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Piscine cytochrome P450 (CYP) enzymes play an important role in the metabolism of xenobiotics. Xenobiotics often act as inducers of CYP1A1 and CYP3A expression and activity in fish. We compared constitutive mRNA expression of CYP1A1, CYP3A27, and CYP3A45 and catalytic activity of CYP1A (7-ethoxyresorufin-O-deethylation, EROD) and CYP3A-like (benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylation, BFCOD) enzymes in the following six rainbow trout tissues: liver, gill, heart, brain, intestine, and gonad. mRNA expression and activity were present in all investigated tissues. The CYP1A1 mRNA expression was higher in the liver, gill, heart, and brain compared to gonad and intestine. The intestine was the main site of CYP3A27 and CYP3A45 expression. The highest EROD and BFCOD activity was observed in liver tissue followed in descending order by heart, brain, gill, intestine, and gonad. Such differences might be related to the role of CYP physiological functions in the specific tissue. Rainbow trout exposure to 50 mg/kg of β-naphthoflavone for 48 h resulted in a 7.5- and 5.9-fold increase in liver EROD and BFCOD activity, respectively. In vitro EROD activity inhibition with ellipticine showed tissue-specific inhibition, while ketoconazole decreased BFCOD activity by 50–98 % in all tissues. Further studies are needed to identify all CYP isoforms that are responsible for these activities and modes of regulation.

Graphical abstract, Highlights • CYP mRNA induction were compared between human and porcine primary hepatocytes. • Both human and porcine primary hepatocytes responded to prototypical CYP inducers. • CYP mRNA induction displayed similar patterns in human and porcine primary hepatocytes., The hepatic cytochrome p450’s (CYP) are of major importance for the metabolism of xenobiotics and knowledge about their regulation is crucial. This knowledge often originates from cell models; primary human hepatocytes (PHH) being the gold standard. However, due to limited availability of high-quality human donor organs, basic knowledge on alternative models are needed. Primary porcine hepatocytes (PPH) have been suggested as an alternative to PHH. Unfortunately, data comparing the response in gene-transcription to standard CYP inducers between PHH and PPH are missing. In the present study we, cultured PHH and PPH under the same conditions, treated them with standard inducers of the CYP1-3 and determined the response in gene and protein expression. The results demonstrated that in both species TCDD and omeprazole caused an increase in CYP1A/B expression. In PPH, CITCO increased the content of CYP1A/B. For the CYP2B/C/D’s, phenobarbital and rifampicin caused increases in expression. For the CYP2D’s, TCDD and omeprazole caused increased gene expression in PPH, which were not the case for PHH. Both phenobarbital, rifampicin and omeprazole increased CYP3A expression in PHH and PPH. Moreover, TCDD increased the gene expression of CYP3A in PPH; this was not the case for PHH. Multivariate data analysis found no difference in gene expression between PHH and PPH for phenobarbital, rifampicin and CITCO. However, differential clustering was observed for TCDD and omeprazole. In conclusion, despite model specificity, there are a high number of similar responses, and experiments investigating mRNA regulation made in PPH permits for a reliable translation into human setting.

The cytochrome P450s (CYPs) constitute a family of enzymes maintaining vital functions in the body and are mostly recognized for their significant role in detoxification. Of the CYP subfamilies, CYP3A, is one of the most active in the clearance of drugs and other xenobiotics. During the last decades, much focus has been on exploring different models for human CYP3A regulation, expression and activity. In that respect, the growing knowledge of the porcine CYP3As is of great interest. Although many aspects of porcine CYP3A regulation and activity are still unknown, the current literature provides a basic understanding of the porcine CYP3As that can be used e.g., when translating results from studies done in the porcine model into human settings. In this review, the current knowledge about porcine CYP3A expression, regulation, activity and metabolic significance are highlighted. Future research needs are also identified.

A review is presented on the strategic use of scattering and imaging tools to design immunologic complexes based on porous silica for oral vaccine formulations., This article summarizes developments attained in oral vaccine formulations based on the encapsulation of antigen proteins inside porous silica matrices. These vaccine vehicles show great efficacy in protecting the proteins from the harsh acidic stomach medium, allowing the Peyer’s patches in the small intestine to be reached and consequently enhancing immunity. Focusing on the pioneering research conducted at the Butantan Institute in Brazil, the optimization of the antigen encapsulation yield is reported, as well as their distribution inside the meso- and macroporous network of the porous silica. As the development of vaccines requires proper inclusion of antigens in the antibody cells, X-ray crystallography is one of the most commonly used techniques to unveil the structure of antibody-combining sites with protein antigens. Thus structural characterization and modelling of pure antigen structures, showing different dimensions, as well as their complexes, such as silica with encapsulated hepatitis B virus-like particles and diphtheria anatoxin, were performed using small-angle X-ray scattering, X-ray absorption spectroscopy, X-ray phase contrast tomography, and neutron and X-ray imaging. By combining crystallography with dynamic light scattering and transmission electron microscopy, a clearer picture of the proposed vaccine complexes is shown. Additionally, the stability of the immunogenic complex at different pH values and temperatures was checked and the efficacy of the proposed oral immunogenic complex was demonstrated. The latter was obtained by comparing the antibodies in mice with variable high and low antibody responses.

Fasting has been shown to regulate the expression of the cytochrome p450 (CYP) enzyme system in the liver. However, the exact mechanism behind the fasting-induced regulation of the CYP’s remains unknown. In the present study we tested the hypothesis that the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), which is a key-regulator of energy metabolism, is responsible for the fasting-induced regulation of the CYP’s. Lox/lox and liver specific PGC-1α (LKO) mice of both sexes, fasted for 18 h and the content of the CYP’s as well as the hepatic metabolome was assessed. Fasting increased the mRNA content of Cyp2a4, Cyp2e1, Cyp3a11 and Cyp4a10. The fasting-induced response in Cyp4a10 mRNA content was different between lox/lox and LKO mice, while the absence of PGC-1α had no effect on the fasting-induced response for the other Cyp’s. Moreover, the fasting-induced response in mRNA content of Sirtinus 1 and Perilipin 2 was different between lox/lox and LKO mice. Only the CYP1A isoform showed a fasting-induced response at the protein level. Absence of hepatic PGC-1α had no effect on the apparent metabolome, where fasting vs fed was the only discriminate in the following multivariate analysis. In conclusion, hepatic PGC-1α is not essential for the fasting-induced regulation of hepatic CYP’s.

Pigs have often been suggested to be a useful model for humans, when investigating CYP dependent events, like drug metabolism. However, comprehensive knowledge about the constitutive expression of the major CYP and corresponding transcription factors is limited. We compared the constitutive mRNA expression of aryl hydrocarbon receptor, constitutive androstane receptor and pregnane X receptor and CYP1A1, CYP1A2, CYP2A, CYP2E1 and CYP3A in liver, adipose tissue, muscle and small intestine in pigs, as well as the expression along the length of the small intestine and colon. Tissue samples were taken from female pigs, and analyzed for gene expression, as well as CYP dependent activity using qPCR and specific probe substrates, respectively. For all investigated transcription factors and CYPs the mRNA expression and activity was highest in the liver. CYP1A1 and CYP3A mRNA expression and activity was shown in all investigated tissues. Along the small intestine and colon the mRNA expression and activity of CYP1A1 and CYP3A was gradually decreased. The results demonstrated, similarity to that reported for humans, and hence adds to the use of pigs as a model for humans.

This study characterized changes in biomarker responses in common carp (Cyprinus carpio) upon exposure to effluent water discharged from a sewage treatment plant (STP) under real conditions. Fish were exposed to contamination in Cezarka pond, which receives all of its water input from the STP in the town of Vodnany, Czech Republic. Five sampling events were performed at day 0, 30, 90, 180, and 360 starting in April 2015. In total, 62 pharmaceutical and personal care products (PPCPs) were detected in the polar organic chemical integrative sampler. Compared to a control pond, the total concentration of PPCPs was 45, 16, 7, and 7 times higher in Cezarka pond at day 30, 90, 180, and 360, respectively. The result of oxidative stress and antioxidant enzyme biomarkers indicated alterations in the liver and intestine tissues of fish from Cezarka pond at day 30 and 360, respectively. High plasma vitellogenin levels were observed in both exposed females (180 and 360 days) and males (360 days) compared with their respective controls. However, only exposed female fish had higher vitellogenin mRNA expression than the control fish in these periods. Exposed female fish showed irregular structure of the ovary with scattered oocytes, which further developed to a vitellogenic stage at day 360. Low white blood cell levels were indicated in all exposed fish. Despite numerous alterations in exposed fish, favorable ecological conditions including high availability of food resulted in a better overall condition of the exposed fish after 1 year of exposure compared to the controls.

The liver hosts a great number of enzymatically driven processes, including detoxification. The super-family of enzymes named cytochrome P450 (CYP) is the major participant in that process. The expression of CYPs is affected by several factors including life-stage (foetal vs. adult). In the present study we investigated the impact of birth-weight (high or low birth weight) and life-stage on constitutive expression of porcine hepatic CYP1A1, CYP1A2, CYP2A19, CYP2B22, CYP2C33, CYP2D25, CYP2E1 and CYP3A29, as well as the transcription factors controlling their expression; aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, C/EBP and hepatocyte nuclear factors 1 and 4. Both RT-PCR and western blotting showed a marked increase in the expression of the adult pigs compared with prenatal pigs. Moreover, CYP2E1 mRNA expression was 7.5 fold higher in foetuses with low birth weight compared with foetuses with high birth weight. Gender did not affect the mRNA expression within the different life-stages. These results indicate a similarity to what is observed in humans and porcine foetuses may therefore be a model for humans when studying expression of CYPs.

The gene expression of the cytochrome P450 (CYP) enzyme family is regulated by numerous factors. Fasting has been shown to induce increased hepatic CYP mRNA in both humans and animals. However, the coordinated regulation of CYP, CYP-regulating transcription factors, and transcriptional co-factors in the liver linking energy metabolism to detoxification has never been investigated. Interleukin-6 (IL-6) has been suggested to be released during fasting and has been shown to regulate CYP expression. The present study investigated the hepatic mRNA content of selected CYP, AhR, CAR, PXR and PPARα in mice fasted for 18 h and subsequently exposed to IL-6. Furthermore, the impact of fasting on PGC-1α, HNF-4α, SIRT1 and SIRT3 mRNA was examined. Fasting induced a marked increase in Cyp2b10, Cyp2e1 and Cyp4a10 mRNA, while CYP1a1, Cyp1a2, Cyp2a4 and Cyp3a11 mRNA levels remained unchanged. In accordance, the mRNA levels of CAR and PPARα were also increased with fasting. The PGC-1α, SIRT1 and SIRT3 mRNA levels were also increased after fasting, while the HNF-4α mRNA levels remained unchanged. In mice subjected to IL-6 injection, the fasting-induced PXR, PPARα and PGC-1α mRNA responses were lower than after saline injection. In conclusion, fasting was demonstrated to be a strong inducer of hepatic CYP mRNA as well as selected transcription factors controlling the expression of the investigated CYP. Moreover, the mRNA levels of transcriptional co-factors acting as energy sensors and co-factors for CYP regulation was also increased in the liver, suggesting crosstalk at the molecular level between regulation of energy metabolism and detoxification.

Higher accumulation of skatole in the fat of male pigs compared with female pigs might be due to gender-related differences in the rate of skatole degradation. In the present study, skatole metabolites and cytochrome P450 (CYP450) isoforms involved in skatole metabolism were for the first time investigated in hepatic S9 fractions from six male and four female pigs (crossbred Landrace× Yorkshire dams and Duroc boar). Surprisingly, the rates of production of major skatole metabolites were similar in male and female pigs. The most abundant metabolite of skatole was 3-hydroxy-3-methyloxindole (HMOI) followed by 3-methyloxindole and indole-3-carbinol in both male and female S9 fractions. Concentrations of formed HMOI and 3-methyloxindole did not differ between the genders (P = 0.124 for HMOI, and P = 0.575 for 3-methyloxindole). Indole-3-carbinol formation was higher in S9 fractions from the females compared with male pigs (P = 0.0001). Enzyme kinetic parameters were similar for both genders (P> 0.05). In both male and female pigs, ellipticine, diallyl sulphide (DAS) and quercetin inhibited HMOI formation, confirming the involvement of CYP1A1 and CYP2E1. The formation of 3-methyloxindole was reduced in the presence of the CYP2E1 inhibitor DAS, and formation of indole-3-carbinol was reduced in the presence of CYP1A1 and CYP2A19 inhibitors. We found only minor differences in skatole metabolism between male and female pigs, particularly the involvement of CYP2C and CYP3A in indole-3-carbinol formation in female but not in male pigs. This is a very essential finding, suggesting the involvement of larger number of CYP450 isoforms in female pigs. On the other hand, indole-3-carbinol is a minor skatole metabolite, and the physiological significance of CYP2C and CYP3A involvement in its formation in female pigs, but not in male pigs, needs to be elucidated. Our results, however, should be interpreted with caution because of the low number of animals and possibility of breed and age effects on skatole metabolism. info:eu-repo/semantics/publishedVersion

High-fat diet (HFD) induces several changes to the pathways regulating energy homeostasis and changes the expression of the hepatic cytochrome p450 (Cyp) enzyme-system. Despite these pervious findings, it is still unclear how the effects of HFD and especially HFD in combination with treadmill running affect hepatic Cyp expression. In this study, we investigated the mRNA and protein expression of selected Cyp's in mice subjected to 16 weeks of HFD and treadmill running. To understand the regulatory mechanisms behind the exercise-induced reversion of the HFD-induced changes in Cyp expression, we used a model in which the exercise-induced myokine and known regulator of hepatic Cyp's, interleukin-6 (IL-6), were knocked out specifically in skeletal muscle. We found that HFD increased the mRNA expression of Cyp1a1 and Cyp4a10, and decreased the expression of Cyp2a4, Cyp2b10, Cyp2e1, and Cyp3a11. HFD in combination with treadmill running reversed the HFD increase in Cyp4a10 mRNA expression. In addition, we observed increased Cyp1a and Cyp3a protein expression as an effect of exercise, whereas Cyp2b expression was lowered as an effect of HFD. IL-6 effected the response in Cyp3a11 and Cyp1a expression. We observed no changes in the content of the aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, or peroxisome proliferation activator receptor alpha. In conclusion, we show that both HFD and exercise in HFD-fed animals can regulate hepatic Cyp expression and that changes in Cyp3a in response to HFD and exercise are dependent on skeletal muscular IL-6.

The physiological effects of the Stevia-derived compounds, rebaudioside A, stevioside and steviol have been the focus of several studies due to their use as sweeteners in food. Despite that, little is known about their potential food-drug interactions. In the present study, IPEC-J2 cells and primary hepatocytes were used to investigate the effect of rebaudioside A, stevioside and steviol on cytochrome p450 (CYP) mRNA expression. Moreover, hepatic microsomes were used to investigate direct interactions between the compounds and specific CYP activity. In IPEC-J2 no changes in mRNA expression of CYP1A1 or CYP3A29 were observed with the Stevia-derived compounds. In primary hepatocytes all three tested compounds induced a significant increase in CYP3A29 expression. The tested compounds had no direct effect on specific CYP activity. In conclusion, rebaudioside A, stevioside and steviol induce only minor or no changes to the CYP expression and activity, and are not likely to cause food-drug interactions.

Primary hepatocytes are widely used in the study of dynamic events like regulation of gene expression, as they are superior to most cell-lines. However, the culturing of the hepatocytes often results in loss of phenotype, e.g. the expression of the cytochrome p450s (CYP). The present study investigated the impact of serum in the culture medium of porcine primary hepatocytes (PPH) on markers of dedifferentiation as well as the impact on CYP induction. The effects were studied in both freshly isolated primary hepatocytes as well as cryopreserved. The exclusion of serum in the culturing media were not introducing significant dedifferentiation as judged by the gene expression of α-fetoprotein, albumin, glucose-6-phosphatase and the constitutive expression of selected transcription factors and CYP. The induction of CYP2B22 and CYP3A29 by phenobarbital and rifampicin, were greater in hepatocytes cultured without serum. The same were not observed for TCDD induced CYP1A2 expression. In conclusion, PPH cultured under serum free conditions results in little or no dedifferentiation, while being more responsive to known CYP inducers. Hence, it can be suggested that PPH cultured under serum free conditions provides a reliable hepatocyte model to investigate CYP gene regulation.

The role of the cross-talk between nuclear receptors in the regulation of Cytochrome P450 expression in the liver is well-documented. Most studies have focused on the cross-talk between the pregnane X receptor (PXR) and other receptors, such as the constitutive androstane receptor. However, cross-talk between PXRs and aryl hydrocarbon receptors (AhRs) has also been suggested, but reports regarding this cross-talk are conflicting. In the present study, we treated HepaRG and primary human hepatocytes (PHHs) with both a strong (TCDD) and weak (3-methylindole; 3MI) AhR activator to investigate their impact on PXR-regulated expression of CYP3A4. Moreover, we investigated the effect of co-activation of PXR, using rifampicin, and AhR, using TCDD and 3MI, on the regulation of CYP3A4 induction. We also investigated whether knockdown of AhR using siRNA affected the basal expression of PXR and CYP3A4 and induction of CYP3A4 by rifampicin, TCDD and 3MI. The results showed that the treatment of HepaRG cells, but not of PHHs, with AhR activators decreased mRNA expression of CYP3A4 and PXR. Moreover, in both HepaRG and PHHs, AhR activation decreased rifampicin-induced expression of CYP3A4 mRNA. Knock-down of AhR in PHHs increased both basal and rifampicin-induced expression of CYP3A4 mRNA. In conclusion, the presented results suggested that the cross-talk between PXR and AhR plays a role in the regulation of CYP3A4 gene expression.

1. Humans and animals are commonly exposed to indole-3-carbinol (I3C) and resveratrol (RES) via food or beverages. Moreover, these compounds have been demonstrated to potentially cause food–drug interactions. However, information about their combined effects is limited. Therefore, we investigated the effects of I3C and RES, both as single compounds and in combination, on cytochrome P450 1A and 3A activity and gene expression. 2. Using porcine microsomes, we demonstrated that RES caused non-competitive inhibition of CYP1A activity and un-competitive inhibition of CYP3A activity. Compared to the effect of single compounds, co-treatment with I3C and RES increased a degree of inhibition of CYP1A activity. 3. In porcine primary hepatocytes, treatment with I3C and RES resulted in induction of CYP1A1, CYP1A2 and CYP3A29 mRNA expression. 4. In conclusion, we demonstrated that both RES and I3C could cause food–drug interactions and that the combined effect could be more potent in doing so.

Cytochrome P450 proteins (CYP450s) are present in most domains of life and play a critical role in the metabolism of endogenous compounds and xenobiotics. The effects of exposure to xenobiotics depend heavily on the expression and activity of drug-metabolizing CYP450s, which is determined by species, genetic background, age, gender, diet, and exposure to environmental pollutants. Numerous reports have investigated the role of different vertebrate CYP450s in xenobiotic metabolism. Model organisms provide powerful experimental tools to investigate Phase I metabolism. The aim of the present review is to compare the existing data on human CYP450 proteins (1-3 families) with those found in pigs, mice, and fish. We will highlight differences and similarities and identify research gaps which need to be addressed in order to use these species as models that mimic human traits. Moreover, we will discuss the roles of nuclear receptors in the cellular regulation of CYP450 expression in select organisms.

Due to limited availability of human liver tissue for the study of cytochrome P450 (CYP450), porcine liver tissue has been suggested as an alternative source to prepare microsomes and hepatocytes. The porcine liver is made by four different lobes. The present study investigated the expression and activity of specific CYP450 isoforms in the four lobes, with the purpose to examine if one lobe of the porcine liver resembles the human more than others. Samples from the four major lobes were taken from female pigs and mRNA expression and activity of CYP1A, 2A, 2C, 2D, 2E and 3A determined. The results showed no differences in specific mRNA expression and activity of any of the investigated CYP450 isoforms. In conclusion, the study shows that all parts of the porcine liver are equally useful as model tissue.

Cytochrome P450 (CYP450) is the major family of enzymes involved in the metabolism of several xenobiotic and endogenous compounds. Among substrates for CYP450 is the tryptophan metabolite skatole (3-methylindole), one of the major contributors to the off-odour associated with boar-tainted meat. The accumulation of skatole in pigs is highly dependent on the hepatic clearance by CYP450s. In recent years, the porcine CYP450 has attracted attention both in relation to meat quality and as a potential model for human CYP450. The molecular regulation of CYP450 mRNA expression is controlled by several nuclear receptors and transcription factors that are targets for numerous endogenously and exogenously produced agonists and antagonists. Moreover, CYP450 expression and activity are affected by factors such as age, gender and feeding. The regulation of porcine CYP450 has been suggested to have more similarities with human CYP450 than other animal models, including rodents. This article reviews the available data on porcine hepatic CYP450s and its implications for boar taint.

International audience; AbstractAbandoning traditional practice of piglet castration will impact the pigmeat sector. As a consequence, there is a need for research aiming at reducing boar taint caused by androstenone and skatole. Skatole is metabolized by cytochrome P450 enzymes (CYP450) in the liver. Skatole hepatic clearance is believed to be hindered by androstenone. Diet ingredients may modify skatole metabolism. Therefore, we tested the effect of hydrolysable tannins. We fed 51 young boars with 1–3 % chestnut wood extract as supplementary diet. After slaughter, the tissues were collected to assess androstenone and skatole accumulation in fat and to measure CYP450 activities, gene, and protein expression in the liver and intestine. Protein expression of two enzymes involved in androstenone metabolism, 3-beta-hydroxysteroid dehydrogenase (3β-HSD) and sulfotransferase family 2A member 1 (SULT2A1), was assessed, and feces collected to evaluate skatole production. Results show that intestinal skatole production in boars supplemented with 3 % of chestnut wood extract was more than halved. The intestinal catalytic activities of CYP450 were tenfold lower than hepatic and were mainly unaffected by tannins. Findings indicate a potential effect of tannins on steroidogenesis, which in the absence of effect on 3β-HSD and SULT2A1 expression suggests lower synthesis of androstenone due to tannins.

International audience; Skatole (3-methylindole) is a product of bacterial fermentation of tryptophan in the intestine. A significant amount of skatole can also be inhaled during cigarette smoking. Skatole is a pulmonary toxin that induces the expression of aryl hydrocarbon receptor (AhR) regulated genes, such as cytochrome P450 1A1 (CYP1A1), in human bronchial cells. The liver has a high metabolic capacity for skatole and is the first organ encountered by the absorbed skatole; however, the effect of skatole in the liver is unknown. Therefore, we investigated the impact of skatole on hepatic AhR activity and AhR-regulated gene expression. Using reporter gene assays, we showed that skatole activates AhR and that this is accompanied by an increase of CYP1A1, CYP1A2 and CYP1B1 expression in HepG2-C3 and primary human hepatocytes. Specific AhR antagonists and siRNA-mediated AhR silencing demonstrated that skatole-induced CYP1A1 expression is dependent on AhR activation. The effect of skatole was reduced by blocking intrinsic cytochrome P450 activity and indole-3-carbinole, a known skatole metabolite, was a more potent inducer than skatole. Finally, skatole could reduce TCDD-induced CYP1A1 expression, suggesting that skatole is a partial AhR agonist. In conclusion, our findings suggest that skatole and its metabolites affect liver homeostasis by modulating the AhR pathway.

Hepatic cytochrome P450 expression and activity are dependent on many factors, including dietary ingredients. In the present study, we investigated the in vivo and in vitro effect of chicory root on hepatic CYP3A and 2C in male pigs. Chicory feeding increased the expression of CYP3A29 mRNA but not CYP2C33.Correspondingly, CYP3A activity was increased by chicory feeding, while CYP2C activity was not affected. Additionally, the in vitro effect of chicory extract on the CYP3A activity was investigated. It was shown that CYP3A activity in the microsomes from male pigs was inhibited, but this effect was eliminated bypre-incubation. In both male and female pigs the CYP3A activity was increased in the presence of chicory after pre-incubation. Furthermore, gender-related differences in mRNA expression and activity were observed. CYP3A mRNA expression was greater in female pigs; this was not reflected on activity. ForCYP2C, no difference in mRNA expression was observed, while CYP2C activity was greater in female pigs. Surprisingly, the expression of the constitutive androstane receptor, pregnane X receptor and aryl hydrocarbon receptor did not differ with feed or gender. In conclusion, chicory root modifies the expression and activity of CYP3A in vivo and in vitro, while CYP2C is not affected.

In vitro inhibition of porcine cytochrome P450 by 17β-estradiol and 17α-estradiol Sexually mature pigs are known to possess high concentrations of testicular steroids, which have been shown to change the activities of cytochrome P450 in vitro. The aim of the present study was to evaluate the regulation of CYP1A and CYP2E1 activity by the steroids dihydrotestosterone (DHT), 3β-androstenol, 17β-estradiol and 17α-estradiol. Catalytic activities of 7-ethoxyresorufin O-deethylase (EROD) and 7-methoxyresorufin O-demethylase (MROD) were used as markers of CYP1A activities, while p-nitrophenol hydroxylase (PNPH) was used as a marker of CYP2E1 activities. Of the steroids tested, only 17β-estradiol and 17α-estradiol inhibited EROD and MROD activities. This inhibition was observed when a steroid concentration of 100 μM was used, while lower concentrations showed no inhibitory effect. PNPH activities were inhibited only by 100 μM of 17β-estradiol. The significance of these results in vivo is unknown because inhibition was only found when concentrations of estrogens higher than physiological levels were used. Nevertheless, the results provided further evidence on the important role of estrogens in regulation of porcine cytochrome P450 activities.

Cytochrome P450 2E1 (CYP2E1) and 2A (CYP2A) are the main enzymes involved in the metabolism of skatole in pigs. In this study, physiological concentrations of androstenone, 17β-oestradiol and testosterone were tested for their ability to regulate CYP2E1 and CYP2A activity in liver microsomes isolated from entire male and female pigs as well as in microsomes from Saccharomyces cerevisiae expressing either human recombinant CYP2E1 or CYP2A6. We found that physiological concentrations of androstenone and oestradiol had the ability to inhibit CYP2E1 activity. The magnitude of this inhibition (approximately 30%) was similar in recombinant human CYP2E1 and microsomes from entire male pigs. This inhibition was only seen when adding the steroid to the assay 15 min before the substrate. Interestingly, CYP2E1 activity in the microsomes from female pigs was not affected. None of the investigated steroids modified the activity of recombinant human CYP2A6. However, CYP2A activity was slightly increased in the microsomes from female pigs in the presence of oestradiol, but the magnitude of this increase was very low (below 10%) and probably irrelevant. Overall, these results indicate that physiological concentrations of androstenone and oestradiol have a potential to inhibit CYP2E1 activities in vitro, and that this inhibition is gender-specific. Further studies are needed to investigate the biochemical mechanisms underlying those differences between the genders.

In pigs, the hepatic cytochrome P450 (CYP) 1A2, 2A and 2E1 activity is important in the regulation of skatole accumulation in adipose tissue. This study investigated gender-related differences in CYP1A2, 2A and 2E1 dependent activity, protein and mRNA expression. This study also investigated the gonadal steroid dependent inhibition of CYP activity in relation to gender and dietary composition. Microsomes were prepared from the liver of female and entire male pigs (Landrace × Yorkshire sire and Duroc boars) reared under similar conditions and slaughtered at an age of 164 days. A group of entire male pigs fed dried chicory root for 16 days prior to slaughter were included in the study. CYP activities were assessed by the use of probe substrates, whilst mRNA and protein expression were analysed by RT-PCR and Western blotting. Furthermore inhibition of CYP dependent activity by gonadal steroids was assessed in vitro. Microsomes from female pigs had greater CYP1A2 and 2A activity, as well as mRNA expression compared to entire male pigs. The antibodies used did not detected differences in protein expression. In vitro inhibition by 17β-oestradiol, oestrone, androstenone and 3β-OH androstenol of CYP2E1 activity in microsomes from entire male pigs as well as inhibition of CYP1A activity in chicory fed entire male pigs was observed. Apart from that no effect of steroids was shown. In conclusion, female pigs show greater CYP activity and mRNA expression. Including chicory in the diet for 16 days changed the gonadal steroid dependent inhibition of CYP activity in entire male pigs.

Effects of aquatic pollutants on fish are of increasing concern. Pharmaceutical-based contaminants are prioritized for further study in environmental risk assessment using several approaches. Dexamethasone (DEX) was one such contaminant recognised for its effect on fish health status. Thus, we carried out an in vivo experiment to identify potential effects of DEX on rainbow trout. Fish were exposed to 3, 30, 300 and 3000ngL(-1) DEX in a semi-static system over a period of 42d. The concentrations of DEX that fish were exposed to was confirmed by LC-LC-MS/MS. Using hepatic microsomes, we determined cytochrome P450 content, activities of ethoxyresorufin O-deethylase (EROD), p-nitrophenol hydroxylase (PNPH), 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase (BFCOD) and benzyloxyquinoline O-debenzylase (BQOD), as well as protein expression. Our results showed that fish do not change the catalytic activity of CYP450-mediated reactions after high DEX concentration exposure. These results disagree with mammalian studies, where DEX is a well-known inducer of CYP450. We showed a significant effect of DEX exposure on CYP450-mediated reactions (EROD, BCFOD, BQOD and PNPH) when expressed as amount of product formed per min per nmol total CYP450 at 3, 30 and 300ngL(-1) after 21d exposure. Moreover, BFCOD and BQ activities showed matching trends in all groups. Western blot analysis showed induction of CYP3A-like protein in the presence of the lowest environmentally relevant concentration of DEX. Based on these findings, continued investigation of the effect of DEX on fish using a battery of complementary biomarkers of exposure and effect is highly relevant.

1. Little is known about the activities and regulation of cytochrome P4503A (CYP3A) enzymes in porcine colon in response to specific feeding components. 2. We added hydrolyzable tannins to the diet of fattening boars and studied its effect on the expression of hepatic and intestinal CYP3A. 3. In total, 51 Landrace × Large White boars were assigned to the following treatment groups: control (without the addition of hydrolysable tannins), T1 (diet-containing 1% hydrolysable tannin extract), T2 (diet-containing 2% hydrolysable tannin extract) and T3 (diet-containing 3% hydrolysable tannin extract). CYP3A expression and activity were measured in microsomes prepared from liver and colon tissue. 4. CYP3A protein expression and activity were increased in the colon of pigs fed 2% and 3% tannins, while no changes were observed with lower tannin concentrations, or in the liver of any treatment groups. Also, it was demonstrated that colon mucosa possess CYP3A activity similar to that measured in the liver. 5. The present results provide the first evidence that tannin supplementation can modulate CYP3A in porcine colon mucosa in vivo. The physiological significance of this finding for the health status of the individual animal needs further investigation.

Chicory (Cichorium intybus) has been shown to induce enzymes of pharmacokinetic relevance (cytochrome P450; CYP). The aim of this study was to investigate the effects of selected secondary plant metabolites with a global extract of chicory root, on the expression of hepatic CYP mRNA (1A2, 2A19, 2C33, 2D25, 2E1 and 3A29), using primary porcine hepatocytes. Of the tested secondary plant metabolites, artemisinin, scoparone, lactucin and esculetin all induced increased expression of specific CYPs, while esculin showed no effect. In contrast, a global extract of chicory root decreased the expression of CYP1A2, 2C33, 2D25 and 3A29 at high concentrations. The results suggest that purified secondary metabolites from chicory affect CYP expression and thereby might affect detoxification in general, and that global extracts of plants can have effects different from individual components.

This study aimed to provide further insights into the mechanism of in vivo regulation of cytochrome P450 (CYP450) 1A, 2A and 2E1 activities in pigs with different levels of testicular steroids. Hepatic mRNA and protein expression and enzymatic activity of CYP1A, CYP2A and CYP2E1 were compared between entire male and castrated pigs. Castration was performed either surgically or immunologically. The pigs were divided into four groups. In the first group, piglets were surgically castrated without anaesthesia. Immunological castration was performed by vaccination with Improvac® (Pfizer Ltd). Vaccinated pigs were subdivided into two groups according to the vaccination regimen: early and standard vaccination. Pigs in the early vaccination group were vaccinated when aged 11 and 15 weeks. Pigs in the standard vaccination group were vaccinated when aged 17 and 21 weeks. In the control group, pigs remained intact throughout the study. Hepatic CYP450 mRNA expression, measured by real-time RT-PCR, differed significantly between groups for all isoforms measured: CYP1A2 (P = 0.002), 2A (P = 0.000) and 2E1 (P = 0.002). Lower CYP450 mRNA in entire male pigs suggests suppression of CYP1A2, CYP2A and CYP2E1 by testicular steroids at the transcriptional level. However, this suppression was not always reflected in decreased protein expression and activities of these isoforms, suggesting that at least some CYP450s (e.g. CYP2E1) are regulated by a post-transcriptional mechanism.

In the present study, porcine liver microsomes prepared by a conventional ultracentrifugation method were compared with microsomes prepared by a calcium aggregation method. Protein concentrations and activities of several cytochrome P450 enzymes were measured. It was concluded that using a calcium aggregation method for microsome preparation resulted in lower activities of porcine 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD), 7-pentoxyresorufin O-depentylase (PROD) and p-nitrophenol hydroxylase (PNPH), compared to ultracentrifugation. Protein concentrations of CYP1A2 and CYP2E1, measured by Western blot, were similar in the microsomes prepared by the two methods, whereas CYP2A protein concentrations were significantly lower in the microsomes prepared by the calcium aggregation method. The choice of homogenization buffer (TRIS with addition of either 250 mM sucrose or 2 mM EDTA) did not affect either individual CYP450 protein concentration or the rates of CYP450-mediated reactions. Freeze/thawing of microsomes did not affect the activities of EROD, MROD, COH and PNPH in the microsomes, indicating the stability of the measured isoforms following three cycles of freezing/thawing. A reduction in the activity of PROD was observed after the third freeze/thawing cycles of the microsomes prepared by both methods.

Cytochrome P450 (CYP) enzymes are widely studied for their involvement in metabolism of drugs and endogenous compounds. In porcine liver, CYP1A2, 2A and 2E1 are important for the metabolism of skatole. Feeding chicory roots to pigs is known to decrease the skatole concentration in plasma and fat. In the present study we investigated the effect of chicory on CYP mRNA and protein expression, as well as their activity. Male pigs were feed dried chicory root for 16 days before liver samples were collected. By the use of RT-PCR and Western blotting we showed that the mRNA and protein expression of CYP1A2 and 2A were increased in chicory fed pigs. The mRNA expression of CYP2E1 was increased, while there was no effect on protein expression. Activity of CYP1A2 and 2A were increased in chicory feed pigs; this was not the case for CYP2E1 activity. In conclusion; oral administration of chicory root for 16 days to pigs increased the mRNA expression of CYP1A2, 2A and 2E1; and the protein expression of CYP1A2 and 2A. The activities of CYP1A2 and 2A were increased.