Factors affecting the efflux of paraquat from rat lung slices

Abstract

In an attempt to reduce the toxicity of paraquat several compounds were examined for their ability to increase the rate of efflux of paraquat from the lung. The compounds were selected because they were known, from in vitro studies, to reduce the accumulation of paraquat into the lung. Histamine (100 μM), promethazine (100 μM), putrescine (100 μM), bromthymol blue (300 μM) and the metabolic inhibitors iodoacetate (1 mM), rotenone (100 μM) and KCN (1 mM) have been shown to reduce the accumulation of paraquat into rat lung slices, as did the incubation of slices under nitrogen. The efflux of paraquat from lung slices prepared from rats dosed intravenously with paraquat was biphasic, having a very fast component and a slow component. The slow component was first order and was characterised by a t 1 2 of 17 h. This half life is similar to that seen in vivo (24h) following intravenous dosing. When lung slices prepared from rats dosed intravenously with paraquat were incubated in the presence of iodoacetate (1 mM) or under nitrogen, the half life of paraquat in the slices was reduced to approximately 3 h. In the presence of rotenone (100 μM) it was reduced to approximately9 h. Histamine (100 μM) and promethazine (100 μM) did not affect the efflux of paraquat from lung slices. Bromthymol blue, a dye which forms “ion pair” complexes with paraquat, also significantly increased the efflux of paraquat from lung slices. The effect of bromthymol blue, however, decreased with time and thus paraquat efflux in the presence of bromthymol blue did not obey first order kinetics. In order to measure cellular viability of lung slices, oxygen comsumption, glucose oxidation and the rate of the efflux of protein from the slices into the incubation medium were determined. Iodoacetate (1 mM) and rotenone (100 μM) almost abolished oxygen consumption and glucose oxidation whereas these activities were inhibited to a lesser extent by bromthymol blue (300 μM) (18% and 30%, respectively). During the first 30 min of incubation in the presence of KCN (1 mM) oxygen consumption was almost abolished but between 30 min and 4 h returned to control levels. The effect of KCN could therefore be divided into 2 phases. Over 4 h incubation glucose oxidation was inhibited by 36%. Iodoacetate (1 mM) and incubation under nitrogen caused the most pronounced increases in the rate of protein efflux from slices. KCN (1 mM) and rotenone (100 μM) also increased the rate of protein efflux but to a lesser extent. We have therefore suggested that the effect of KCN (1 mM) on cellular viability, while severe, may be less than that of iodoacetate (1 mM) or incubation under nitrogen. We have concluded from these studies that: (1) the reduction in the accumulation of paraquat or increase in its efflux produced by metabolic inhibitors may be a consequence of lung cell damage; (2) bromthymol blue and putrescine cause an increase in the efflux of paraquat from the lung in vitro without damaging the tissue suggesting that in principle this approach may be useful in vivo; and (3) histamine and promethazine reduce the uptake of paraquat into the lung by direct competition with paraquat since they neither increase the rate of efflux of paraquat from the lung nor reduce cellular viability.