ABCG2/BCRP decreases the transfer of a food-born chemical carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in perfused term human placenta

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Abstract

We have studied the role of ATP binding cassette (ABC) transporters in fetal exposure to carcinogens using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) a known substrate for ABC transporters as a model compound. In perfusion of human term placenta, transfer of 14C-PhIP (2 μM) through the placenta resulted in fetal-to-maternal concentration ratio (FM ratio) of 0.72 ± 0.09 at 6 h. The specific ABCG2 inhibitor KO143 increased the transfer of 14C-PhIP from maternal to fetal circulation (FM ratio 0.90 ± 0.08 at 6 h, p < 0.05) while the ABCC1/ABCC2 inhibitor probenecid had no effect (FM ratio at 6 h 0.75 ± 0.10, p = 0.84). There was a negative correlation between the expression of ABCG2 protein in perfused tissue and the FM ratio of 14C-PhIP (R =  0.81, p < 0.01) at the end of the perfusion. The expression of ABCC2 protein did not correlate with FM ratio of PhIP (R: − 0.11, p = 0.76). In addition, PhIP induced the expression of ABC transporters in BeWo cells at mRNA level. In conclusion, our data indicates that ABCG2 decreases placental transfer of 14C-PhIP in perfused human placenta. Also, PhIP may modify ABC transporter expression in choriocarinoma cells.

Introduction

Exposure to a large variety of chemicals in highly variable doses cannot be avoided in an industrialized environment. There are currently increasing concern about fetal exposure to such pollutants and other foreign chemicals affecting health later in life. This idea, called ‘fetal origins of disease’, is gaining ground as more studies show how early exposure can alter developmental patterns and lead to diseases, sometimes decades later in life (for reviews: see e.g. Hilakivi-Clarke and de Assis, 2006, Gluckman et al., 2005, Ozanne and Constancia, 2007). The most well-known example of transplacental carcinogen is the synthetic estrogen, diethylstilbestrol (DES), but animal studies have also identified many other transplacental carcinogens potentially harmful also in humans (for a review: see e.g. Miller, 2004).

PhIP (2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) is the most abundant heterocyclic amine in cooked meat. It is considered to be highly relevant considering the total mutagenicity of food because of its abundance although it is not the most mutagenic compound in meat (Felton et al., 1986). Epidemiological studies have shown that exposure to PhIP is associated with an increased risk of breast cancer in women (Zheng et al., 1998, Sinha et al., 2000). Also, transplacental exposure and exposure via breast milk have been associated with mammary carcinomas in Sprague–Dawley rats (Hasegawa et al., 1995). However, there is no data on transplacental exposure or effects in humans. Similarly to many other chemical carcinogens PhIP has to be metabolically activated oxidization to N-hydroxylamine derivatives being the first reaction (Malfatti et al., 1999, Schut and Snyderwine, 1999, Felton et al., 2004, Sugimura et al., 2004). PhIP is mainly metabolized to genotoxic N-hydroxy derivatives in humans by hepatic CYP1A2 with high specificity and catalytic activity. CYP1A1 and CYP1B1 have also been reported to take part in the metabolism of PhIP (Crofts et al., 1998).

Most of the foreign chemicals that have been studied so far cross the placenta at least to some extent by simple passive diffusion (Audus, 1999). However, placenta is not merely a passive barrier but is known to express both xenobiotic metabolizing enzymes and active transporter proteins, which may modify the transfer processes. Transporter proteins are found both in the brush-border (apical) membrane facing maternal blood space and basolateral (basal) membrane facing fetal capillaries where they transfer compounds in and out of syncytiotrophoblasts (Syme et al., 2004, Evseenko et al., 2006, Myllynen et al., 2007a). Some transporters are also expressed in fetal capillary endothelium. The selection of transporters found in apical and basal membranes of trophoblasts differs leading to a polarized transport in the trophoblast layer (Syme et al., 2004, Evseenko et al., 2006, Myllynen et al., 2007a). Depending on their function and localization placental transporters may either increase or decrease placental transfer of foreign chemicals (Smit et al., 1999, Lankas et al., 1998). ABCB1 (also known as MDR1, multidrug resistance protein 1, p-glycoprotein), ABCC2 (also known as MRP2, multiresistance associated protein 2) and ABCG2 (also known as breast cancer resistance protein, BCRP) are efflux transporters that are localized in the brush-border membrane facing maternal circulation of the trophoblast layer and therefore they may prevent the entry of foreign compounds into the fetal compartment (Smit et al., 1999, Lankas et al., 1998, Jonker et al., 2000).

It has been established that there are interindividual variation in the toxicokinetics of chemical carcinogens. Many of the known differences are based on variation in metabolism due to genetic enzyme polymorphisms or changes in enzyme activities caused by exposure to pharmaceutical drugs or other foreign chemicals (for a review: see e.g. Autrup, 2000). Another group of important players is transporter proteins which have also been shown to contribute significantly to interindividual differences in pharmaco/toxicokinetics (Ishikawa et al., 2004, Shitara et al., 2006). Similarly to metabolizing enzymes, transporter genes from several protein families are polymorphic leading to variation in expression (Tanabe et al., 2001, Hitzl et al., 2004, Kobayashi et al., 2005). Also, the expression of transporter proteins can be up- or down-regulated in a similar manner as in the expression of xenobiotic metabolizing enzymes (Klaassen and Slitt, 2005). Therefore, at least in theory, environmental compounds could affect placental functions by altering the functions of placental transporter proteins.

In this study we tested the hypothesis that ABCG2 and/or ABCC2 can modify the fetal exposure to harmful foreign compounds including chemical carcinogens. PhIP was selected as a model compound because it is a known substrate for the human ABCG2 transporter (Pavek et al., 2005). PhIP and its metabolites are also substrates for Abcc2/Mrp2 in rodents (Dietrich et al., 2001a, Dietrich et al., 2001b), but data from studies of the human ABCC2 are contradictory. Based on studies in Caco-2 colon carcinoma cells (Walle and Walle, 1999), PhIP is a substrate for human ABCC2. On the other hand, van Herwaarden et al. (2003) concluded based on studies in human MRP2-transduced MDCK-II cells that PhIP is not a substrate for ABCC2/MRP2.

Section snippets

Chemicals

PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) and 14C-PhIP were purchased from Toronto Research Chemicals (specific activity 368.9 MBq/mmol). The ABCC1/ABCC2 inhibitor probenecid was purchased from Sigma (St Louis, Mo, USA). The ABCG2 inhibitor KO143 (Allen et al., 2002) was a gift from Dr. Schinkel (The Netherlands Cancer Institute, Amsterdam, The Netherlands) and the p-glycoprotein/ABCG2 inhibitor GF120918 from GlaxoSmithKline (Brentford, UK).

Placental perfusions

The official Ethics Committee of The

Viability of the perfused placentas and placental transfer of reference compound antipyrine

Altogether 16 instances of perfusion fulfilled the criteria of a successful perfusion: 1) a leaking of less than 2 mL/h from the fetal-to-maternal circulation, suggesting an intact fetomaternal barrier (Pienimäki et al., 1995), 2) pH at nearly physiological levels and 3) tissue consumed glucose (Table 2). Placental transfer of the reference compound antipyrine was used to confirm the overlap of maternal and fetal circulations. Antipyrine crossed the placenta in a manner and amount comparable to

Discussion

Previously, it has been shown that pharmacological inhibition of ABCB1 or ABCG2 may increase fetal exposure to pharmaceutical drugs (see e.g. Smit et al., 1999, Lankas et al., 1998, Jonker et al., 2000, Mölsä et al., 2005, Zhang et al., 2007). However, the relevance of transporter proteins in developmental toxicity of chemical carcinogens has been an unexplored area. This is the first study indicating that the placental transporter protein can modify fetal exposure to a food-born chemical

Acknowledgments

We are indebted to the nursing personnel in the Delivery Room of the Department of Obstetrics and Gynecology University Hospital of Oulu, Finland for their co-operation. We thank Mr. Esa Kerttula and Ms. Ritva Tauriainen for their practical help. We are grateful to Dr. Risto Bloigu, the Biostatistician of Medical Faculty, University of Oulu, for his help in statistical analysis. We also thank GlaxoSmithKline for providing GF120918 and Dr. Alfred Schinkel for KO143. The project was financially

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