Placental transfer and DNA binding of benzo(a)pyrene in human placental perfusion

Toxicology Letters 197 (2010) 75–81

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Placental transfer and DNA binding of benzo(a)pyrene in human placental perfusion Vesa Karttunen a , Päivi Myllynen b , Gabriela Prochazka c , Olavi Pelkonen b , Dan Segerbäck c , Kirsi Vähäkangas a,∗ a b c

Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland Department of Pharmacology and Toxicology, University of Oulu, Finland Department of Biosciences and Nutrition, Karolinska Institute, Huddinge, Sweden

a r t i c l e

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Article history: Received 11 January 2010 Received in revised form 28 April 2010 Accepted 30 April 2010 Available online 11 May 2010 Keywords: ABCB1/P-glycoprotein Placental xenobiotic metabolism BPDE–DNA adducts EROD activity BeWo cells

a b s t r a c t Benzo(a)pyrene (BP) is the best studied polycyclic aromatic hydrocarbon, classified as carcinogenic to humans. The carcinogenic metabolite, benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), binds covalently to DNA. The key enzyme in this metabolic reaction is CYP1A1, which has also been found in placenta and human trophoblastic cells. By using human placental perfusion we confirmed that BP added to the maternal circulation in concentrations of 0.1 and 1 ␮M reaches fetal compartment but somewhat slower than the freely diffusible reference substance antipyrine. A well-known P-glycoprotein (ABCB1/P-gp) antagonist verapamil did not affect the transfer more than it did in the case of antipyrine, indicating that ABCB1/P-gp does not have a role in BP transfer. In one of the two placentas perfused for 6 h with the higher concentration of BP (1 ␮M) BPDE specific DNA adducts were found in placental tissue after the perfusion, but not before. The ability of human trophoblastic cells to activate BP to BPDE–DNA adducts was confirmed in human trophoblastic BeWo cells. This study shows that maternal exposure to BP leads to the exposure of the fetus to BP and/or its metabolites and that placenta itself can activate BP to DNA adducts. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Pregnant mothers are inevitably exposed to chemical carcinogens such as polycyclic aromatic hydrocarbons (PAH) in food and urban air (Frey-Hattemer and Travis, 1991). The best known PAH is benzo(a)pyrene (BP), which has been classified as carcinogenic to humans by the International Agency for Research on Cancer (Straif et al., 2005). The exposure levels vary due to lifestyle factors such as diet and smoking. The most common sources of BP, for those not occupationally exposed (see e.g. Pyy et al., 1997), are cigarette smoke, flamed, grilled and smoked food, air pollution and exhaust (Dybing et al., 2008; Miller and Ramos, 2001). BP requires metabolic activation to benzo(a)pyrene7,8-dihydrodiol-9,10-epoxide (BPDE) which can bind covalently to DNA and is putatively the most carcinogenic metabolite (for reviews see Pelkonen and Nebert, 1982; Baird et al., 2005). Toxicokinetics of chemical carcinogens including BP varies from one person to another (Wogan et al., 2004). Such interindividual dif-

∗ Corresponding author at: Faculty of Health Sciences, School of Pharmacy/Toxicology, University of Eastern Finland, PO Box 1627, FI-70211 Kuopio, Finland. Tel.: +358 40 745 5254; fax: +358 17 16 2424. E-mail address: kirsi.vahakangas@uef.fi (K. Vähäkangas). 0378-4274/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2010.04.028

ferences are based, at least partly, on genetic polymorphisms or changes in metabolic enzymes due to exogenous reasons and transporter proteins (Mao, 2008). Important properties of drugs and other chemicals that determine placental transfer by passive diffusion include molecular weight, pKa , lipid solubility and protein binding (for a review, see e.g. Pacifici and Nottoli, 1995). It was earlier widely believed that all chemical compounds cross the placenta only by passive diffusion. However, lately many placental transporter proteins have been identified (Ganapathy and Prasad, 2005; Syme et al., 2004; Vähäkangas and Myllynen, 2009). The efflux transporters P-glycoprotein (ABCB1/P-gp) and breast cancer resistance protein (ABCG2/BCRP), localize in the apical surface of syncytiotrophoblast, and may thus prevent the entry of foreign compounds into the fetoplacental unit (Jonker et al., 2000; Mao, 2008; Smit et al., 1999). In animal studies, the importance of ABCB1/P-gp for avermectin teratogenicity has been shown in ABCB1/P-gp deficient mice (Lankas et al., 1998). Another set of proteins possibly affecting the kinetics of xenobiotics are the drug metabolizing enzymes. Such enzymes are also expressed in the placenta although the selection and the activities are much more restricted than in the maternal liver (Hakkola et al., 1998; Huuskonen et al., 2008; Myllynen et al., 2007). Of the cytochrome P450 (CYP) enzymes, only the activity of CYP1A1

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V. Karttunen et al. / Toxicology Letters 197 (2010) 75–81 Table 1 Human placental perfusions carried out for this study. Number of perfusions (T/M)a

2/2 4/4 4/4 0/2 a

Concentrations of added compounds BP (␮M)

Verapamil (␮M)

Antipyrine (␮g/ml)

– 0.1 0.1 1

– – 1 –

100 100 100 100

T = number of perfusions used for transfer studies; M = number of perfusions used for metabolism studies; BP = benzo(a)pyrene.

is clearly measurable with significant interindividual variation (Vähäkangas et al., 1989). It has been long known that cigarette smoking induces placental CYP1A1, one of the main enzymes catalyzing BP metabolism (Pelkonen et al., 1984; Vähäkangas et al., 1989). Activation of BP in the placenta may lead to DNA damage, especially in the placenta itself. Most chemical compounds cross the placenta (Myllynen et al., 2007; Pacifici and Nottoli, 1995). Evidence from human studies indicates that human fetus is exposed also to BP. BPDE–DNA adducts have been found in the placentas of smoking mothers (Manchester et al., 1992), and in cord blood (Perera, 1997; Perera et al., 1999; Phillips, 2002). Environmental pollution, maternal smoking and exposure of pregnant women to passive smoking have been associated with increased mutation frequency at the HPRT locus of newborns (Grant, 2005; Perera et al., 2002). Human placental perfusion method has been widely used in scientific research for transplacental transfer in human placenta. However, it is not a validated test system for toxicological risk assessment. Although drugs have been studied in the placental perfusion, very little data on environmental toxic compounds exist. A recent paper on BP transfer was published by Mathiesen et al. (2009) showing a relatively poor transport of BP through human placenta. Most of the experimental studies on the placental transfer of drugs using isolated perfused human placental cotyledon have been carried out using relatively high concentrations, while the environmental exposures occur at low levels. In this study we have pursued toxicokinetics and DNA binding of BP in the perfused human placenta at low, environmentally more relevant concentrations. 2. Materials and methods 2.1. Chemicals BP and verapamil were from Sigma–Aldrich (Steinheim, Germany) and generally labelled 3 H-BP, specific activity 86.0 Ci/mmol; 3.18 TBq/mmol, from Amersham Biosciences (UK). Antipyrine was ordered from Aldrich Chemie (Steinheim, Germany). Ribonuclease A and T1, prostatic acid phosphatase, snake venom phosphodiesterase I and nuclease P1 were obtained from Sigma–Aldrich. Molecular biology grade phenol was purchased from VWR International (Darmstadt, Germany). The polyethyleneimine thin-layer chromatography plates were from Machery-Nagel (Düren, Germany) and proteinase K was from Roche (Rotkreutz, Switzerland). [32 P]ATP was provided by GE Healthcare (Uppsala, Sweden) and the polynucleotide kinase by US Biochemicals (Cleveland, OH, USA). All other chemicals were of analytical grade and obtained from VW International or Sigma–Aldrich. 2.2. Human placental perfusion Placentas for perfusions were received from healthy mothers after uncomplicated pregnancies from the Department of Gynecology and Obstetrics, University Hospital of Oulu. The tissues were collected immediately after either normal vaginal delivery or cesarean section, performed at term. All mothers reported that they were non-smokers. The Ethics Committee of The Northern Ostrobothnia Hospital District approved the study protocol. Oral and written information about the study was given to the mothers and a written informed consent was obtained from them. Preparation for perfusion was successful in altogether 18 placentas. However, 6 of these perfusions were omitted because of too big a leak during the perfusion and thus the final number of perfusion was 12. We used a dual recirculating placental number of perfusions (Schneider et al., 1972; Pienimäki et al., 1995; Annola et al., 2008; Myllynen et al., 2003) where both the maternal and fetal sides of the placenta are perfused separately. Krebs–Ringerphosphate buffer with heparin was injected into cord vessels within 10 min after the

delivery of the placenta. A single chorionic artery and vein in an intact peripheral cotyledon were cannulated and the lobe was placed in the perfusion apparatus. On the maternal side, two cannulas were placed into the intervillous space through the basal plate. The perfusate was RPMI 1640 cell culture medium (Gibco) with penicillin–streptomycin, l-glutamine, sodium puryvate, non-essential amino acids, heparin 25 IU/ml (Leo Pharma, Malmö, Sweden), albumin 2 g/l (SPR, Finland) and Dextran T 40 1 g/l (Sigma Chemical Company, St Louis, MO, USA). Final maternal volume was 200 ml and the fetal volume 120 ml. The perfusion flow was 3 ml/min on the fetal side and 9 ml/min on the maternal side. Using a membrane oxygenator, the maternal perfusate was gassed with 95% O2 /5% CO2 and the fetal perfusate with 95% N2 /5% O2 . To stabilize the condition of the placenta and to reverse hypoxia, preperfusion was carried out for 30–60 min. Silicone tubing in the perfusion equipment was changed after every perfusion. Ten successful perfusions were carried out using BP alone or in combination with verapamil (Table 1). Two control perfusions for adduct studies using vehicle for BP were also carried out. Antipyrine (100 ␮g/ml; Aldrich Chemie, Mannheim, Germany), transferred by passive diffusion (Schneider et al., 1972) was used as a reference compound in all perfusions. Studied compounds were added into the maternal perfusate simultaneously, and perfusion was continued for 6 h. All the perfused placentas in the transfer studies using 0.1 ␮M BP with or without verapamil fulfilled the criteria for a successful perfusion (Pienimäki et al., 1995, 1997). The leak from the fetal to the maternal circulation was less than 3 ml/h, and the fetal pressure, monitored continuously, was stable and less than 70 mmHg at all times. The placentas also consumed glucose during the perfusions. The two perfused placentas with 1 ␮M BP were only analyzed for BPDE–DNA adducts. 2.3. Analysis of study compounds from perfusion fluid 2.3.1. Benzo(a)pyrene analysis Placental transfer was studied using 3 H-BP (Amersham Biosciences). To enhance the counting efficiency, 500 ␮l samples from the perfusion media were first flushed under a nitrogen stream for 10 min. The samples were then bleached using 100 ␮l of strong hydrogen peroxide solution (>30%) and incubated for 30 min at room temperature. Thereafter, 250 ␮l 4 M HCl and 20 ml of liquid scintillation cocktail (HiSafe 2, PerkinElmer) were added and the samples were incubated overnight in the dark at room temperature. Radioactivity was quantified using LKB Wallace 1215 Rackbeta II liquid scintillation counter. All samples were measured in duplicate. 2.3.2. Antipyrine analysis Antipyrine concentrations were analyzed using HPLC as described previously (Myllynen, 2003). In brief, the precolumn was LiChrospher100 RP-18, 4-4 i.d.; 5 ␮m. The main column used was LiChrocart® 125-4. The HPLC consisted of a MerckHitachi L-6200 gradient pump and an L-4250 UV–vis detector. The mobile phase was a mixture of acetonitrile (20%) and 20 mM KH2 PO4 buffer solution (80%). The HPLC analysis was isocratic and carried out at room temperature at a flow rate of 1.0 ml/min. Standard curve of antipyrine was determined every time we analyzed antipyrine samples. The measured concentrations were quantified with the standard curve. 2.4. Tissue analysis Tissue samples before and after perfusion were homogenized into the buffer A (100 mM NaCl, 50 mM Tris, 1% SDS, 10 mM EDTA). The homogenates were then incubated with proteinase K to further solubilise the tissue overnight and quantified using liquid scintillation counting. The amount of BP in tissue was determined from 7/8 placentas that were used for transfer studies. 2.5. Ethoxyresorufin O-deethylase (EROD) activity assay Microsomal fractions from the placentas were prepared as described previously (Myllynen et al., 1998). The final microsomal pellet was suspended in 0.1 M phosphate buffer pH 7.4. The protein content was measured by the Bradford method (Kruger, 1994). The fluorometric end point assay of Burke et al. (1977) was used for measuring EROD activity using 1 mg of microsomal protein for the reaction. Enzyme activities are expressed as pmol/mg protein/min.

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2.6. Cell culture experiments BeWo human choriocarcinoma cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). The BeWo cells were maintained in F12K medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 ␮g/ml streptomycin in a humidified incubator (5% CO2 /95% air, 37 ◦ C). The plates used for exposure were BD FalconTM 10 cm polystyrene dishes. The cells were plated at an initial concentration of 105 cells/ml and maintained in a humidified incubator for three days, after which the cells were exposed for 24 h to 1 ␮M BP and verapamil (0.1 and 1 ␮M). The compounds were added to the cell culture media in dimethylsulfoxide (DMSO), volume <0.5%. The control cells were treated with vehicle. After exposure the cells were removed to buffer A (100 mM NaCl, 50 mM Tris, 1% SDS, 10 mM EDTA) and DNA was isolated using phenol–chloroform extraction/ethanol precipitation method (Lagerqvist et al., 2008). The cells were tested for Mycoplasma by using MycoAlertTM Mycoplasma Detection Kit (Cambrex) before the experiments. 2.7. DNA adduct analysis 2.7.1. Synchronous fluorescence spectrophotometry BPDE–DNA adducts were analyzed in placental tissue after perfusions and cell culture experiments using synchronous fluorescence spectrophotometry (SFS, Vähäkangas et al., 1985). DNA was isolated using the phenol/chloroform precipitation method. One hundred ␮g of DNA was hydrolyzed in 0.1 M HCl at 90–95 ◦ C for 3 h before fluorescence analysis using synchronous scanning with a constant 34 nm difference between emission and excitation. In SFS, the level of adducts is linearly correlated with the height of the peak at 345 nm excitation. One fluorescence unit equals approximately 1.1 fmol BPDE/␮g DNA. The sensitivity of SFS is about 1 adduct/107 normal nucleotides. 2.7.2. 32 P-postlabelling The BPDE–DNA adducts were also analyzed by the 32 P-postlabelling assay using the so-called dinucleotide monophosphate version of the method (Randerath et al., 1989), with some modifications (Plna et al., 1999). Four ␮g samples of DNA were analyzed, and adducts were separated by thin-layer chromatography (TLC), and visualized and quantified using Phosphoimage analysis. The sensitivity of the assay is about 1 adduct/109 nucleotides. A duplicate sample of the external standard was included in each analysis and used for the DNA adduct calculation. As external standard, DNA treated in vitro with BPDE was used with an adduct level of 111/108 normal nucleotides (Phillips and Castegnaro, 1999). 2.8. Calculations Statistical analysis was performed using the one-way or two-way ANOVA. Values of p less than 0.05 were taken as statistically significant. The transfer percentage from maternal to fetal circulation was calculated by the following formula: 100 × Fc × Fv/[(Fc × Fv) + (Mc × Mv)], where Fc is fetal concentration, Mc is maternal concentration, Fv is fetal perfusate volume, and Mv is maternal perfusate volume. FM ratio, calculated for the figures, is the ratio of fetal concentration to maternal concentration. Transfer index was calculated by dividing the transfer percentage of BP with the transfer percentage of antipyrine. All values are expressed as a mean ± s.d. Recovery of BP was calculated by the following formula: 100 × (FBP + MBP + TISBP + SAMBP )/5050 ng, where FBP is the amount of BP in the fetal reservoir at the end of perfusion, MBP is the amount of BP in the maternal reservoir at the end of perfusion, TISBP is the amount of BP in tissue after perfusion, and SAMBP is the amount of BP which is gone with the samples taken during perfusion. 5050 ng is the total amount of BP added in every perfusion.

3. Results 3.1. Placental transfer of 3 H-benzo(a)pyrene The overall transfer rate of 3 H-BP was significantly slower than the transfer rate of antipyrine in all of the perfusions (Fig. 1). However, similarly to the reference compound antipyrine, radioactivity representing both 3 H-BP and its possible metabolites was detectable in the fetal circulation already in the first 15 min sample even though the concentration at this time point was still low (transfer percentage between 1% and 7%, Fig. 1). 3 H-BP was steadily transferred to fetal circulation throughout the perfusions and at the end of the perfusions significant amounts of 3 H-BP were found in the fetal circulation (Figs. 1 and 2). However, considerable interindividual variation in the placental transfer of 3 H-BP was seen already in the first samples and at the end feto-maternal ratios varied from 0.27 to 1.1 (Fig. 2B). The recovery of BP in perfusions was 85.1 ± 30.6% (Table 2) and indicates some binding of BP to perfusion equipment.

Fig. 1. Placental transfer of the reference compound antipyrine (open circles) in comparison with 3 H-benzo(a)pyrene (squares) (n = 4). FM ratio = feto-maternal concentration ratio, **p < 0.01; ***p < 0.001. Values are expressed as a mean ± s.d.

Transfer data of 3 H-BP was compared with the transfer of antipyrine, a passively diffusing compound (Schneider et al., 1972). Transfer of antipyrine through the placenta is flow limited and can thus be used to normalize variation due to flow conditions in the perfusions. However, normalization did not lead to disappearance of the individual differences in BP transfer (Fig. 2C). 3.2. Verapamil and placental transfer of antipyrine and 3 H-benzo(a)pyrene

The effect of verapamil, inhibiting mainly ABCB1/P-gp but to some extent also ABCC1-2/MRP 1-2 (Vähäkangas and Myllynen, 2009), on the transfer of 3 H-BP was also studied. Transfer of 3 HBP appeared to be slightly slower in the perfusions with 1 ␮M verapamil than in perfusions without verapamil (Fig. 3A). This difference was most pronounced during the first 3 h of the perfusion. However, due to the high interindividual variation in 3 H-BP transfer, there were no statistically significant differences between the groups with and without verapamil. A similar effect by verapamil on the transfer of antipyrine was noted (Fig. 3B). 3.3. Tissue binding The amount of BP in tissue after perfusion varied a lot between different perfusions. In most perfusions we found at least a small amount of BP in tissue. In one perfused tissue no BP was found and in one case no tissue was available. The proportion of BP in tissue after perfusion, when comparing to the amount of BP added to perfusion, was 4.2 ± 4.8%. Table 2 Recovery of benzo(a)pyrene in placental perfusions. Perfusion

1 2 3 4 5 6 7 8 Mean ± s.d.

Added compounds

Recovery (%)

BP (␮M)

VER (␮M)

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

0 0 0 0 1 1 1 1

45 77 109 144a 88 67 101 51 85.1 ± 30.6

BP = benzo(a)pyrene; VER = verapamil. a This does not include BP in the tissue due to accidental loss of the tissue.

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Fig. 3. (A) Effect of verapamil on placental transfer of benzo(a)pyrene (BP) (n = 4). Verapamil was added concurrently with the other compounds. (B) Effect of verapamil on placental transfer of antipyrine. FM ratio = feto-maternal concentration ratio. Values are expressed as a mean ± s.d.

3.4. DNA adducts in the perfused placentas

Fig. 2. Placental transfer of 3 H-benzo(a)pyrene (BP). Different symbols represent the four different perfusions carried out. (A) Appearance of BP into the fetal circulation. (B) Feto-maternal concentration ratio (FM ratio) of BP. (C) Transfer index of BP. Transfer index was calculated by dividing the transfer percentage of BP with the transfer percentage of antipyrine. Transfer percentage is the percentage of a compound transferred into the fetal circulation from the initial concentration in maternal circulation measured immediately after the addition of the compound into the maternal circulation.

In one of the two perfusions with 1 ␮M BP, high levels of DNA adducts were detected both by SFS and 32 P-postlabelling. Using SFS there was one peak corresponding to approximately 1.28 fmol BPDE/␮g DNA in the perfused area (data not shown). In the control sample, taken from the same placenta before perfusion, no adducts were detected. By the 32 P-postlabelling assay, spots co-migrating with the major adducts detected in BPDEtreated DNA were observed on the TLC plates (Fig. 4B). In addition, some minor exposure related spots were detected which were not observed in DNA treated in vitro with BPDE. BPDE–DNA adduct level (corrected for recovery with the use of the external standard) was 0.485 ± 0.144 adducts/106 normal nucleotides (1.570 ± 0.466 fmol adduct/␮g DNA) and in very good agreement with the SFS data. Only background levels of bulky DNA adducts from the same placenta before perfusion were found. In the second perfusion with 1 ␮M BP no DNA adducts derived from BP were found using either of the available methods. In eight perfusions with 0.1 ␮M of BP no DNA adducts could be detected. By 32 P-postlabelling analysis distinct adduct spots were observed in all placentas before perfusion (Fig. 4A, left panel). Adduct levels varied between the 10 placentas used. The mean level of bulky DNA adducts (sum of the major spots) from all tested 10 placentas before perfusion was

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Table 3 Ethoxyresorufin-O-deethylase (EROD) activities in the placentas before and after perfusions. Perfusion

EROD before perfusion (pmol/mg/min)

BP 1 ␮M

1.85 0.688

2.03 0.813

BP 0.1 ␮M

0 0.232 0.253 0.170

0.144 0.217 0.132 10.82

BP 0.1 ␮M + VER 1 ␮M

0.201 0.417 0.225 0.055

0.03 1.90 4.45 0.407

EROD after perfusion (pmol/mg/min)

BP = benzo(a)pyrene; VER = verapamil.

3.6. DNA adduct formation in BeWo cells BeWo (human choriocarcinoma) cells activated BP to BPDE–DNA adducts detectable by SFS analysis. Verapamil did not affect the level of BPDE–DNA adducts (data not shown). 4. Discussion

Fig. 4. 32 P-postlabelling analysis of perfused human placenta. (A) Bulky DNA adducts in placentas. Example of TLC separation of two different placentas before perfusion (left panels) and after perfusion of same placentas with 0.1 ␮M BP (right panel). Phosphoimager screen was exposed for 24 h. (B) DNA adduct in the BPDEtreated DNA used as external standard (1) and in placenta perfused with 1 ␮M BP (2) Phosphoimager screen was exposed for 2 h.

1.5 ± 0.7 adduct/108 normal nucleotides or 0.047 fmol adduct/␮g DNA (range 0.41–2.65 adduct/108 normal nucleotides). The same adduct pattern was also seen after perfusion with BP (Fig. 4A, right panel) and there was no statistically significant difference between the bulky DNA adduct levels before and after perfusions. Some samples had before the perfusion an adduct spot which co-migrated with the major spot in the BPDE-treated DNA. 3.5. Placental EROD activities Placental EROD activities were measured in the placentas perfused with BP alone and those with BP plus verapamil before and after the perfusions (Table 3). In average, EROD activities were 0.19 ± 0.14 pmol/min/mg (range 0–1.85) before perfusions and 2.26 ± 3.8 pmol/min/mg (range 0.03–10.82) (p > 0.05) protein after perfusions. However, a higher EROD activity was not associated with a decreased transfer: rather the trend seemed to be the opposite (data not shown). Among the perfusions with 1 ␮M of BP, EROD activity was higher in the placenta with a higher level of BPDE–DNA adducts.

The transfer of BP through the human placenta has been studied only in one published paper before the current study (Mathiesen et al., 2009). They suggest that BP transfer through human placenta increases if a physiological concentration of albumin, especially if human serum albumin is used. In our study, a closely similar ratio of BP to albumin was used and the resulting feto-maternal concentration ratio within 6 h of perfusion is in accordance with the one of Mathiesen et al. (2009). It thus seems that albumin aids the transfer of BP through human placenta. However, in an early study by Kihlström (1986) placental transfer of BP in in situ perfusion in guinea pig, albumin in perfusion fluid slowed down the transfer of BP and dextran inhibited it altogether. This is a clear indication of the need to use human system for placental transfer studies as it is in line also by the anatomical and physiological differences in the placenta between different species (Benirschke et al., 2006). People are exposed to BP from many sources like diet, air pollution and various exhausts. Tobacco smoke is the most important source for smokers and the nature of BP exposure is long term. The total dietary BP intake due to consumption of various food items has been estimated to be 124 ng/person/day (Lee and Shim, 2007). The BP concentration used in perfusions in transfer studies, 0.1 ␮M (5050 ng), is clearly higher, but practical for BP analysis from perfusion fluid. In our study the transfer of BP through the placenta, while unequivocally measurable, differed from that of antipyrine with a slower appearance to the fetal circulation. Because BP is lipophilic, there was a possibility that BP accumulates in the placenta. We have shown earlier (AlaKokko et al., 1995) that more lipophilic bupivacaine penetrates the placenta more slowly than lidocaine and antipyrine. Bupivacaine also accumulates in the placental tissue. However, the proportion of BP in the perfused placental tissue in this study was less than 10% which does not explain the different kinetics. The binding to tubings of the perfusion system may be a partial explanation because the recovery of BP was generally less than 100%. Short perfusion time and variation between perfusions does not indicate systematic loss of tritium to water. The main mechanism by which antipyrine goes through the placenta is passive diffusion (Bassily et al., 1995; Schneider et al., 1972). Because BP transfer was slower, there was a possibility that one or more transporters are involved with BP transport in human placenta. Many efflux transporters such as ABCB1/P-gp are found

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in the maternal blood facing brush border of syncytiotrophoblast of human placenta (Pacifici and Nottoli, 1995). ABCB1/P-gp is believed to prevent entry of some foreign compounds into the fetoplacental unit (Lankas et al., 1998; Smit et al., 1999). Lankas et al. (1998) found that the pesticide, avermectin, accumulates on the fetal side and is teratogenic in ABCB1/P-gp deficient mice. In the literature exists an indication that BP can be a ligand for ABCB1/P-gp. Yeh et al. (1992) reported that ABCB1/P-gp inhibits the access of BP to MCF-7 breast adenocarcinoma cells. One of the inhibitors used in their study was verapamil. Also in our study BP was perfused with this known ABCB1/P-gp antagonist verapamil (Kim, 2002) but there was no significant effect on the feto-maternal ratio of BP. Actually BP was transferred slightly slower with than without verapamil. Adding verapamil probably changed the flow by lowering perfusion pressure because verapamil is a known vasodilator (Benowitz, 1995). This is supported by the fact that verapamil affected also antipyrine kinetics so that antipyrine transfer was slower with than without verapamil. Our results support an earlier study where ABCB1/P-gp does not inhibit the transfer of BP through cell membrane (Schuetz et al., 1998). Furthermore, using concentrations that might be clinically achievable there was not any significant effect on the kinetics of BP. The interplacental variation in the transfer of BP is not unexpected considering the individual differences e.g. due to polymorphisms and induction by exogenous chemicals of transporter proteins and metabolic enzymes. There may be also variation in the preparation and the perfusion procedure of different placentas. However, in these perfusions the differences in the transfer of the reference compound, antipyrine, were not as great. CYP1A1 is a key enzyme in BP activation (Pelkonen et al., 1984) and the only CYP enzyme with a consistently measurable activity in the placenta. A highly variable CYP1A1-specific EROD activity was detected, but it seemed not to affect the transport of BP (see below). The difficulty in comparing EROD activities before and after perfusion is that they have been measured from different parts of the same placenta. It is feasible to think that EROD activity may vary in different parts of the same placenta because placental tissue is not homogenous (Castellucci and Kaufmann, 2006). Circulation is not equally distributed and calcifications may also occur in some parts of the placenta (Benirschke et al., 2006). The effect of perfusion itself on the enzyme activities in the placenta was not studied. BP–DNA adducts have been found after in vivo ambient or cigarette smoke exposure in several studies in human cord blood (e.g. Perera et al., 2005; Tang et al., 2006; Topinka et al., 2009; Wang et al., 2008) and placenta (for a review see Phillips, 2002). Correlation between the maternal and fetal adduct levels is not good, at least partly because of polymorphic variation in the PAHmetabolizing enzymes in maternal and fetal tissues (Wang et al., 2008). However, from in vivo studies it remains unclear what is the role of the placenta itself in the activation of BP. In this study we have confirmed the ability of human placenta to activate BP as shown by the existence of BPDE–DNA adducts after the perfusion in at least one perfused placenta. The fact that we found BPDE–DNA adducts also in BP-treated human trophoblastic BeWo cells supports this result. In the literature, it has been shown before that the BeWo cells can metabolize xenobiotics (Zhang et al., 1995; Avery et al., 2003). Even a high EROD activity indicating CYP1A1 induction did not significantly affect the levels of BP in fetal or maternal circulations in our perfusions. Furthermore, the efflux transporters known to be expressed in the placenta (Vähäkangas and Myllynen, 2009) are probably not able to significantly prevent BP transport based on this study and the in vivo occurrence of BPDE–DNA adducts in cord blood (Perera et al., 1999; Topinka et al., 2009). It is thus very likely that any maternal exposure to BP leads also to the exposure of the fetus to BP and/or its metabolites. The origin of the bulky adducts detected in all placentas (even

before perfusion) is unknown, but a potential source is exposure to BP from sources other than main stream tobacco smoke. However, it is also possible that the spots represent endogenous DNA modifications of unknown origin. In conclusion, based on the results of this study and those from the literature, BP is transferred from maternal to fetal circulation and ABCB1/P-gp may not protect human fetus from BP. Our study supports the data from the literature (see e.g. Barr et al., 2007) showing a high interindividual variation in the exposure of fetus. This study shows that maternal exposure to BP leads to the exposure of the fetus to BP and/or its metabolites and that placenta itself can activate BP to DNA adducts. Conflict of interest statement None of the authors have any conflicts of interest to disclose. Acknowledgements We are grateful to the nursing personnel in the Delivery Room of the Department of Obstetrics and Gynecology for their cooperation. We also thank Ms. Anna Malinen, Ms. Kaisa Penttilä, Ms. Ritva Tauriainen, Mr. Esa Kerttula, Ms. Päivi Asikainen and Dr. Kirsi Annola for practical help. Financially this project was supported by the Finnish Academy, Children and Genotoxicity Network (EUproject QLK4-CT-2002-02198, coordinator Lisbeth Knudsen) and the Swedish Animal Welfare Agency. The study sponsors did not have any role in a study design, in the collection, analysis, and interpretation of data, in the writing of the report or in the decision to submit the paper for publication. Authors belong to the EU networks NewGeneris (FOOD-CT-2005 016320) and ReProTect (LSHB-CT-2004-503257). References AlaKokko, T.I., Pienimäki, P., Herva, R., Hollmén, A.I., Pelkonen, O., Vähäkangas, K., 1995. Transfer of lidocaine and bupivacaine across the isolated perfused human placenta. Pharmacol. Toxicol. 77, 142–145. Annola, K., Karttunen, V., Keski-Rahkonen, P., Myllynen, P., Segerbäck, D., Heinonen, S., Vähäkangas, K., 2008. Transplacental transfer of acrylamide and glycidamide are comparable to that of antipyrine in perfused human placenta. Toxicol. Lett. 182, 50–56. Avery, M.L., Meek, C.E., Audus, K.L., 2003. The presence of inducible cytochrome P450 types 1A1 and 1A2 in the BeWo cell line. Placenta 24, 45–52. Baird, W.M., Hooven, L.A., Mahadevan, B., 2005. Carcinogenic polycyclic aromatic hydrocarbon–DNA adducts and mechanism of action. Environ. Mol. Mutagen. 45, 106–114. Barr, D.B., Bishop, A., Needham, L.L., 2007. Concentrations of xenobiotic chemicals in the maternal–fetal unit. Reprod. Toxicol. 23, 260–266. Bassily, M., Ghabrial, H., Smallwood, R.A., Morgan, D.J., 1995. Determinants of placental drug transfer: studies in the isolated perfused human placenta. J. Pharm. Sci. 84, 1054–1060. Benirschke, K., Kaufmann, P., Baergen, R.N., 2006. Pathology of the Human Placenta, fifth ed. Springer Science+Business Media, New York. Benowitz, N.L., 1995. Antihypertensive agents. In: Katzung, B.G. (Ed.), Basic & Clinical Pharmacology, sixth ed. Prentice Hall International, London, pp. 147–170. Burke, M.D., Prough, R.A., Mayer, R.T., 1977. Characteristics of a microsomal cytochrome P-448-mediated reaction: ethoxyresofurin O-deethylation. Drug Metab. Dispos. 5, 1–8. Castellucci, M., Kaufmann, P., 2006. Basic structure of the villous trees. In: Benirschke, K., Kaufmann, P., Baergen, R.N. (Eds.), Pathology of the Human Placenta, fifth ed. Springer Science+Business Media, New York, pp. 50–99. Dybing, E., OˇıBrien, J., Renwick, A.G., Sanner, T., 2008. Risk assessment of dietary exposures to compounds that are genotoxic and carcinogenic—an overview. Toxicol. Lett. 180, 110–117. Frey-Hattemer, H.A., Travis, C.C., 1991. Benzo-a-pyrene: environmental partitioning and human exposure. Toxicol. Ind. Health 7, 141–157. Ganapathy, V., Prasad, P.D., 2005. Role of transporters in placental transfer of drugs. Toxicol. Appl. Pharmacol. 207, 381–387. Grant, S.G., 2005. Qualitatively and quantitatively similar effects of active and passive maternal tobacco smoke exposure on in utero mutagenesis at the HPRT locus. BMC Pediatr. 5, 20. Hakkola, J., Pelkonen, O., Pasanen, M., Raunio, H., 1998. Xenobiotic-metabolizing cytochrome P450 enzymes in the human feto-placental unit: role in intrauterine toxicity. Crit. Rev. Toxicol. 28, 35–72.

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