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

Abstract

Boar taint is the unfavourable odour and taste from pork fat, which results in part from the accumulation of skatole (3-methylindole, 3MI). The key enzymes in skatole metabolism are thought to be cytochrome P450 2E1 (CYP2E1) and cytochrome 2A (CYP2A); however, the cytochrome P450 (CYP450) isoform responsible for the production of the metabolite 6-hydroxy-3-methylindole (6-OH-3MI, 6-hydroxyskatole), which is thought to be involved in the clearance of skatole, has not been established conclusively. The aim of this study was to characterize the role of porcine CYP450s in skatole metabolism by expressing them individually in the human embryonic kidney HEK293-FT cell line. This system eliminates the problems of the lack of specificity of antibodies, inhibitors and substrates for CYP450 isoforms in the pig, and contributions of any other CYP450s that would be present. The results show that pig CYP1A1, CYP2A19, CYP2C33v4, CYP2C49, CYP2E1 and CYP3A and human CYP2E1 (hCYP2E1) are all capable of producing the major skatole metabolite 3-methyloxyindole (3MOI), as well as indole-3-carbinol (I3C), 5-hydroxy-3-methylindole (5-OH-3MI), 6-OH-3MI, 2-aminoacetophenone (2AAP) and 3-hydroxy-3-methyloxindole. CYP2A19 produced the highest amount of the physiologically important metabolite 6-OH-3MI, followed by porcine CYP2E1 and CYP2C49; CYP2A19 also produced more 6-OH-3MI than hCYP2E1. Co-transfection with CYB5A increased the production of skatole metabolites by some of the CYP450s, suggesting that CYB5A plays an important role in the metabolism of skatole. We also show the utility of this expression system to check the specificity of selected substrates and antibodies for porcine CYP450s. Further information regarding the abundance of different CYP450 isoforms is required to fully understand their contribution to skatole metabolism in vivo in the pig.Keywords: boar taint, skatole, 3-methylindole, cytochrome b5A, cytochrome P450 ImplicationsStudies on the metabolism and clearance of skatole, a major component of boar taint, have been hampered by the lack of specific tools to measure the activity of the cytochrome P450 (CYP450) enzymes involved. Here we have used a defined cellbased system to individually express six different isoforms of porcine CYP450 and cytochrome b5A, and measure their role in the metabolism of skatole. Our results show that CYP2A19 is the primary isoform responsible for the formation of 6-hydroxyskatole, although its importance to in vivo clearance will also be affected by its level of expression in vivo.

Gender-related differences in the formation of skatole metabolites by specific CYP450 in porcine hepatic S9 fractions

“…This, however, needs further investigation. CYP2A19 was not involved in HMOI formation (as supported by Wiercinska et al, 2012). CYP2E1 was involved in the formation of 3MOI in both male and female pigs.…”

“…CYP2C (sulphaphenazole) and CYP1A2 (furafylline) were not involved in the formation of HMOI in either male or female pigs. On the contrary, involvement of CYP2C and CYP1A2 was demonstrated by previous in vitro studies (Wiercinska et al, 2012). The disagreement between these studies might be because of the different in vitro systems used.…”

“…In these studies, CYP2A and CYP2E1 were identified as major isoforms involved in skatole metabolism. Further in vitro studies on primary cultured pig hepatocytes (Terner et al, 2006), individual purified porcine enzymes (Matal et al, 2009) and individually expressed enzymes (Wiercinska et al, 2012) confirmed an involvement of CYP2A and CYP2E1, and revealed that other isoforms (CYP1A, CYP2C, CYP3A) are also involved in skatole metabolism. Moreover, genderrelated differences in the rate of skatole metabolism are frequently suggested as a cause of the higher levels of skatole in male pigs compared with the female pigs.…”

Skatole metabolites in urine as a biological marker of pigs with enhanced hepatic metabolism

“…This finding was later confirmed by Borrisser Pairó et al (2015). In contrast, Wiercinska et al (2011) demonstrated formation of I3C by porcine CYP2E1 expressed in the human embryonic kidney HEK293-FT cell line. MOI and HMOI were repeatedly shown to be produced by several CYP450 isoforms, including CYP2E1 and CYP2A19 (Zamaratskaia and Squires, 2009).…”

“…The importance of cytochrome P450 (CYP450; especially CYP1A, CYP2A and CYP2E1) in skatole metabolism is well established (Babol et al, 1998;Diaz and Squires, 2000;Zamaratskaia et al, 2005;Matal et al, 2009;Wiercinska et al, 2011). Currently, seven skatole metabolites are known, indole-3-carbinol (I3C), 2-aminoacetophenone (AAP), 3-hydroxy-3-methyloxindole (HMOI), 3-methyloxyindole (MOI), 3-hydroxy-3-methylindolenine, 5-hydroxy-3-methylindole and 6-hydroxy-3-methylindole (6-OH-3MI) (Baek et al, 1997;Diaz et al, 1999;Borrisser Pairó et al, 2015).…”