Martin Krøyer Rasmussen

Aarhus University, Aarhus, Central Jutland, Denmark

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Publications (16)31.47 Total impact

  • Martin Krøyer Rasmussen, Galia Zamaratskaia, Bo Ekstrand
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    ABSTRACT: 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.
    Toxicology in vitro : an international journal published in association with BIBRA. 06/2014;
  • Martin Krøyer Rasmussen, Christina Lindgaard Klausen, Bo Ekstrand
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    ABSTRACT: 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.
    Food Chemistry 03/2014; 146:255-63. · 3.33 Impact Factor
  • Martin Krøyer Rasmussen, Galia Zamaratskaia, Bo Ekstrand
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    ABSTRACT: 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.
    Toxicology in Vitro. 01/2014;
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    Martin Krøyer Rasmussen, Galia Zamaratskaia
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    ABSTRACT: 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.
    Computational and Structural Biotechnology Journal. 01/2014;
  • Martin Krøyer Rasmussen, Bo Ekstrand
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    ABSTRACT: In pigs the endogenously produced compound androstenone is metabolised in the liver in two steps by 3β-hydroxysteroid dehydrogenase (3β-HSD) and sulfotransferase 2A1 (SULT2A1). The present study investigated the effect of selected sex-steroids (0.01-1 μM androstenone, testosterone and estradiol), skatole (1-100 μM) and secondary plant metabolites (1-100 μM) on the expression of 3β-HSD and SULT2A1 mRNA. Additionally the effect of a global methanolic extract of dried chicory root was investigated and compared to previous obtained in vivo effects. Primary hepatocytes were isolated from the livers of piglets (crossbreed: Landrace X Yorkshire and Duroc) and cultured for 24 hours before treatment for an additionally 24 hours. RNA was isolated from the hepatocytes and specific gene-expression determined by RT-PCR using TaqMan probes. The investigated sex-steroids had no effect on the mRNA expression of 3β-HSD and SULT2A1, while skatole decreased the content of SULT2A1 30 % compared to control. Of the investigated secondary plant metabolites artemisinin and scoparone (found in Artemisia sp.) lowered the content of SULT2A1 by 20 and 30 % compared to control, respectively. Moreover, we tested three secondary plant metabolites (lactucin, esculetin and esculin) found in chicory root. Lactucin increased the mRNA content of both 3β-HSD and SULT2A1 by 200 % compared to control. An extract of chicory root was shown to decrease the expression of both 3β-HSD and SULT2A1. It is concluded that the gene-expression of enzymes with importance for androstenone metabolism are regulated by secondary plant metabolites in a complex manner.
    Gene 12/2013; · 2.20 Impact Factor
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    Martin Krøyer Rasmussen, Bo Ekstrand, Galia Zamaratskaia
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    ABSTRACT: 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.
    International Journal of Molecular Sciences 01/2013; 14(9):17926-42. · 2.46 Impact Factor
  • Martin Krøyer Rasmussen, Galia Zamaratskaia, Bente Andersen, Bo Ekstrand
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    ABSTRACT: 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 by pre-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. For CYP2C, 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.
    Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 08/2012; 50(11):4175-9. · 2.99 Impact Factor
  • Martin Krøyer Rasmussen, Carl Brunius, Bo Ekstrand, Galia Zamaratskaia
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    ABSTRACT: The present study investigated the effect of surgical (SC) and immunological castration on the steroid metabolizing enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and sulfotransferase 2A1 (SULT2A1) in male pigs. Thirty-two male pigs were divided in four groups; in one group the pigs were SC before the age of 7 days, two groups were injected with Improvac(®) a vaccine against gonadotropin releasing hormone (immunological castration), while the pigs in the last group remained entire males (EMs). Immunological castration was in one group performed by vaccine injection at ages 11 and 14 weeks, while the other group received injections at ages 17 and 21 weeks. Plasma, adipose and liver tissue were collected at the time of slaughter. Plasma was analyzed for concentrations of testosterone and oestradiol. The adipose tissue was analyzed for the concentration of androstenone, while the liver tissue was analyzed for mRNA and protein expression of 3β-HSD and SULT2A1. Independent of method, all castrated pigs showed greater mRNA and protein expression of 3β-HSD and lower levels of all steroids in plasma compared with EMs. Moreover, there was a strong correlation between mRNA and protein expression of 3β-HSD and steroid levels. The same was not valid for expression of SULT2A1. It is concluded that steroid levels can increase expression of the steroid metabolizing enzyme 3β-HSD and thereby influence steroid metabolism, e.g. of androstenone.
    Molecular Biology Reports 04/2012; 39(8):7927-32. · 2.51 Impact Factor
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    ABSTRACT: In the present study, we investigated hepatic mRNA expression and activities of CYP3A and 2C in entire, surgically castrated and pigs vaccinated with Improvac. Additionally, we examined the mRNA expression of the two nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR), known to regulate CYP3A and 2C mRNA expression, respectively. Activities of CYP3A and 2C were estimated as a rate of 7-benzyloxy-4-trifluoromethylcoumarin and 7-benzyloxyquinoline metabolism (CYP3A) and tolbutamide metabolism (CYP2C). We found no effect of Improvac treatment or surgical castration on either CYP3A or 2C activities. Similarly, the mRNA expressions of CYP3A29, 2C33 and PXR were not changed. CAR mRNA expression differed only between entire and surgically castrated male pigs (p=0.005), being greater in surgically castrated pigs. Our results indicated that neither CYP3A nor 2C are affected by Improvac.
    Vaccine 04/2012; 30(24):3515-8. · 3.77 Impact Factor
  • Martin Krøyer Rasmussen, Carl Brunius, Galia Zamaratskaia, Bo Ekstrand
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    ABSTRACT: The present study investigated the in vivo effect of chicory root on testicular steroid concentrations and androstenone metabolizing enzymes in entire male pigs. Furthermore, the effect on skatole and indole concentrations in plasma and adipose tissue was investigated. The pigs were divided into two groups; one receiving experimental feed containing 10% dried chicory root for 16 days before slaughter, the control group was fed a standard diet. Plasma, adipose and liver tissue samples were collected at slaughter. Plasma was analyzed for the concentration of testosterone, estradiol, insulin-like growth factor 1 (IGF-1), skatole and indole. Adipose tissue was analyzed for the concentration of androstenone, skatole and indole, while the liver tissue was analyzed for mRNA and protein expressions of 3β-hydroxysteroid dehydrogenase (3β-HSD), sulfotransferase 2A1 and heat-shock protein 70 (HSP70). The results showed that the androstenone concentrations in the adipose tissue of chicory fed pigs were significantly (p<0.05) lower and indole concentrations were higher (p<0.05) compared to control fed pigs. Moreover the chicory root fed pigs had increased mRNA and protein expression of 3β-HSD and decreased HSP70 expression (p<0.05). Testosterone and IGF-1 concentrations in plasma as well as skatole concentrations in adipose tissue were not altered by dietary intake of chicory root. It is concluded that chicory root in the diet reduces the concentration of androstenone in adipose tissue via induction of 3β-HSD, and that these changes were not due to increased cellular stress.
    The Journal of steroid biochemistry and molecular biology 02/2012; 130(1-2):90-5. · 3.98 Impact Factor
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    ABSTRACT: 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.
    animal 02/2012; 6(2):271-7. · 1.65 Impact Factor
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    ABSTRACT: 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.
    Interdisciplinary toxicology 06/2011; 4(2):78-84.
  • M K Rasmussen, G Zamaratskaia, B Ekstrand
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    ABSTRACT: 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.
    Reproduction in Domestic Animals 02/2011; 46(1):149-54. · 1.39 Impact Factor
  • M K Rasmussen, G Zamaratskaia, B Ekstrand
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    ABSTRACT: 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.
    Reproduction in Domestic Animals 11/2010; 46(4):616-23. · 1.39 Impact Factor
  • Martin Krøyer Rasmussen, Galia Zamaratskaia, Bo Ekstrand
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    ABSTRACT: 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.
    Toxicology Letters 11/2010; 200(1-2):88-91. · 3.15 Impact Factor
  • Martin Krøyer Rasmussen, Bo Ekstrand, Galia Zamaratskaia
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    ABSTRACT: 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.
    Toxicology in Vitro 10/2010; 25(1):343-6. · 2.65 Impact Factor