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This study was designed to investigate the expression of different isoenzymes of glutathione transferase (GST) in the rat ovary and to follow possible changes in the pattern of expression during maturation and the different stages of the oestrus cycle. The GST subunits present in the rat ovary (as analyzed by HPLC of affinity-purified GSTs) were A3, A4, M1, M2, M3 and P1. The most abundant subunit in the ovary was A3, but the mu enzymes demonstrated the largest increase (3.5-fold) when the mature ovary was compared to the immature organ. An overall decrease in GST isoenzyme content during dioestrus was observed, but there were no other recurrent changes during the oestrus cycle. Treatment of immature rats with pregnant mare's serum gonadotropin clearly demonstrated that the mu and alpha isoenzymes are up-regulated by gonadotropin stimulation, i.e. a 4-6 fold increase was seen. This treatment also elevated the P1 subunit 3-fold. At present, it is only possible to speculate concerning the mechanism(s) underlying these variations in ovarian GST expression in connection with hormonal changes. The functional significance of these variations is not yet known.

A novel protein that binds to a glutathione-Sepharose affinity column has been detected in mature, but not immature, rat ovary. This protein could be resolved from all identifiable components when the affinity-purified material, containing primarily glutathione transferases, was analyzed on reversed phase-HPLC. The unidentified protein migrated with an apparent molecular weight of 34 kDa on SDS-PAGE. After purification by affinity chromatography and subsequent preparative electrophoresis, the protein was subjected to N-terminal amino acid sequence analysis. The sequence obtained demonstrated a high degree of homology with an internal amino acid sequence in human carbonyl reductase (EC 1.1.1.184).

The capacity of ovarian granulosa cells from rat and pig to release reactive metabolites produced from polycyclic aromatic hydrocarbons with the ability to cause mutations in neighbouring cells has been studied. For this purpose we have used cocultivation with V79 Chinese hamster cells as a detection system. The cells were treated with two different polycyclic aromatic hydrocarbons (PAHs), 7,12-dimethylbenz(a)anthracene (DMBA) or (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol). The resulting mutation frequency in the V79 cells after cocultivation, as a function of granulosa cell number, DMBA, or BP-7,8-diol concentration and time, was determined. The progesterone concentration in the medium after cocultivation was also analyzed as a measure of the differentiation of the granulosa cells. These studies demonstrate an increasing frequency of mutations in the V79 cells with an increasing number of granulosa cells. The maximal number of mutations were achieved with a DMBA or BP-7,8-diol concentration of 5 or 2 microM, respectively. The optimal cocultivation time was 24 h. These results clearly show that the granulosa cells can bioactivate PAHs to reactive metabolites with the capacity to migrate into surrounding cells and cause mutations in these cells. Compounds metabolized to mutagenic products by granulosa cells might thus cause mutations in the neighbouring germ cells, with possible consequences for the offspring.

Alterations in cellular GSH and its compartmentation were investigated as a possible mechanism of toxicity of the anthracycline derivative daunorubicin in neonatal heart cells. Cultured beating heart cells from neonatal rats were exposed to daunorubicin at therapeutically relevant concentrations and the resulting changes in cellular GSH as well as cytosolic and mitochondrial pools of GSH were determined. Toxicity was estimated as an increased permeability of the plasma membrane to cytosolic enzymes, e.g., lactate dehydrogenase. Control heart cells were found to contain 12.2 +/- 1.8 nmoles GSH/10(6) cells. Daunorubicin caused a rapid initial decrease followed by a transient increase in cellular GSH. The extent of the latter increase was dependent on the concentration of daunorubicin. High concentrations of daunorubicin gave only a slight increase followed by a pronounced decrease in cellular GSH. By applying a digitonin-based method the effect of daunorubicin on the cytosolic and mitochondrial pools of GSH were separated. The concentration of cytosolic and mitochondrial reduced GSH was estimated to be 8.9 +/- 1.5 nmoles/10(6) cells and 3.3 +/- 0.6 nmoles/10(6) cells, respectively. The results indicate that daunorubicin caused a decrease of cytosolic GSH and, after a short lag period, a release of alctate dehydrogenase. No decrease of mitochondrial GSH occurred under these conditions indicating that daunorubicin influences selectively cytosolic GSH. No lipid peroxidation products were detected in DRB-treated cells under conditions when lactate dehydrogenase was released. Likewise, addition of the iron-chelator desferrioxamin did not influence the release of lactate dehydrogenase, whereas dithiothreitol offered partial protection.(ABSTRACT TRUNCATED AT 250 WORDS) Free Radic Res Commun