Differential toxicity as a result of apical and basolateral treatment of LLC-PK1 monolayers with S-(1,2,3,4,4-pentachlorobutadienyl)glutathione and N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine.
Monolayers of LLC-PK1 cells, a cell line with features typical of proximal tubular epithelial cells, were treated at the apical and basolateral side with S-(1,2,3,4,4-pentachlorobutadienyl)glutathione (PCBD-GSH) and N-acetyl-S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBD-NAC). Apical treatment with PCBD-GSH (greater than 20 microM) resulted in cytotoxicity, which could be inhibited by acivicin and aminooxyacetic acid (AOAA), inhibitors of gamma-glutamyltranspeptidase (gamma GT) and beta-lyase respectively. In contrast apical treatment with PCBD-NAC was only toxic at high concentrations (greater than 850 microM), and this effect could hardly be inhibited by AOAA. Basolateral treatment of confluent LLC-PK1 monolayers, grown on porous membranes, with PCBD-GSH gave a much smaller response than apical treatment, consistent with the fact that gamma GT is predominantly present at the apical side. Basolateral treatment even with high concentrations of PCBD-NAC (1.1 mM) did not show an increase in cytotoxicity when compared to the effect after apical treatment. These results suggest the absence of an organic anion transporter, by which these conjugates in vivo are transported into the cells from the basolateral side. This supposition was substantiated in a study of transcellular transport of the model ions tetraethyl ammonium (TEA) and para-aminohippurate (PAH), in LLC-PK1 monolayers, grown as indicated above. No active PAH transport could be demonstrated, whereas an active TEA transport was present. The absence of an organic anion transporter limits the usefulness of LLC-PK1 cells for the study of nephrotoxicity of compounds, like PCBD-NAc, needing this transport to enter the cells. However, the finding of an active basolateral organic cation transporter, together with the presence of gamma GT, dipeptidase and beta-lyase, makes this system especially interesting for testing all compounds that use this transporter or these enzymes in order to elicit toxicity.
Mertens
J J
JJ
Dept. of Toxicology, Agricultural University Wageningen, The Netherlands.
Weijnen
J G
JG
van Doorn
W J
WJ
Spenkelink
B
B
Temmink
J H
JH
van Bladeren
P J
PJ
D000111:Acetylcysteine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000633:toxicity; D000625:Aminooxyacetic Acid / Q000494:pharmacology; D000818:Animals; D027321:Anion Transport Proteins; D000838:Anions; D001692:Biological Transport; D002070:Butadienes / Q000008:administration & dosage / Q000494:pharmacology / Q000633:toxicity*; D002352:Carrier Proteins / Q000378:metabolism; D002412:Cations; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D005978:Glutathione / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000633:toxicity; D007555:Isoxazoles / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D007674:Kidney Diseases / Q000139:chemically induced*; D013552:Swine; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000378:metabolism; D010130:p-Aminohippuric Acid / Q000378:metabolism
D027321:Anion Transport Proteins; D000838:Anions; D002070:Butadienes; D002352:Carrier Proteins; D002412:Cations; D007555:Isoxazoles; D013757:Tetraethylammonium Compounds; D000625:Aminooxyacetic Acid; D019789:Tetraethylammonium; C050500:S-(1,2,3,4,4-pentachloro-1,3-butadienyl)glutathione; C049739:N-acetyl-S-pentachloro-1,3-butadienylcysteine; D005978:Glutathione; C006303:acivicin; D000111:Acetylcysteine; D010130:p-Aminohippuric Acid