Evaluation of the induction of rat hepatic and pancreatic glutathione S-transferases by treatment with the cruciferous nitrile, cyanohydroxybutene

Cyanohydroxybutene (CHB) is a nitrile derived from glucosinolates commonly found in cruciferous plants. Glucosinolate derivatives are believed to provide protection against carcinogens by inducing detoxifying including the glutathione S-transferase isoenzymes (GSTs). The GSTs catalyze the conjugation of glutathione (GSH) to electrophilic xenobiotics. As peroxidases, they detoxify organic hydroperoxides. Each isoenzyme has a unique level of activity in detoxifying xenobiotics, including certain carcinogens. The amount of individual isoenzymes within tissues and cells, particularly in response to treatment with inducers, is important when considering mechanisms of cancer chemoprevention and chemotherapy. At 200 mg/kg per os, CHB is selectively toxic to the rat exocrine pancreas. At this and non-toxic doses, CHB causes the sustained elevation of GSH in the liver and pancreas. The elevation, which follows a brief depletion phase, is associated with the induction of the hepatic GSH synthesizing enzyme, $\gamma$-glutamylcysteine synthetase, and an increase in the cellular uptake of GSH precursors. CHB also induces hepatic and pancreatic GST and quinone reductase activities and hepatic GSH reductase activity.Because elevated GST activity does not reflect individual isoenzyme induction and because pancreatic GST induction has not been evaluated thoroughly, objectives here were (1) to characterize rat pancreatic GSTs by chromatography and immunohistochemistry (IHC) and (2) to use these methods for assessing the CHB-mediated induction of rat hepatic and pancreatic GSTs. Finally, because initial GSH depletion by CHB may instigate enzyme induction, (3) the depletion of GSH by CHB in vitro was investigated.GSTs were extracted from the pancreatic cytosol of male Fisher 344 rats by affinity chromatography (AC), and the isoenzyme and subunit content was assessed by chromatofocusing, SDS-PAGE, and reverse phase HPLC. Rats were dosed by gavage with either (1) 100 mg CHB/kg/day or (2) 50 mg CHB/kg/day for 7 days. Hepatic and pancreatic GSH levels and GST activity toward chlorodinitrobenzene (CDNB) were measured. Hepatic GSTs in (1) and pancreatic GSTs in (2) were extracted by AC and assessed for subunit content by HPLC. GSH and GST activity were elevated 1.3- to 2.0-fold over control values. Hepatic GST subunit 4 was induced less than subunits 1, 2, and 3, while pancreatic GST subunit 2 was induced more than subunits 3, 4, 7, and 8. Pancreatic subunit 6 was not induced, and subunit 1 was slightly induced. IHC did not demonstrate any difference between the hepatic and pancreatic GSTs of CHB-treated and control rats; GST induction apparently occurred within cells that normally contain GSTs.GSH, with or without rat hepatic cytosol and microsomes, was incubated with CHB at 37$\sp\circ$C. GSH concentrations were measured from 0 to 3 hours, and oxidized GSH was measured at 3 hours. CHB oxidized GSH non-enzymatically. Oxidation was prevented by the addition of both cytosol and an NADPH generating system. This suggested that GSH oxidation by CHB might not have been detected in vivo because of cytosolic GSH reductase activity. (Abstract shortened by UMI.)U of I OnlyETDs are only available to UIUC Users without author permissio