Human Organic Anion Transporter hOAT4 is a Transporter of Perfluorooctanoic Acid

Perfluorooctanoic acid is a class of synthetic fluorochemicals used in a variety of applications such as processing aids for fluoropolymer production, surfactants and water-repellent coatings [1,2]. It poses special public health concerns due to its long-term persistence and bioaccumulation in the environment, including humans [3–5]. There have been a number of reports regarding the health effects of perfluorooctanoic acid. It was found to be a carcinogen for rodents [6] while it is not genotoxic in umu test [7]. Furthermore, a developmental toxicity study of perfluorooctanoic acid in mice revealed early total loss and delays in general growth and development [8]. Species and sex differences have been reported for the toxicokinetics of perfluorooctanoic acid. Human serum elimination half-life of perfluorooctanoic acid is reported to be 3.8 years [9]. However, its serum elimination half-lives are much shorter in male Wistar rats (5.7 days) [10] suggesting large differences in perfluorooctanoic acid elimination kinetics among species. In clear contrast to rats, renal clearance of perfluorooctanoic acid in humans is negligible [11]. Although several organic anion transporters (OATs) are found to mediate transport of perfluorooctanoic acid in rats and humans, there were no kinetically significant difference in their activities [12,13]. Assuming that perfluorooctanoic acid transport from serum to tubular epithelial cells would be expected to occur in similar manners in both species, it is reasonable to predict a critical role for the re-absorption process as a determinant for the large species differences observed in the renal excretion of perfluorooctanoic acid. OAT4 (SLC22A11), a transporter that is only expressed in humans, is an apical type isoform in proximal tubules and mediates the re-absorption of organic anions [14]. Therefore, it may contribute to the long half-life of perfluorooctanoic acid in humans. In the present study, we aimed to characterize the perfluorooctanoic acid transport activity of human OAT4. Materials. p-[Glycyl-1-14C]aminohippurate (PAH, 1.9 GBq/mmol) and [6,7-3H(N)]estrone sulphate, ammonium salt (ES, 2.1 TBq/mmol) were from Perkin-Elmer Life Sciences Inc. (Boston, MA). We also commissioned Perkin Elmer Life Sciences to synthesize [1-14C]perfluorooctanoic acid (34 mCi/mmol). Foetal bovine serum and trypsin were purchased from Invitrogen (Carlsbad, CA). All other compounds used were of the highest purity available. Cell culture and transfection. HEK 293 cells (American Type Culture Collection CRL-1573), a transformed cell line derived from human embryonic kidney, were cultured in complete medium consisting of Medium 199 (Invitrogen, Carlsbad, CA) with 10% foetal bovine serum (Thermo. Electron Co., Waltham, MA) in an atmosphere of 5% CO2, 95% air at 37°. Full-length cDNAs of human (h) OAT1 (SLC22A6) [15], hOAT3 (SLC22A8) [15] and hOAT4 [16] were subcloned into pBK-CMV plasmid vector (Stratagene, La Jolla, CA). HEK 293 cells were transfected with 0.8 µg hOAT1 cDNA, hOAT3 cDNA, hOAT4 or empty vector using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. G418 (Nacalai Tesque, Kyoto, Japan) (0.5 mg/ml) – resistant cells were removed. Cells expressing hOAT1 (HEK-hOAT1) were selected by measuring p-[14C]aminohippurate uptake, and cells expressing hOAT3 (HEK-hOAT3) and hOAT4 (HEK-hOAT4) were selected by measuring [3H]estrone sulphate uptake. Expression of hOAT4 mRNA was analysed by reverse transcription-PCR (forward primer, 5′-TAAGCCAGTCCATCTTCATGTCCG-3′; reverse primer, 5′-TTGCCCTTAATAATCAGCCACCGG-3′), as previously reported [17]. Cells transfected with empty vector (HEK-pBK) were used as control cells. These transfectants were maintained in complete medium with G418 (0.5 mg/ml). Uptake experiments. Uptake experiments were performed as described previously [18] with some modifications. HEK cells were seeded on poly-d-lysine-coated 24-well plates at a density of 2 × 105 cells/well for the uptake of [1-14C]perfluorooctanoic acid. At 48 hrs after seeding, the uptake of these compounds by HEK cells was examined. The composition of the incubation medium was as follows (in mM): 145 NaCl, 3 KCl, 1 CaCl2, 0.5 MgCl2, 5 d-glucose and 5 HEPES (pH 7.4). The cells were pre-incubated with 0.2 ml of the incubation medium for 10 min. at 37°. After the pre-incubation, the medium was replaced with 0.2 ml of incubation medium containing [1-14C]perfluorooctanoic acid. At the end of the incubation period of 15 min., the medium was aspirated and then cells were washed two times with 1 ml of ice-cold incubation medium. The cells were lysed in 0.25 ml of 0.5 N NaOH solution and the radioactivity in aliquots was determined in 3 ml of ACSII (Amersham International, Buckingham shire, UK). The protein contents of the solubilized cells were determined by the method of Bradford [19] using the Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA) with bovine γ-globulin as a standard. Statistical analysis. Each uptake experiment was performed in triplicate. Data are expressed as means and standard errors of the mean. Statistical differences were determined using Student's unpaired t-test. Differences were considered significant at P < 0.01. Uptake of perfluorooctanoic acid by OATs transfected into HEK293 cells. Firstly, we confirmed expression of hOAT4 mRNA (fig. 1). Strong expression of hOAT4 mRNA was observed in pBK-hOAT4-transfected HEK 293 cells while weak expression was detected in mock-transfected cells. Secondly, we examined the [3H]ES uptake rate into HEK293 cells transfected with hOAT4 (fig. 2A). We confirmed the functional expression of hOAT4 using its optimal substrate. Thirdly, we examined [14C]perfluorooctanoic acid uptake by hOATs expressed in HEK293 cells. The uptake for 15 min. was stimulated in cells transfected with human OAT1, OAT3, and OAT4 (fig. 2B). The uptake of [14C]perfluorooctanoic acid by HEK-hOATs was 1.6–2.7-fold higher than that by control cells (P < 0.01 for each cells). Moreover, the accumulation of perfluorooctanoic acid was significantly greater in HEK-hOAT4 than in HEK-hOAT1 (P < 0.01). To investigate the contribution of possible non-specific binding of perfluorooctanoic acid on cell membrane, HEK293 cells were washed with bovine serum albumin after incubation with perfluorooctanoic acid (fig. 2C). BSA washing reduced perfluorooctanoic acid uptake by 30% at most, suggesting that perfluorooctanoic acid uptake in transfected cells was mediated by transporters. Expression of hOAT4 mRNA in hOAT4 cDNA-transfected HEK293 cells. RT-PCR products were electrophoresed in 1.5% agarose gel. HEK-pBK: cells transfected with empty vector; HEK-hOAT4: cells transfected with hOAT4 cDNA; pBK-hOAT4: PCR products from plasmid vector. Transport activity of hOAT4 expressed in HEK293 cells. (A) HEK293 cells transfected with the pBK vector alone (mock, white bar) and hOAT4 (black bar) were incubated in a solution containing 20 nM [3H]ES at 37° for 15 min. (B) [14C]perfluorooctanoic acid uptake by hOAT1, hOAT3, and hOAT4 expressed in HEK293 cells. HEK293 cells transfected with pBK were incubated in a solution containing 10 µM [14C]perfluorooctanoic acid at 37° for 15 min. (C) Effect of BSA washing on [14C]perfluorooctanoic acid uptake by HEK293 cells. HEK293 cells were incubated in a solution containing 10 µM [14C]perfluorooctanoic acid at 37° for 15 min., and then washed with incubation medium containing 1%, 3%, and 5% of bovine serum albumin. Each value represents the mean ± S.E.M. of three determinations. *P < 0.01, **P < 0.001 versus mock. In the present study, we found that human OAT4 transported perfluorooctanoic acid. The uptake by hOAT4 was greater than that by hOAT1. Previously, we have found that the transport activities of hOAT1 and hOAT3 for perfluorooctanoic acid were nearly equal to those for PAH which is a good substrate for both OAT1 and OAT3 [12]. Therefore, hOAT1 and hOAT3 could involve in perfluorooctanoic acid transport from serum to tubular epithelial through the basolateral membrane, while only a small amount of perfluorooctanoic acid is detected in urine in humans [20]. Perfluorooctanoic acid transport activity by hOAT4 suggests that re-absorption of perfluorooctanoic acid from tubule might cause poor excretion into urine. Further studies are required to evaluate the contributions of OAT4 to species-difference in the toxicokinetics of perfluorooctanoic acid. In addition, hOAT4 mRNA is abundantly expressed in the placenta as well as in the kidney [21]. hOAT4 is expressed in basal (foetal facing) membrane and could be involved in elimination of toxic substances from foetuses. Moderate partition of perfluorooctanoic acid from maternal blood to foetus was observed in humans [22]. The contribution of OAT4 to distribution of perfluorooctanoic acid warrants further investigation. In conclusion, we have demonstrated that OAT4 have perfluorooctanoic acid transport. This study was mainly supported by a grant (H15-Chemistry-004) from the Ministry of Health, Labor and Welfare of Japan.