L-THP attenuated OXA-induced toxicity and reduced the accumulation of OXA in OCT2- and OCTN1-transfected cells
Our previous study demonstrated that L-THP was a strong inhibitor of OCT2, we studied the inhibitory effect of L-THP on OXA-induced toxicity in mock and MDCK-hOCTN1 cells, as well as MDCK-hOCT2 cells. As we described above, 40 μM of OXA reduced the cell viability to 76%, 50%, and 12% of the control (vehicle treatment) in the mock, MDCK-hOCTN1, and MDCK-hOCT2 cells, respectively, while the LDH activities in the medium were increased to 1.1, 1.7, and 4.0 folds, respectively. In the MDCK-hOCTN1 and MDCK-hOCT2 cells, 1-100 μM of L-THP attenuated OXA-induced toxicity in a concentration-dependent manner, with increase in cell viability and reduction of LDH leakage in the cell medium; particularly, in the MCDK-OCT2 cells, the cell viability was increased to 64% (vs 12%) and the LDH was reduced to half of that in the OXA group (Figure 2a-2f ).
On the other hand, the cellular accumulation of OXA (40 μM, 30 min incubation) in the MDCK-hOCTN1 and MDCK-hOCT2 cells was approximately 2.0 and 5.4 folds that in mock cells, respectively, whereas co-incubation with 100 μM L-THP reduced the OXA accumulation in the MDCK-hOCTN1 and MDCK-hOCT2 cells to 1.1 and 2.8 folds that in the mock cells (Figure 3 ). The results indicate that L-THP significantly inhibits OCT2- and OCTN1-mediated OXA uptake, exhibiting a considerably stronger inhibition of hOCT2 than hOCTN1, which implies that L-THP can significantly inhibit OCT2- and OCTN1-mediated OXA uptake and subsequently attenuate OXA-induced toxicity.
L-THP did not inhibit cellular accumulation of OXA in MDCK-hMRP2
Since MRP2 is the most important transporter mediating the efflux of OXA from DRG cells, we evaluated the effect of L-THP on MRP2 to elucidate whether L-THP would reduce OXA efflux by inhibiting MRP2. As shown inFigure 4 , the accumulation of OXA in MDCK-MRP2 cells was approximately half of that in the mock cells, and 1-100 μM L-THP did not increase the accumulation, which indicates that L-THP does not decrease OXA efflux by inhibition of MRP2.