RBE4 cells are highly resistant to paraquat‐induced cytotoxicity: studies on uptake and efflux mechanisms

Abstract

Paraquat (PQ) is a widely used, highly toxic and non-selective contact herbicide, which has been associated with central neurotoxic effects, namely the development of Parkinson's disease, but whose effects to the blood-brain barrier (BBB) itself have rarely been studied. This work studied the mechanisms of PQ uptake and efflux in a rat's BBB cell model, the RBE4 cells. PQ is believed to enter cells using the basic or neutral amino acid or polyamine transport systems or through the choline-uptake system. In contrast, PQ efflux from cells is reported to be mediated by P-glycoprotein. Therefore, we evaluated PQ-induced cytotoxicity and the effect of some substrates/blockers of these transporters (such as arginine, L-valine, putrescine, hemicholinium-3 and GF120918) on such cytotoxicity. RBE4 cells were shown to be extremely resistant to PQ after 24 h of exposure; even at concentrations as high as 50 mM approximately 45% of the cells remained viable. Prolonging exposure until 48 h elicited significant cytotoxicity only for PQ concentrations above 5 mM. Although hemicholinium-3, a choline-uptake system inhibitor, significantly protected cells against PQ-induced toxicity, none of the effects were observed for arginine, L-valine or putrescine. Meanwhile, inhibiting the efflux pump P-glycoprotein using GF120918 significantly enhanced PQ-induced cytotoxicity. In conclusion, PQ used the choline-uptake system, instead of the transporters for the basic or neutral amino acids or for the polyamines, to enter RBE4 cells. P-glycoprotein extrudes PQ back to the extracellular medium. However, this efflux mechanism only partially explains the observed RBE4 resistance to PQ.

An Interspecies Molecular and Functional Study of Organic Cation Transporters at the Blood-Brain Barrier: From Rodents to Humans

“…Knowing that the concentration/extent of a compound within the ISF, although primarily controlled by the BBB, is also dependent of the transport processes present at the level of glial and neuronal cell membranes, as known at the synaptic cleft, the presence of OCTs on these cells appears to more likely support the pharmacokinetic-pharmacodynamic interspecies variability for OCT substrates for the brain tissue. Indeed, our results support that the absence of OCTs/MATE1 at the BBB helps in the prevention of the brain uptake of their substrates like MPP + or paraquat, even though paraquat may be able to reach the CNS via LAT1 (SLC7A5) BBB transport [4,5], it is also extruded from the CNS wherever a BBB lies, probably via P-gp, since paraquat is also a recognized substrate of this ABC efflux transporter [51][52][53].…”

Cellular Senescence Is Induced by the Environmental Neurotoxin Paraquat and Contributes to Neuropathology Linked to Parkinson’s Disease

“…It is theoretically possible that senescence induction in peripheral tissues could contribute to PQ-mediated brain pathologies, but this seems unlikely, as the chronic, low-dose PQ regimen utilized in our studies is known to result in receptor-mediated uptake and immune response in the brain ( McCormack and Di Monte, 2003 ) without activating peripheral immune cells or disrupting blood-brain barrier integrity ( Vilas-Boas et al, 2014 ; Watson et al, 2013 ).…”

Development of Novel Rifampicin-Derived P-Glycoprotein Activators/Inducers. Synthesis, In Silico Analysis and Application in the RBE4 Cell Model, Using Paraquat as Substrate

“…A simultaneous exposure to RedRif and PQ for 48 h was also performed, to evaluate P-gp activation effects. We recently reported that RBE4 cells are highly resistant to PQ toxicity, which implies that all PQ exposures need to last 48 h, the time necessary for PQ to induce a significant toxic effect on RBE4 cells [33]. RedRif significantly protected RBE4 cells against PQ-induced cytotoxicity.…”

“…Three days after seeding, cells were treated with 0.1 to 50 µM of Rif, MeORif, PerAcRif or RedRif, and cytotoxicity was evaluated after 24, 48 and 72 h by the NR uptake assay and by the MTT reduction assay. PQ cytotoxicity profile in this cell line has been previously established [33]. Each experiment was performed in triplicate and independently repeated at least 3 times.…”