Inhibition of organic cation transporter (OCT) activities by carcinogenic heterocyclic aromatic amines

Highlights

• Various HAAs inhibit activity of the organic cation transporters OCT1, OCT2 and OCT3.

• Trp-P-1 and Trp-P-2 are ones of the most active HAAs against OCT activities.

• HAA IC₅₀ towards OCTs are higher than expected HAA concentrations in humans.

• Trp-P-1 and Trp-P-2 are not substrates for OCT1 and OCT2.

Abstract

Carcinogenic heterocyclic aromatic amines (HAAs) interact with some drug transporters, like the efflux pump BCRP and the organic anion transporters OAT1 and OAT3. The present study was designed to determine whether they can also target activities of the organic cation transporters (OCTs), using mainly OCT1-, OCT2- and OCT3-overexpressing HEK293 cells. Fifteen HAAs were demonstrated to differently alter OCT activities; with a cut-off of at least 50% reduction of transporter activity by 100 μM HAAs, 5/15 HAAs, including Trp-P-1 and Trp-P-2, inhibited activities of OCT1, OCT2 and OCT3, whereas 7/15 HAAs, including PhIP and MeIQx, blocked those of OCT2 and OCT3, 1/15 HAAs reduced those of OCT1 and OCT2 and 2/15 HAAs, including AαC, only that of OCT2. IC₅₀ values of Trp-P-1 and Trp-P-2 towards OCT activities were found to be in the 2–6 μM range, likely not relevant for human exposure to HAAs through smoking or the diet. Trp-P-1 and Trp-P-2 additionally failed to trans-stimulate OCT1 and OCT2 activities and exhibited similar accumulation in OCT1/2-transduced HEK293 cells and control HEK293-MOCK cells. These data demonstrate that HAAs, notably Trp-P-1 and Trp-P-2, interact with OCT1/2, without however being transported, thus likely discarding a major role for OCT1/2 in HAA systemic toxicokinetics.

Introduction

Heterocyclic aromatic amines (HAAs) are chemicals containing at least one heterocyclic ring, as well as at least one amine (nitrogen-containing) group; typically, it is a nitrogen atom of an amine group that also makes the ring heterocyclic. Various HAAs can be found in cooked meat and cigarette smoke and exert mutagenic and carcinogenic effects for most of them (Sugimura et al., 2004; Talhout et al., 2011; Turesky and Le Marchand, 2011). Such HAAs belong to two main classes: pyrolytic/aminocarbolins and thermic/aminoimidazoarenes (Cheng et al., 2006). Pyrolytic HAAs come from high-temperature (>250 °C)-related pyrolysis of proteins or amino acids and notably comprise 2-amino-9H-pyrido[2,3-b]indole (AαC), the major carcinogenic HAA found in tobacco smoke (Zhang et al., 2011), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-6-methyl[1,2-a:3′,2“-d]imidazole (Glu-P-1), 2-aminodipyrido[1,2-a:3’,2”-d]imidazole (Glu-P-2), 3-amino-1,4-dimethyl-5H-pyrido [4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]-indole (Trp-P-2) and the non-mutagenic β-carbolins methyl-9H-pyrido[3,4-b]indole (Harmane) and 9H-pyrido[3,4-b]indole (Norharmane). Thermic/aminoimidazoarenes HAAs such as 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) and 2-amino-3-methyl-imidazo [4, 5-f] quinoline (IQ) are formed at lower temperature (150–250 °C) than pyrolytic HAAs, through notably the Maillard reaction between hexoses and amino acids during meat cooking (Turesky, 2007).

To be mutagenic or carcinogenic, dietary and environmental HAAs require metabolic activation (Turesky, 2002). In humans, this occurs primarily in the liver through the phase I enzyme cytochrome P-450 (CYP) 1A2-mediated N-oxidation of the exocyclic amine groups of HAAs, to form N-hydroxy-HAA derivatives (Langouet et al., 2001). In extrahepatic tissues, CYP1A1 and CYP1B1 contribute also to the bioactivation of HAAs (Shimada et al., 1996). Subsequent acetylation or sulfation of N-hydroxy-HAAs by some phase II drug metabolizing enzymes like N-acetyl-transferase or sulfo-transferase produces highly unstable esters, which react with DNA to form mutagenic adducts, through notably the formation of arylnitrenium ions (Turesky and Le Marchand, 2011). These reactive species can however be detoxified by glutathione -S-transferases (Coles et al., 2001), whereas other phase II conjugating enzymes such as UDP-glucuronosyl-transferases directly inactivate N-hydroxy-HAAs (Stillwell et al., 1999).

In addition to drug metabolizing enzymes, ATP-binding cassette (ABC) drug transporters can participate to disposition and detoxification of HAAs. Indeed, PhIP and its metabolites are eliminated in vivo by the ABC efflux pumps P-glycoprotein (P-gp/ABCB1), encoded by the multidrug resistance gene 1 (MDR1), multidrug resistance-associated protein 2 (MRP2/ABCC2), and breast cancer resistance protein (BCRP/ABCG2) (van Herwaarden et al., 2003; Walle and Walle, 1999). BCRP also handles Trp-P-1 and IQ (van Herwaarden et al., 2006). Because ABC transporters are notably localized at the apical domain of enterocytes and at the canalicular domain of hepatocytes, they likely reduce systemic exposure to HAAs by preventing their intestinal absorption and by contributing to their biliary elimination (Dietrich et al., 2001; Vlaming et al., 2014). Besides ABC efflux pumps, solute carrier (SLC) transporters, which mainly mediate drug uptake into cells through facilitated diffusion or secondary active transport (Giacomini et al., 2010), are presumed to also interact with HAAs. Indeed, PhIP and AαC have been shown to block activities of the renal organic anion transporter (OAT) 3 (SLC22A8), without probably being handled by this transporter (Sayyed et al., 2017); OAT3 activity is also inhibited by Trp-P-2, whereas PhIP blocks that of OAT1 (SLC22A6). Trp-P-1 and Trp-P-2 have additionally been postulated to be transported by the dopamine active transporter (DAT/SLC6A3) and the serotonin transporter (SERT/SLC6A4) (Hashimoto et al., 2002; Naoi et al., 1989). Whether HAAs may interact with other SLC drug transporters remains unknown, but is probably important to determine, owing to the now well-established role of SLC transporters in xenobiotic disposition (Zhou et al., 2017). In this context, it is likely important to consider the putative interactions of HAAs with organic cation transporters (OCTs) (Jonker and Schinkel, 2004), such as OCT1 (SLC22A1) and OCT2 (SLC22A2), expressed mainly at the basolateral pole of hepatocytes and proximal tubular cells, respectively, and OCT3 (SLC22A3), found in various tissues and organs. Indeed, such transporters handle various amine drugs and biogenic amines (Nies et al., 2011b), thus suggesting that they may interact with additional amines such as HAAs. The fact that dopamine, a physiological amine substrate for OCT1 and OCT2 (Grundemann et al., 1999), can inhibit cellular uptake of Trp-P-1 and Trp-P-2 (Hashimoto et al., 2002) likely supports this hypothesis. The present study was therefore designed to determine whether HAAs may interact with OCTs using mainly OCT1-, OCT2- and OCT3-overexpressing cells. Our data demonstrate that various HAAs can inhibit activities of OCTs. It is notably the case for the pyrolytic HAAs Trp-P-1 and Trp-P-2, which inhibit activities of OCT1, OCT2 and OCT3, without however being transported by OCT1 and OCT2, thus making unlikely a contribution of these SLC transporters to their toxicokinetics.