Dual protective role for glutathione S-transferase class pi against VCD-induced ovotoxicity in the rat ovary

Abstract

The occupational chemical 4-vinylcyclohexene diepoxide (VCD) selectively destroys ovarian small pre-antral follicles in rats and mice via apoptosis. Detoxification of VCD can occur through glutathione conjugation, catalyzed by glutathione S-transferase (GST) enzymes. Further, GST class pi (GSTp) can negatively regulate JNK activity through protein:protein interactions in extra-ovarian tissues. Dissociation of this protein complex in the face of chemical exposure releases the inhibition of pro-apoptotic JNK. Increased JNK activity during VCD-induced ovotoxicity has been shown in isolated ovarian small pre-antral follicles following in vivo dosing of rats (80mg/kg/day; 15days, i.p.). The present study investigated the pattern of ovarian GSTp expression during VCD exposure. Additionally, the effect of VCD on an ovarian GSTp:JNK protein complex was investigated. PND4 F344 rat ovaries were incubated in control medium+/-VCD (30muM) for 2-8days. VCD increased ovarian GSTp mRNA (P <0.05) relative to control on d4-d8; whereas GSTp protein was increased (P<0.05) on d6-d8. A GSTp:JNK protein complex was detected by immunoprecipitation and Western blotting in ovarian tissues. Relative to control, the amount of GSTp-bound JNK was increased (P=0.09), while unbound JNK was decreased (P<0.05) on d6 of VCD exposure. The VCD-induced decrease in unbound JNK was preceded by a decrease in phosphorylated c-Jun which occurred on d4. These findings are in support of a possible dual protective role for GSTp in the rat ovary, consisting of metabolism of VCD and inhibition of JNK-initiated apoptosis.

Figure 1. Temporal effect of VCD on GSTp mRNA

Ovaries from PND4 F344 rats were cultured with control (CT) medium or medium containing 30 μM VCD for 2-8d. Following incubation, total RNA was isolated, reverse transcribed to cDNA and analyzed for GSTp or Actb mRNA expression by RT-PCR. Values are percentage of control ± SE; n=3 (8-10 ovaries per pool). *P < 0.05; different from control.

Figure 2. Temporal effect of VCD on GSTp protein

Ovaries from PND4 F344 rats were cultured with control (CT) medium or medium containing 30 μM VCD for 4-8d. Following incubation, total protein was isolated and Western blotting carried out for GSTp and ACTB. (A) Values are normalized to ACTB protein and expressed as a percentage of control mean ± S.E.; n=3; * P <0.05; Different from control. (B) Representative Western blot is shown on d6; control = c; VCD = v.

Figure 3. Effect of VCD on GSTp:JNK protein complex formation

Ovaries from PND4 F344 rats were cultured with control (CT) medium or medium containing 30 μM VCD for 4d (D4) or 6d (D6). Following incubation, total protein was isolated and immunoprecipitation carried out using an anti-GSTp antibody as described in materials and methods. (A) Representative Western blot of GSTp immunoprecipitation, followed by detection of JNK protein (control ovary; d6). (B) Ratio of JNK protein bound to GSTp on D4 and D6. (C) JNK protein relative to total GSTp. Values are percentage of control ± SE. * P <0.05; ^# P < 0.1; Different from control.

Figure 4. Effect of VCD on unbound JNK and p-c-Jun protein levels

Ovaries from PND4 F344 rats were cultured with control (CT) medium or medium containing 30 μM VCD for 4d (D4) or 6d (D6). Following incubation, total protein was isolated and immunoprecipitation carried out using an anti-GSTp antibody as described in materials and methods. JNK or p-c-Jun proteins were detected by Western blotting. Image J software was used to measure integrated density of protein bands. (A) Representative Western blot for JNK, p-c-Jun and ACTB on d6; control = c; VCD = v. (B) Unbound JNK protein level, normalized to ACTB. (C) Unbound p-c-Jun protein level, normalized to ACTB. Values are expressed as percentage of control ± SE; n=3; *P <0.05; different from control.