Evaluation of oxidative damage and renal distal tubule cell stress response following exposure to lindane

Lindane, or γ-hexachlorocyclohexane, is a chlorinated hydrocarbon pesticide that was banned from U.S. production in 1976, but until recently continued to be imported and applied for occupational and domestic purposes. Lindane is known to cause central nervous system (CNS), immune, cardiovascular, reproductive, liver, and kidney toxicity. The mechanism for which lindane interacts with the CNS has been elucidated, and involves antagonism of the γ-aminobutyric acid/benzodiazepine (GABAA/BZD) receptor. Antagonism of this receptor results in the inhibition of Cl- channel flux, with subsequent convulsions, seizures, and paralysis. This response makes lindane a desirable defense against arthropod pests in agriculture and the home. However, formulation and application of this compound can contribute to human toxicity. In conjunction with this exposure scenario, workers may be subject to both heat and physical stress that may increase their susceptibility to pesticide toxicity by altering their cellular stress response. The kidneys are responsible for maintaining osmotic homeostasis, and are exposed to agents that undergo urinary excretion. The mechanistic action of lindane on the kidneys is not well understood. Lindane, in other organ systems, has been shown to cause cellular damage by generation of free radicals and oxidative stress. Previous research in our laboratory has shown that lindane causes apoptosis in distal tubule cells, and delays renal stress response under hypertonic stress. Characterizing the mechanism of action of lindane under conditions of physiologic stress is necessary to understand the potential hazard cyclodiene pesticides and other organochlorine compounds pose to exposed individuals under baseline conditions, as well as under conditions of physiologic stress. We demonstrated that exposure to lindane results in oxidative damage and dysregulation of glutathione response in renal distal tubule (MDCK) cells. We showed that under conditions of hypertonic stress, lindane-induced oxidative stress resulted in early onset apoptosis and corresponding down-regulated expression of the anti-apoptotic protein, Bcl-xL. Thus, the interaction of lindane with renal peripheral benzodiazepine receptors (PBR) is associated with attenuation of cellular protective proteins, making the cell more susceptible to injury or death. ^

The characterization of an epithelial chloride channel and purification of a channel component

A membrane fraction (M$\sb{\rm PS}$), enriched in Cl$\sp-$ channels, has been isolated from bovine tracheal epithelia and renal cortex homogenates by hydrophobic chromatography. The tracheal fraction shows a 37 fold enrichment of Cl$\sp-$ channels over crude tracheal homogenates by net Cl$\sp-$ measurements in membrane vesicles. Alkaline phosphatase and (Na$\sp+$ + K$\sp+$)-ATPase are not found in these membranes, suggesting that they are not apical or basolateral plasma membranes. The M$\sb{\rm PS}$ fraction exhibits a protein profile unlike that of other membrane fractions with major proteins of 200 kDa and 42 kDa, proteins of 30 to 35 kDa, and lesser amounts of other proteins. Reconstitution of M$\sb{\rm PS}$ fractions from both trachea and kidney into planar lipid bilayers demonstrates the presence of a single type of anion channel. The current-voltage relationship of this channel is linear with a slope conductance of 84 pS in symmetrical 400 mM KCl, and is identical to that of the predominant anion channel observed in tracheal apical membranes under similar conditions (Valdivia, Dubinsky, and Coronado. Science, 1988). In addition, the voltage dependence, selectivity sequence of Cl$\sp- >$ Br$\sp- \ge$ I$\sp-$, and inhibition by low concentrations of the Cl$\sp-$ channel blocker, DIDS, correspond to those of the predominant apical membrane channel. Thus, although the M$\sb{\rm PS}$ fraction appears to be of subcellular origin, it may be functionally related to an apical membrane Cl$\sp-$ permeability. When renal M$\sb{\rm PS}$ membranes were treated with the detergent octyl-glucoside (OG, 2%) and centrifuged, the supernatant, sM$\sb{\rm PS}$, showed a 2 to 7-fold enrichment in specific Cl$\sp-$ flux activity compared with the detergent treated M$\sb{\rm PS}$. These solubilized proteins were then size fractionated on a Superose 12 HPLC gel filtration column, followed by fractionation on a Mono Q HPLC anion exchange column. Fractions that eluted in high salt consistently exhibited significant Cl$\sp-$ flux activity. These fractions had protein profiles consisting of a major band at 34 kDa, a band at 66 kDa, and variable faint bands. Fractions eluting in lower salt had protein profiles consisting of a single band at 34 kDa, and often had little or no Cl$\sp-$ flux activity. However, co-reconstitution of the low salt, solely 34 kDa protein-containing Mono Q fractions with sM$\sb{\rm PS}$ resulted in an enhancement of flux activity compared to that of sM$\sb{\rm PS}$ reconstituted alone. Flux assays of active Mono Q fractions showed that the channel retained its DIDS sensitivity. Applying sM$\sb{\rm PS}$ to a DIDS-affinity column and eluting with salt resulted in fractions with protein profiles again consisting of at least one major band at 34 kDa, a band at 66 kDa, and variable faint bands. Co-reconstitution with sM$\sb{\rm PS}$ again resulted in an enhancement of activity. Thus, the 34 kDa protein appears to be a component of the M$\sb{\rm PS}$ Cl$\sp-$ channel. ^