The Journal of Maternal-Fetal & Neonatal Medicine
ISSN: 1476-7058 (Print) 1476-4954 (Online) Journal homepage: http://www.tandfonline.com/loi/ijmf20
Overlapping between CYP3A4 and CYP3A7 expression in the fetal human liver during development Daniela Fanni, Vassillios Fanos, Rossano Ambu, Federica Lai, Clara Gerosa, Pietro Pampaloni, Peter Van Eyken, Giancarlo Senes, Massimo Castagnola & Gavino Faa To cite this article: Daniela Fanni, Vassillios Fanos, Rossano Ambu, Federica Lai, Clara Gerosa, Pietro Pampaloni, Peter Van Eyken, Giancarlo Senes, Massimo Castagnola & Gavino Faa (2015) Overlapping between CYP3A4 and CYP3A7 expression in the fetal human liver during development, The Journal of Maternal-Fetal & Neonatal Medicine, 28:11, 1291-1295, DOI: 10.3109/14767058.2014.951625 To link to this article: http://dx.doi.org/10.3109/14767058.2014.951625
Published online: 10 Sep 2014.
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Date: 05 November 2015, At: 18:14
http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, 2015; 28(11): 1291–1295 ! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2014.951625
ORIGINAL ARTICLE
Overlapping between CYP3A4 and CYP3A7 expression in the fetal human liver during development Daniela Fanni1, Vassillios Fanos2, Rossano Ambu1, Federica Lai1, Clara Gerosa1, Pietro Pampaloni1, Peter Van Eyken3, Giancarlo Senes1, Massimo Castagnola4, and Gavino Faa1 1
Department of Surgical Sciences, Divison of Pathology, University Hospital San Giovanni di Dio, University of Cagliari, Cagliari, Italy, Department of Pediatrics and Clinical Medicine, NICU, Puericulture Institute and Neonatal Section, University of Cagliari, Cagliari, Italy, 3 Pathology Department, ZOL (Ziekenhuis Oost-Limburg), Genk, Belgium, and 4Faculty of Medicine, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy
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Abstract
Keywords
Objective: The cytochrome P450 (CYP450) superfamily is implicated in important life processes, including metabolism of many molecules. CYP3A account for the largest portion of CYP450 proteins in human, including CYP3A4, CYP3A5 and CYP3A7. The purpose of this study was to investigate the immunohistochemical expression of CYP3A4 and CYP3A7 in human liver at different post-conceptional (PC) ages. Methods: Human liver samples from 30 fetuses and newborns were, clustered according with the PC age, routinely processed for immunohistochemical analysis of CYP3A4 and CYP3A7. Results: CYP3A4 was positive in all but two cases, CYP3A7 was positive in all but one case, which was negative also for CYP3A4. Conclusions: Our data on immunohistochemical detection of CYP3A4 and CYP3A7 during development show that CYP3A4 expression is not restricted to the post-natal age, being the immunostaining for both CYP3A4 and CYP3A7 identical after 25 weeks of PC age, thus the relationship between these CYP450 isoforms should be considered much more complex than previous thought. A high interindividual variability was observed among subjects at all gestational age. The variable CYP3A expression suggests the existence of a marked interindividual variability in drug metabolism during the intrauterine life and in perinatal period.
CYP3A4, CYP3A7, cytochrome P450, fetus, immunohistochemistry, liver, newborns
Introduction The cytochrome P450 (CYP450) superfamily is formed by 57 genes and 58 pseudogenes, encoding for 57 functional monooxygenases critical for human metabolism [1,2]. The myriad of enzymes encoded by the 57 CYP genes are implicated in important life processes, including metabolism of drugs, foreign chemicals, arachidonic acid, cholesterol, biosynthesis of bile acids, steroids, vitamin D3 and hydroxylation of retinoic acid [3]. The expression of CYP450 enzymes in the human body is characterized by a marked substrate and tissue specificity. The most important isoenzymes for drug metabolism are localized in the liver; seven isoenzymes are (CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) being responsible for the metabolism of more than 95% of drugs in adulthood [1,4].
Address for correspondence: Dr Daniela Fanni, MD, PhD, Pathologist, Department of Pathology, University of Cagliari, ‘‘S. Giovanni di Dio’’ Hospital, via Ospedale 60, 09124 Cagliari, Italy. Tel: 0390706092372. Fax: 0396092115. E-mail: [email protected]
History Received 14 April 2014 Accepted 17 May 2014 Published online 10 September 2014
CYP450 isoforms involved in drug metabolism have been also reported in kidney, lung, brain, breast, prostate and in the small intestine [5]. The protein products of human CYP4503A gene family (CYP3A) account for the largest portion of CYP450 proteins in human liver, including CYP3A4 and CYP3A7. CYP3A4 has been reported to be undetectable during gestation and at birth, dramatically increasing after one postnatal week and reaching adult levels around one month after birth. At least 50% of currently used drugs in NICU centers are substrates of CYP3A4 including antibiotics, antivirals, antifungals, immunomodulators, benzodiazepines, proton pump inhibitors, steroid hormones and acetaminophen [6–9]. It has been claimed that the low content of CYP3A4 in the neonate at birth might be the main responsible for the impaired metabolism of cisapride in the perinatal period, resulting in accumulation of the drug and cardiac toxicity [10]. CYP3A7 has been reported to be predominantly expressed in the fetal liver, the CYP3A7 gene being silenced in the perinatal period, normally within the first postnatal week.
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Catalytic activity is detectable as early as 50–60 d of gestation in embryonic liver, where it catalyzes 16-alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, an intermediate metabolite in the estriol biosynthesis [11]. CYP3A7 expression is characterized by interindividual variability; in some adult livers, the enzymatic activity does not end at birth, but it continues in adulthood, being detectable even extrahepatically. The function of this CYP enzyme is mainly associated to hydroxylation of retinoic acid and to 16 alpha-hydroxylation of steroids, playing a key role in normal development, as well as in carcinogenesis [12]. CYP3A4 represents 30–40% of the total CYP content in human adult liver and in small intestine mucosa cells. CYP3A7 is the major isoform detected in embryonic, fetal and newborn liver, being also detectable at low levels in adult liver cells [13]. A marked interindividual variability has been reported regarding CYP3A isoforms’ expression and activity: in particular, the variable expression of CYP3A7 in fetal liver, starting from the ninth week of gestation, suggests that significant interindividual differences in drug metabolism may already exist at the prenatal stage, these differences contributing to individual pharmacological responses in the intrauterine life [14]. Since few data are available regarding the expression of the CYP enzymes in the newborn liver, the purpose of this study was to investigate the immunohistochemical expression of CYP3A4 and CYP3A7 in human liver during gestation and in newborns at different post-conceptional (PC) ages.
national standards of the responsible committee on human experimentation. The histological examination was performed by three observers (D. F., F. L. and G. F.), who independently assigned two different scores. The first one regarded the distribution: score 1 for focal distribution, score 2 for zonal distribution and score 3 for diffuse distribution. The second score concerned the intensity of immunostaining, that was scored as weak (score 1), medium (score 2) and strong (score 3). In both score systems, score 0 was assigned for negativity. The sum between the distribution score and the intensity score gave rise to the following final grading system: grade 0 for negative stain, grade 1 for an amount from 1 to 3, grade 2 from 4 to 5 and grade 3 more than 5. All cases were then discussed at multisession microscope until all three observers approved the given scores. The percentages of the different immunoreactivities for CYP3A4 and CYP3A7 were calculated and compared in the different groups in order to evaluate the presence of significant differences in the degree of immunostaining.
Methods
CYP3A4
The expression of CYP3A4 and CYP3A7 was evaluated in liver samples from 30 human fetuses and newborns with different PC age, that is, the sum between gestational age and the post-natal age. Patients were clustered into three groups of 10, according with the PC age: group 1 included 10 cases with PC age lower than 24 weeks, group 2 incorporated 10 cases from 25 to 28 weeks of PC age and group 3 integrated cases from 29 to 41 weeks of PC age. Liver samples were fixed in 10% formalin, dehydrated and embedded in paraffin. Tissue sections were then dewaxed, rehydrated through graded alcohols and pre-treated for 10 min with the heat-induced epitope retrieval in buffer pH 6.00 (EnVisionÔ FLEX Target Retrieval Solution Low pH – Dako Denmark A/S, Glostrup, Denmark, code: K8005), for immunohistochemical analysis of CYP3A4 and in buffer pH 9.00 (EnVisionÔ FLEX Target Retrieval Solution High pH – Dako Denmark A/S, code: K8004) and for immunohistochemical analysis of CYP3A7. Slides were then incubated for 20 min at room temperature with anti-CYP450 3A4 rabbit polyclonal antibody at 1:1000 dilution and with anti-CYP450 3A7 mouse monoclonal antibody clone F19 P2 H2 at 1:50 dilution (all produced by Abcam, Cambridge, UK, catalog numbers: ab3572, ab108624 and ab55840, respectively). Staining procedures were performed by EnvisionÔ FLEX+ (Dako Denmark A/S, code: K8002) Detection System and AutostainerLink 48 instrument following dealer’s instructions. Negative controls samples were incubated without mouse anti-CYP3A4 and CYP3A7 polyclonal antibody. All procedures performed were in accordance with the ethical
Results Immunoreactivity for CYP3A4 and CYP3A7 was detected in the majority of fetal livers. Four cases, two belonging to group 2 and two to group 3, showed a diffuse and strong positivity for the two markers, reporting the final grading score of 3 either for CYP3A4 and for CYP3A7. Just three cases were negative at list for a CYP’s markers.
CYP3A4 was positive in all but two cases (Table 1), case N 7, belonging to group 1, with 20 weeks of PC age, and case N 19, belonging to group 2 with 28 weeks of PC age. Group 1 livers showed a diffuse distribution of CYP3A4 (score 3) in all but three cases: N 7, which was negative, N 3 with score 1 and N 6 with score 2. The intensity and the final grading score system were extremely variable ranging from 0 (N 7) to 1 (N 3 and N 6), to 2 in 5 (N 1, 2, 4, 5 and 9) and to 3 (N 8 and N 10) (Figures 1 and 2). In group 2 livers, the reactivity was diffuse (score 3) in all cases but three cases: N 19, which was negative, N 12 with score 2 and N 20 with score 1. The intensity and the final grading score system were extremely variable ranging from 0 (N 19) to 1 (N 20), 2 (N 12, 14, 15 and 18) and 3 (N 11, 13, 16 and 17). In group 3 livers, all cases showed immunostaining for CYP3A4. The reactivity was diffuse (score 3) in all but three cases: N 25 and N 28 with score 2 and N 27 with score 1. The intensity of the final score grading system was again variable ranging from 2 (N 22, 24, 27 and 28) to 3 (N 21, 23, 25, 29 and 30). The grading score resulted identical of the intensity score but in case N 27, at 35 weeks of PC age, in which because of the focal distribution (score 1) the final grading score was 1. CYP3A7 CYP3A7 was detected in all but one case (N 19) belonging to group 3, with 32 weeks of PC age, which was negative also for CYP3A4 (Table 2).
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Table 1. CYP3A4. N
Distribution
Intensity
Final grading
PC age in weeks
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
3 3 1 3 3 2 0 3 3 3 3 2 3 3 3 3 3 3 0 2 3 3 3 3 2 3 1 2 3 3
2 2 1 2 2 1 0 3 2 3 3 2 3 2 2 3 3 2 0 1 3 2 3 2 3 3 2 2 3 3
2 2 1 2 2 1 0 3 2 3 3 2 3 2 2 3 3 2 0 1 3 2 3 2 2 3 1 2 3 3
13 14 15 16 17 20 20 21 23 23 25 26 26 27 27 27 27 28 28 28 29 30 30 31 32 32 35 38 38 41
Figure 2. Immunostaining for CYP3A4 show a strong reactivity in the cytoplasm of periterminal zone hepatocytes; case N 10, 23 weeks, 40 .
Table 2. CYP3A7. N
Distribution
Intensity
Final grading
PC age in weeks
Figure 1. Immunostaining for CYP3A4 show a strong reactivity in the cytoplasm of periportal zone hepatocytes; case N 10, 23 weeks, 40 .
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
2 3 2 3 3 2 3 3 3 3 3 1 3 3 3 2 3 3 0 3 3 3 3 3 2 3 1 3 3 2
2 3 1 3 2 2 3 2 3 2 2 1 3 3 3 2 3 1 0 2 3 3 3 2 2 3 2 2 3 2
2 3 1 3 2 2 3 2 3 2 2 1 3 3 3 2 3 2 0 2 3 3 3 2 2 3 1 2 3 2
13 14 15 16 17 20 20 21 23 23 25 26 26 27 27 27 27 28 28 28 29 30 30 31 32 32 35 38 38 41
The group 1 livers showed a diffuse distribution (score 3) of CYP3A7 in all but three cases: N 1, N 3 and N 6, characterized by zonal distribution (score 2). None showed focal distribution (score 1). The intensity and the grading score were extremely variable ranging from 1 (N 3) to 2 (N 1, 5, 6, 8 and 10) and 3 (N 2, 4, 7 and 9). In group 2 livers, the reactivity was diffuse (score 3) in all but three cases: N 19, which was negative, N 12, which showed a focal (score 1) and case N 16 with zonal distribution (score 2). The intensity score was extremely variable ranging from 0 (N 19) to 1 (N 12 and 18),
2 (N 11, 16 and 20), 3 (N 13, 14, 15 and 17). The grading score resulted identical of the intensity score but in case N 18, in which because of the diffuse distribution (score 3), the final grading score was 2. In group 3 livers, seven cases (N 21, 22, 23, 24, 26, 28 and 29) showed a diffuse reactivity (score 3) (Figures 3 and 4), case N 25, at 32 weeks of PC age, and case 30, at 41 weeks of PC age, a zonal positivity (score 2), and case N 27, at 35 weeks of gestational age, score 1. The intensity score were again variable, ranging from 1 in case N 27, at 35 weeks of gestational age, to 2 in five (cases N 24, 25, 27, 28 and 30), to 3 in five cases (N 21, 22, 23, 26 and 29). The grading score
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Figure 3. Immunostaining for CYP3A7 show a strong reactivity in the cytoplasm of periportal zone hepatocytes; case N 21, 29 weeks, 40 .
Figure 5. Pie charts illustrating the percentage of the different final grading score resulted with CYP3A4 and CYP3A7 reactivity in the three liver groups.
Figure 4. Immunostaining for CYP3A7 show a strong reactivity in the cytoplasm of periterminal zone hepatocytes; case N 21, 29 weeks, 40 .
resulted identical of the intensity score but in case of N 27, at 35 weeks of PC age, in which because of the focal distribution (score 1) the final grading score was 1. When the percentage of the different immunoreactivities for CYP3A4 and CYP3A7 were compared in the different groups, significant differences in the degree of immunostaining were restricted to fetuses of group 1 (Figure 5).
Discussion The human CYP3A subfamily consists of four genes that encode for CYP3A4, CYP3A7, CYP3A5 and CYP3A43. CYP3A4 and CYP3A7 are the most abundant hepatic members of the P450 family and account for the majority of the oxidative metabolism of clinically relevant drugs in humans [15–19]. CYP3A7 is reported to be the dominant enzyme in fetal liver, with a subsequent age-related shift between CYP3A7 and CYP3A4 [20]. Our data on the immunohistochemical detection of CYP3A4 and CYP3A7 during development show, for the first time, that the relationship between these two CYP450 isoforms should be
considered much more complex than previously thought. First of all, according to our data, CYP3A4 expression is not restricted to the postnatal age, being detected in this study in more than 90% of fetal and neonatal livers at all gestational ages tested (Figure 5). Moreover, in a percentage of cases ranging from 20% in group 1 up to 50% of group 3 neonates, the expression of CYP3A4 was strong and diffuse (Figure 5). CYP3A4 was not restricted to the periportal liver zone (zone 1 according to Rappaport) (Figure 1), immunostaining for the enzyme being detected in all acinar zone, including the hepatocytes surrounding the central (terminal) vein (zone 3 of Rappaport) (Figure 2). When the grading of CYP3Aa was compared with that of CYP3A7 immunostaining, the degree of liver immunoreactivity for the two isoforms was identical in groups 2 and 3 subjects (Figure 5). Mild differences were detected in group 1 fetuses: in very preterm infants (below 24 weeks of PC age), CYP3A7 expression was stronger and more diffuse (Figure 5). In this group, 40% of subjects showed a grade 3 immunostaining for CYP3A7, compared with 20% showing the same degree of immunoreactivity for CYP3A4. Taken together, these data clearly show that the previously reported schemes based on restriction of CYP3A7 to the prenatal life, with a shift between CYP3A7 and CYP3A4 occurring immediately after birth [20] cannot be accepted in the human liver.
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DOI: 10.3109/14767058.2014.951625
The second relevant finding emerging from our study is the high interindividual variability observed among subjects at all gestational age. The final grade of immunostaining for CYP3A4 ranged from 0 (negative stain) up to 3 (strong and diffuse reactivity) in groups 1 and 2 (Table 1). In particular, livers from fetuses with similar gestational age (cases 7 and 8 in Table 1) frequently showed striking differences in CYP3A4 expression. The interindividual variability in CYP3A4 immunoreactivity was well evidenced in three newborn of 28 week of gestation (cases 18, 19 and 20 in Table 1): one did not express the enzyme (grade 0), another showed grade 1 and the other had a final grading of 2. A marked interindividual variability was also found when CYP3A4 and CYP3A7 immunoreactivity was compared in the same liver. In some subjects, CYP3A7 was the dominant isoform being strong and diffusely detected (grade 3), contrasting with the absence of the expression for CYP3A4 (case 7, Tables 1 and 2). In other subjects (cases 13, 17 and 29), both isoforms were highly expressed, immunostaining for CYP3A4 and CYP3A7 being detected in the cytoplasm of the majority of hepatocytes. In one case (N 19), both CYP3A4 and CYP3A7 were not detected in liver cells (Tables 1 and 2). The spectrum of the complex relationship between the two CYP450 isoforms was completed by few cases in which CYP3A4 expression, generally considered typical of postnatal life, was higher than CYP3A7 (cases 8, 12, 16 and 30 in Tables 1 and 2). These data clearly evidence that gestational age is not the most important covariable during fetal life regarding the hepatic expression of CYP3A4 and CYP3A7. High and low expression of both enzymes detected al all gestational age. On the contrary, our data suggest that other covariables contribute to a high phenotypic interindividual variability in CYP3A and CYP3A7 isoforms expression during intrauterine life. From a practical point of view, this variable CYP3A4 and CYP3A7 expression suggests the existence of a marked interindividual variability in drug metabolism during the intrauterine life and neonates. As a consequence, the need of an individualized tailored therapeutic approach in NICU centers appears mandatory.
Declaration of interest The authors have no declarations of interest to report.
References 1. Yang X, Zhang B, Molony C, et al. Systematic genetic and genomic analysis of cytochrome P450 enzyme activities in human liver. Genome Res 2010;20:1020–36.
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2. Wang D, Jiang Z, Shen Z, et al. Functional evaluation of genetic and environmental regulators of p450 mRNA levels. PLoS One 2011;6:e24900. 3. Nebert DW, Russell DW. Clinical importance of the cytochromes P450. Lancet. 2002;360:1155–62. 4. Gow PJ, Ghabrial H, Smallwood RA, et al. Neonatal hepatic drug elimination. Pharmacol Toxicol 2001;88:3–15. 5. Lamba JK, Lin YS, Schuetz EG, Thummel KE. Genetic contribution to variable human CYP3A-mediated metabolism. Adv Drug Deliv Rev 2002;54:1271–94. 6. Buratti S, Lavine JE. Drugs and the liver: advances in metabolism, toxicity, and therapeutics. Curr Opin Pediatr 2002;14:601–7. 7. Gellner K, Eiselt R, Hustert E, et al. Genomic organization of the human CYP3A locus: identification of a new, inducible CYP3A gene. Pharmacogenetics 2001;11:111–21. 8. Zanger UM, Klein K, Richter T, et al. Impact of genetic polymorphism in relation to other factors on expression and function of human drug-metabolizing p450s. Toxicol Mech Methods 2005;15:121–4. 9. Yang J, Xu J, Chen X, et al. Decrease of plasma platelet-activating factor acetylhydrolase activity in lipopolysaccharide induced mongolian gerbil sepsis model. PLoS One 2010;5:e9190. 10. Treluyer JM, Rey E, Sonnier M, et al. Evidence of impaired cisapride metabolism in neonates. Br J Clin Pharmacol 2001;52: 419–25. 11. Blake MJ, Castro L, Leeder JS, Kearns GL. Ontogeny of drug metabolizing enzymes in the neonate. Semin Fetal Neonatal Med 2005;10:123–38. 12. Daly AK. Significance of the minor cytochrome P450 3A isoforms. Clin Pharmacokinet 2006;45:13–31. 13. de Wildt SN, Kearns GL, Leeder JS, van den Anker JN. Cytochrome P450 3A: ontogeny and drug disposition. Clin Pharmacokinet 1999;37:485–505. 14. Hakkola J, Raunio H, Purkunen R, et al. Cytochrome P450 3A expression in the human fetal liver: evidence that CYP3A5 is expressed in only a limited number of fetal livers. Biol Neonate 2001;80:193–201. 15. Johnson TN, Rostami-Hodjegan A, Tucker GT. Prediction of the clearance of eleven drugs and associated variability in neonates, infants and children. Clin Pharmacokinet 2006;45: 931–56. 16. Bjorkman S. Prediction of cytochrome p450-mediated hepatic drug clearance in neonates, infants and children: how accurate are available scaling methods? Clin Pharmacokinet 2006;45: 1–11. 17. Kearns GL, Robinson PK, Wilson JT, et al. Cisapride disposition in neonates and infants: in vivo reflection of cytochrome P450 3A4 ontogeny. Clin Pharmacol Ther 2003;74:312–25. 18. de Wildt SN, Kearns GL, Hop WC, et al. Pharmacokinetics and metabolism of oral midazolam in preterm infants. Br J Clin Pharmacol 2002;53:390–2. 19. Smits A, Kulo A, de Hoon JN, Allegaert K. Pharmacokinetics of drugs in neonates: pattern recognition beyond compound specific observations. Curr Pharmaceut Design 2012;18:3119–46. 20. Lacroix D, Sonnier M, Moncion A, et al. Expression of CYP3A in the human liver – evidence that the shift between CYP3A7 and CYP3A4 occurs immediately after birth. Eur J Biochem 1997;247: 625–34.
ISSN: 1476-7058 (Print) 1476-4954 (Online) Journal homepage: http://www.tandfonline.com/loi/ijmf20
Overlapping between CYP3A4 and CYP3A7 expression in the fetal human liver during development Daniela Fanni, Vassillios Fanos, Rossano Ambu, Federica Lai, Clara Gerosa, Pietro Pampaloni, Peter Van Eyken, Giancarlo Senes, Massimo Castagnola & Gavino Faa To cite this article: Daniela Fanni, Vassillios Fanos, Rossano Ambu, Federica Lai, Clara Gerosa, Pietro Pampaloni, Peter Van Eyken, Giancarlo Senes, Massimo Castagnola & Gavino Faa (2015) Overlapping between CYP3A4 and CYP3A7 expression in the fetal human liver during development, The Journal of Maternal-Fetal & Neonatal Medicine, 28:11, 1291-1295, DOI: 10.3109/14767058.2014.951625 To link to this article: http://dx.doi.org/10.3109/14767058.2014.951625
Published online: 10 Sep 2014.
Submit your article to this journal
Article views: 66
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ijmf20 Download by: [University of Florida]
Date: 05 November 2015, At: 18:14
http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, 2015; 28(11): 1291–1295 ! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2014.951625
ORIGINAL ARTICLE
Overlapping between CYP3A4 and CYP3A7 expression in the fetal human liver during development Daniela Fanni1, Vassillios Fanos2, Rossano Ambu1, Federica Lai1, Clara Gerosa1, Pietro Pampaloni1, Peter Van Eyken3, Giancarlo Senes1, Massimo Castagnola4, and Gavino Faa1 1
Department of Surgical Sciences, Divison of Pathology, University Hospital San Giovanni di Dio, University of Cagliari, Cagliari, Italy, Department of Pediatrics and Clinical Medicine, NICU, Puericulture Institute and Neonatal Section, University of Cagliari, Cagliari, Italy, 3 Pathology Department, ZOL (Ziekenhuis Oost-Limburg), Genk, Belgium, and 4Faculty of Medicine, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy
Downloaded by [University of Florida] at 18:14 05 November 2015
2
Abstract
Keywords
Objective: The cytochrome P450 (CYP450) superfamily is implicated in important life processes, including metabolism of many molecules. CYP3A account for the largest portion of CYP450 proteins in human, including CYP3A4, CYP3A5 and CYP3A7. The purpose of this study was to investigate the immunohistochemical expression of CYP3A4 and CYP3A7 in human liver at different post-conceptional (PC) ages. Methods: Human liver samples from 30 fetuses and newborns were, clustered according with the PC age, routinely processed for immunohistochemical analysis of CYP3A4 and CYP3A7. Results: CYP3A4 was positive in all but two cases, CYP3A7 was positive in all but one case, which was negative also for CYP3A4. Conclusions: Our data on immunohistochemical detection of CYP3A4 and CYP3A7 during development show that CYP3A4 expression is not restricted to the post-natal age, being the immunostaining for both CYP3A4 and CYP3A7 identical after 25 weeks of PC age, thus the relationship between these CYP450 isoforms should be considered much more complex than previous thought. A high interindividual variability was observed among subjects at all gestational age. The variable CYP3A expression suggests the existence of a marked interindividual variability in drug metabolism during the intrauterine life and in perinatal period.
CYP3A4, CYP3A7, cytochrome P450, fetus, immunohistochemistry, liver, newborns
Introduction The cytochrome P450 (CYP450) superfamily is formed by 57 genes and 58 pseudogenes, encoding for 57 functional monooxygenases critical for human metabolism [1,2]. The myriad of enzymes encoded by the 57 CYP genes are implicated in important life processes, including metabolism of drugs, foreign chemicals, arachidonic acid, cholesterol, biosynthesis of bile acids, steroids, vitamin D3 and hydroxylation of retinoic acid [3]. The expression of CYP450 enzymes in the human body is characterized by a marked substrate and tissue specificity. The most important isoenzymes for drug metabolism are localized in the liver; seven isoenzymes are (CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) being responsible for the metabolism of more than 95% of drugs in adulthood [1,4].
Address for correspondence: Dr Daniela Fanni, MD, PhD, Pathologist, Department of Pathology, University of Cagliari, ‘‘S. Giovanni di Dio’’ Hospital, via Ospedale 60, 09124 Cagliari, Italy. Tel: 0390706092372. Fax: 0396092115. E-mail: [email protected]
History Received 14 April 2014 Accepted 17 May 2014 Published online 10 September 2014
CYP450 isoforms involved in drug metabolism have been also reported in kidney, lung, brain, breast, prostate and in the small intestine [5]. The protein products of human CYP4503A gene family (CYP3A) account for the largest portion of CYP450 proteins in human liver, including CYP3A4 and CYP3A7. CYP3A4 has been reported to be undetectable during gestation and at birth, dramatically increasing after one postnatal week and reaching adult levels around one month after birth. At least 50% of currently used drugs in NICU centers are substrates of CYP3A4 including antibiotics, antivirals, antifungals, immunomodulators, benzodiazepines, proton pump inhibitors, steroid hormones and acetaminophen [6–9]. It has been claimed that the low content of CYP3A4 in the neonate at birth might be the main responsible for the impaired metabolism of cisapride in the perinatal period, resulting in accumulation of the drug and cardiac toxicity [10]. CYP3A7 has been reported to be predominantly expressed in the fetal liver, the CYP3A7 gene being silenced in the perinatal period, normally within the first postnatal week.
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J Matern Fetal Neonatal Med, 2015; 28(11): 1291–1295
Catalytic activity is detectable as early as 50–60 d of gestation in embryonic liver, where it catalyzes 16-alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, an intermediate metabolite in the estriol biosynthesis [11]. CYP3A7 expression is characterized by interindividual variability; in some adult livers, the enzymatic activity does not end at birth, but it continues in adulthood, being detectable even extrahepatically. The function of this CYP enzyme is mainly associated to hydroxylation of retinoic acid and to 16 alpha-hydroxylation of steroids, playing a key role in normal development, as well as in carcinogenesis [12]. CYP3A4 represents 30–40% of the total CYP content in human adult liver and in small intestine mucosa cells. CYP3A7 is the major isoform detected in embryonic, fetal and newborn liver, being also detectable at low levels in adult liver cells [13]. A marked interindividual variability has been reported regarding CYP3A isoforms’ expression and activity: in particular, the variable expression of CYP3A7 in fetal liver, starting from the ninth week of gestation, suggests that significant interindividual differences in drug metabolism may already exist at the prenatal stage, these differences contributing to individual pharmacological responses in the intrauterine life [14]. Since few data are available regarding the expression of the CYP enzymes in the newborn liver, the purpose of this study was to investigate the immunohistochemical expression of CYP3A4 and CYP3A7 in human liver during gestation and in newborns at different post-conceptional (PC) ages.
national standards of the responsible committee on human experimentation. The histological examination was performed by three observers (D. F., F. L. and G. F.), who independently assigned two different scores. The first one regarded the distribution: score 1 for focal distribution, score 2 for zonal distribution and score 3 for diffuse distribution. The second score concerned the intensity of immunostaining, that was scored as weak (score 1), medium (score 2) and strong (score 3). In both score systems, score 0 was assigned for negativity. The sum between the distribution score and the intensity score gave rise to the following final grading system: grade 0 for negative stain, grade 1 for an amount from 1 to 3, grade 2 from 4 to 5 and grade 3 more than 5. All cases were then discussed at multisession microscope until all three observers approved the given scores. The percentages of the different immunoreactivities for CYP3A4 and CYP3A7 were calculated and compared in the different groups in order to evaluate the presence of significant differences in the degree of immunostaining.
Methods
CYP3A4
The expression of CYP3A4 and CYP3A7 was evaluated in liver samples from 30 human fetuses and newborns with different PC age, that is, the sum between gestational age and the post-natal age. Patients were clustered into three groups of 10, according with the PC age: group 1 included 10 cases with PC age lower than 24 weeks, group 2 incorporated 10 cases from 25 to 28 weeks of PC age and group 3 integrated cases from 29 to 41 weeks of PC age. Liver samples were fixed in 10% formalin, dehydrated and embedded in paraffin. Tissue sections were then dewaxed, rehydrated through graded alcohols and pre-treated for 10 min with the heat-induced epitope retrieval in buffer pH 6.00 (EnVisionÔ FLEX Target Retrieval Solution Low pH – Dako Denmark A/S, Glostrup, Denmark, code: K8005), for immunohistochemical analysis of CYP3A4 and in buffer pH 9.00 (EnVisionÔ FLEX Target Retrieval Solution High pH – Dako Denmark A/S, code: K8004) and for immunohistochemical analysis of CYP3A7. Slides were then incubated for 20 min at room temperature with anti-CYP450 3A4 rabbit polyclonal antibody at 1:1000 dilution and with anti-CYP450 3A7 mouse monoclonal antibody clone F19 P2 H2 at 1:50 dilution (all produced by Abcam, Cambridge, UK, catalog numbers: ab3572, ab108624 and ab55840, respectively). Staining procedures were performed by EnvisionÔ FLEX+ (Dako Denmark A/S, code: K8002) Detection System and AutostainerLink 48 instrument following dealer’s instructions. Negative controls samples were incubated without mouse anti-CYP3A4 and CYP3A7 polyclonal antibody. All procedures performed were in accordance with the ethical
Results Immunoreactivity for CYP3A4 and CYP3A7 was detected in the majority of fetal livers. Four cases, two belonging to group 2 and two to group 3, showed a diffuse and strong positivity for the two markers, reporting the final grading score of 3 either for CYP3A4 and for CYP3A7. Just three cases were negative at list for a CYP’s markers.
CYP3A4 was positive in all but two cases (Table 1), case N 7, belonging to group 1, with 20 weeks of PC age, and case N 19, belonging to group 2 with 28 weeks of PC age. Group 1 livers showed a diffuse distribution of CYP3A4 (score 3) in all but three cases: N 7, which was negative, N 3 with score 1 and N 6 with score 2. The intensity and the final grading score system were extremely variable ranging from 0 (N 7) to 1 (N 3 and N 6), to 2 in 5 (N 1, 2, 4, 5 and 9) and to 3 (N 8 and N 10) (Figures 1 and 2). In group 2 livers, the reactivity was diffuse (score 3) in all cases but three cases: N 19, which was negative, N 12 with score 2 and N 20 with score 1. The intensity and the final grading score system were extremely variable ranging from 0 (N 19) to 1 (N 20), 2 (N 12, 14, 15 and 18) and 3 (N 11, 13, 16 and 17). In group 3 livers, all cases showed immunostaining for CYP3A4. The reactivity was diffuse (score 3) in all but three cases: N 25 and N 28 with score 2 and N 27 with score 1. The intensity of the final score grading system was again variable ranging from 2 (N 22, 24, 27 and 28) to 3 (N 21, 23, 25, 29 and 30). The grading score resulted identical of the intensity score but in case N 27, at 35 weeks of PC age, in which because of the focal distribution (score 1) the final grading score was 1. CYP3A7 CYP3A7 was detected in all but one case (N 19) belonging to group 3, with 32 weeks of PC age, which was negative also for CYP3A4 (Table 2).
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Table 1. CYP3A4. N
Distribution
Intensity
Final grading
PC age in weeks
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
3 3 1 3 3 2 0 3 3 3 3 2 3 3 3 3 3 3 0 2 3 3 3 3 2 3 1 2 3 3
2 2 1 2 2 1 0 3 2 3 3 2 3 2 2 3 3 2 0 1 3 2 3 2 3 3 2 2 3 3
2 2 1 2 2 1 0 3 2 3 3 2 3 2 2 3 3 2 0 1 3 2 3 2 2 3 1 2 3 3
13 14 15 16 17 20 20 21 23 23 25 26 26 27 27 27 27 28 28 28 29 30 30 31 32 32 35 38 38 41
Figure 2. Immunostaining for CYP3A4 show a strong reactivity in the cytoplasm of periterminal zone hepatocytes; case N 10, 23 weeks, 40 .
Table 2. CYP3A7. N
Distribution
Intensity
Final grading
PC age in weeks
Figure 1. Immunostaining for CYP3A4 show a strong reactivity in the cytoplasm of periportal zone hepatocytes; case N 10, 23 weeks, 40 .
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
2 3 2 3 3 2 3 3 3 3 3 1 3 3 3 2 3 3 0 3 3 3 3 3 2 3 1 3 3 2
2 3 1 3 2 2 3 2 3 2 2 1 3 3 3 2 3 1 0 2 3 3 3 2 2 3 2 2 3 2
2 3 1 3 2 2 3 2 3 2 2 1 3 3 3 2 3 2 0 2 3 3 3 2 2 3 1 2 3 2
13 14 15 16 17 20 20 21 23 23 25 26 26 27 27 27 27 28 28 28 29 30 30 31 32 32 35 38 38 41
The group 1 livers showed a diffuse distribution (score 3) of CYP3A7 in all but three cases: N 1, N 3 and N 6, characterized by zonal distribution (score 2). None showed focal distribution (score 1). The intensity and the grading score were extremely variable ranging from 1 (N 3) to 2 (N 1, 5, 6, 8 and 10) and 3 (N 2, 4, 7 and 9). In group 2 livers, the reactivity was diffuse (score 3) in all but three cases: N 19, which was negative, N 12, which showed a focal (score 1) and case N 16 with zonal distribution (score 2). The intensity score was extremely variable ranging from 0 (N 19) to 1 (N 12 and 18),
2 (N 11, 16 and 20), 3 (N 13, 14, 15 and 17). The grading score resulted identical of the intensity score but in case N 18, in which because of the diffuse distribution (score 3), the final grading score was 2. In group 3 livers, seven cases (N 21, 22, 23, 24, 26, 28 and 29) showed a diffuse reactivity (score 3) (Figures 3 and 4), case N 25, at 32 weeks of PC age, and case 30, at 41 weeks of PC age, a zonal positivity (score 2), and case N 27, at 35 weeks of gestational age, score 1. The intensity score were again variable, ranging from 1 in case N 27, at 35 weeks of gestational age, to 2 in five (cases N 24, 25, 27, 28 and 30), to 3 in five cases (N 21, 22, 23, 26 and 29). The grading score
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Figure 3. Immunostaining for CYP3A7 show a strong reactivity in the cytoplasm of periportal zone hepatocytes; case N 21, 29 weeks, 40 .
Figure 5. Pie charts illustrating the percentage of the different final grading score resulted with CYP3A4 and CYP3A7 reactivity in the three liver groups.
Figure 4. Immunostaining for CYP3A7 show a strong reactivity in the cytoplasm of periterminal zone hepatocytes; case N 21, 29 weeks, 40 .
resulted identical of the intensity score but in case of N 27, at 35 weeks of PC age, in which because of the focal distribution (score 1) the final grading score was 1. When the percentage of the different immunoreactivities for CYP3A4 and CYP3A7 were compared in the different groups, significant differences in the degree of immunostaining were restricted to fetuses of group 1 (Figure 5).
Discussion The human CYP3A subfamily consists of four genes that encode for CYP3A4, CYP3A7, CYP3A5 and CYP3A43. CYP3A4 and CYP3A7 are the most abundant hepatic members of the P450 family and account for the majority of the oxidative metabolism of clinically relevant drugs in humans [15–19]. CYP3A7 is reported to be the dominant enzyme in fetal liver, with a subsequent age-related shift between CYP3A7 and CYP3A4 [20]. Our data on the immunohistochemical detection of CYP3A4 and CYP3A7 during development show, for the first time, that the relationship between these two CYP450 isoforms should be
considered much more complex than previously thought. First of all, according to our data, CYP3A4 expression is not restricted to the postnatal age, being detected in this study in more than 90% of fetal and neonatal livers at all gestational ages tested (Figure 5). Moreover, in a percentage of cases ranging from 20% in group 1 up to 50% of group 3 neonates, the expression of CYP3A4 was strong and diffuse (Figure 5). CYP3A4 was not restricted to the periportal liver zone (zone 1 according to Rappaport) (Figure 1), immunostaining for the enzyme being detected in all acinar zone, including the hepatocytes surrounding the central (terminal) vein (zone 3 of Rappaport) (Figure 2). When the grading of CYP3Aa was compared with that of CYP3A7 immunostaining, the degree of liver immunoreactivity for the two isoforms was identical in groups 2 and 3 subjects (Figure 5). Mild differences were detected in group 1 fetuses: in very preterm infants (below 24 weeks of PC age), CYP3A7 expression was stronger and more diffuse (Figure 5). In this group, 40% of subjects showed a grade 3 immunostaining for CYP3A7, compared with 20% showing the same degree of immunoreactivity for CYP3A4. Taken together, these data clearly show that the previously reported schemes based on restriction of CYP3A7 to the prenatal life, with a shift between CYP3A7 and CYP3A4 occurring immediately after birth [20] cannot be accepted in the human liver.
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DOI: 10.3109/14767058.2014.951625
The second relevant finding emerging from our study is the high interindividual variability observed among subjects at all gestational age. The final grade of immunostaining for CYP3A4 ranged from 0 (negative stain) up to 3 (strong and diffuse reactivity) in groups 1 and 2 (Table 1). In particular, livers from fetuses with similar gestational age (cases 7 and 8 in Table 1) frequently showed striking differences in CYP3A4 expression. The interindividual variability in CYP3A4 immunoreactivity was well evidenced in three newborn of 28 week of gestation (cases 18, 19 and 20 in Table 1): one did not express the enzyme (grade 0), another showed grade 1 and the other had a final grading of 2. A marked interindividual variability was also found when CYP3A4 and CYP3A7 immunoreactivity was compared in the same liver. In some subjects, CYP3A7 was the dominant isoform being strong and diffusely detected (grade 3), contrasting with the absence of the expression for CYP3A4 (case 7, Tables 1 and 2). In other subjects (cases 13, 17 and 29), both isoforms were highly expressed, immunostaining for CYP3A4 and CYP3A7 being detected in the cytoplasm of the majority of hepatocytes. In one case (N 19), both CYP3A4 and CYP3A7 were not detected in liver cells (Tables 1 and 2). The spectrum of the complex relationship between the two CYP450 isoforms was completed by few cases in which CYP3A4 expression, generally considered typical of postnatal life, was higher than CYP3A7 (cases 8, 12, 16 and 30 in Tables 1 and 2). These data clearly evidence that gestational age is not the most important covariable during fetal life regarding the hepatic expression of CYP3A4 and CYP3A7. High and low expression of both enzymes detected al all gestational age. On the contrary, our data suggest that other covariables contribute to a high phenotypic interindividual variability in CYP3A and CYP3A7 isoforms expression during intrauterine life. From a practical point of view, this variable CYP3A4 and CYP3A7 expression suggests the existence of a marked interindividual variability in drug metabolism during the intrauterine life and neonates. As a consequence, the need of an individualized tailored therapeutic approach in NICU centers appears mandatory.
Declaration of interest The authors have no declarations of interest to report.
References 1. Yang X, Zhang B, Molony C, et al. Systematic genetic and genomic analysis of cytochrome P450 enzyme activities in human liver. Genome Res 2010;20:1020–36.
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2. Wang D, Jiang Z, Shen Z, et al. Functional evaluation of genetic and environmental regulators of p450 mRNA levels. PLoS One 2011;6:e24900. 3. Nebert DW, Russell DW. Clinical importance of the cytochromes P450. Lancet. 2002;360:1155–62. 4. Gow PJ, Ghabrial H, Smallwood RA, et al. Neonatal hepatic drug elimination. Pharmacol Toxicol 2001;88:3–15. 5. Lamba JK, Lin YS, Schuetz EG, Thummel KE. Genetic contribution to variable human CYP3A-mediated metabolism. Adv Drug Deliv Rev 2002;54:1271–94. 6. Buratti S, Lavine JE. Drugs and the liver: advances in metabolism, toxicity, and therapeutics. Curr Opin Pediatr 2002;14:601–7. 7. Gellner K, Eiselt R, Hustert E, et al. Genomic organization of the human CYP3A locus: identification of a new, inducible CYP3A gene. Pharmacogenetics 2001;11:111–21. 8. Zanger UM, Klein K, Richter T, et al. Impact of genetic polymorphism in relation to other factors on expression and function of human drug-metabolizing p450s. Toxicol Mech Methods 2005;15:121–4. 9. Yang J, Xu J, Chen X, et al. Decrease of plasma platelet-activating factor acetylhydrolase activity in lipopolysaccharide induced mongolian gerbil sepsis model. PLoS One 2010;5:e9190. 10. Treluyer JM, Rey E, Sonnier M, et al. Evidence of impaired cisapride metabolism in neonates. Br J Clin Pharmacol 2001;52: 419–25. 11. Blake MJ, Castro L, Leeder JS, Kearns GL. Ontogeny of drug metabolizing enzymes in the neonate. Semin Fetal Neonatal Med 2005;10:123–38. 12. Daly AK. Significance of the minor cytochrome P450 3A isoforms. Clin Pharmacokinet 2006;45:13–31. 13. de Wildt SN, Kearns GL, Leeder JS, van den Anker JN. Cytochrome P450 3A: ontogeny and drug disposition. Clin Pharmacokinet 1999;37:485–505. 14. Hakkola J, Raunio H, Purkunen R, et al. Cytochrome P450 3A expression in the human fetal liver: evidence that CYP3A5 is expressed in only a limited number of fetal livers. Biol Neonate 2001;80:193–201. 15. Johnson TN, Rostami-Hodjegan A, Tucker GT. Prediction of the clearance of eleven drugs and associated variability in neonates, infants and children. Clin Pharmacokinet 2006;45: 931–56. 16. Bjorkman S. Prediction of cytochrome p450-mediated hepatic drug clearance in neonates, infants and children: how accurate are available scaling methods? Clin Pharmacokinet 2006;45: 1–11. 17. Kearns GL, Robinson PK, Wilson JT, et al. Cisapride disposition in neonates and infants: in vivo reflection of cytochrome P450 3A4 ontogeny. Clin Pharmacol Ther 2003;74:312–25. 18. de Wildt SN, Kearns GL, Hop WC, et al. Pharmacokinetics and metabolism of oral midazolam in preterm infants. Br J Clin Pharmacol 2002;53:390–2. 19. Smits A, Kulo A, de Hoon JN, Allegaert K. Pharmacokinetics of drugs in neonates: pattern recognition beyond compound specific observations. Curr Pharmaceut Design 2012;18:3119–46. 20. Lacroix D, Sonnier M, Moncion A, et al. Expression of CYP3A in the human liver – evidence that the shift between CYP3A7 and CYP3A4 occurs immediately after birth. Eur J Biochem 1997;247: 625–34.
