Proposal for fluorescence-based in vitro assay using human and zebrafish monoamine transporters to detect biological activities of antidepressants in wastewater

摘要

Antidepressants are among the most commonly detected Pharmaceuticals in the aquatic environment. As they modulate neurotransmission in nervous systems, behavioural abnormalities among aquatic species are of concern. It is possible to measure the concentrations of selected antidepressants by chemical analysis, but other non-target antidepressants and active metabolites might also be present. Here, we propose an "in vitro monoamine transporter inhibition assay" to measure the biological activity of antidepressants, particularly monoamine transporter inhibitors, in wastewater. We used APP, a fluorescent substrate for monoamine transporters, to measure the activity of wastewater extracts at inhibiting APP uptake through the human serotonin transporter (hSERT), norepinephrine transporter (hNET), and dopamine transporter, and the zebrafish SERT (zSERT). We confirmed that the assay could measure the biological activity of test antidepressants. Interestingly, the IC_(50) values of antidepressants (the concentration that gave a 50% reduction of APP uptake) for the zSERT were smaller than those for the hSERT. For example, IC_(50) value of desipramine for the zSERT was 1 /200 of that for the hSERT. These results indicate that antidepressants inhibited zSERT more strongly than hSERT. Then we applied the assay to extracts of effluent from municipal wastewater treatment plants and detected biological activity of antidepressants specifically against the hSERT, hNET, and zSERT for the first time. For the hSERT, antidepressant-equivalent quantities (EQs) ranged from 2.2 × 10~1 to 2.5 × 10~2 ng-clomipramine-EQ/L For the hNET, EQs ranged from below limit of detection to 8.2 × 10~1 ng-desipramine-EQ/L For the zSERT, EQs ranged from 2.8 × 10~2 to 3.3 × 10~2 ng-duloxetine-EQ/L. The in vitro monoamine transporter inhibition assay is thus useful for measuring the biological activity of antidepressants in the aquatic environment