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Female infertility induced by vanadium inhalation in a murine model
Abstracts / Toxicology Letters 259S (2016) S73–S247
fertilization and zygote development was assessed using mating and in vitro fertilization techniques. Results: Estrous cyclicity assessed as the number of days spent per stage, ovulation rate assessed by a superovulation protocol, and oocyte viability assessed with propidium/Hoechst staining were similar in control mice and DEHP-treated mice. Exposure to DEHP and then in vivo mating revealed significantly increased number of unfertilized oocytes, increased number of zygotes at the 1-cell stage and decreased number of zygotes at the 4-cell stage, compared to control. However, exposure to DEHP and in vitro fertilization revealed no alterations in fertilization and zygote development compared to control. To provide evidence on a possible mechanism of toxicity, a group of mice were exposed to 5-fluorouracil (5FU), a compound that inhibits cell division, which caused similar effects to those observed in in vivo mated female mice. Conclusion: DEHP mainly targets zygote development at the 1cell stage via unknown effects on the oviduct of the exposed female. Our data further suggest that DEHP may delay zygote development via disrupting cell division (cleavage). Conacyt México CB-167678. http://dx.doi.org/10.1016/j.toxlet.2016.07.563 PP25.8 The biomarker research of chronic prostatitis Z. Sun, L. Zhou, Y. Jia National Evaluation Centre for the Toxicology of Fertility Regulating Drug, Department of Pharmacology and Toxicology, Shanghai Institute of Planned Parenthood Research, 2140 Xietu Road, Xuhui District, Shanghai 200032, PR China Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is an important public health problem. CP/CPPS is a poorly understood entity characterized by pelvic or perineal pain, irritative voiding symptoms, and sexual dysfunction, and, from a clinical point of view, is truly lacking a cause that would allow a more rationaldriven therapy. Currently, there is not a “golden standard” in diagnosis of prostatitis, while the detection of biomarkers can be helpful for the diagnosis, treatment and prognostic monitoring of prostatitis. Following significant improvements of the methods of detection in biological fluids, a number of prostate inflammation biomarkers were identified and quantified, in peripheral blood, urine and seminal plasma. In fact, white blood cells (WBC) count in expressed prostatic secretions (EPS) has long been considered as the marker of prostatitis. However, it does not appear to be the optimal marker of inflammation and the current categorization of chronic prostatitis/chronic pelvic pain syndrome III B and asymptomatic inflammatory prostatitis as inflammatory or non-inflammatory based on WBC count appears to offer little clinically useful information. And whilst WBC can be found in the prostatic fluid or seminal plasma of asymptomatic men as well as in that of men with pelvic pain. Also, the measures of the NIH-CP Symptom Index in symptomatic men show no correlation with WBC in EPS or seminal plasma. As the prostatitis biomarkers, cytokines/chemokine may have high sensitivity and good specificity. Interleukin 8 (IL-8) is a proinflammatory cytokine and plays an important role in different inflammatory diseases. Significant correlations between IL-8 levels and symptom score results were found. IL-8 values strongly correlated with CP/CPPS. Moreover, the patients with higher levels of IL-8 reported the worst symptoms. The study has shown that IL-8 was significantly elevated compared to controls in patients
S235
with CP/CPPS IIIA, CP/CPPS IIIB and benign prostatic hyperplasia (BPH). IL-8 is a reliable biomarker in seminal plasma for chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), and for BPH, and also the IL-8 levels were correlated with symptom scores and serum PSA values, increasing its value as a biomarker for prostate inflammation. Monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein-1␣ (MIP-1␣) recruit monocytes and macrophages via their release from fibroblasts and macrophages in joints of patients with rheumatoid arthritis and perpetuate the inflammatory process. For MCP-1 and MIP-1␣, chronic pelvic pain syndrome subtypes had statistically higher levels than the control group and patients with benign prostatic hyperplasia. MCP-1 and MIP-1␣ within the prostatic fluid in both chronic pelvic pain syndrome subtypes provide candidate future biomarkers for chronic pelvic pain syndrome. In addition, macrophage inflammatory protein-1␣ increase in expressed prostatic secretions provides a new marker for clinical pain in chronic pelvic pain syndrome patients. Given these findings prostatic dysfunction likely has a role in the pathophysiology of this syndrome. This work was supported by Shanghai experimental animal scientific and technological innovative action plan (No: 14140901302); Shanghai professional service platform of nonclinical evaluation of drug against male reproductive and urinary diseases (No: 15DZ2290400). http://dx.doi.org/10.1016/j.toxlet.2016.07.564 PP25.9 Female infertility induced by vanadium inhalation in a murine model N. Meléndez-García, P. Bizarro, M. Altamirano-Lozano, E. Rendón-Huerta, R. Guerrero-Alquicira, T. Fortoul Departamento de Biología Celular y Tisular, Facultad de Medicina, UNAM, México City, Mexico Introduction: Epidemiological studies have proven a relationship between maternal exposure to PM2.5 and pregnancy outcomes (low birth weight, intrauterine growth retardation, preterm birth). Particulate matter is composed, among others substances, by metals which produces miscarriages, menstrual disorders and stillbirth in occupationally exposed women. In animal models, particularly vanadium inhalation induces anestrus, lowers progesterone, and estrogen serum concentrations in female mice. However, it has not been evaluated if these alterations produce infertility in exposed females. Metals could interfere with the endocrine system but also in delocalizing expression of connexins and decreasing its expression, which are for important embryo’s implantation. Objective: To evaluate vanadium inhalation effect on connexins in female mice fertility. Materials and methods: 15 CD1 female mice inhaled vanadium pentoxide (V2 O5 ) 0.02 M. The postcoitusV-group inhaled vanadium just 1 h at 5th day after mating, meanwhile V4-WK group inhaled V 1 h, twice a week per four weeks and control group inhaled only the vehicle. Ovaries and uterus were collected and processed for routine histological technique, and also Cx43 and cx26 immunohistochemistry analysis was performed. The number of implantations and corpus luteum per dam were counted. Results: Control females presented 14 implantation sites, whereas in exposed mice during 4-WKs there were not any implantation sites, therefore we were not able to analyze connexin expression in implantation sites. Because of that we included a poscoitusV-group. Embryo reabsorptions were observed in this
S236
Abstracts / Toxicology Letters 259S (2016) S73–S247
former group and decreased stain for both connexins in epithelium and in the decidua. Conclusions: Vanadium inhalation prevents embryo implantation in females exposed for 4 WKs before, this finding indicates that lower levels of progesterone and estradiol induced by vanadium (reported in previous works) have the potential to induce infertility. In addition, embryo resorption in mice exposed just 1 h at 5th dpc suggests that embryo implantation is very sensitive to vanadium and maybe to other air pollutants. Hence, vanadium should be considered as an endocrine disruptor and reprotoxic. Financial support: CVU 630707. Posgrado en Ciencias Biológicas, UNAM. CONACyT. http://dx.doi.org/10.1016/j.toxlet.2016.07.565 PP25.10 Bisphenol-A does not alter mRNA levels of genes encoding proteins involved in communication and expansion of the cumulus cells–oocyte complex ˜ S.P. García-Zepeda, D.G. Acuna-Hernández, R. Santacruz-Márquez, I. Hernández-Ochoa Departamento de Toxicología, Cinvestav-IPN, Ciudad de México, Mexico Introduction: Cumulus cells maintain a close association with the oocyte through gap junction intercellular communication (GJIC) to facilitate the transfer of factors needed for the proper function of the female gamete. Upon signaling pathways triggering ovulation, luteinizing hormone (LH) induces mRNA levels of genes encoding proteins involved in cumulus cells (CC) expansion, including Prostaglandin-endoperoxide synthase 2 (Cox2), Hyaluronan synthase 2 (Has2), Tumor necrosis factor alpha induced protein 6 (Tsg6), Versican (Vcan), and CD44 antigen (Cd44). Objective: Because it has been shown that exposure to the worldwide used plasticizer bisphenol-A (BPA) decreases communication via GAP junctions, and that BPA alters CC expansion, this study evaluated whether BPA alters mRNA levels of genes encoding proteins involved in communication and expansion of the cumulus cells–oocyte complex (COC). Materials: Female mice were orally dosed with BPA (20 and 50 g/kg bw/day), vehicle (corn oil) or diethylstilbestrol (DES) as a positive control for 12–16 days. At the end of treatments, females received ip 5 IU of equine chorionic gonadotropin (eCG), and 48 h later were either sacrificed to collect COC or were administrated ip with 5 IU human chorionic gonadotropin (hCG) to collect COC at 10 h later. COC from eCG-primed mice were used to evaluate Luteinizing hormone/chorionic gonadotropin receptor (Lhcgr) mRNA levels, whereas COC from hCG-primed mice were used to evaluate Gap junction protein alpha 1 (Cx43), Gap junction protein alpha 4 (Cx37), Cox2, Has2, Tsg6, Vcan, and Cd44 mRNA levels. Results: The data showed similar mRNA levels of all genes related to communication and expansion of COC. Conclusion: These data suggest that transcription of main genes related to communication and expansion of murine COC is not a target of environmentally relevant doses of BPA. Financial support: Conacyt-México CB-167678. http://dx.doi.org/10.1016/j.toxlet.2016.07.566
PP25.11 Temephos decreases ovarian antral follicle growth in an in vitro system L. Millán-Mejía, S.P. García-Zepeda, I. Hernández-Ochoa Departamento de Toxicología, Cinvestav-IPN, Ciudad de México, Mexico Introduction: Temephos (phosphorothioate O,O,O,Otetramethyl O, di-p-; o -thio-phenyl) is a no systemic organophosphate pesticide (OP) used worldwide in public health campaigns to control Aedes aegypti mosquito, a transmitter of dengue and zika viruses. Recently, in vitro studies demonstrated that this OP exerts cytotoxic and genotoxic effects on cellular systems possessing metabolic activity. In spite of its extensive use, the potential detrimental effects of temephos on antral follicles, which are functional units in the ovary that releases a fertilizable oocyte and that possess a high metabolic and endocrine activities, remain to be elucidated. Objective: The present study tested the hypothesis that environmentally relevant concentrations of temephos inhibit antral follicle growth. Materials and methods: Ovarian antral follicles isolated from CD-1 female mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or temephos (0, 0.5, 1, 2, 5, and 10 M) for up to 7 days. Follicle growth, measured as follicle diameters every 24 h, was compared in vehicle control and temephos-treated follicles using general linear models for repeated measures. Results: Ovarian antral follicles cultured with 10 M temephos had significantly decreased growth compared to vehicle control at days 6 and 7. Conclusion: Temephos could have potential endocrine disrupting effects on the ovary. Financial support: This study was partially supported by the Pesticide Toxicology Network (Conacyt-Mexico #253789/271775), and Conacyt-Mexico CB-167678. http://dx.doi.org/10.1016/j.toxlet.2016.07.567 PP25.12 Genotoxic damage in sperm cells from Long–Evans rats exposed to arsenic and its relationship with poor quality sperm ˜ L. Nava, J. Jimenez, N. Betancourt, R. Alcaraz, O. M.S. Nino, Sambrano, J.M. Moran, E. Olivas Department of Cell Biology and Ultrastructure, Universidad Autónoma de Coahuila, Torreón, Coahuila, Mexico Introduction: Day by day the groundwater in the Laguna in Coahuila and Durango States, Northern Mexico is contaminated with arsenic (As) due to the gradual lowering of the groundwater sources, then exposing people to high concentrations of As in drinking water. Several animal studies have described the effects of As in the male reproductive function, resulting in low sperm quality as well as a genotoxic effect in lymphocytes. In this study, we assessed the genotoxic effects of As in rats exposed to As2 O3 in drinking water. Materials and methods: An experimental design was made with 9 male Long–Evans rats 15 weeks old, and 215–240 g. The animals (n = 9) were randomly divided into 3 groups (n = 3 each). The control group received distilled water and groups 1 and 2 received a solution of As (As2 O3 ) dissolved in distilled water at concentrations of 0.025, and 0.05 mg L−1 , respectively for five days a week, for
fertilization and zygote development was assessed using mating and in vitro fertilization techniques. Results: Estrous cyclicity assessed as the number of days spent per stage, ovulation rate assessed by a superovulation protocol, and oocyte viability assessed with propidium/Hoechst staining were similar in control mice and DEHP-treated mice. Exposure to DEHP and then in vivo mating revealed significantly increased number of unfertilized oocytes, increased number of zygotes at the 1-cell stage and decreased number of zygotes at the 4-cell stage, compared to control. However, exposure to DEHP and in vitro fertilization revealed no alterations in fertilization and zygote development compared to control. To provide evidence on a possible mechanism of toxicity, a group of mice were exposed to 5-fluorouracil (5FU), a compound that inhibits cell division, which caused similar effects to those observed in in vivo mated female mice. Conclusion: DEHP mainly targets zygote development at the 1cell stage via unknown effects on the oviduct of the exposed female. Our data further suggest that DEHP may delay zygote development via disrupting cell division (cleavage). Conacyt México CB-167678. http://dx.doi.org/10.1016/j.toxlet.2016.07.563 PP25.8 The biomarker research of chronic prostatitis Z. Sun, L. Zhou, Y. Jia National Evaluation Centre for the Toxicology of Fertility Regulating Drug, Department of Pharmacology and Toxicology, Shanghai Institute of Planned Parenthood Research, 2140 Xietu Road, Xuhui District, Shanghai 200032, PR China Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is an important public health problem. CP/CPPS is a poorly understood entity characterized by pelvic or perineal pain, irritative voiding symptoms, and sexual dysfunction, and, from a clinical point of view, is truly lacking a cause that would allow a more rationaldriven therapy. Currently, there is not a “golden standard” in diagnosis of prostatitis, while the detection of biomarkers can be helpful for the diagnosis, treatment and prognostic monitoring of prostatitis. Following significant improvements of the methods of detection in biological fluids, a number of prostate inflammation biomarkers were identified and quantified, in peripheral blood, urine and seminal plasma. In fact, white blood cells (WBC) count in expressed prostatic secretions (EPS) has long been considered as the marker of prostatitis. However, it does not appear to be the optimal marker of inflammation and the current categorization of chronic prostatitis/chronic pelvic pain syndrome III B and asymptomatic inflammatory prostatitis as inflammatory or non-inflammatory based on WBC count appears to offer little clinically useful information. And whilst WBC can be found in the prostatic fluid or seminal plasma of asymptomatic men as well as in that of men with pelvic pain. Also, the measures of the NIH-CP Symptom Index in symptomatic men show no correlation with WBC in EPS or seminal plasma. As the prostatitis biomarkers, cytokines/chemokine may have high sensitivity and good specificity. Interleukin 8 (IL-8) is a proinflammatory cytokine and plays an important role in different inflammatory diseases. Significant correlations between IL-8 levels and symptom score results were found. IL-8 values strongly correlated with CP/CPPS. Moreover, the patients with higher levels of IL-8 reported the worst symptoms. The study has shown that IL-8 was significantly elevated compared to controls in patients
S235
with CP/CPPS IIIA, CP/CPPS IIIB and benign prostatic hyperplasia (BPH). IL-8 is a reliable biomarker in seminal plasma for chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), and for BPH, and also the IL-8 levels were correlated with symptom scores and serum PSA values, increasing its value as a biomarker for prostate inflammation. Monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein-1␣ (MIP-1␣) recruit monocytes and macrophages via their release from fibroblasts and macrophages in joints of patients with rheumatoid arthritis and perpetuate the inflammatory process. For MCP-1 and MIP-1␣, chronic pelvic pain syndrome subtypes had statistically higher levels than the control group and patients with benign prostatic hyperplasia. MCP-1 and MIP-1␣ within the prostatic fluid in both chronic pelvic pain syndrome subtypes provide candidate future biomarkers for chronic pelvic pain syndrome. In addition, macrophage inflammatory protein-1␣ increase in expressed prostatic secretions provides a new marker for clinical pain in chronic pelvic pain syndrome patients. Given these findings prostatic dysfunction likely has a role in the pathophysiology of this syndrome. This work was supported by Shanghai experimental animal scientific and technological innovative action plan (No: 14140901302); Shanghai professional service platform of nonclinical evaluation of drug against male reproductive and urinary diseases (No: 15DZ2290400). http://dx.doi.org/10.1016/j.toxlet.2016.07.564 PP25.9 Female infertility induced by vanadium inhalation in a murine model N. Meléndez-García, P. Bizarro, M. Altamirano-Lozano, E. Rendón-Huerta, R. Guerrero-Alquicira, T. Fortoul Departamento de Biología Celular y Tisular, Facultad de Medicina, UNAM, México City, Mexico Introduction: Epidemiological studies have proven a relationship between maternal exposure to PM2.5 and pregnancy outcomes (low birth weight, intrauterine growth retardation, preterm birth). Particulate matter is composed, among others substances, by metals which produces miscarriages, menstrual disorders and stillbirth in occupationally exposed women. In animal models, particularly vanadium inhalation induces anestrus, lowers progesterone, and estrogen serum concentrations in female mice. However, it has not been evaluated if these alterations produce infertility in exposed females. Metals could interfere with the endocrine system but also in delocalizing expression of connexins and decreasing its expression, which are for important embryo’s implantation. Objective: To evaluate vanadium inhalation effect on connexins in female mice fertility. Materials and methods: 15 CD1 female mice inhaled vanadium pentoxide (V2 O5 ) 0.02 M. The postcoitusV-group inhaled vanadium just 1 h at 5th day after mating, meanwhile V4-WK group inhaled V 1 h, twice a week per four weeks and control group inhaled only the vehicle. Ovaries and uterus were collected and processed for routine histological technique, and also Cx43 and cx26 immunohistochemistry analysis was performed. The number of implantations and corpus luteum per dam were counted. Results: Control females presented 14 implantation sites, whereas in exposed mice during 4-WKs there were not any implantation sites, therefore we were not able to analyze connexin expression in implantation sites. Because of that we included a poscoitusV-group. Embryo reabsorptions were observed in this
S236
Abstracts / Toxicology Letters 259S (2016) S73–S247
former group and decreased stain for both connexins in epithelium and in the decidua. Conclusions: Vanadium inhalation prevents embryo implantation in females exposed for 4 WKs before, this finding indicates that lower levels of progesterone and estradiol induced by vanadium (reported in previous works) have the potential to induce infertility. In addition, embryo resorption in mice exposed just 1 h at 5th dpc suggests that embryo implantation is very sensitive to vanadium and maybe to other air pollutants. Hence, vanadium should be considered as an endocrine disruptor and reprotoxic. Financial support: CVU 630707. Posgrado en Ciencias Biológicas, UNAM. CONACyT. http://dx.doi.org/10.1016/j.toxlet.2016.07.565 PP25.10 Bisphenol-A does not alter mRNA levels of genes encoding proteins involved in communication and expansion of the cumulus cells–oocyte complex ˜ S.P. García-Zepeda, D.G. Acuna-Hernández, R. Santacruz-Márquez, I. Hernández-Ochoa Departamento de Toxicología, Cinvestav-IPN, Ciudad de México, Mexico Introduction: Cumulus cells maintain a close association with the oocyte through gap junction intercellular communication (GJIC) to facilitate the transfer of factors needed for the proper function of the female gamete. Upon signaling pathways triggering ovulation, luteinizing hormone (LH) induces mRNA levels of genes encoding proteins involved in cumulus cells (CC) expansion, including Prostaglandin-endoperoxide synthase 2 (Cox2), Hyaluronan synthase 2 (Has2), Tumor necrosis factor alpha induced protein 6 (Tsg6), Versican (Vcan), and CD44 antigen (Cd44). Objective: Because it has been shown that exposure to the worldwide used plasticizer bisphenol-A (BPA) decreases communication via GAP junctions, and that BPA alters CC expansion, this study evaluated whether BPA alters mRNA levels of genes encoding proteins involved in communication and expansion of the cumulus cells–oocyte complex (COC). Materials: Female mice were orally dosed with BPA (20 and 50 g/kg bw/day), vehicle (corn oil) or diethylstilbestrol (DES) as a positive control for 12–16 days. At the end of treatments, females received ip 5 IU of equine chorionic gonadotropin (eCG), and 48 h later were either sacrificed to collect COC or were administrated ip with 5 IU human chorionic gonadotropin (hCG) to collect COC at 10 h later. COC from eCG-primed mice were used to evaluate Luteinizing hormone/chorionic gonadotropin receptor (Lhcgr) mRNA levels, whereas COC from hCG-primed mice were used to evaluate Gap junction protein alpha 1 (Cx43), Gap junction protein alpha 4 (Cx37), Cox2, Has2, Tsg6, Vcan, and Cd44 mRNA levels. Results: The data showed similar mRNA levels of all genes related to communication and expansion of COC. Conclusion: These data suggest that transcription of main genes related to communication and expansion of murine COC is not a target of environmentally relevant doses of BPA. Financial support: Conacyt-México CB-167678. http://dx.doi.org/10.1016/j.toxlet.2016.07.566
PP25.11 Temephos decreases ovarian antral follicle growth in an in vitro system L. Millán-Mejía, S.P. García-Zepeda, I. Hernández-Ochoa Departamento de Toxicología, Cinvestav-IPN, Ciudad de México, Mexico Introduction: Temephos (phosphorothioate O,O,O,Otetramethyl O, di-p-; o -thio-phenyl) is a no systemic organophosphate pesticide (OP) used worldwide in public health campaigns to control Aedes aegypti mosquito, a transmitter of dengue and zika viruses. Recently, in vitro studies demonstrated that this OP exerts cytotoxic and genotoxic effects on cellular systems possessing metabolic activity. In spite of its extensive use, the potential detrimental effects of temephos on antral follicles, which are functional units in the ovary that releases a fertilizable oocyte and that possess a high metabolic and endocrine activities, remain to be elucidated. Objective: The present study tested the hypothesis that environmentally relevant concentrations of temephos inhibit antral follicle growth. Materials and methods: Ovarian antral follicles isolated from CD-1 female mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or temephos (0, 0.5, 1, 2, 5, and 10 M) for up to 7 days. Follicle growth, measured as follicle diameters every 24 h, was compared in vehicle control and temephos-treated follicles using general linear models for repeated measures. Results: Ovarian antral follicles cultured with 10 M temephos had significantly decreased growth compared to vehicle control at days 6 and 7. Conclusion: Temephos could have potential endocrine disrupting effects on the ovary. Financial support: This study was partially supported by the Pesticide Toxicology Network (Conacyt-Mexico #253789/271775), and Conacyt-Mexico CB-167678. http://dx.doi.org/10.1016/j.toxlet.2016.07.567 PP25.12 Genotoxic damage in sperm cells from Long–Evans rats exposed to arsenic and its relationship with poor quality sperm ˜ L. Nava, J. Jimenez, N. Betancourt, R. Alcaraz, O. M.S. Nino, Sambrano, J.M. Moran, E. Olivas Department of Cell Biology and Ultrastructure, Universidad Autónoma de Coahuila, Torreón, Coahuila, Mexico Introduction: Day by day the groundwater in the Laguna in Coahuila and Durango States, Northern Mexico is contaminated with arsenic (As) due to the gradual lowering of the groundwater sources, then exposing people to high concentrations of As in drinking water. Several animal studies have described the effects of As in the male reproductive function, resulting in low sperm quality as well as a genotoxic effect in lymphocytes. In this study, we assessed the genotoxic effects of As in rats exposed to As2 O3 in drinking water. Materials and methods: An experimental design was made with 9 male Long–Evans rats 15 weeks old, and 215–240 g. The animals (n = 9) were randomly divided into 3 groups (n = 3 each). The control group received distilled water and groups 1 and 2 received a solution of As (As2 O3 ) dissolved in distilled water at concentrations of 0.025, and 0.05 mg L−1 , respectively for five days a week, for