- Email: [email protected]
Ontogeny of CYP3A in the liver of the Göttingen minipig: An immunohistochemical and functional evaluation
Abstracts / Reproductive Toxicology 48 (2014) 23–36
P-30 Ontogeny of CYP3A in the liver of the Göttingen minipig: An immunohistochemical and functional evaluation Els M. Van Peer ∗ , Evy Verbueken, Moayad A. Saad, Casper Pype, Christophe R. Casteleyn, Chris J. Van Ginneken, Steven J. Van Cruchten University of Antwerp, Applied Veterinary Morphology, Universiteitsplein 1, 2610 Wilrijk, Belgium Despite the increasing use of the minipig as a non-rodent species in general and juvenile toxicity studies, knowledge on the ontogeny of their biotransformation processes is scarce. The cytochrome P450 enzyme family (CYP) is one of the most important groups of biotransformation enzymes, with CYP3A being the largest subfamily expressed in liver of humans. In this study, we assessed the expression and the activity of CYP3A in livers of foetal, neonatal, juvenile and adult Göttingen minipigs. For immunohistochemistry, we used a rabbit polyclonal anti-human anti-CYP3A4 antibody with porcine cross reactivity. Liver samples were fixed in 4% (w/v) paraformaldehyde (24 h) in distilled water and routinely processed to paraffin blocks. For CYP3A activity, the Vivid® 3A4 Red substrate BOMR (benzyloxy-methyl-resorufin) was used in liver microsomes from the different age groups. The highly fluorescent metabolite (resorufin) resulting from oxidation of BOMR by CYP3A was quantified with a fluorescent plate reader. Human liver microsomes, pooled from 20 different donors (adult, mixed gender) and selected for high CYP3A4 activity, were used as a positive control. This study showed low immunoreactivity for CYP3A in the livers of foetal (84, 86 and 108 days of gestation) and neonatal (Day 1 and Day 3) piglets, which was also reflected by low CYP3A activity in these lowest age groups. A zonal distribution with more intensively stained hepatocytes surrounding the central vein of each lobule was noticed in a few 7-day-old animals but was clearly present at 14 days of age. The animals of 28 days of age and older showed a more pronounced zonal pattern of highly immunoreactive hepatocytes close to the central vein and a moderate staining of the hepatocytes adjacent to the interlobular septa. A similar increase in CYP3A activity was noted during postnatal development, with the highest activity present in piglets at weaning age (28 days of age) and in adult animals. CYP3A activity in human liver microsomes was similar to that observed for the porcine liver microsomes at 28 days of age and in adult pigs. In conclusion, immunohistochemical analysis of liver tissues revealed a low CYP3A expression in foetal and neonatal pigs, with an increasing expression during postnatal development. A similar trend was observed for CYP3A activity. http://dx.doi.org/10.1016/j.reprotox.2014.07.058 P-31 Lack of in vitro CYP3A activity in early zebrafish embryos Evy Verbueken ∗ , Moayad A. Saad, Casper Pype, Els M. Van Peer, Christophe R. Casteleyn, Chris J. Van Ginneken, Steven J. Van Cruchten University of Antwerp, Applied Veterinary Morphology, Universiteitsplein 1, 2610 Wilrijk, Belgium The zebrafish embryo is increasingly used to investigate developmental toxicity. However, knowledge of the ontogeny of cytochrome P450 (CYP) activity in zebrafish is scarce. This
35
information is pivotal, especially for xenobiotics that require bioactivation to exert their teratogenic potential, as these latter compounds may be missed if the CYPs are not mature yet during (part of) zebrafish organogenesis. The aim of this preliminary study was to investigate CYP3A activity in zebrafish embryos at 24 hpf (hours post-fertilisation) as CYP3A plays an important role in the biotransformation of xenobiotics in man. To avoid in vivo issues regarding absorption of the compound, we investigated the intrinsic CYP3A activity of zebrafish embryos, i.e. in microsomes from whole embryo homogenates. To assess CYP3A activity, we used the Vivid® CYP3A4 Red Screening Kit from Life TechnologiesTM containing the fluorogenic substrate BOMR (benzyloxymethyl resorufin). BOMR is metabolised by human CYP3A4 into the highly fluorescent metabolite resorufin. Therefore, we included pooled human liver microsomes of mixed genders (GIBCO® ) as a positive control. As data on BOMR biotransformation are lacking for adult zebrafish, including possible gender differences, we also investigated isolated liver microsomes from adult male and female zebrafish. As a negative control, we used Insect Cell Control SupersomesTM (BD Biosciences), which are microsomes that contain no CYPs. In our study, we used the same microsomal protein concentration (200 g/ml) for all groups. Controls and zebrafish microsomes were exposed to the BOMR substrate for 90 min at 28 ◦ C during which the formation of resorufin was measured by a TECAN GENios microplate reader. The amount of resorufin was expressed as picomole resorufin per milligram microsomal protein per minute (pmol mg−1 min−1 ). Our study showed that the amount of resorufin formed in male zebrafish (29.75 pmol mg−1 min−1 ) was more than twice as high as in female zebrafish (11.85 pmol mg−1 min−1 ). The latter was in the same order of magnitude as resorufin formation in humans (10.88 pmol mg−1 min−1 ). However, we were not able to detect any resorufin levels in the zebrafish embryos at 24 hpf. The results from this in vitro study suggest that CYP3A activity is lacking in early zebrafish embryos but is present in adult male and female zebrafish. http://dx.doi.org/10.1016/j.reprotox.2014.07.059 P-32 Developmental toxicity assessment of a new turf herbicide, methiozolin, by oral administration in rabbits Wook-Joon Yu 1,∗ , Jinsoo Lee 1 , Sun-Young Lee 1 , Jeong-Dong Park 1 , Eun Ju Jeong 1 , Moon-Koo Chung 1 , Ki-Hwan Hwang 2 , Suk-Jin Koo 2 1
Korea Institute of Toxicology, Daejeon, Republic of Korea 2 Moghu Research Center Ltd., Daejeon, Republic of Korea Methiozolin [5-(2,6-difluorobenzly)oxymethyl-5-methyl-3-(3methylthiophen-2-yl)-1,2-isoxazoline] is a new turf herbicide that was recently registered in 2010 in Republic of Korea. The mode of action of methiozolin might be directly or indirectly associated with cell wall biosynthesis and potentially plastoquinone biosynthesis in susceptible plants, but the primary site of herbicidal action is still unclear. To support the safety assessment of this new turf herbicide, this study was conducted to evaluate the potential maternal and prenatal developmental toxicity effect of methiozolin in pregnant New Zealand White rabbits. Methiozolin was orally administered to artificially inseminated rabbits (25 females per group) once a day from gestation day (GD) 6 to 28 at dose levels of 0, 125, 250 and 500 mg/kg/day, followed by cesarean section on GD 29. Maternal toxicity including abortion, decreased mean body weight/weight
P-30 Ontogeny of CYP3A in the liver of the Göttingen minipig: An immunohistochemical and functional evaluation Els M. Van Peer ∗ , Evy Verbueken, Moayad A. Saad, Casper Pype, Christophe R. Casteleyn, Chris J. Van Ginneken, Steven J. Van Cruchten University of Antwerp, Applied Veterinary Morphology, Universiteitsplein 1, 2610 Wilrijk, Belgium Despite the increasing use of the minipig as a non-rodent species in general and juvenile toxicity studies, knowledge on the ontogeny of their biotransformation processes is scarce. The cytochrome P450 enzyme family (CYP) is one of the most important groups of biotransformation enzymes, with CYP3A being the largest subfamily expressed in liver of humans. In this study, we assessed the expression and the activity of CYP3A in livers of foetal, neonatal, juvenile and adult Göttingen minipigs. For immunohistochemistry, we used a rabbit polyclonal anti-human anti-CYP3A4 antibody with porcine cross reactivity. Liver samples were fixed in 4% (w/v) paraformaldehyde (24 h) in distilled water and routinely processed to paraffin blocks. For CYP3A activity, the Vivid® 3A4 Red substrate BOMR (benzyloxy-methyl-resorufin) was used in liver microsomes from the different age groups. The highly fluorescent metabolite (resorufin) resulting from oxidation of BOMR by CYP3A was quantified with a fluorescent plate reader. Human liver microsomes, pooled from 20 different donors (adult, mixed gender) and selected for high CYP3A4 activity, were used as a positive control. This study showed low immunoreactivity for CYP3A in the livers of foetal (84, 86 and 108 days of gestation) and neonatal (Day 1 and Day 3) piglets, which was also reflected by low CYP3A activity in these lowest age groups. A zonal distribution with more intensively stained hepatocytes surrounding the central vein of each lobule was noticed in a few 7-day-old animals but was clearly present at 14 days of age. The animals of 28 days of age and older showed a more pronounced zonal pattern of highly immunoreactive hepatocytes close to the central vein and a moderate staining of the hepatocytes adjacent to the interlobular septa. A similar increase in CYP3A activity was noted during postnatal development, with the highest activity present in piglets at weaning age (28 days of age) and in adult animals. CYP3A activity in human liver microsomes was similar to that observed for the porcine liver microsomes at 28 days of age and in adult pigs. In conclusion, immunohistochemical analysis of liver tissues revealed a low CYP3A expression in foetal and neonatal pigs, with an increasing expression during postnatal development. A similar trend was observed for CYP3A activity. http://dx.doi.org/10.1016/j.reprotox.2014.07.058 P-31 Lack of in vitro CYP3A activity in early zebrafish embryos Evy Verbueken ∗ , Moayad A. Saad, Casper Pype, Els M. Van Peer, Christophe R. Casteleyn, Chris J. Van Ginneken, Steven J. Van Cruchten University of Antwerp, Applied Veterinary Morphology, Universiteitsplein 1, 2610 Wilrijk, Belgium The zebrafish embryo is increasingly used to investigate developmental toxicity. However, knowledge of the ontogeny of cytochrome P450 (CYP) activity in zebrafish is scarce. This
35
information is pivotal, especially for xenobiotics that require bioactivation to exert their teratogenic potential, as these latter compounds may be missed if the CYPs are not mature yet during (part of) zebrafish organogenesis. The aim of this preliminary study was to investigate CYP3A activity in zebrafish embryos at 24 hpf (hours post-fertilisation) as CYP3A plays an important role in the biotransformation of xenobiotics in man. To avoid in vivo issues regarding absorption of the compound, we investigated the intrinsic CYP3A activity of zebrafish embryos, i.e. in microsomes from whole embryo homogenates. To assess CYP3A activity, we used the Vivid® CYP3A4 Red Screening Kit from Life TechnologiesTM containing the fluorogenic substrate BOMR (benzyloxymethyl resorufin). BOMR is metabolised by human CYP3A4 into the highly fluorescent metabolite resorufin. Therefore, we included pooled human liver microsomes of mixed genders (GIBCO® ) as a positive control. As data on BOMR biotransformation are lacking for adult zebrafish, including possible gender differences, we also investigated isolated liver microsomes from adult male and female zebrafish. As a negative control, we used Insect Cell Control SupersomesTM (BD Biosciences), which are microsomes that contain no CYPs. In our study, we used the same microsomal protein concentration (200 g/ml) for all groups. Controls and zebrafish microsomes were exposed to the BOMR substrate for 90 min at 28 ◦ C during which the formation of resorufin was measured by a TECAN GENios microplate reader. The amount of resorufin was expressed as picomole resorufin per milligram microsomal protein per minute (pmol mg−1 min−1 ). Our study showed that the amount of resorufin formed in male zebrafish (29.75 pmol mg−1 min−1 ) was more than twice as high as in female zebrafish (11.85 pmol mg−1 min−1 ). The latter was in the same order of magnitude as resorufin formation in humans (10.88 pmol mg−1 min−1 ). However, we were not able to detect any resorufin levels in the zebrafish embryos at 24 hpf. The results from this in vitro study suggest that CYP3A activity is lacking in early zebrafish embryos but is present in adult male and female zebrafish. http://dx.doi.org/10.1016/j.reprotox.2014.07.059 P-32 Developmental toxicity assessment of a new turf herbicide, methiozolin, by oral administration in rabbits Wook-Joon Yu 1,∗ , Jinsoo Lee 1 , Sun-Young Lee 1 , Jeong-Dong Park 1 , Eun Ju Jeong 1 , Moon-Koo Chung 1 , Ki-Hwan Hwang 2 , Suk-Jin Koo 2 1
Korea Institute of Toxicology, Daejeon, Republic of Korea 2 Moghu Research Center Ltd., Daejeon, Republic of Korea Methiozolin [5-(2,6-difluorobenzly)oxymethyl-5-methyl-3-(3methylthiophen-2-yl)-1,2-isoxazoline] is a new turf herbicide that was recently registered in 2010 in Republic of Korea. The mode of action of methiozolin might be directly or indirectly associated with cell wall biosynthesis and potentially plastoquinone biosynthesis in susceptible plants, but the primary site of herbicidal action is still unclear. To support the safety assessment of this new turf herbicide, this study was conducted to evaluate the potential maternal and prenatal developmental toxicity effect of methiozolin in pregnant New Zealand White rabbits. Methiozolin was orally administered to artificially inseminated rabbits (25 females per group) once a day from gestation day (GD) 6 to 28 at dose levels of 0, 125, 250 and 500 mg/kg/day, followed by cesarean section on GD 29. Maternal toxicity including abortion, decreased mean body weight/weight