E. J. Squires

Metabolism of the 16-androstene steroids in primary cultured porcine hepatocytes

The hepatic metabolism of the 16-androstene steroids was investigated using isolated porcine hepatocytes. This study demonstrated that the liver is capable of producing both phase I and phase II steroid metabolites from 16-androstene steroid precursors. 16-Androstene metabolites were recovered by solid-phase extraction and identified by gas chromatography-mass spectrometry (GC-MS). When 5alpha-androstenone was provided as a substrate, both 3beta- and 3alpha-androstenol were produced as well as a metabolite that showed evidence of hydroxylation. Incubations with the various 16-androstene steroids produced metabolic profiles which suggested that the major role of the liver is phase II conjugation. Sulfoconjugated 16-androstene steroids included androstadienol, 5alpha-androstenone, 3beta-, 3alpha-androstenol, and possibly the hydroxylated metabolite of 5alpha-androstenone. It was determined that hydroxysteroid sulfotransferase (HST) is the likely candidate for the sulfoconjugation of the 16-androstene steroids within the liver. Despite the capacity of the hepatocytes to sulfoconjugate the 16-androstene steroids, the principle metabolites produced from incubations with 5alpha-androstenone, 3beta-, and 3alpha-androstenol were glucuronide conjugates, accounting for approximately 68% of all phase II metabolism. These findings underline the importance of steroid conjugation and suggest that hepatic metabolism of the 16-androstene steroids may influence the levels of 5alpha-androstenone present in the circulation, and thus, capable of accumulating in fat.

Cytochrome P450c17 from porcine and bovine adrenal catalyses the formation of 5,16-androstadien-3β-ol from pregnenolone in the presence of cytochrome b5

The synthesis of 5,16-androstadien-3 beta-ol from pregnenolone occurs via a cytochrome P450-dependent reaction (andien-beta synthase) that is analogous to the C17-hydroxylase/lyase reaction. It is not known whether the andien-beta synthase activity in adult porcine testis involves cytochrome P450c17 or is unique to porcine testis. Andien-beta synthase activity in testis microsomes was inhibited by high pH and concentration of salt, while C17-hydroxylase/lyase activity was stimulated under these conditions. Cytochrome P450c17 purified from adult porcine testis and adrenal glands and bovine adrenal glands had only C17-hydroxylase/lyase activity in the absence of cytochrome b5. However, when cytochrome b5 isolated from porcine testis was added, andien-beta synthase activity was detected in all three preparations of cytochrome P450c17, with the highest activity found in the porcine preparations. The andien-beta synthase activity was further increased from 2.5 to 6 times when NADH cytochrome b5 reductase was added along with cytochrome b5. Levels of mRNA for cytochrome b5 relative to cytochrome P450c17 mRNA were five times higher in porcine testis than in porcine adrenal. It appears that the andien-beta synthase activity is catalysed by cytochrome P450c17, which is not unique to the porcine testis and is dependent upon adequate levels of cytochrome b5.

Individual and combined effects of t‐2 toxin and das in laying hens

1. The individual and combined effects of T-2 toxin and 4,15-diacetoxyscirpenol (DAS) on laying hens were investigated in an experiment consisting of a 2 x 2 completely randomised factorial design with dietary concentrations of 0 and 2 mg/kg T-2 toxin and 0 and 2 mg/kg DAS. 2. Individually, T-2 toxin and DAS induced oral lesions in half of the hens and decreased significantly egg production and food intake. 3. The effects of T-2 toxin and DAS were additive for reduced food consumption and incidence of oral lesions. However, a synergism for reduced egg production was observed during the last experimental period. 4. No effects on body weight were observed during this study. Mild changes in selected plasma enzymes activities and no change in liver malondialdehyde content were detected. 5. The combination of T-2 toxin and DAS was more toxic than the single mycotoxins, for some parameters, and therefore, may pose a greater economic threat to the poultry industry than either of the toxins individually.

Effect of dietary flaxseed, flax oil and n-3 fatty acid supplement on hepatic and plasma characteristics relevant to fatty liver haemorrhagic syndrome in laying hens

1. Two experiments were carried out to investigate the effect of dietary flaxseed, flax oil and n-3 fatty acid supplementation (Dry n-3®) on hepatic fat content, plasma triglycerides, hepatic haemorrhage score, egg production, food intake and body weight in an inbred line of Single Comb White Leghorns (UCD-003) predisposed to fatty liver haemorrhagic syndrome (FLHS) and normal SCWL hens. 2. Feeding diets containing 100 g/kg ground flaxseed, 40 g/kg flax oil, or 100 g/kg Dry n-3® reduced body weight and significantly reduced hepatic fat content compared to feeding the control diet with animal and vegetable oil as a fat source. 3. Hepatic malondialdehyde, an indicator of lipid peroxidation within the liver, was not significantly affected by dietary treatment. 4. Normal SCWL hens tended to have higher egg production, greater body weight, greater food intake and higher blood triglyceride concentrations than UCD-003 hens, although the strain effects were not significant. Liver weight as a percent of body weight was significantly lower in normal SCWL hens. Treatments by strain interactions were not found. 5. The result suggested that dietary flaxseed, flax oil and Dry n-3® decrease hepatic fat content and reduce body weight, 2 of the predisposing factors believed to contribute to FLHS onset. However, haemorrhages were still apparent in both strains regardless of treatment, indicating that other unknown underlying mechanisms may also be responsible for FLHS.

Synthesis of free and sulphoconjugated 16-androstene steroids by the Leydig cells of the mature domestic boar

This study examined the involvement of sulphoconjugation in the biosynthesis of the 16-androstene steroids in Leydig cells of the mature boar, since the formation of steroid sulphoconjugates can reduce the levels of these steroids that accumulate in fatty tissue. Leydig cells were purified from testes of mature male pigs and incubated with pregnenolone, or various individual 16-androstene steroids for 10 min, 1, 4 and 8h. Sulphoconjugated steroids were recovered by solid-phase extraction followed by solvolysis. Profiles of unconjugated and sulphoconjugated steroids were analysed by HPLC. Steroids present in the sulphoconjugated fractions were purified, derivatised as O-methoxime/trimethylsilyl ethers (MO-TMS), and subsequently identified using gas chromatography-mass spectrometry (GC-MS). The principal metabolite produced from incubations with pregnenolone, androstadienol, androstadienone and 5alpha-androstenone was 3beta-androstenol. 16-Androstene steroids that were sulphoconjugated included 5alpha-androstenone, 3beta-androstenol and 3alpha-androstenol. Approximately 70% of the total amount of each 16-androstene steroid was in its sulphoconjugated form after incubations for 4h or more. The finding that sulphoconjugated 5alpha-androstenone was present in large amounts suggests that this steroid may be converted from a 3-keto to a 3-enol form which is subsequently sulphoconjugated. These findings emphasise the need to consider the impact of sulphoconjugation of the 16-androstene steroids and their role in contributing to boar taint.

Phase II in vitro metabolism of 3-methylindole metabolites in porcine liver.

1. The Phase II in vitro metabolism of 3-methylindole (3MI) metabolites was investigated in pigs to determine the possible relationship between 3MI Phase II metabolism and 3MI accumulation in fat. Sulphation and glucuronidation of five of the seven major metabolites found to be produced by porcine microsomes was investigated using porcine cytosol and microsomes, respectively. The possible formation of glutathione conjugates was also investigated using microsomally activated 3MI intermediate(s). 2. No sulphation or glucuronidation was observed for metabolites 3-hydroxy-3-methyloxindole, 3-methyloxindole, indole-3-carbinol or 2-aminoacetophenone; however, 5-hydroxy-3-methylindole (5-OH-3MI) was conjugated with both sulphate and glucuronic acid. 3. The enzyme responsible for sulphation of 5-OH-3MI was identified as the thermostable form of phenol-sulphotransferase (TS-PST) based on its susceptibility to TS-PST inhibitors and the correlation between sulphation of 5-OH-3MI and sulphation of the prototype substrate p -nitrophenol (r = 0.94, p < 0.001). 4. A 3MI-glutathione adduct was identified in microsomal incubations containing 3MI and glutathione. 5. Sulphation of 5-OH-3MI was high in pigs with low levels of 3MI in fat. No relationship was observed between 3MI levels in fat and either glutathione transferase or glucuronidation activities in liver.

Assessment of factor V, VII and X activities, the key coagulant proteins of the tissue factor pathway in poultry plasma.

1. Assay methods were developed for key components of the tissue factor pathway of blood coagulation, namely Factor V, Factor VII and Factor X. Using these assays, plasma from healthy laying hens, cockerels and broilers was shown to contain functional and equivalent amounts of each of these clotting factors. 2. The plasma activities for Factor V, Factor VII and Factor X can only be accurately determined when chicken tissue factor is used to initiate the coagulation mechanism in poultry plasma. Neither human tissue factor nor rabbit tissue factor forms a fully functional enzyme reactive complex with chicken Factor VII. 3. The overall tissue factor pathway coagulation mechanism was evaluated in plasma from laying hens, cockerels and broilers using the one-stage prothrombin time assay. As long as sufficient tissue factor was used, the overall clotting time results obtained with human recombinant tissue factor were not significantly different from those obtained with chicken tissue factor. 4. We conclude that poultry plasma does possess a fully functional tissue factor coagulation mechanism, but homologous chicken tissue factor must be used for in vitro assays of the components of this pathway.

Cobalt-induced polycythaemia causing right ventricular hypertrophy and ascites in meat-type chickens.

Cobalt increases the red cell mass in both man and animals by increasing the production of erythropoietin. Since meat-type chickens can develop pulmonary hypertension from increased erythropoiesis and polycythaemia, two trials were conducted to investigate the role of cobalt on broiler chicken erythropoiesis and pulmonary hypertension. The results showed that feeding cobaltous chloride at 500 parts/10(6) to meat-type chickens from 1-day-old for 42 days significantly increased haemoglobin content and, to a lesser extent red blood cell count, and haematocrit. No effect was observed on mean corpuscular volume. Increased haemoglobin content was linearly correlated with pulmonary hypertension as measured by the right ventricle weight to total ventricle weight ratio (RV:TV). Levels of malondialdehyde in cardiac tissue were also correlated with the RV:TV ratio, suggesting that peroxidative damage may be related to ventricular hypertrophy. Chickens fed cobalt showed a significantly higher incidence of right ventricular hypertrophy and right ventricular failure and 18.3% developed ascites.

The roles of different porcine cytochrome P450 enzymes and cytochrome b5A in skatole metabolism.

Boar taint is the unfavourable odour and taste from pork fat, which results in part from the accumulation of skatole (3-methylindole, 3MI). The key enzymes in skatole metabolism are thought to be cytochrome P450 2E1 (CYP2E1) and cytochrome 2A (CYP2A); however, the cytochrome P450 (CYP450) isoform responsible for the production of the metabolite 6-hydroxy-3-methylindole (6-OH-3MI, 6-hydroxyskatole), which is thought to be involved in the clearance of skatole, has not been established conclusively. The aim of this study was to characterize the role of porcine CYP450s in skatole metabolism by expressing them individually in the human embryonic kidney HEK293-FT cell line. This system eliminates the problems of the lack of specificity of antibodies, inhibitors and substrates for CYP450 isoforms in the pig, and contributions of any other CYP450s that would be present. The results show that pig CYP1A1, CYP2A19, CYP2C33v4, CYP2C49, CYP2E1 and CYP3A and human CYP2E1 (hCYP2E1) are all capable of producing the major skatole metabolite 3-methyloxyindole (3MOI), as well as indole-3-carbinol (I3C), 5-hydroxy-3-methylindole (5-OH-3MI), 6-OH-3MI, 2-aminoacetophenone (2AAP) and 3-hydroxy-3-methyloxindole. CYP2A19 produced the highest amount of the physiologically important metabolite 6-OH-3MI, followed by porcine CYP2E1 and CYP2C49; CYP2A19 also produced more 6-OH-3MI than hCYP2E1. Co-transfection with CYB5A increased the production of skatole metabolites by some of the CYP450s, suggesting that CYB5A plays an important role in the metabolism of skatole. We also show the utility of this expression system to check the specificity of selected substrates and antibodies for porcine CYP450s. Further information regarding the abundance of different CYP450 isoforms is required to fully understand their contribution to skatole metabolism in vivo in the pig.

Liposome‐mediated DNA transfer to chicken sperm cells

Abstract The efficacy of using liposomes to transfer DNA to chicken sperm cells was investigated. Liposomes were prepared from dilauroyl (12:0) phosphatidylcholine (DLPC), dimyristoyl (14:0) phosphatidyl choline (DMPC), dipalmitoyl (16:0) phosphatidylcholine (DPPC), egg yolk phosphatidylcholine (EYPC) or lipids extracted from sperm cell membranes. The efficiency of trapping of DNA into the liposomes, transfer of the DNA from the liposomes to the sperm cells and the effect of the liposomes on the fertilizing ability of the sperm cells were determined. Increasing the concentration of lipid in the liposome preparations increased the trapping efficiency of DNA into liposomes but lowered the transfer of DNA to sperm. Including stearylamine (SA) in the liposomes increased the incorporation of DNA into the liposomes and the DNA transfer to sperm cells, while including lauroyllysophosphatidylcholine (LPC) along with SA resulted in the highest transfer efficiency from liposomes to sperm. The transfer of DNA from l...