Martin Krøyer Rasmussen

Gender-related Differences in Cytochrome P450 in Porcine Liver – Implication for Activity, Expression and Inhibition by Testicular Steroids

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.

Comparison of cytochrome P450 concentrations and metabolic activities in porcine hepatic microsomes prepared with two different methods

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 2mM 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.

Exercise-induced regulation of phospholemman (FXYD1) in rat skeletal muscle: implications for Na+/K+-ATPase activity

Background:  Na+/K+‐ATPase activity is upregulated during muscle exercise to maintain ionic homeostasis. One mechanism may involve movement of α‐subunits to the outer membrane (translocation).

In vivo effect of dried chicory root (Cichorium intybus L.) on xenobiotica metabolising cytochrome P450 enzymes in porcine liver

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.

Na+–K+ pump location and translocation during muscle contraction in rat skeletal muscle

Muscle contraction may up-regulate the number of Na+–K+ pumps in the plasma membrane by translocation of subunits. Since there is still controversy about where this translocation takes place from and if it takes place at all, the present study used different techniques to characterize the translocation. Electrical stimulation and biotin labeling of rat muscle revealed a 40% and 18% increase in the amounts of the Na+–K+ pump α2 subunit and caveolin-3 (Cav-3), respectively, in the sarcolemma. Exercise induced a 36% and 19% increase in the relative amounts of the α2 subunit and Cav-3, respectively, in an outer-membrane-enriched fraction and a 41% and 17% increase, respectively, in sarcolemma giant vesicles. The Na+–K+ pump activity measured with the 3-O-MFPase assay was increased by 37% in giant vesicles from exercised rats. Immunoprecipitation with Cav-3 antibody showed that 17%, 11% and 14% of the α1 subunits were associated with Cav-3 in soleus, extensor digitorum longus, and mixed muscles, respectively. For the α2, the corresponding values were 17%, 5% and 16%. In conclusion; muscle contraction induces translocation of the α subunits, which is suggested to be caused partly by structural changes in caveolae and partly by translocation from an intracellular pool.

Expression and activities of hepatic cytochrome P450 (CYP1A, CYP2A and CYP2E1) in entire and castrated male pigs

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 Vitro Cytochrome P450 2E1 and 2A Activities in the Presence of Testicular Steroids

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.

Angiotensin I–converting enzyme–inhibitory peptides from bovine collagen: insights into inhibitory mechanism and transepithelial transport

The inhibitory mechanism and transepithelial transport of angiotensin I-converting enzyme (ACE)-inhibitory peptides (VGPV and GPRGF) derived from Alcalase®- and papain-hydrolyzed bovine collagen were investigated. The inhibitory mechanism of VGPV and GPRGF was experimentally determined to be non-competitive and the results were supported by molecular docking data. In silico and in vitro gastrointestinal digestion indicated that VGPV remained resistant to digestive enzymes, while GPRGF was degraded into smaller ACE-inhibitory peptides (GPR and GF). VGPV and GPRGF were transported across monolayers of human intestinal epithelial Caco-2 cells through paracellular pathway and retained their ACE-inhibitory activities. The present study suggests that VGPV and GPRGF may possibly be absorbed and exert antihypertensive effects in vivo.

Regulation of porcine hepatic cytochrome p450 - implication for boar taint.

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.

Regulation of 3β-Hydroxysteroid Dehydrogenase/Δ5-Δ4 Isomerase: A Review

This review focuses on the expression and regulation of 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD), with emphasis on the porcine version. 3β-HSD is often associated with steroidogenesis, but its function in the metabolism of both steroids and xenobiotics is more obscure. Based on currently available literature covering humans, rodents and pigs, this review provides an overview of the present knowledge concerning the regulatory mechanisms for 3β-HSD at all omic levels. The HSD isoenzymes are essential in steroid hormone metabolism, both in the synthesis and degradation of steroids. They display tissue-specific expression and factors influencing their activity, which therefore indicates their tissue-specific responses. 3β-HSD is involved in the synthesis of a number of natural steroid hormones, including progesterone and testosterone, and the hepatic degradation of the pheromone androstenone. In general, a number of signaling and regulatory pathways have been demonstrated to influence 3β-HSD transcription and activity, e.g., JAK-STAT, LH/hCG, ERα, AR, SF-1 and PPARα. The expression and enzymic activity of 3β-HSD are also influenced by external factors, such as dietary composition. Much of the research conducted on porcine 3β-HSD is motivated by its importance for the occurrence of the boar taint phenomenon that results from high concentrations of steroids such as androstenone. This topic is also examined in this review.