Nivedita Sen

Effect of phosphatidylinositol-3 kinase inhibition on ovotoxicity caused by 4-vinylcyclohexene diepoxide and 7, 12-dimethylbenz[a]anthracene in neonatal rat ovaries.

4-vinylcyclohexene diepoxide (VCD) is an ovotoxicant that specifically destroys primordial and small primary follicles in the ovaries of mice and rats. In contrast, 7,12-dimethylbenz[a]anthracene (DMBA) is ovotoxic to all ovarian follicle classes. This study investigated phosphatidylinositol-3 kinase signaling involvement in VCD- and DMBA-induced ovotoxicity. Postnatal day (PND) 4 Fischer 344 (F344) rat whole ovaries were cultured for 2-12 days in vehicle control, VCD (30 microM), or DMBA (1 microM), +/-PI3 kinase inhibitor LY294002 (20 microM) or its inactive analog LY303511 (20 microM). Following culture, ovaries were histologically evaluated, and healthy follicles were classified and counted. PI3 kinase inhibition had no effect on primordial follicle number, but reduced (P<0.05) small primary and larger follicles beginning on day 4. VCD caused primordial and small primary follicle loss (P<0.05) beginning on day 6. With PI3 kinase inhibition, VCD did not affect primordial follicles (P>0.05) at any time, but did cause loss (P<0.05) of small primary follicles. DMBA exposure caused primordial and small primary follicle loss (P<0.05) on day 6. Further, DMBA-induced primordial and small primary follicle loss was greater with PI3 kinase inhibition (P<0.05) than with DMBA alone. These results support that (1) PI3 kinase mediates primordial to small primary follicle recruitment, (2) VCD, but not DMBA, enhances ovotoxicity by increasing primordial to small primary follicle recruitment, and (3) in addition to xenobiotic-induced ovotoxicity, VCD is also a useful model chemical with which to elucidate signaling mechanisms involved in primordial follicle recruitment.

Involvement of the KIT/KITL Signaling Pathway in 4-Vinylcyclohexene Diepoxide-Induced Ovarian Follicle Loss in Rats

Abstract Repeated daily dosing of rats with the occupational chemical 4-vinylcyclohexene diepoxide (VCD) depletes the ovary of primordial and primary follicles through an increase in the natural process of atresia. Additionally, in vitro exposure of Postnatal Day 4 (PND 4) rat ovaries to VCD causes similar follicular depletion. This study was designed to investigate survival signaling pathways that may be associated with VCD-induced ovotoxicity in small preantral follicles. Female Fischer 344 rats (PND 28) were dosed daily (80 mg/kg/day VCD i.p.; 12 days in vivo), and PND 4 ovaries were cultured (VCD 20 or 30 μM; 8 days in vitro). Microarray analysis identified a subset of 14 genes whose expression was increased or decreased by VCD in both experiments (i.e., via both exposure routes). Particularly, the analysis showed that relative to controls, VCD did not affect mRNA expression of growth and differentiation factor 9 (Gdf9), whereas there were decreases in mRNA encoding bone morphogenic protein receptor 1a (Bmpr1a) and Kit. To confirm findings from microarray, the genes Gdf9, Bmpr1a, and Kit were further examined. When growth factors associated with these pathways were added to ovarian cultures during VCD exposure, GDF9 and BMP4 had no effect on VCD-induced ovotoxicity; however, KITL attenuated this follicle loss. Additionally, there was a decrease in Kit and an increase in Kitl expression (mRNA and protein) following VCD exposure, relative to control. These results support that VCD compromises KIT/KITL signaling, which is critical for follicular survival in primordial and primary follicles.

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

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.

Inhibition of PIK3 Signaling Pathway Members by the Ovotoxicant 4-Vinylcyclohexene Diepoxide in Rats

4-Vinylcyclohexene diepoxide (VCD), an occupational chemical that specifically destroys primordial and small primary follicles in the ovaries of rats and mice, is thought to target an oocyte-expressed tyrosine kinase receptor, Kit. This study compared the temporal effect of VCD on protein distribution of KIT and its downstream PIK3-activated proteins, AKT and FOXO3. Postnatal Day 4 Fischer 344 rat ovaries were cultured in control media ± VCD (30 μM) for 2–8 days (d2–d8). KIT, AKT, phosphorylated AKT, FOXO3, and pFOXO3 protein levels were assessed by Western blotting and/or immunofluorescence staining with confocal microscopy. Phosphorylated AKT was decreased (P < 0.05) in oocyte nuclei in primordial (39% decrease) and small primary (37% decrease) follicles within 2 days of VCD exposure. After d4, VCD reduced (P < 0.05) oocyte staining for KIT (primordial, 44% decrease; small primary, 39% decrease) and FOXO3 (primordial, 40% decrease; small primary, 36% decrease) protein. Total AKT and pFOXO3 were not affected by VCD at any time. Akt1 mRNA, as measured by quantitative RT-PCR, was reduced (P < 0.05) by 23% on d4 of VCD exposure, but returned to control levels on d6 and d8. VCD exposure reduced Foxo3a mRNA by 26% on d6 (P < 0.05) and by 23% on d8 (P < 0.1). These results demonstrate that the earliest observed effect of VCD is an inhibition of phosphorylation and nuclear localization of AKT in the oocyte of primordial and small primary follicles. This event is followed by reductions in KIT and FOXO3 protein subcellular distribution prior to changes in mRNA. Thus, these findings further support that VCD induces ovotoxicity by directly targeting the oocyte through posttranslational inhibition of KIT-mediated signaling components.

Glutathione S-transferase class mu regulation of apoptosis signal-regulating kinase 1 protein during VCD-induced ovotoxicity in neonatal rat ovaries

4-Vinylcyclohexene diepoxide (VCD) destroys ovarian primordial and small primary follicles via apoptosis. In mice, VCD exposure induces ovarian mRNA expression of glutathione S-transferase (GST) family members, including isoform mu (Gstm). Extra-ovarian GSTM negatively regulates pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1) through protein complex formation, which dissociates during stress, thereby initiating ASK1-induced apoptosis. The present study investigated the ovarian response of Gstm mRNA and protein to VCD. Induction of Ask1 mRNA at VCD-induced follicle loss onset was determined. Ovarian GSTM:ASK1 protein complex formation was investigated and VCD exposure effects thereon evaluated. Phosphatidylinositol-3 kinase (PI3K) regulation of GSTM protein was also studied. Postnatal day (PND) 4 rat ovaries were cultured in control media ± 1) VCD (30 μM) for 2-8 days; 2) VCD (30 μM) for 2 days, followed by incubation in control media for 4 days (acute VCD exposure); or 3) LY294002 (20 μM) for 6 days. VCD exposure did not alter Gstm mRNA expression, however, GSTM protein increased (P<0.05) after 6 days of both the acute and chronic treatments. Ask1 mRNA increased (0.33-fold; P<0.05) relative to control after 6 days of VCD exposure. Ovarian GSTM:ASK1 protein complex formation was confirmed and, relative to control, the amount of GSTM bound to ASK1 increased 33% (P<0.05) by chronic but with no effect of acute VCD exposure. PI3K inhibition increased (P<0.05) GSTM protein by 40% and 71% on d4 and d6, respectively. These findings support involvement of GSTM in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1.

Ovarian expressed microsomal epoxide hydrolase: role in detoxification of 4-vinylcyclohexene diepoxide and regulation by phosphatidylinositol-3 kinase signaling

4-vinylcyclohexene diepoxide (VCD) is a metabolite of 4-vinylcyclohexene (VCH) which has the potential to be formed in the ovary through CYP2E1 activity. VCD specifically destroys primordial and small primary follicles in the rodent ovary. Mouse ovaries exposed to VCD demonstrate increased mRNA and protein expression of microsomal epoxide hydrolase (mEH), and an inactive tetrol metabolite (4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane) can be formed in mouse ovarian follicles, potentially through detoxification action of mEH. In contrast, mEH can bioactivate another ovotoxic chemical, 7,12-dimethylbenz[a]anthracene (DMBA) to a more toxic compound, DMBA-3,4-diol-1,2-epoxide. Thus, the present study evaluated a functional role for mEH during detoxification of VCD. Additionally, because inhibition of the phosphatidyinositol-3 kinase (PI3K) signaling pathway in a previous study protected primordial follicles from VCD-induced destruction, but accelerated DMBA-induced ovotoxicity, a role for PI3K in ovarian mEH regulation was evaluated. Using a post-natal day (PND) 4 Fischer 344 rat whole ovary culture system inhibition of mEH using cyclohexene oxide during VCD exposure resulted in a greater (P<0.05) loss of primordial and small primary follicles relative to VCD-treated ovaries. Also, relative to controls, meh mRNA was increased (P<0.05) on day 4 of VCD (30 μM) exposure, followed by increased (P<0.05) mEH protein after 6 days. Furthermore, inhibition of PI3K signaling increased mEH mRNA and protein expression. Thus, these results support a functional role for mEH in the rat ovary, and demonstrate the involvement of PI3K signaling in regulation of ovarian xenobiotic metabolism by mEH.

Short term exposure to di-n-butyl phthalate (DBP) disrupts ovarian function in young CD-1 mice

Di-n-butyl phthalate (DBP) is present in many beauty and medical products. Human exposure estimates range from 0.007-0.01 mg/kg/day in the general population and up to 0.233 mg/kg/day in patients taking DBP-coated medications. Levels of phthalates tend to be higher in women, thus, evaluating ovarian effects of DBP exposure is of great importance. Mice were given corn oil (vehicle) or DBP at 0.01, 0.1, and 1000 mg/kg/day (high dose) for 10 days to test whether DBP causes ovarian toxicity. Estrous cyclicity, steroidogenesis, ovarian morphology, and apoptosis and steroidogenesis gene expression were evaluated. DBP exposure decreased serum E2 at all doses, while 0.1DBP increased FSH, decreased antral follicle numbers, and increased mRNA encoding pro-apoptotic genes (Bax, Bad, Bid). Interestingly, mRNAs encoding the steroidogenic enzymes Hsd17b1, Cyp17a1 and Cyp19a1 were increased in all DBP-treated groups. These novel findings show that DBP can disrupt ovarian function in mice at doses relevant to humans.

Inhibition of Ovarian KIT Phosphorylation by the Ovotoxicant 4-Vinylcyclohexene Diepoxide in Rats

In vitro exposure of Postnatal Day 4 (PND4) rat ovaries to the occupational chemical 4-vinylcyclohexene diepoxide (VCD) destroys specifically primordial and primary follicles via acceleration of atresia. Because oocyte-expressed c-kit (KIT) plays a critical role in follicle survival and activation, a direct interaction of VCD with KIT as its mechanism of ovotoxicity was investigated. PND4 rat ovaries were cultured with and without VCD (30 μM) for 2 days. When assessed by Western analysis or mobility shift detection, phosphorylated KIT (pKIT) was decreased (P < 0.05) by VCD exposure, while total KIT protein was unaffected. Anti-mouse KIT2 (ACK2) antibody binds KIT and blocks its signaling pathways, whereas anti-mouse KIT 4 (ACK4) antibody binds KIT but does not block its activity. PND4 rat ovaries were incubated for 2 days with and without VCD with and without ACK2 (80 μg/ml) or ACK4 (80 μg/ml). ACK2 decreased pKIT; however, ACK4 had no effect. Conversely, ACK2 did not affect a VCD-induced decrease in pKIT, whereas ACK4 further reduced it. Because ACK2 and ACK4 (known to directly bind KIT) affect VCD responses, these results support the fact that VCD interacts directly with KIT. The effect of these antibodies on VCD-induced follicle loss was measured after 8 days of incubation. ACK2 further reduced (P < 0.05) VCD-induced follicle loss, whereas ACK4 did not affect it. These findings demonstrate that VCD induces ovotoxicity by direct inhibition of KIT autophosphorylation of the oocyte. The data also further support the vital function of KIT and its signaling pathway in primordial follicle survival and activation, as well as its role in VCD-induced ovotoxicity.

Distribution and responsiveness of rat anti-Müllerian hormone during ovarian development and VCD-induced ovotoxicity

Anti-Müllerian hormone (AMH) is produced by granulosa cells in primary to small antral follicles of the adult ovary and helps maintain primordial follicles in a dormant state. The industrial chemical, 4-vinylcyclohexene diepoxide (VCD) causes specific ovotoxicity in primordial and small primary follicles of mice and rats. Previous studies suggest that this ovotoxicity involves acceleration of primordial to primary follicle recruitment via interactions with the Kit/Kit ligand signaling pathway. Because of its accepted role in inhibiting primordial follicle recruitment, the present study was designed to investigate a possible interaction between AMH and VCD-induced ovotoxicity. Protein distribution of AMH was compared in neonatal and adult F344 rat ovaries. AMH protein was visualized by immunofluorescence microscopy in large primary and secondary follicles of the adult ovary, but in small primary follicles in neonatal rat ovaries. In cultured postnatal day (PND) 4 F344 rat ovaries, VCD exposure (30 μM, 2-8 days) decreased (P<0.05) AMH mRNA (d4-8) and protein (d6-8). Recombinant AMH (100-400 mg/ml) in PND4 ovaries cultured 8 days±VCD (30 μM) caused an increase (P<0.05) in primordial, and a decrease (P<0.05) in small primary follicles, supporting that AMH retarded primordial follicle recruitment. However, no concentration of AMH had an effect on VCD-induced ovotoxicity. Whereas, VCD caused a reduction in expression of AMH (d4-d8), it followed previously reported initial disruptions in Kit signaling induced by VCD (d2). Thus, collectively, these results do not support a mechanism whereby VCD causes ovotoxicity via generalized activation of primordial follicle recruitment, but instead provide further support for the specificity of other intracellular mechanisms involved in VCD-induced ovotoxicity.

Effects of oral exposure to the phthalate substitute acetyl tributyl citrate on female reproduction in mice

Acetyl tributyl citrate (ATBC), is a phthalate substitute used in food and medical plastics, cosmetics and toys. Although systemically safe up to 1000 mg kg−1 day−1, its ability to cause reproductive toxicity in females at levels below 50 mg kg−1 day−1 has not been examined. This study evaluated the effects of lower ATBC exposures on female reproduction using mice. Adult CD‐1 females (n = 7–8 per treatment) were dosed orally with tocopherol‐stripped corn oil (vehicle), 5 or 10 mg kg−1 day−1 ATBC daily for 15 days, and then bred with a proven breeder male. ATBC exposure did not alter body weights, estrous cyclicity, and gestational and litter parameters. Relative spleen weight was slightly increased in the 5 mg kg−1 day−1 group. ATBC at 10 mg kg−1 day−1 targeted ovarian follicles and decreased the number of primordial, primary and secondary follicles present in the ovary. These findings suggest that low levels of ATBC may be detrimental to ovarian function, thus, more information is needed to understand better the impact of ATBC on female reproduction. Copyright