Kristina Pogrmic-Majkic

Upregulation of Peripubertal Rat Leydig Cell Steroidogenesis Following 24 h In Vitro and In Vivo Exposure to Atrazine

Atrazine is currently one of the most widely used herbicides in the United States and elsewhere. Here we examined 24 h in vitro and in vivo effects of atrazine on androgen production and on expression and activity of steroidogenic enzymes and regulatory proteins involved in cyclic adenosine monophosphate (cAMP)-signaling pathway in peripubertal rat Leydig cells. When in vitro added, 1-50 μM atrazine increased basal and human chorion gonadotropin-stimulated testosterone production and accumulation of cAMP in the medium of treated cells. The stimulatory action of atrazine on androgen production but not on cAMP accumulation was abolished in cells with inhibited protein kinase A. Atrazine also stimulated the expression of mRNA transcripts for steroidogenic factor-1, steroidogenic acute regulatory protein, cytochrome P450 (CYP)17A1, and 17β-hydroxysteroid dehydrogenase (HSD), as well as the activity of CYP17A1 and 17βHSD. The stimulatory effects of atrazine on cAMP accumulation and androgen production were also observed during the first 3 days of in vivo treatment (200 mg/kg body weight, by gavage) followed by a decline during further treatment. These results indicate that atrazine has a transient stimulatory action on cAMP signaling pathway in Leydig cells, leading to facilitated androgenesis.

Atrazine acts as an endocrine disrupter by inhibiting cAMP-specific phosphodiesterase-4

Atrazine, one of the most commonly used herbicides worldwide, acts as an endocrine disruptor, but the mechanism of its action has not been characterized. In this study, we show that atrazine rapidly increases cAMP levels in cultured rat pituitary and testicular Leydig cells in a concentration-dependent manner, but less effectively than 3-isobutyl-1-methylxanthine, a competitive non-specific inhibitor of phosphodiesterases (PDEs). In forskolin (an activator of adenylyl cyclase)- and probenecid (an inhibitor of cyclic nucleotide transporters)-treated cells, but not in 3-isobutyl-1-methylxanthine-treated cells, atrazine further increased cAMP levels, indicating that inhibition of PDEs accounts for accumulation of cAMP. In contrast to cAMP, atrazine did not alter cGMP levels, further indicating that it inhibits cAMP-specific PDEs. Atrazine-induced changes in cAMP levels were sufficient to stimulate prolactin release in pituitary cells and androgen production in Leydig cells, indicating that it acts as an endocrine disrupter both in cells that secrete by exocytosis of prestored hormones and in cells that secrete by de novo hormone synthesis. Rolipram abolished the stimulatory effect of atrazine on cAMP release in both cell types, suggesting that it acts as an inhibitor of PDE4s, isoforms whose mRNA transcripts dominate in pituitary and Leydig cells together with mRNA for PDE8A. In contrast, immortalized lacto-somatotrophs showed low expression of these mRNA transcripts and several fold higher cAMP levels compared to normal pituitary cells, and atrazine was unable to further increase cAMP levels. These results indicate that atrazine acts as a general endocrine disrupter by inhibiting cAMP-specific PDE4s.

Atrazine effects on antioxidant status and xenobiotic metabolizing enzymes after oral administration in peripubertal male rat

Previously, we reported that in vivo applied atrazine from postnatal day 23 to 50 induced strong inhibition of testicular steroidogenesis. Therefore, the aim of the present study was to investigate, in the same experimental model, the oxidative status in androgen-producing testicular interstitial compartment characterized by diminished steroidogenesis. In parallel, we determined activities of antioxidative and cytochrome P450 (CYP) xenobiotic-metabolizing enzymes in liver. To confirm the results on atrazine induced-inhibition of testicular androgenesis, we measured ex vivo production of androgen in Leydig cells. The results revealed decreased activity of antioxidant enzymes, especially glutathione S-transferase (GST), but also glutathione peroxidase (GSH-Px) and catalase (CAT) in testicular interstitial cells, in parallel with strongly diminished ex vivo basal and agonist-stimulated Leydig cell androgenesis. In liver, atrazine increased the activity of GSH-Px, GST, and CYP1A1/2 enzyme, but not lipid peroxidation. These results indicate that atrazine markedly affects both antioxidant status and androgenesis in peripubertal rats.

Involvement of ERK1/2 signaling pathway in atrazine action on FSH-stimulated LHR and CYP19A1 expression in rat granulosa cells

Worldwide used herbicide atrazine is linked to reproductive dysfunction in females. In this study, we investigated the effects and the mechanism of atrazine action in the ovary using a primary culture of immature granulosa cells. In granulosa cells, follicle-stimulating hormone (FSH) activates both cyclic adenosine monophosphate (cAMP) and extracellular-regulated kinase 1/2 (ERK1/2) cascades, with cAMP pathway being more important for luteinizing hormone receptor (LHR) and aromatase (CYP19A1) mRNA expression. We report that 48h after atrazine exposure the FSH-stimulated LHR and CYP19A1 mRNA expression and estradiol synthesis were decreased, with LHR mRNA being more sensitive to atrazine than CYP19A1 mRNA. Inadequate acquisition of LHR in the FSH-stimulated and atrazine-exposed granulosa cells renders human chorionic gonadotropin (hCG) ineffective to stimulate amphiregulin (Areg), epiregulin (Ereg), and progesterone receptor (Pgr) mRNA expression, suggesting anti-ovulatory effect of atrazine. To dissect the signaling cascade involved in atrazine action in granulosa cells, we used U0126, a pharmacological inhibitor of ERK1/2. U0126 prevents atrazine-induced decrease in LHR and CYP19A1 mRNA levels and estradiol production in the FSH-stimulated granulosa cells. ERK1/2 inactivation restores the ability of hCG to induce expression of the ovulatory genes in atrazine-exposed granulosa cells. Cell-based ELISA assay revealed that atrazine does not change the FSH-stimulated ERK1/2 phosphorylation in granulosa cells. The results from this study reveal that atrazine does not affect but requires ERK1/2 phosphorylation to cause decrease in the FSH-induced LHR and CYP19A1 mRNA levels and estradiol production in immature granulosa cells, thus compromising ovulation and female fertility.

Atrazine Enhances Progesterone Production Through Activation of Multiple Signaling Pathways in FSH-Stimulated Rat Granulosa Cells: Evidence for Premature Luteinization

ABSTRACT Premature luteinization is a possible cause of infertility in women. It is currently unknown whether environmental chemicals can induce changes associated with premature luteinization. Using rat granulosa cells (GC) in vitro, we demonstrated that exposure to atrazine (ATR), a widely used herbicide, causes GC phenotype that resembles that of human premature luteinization. At the end of the 48-h stimulation with FSH, ATR-exposed GC showed (1) higher levels of progesterone, (2) overexpression of luteal markers (Star and Cyp11a1), and (3) an increase in progesterone:estradiol ratio above 1. Mechanistic experiments were conducted to understand the signaling events engaged by ATR that lead to this phenotype. Western blot analysis revealed prolonged phosphorylation of protein kinase B (AKT) and cAMP response element-binding protein (CREB) in ATR- and FSH-exposed GC. An increased level of ERK1/2-dependent transcriptional factor CCATT/enhancer-binding protein beta (CEBPB) was observed after 4 h of ATR exposure. Inhibitors of PI3K (wortmannin) and MEK (U0126) prevented ATR-induced rise in progesterone level and expression of luteal markers in FSH-stimulated GC. Atrazine intensified AKT and CEBPB signaling and caused Star overexpression in forskolin-stimulated GC but not in epidermal growth factor (EGF)-stimulated GC. In the presence of rolipram, a specific inhibitor of phosphodiesterase 4 (PDE4), ATR was not able to further elevate AKT phosphorylation, CEBPB protein level, and Star mRNA in FSH-stimulated GC, suggesting that ATR inhibits PDE4. Overall, this study showed that ATR acts as a FSH sensitizer leading to enhanced cAMP, AKT, and CEBPB signaling and progesterone biosynthesis, which promotes premature luteinization phenotype in GC.

Acute effects of hexabromocyclododecane on Leydig cell cyclic nucleotide signaling and steroidogenesis in vitro

Hexabromocyclododecane (HBCDD), an additive brominated flame retardant routinely added to various consumer products, was reported to have toxic effects upon biota, including endocrine disruption. In this study, the potential toxicity of HBCDD was tested in peripubertal rat Leydig cells in vitro during 6h exposure. HBCDD inhibited human chorionic gonadotropin- and forskolin-supported cAMP accumulation and steroidogenesis. It also inhibited basal cAMP production, but elevated basal steroidogenesis. The expression of several cAMP-dependent genes, including steroidogenic acute regulatory protein, cholesterol side chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase, was also inhibited by HBCDD treatment. Nevertheless, this was not accompanied by a decrease in steroidogenic acute regulatory protein expression, as documented by western blot analysis, and activity of steroidogenic enzymes, as documented by unaffected steroidogenesis in the presence of permeable 22(R)-hydroxycholesterol. However, HBCDD caused significant decrease in mitochondrial membrane potential in untreated and human chorionic gonadotropin-treated cells. This indicates that HBCDD acute toxicity in Leydig cells reflects changes in mitochondrial membrane potential-dependent cAMP production and basal and cAMP-regulated cholesterol transport. This in turn facilitates basal but inhibits cAMP-dependent steroidogenesis. Acute effects of HBCDD treatment on transcription are also indicative of its sustained effects on Leydig cell function.

Atrazine blocks ovulation via suppression of Lhr and Cyp19a1 mRNA and estradiol secretion in immature gonadotropin-treated rats.

We investigated whether in vivo exposure to herbicide atrazine (ATR) exerts anovulatory effect by direct action in the ovary. Female rats were given ATR (50mg/kg body weight) during equine chorionic gonadotropin (eCG) priming. Forty eight hours after eCG administration, the animals were injected with human CG (hCG) to induce ovulation. ATR blocked ovulation and prevented expression of epiregulin and progesterone receptor mRNA in hCG-treated animals. During eCG-induced follicular growth, ATR suppressed luteinizing hormone receptor (Lhr) and aromatase expression in granulosa cells and decreased estradiol (E2) serum levels. ATR increased cytochrome p450 1b1 (Cyp1b1) mRNA expression after both in vivo and in vitro exposures. In vitro addition of beta-naphthoflavone, a known Cyp1b1 mRNA inductor, suppressed follicle-stimulating hormone-induced Lhr expression. Collectively, these data indicate that under in vivo conditions, ATR may act directly on granulosa cells by decreasing E2 levels and Lhr mRNA, thus leading to inhibition of ovulation.

Erk-Creb pathway suppresses glutathione-S-transferase pi expression under basal and oxidative stress conditions in zebrafish embryos

Transcriptional activation of phase II enzymes including glutathione-S-transferase pi class (Gst Pi) is important for redox regulation and defense from xenobiotics. The role of extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) in regulation of Gst Pi expression has been described using adult mammalian cells. Whether these signaling pathways contribute to Gst Pi expression during embryogenesis is unknown. Using zebrafish embryo model, we provide novel evidence that Erk signaling acts as a specific suppressor of gstp1-2 mRNA during early embryogenesis. Addition of Erk inhibitor U0126 enhanced gstp1-2 mRNA expression during transition from blastula to the segmentation stage and from pharyngula until the hatching stage. Basal Erk activity did not affect gstp1-2 expression in tert-butylhydroquinone-exposed embryos. Addition of phorbol 12-myristate 13-acetate increased Erk activity leading to suppression of gstp1-2 mRNA. Activation of cAMP/Creb pathway by forskolin prevented gstp1-2 expression, whereas U0126 suppressed Creb phosphorylation, thus setting up Creb as a proximal transmitter of Erk inhibitory effect. Collectively, these findings suggest that Erk-Creb pathway exerts suppressive effect on gstp1-2 mRNA in a narrow developmental window. This study also provides a novel link between Erk and gstp1-2 expression, setting apart a possible differential regulation of gstp1-2 in adult and embryonic cells.

Atrazine activates multiple signaling pathways enhancing the rapid hCG-induced androgenesis in rat Leydig cells

Atrazine (ATR) is an endocrine disruptor that affects steroidogenic process, resulting in disruption of reproductive function of the male and female gonads. In this study, we used the primary culture of peripubertal Leydig cells to investigate the effect of ATR on the rapid androgen production stimulated by human chorionic gonadotropin (hCG). We demonstrated that ATR activated multiple signaling pathways enhancing the rapid hCG-stimulated androgen biosynthesis in Leydig cells. Low hCG concentration (0.25ng/mL) caused cAMP-independent, but ERK1/2-dependent increase in androgen production after 60min of incubation. Co-treatment with ATR for 60min enhanced the cAMP production in hCG-stimulated cells. Accumulation of androgens was prevented by addition of U0126, N-acetyl-l-cysteine and AG1478. Co-treatment with hCG and ATR for 60min did not alter steroidogenic acute regulatory protein (Star) mRNA level in Leydig cells. After 120min, hCG further increased androgenesis in Leydig cells that was sensitive to inhibition of the cAMP/PKA, ERK1/2 and ROS signaling pathways. Co-treatment with ATR for 120min further enhanced the hCG-induced androgen production, which was prevented by inhibition of the calcium, PKC and EGFR signaling cascades. After 120min, ATR enhanced the expression of Star mRNA in hCG-stimulated Leydig cells through activation of the PKA and PKC pathway. Collectively, these data suggest that exposure to ATR caused perturbations in multiple signaling pathways, thus enhancing the rapid hCG-dependent androgen biosynthesis in peripubertal Leydig cells.

Differential expression of CYP1A1 and CYP1A2 genes in H4IIE rat hepatoma cells exposed to TCDD and PAHs

Rat hepatoma cells H4IIE were treated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons (PAHs) (dibenz(a,h)anthracene, benzo(a)pyrene, benz(a)anthracene, chrysene), low-concentration mixtures of PAHs and TCDD, and environmental mixtures contaminated by PAHs and their derivatives. Expression of the gene battery comprising cytochrome P450 Cyp1a1, Cyp1a2, Cyp1b1, and glutathione-s-transferase Gsta2 and Gstp was investigated using quantitative real time polymerase chain reaction (qRT-PCR) analysis. The results revealed that TCDD induce Cyp1a1>Cyp1a2>Cyp1b1, while PAHs and PAH-containing environmental mixtures induce Cyp1a2>Cyp1a1>Cyp1b1 gene expression pattern. While low-concentration mixtures elicited a more pronounced response in comparison to single treatments, the typical gene expression patterns were not observed. In all samples, Gsta2 was predominantly expressed relative to Gstp. These findings indicate that differential Cyp1a1 and Cyp1a2 expression in the H4IIE cells might be used for detection of PAHs in highly contaminated environmental mixtures, but not in low-concentration mixtures of these compounds.