The cholestatic agent, α-naphthylisothiocyanate, stimulates superoxide release by rat neutrophils in vitro

Studies in rats indicate that neutrophils (polymorphonuclear leukocytes (PMNs) are associated with areas of tissue damage after treatment with the hepatotoxicant, α-naphthylisothiocyanate (ANIT). Several synthetic and naturally occurring substances stimulate PMNs to release cytotoxic mediators, such as superoxide (0₂^-). The purpose of the present study was to test the hypothesis that ANIT stimulates the release of 0₂^- from isolated rat PMNs. PMNs derived from rat peritoneum were treated with ANIT in vitro and tested for release of 0₂^-. ANIT caused the release of 0₂^- from PMNs in a concentration-dependent manner. Maximal 0₂^- release (10 ± 1 nmoles/30 minutes/2 x 10⁶ cells) was achieved by an ANIT concentration of 110 μM. This ANIT-induced 0₂^- release was significantly reduced or blocked completely by preincubation of PMNs for 10 minutes with 10 μM or 100 μM SKF 525A, respectively. The β-isomer of ANIT, which does not cause cholestasis in vivo, did not stimulate 0₂^- release. ANIT-stimulated 0₂^- production decreased sharply after 5 minutes of incubation with ANIT and ceased entirely between 10 to 15 minutes. Shortly after this decrease in 0₂^- production was an increase in the extracellular activity of lactate dehydrogenase. PMNs exposed to ANIT also failed to exclude trypan blue dye, either in the presence or in the absence of superoxide dismutase and catalase, suggesting a direct, oxygen radical-independent, cytotoxic effect of ANIT on PMNs. Release of the lysosomal enzyme, β-glucuronidase, occurred within 5 minutes of incubation of isolated PMNs with ANIT (110 μM). These results indicate that exposure of rat PMNs to the hepatotoxicant, ANIT, causes the release of cytotoxic agents, whereas its less hepatotoxic β-isomer does not.