Melatonin and Vitamins as Protectors against the Reproductive Toxicity of Bisphenols: Which Is the Most Effective? A Systematic Review and Meta-Analysis.

Bisphenols such as bisphenol A (BPA), S (BPS), C (BPC), F (BPF), AF (BPAF), tetrabromobisphenol, nonylphenol, and octylphenol are plasticizers used worldwide to manufacture daily-use articles. Exposure to these compounds is related to many pathologies of public health importance, such as infertility. Using a protector compound against the reproductive toxicological effects of bisphenols is of scientific interest. Melatonin and vitamins have been tested, but the results are not conclusive. To this end, this systematic review and meta-analysis compared the response of reproductive variables to melatonin and vitamin administration as protectors against damage caused by bisphenols. We search for controlled studies of male rats exposed to bisphenols to induce alterations in reproduction, with at least one intervention group receiving melatonin or vitamins (B, C, or E). Also, molecular docking simulations were performed between the androgen (AR) and estrogen receptors (ER), melatonin, and vitamins. About 1234 records were initially found; finally, 13 studies were qualified for review and meta-analysis. Melatonin plus bisphenol improves sperm concentration and viability of sperm and increases testosterone serum levels compared with control groups; however, groups receiving vitamins plus bisphenols had lower sperm concentration, total testis weight, and testosterone serum levels than the control. In the docking analysis, vitamin E had the highest negative MolDock score, representing the best binding affinity with AR and ER, compared with other vitamins and melatonin in the docking. Our findings suggest that vitamins could act as an endocrine disruptor, and melatonin is most effective in protecting against the toxic effects of bisphenols.

Vitamin E (α-Tocopherol) Does Not Ameliorate the Toxic Effect of Bisphenol S on the Metabolic Analytes and Pancreas Histoarchitecture of Diabetic Rats.

This study investigated whether the coadministration of vitamin E (VitE) diminishes the harmful effects provoked by plasticizer bisphenol S (BPS) in the serum metabolites related to hepatic and renal metabolism, as well as the endocrine pancreatic function in diabetic male Wistar rats. Rats were divided into five groups (n = 5-6); the first group was healthy rats (Ctrl group). The other four groups were diabetic rats induced with 45 mg/kg bw of streptozotocin: Ctrl-D (diabetic control); VitE-D (100 mg/kg bw/d of VitE); BPS-D (100 mg/kg bw/d of BPS); The animals from the VitE + BPS-D group were administered 100 mg/kg bw/d of VitE + 100 mg/kg bw/d of BPS. All compounds were administered orally for 30 days. Body weight, biochemical assays, urinalysis, glucose tolerance test, pancreas histopathology, proximate chemical analysis in feces, and the activity of antioxidants in rat serum were assessed. The coadministration of VitE + BPS produced weight losses, increases in 14 serum analytes, and degeneration in the pancreas. Therefore, the VitE + BPS coadministration did not have a protective effect versus the harmful impact of BPS or the diabetic metabolic state; on the contrary, it partially aggravated the damage produced by the BPS. VitE is likely to have an additive effect on the toxicity of BPS.

Bisphenols as promoters of the dysregulation of cellular junction proteins of the blood-testis barrier in experimental animals: A systematic review of the literature.

Daily, people are exposed to chemicals and environmental compounds such as bisphenols (BPs). These substances are present in more than 80% of human fluids. Human exposure to BPs is associated with male reproductive health disorders. Some of the main targets of BPs are intercellular junction proteins of the blood-testis barrier (BTB) in Sertoli cells because BPs alter the expression or induce aberrant localization of these proteins. In this systematic review, we explore the effects of BP exposure on the expression of BTB junction proteins and the characteristics of in vivo studies to identify potential gaps and priorities for future research. To this end, we conducted a systematic review of articles. Thirteen studies met our inclusion criteria. In most studies, animals treated with bisphenol-A (BPA) showed decreased occludin expression at all tested doses. However, bisphenol-AF treatment did not alter occludin expression. Cx43, ZO-1, ß-catenin, nectin-3, cortactin, paladin, and claudin-11 expression also decreased in some tested doses of BP, while N-cadherin and FAK expression increased. BP treatment did not alter the expression of α and γ catenin, E-cadherin, JAM-A, and Arp 3. However, the expression of all these proteins was altered when BPA was administered to neonatal rodents in microgram doses. The results show significant heterogeneity between studies. Thus, it is necessary to perform more research to characterize the changes in BTB protein expression induced by BPs in animals to highlight future research directions that can inform the evaluation of risk of toxicity in humans.

Maternal perinatal exposure to bisphenol S induces an estrogenic like effect in glucose homeostasis in male offspring.

Bisphenol S (BPS) has been introduced into the industry as a safer alternative to bisphenol A (BPA). However, the recent studies have reported a possible association between BPS and disturbed glucose homeostasis, indicating that it may be a risk factor for type 1 and type 2 diabetes mellitus, obesity, and gestational diabetes mellitus. Nevertheless, the role of BPS in glucose metabolism remains controversial. In this study, we investigated the glucose metabolism of male Wistar rats born from dams that were BPS-exposed (groups: BPS-L (0.05 mg/kg/day), BPS-H (20 mg/kg/day)) during pregnancy and lactation. We observed that both BPS treated groups of animals presented a significant decrease in anogenital distance/weight1/3 , as compared to control animals, although no alterations in testosterone levels were observed. Furthermore, the BPS-L group presented a significant decrease in body weight from postnatal day (PND) 21 to adult stage. In addition, a significant increase in glucose tolerance, pancreatic ß-cell proliferation, the frequency of small islets, and the average ß-cell size at PND 36 was observed in this group. However, no changes in insulin serum levels and percentage of ß-cells were recorded. Furthermore, these changes were not preserved at the adult stage (PND 120). The results suggest that the administration of low doses of BPS during the perinatal period induced an estrogenic like effect, with males apparently becoming more female-like in their responses to a glucose challenge.

Effects of bisphenols on Blood-Testis Barrier protein expression in vitro: A systematic review and meta-analysis.

Bisphenols are a group of environmental endocrine-disrupting chemicals that produce alterations in the expression of intercellular junction proteins of the Blood-Testis Barrier (BTB) involved in spermatogenesis. The association between bisphenol exposure and BTB protein expression is controversial. Therefore, we performed this systematic review and meta-analysis to clarify bisphenol effects on Sertoli cell BTB protein expression in vitro. The Standardized Mean Difference (SMD) with a 95 % confidence interval (95 % CI) was used to evaluate the association between alterations in the BTB protein expression and bisphenol exposure in vitro. Six articles were included in the meta-analysis. Bisphenol-A (BPA) exposure at 200 µM was associated with significant decrease in BTB protein expression (SMD = -2.70, 95 %CI: -3.59, -1.80, p het = 0.46, p = <0.00001). In the moderate (40-50 µM) and low dose (<25 µM), no significant associations were obtained. We also found a non-monotonic dose-response curve of bisphenol effect in ZO-1 protein expression; low and high doses presented a significant decrease compared to control, while moderate dose presented no change. The current temporary Tolerable Daily Intake (tTDI) of BPA is 4 µg/kg bw/day. The 5-25 µM doses of BPA are equivalent to ∼1-5 mg/kg bw, respectively. Although the low dose group (<25 µM) assessed doses below the previous NOAEL value, these doses are above the current tTDI. Thus, it is necessary to conduct more studies with lower bisphenol concentrations to avoid underestimating the potential adverse effects of bisphenols at doses below tTDI.

Parental perinatal exposure to bisphenol A reduces the threshold to disrupt blastocyst implantation via decreasing talin, occudin and E-cadherin levels.

The aim was to evaluate the effect of perinatal BPA exposure of one or both parents on the implantation index and expression of talin, occludin and E-cadherin in the uterine epithelial cells (UEC) of the offspring. Pregnant Wistar dams (F0) received BPA or vehicle from gestational day (GD) 6 to lactation day 21. F1 animals were mated forming four groups: Control dam-Control sire (C♀-C♂), BPA dam -Control sire (B♀-C♂), Control dam -BPA sire (C♀-B♂), BPA dam -BPA sire (B♀-B♂). F1 dams were sacrificed at GD 6. Significantly decreased number of implantation sites was observed in the B♀-B♂ group as compared to the C♀-C♂ group, which correlated with decreased talin apical/basal expression ratio, occludin apical expression, and E-cadherin apical/lateral expression ratio in the UEC. Furthermore, decreased E-cadherin expression in the blastocyst was observed. Our data suggest that reduced protein expressions in F1 BPA offspring could result from decreased progesterone serum levels.

Perinatal administration of bisphenol A alters the expression of tight junction proteins in the uterus and reduces the implantation rate.

We studied the effect of bisphenol-A (BPA) administration to rats, during the perinatal period, on the fertility of F1 generation and on the expression of tight junction (TJ) proteins in the uterus during early pregnancy. Pregnant Wistar dams (F0) received: BPA-L (0.05mg/kg/day), BPA-H (20mg/kg/day) or vehicle, from gestational day (GD) 6 to lactation day 21. F1 female pups were mated at 3 months of age and sacrificed at GD 1, 3, 6, and 7. Serum hormonal levels, ovulation rate, number of implantation sites and expression of TJ proteins in the uterus of F1 females were evaluated. BPA treatment induced no change in ovulation rate, but induced alterations in progesterone (P4) and estradiol (E2) serum levels, and in implantation rate. With regards to TJ proteins, BPA-H increased claudin-1 during all GDs; eliminated the peaks of claudins -3 and -4 at GD 3 and 6, respectively; and decreased claudin-7 at GD 6, ZO-1 from GD 1-6, and claudin-3 at GD 7 in stromal cells. BPA-L instead, eliminated claudin-3 peak at GD 3, increased claudin-4 and decreased claudin-7 from GD 1-6, decreased claudin-1 at GD 3 and 7 and claudin-4 at GD 7 in stromal cells. BPA-L also decreased ZO-1 at GDs 1 and 3 and increased ZO-1 at GD 6. Thus, BPA treatment during perinatal period perturbed, when the animals reached adulthood and became pregnant, the particular expression of TJ proteins in the uterine epithelium and reduced in consequence the number of implantation sites.

Prevalencia de asimetrías mandibulares en radiografías panorámicas de población de Bogotá-Colombia / Mandibular asymmetries prevalence in panoramic radiographies in a Bogotá­ Colombia population

La simetría mandibular es esencial para determinar los patrones no solo de belleza facial sino de función masticatoria; las alteraciones de crecimiento y desarrollo, patologías genéticas, traumáticas o neoplásicas o por características propias de cada población, que afectan a la mandíbula, generan consecuencias que llevan a tratamientos inclusive quirúrgicos para su corrección. El análisis de Thilander en radiografías panorámicas, permite la valoración de la magnitud de las asimetrías mandibulares, como parte de los exámenes complementarios en el proceso diagnóstico. Determinar la frecuencia de asimetrías condilares, de cuerpo y rama mandibular en radiografías panorámicas digitales. Estudio descriptivo de corte transversal en 500 radiografías panorámicas digitales, de adecuado contraste y densidad, obtenidas de pacientes mayores de 18 años de edad, con dentición completa; se registró sexo, edad y mediante el programa CliniView 9.1 se tomaron las medidas mandibulares propuestas por Thilander calculando las diferencias con la fórmula de Bezzur. La prevalencia de asimetrías condilares patológicas en la población mayor de 18 años es del 6 %. Se encontró una diferencia significativa en las medidas verticales entre los dos lados sin que en ellas tenga influencia el género o la edad. Las diferencias en la altura de la rama mandibular se encuentran en la mayoría de los casos a expensas del cóndilo y las discrepancias entre los lados derecho e izquierdo, deben considerarse como elemento diagnóstico en posibles patologías articulares.

The mandibular symmetry is essential to determine the patterns, not only of facial beauty, but also in the masticatory function. The growth and development alterations, genetic pathologies, traumatic, neoplasic or the characteristics of each population, that affect the jaw, generate consequences that may lead to even surgical treatments for correction. Thilander analysis on panoramic x-rays, allows the assessment of the magnitude of mandibular asymmetries, as part of the complementary exams in the diagnostic process. The objective of the study was to determine the frequency of condylar asymmetries in mandibular body and ramus in digital panoramics x-rays. A cross-sectional study was carried out in 500 digital panoramic x-rays with adequate contrast and density, obtained from patients over 18 years of age, with full dentition. Sex and age were registered by 9.1 CliniView program. Proposed mandibular measurements were recorded by Thilander, calculating the differences with the Bazzur's formula. The prevalence of pathological condylar asymmetries in the population over 18 years is 6 %. A significant difference was found in the vertical measurements between the two sides, though there was no influence by either sex or age. The differences in the height of the mandibular ramus are, in most cases, at the expense of the condyle. The discrepancies between the left and right sides must be considered as diagnostic element in possible articular pathologies.

Serotonin in testes of bat Myotis velifer during annual reproductive cycle: expression, localization, and content variations.

The mechanism of reproduction in mammals is very complex and in some cases is quite particular. For example in some bat species, the male presents a reproductive mechanism characterized by an annual testicular cycle that goes from recrudescence to regression (spermatogenesis to inactivity period, respectively). After recrudescence, the spermatozoa arrive at epididymis and wait to be expelled at the time of ejaculation during the mating period, which occurs some months later. Because serotonin (5-HT) has gained reproductive importance in the last years, the aim of the present study was to analyze the expression of this indolamine and both tryptophan hydroxylase and monoamine oxidase isoform A-enzymes involved in its metabolism-in Myotis velifer testes, a seasonal reproductive bat species that shows temporal asynchrony in its sexual cycle, across the principal periods of their reproductive cycle. By using both Falck-Hillarp histochemistry and immunofluorescence techniques, we found serotonin in vesicles of Leydig cells and probably Sertoli cells too; interestingly, both intracellular localization and concentration was variable across the different stages of the reproductive cycle, being lower during spermatogenesis phase and increasing during the mating phase. These results suggest that 5-HT is present in bat testes and it could play an important role in testicular function during their reproductive cycle.

Agonistic activity of ICI 182 780 on activation of GSK 3ß/AKT pathway in the rat uterus during the estrous cycle.

We examined the ability of ICI 182,780 (ICI) to block uterine cell proliferation via protein kinase b/AKT pathway in the uterus of the rat during the estrous cycle. Intact rats, with regular estrous cycles, received a subcutaneous (s.c.) injection of either vehicle or ICI at 08:00 h on the day of proestrus or at 00:00 h on the day of estrus and sacrificed at 13:00 h of metaestrus. Estradiol (E2) and progesterone (P4) plasma levels were measured by radioimmunoassay. Both ICI treatments, induced a significant decrease (p<0.01) in uterine estrogen receptor alpha (ERα) content, had no effect on uterine progesterone receptor (PR) protein expression and caused marked nuclear localization of cyclin D1, in both luminal and glandular uterine epithelium, as compared to vehicle-treated animals. Furthermore, we detected that ICI treatment induced glycogen synthase kinase (Gsk3-ß) Ser 9 phosphorylation, which correlates with cyclin D1 nuclear localization. However, some differences were observed between the two different time schedules of administration. We observed that the administration of ICI at 08:00 h on proestrus day produced a 15% inhibition of luminal epithelial cell proliferation, reduced uterine wet weight by 21% and caused reduction of Akt phosphorylation at Ser 473 as compared to vehicle-treated animals, whereas ICI treatment at 00:00 h on estrus day had no effect on these parameters. The overall results indicate that ICI may exert agonistic and antagonistic effects on uterine cell proliferation through differential activation of the Akt pathway depending on the administration period during the estrous cycle, and indicates that the mechanism of cell proliferation during the physiological conditions of the estrous cycle, is under a different and more complex regulation than in the ovariectomized + E2 animal model.

Synergistic anticancer activity of Thiazolo[5,4-b]quinoline derivative D3CLP in combination with cisplatin in human cervical cancer cells.

BACKGROUND: D3CLP (9-[(3-chloro)phenylamine]-2-[3-(diethylamine)propylamine]thiazolo[5,4-b]quinoline) is a potent cytotoxic thiazolo[5,4-b]quinoline synthetic derivative that induces apoptosis of leukemia cells, while it displays low toxicity towards non-tumoral cells. The aim of this study was to determine if D3CLP can enhance the cytotoxicity of other antineoplastic drugs. MATERIALS AND METHODS: Leukemia, breast and cervical cancer cell lines were exposed to D3CLP-alone or in combination with imatinib, tamoxifen or cisplatin, respectively. Cell viability after treatment was evaluated by the MTT assay, and cell death by the TUNEL assay. The effects of combined treatments were analyzed by combination index and isobolographic analysis. RESULTS: Antiproliferative activity results indicate that D3CLP in combination with antineoplastic drugs induced a synergistic effect, at 3:1 and 1:1 ratios for D3CLP plus imatinib in K-562 leukemia cells, and at a 3:1 ratio for D3CLP with cisplatin in HeLa cells, as determined by their combination index. Furthermore, isobolographic analysis demonstrated a significant synergism for a 3:1 combination ratio of D3CLP with cisplatin in HeLa cells. In addition, TUNEL assay suggests cell death by apoptosis of HeLa cells after treatment with D3CLP and its combination with cisplatin at a 3:1 ratio. CONCLUSION: Overall the results indicate that D3CLP, in combined preparation with antineoplastic drugs, is a good candidate for pre-clinical studies in the treatment of different carcinoma cell types.

Evidence of 5-HT components in human sperm: implications for protein tyrosine phosphorylation and the physiology of motility.

Serotonin (5-hydroxytryptamine; C(10)H(12)N(2)O (5-HT)) is produced in the CNS and in some cells of peripheral tissues. In the mammalian male reproductive system, both 5-HT and tryptophan hydroxylase (TPH) have been described in Leydig cells of the testis and in principal cells of the caput epididymis. In capacitated hamster sperm, it has been shown that 5-HT promotes the acrosomal reaction. The aim of this work was to explore the existence of components of the serotoninergic system and their relevance in human sperm physiology. We used both immunocytochemistry and western blot to detect serotoninergic markers such as 5-HT, TPH1, MAO(A), 5-HT(1B), 5-HT(3), and 5HT(T); HPLC for TPH enzymatic activity; Computer Assisted Semen Analysis assays to measure sperm motility parameters and pharmacological approaches to show the effect of 5-HT in sperm motility and tyrosine phosphorylation was assessed by western blot. We found the presence of serotoninergic markers (5-HT, TPH1, MAO(A), 5-HT(1B), 5-HT(2A), 5-HT(3), 5-HT(T), and TPH enzymatic activity) in human sperm. In addition, we observed a significant increase in tyrosine phosphorylation and changes in sperm motility after 5-HT treatment. In conclusion, our data demonstrate the existence of components of a serotoninergic system in human sperm and support the notion for a functional role of 5-HT in mammalian sperm physiology, which can be modulated pharmacologically.

Post-burn hypertrophic scars are characterized by high levels of IL-1ß mRNA and protein and TNF-α type I receptors.

Post-burn hypertrophic scars are characterized by increased collagen synthesis and hyperplasia, and may be associated with erythema, pain, dysesthesia, pruritus, and skin border elevation. Although the etiopathogenesis of hypertrophic scarring remains unclear, proinflammatory and profibrogenic cytokines are known to play an important role in general skin dysfunction. This study assessed mRNA expression, proteins, and type I receptors of tumor necrosis factor-alpha (TNF-α) and interleukin 1-beta (IL-1ß) in normal skin, normotrophic and post-burn hypertrophic scars. Skin biopsies were obtained from 10 hypertrophic and 9 normotrophic scars, and 4 normal skin sites. Only post-burn scars covering more than 10% of the body were included. Ex vivo histopathological analysis evaluated scar maturity, in situ hybridization assessed mRNA expression, and cytokine protein and cytokine/cell colocalization were performed using single- and double-label immunohistochemistry, respectively. IL-1ß is overexpressed in hypertrophic scars at the post-transcriptional level, associated primarily with keratinocytes and CD1a(+) cells. Type I receptors for TNF-α are overexpressed in blood vessels of hypertrophic scars. The coordinated overexpression of IL-1ß and TNF-α type I receptor may maintain the fibrogenic phenotypes of hypertrophic scars, even those in "remission".

Polymorphisms in the hypoxia-inducible factor 1 alpha gene in Mexican patients with preeclampsia: A case-control study.

BACKGROUND: Although the etiology of preeclampsia is still unclear, recent work suggests that changes in circulating angiogenic factors play a key role in its pathogenesis. In the trophoblast of women with preeclampsia, hypoxia-inducible factor 1 alpha (HIF-1α) is over-expressed, and induces the expression of non-angiogenic factors and inhibitors of trophoblast differentiation. This observation prompted the study of HIF-1α and its relation to preeclampsia. It has been described that the C1772T (P582S) and G1790A (A588T) polymorphisms of the HIF1A gene have significantly greater transcriptional activity, correlated with an increased expression of their proteins, than the wild-type sequence. In this work, we studied whether either or both HIF1A variants contribute to preeclampsia susceptibility. RESULTS: Genomic DNA was isolated from 150 preeclamptic and 105 healthy pregnant women. Exon 12 of the HIF1A gene was amplified by PCR, and the genotypes of HIF1A were determined by DNA sequencing.In preeclamptic women and controls, the frequencies of the T allele for C1772T were 4.3 vs. 4.8%, and the frequencies of the A allele for G1790A were 0.0 vs. 0.5%, respectively. No significant differences were found between groups. CONCLUSION: The frequency of the C1772T and G1790A polymorphisms of the HIF1A gene is very low, and neither polymorphism is associated with the development of preeclampsia in the Mexican population.

Persistence of decidual NK cells and KIR genotypes in healthy pregnant and preeclamptic women: a case-control study in the third trimester of gestation.

BACKGROUND: Natural Killer (NK) cells are the most abundant lymphocytes in the decidua during early gestation. The interactions of NK cells with the extravillous cytotrophoblast have been associated with a normal spiral artery remodeling process, an essential event for a successful pregnancy. Recent data indicate that alterations in the amount of decidual NK (dNK) cells contribute to the development of preeclampsia (PE). Moreover, genetic studies suggest that Killer Immunoglobulin-like Receptors (KIR) expressed in dNK cells influence the susceptibility to PE. Although dNK cells have been well characterized during early pregnancy, they have been scarcely studied in the third trimester of gestation. The aim of this work was to characterize dNK cells at the last trimester of gestation and to analyze the KIR genotype of healthy and PE women. METHODS: Decidual samples were obtained during Caesarean section from control (n = 10) and PE (n = 9) women. Flow cytometric analysis of CD3, CD56, CD16 and CD9 was used to characterize and quantify dNK cells in both groups. Cell surface markers from decidual leukocytes were compared with PBMC from healthy donors.KIR genotyping was performed in genomic DNA (control, n = 86; PE, n = 90) using PCR-SSP. RESULTS: The results indicate that dNK cells persist throughout pregnancy. They represented 20% of total leukocytes in control and PE groups, and they expressed the same cell surface markers (CD3-, CD56+, CD16- and CD9+) as dNK in the first trimester of gestation. There were no significant differences in the percentage of dNK cells between control and PE groups. The analysis of KIR gene frequencies and genotypes was not statistically different between control and PE groups. The ratio of activating to inhibitory genes indicated that the overall inhibitory balance (0.2-0.5) was more frequent in the PE group (control, 31.3% vs PE, 45.5%), and the activating balance (0.6-1.1) was more frequent in the control group (control, 68.6% vs PE, 54.4%). However this difference was not significant. CONCLUSION: We demonstrated the persistence of dNK cells in PE and control women at the third trimester of pregnancy; these dNK cells had a similar phenotype to those found during early pregnancy. The predominance of a KIR inhibitory balance in the PE group could be associated to the physiopathology of PE.

Administration of bisphenol A to dams during perinatal period modifies molecular and morphological reproductive parameters of the offspring.

Bisphenol A (BPA) is an estrogenic agonist compound that induces changes in diverse reproductive parameters in rats. The aim of the present study was to determine the effects of BPA given in drinking water containing 10mg/L (approximate dose 1.2mg/kg BW/day), administered chronically to rats during pregnancy and lactation, on reproductive tract parameters of the offspring. 79.2% of the female offspring from BPA-treated mothers presented irregular estrous cycles. As compared to the control group, a significant increase in the thickness of the uterine epithelia and stroma was observed in the BPA group. Additionally, 60% of the female offspring from BPA mothers did not undergo abundant uterine epithelial apoptosis during the estrus phase of the cycle while control animals did. In addition, a down regulation of ERα expression was observed in epithelial cells on estrus day. The results indicate that BPA, when administered chronically in water beverages to dams, modifies the reproductive cycle of the offspring during young adulthood.

Differential expression of estrogen receptor alpha gene in the ampullae and isthmus regions of the rabbit oviduct during early pregnancy.

We characterized the expression pattern of estrogen receptor alpha (ERalpha) gene in two regions of the oviduct, ampullae and isthmus, of the rabbit (Oryctolagus cuniculus) during early pregnancy (1-4 days) by RT-PCR and immunohistochemistry. In both regions of the oviduct, ERalpha mRNA was increased (P<0.01) in pregnant rabbits as compared with non-pregnant animals (NG). In the ampullae, the greatest amount of ERalpha mRNA was detected on the third day of pregnancy (1.01+/-0.02 relative amount), followed by a decrease on Day 4. In the isthmus, an increase in ERalpha mRNA was observed during the first 4 days of pregnancy, with the greatest amount on Day 3 (1.49+/-0.3 relative amount). A marked ERalpha immunostaining was detected in epithelial, stromal and smooth muscle cells of the ampullae on the second and third days of pregnancy as compared with the NG group. In contrast, a significant increase in ERalpha immunostaining was observed on the first and second days of pregnancy with a reduction on the third and fourth days in the epithelial, stromal and smooth muscle cells of the isthmus. The overall results suggest there is differential expression pattern for the ERalpha gene in the ampullae and isthmus during early pregnancy of the rabbit, and that these variations are related to specific functions of ERalpha in the reproductive tract during early pregnancy.

Progesterone receptor immunoreactivity differs in the uterus of pseudopregnant and medroxyprogesterone acetate-treated rabbits.

Progesterone receptor (PR) plays an important role in mammals pregnancy which is characterized by greater progesterone plasma concentrations. We assessed PR protein distribution in the rabbit uterus by immunohistochemistry in two progestational conditions: pseudopregnancy (intact adult animals treated with hCG) and after application of a synthetic progestin, medroxyprogesterone acetate (MPA), to ovariectomized animals (OVX). PR immunoreactivity in uterine epithelium of pseudopregnant rabbits was increased in relation to non-pseudopregnant (NP) rabbits. Amounts were similar on Days 1, 3, and 5 of treatment, and was greater on Day 7 (P<0.001). In contrast, a significant diminution in PR immunoreactivity was observed in stroma cells from Days 1 to 7 (P<0.001). In OVX rabbits treated with MPA, an increase in PR immunoreactivity was observed in the uterine epithelium on Days 1 to 5 of treatment, reaching a maximum on Day 3 (P<0.001). In contrast, in stromal cells a diminution in PR immunoreactivity was observed when compared to the OVX group on Days 1, 3 and 7 of MPA treatment (P<0.001), and there was a slight increase on Day 5. Results suggest a differential time course and tissue specific immunoreactivity for PR in the uterus of the rabbit in two progestational conditions. The present study indicated synthetic progestins have different mechanisms of receptor regulation than those of natural hormones and it should be taken into account in reproductive applications.

Different expression of alpha and beta mitochondrial estrogen receptors in the aging rat brain: interaction with respiratory complex V.

Recent evidence suggests that hormonal effects on mitochondria could be mediated by mitochondrial estrogen receptors (mtERs). These receptors are new candidates for the beneficial estrogenic effects on mitochondria in different physiological conditions. The aim of this investigation was to study mtER expression during brain aging. We analyzed mtERalpha and mtERbeta expression in cortical, hippocampal and hypothalamic mitochondria of young adult (3months) and aged (18 months) female Wistar rats by Western blot. In addition, we explored the interaction of mtERbeta with respiratory complex V by using coimmunoprecipitation assays. The results show that mtERalpha and mtERbeta are present in young and aged brain mitochondria. We also demonstrate that mtERs are expressed as variants and have a brain region specific distribution. The predominant mtER variants detected were of 61 and 55KDa for mtERalpha and of 63 and 52KDa for mtERbeta. However, we did not observe differences in the mtERalpha or beta content between the two age groups studied. Additionally, we show that mtERbeta interacts with complex V. The overall results demonstrate that there is a differential expression of mtERalpha and mtERbeta variants in different brain areas, indicating that they may participate in different functions in the brain during aging.

Coumarin A/AA induces apoptosis-like cell death in HeLa cells mediated by the release of apoptosis-inducing factor.

It has been demonstrated that naturally occurring coumarins have strong biological activity against many cancer cell lines. In this study, we assessed the cytotoxicity induced by the naturally isolated coumarin A/AA in different cancer cell lines (HeLa, Calo, SW480, and SW620) and in normal peripheral-blood mononuclear cells (PBMCs). Cytotoxicity was evaluated using the MTT assay. The results demonstrate that coumarin A/AA was cytotoxic in the four cancer cell lines tested and importantly was significantly less toxic in PBMCs isolated from healthy donors. The most sensitive cancer cell line to coumarin A/AA treatment was Hela. Thus, the programmed cell death (PCD) mechanism induced by this coumarin was further studied in this cell line. DNA fragmentation, histomorphology, cell cycle phases, and subcellular distribution of PCD proteins were assessed. The results demonstrated that DNA fragmentation, but not significant cell cycle disruptions, was part of the PCD activated by coumarin A/AA. Interestingly, it was found that apoptosis-inducing factor (AIF), a proapoptotic protein of the mitochondrial intermembrane space, was released to the cytoplasm in treated cells as detected by the western blot analysis in subcellular fractions. Nevertheless, the active form of caspase-3 was not detected. The overall results indicate that coumarin A/AA induces a caspase-independent apoptotic-like cell death program in HeLa cells, mediated by the early release of AIF and suggest that this compound may be helpful in clinical oncology.