Polycyclic aromatic hydrocarbons are ubiquitous contaminants in the environment. Benzo[a]pyrene (BaP), a prototypical member of this class of chemicals, has been extensively studied for its toxic effects in laboratory animals and human populations. BaP toxicity is often mediated by oxidative metabolism to reactive intermediates that interact with macromolecules leading to alterations in target cell structure and function. More recent evidence suggests that disruption of cellular signaling pathways involved in the regulation of growth and differentiation contribute significantly to the toxicity of BaP and its metabolites. This review summarizes recent advances in our understanding of biological mechanisms of BaP toxicity at the molecular level, and the role of metabolic intermediates in carcinogenesis, atherogenesis, and teratogenesis.

Methoxychlor (MXC) is an organochlorine pesticide and reproductive toxicant. While in vivo studies indicate that MXC exposure increases antral follicle atresia, in part by altering apoptotic regulators (Bcl-2 and Bax), they do not distinguish whether MXC does so via direct or indirect mechanisms. Therefore, we utilized an in vitro follicle culture system to test the hypothesis that MXC is directly toxic to antral follicles, and that overexpression of anti-apoptotic Bcl-2, or deletion of pro-apoptotic Bax, protects antral follicles from MXC-induced toxicity. Antral follicles were isolated from wild-type (WT), Bcl-2 overexpressing (Bcl-2 OE), or Bax deficient (BaxKO) mice, and exposed to dimethylsulfoxide (control) or MXC (1-100 microg/ml) for 96 h. Follicle diameters were measured every 24 h to assess growth. After 96 h, follicles were histologically evaluated for atresia or collected for quantitative PCR analysis of Bcl-2 and Bax mRNA levels. MXC (10-100 microg/ml) significantly inhibited antral follicle growth at 72 and 96 h, and increased atresia (100 microg/ml) compared to controls at 96 h. Furthermore, MXC increased Bax mRNA levels between 48-96 h and decreased Bcl-2 mRNA levels at 96 h. While MXC inhibited growth of WT antral follicles beginning at 72 h, it did not inhibit growth of Bcl-2 OE or BaxKO follicles until 96 h. MXC also increased atresia of small and large WT and BaxKO antral follicles over controls, but it did not increase atresia of large Bcl-2 OE antral follicles over controls. These data suggest that MXC directly inhibits follicle growth partly by Bcl-2 and Bax pathways, and increases atresia partly through Bcl-2 pathways.

The mammalian ovary contains antral follicles, which are responsible for the synthesis and secretion of hormones that regulate estrous cyclicity and fertility. The organochlorine pesticide methoxychlor (MXC) causes atresia (follicle death via apoptosis) of antral follicles, but little is known about the mechanisms by which MXC does so. Oxidative stress is known to cause apoptosis in nonreproductive and reproductive tissues. Thus, we tested the hypothesis that MXC inhibits growth and induces atresia of antral follicles through an oxidative stress pathway. To test this hypothesis, antral follicles isolated from 39-day-old CD-1 mice were cultured with vehicle control (dimethylsulfoxide [DMSO]), MXC (1-100 microg/ml), or MXC + the antioxidant N-acetyl cysteine (NAC) (0.1-10 mM). During culture, growth was monitored daily. At the end of culture, follicles were processed for quantitative real-time polymerase chain reaction of Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPX), and catalase (CAT) mRNA expression or for histological evaluation of atresia. The results indicate that exposure to MXC (1-100 microg/ml) inhibited growth of follicles compared to DMSO controls and that NAC (1-10 mM) blocked the ability of MXC to inhibit growth. MXC induced follicular atresia, whereas NAC (1-10 mM) blocked the ability of MXC to induce atresia. In addition, MXC reduced the expression of SOD1, GPX, and CAT, whereas NAC reduced the effects of MXC on their expression. Collectively, these data indicate MXC causes slow growth and increased atresia by inducing oxidative stress.

Smad3 is an important mediator of the TGF beta signaling pathway. Interestingly, Smad3-deficient (Smad3-/-) mice have reduced fertility compared with wild-type (WT) mice. To better understand the molecular mechanisms underlying the reduced fertility in Smad3-/- animals, this work tested the hypothesis that Smad3 deficiency interferes with three critical aspects of folliculogenesis: growth, atresia, and differentiation. Growth was assessed by comparing the size of follicles, expression of proliferating cell nuclear antigen, and expression of cell cycle genes in Smad3-/- and WT mice. Atresia was assessed by comparing the incidence of atresia and expression of bcl-2 genes involved in cell death and cell survival in Smad3-/- and WT mice. Differentiation was assessed by comparing the expression of FSH receptor (FSHR), estrogen receptor (ER) alpha, ER beta, and inhibin alpha-, beta(A)-, and beta(B)-subunits in Smad3-/- and WT mice. Because growth, atresia, and differentiation are regulated by hormones, estradiol, FSH, and LH levels were compared in Smad3-/- and WT mice. Moreover, because alterations in folliculogenesis can affect the ability of mice to ovulate, the number of corpora lutea and ovulated eggs in response to gonadotropin treatments were compared in Smad3-/- and WT animals. The results indicate that Smad3 deficiency slows follicle growth, which is characterized by small follicle diameters, low levels of proliferating cell nuclear antigen, and low expression of cell cycle genes (cyclin-dependent kinase 4 and cyclin D2). Smad3 deficiency also causes atretic follicles, degenerated oocytes, and low expression of bcl-2. Furthermore, Smad3 deficiency affects follicular differentiation as evidenced by decreased expression of ER beta, increased expression of ER alpha, and decreased expression of inhibin alpha-subunits. Smad3 deficiency causes low estradiol and high FSH levels. Finally, Smad3-/- ovaries have no corpora lutea, and they do not ovulate after ovulatory induction with exogenous gonadotropins. Collectively, these data provide the first evidence that reduced fertility in Smad3-/- mice is due to impaired folliculogenesis, associated with altered expression of genes that control cell cycle progression, cell survival, and cell differentiation. The findings that Smad3-/- follicles have impaired growth, increased atresia, and altered differentiation in the presence of high FSH levels, normal expression of FSHR, and lower expression of cyclin D2, suggest a possible interaction between Smad3 and FSH signaling downstream of FSHR in the mouse ovary.

Although it was recently recognized that sterol carrier protein-2 (SCP-2) interacts with fatty acids, little is known regarding the specificity of SCP-2 for long-chain fatty acids or branched-chain fatty-acid-like molecules. Likewise the location of the fatty-acid binding site within SCP-2 is unresolved. A fluorescent cis-parinaric acid displacement assay was used to show that SCP-2 optimally interacted with 14-22 carbon chain lipidic molecules: polyunsaturated fatty acids > monounsaturated, saturated > branched-chain isoprenoids > branched-chain phytol-derived fatty acids. In contrast, the other major fatty-acid binding protein in liver, fatty-acid binding protein (L-FABP), displayed a much narrower carbon chain preference in general: polyunsaturated fatty acids > branched-chain phytol-derived fatty acids > 14- and 16-carbon saturated > branched-chain isoprenoids. However, both SCP-2 and L-FABP displayed a very similar unsaturated fatty-acid specificity profile. The presence and location of the SCP-2 lipid binding site were investigated by fluorescence energy transfer. The distance between the SCP-2 Trp50 and bound cis-parinaric acid was determined to be 40 A. Thus, the SCP-2 fatty-acid binding site appeared to be located on the opposite side of the SCP-2 Trp50. These findings not only contribute to our understanding of the SCP-2 ligand binding site but also provide evidence suggesting a potential role for SCP-2 and/or L-FABP in metabolism of branched-chain fatty acids and isoprenoids.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that binds various environmental contaminants. Despite our knowledge regarding the role of the AhR in mediating toxicity, little is known about the physiological role of the AhR. Previous studies indicate that the AhR may regulate folliculogenesis, because AhR-deficient (AhRKO) mice have fewer preantral and antral follicles than wild-type (WT) mice during postnatal life. Thus, the first objective of the present study was to test the hypothesis that AhR deficiency reduces the numbers of preantral and antral follicles by slowing growth and/or increasing atresia of follicles. Because alterations in follicular growth or atresia can affect the ability to ovulate, the second objective was to test whether AhR deficiency reduces the number of ovulated eggs. To test these hypotheses, follicular growth was compared in WT and AhRKO ovaries using morphometric techniques and by measuring the ability of the ovary and follicles to grow in response to eCG. Atresia was compared in WT and AhRKO ovaries using morphometric techniques, TUNEL assays, and 3'-end labeling of fragmented DNA. Ovulation was compared in WT and AhRKO mice by assessing the number of corpora lutea per ovary. The results indicate that follicular growth and ovulation were reduced in AhRKO ovaries compared to WT ovaries. The WT ovaries had a 1.5-fold increase in the number of preantral and antral follicles between Postnatal Days 32 and 45, were more responsive to eCG, and contained more corpora lutea than AhRKO ovaries. In contrast, no significant difference was observed in the incidence of atresia in WT and AhRKO ovaries. Taken together, these results suggest that the AhR may regulate growth, but not atresia, of preantral and antral follicles in the mouse ovary.

The aryl hydrocarbon receptor (AHR) is a known transcription factor. Although studies indicate that Ahr-deficient (AhRKO) mice have defects in female reproduction, only a few studies have examined the role of AHR in the ovary. Previous studies have suggested, without directly testing, that AhRKO mice have slower follicular growth than wild-type (WT) mice. Therefore, the first objective of the present study was to examine whether AhRKO follicles grow slower than WT follicles and if so, to determine whether the mechanism by which Ahr affects follicular growth is through effects on antrum size, granulosa cell proliferation, and regulators of cell cycle progression. Since estradiol (E(2)) is critical for the normal growth of ovarian follicles, the second objective of the present study was to determine the role of Ahr in regulating E(2) production and responsiveness. The third objective of the present study was to determine whether E(2) replacement restores follicular growth of AhRKO follicles to WT levels in vitro. We found that AhRKO follicles grew slower than WT follicles in vitro. While AhRKO and WT follicles had similar antrum sizes, AhRKO follicles showed decreased granulosa cell proliferation and reduced mRNA and protein levels of cell cycle regulators, as compared to WT follicles. Furthermore, the AhRKO mice had lower serum and follicle-produced E(2) levels and showed decreased Esr1 and Esr2 mRNA levels compared to WT mice. Finally, E(2) treatment of AhRKO follicles restored follicular growth to WT levels in vitro. Collectively, these findings suggest that the AHR affects follicular growth via mechanisms that involve E(2) regulation and responsiveness.

Pancreatic ductal adenocarcinoma (PDAC) remains a devastating human malignancy with poor prognosis and low survival rates. Several cellular mechanisms have been linked with pancreatic carcinogenesis and also implicated in inducing tumor resistance to known therapeutic regimens. Of various factors, immune evasion mechanisms play critical roles in tumor progression and impeding the efficacy of cancer therapies including PDAC. Among immunosuppressive cell types, myeloid-derived suppressor cells (MDSCs) have been extensively studied and demonstrated to not only support PDAC development but also hamper the anti-tumor immune responses elicited by therapeutic agents. Notably, recent efforts have been directed in devising novel approaches to target MDSCs to limit their effects. Multiple strategies including immune-based approaches have been explored either alone or in combination with therapeutic agents to target MDSCs in preclinical and clinical settings of PDAC. The current review highlights the roles and mechanisms of MDSCs as well as the implications of this immunomodulatory cell type as a potential target to improve the efficacy of therapeutic regimens for PDAC.