Protective role for ovarian glutathione S-transferase isoform pi during 7,12-dimethylbenz[a]anthracene-induced ovotoxicity

Abstract

7,12-dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles at all developmental stages. This study investigated a role for the glutathione S-transferase (Gst) isoforms alpha (a), mu (m) and pi (p) and the transcription factors, Ahr and Nrf2, during DMBA-induced ovotoxicity, and their regulation by phosphatidylinositol-3 kinase (PI3K) signaling. Negative regulation of JNK by GSTP during DMBA exposure was also studied. Post-natal day (PND) 4 Fischer 344 rat ovaries were exposed to vehicle control (1% DMSO) ± DMBA (1 μM) or vehicle control (1% DMSO) ± LY294002 (PI3K inhibitor; 20 μM) for 1, 2, 4, or 6 days. Total RNA or protein was isolated, followed by RT-PCR or Western blotting to determine mRNA or protein level, respectively. Immunoprecipitation using an anti-GSTP antibody was performed to determine interaction between GSTP and JNK, followed by Western blotting to determine JNK and p-c-Jun protein level. DMBA had no impact on Gsta, Gstm or Nrf2 mRNA level, but increased Gstp mRNA and protein after 2 days. Ahr mRNA and protein increased after 2 and 4 days of DMBA exposure, respectively and DMBA increased NRF2 protein level after 4 days. JNK bound to GSTP was increased during DMBA exposure, with a concomitant decrease in unbound JNK and p-c-Jun. Ahr and Gstp mRNA were decreased (2 days) and increased (4 days) by PI3K inhibition, while Gstm mRNA increased (P < 0.05) after both time points, and there was no effect on Nrf2 mRNA. PI3K inhibition increased AHR, NRF2 and GSTP protein level. These findings support involvement of ovarian GSTP during DMBA exposure, and indicate a regulatory role for the PI3K signaling pathway on ovarian xenobiotic metabolism gene expression.

Phase III biotransformation enzyme involvement in the ovarian response to ovotoxic environmental exposures

“…Similar to ABCC1, ABCB1 protein expression significantly decreased with inhibition of PI3K and yet again no impact of kit ligand treatment was observed. Thus, similar to our previous findings that suggest that NRF2, EPHX1, GSTM, GSTP, and AHR are downstream of the PI3K pathway [55,57], these data support that ABCC1 and ABCB1 are regulated by PI3K. Lack of any effect of kit ligand to overcome the impact of PI3K deficiency on ABCB1 and ABCC1 is an area for further investigation and may yield insight into specific regulators of these phase III drug transporters.…”

“…ABCB1 and ABCC1 were discovered on the basis of drug resistance to chemotherapy agents in tumors and therefore could encompass potential roles in conservation of the ovary [36]. We hypothesized that phase III chemical metabolism involving ABCB1 and ABCC1 may be an important factor for protection of the ovary during PM exposure and that ABCB1 and ABCC1 are regulated via the PI3K pathway as we have previously observed with Phase I and II chemical biotransformation proteins [55,57].…”

“…We and others have demonstrated that the ovary has the capacity for phase I [52][53][54] and phase II [55][56][57][58][59][60][61] chemical metabolism. We have also discovered that both the xenobiotic biotransformation enzymes transcriptional regulators, arylhydrocarbon receptor (AHR) and nuclear factor erythroid-related factor 2 (NRF2), as well as phase I (microsomal epoxide hydrolase (EPHX1)) and phase II (Glutathione S-transferase isoforms pi (GSTP) and mu (GSTM)) enzymes are downstream of the phosphatidylinositol-3 kinase (PI3K) pathway [55,57].…”

“…In the late follicular phase, the preovulatory follicle primarily produces E2 [49]. The follicle is dependent on the interaction between the GC and TC and therefore, production of 17β-estradiol is referred to as the two-cell-two gonadotropin theory [34,[53][54][55][56][57][58][59]. As the follicle approaches ovulation, the GC increase the synthesis of P4, is required for luteinization initiation [2,49].…”

“…We have also discovered that both the xenobiotic biotransformation enzymes transcriptional regulators, arylhydrocarbon receptor (AHR) and nuclear factor erythroid-related factor 2 (NRF2), as well as phase I (microsomal epoxide hydrolase (EPHX1)) and phase II (Glutathione S-transferase isoforms pi (GSTP) and mu (GSTM)) enzymes are downstream of the phosphatidylinositol-3 kinase (PI3K) pathway [55,57]. Indeed, inhibition of PI3K prevented ovotoxicity induced by 4-vinylcyclohexene diepoxide (VCD) but accelerated that of dimethylbenz[a]anthracene (DMBA) [62], potentially due to increased EPHX1 [57].…”