title: Several transport systems contribute to the intestinal uptake of Paraquat, modulating its cytotoxic effects.
abstract: Paraquat (PQ) is an extremely toxic herbicide upon oral ingestion that lacks a specific antidote. In case of intoxication, treatment primarily relies on limiting its intestinal absorption. In this study, we elucidate the intestinal transport mechanisms of PQ uptake using Caco-2 cells as a model of the human intestinal epithelium. The cells were incubated with a wide range of PQ concentrations (0-5000μM) for 24h with or without simultaneous exposure to different transporters substrates/inhibitors including, choline or hemicolinium-3 (for choline carrier-mediated transport system inhibition) and putrescine, trifluoperazine, valine, lysine, arginine or N-ethylmaleimide (for basic amino acid transport systems inhibition). PQ cytotoxicity was evaluated by the MTT reduction assay and correlated with PQ intracellular levels quantified by gas chromatography-ion trap-mass spectrometry (GC-IT/MS). Potential interactions of PQ with the substrates/inhibitors of the transport systems were investigated and discarded by infrared spectroscopy. Our results showed a significant reduction in PQ intracellular accumulation and, consequently, in PQ cytotoxicity, in the presence of both choline and hemicolinium-3, demonstrating that the choline carrier-mediated transport system is partially involved in PQ intestinal uptake. Likewise, PQ cytotoxicity and intracellular accumulation were significantly attenuated by simultaneous exposure to putrescine, trifluoperazine, valine, lysine, arginine and N-ethylmaleimide. These data suggested the involvement of more than one of the basic amino acids transport systems, including the y(+), b(0,+) or y(+)L systems. In conclusion, this study demonstrated that several transport systems mediate PQ intestinal absorption and, therefore, their modulation may provide alternative efficient pathways for limiting PQ toxicity in intoxication scenarios.
authors.affiliation: REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal. Electronic address: rsilva@ff.up.pt.
authors.forename: Renata
authors.initials: R
authors.lastname: Silva
authors.affiliation: REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal. Electronic address: helenacarmo@ff.up.pt.
authors.forename: Helena
authors.initials: H
authors.lastname: Carmo
authors.affiliation: REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal. Electronic address: vvilasboas@ff.up.pt.
authors.forename: Vânia
authors.initials: V
authors.lastname: Vilas-Boas
authors.affiliation: REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal. Electronic address: daniel.barbosa@ff.up.pt.
authors.forename: Daniel José
authors.initials: DJ
authors.lastname: Barbosa
authors.affiliation: REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal. Electronic address: aicram_monteiro@hotmail.com.
authors.forename: Márcia
authors.initials: M
authors.lastname: Monteiro
authors.affiliation: REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal. Electronic address: pguedes@ff.up.pt.
authors.forename: Paula Guedes
authors.initials: PG
authors.lastname: de Pinho
authors.affiliation: REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal. Electronic address: mlbastos@ff.up.pt.
authors.forename: Maria
authors.initials: M
authors.lastname: de Lourdes Bastos
authors.affiliation: REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal. Electronic address: remiao@ff.up.pt.
authors.forename: Fernando
authors.initials: F
authors.lastname: Remião
mesh_terms: D026962:Amino Acid Transport System ASC / Q000378:metabolism; D027182:Amino Acid Transport System y+ / Q000378:metabolism; D027241:Amino Acid Transport System y+L / Q000378:metabolism; D018938:Caco-2 Cells; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D008401:Gas Chromatography-Mass Spectrometry; D006540:Herbicides / Q000378:metabolism* / Q000633:toxicity; D006801:Humans; D007408:Intestinal Absorption; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D007700:Kinetics; D049990:Membrane Transport Modulators / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000187:drug effects / Q000378:metabolism*; D015778:Minor Histocompatibility Antigens / Q000378:metabolism; D010269:Paraquat / Q000378:metabolism* / Q000633:toxicity; D013055:Spectrophotometry, Infrared
chemical_list: D026962:Amino Acid Transport System ASC; D027182:Amino Acid Transport System y+; D027241:Amino Acid Transport System y+L; D006540:Herbicides; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D015778:Minor Histocompatibility Antigens; C420506:SLC1A5 protein, human; C065637:choline transporter; D010269:Paraquat
keywords: Arginine; Choline; Lysine; Paraquat intestinal uptake; Putrescine; y(+) transport system