Differential gene expression of CYP3A isoforms in equine liver and intestines

Tydén, E., Löfgren, M., Pegolo, S., Capolongo, F., Tjälve, H., Larsson, P. Differential gene expression of CYP3A isoforms in equine liver and intestines. J. vet. Pharmacol. Therap.  35 , 588–595. Recently, seven CYP3A isoforms – CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP129 – have been isolated from the horse genome. In this study, we have examined the hepatic and intestinal gene expression of these CYP3A isoforms using TaqMan probes. We have also studied the enzyme activity using luciferin‐isopropyl acetal (LIPA) as a substrate. The results show a differential gene expression of the CYP3A isoforms in the liver and intestines in horses. In the liver, CYP3A89, CYP3A94, CYP3A96 and CYP3A97 were highly expressed, while in the intestine there were only two dominating isoforms, CYP3A93 and CYP3A96. The isoform CYP3A129 was not detected in the liver or the intestine, although this gene consists of a complete set of exons and should therefore code for a functional protein. It is possible that this gene is expressed in tissues other than the liver and intestines. In the intestine, both CYP3A96 and CYP3A93 showed the highest gene expression in the duodenum and the proximal parts of the jejunum. This correlated with a high protein expression in these tissues. Studies of the enzyme activity showed the same K_m for the LIPA substrate in the liver and the intestine, while the maximum velocity (V_max) in the liver was higher than in the intestine. Our finding of a differential gene expression of the CYP3A isoforms in the liver and the intestines contributes to a better understanding of drug metabolism in horses.     

Characterization of equine cytochrome P450: role of CYP3A in the metabolism of diazepam

Research on drug metabolism and pharmacokinetics in large animal species including the horse is scarce because of the challenges in conducting in vivo studies. The metabolic reactions catalyzed by cytochrome P450s (CYPs) are central to drug pharmacokinetics. This study elucidated the characteristics of equine CYPs using diazepam (DZP) as a model compound as this drug is widely used as an anesthetic and sedative in horses, and is principally metabolized by CYPs. Diazepam metabolic activities were measured in vitro using horse and rat liver microsomes to clarify the species differences in enzyme kinetic parameters of each metabolite (temazepam [TMZ], nordiazepam [NDZ], p‐hydroxydiazepam [p‐OH‐DZP], and oxazepam [OXZ]). In both species microsomes, TMZ was the major metabolite, but the formation rate of p‐OH‐DZP was significantly less in the horse. Inhibition assays with a CYP‐specific inhibitors and antibody suggested that CYP3A was the main enzyme responsible for DZP metabolism in horse. Four recombinant equine CYP3A isoforms expressed in Cos‐7 cells showed that CYP3A96, CYP3A94, and CYP3A89 were important for TMZ formation, whereas CYP3A97 exhibited more limited activity. Phylogenetic analysis suggested diversification of CYP3As in each mammalian order. Further study is needed to elucidate functional characteristics of each equine CYP3A isoform for effective use of diazepam in horses.     

A gene expression estimator of intramuscular fat percentage for use in both cattle and sheep

Background

The expression of genes encoding proteins involved in triacyglyceride and fatty acid synthesis and storage in cattle muscle are correlated with intramuscular fat (IMF)%. Are the same genes also correlated with IMF% in sheep muscle, and can the same set of genes be used to estimate IMF% in both species?

Results

The correlation between gene expression (microarray) and IMF% in the longissimus muscle (LM) of twenty sheep was calculated. An integrated analysis of this dataset with an equivalent cattle correlation dataset and a cattle differential expression dataset was undertaken. A total of 30 genes were identified to be strongly correlated with IMF% in both cattle and sheep. The overlap of genes was highly significant, 8 of the 13 genes in the TAG gene set and 8 of the 13 genes in the FA gene set were in the top 100 and 500 genes respectively most correlated with IMF% in sheep, P-value = 0. Of the 30 genes, CIDEA, THRSP, ACSM1, DGAT2 and FABP4 had the highest average rank in both species. Using the data from two small groups of Brahman cattle (control and Hormone growth promotant-treated [known to decrease IMF% in muscle]) and 22 animals in total, the utility of a direct measure and different estimators of IMF% (ultrasound and gene expression) to differentiate between the two groups were examined. Directly measured IMF% and IMF% estimated from ultrasound scanning could not discriminate between the two groups. However, using gene expression to estimate IMF% discriminated between the two groups. Increasing the number of genes used to estimate IMF% from one to five significantly increased the discrimination power; but increasing the number of genes to 15 resulted in little further improvement.

Conclusion

We have demonstrated the utility of a comparative approach to identify robust estimators of IMF% in the LM in cattle and sheep. We have also demonstrated a number of approaches (potentially applicable to much smaller groups of animals than conventional methods) to using gene expression to rank animals for IMF% within a single farm/treatment, or to estimate differences in IMF% between two farms/treatments.     

In ovo leptin administration affects hepatic lipid metabolism and microRNA expression in newly hatched broiler chickens

Background

A leptin-like immunoreactive substance has been found in chicken eggs and has been implicated in serving as a maternal signal to program offspring growth and metabolism. In the present study, we investigated the effects of in ovo leptin administration on hatch weight, serum and hepatic concentrations of metabolites and hormones, as well as on the expression of genes involved in hepatic lipid metabolism and the predicted microRNAs (miRNAs) targeting the affected genes. To this end we injected fertile eggs with either 0.5 μg of recombinant murine leptin or vehicle (PBS) before incubation.

Results

Prenatally leptin-exposed chicks showed lower hatch weight, but higher liver weight relative to the body weight, compared to the control group. In ovo leptin treatment increased the hepatic content and serum concentration of leptin in newly hatched chickens. The hepatic contents of triglycerides (TG) and total cholesterol (Tch) were decreased, whereas the serum levels of TG, Tch and apolipoprotein B (ApoB) were increased. The hepatic mRNA expression of sterol regulator element binding protein 1 (SREBP-1c), SREBP-2, hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and cholesterol 7α-hydroxylase 1 (CYP7A1) was significantly up-regulated, as was the protein content of both SREBP-1c and SREBP-2 in hepatic nuclear extracts of leptin-treated chickens. Moreover, out of 12 miRNAs targeting SREBP-1c and/or HMGCR, five were significantly up-regulated in liver of leptin-treated chicks, including gga-miR-200b and gga-miR-429, which target both SREBP-1c and HMGCR.

Conclusions

These results suggest that leptin in ovo decreases hatch weight, and modifies hepatic leptin secretion and lipid metabolism in newly hatched broiler chickens, possibly via microRNA-mediated gene regulation.     

绵羊BTG2基因CDs区克隆、生物信息学分析及组织mRNA表达研究

本研究旨在克隆小尾寒羊B细胞易位基因2(B-cell Translocation Gene 2,BTG2)基因编码CDs区,并对其进行生物信息学分析,同时检测该基因在羔羊期各组织中的mRNA表达情况。通过RT-PCR和TA克隆方法获得BTG2基因CDs区序列;利用在线软件对该基因进行生物信息分析;采用qPCR检测该基因在3、40日龄绵羊的各组织中的表达情况。结果表明:成功获得小尾寒羊BTG2基因CDs区序列453 bp;绵羊与牛、猪、虎鲸、人、大鼠、小鼠同源性分别为98.9%、90.29%、94.04%、88.52%和85.65%、86.98%,且绵羊与牛的亲缘关系最近,与小鼠的亲缘关系最远。理化性质结果显示BTG2基因编码150个氨基酸,蛋白分子式为C748H1190N208O213S8,分子质量为17 ku,理论等电点(pI)为9.14,半衰期为30 h,属于不稳定亲水性蛋白,且不存在跨膜结构和信号肽序列,蛋白结构主要通过α-螺旋、无规则卷曲和β股组成,为混合型蛋白;qPCR检测到BTG2基因mRNA在不同日龄羔羊心脏、肝脏、脾脏、肺脏、肾脏、肠和肌肉均有表达,且均在肝脏中表达量最高;在心脏、肝脏、脾脏、肺脏组织中,40日龄BTG2表达量极显著高于3日龄,而在肾脏、肠和肌肉中,40日龄极显著低于3日龄。结果表明,BTG2广泛存在各组织中,且随着日龄增加,在各组织中的表达趋势不同,提示其功能的重要性和多重性,并且BTG2在肝组织中表达量最高,说明BTG2可能与脂代谢以及糖代谢等调控功能相关。     

Growth,digestive and absorptive capacity and antioxidant status in intestine and hepatopancreas of sub-adult grass carp Ctenopharyngodonidella fed graded levels of dietary threonine

Background

This study was carried out to investigate effects of threonine levels on growth, digestive and absorptive capacity and antioxidant status in intestine and hepatopancreas of sub-adult grass carp (Ctenopharyngodonidella).

Results

Weight gain, specific growth rate, feed intake and feed efficiency were significantly improved by dietary threonine (P < 0.05). Intestinal activities of trypsin, chymotrypsin, alpha-amylase, lipase, alkaline phosphatase, γ-glutamyl transpeptidase and creatine kinase took the similar trends. Contents of malondialdehyde and protein carbonyl in intestine and hepatopancreas were significantly decreased by dietary optimal threonine supplementation (P < 0.05). Anti-superoxide anion capacity, anti-hydroxyl radical capacity, glutathione content and activities of superoxide dismutase, catalase and glutathione-S-transferase in intestine and hepatopancreas were enhanced by dietary threonine (P < 0.05).

Conclusions

Dietary threonine could improve growth, enhance digestive and absorptive capacity and antioxidant status in intestine and hepatopancreas of sub-adult grass carp. The dietary threonine requirement of sub-adult grass carp (441.9-1,013.4 g) based on weight gain was 11.6 g/kg diet or 41.5 g/kg of dietary protein by quadratic regression analysis.     

The genes of all seven CYP3A isoenzymes identified in the equine genome are expressed in the airways of horses

In the present study, we examined the gene expression of cytochrome P450 3A (CYP3A) isoenzymes in the tracheal and bronchial mucosa and in the lung of equines using TaqMan probes. The results show that all seven CYP3A isoforms identified in the equine genome, that is, CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP3A129, are expressed in the airways of the investigated horses. Though in previous studies, CYP3A129 was found to be absent in equine intestinal mucosa and liver, this CYP3A isoform is expressed in the airways of horses. The gene expression of the CYP3A isoenzymes varied considerably between the individual horses studied. However, in most of the horses CYP3A89, CYP3A93, CYP3A96, CYP3A97 and CYP3A129 were expressed to a high extent, while CYP3A94 and CYP3A95 were expressed to a low extent in the different parts of the airways. The CYP3A isoenzymes present in the airways may play a role in the metabolic degradation of inhaled xenobiotics. In some instances, the metabolism may, however, result in bioactivation of the xenobiotics and subsequent tissue injury.     

Cytochrome P450-mediated hepatic metabolism of new fluorescent substrates in cats and dogs

This study aimed to investigate the biotransformation of cat liver microsomes in comparison to dogs and humans using a high throughput method with fluorescent substrates and classical inhibitors specific for certain isozymes of the human cytochrome P450 (CYP) enzyme family. The metabolic activities associated with CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A were measured. Cat liver microsomes metabolized all substrates selected for the assessment of cytochrome P450 activity. The activities associated with CYP3A and CYP2B were higher than the activities of the other measured CYPs. Substrate selectivity could be demonstrated by inhibition studies with α-naphthoflavone (CYP1A), tranylcypromine/quercetine (CYP2C), quinidine (CYP2D), diethyldithiocarbamic acid (CYP2E) and ketoconazole (CYP3A) respectively. Other prototypical inhibitors used for characterization of human CYP activities such as furafylline (CYP1A), tranylcypromine (CYP2B) and sulfaphenazole (CYP2C) did not show significant effects in cat and dog liver microsomes. Moreover, IC50-values of cat CYPs differed from dog and human CYPs underlining the interspecies differences. Gender differences were observed in the oxidation of 7-ethoxy-4-trifluoromethylcoumarin (CYP2B) and 3-[2-(N, N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (CYP2D), which were significantly higher in male cats than in females. Conversely, oxidation of the substrates dibenzylfluorescein (CYP2C) and 7-methoxy-4-trifluoromethylcoumarin (CYP2E) showed significant higher activities in females than in male cats. Overall CYP-activities in cat liver microsomes were lower than in those from dogs or humans, except for CYP2B. The presented difference between feline and canine CYP-activities are useful to establish dose corrections for feline patients of intensively metabolized drugs licensed for dogs or humans.     

Determination of the unsaturated disaccharides of hyaluronic acid in equine synovial fluid by high-performance liquid chromatography and fluorescence detection

Background

The purpose of this study was to develop and validate an analytical method to determine the presence of hyaluronic acid derived disaccharides in equine synovial fluid.

Findings

A high-performance liquid chromatography method for the determination of hyaluronic acid derived unsaturated disaccharides in equine synovial fluid was developed and validated. The method is based on the measurement of unsaturated disaccharides released by digestion of linear hyaluronic acid molecules. The method showed linearity (r² = 0.996) over the full working concentration range 0.89-30 mg/l. Relative standard deviation of intra- and inter-day precision ranged from of 4.3-6.7% and 7.1-7.8% respectively. The detection limit was 0.3 mg/l corresponding to 20 mg/l in synovial fluid. Accuracy of the assay was 97-103%. This method was evaluated by determining the concentration of unsaturated disaccharides from hyaluronic acid in synovial fluid of horses with lameness in the metacarpo-/metatarsophalangeal joint localized with positive response to intra-articular anesthesia.

Conclusions

The described method is valid for determination of hyaluronic acid derived disaccharides in equine synovial fluid. This method was applied to a larger research project dealing with a new form of intra-articular therapy in horses with arthritic diseases.     

Tumor slices as a model to evaluate doxorubicin in vitro treatment and expression of trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 in canine mammary gland cancer

Background

In women with breast cancer submitted to neoadjuvant chemotherapy based in doxorubicin, tumor expression of groups of three genes (PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2) have classified them as responsive or resistant. We have investigated whether expression of these trios of genes could predict mammary carcinoma response in dogs and whether tumor slices, which maintain epithelial-mesenchymal interactions, could be used to evaluate drug response in vitro.

Methods

Tumors from 38 dogs were sliced and cultured with or without doxorubicin 1 μM for 24 h. Tumor cells were counted by two observers to establish a percentage variation in cell number, between slices. Based on these results, a reduction in cell number between treated and control samples ≥ 21.7%, arbitrarily classified samples, as drug responsive. Tumor expression of PRSS11, MTSS1, CLPTM1 and SMYD2, was evaluated by real time PCR. Relative expression results were then transformed to their natural logarithm values, which were spatially disposed according to the expression of trios of genes, comprising PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. Fisher linear discrimination test was used to generate a separation plane between responsive and non-responsive tumors.

Results

Culture of tumor slices for 24 h was feasible. Nine samples were considered responsive and 29 non-responsive to doxorubicin, considering the pre-established cut-off value of cell number reduction ≥ 21.7%, between doxorubicin treated and control samples. Relative gene expression was evaluated and tumor samples were then spatially distributed according to the expression of the trios of genes: PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. A separation plane was generated. However, no clear separation between responsive and non-responsive samples could be observed.

Conclusion

Three-dimensional distribution of samples according to the expression of the trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 could not predict doxorubicin in vitro responsiveness. Short term culture of mammary gland cancer slices may be an interesting model to evaluate chemotherapy activity.     

Poultry fat decreased fatty acid transporter protein mRNA expression and affected fatty acid composition in chickens

Background

A study was undertaken to examine the effects of poultry fat (PF) compared with those of soybean oil (SBO) on intestinal development, fatty acid transporter protein (FATP) mRNA expression, and fatty acid composition in broiler chickens. A total of 144 day-old male commercial broilers were randomly allocated to 2 treatment groups (6 replicates of 12 chicks for each treatment) and fed isocaloric diets containing 3.0% PF or 2.7% SBO at 0 to 3 wk and 3.8% PF or 3.5% SBO at 4 to 6 wk, respectively.

Results

PF had no influence on intestinal morphology, weight, or DNA, RNA, or protein concentrations at 2, 4, and 6 wk of age. However, compared with SBO, PF significantly decreased FATP mRNA abundance at 4 wk (P = 0.009) and 6 wk of age (P < 0.001); decreased liver fatty acid-binding protein (L-FABP) mRNA abundance at 6 wk of age (P = 0.039); and decreased C18:2 (P = 0.015), C18:3 (P < 0.001), C20:2 (P = 0.018), Σ-polyunsaturated fatty acids (Σ-PUFA) (P = 0.020), and the proportion of PUFA (P < 0.001) in the intestinal mucosa and decreased C18:2 (P = 0.010), C18:3 (P < 0.001), C20:2 (P < 0.001), Σ-PUFA (P = 0.005), and the proportion of PUFA (P < 0.001) in breast muscle at 6 wk of age.

Conclusions

PF decreases FATP and L-FABP mRNA expression and decreased the proportion of PUFA in the intestinal mucosa and breast muscle.     

Physiological indicators of stress and meat and carcass characteristics in tail bitten slaughter pigs

Background

Tail biting is a common welfare problem in pig production and in addition to being a sign of underlying welfare problems, tail biting reduces welfare in itself. The aim of this study was to evaluate the effects of tail biting on different pre and post mortem indicators of stress in slaughter pigs and on carcass and meat characteristics. A total of 12 tail bitten (TB) and 13 control (C) pigs from a farm with a long-term tail biting problem were selected for salivary cortisol analyses before and after transport to the slaughterhouse. After stunning, samples were taken for the analysis of serum cortisol, blood lactate, intestinal heat shock protein 70 (HSP70), and meat quality characteristics. In addition, body temperature immediately after and muscle temperature 35 min after stunning were measured, as well as lean meat percentage and carcass weight.

Results

TB pigs showed a lower cortisol response to the transport-induced stress than C pigs and also had a lower serum cortisol concentration after stunning. HSP70 content in the small intestine was higher in the TB pigs than in C pigs. TB pigs had a considerably lower carcass weight therefore produced a lower total amount of lean meat per carcass than C pigs.

Conclusions

This study suggests that prolonged or repeated stress in the form of tail biting causes a blunted stress response, possibly a sign of hypocortisolism. In addition, it underlines the importance of reducing tail biting, both from an animal welfare and an economic point-of-view.     

Expression and localisation of calbindin D28k in all intestinal segments of the laying hen

1. The aim of the present study was to investigate expression and localisation of a 28-kDa calcium-binding protein (CaBP-D28k) related to active calcium (Ca) absorption, in the entire intestine of egg-laying hens. 2. Western blotting analysis showed that the entire intestine expressed CaBP-D28k to the following degree: duodenum > jejunum > caecum > ileum > colon. Immunohistochemistry showed strong CaBP-D28k localisation in enterocytes along the villus tip-crypt axis in the duodenum and in villus tips in the caecum and colon. The jejunum and ileum had moderate localisation with respect to the number of immunoreactive cells and staining intensity. 3. These results suggest that laying hens actively absorb Ca in both the large and small intestines.