone and the sex steroids. In this study, we established a sensitive and flexible single-stranded conformational polymorphism technique capable of detecting a single nucleotide polymorphism. We then used this method to identify a substitution from T to A at nucleotide 1317 of CYP17, which caused a change in the amino acid sequence from Leu(439) to His(439). This mutation, however, did not alter the enzyme activity of CYP17 in the biosynthesis of androstenone or sex steroids. Other polymorphisms previously suggested for CYP17, which are vital for the functional interaction of CYP17 with CYB5 in human, were not observed. This study suggests that the synthesis of androstenone in pig testis is not directly affected by any polymorphisms in the coding region of the porcine CYP17 gene.

A novel polymorphism in the 5' untranslated region of the porcine cytochrome b5 (CYB5) gene is associated with decreased fat androstenone level.

Raising intact male pigs would have a significant economic impact on the pork industry; however, the presence of 16-androstene (a major cause of boar taint) in meat from male pigs would be highly objectionable to consumers. In pigs, a positive correlation has been found between cytochrome b5 (CYB5) and production of 16-androstene. The search for polymorphism of CYB5 and functional analysis of polymorphism found should have an important impact on the efforts to develop genetic markers to select for low androstenone levels in fat from pigs. The aim of this study was to search the porcine CYB5 gene for mutations, examine its expression, identify genetic polymorphisms, and study how a genetic variation in this enzyme translates into interindividual variation in androstenone levels in fat from pig testis. We have identified a single nucleotide polymorphism (SNP) (G --> T) at base 8 up-stream of ATG in the CYB5 5' untranslated region which is associated with a lower fat androstenone level. Of the 229 testis samples tested, 84.8% were homozygous for the variant G, 12.4% were heterozygous, and 2.8% were homozygous for the variant T. Functional analysis of this mutation revealed that an individual homozygous for the T allele showed significantly lower CYB5 activity than an individual homozygous for the G allele. Thus, this may be at least partially responsible for a lower level of androstenone in pigs. Our findings provide an important genetic basis toward the goal of predicting the androstenone status in pigs and developing genetic markers for low androstenone.

Molecular cloning and functional analysis of porcine SULT1A1 gene and its variant: a single mutation SULT1A1 causes a significant decrease in sulfation activity.

The characterization of the SULT1A1 gene and its variants should have an important impact on the efforts to develop genetic markers to select for low skatole in pigs. Raising intact male pigs would have a significant economic impact on the pork industry; however, the presence of skatole (a major cause of boar taint) in meat from male pigs would be highly objectionable to consumers. It has been shown that the phase II metabolism of skatole metabolites by phenol sulfotransferase is related to the clearance of skatole in the liver. The aim of this study was to isolate and characterize the SULT1A1 gene from pig liver, examine its expression, identify genetic polymorphisms, and study how a genetic variation in this enzyme translates into interindividual variation in skatole levels. We show here that a substitution of A-->G at base 546 of SULT1A1 causes a significant decrease in its sulfation activity and thus may be at least partially responsible for a higher level of skatole in pigs. Our findings provide an important basis toward the goal of making it possible to predict the sulfation status in pigs and the development of genetic markers for low skatole.