Methylmercury (MeHg) is a pervasive environmental contaminant with potent neurotoxic, teratogenic and likely carcinogenic activity, for which the underlying molecular mechanisms remain largely unclear. Base excision repair (BER) is important in mitigating the pathogenic effects of oxidative stress, which has also been implicated in the mechanism of MeHg toxicity, however the importance of BER in MeHg toxicity is currently unknown. Accordingly, we addressed this question using: (1) spontaneously- and Simian virus 40 (SV40) large T antigen-immortalized oxoguanine glycosylase 1-null (Ogg1-/-) murine embryonic fibroblasts (MEFs); and, (2) human Ogg1 (hOgg1)- or formamidopyrimidine glycosylase (Fpg)-expressing human embryonic kidney (HEK) cells; reciprocal in vitro cellular models with deficient and enhanced ability to repair oxidatively damaged DNA respectively. When spontaneously-immortalized wild-type and Ogg1-/- MEFs were exposed to environmentally relevant, low micromolar concentrations of MeHg, both underwent cell cycle arrest but Ogg1-/- cells exhibited a greater sensitivity to MeHg than wild-type controls with reduced clonogenic survival and increased apoptosis, DNA damage and DNA damage response activation. Antioxidative catalase alleviated the MeHg-initiated DNA damage in both wild-type and Ogg1-/- cells, but failed to block MeHg-mediated apoptosis at micromolar concentrations. As in spontaneously immortalized MEFs, MeHg induced cell cycle arrest in SV40 large T antigen-immortalized MEFs, with increased sensitivity to MeHg persisting in the Ogg1-/- MEFs. Importantly, cells seeded at a higher density exhibited compromised proliferation, which protected against MeHg-mediated cell cycle arrest and DNA damage. In the reciprocal model of enhanced DNA repair, hOgg1- and Fpg-expressing cells appeared paradoxically more sensitive than wild-type controls to acute MeHg exposure for all cellular and biochemical parameters, potentially due to the accumulation of toxic intermediary abasic sites. Accordingly, our results provide the first evidence that Ogg1 status represents a critical determinant of risk for MeHg toxicity independent of cellular immortalization method, with variations in cellular proliferation and interindividual variability in antioxidative and DNA repair capacities constituting important determinants of risk for environmentally-initiated oxidatively damaged DNA and its pathological consequences.

Methylmercury (MeHg) is a pervasive environmental contaminant with potent neurotoxic, teratogenic and likely carcinogenic activity, for which the underlying molecular mechanisms remain largely unclear. Base excision repair (BER) is important in mitigating the pathogenic effects of oxidative stress, which has also been implicated in the mechanism of MeHg toxicity, however the importance of BER in MeHg toxicity is currently unknown. Accordingly, we addressed this question using: (1) spontaneously- and Simian virus 40 (SV40) large T antigen-immortalized oxoguanine glycosylase 1-null (Ogg1-/-) murine embryonic fibroblasts (MEFs); and, (2) human Ogg1 (hOgg1)- or formamidopyrimidine glycosylase (Fpg)-expressing human embryonic kidney (HEK) cells; reciprocal in vitro cellular models with deficient and enhanced ability to repair oxidatively damaged DNA respectively. When spontaneously-immortalized wild-type and Ogg1-/- MEFs were exposed to environmentally relevant, low micromolar concentrations of MeHg, both underwent cell cycle arrest but Ogg1-/- cells exhibited a greater sensitivity to MeHg than wild-type controls with reduced clonogenic survival and increased apoptosis, DNA damage and DNA damage response activation. Antioxidative catalase alleviated the MeHg-initiated DNA damage in both wild-type and Ogg1-/- cells, but failed to block MeHg-mediated apoptosis at micromolar concentrations. As in spontaneously immortalized MEFs, MeHg induced cell cycle arrest in SV40 large T antigen-immortalized MEFs, with increased sensitivity to MeHg persisting in the Ogg1-/- MEFs. Importantly, cells seeded at a higher density exhibited compromised proliferation, which protected against MeHg-mediated cell cycle arrest and DNA damage. In the reciprocal model of enhanced DNA repair, hOgg1- and Fpg-expressing cells appeared paradoxically more sensitive than wild-type controls to acute MeHg exposure for all cellular and biochemical parameters, potentially due to the accumulation of toxic intermediary abasic sites. Accordingly, our results provide the first evidence that Ogg1 status represents a critical determinant of risk for MeHg toxicity independent of cellular immortalization method, with variations in cellular proliferation and interindividual variability in antioxidative and DNA repair capacities constituting important determinants of risk for environmentally-initiated oxidatively damaged DNA and its pathological consequences.

El objetivo de la presente tesis doctoral ha sido estudiar comparativamente los efectos de dietas liquidas control, de composición definida, conteniendo aceite de oliva o, de maíz como fuente lipídica, respectivamente de ácidos grasos monoinsaturados y poliinsaturados, sobre el metabolismo oxidativo de ratas. La presencia de dobles enlaces en la molécula de un ácido graso constituyen puntos vulnerables a las reacciones de oxidación generando los peróxidos lipídicos, compuestos potencialmente tóxicos que pueden producir graves daños celulares (alteración de la permeabilidad celular, alteración de las prostaglandinas, etc.). Frente a este daño oxidativo, los organismos aeróbicos cuentan con mecanismos de defensa intracelulares, sistemas antioxidantes tanto enzimáticos como no enzimáticos. Entre los antioxidantes destaca el alfa-tocoferol como antioxidante lipofílico mayoritario. En nuestro estudio también hemos considerado las posibles interacciones de los productos de la peroxidación lipídica con el DNA en hígado de ratas, a través de la inducción de la 8-oxo-2' deoxiguanosina, un marcador del daño oxidativo del DNA. Se observó que la actividad de las enzimas antioxidantes superoxido dismutasa (SOD), catalasa (CAT), glutatión peroxidasa (GPX), aumentaban en el hígado y en la grasa blanca de las ratas control alimentadas con aceite de oliva al comparar con los grupos control maíz. Sin embargo, en la grasa parda y en las células sanguíneas las actividades enzimáticas presentaban una tendencia a disminuir en los grupos control alimentados con aceite de oliva. Cuando se estudiaron los niveles del glutatión reducido (GSH) se observó que, tanto en el hígado como en las células sanguíneas de las ratas control alimentadas con aceite de maíz, los niveles de este tripéptido se habían reducido. Sin embargo, en los hepatocitos de las mismas ratas estos niveles habían aumentado. Esto pone de manifiesto el efecto protector de la vitamina E como antioxidante. Se obse / Drehmer Rieger, E. (2007). Estudio comparativo de aceite de oliva y maíz sobre el metabolismo oxidativo de rata [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/4181 / Palancia

A sensitive and specific assay aimed at measuring the oxidized nucleic acids, 8-oxoguanine (8-oxoGua), fapy-guanine (Fapy-Gua), 8-oxoguanosine (8-oxoGuo), 8-oxo-2′-deoxyguanosine (8-oxodG) has been developed by coupling reversed phase liquid chromatography (HPLC) with electrospray tandem mass spectrometry detection (MS/MS) and isotope dilution. The HPLC-MS/MS approach with multiple reaction monitoring (MRM) allowed for the sensitive determination of 8-oxoGua, Fapy-Gua, 8-oxoGuo, and 8-oxodG in human urine samples. There is no sample preparation needed except for the addition of buffer and 13C- and 15N-labeled internal standards to the urine prior to sample injection into the HPLC-MS/MS system. This method was tested in urine samples from non-smokers, smokers, non-smokers with chronic kidney disease (CKD) and smokers with CKD, to assess the level of oxidative damage to nucleic acids. Markers of both RNA and DNA damage were significantly increased in the smokers with and without CKD compared to their respective control subjects. These findings suggest that a highly specific and sensitive analytical method such as isotope dilution HPLC-MS/MS may represent a valuable tool for the measurement of oxidative stress in human subjects.

No description available.

O objetivo deste estudo foi avaliar a influência da exposição a níveis normais ou reduzidos de testosterona durante a vida intrauterina no desenvolvimento psicológico, social e sexual dos pacientes com DDS 46, XY. Pacientes e métodos: Trata-se de um estudo retrospectivo. Os 53 participantes são pacientes portadores de DSD 46,XY devido a defeitos de produção de testosterona ou deficiência da 5alfa-RD2, todos eles com genitália ambígua que resultou na atribuição do sexo feminino ao nascimento. Os pacientes foram divididos em dois grupos: Grupo 1 (G1) - pacientes com DDS 46, XY, devido a defeito na produção de testosterona, constituído por 29 pacientes, 8 deles com deficiência de 17beta-HSD3, 7 com hipoplasia das células de Leydig, 7 com disgenesia gonadal parcial, 6 com deficiência 17alfa-hidroxilase e 1 com deficiência 3beta-HSD2; Grupo 2 (G2) - constituído por 24 pacientes com deficiência de 5alfa-RD2. Foi utilizado um questionário com 32 perguntas abrangendo aspectos do desenvolvimento psicológico, social e sexual destes pacientes. Resultados: Foi encontrada uma diferença significativa nos seguintes aspectos do desenvolvimento psicológico, social e sexual dos participantes do estudo: maior incidência de masturbação, fantasias eróticas e desejo de ter filhos em pacientes com deficiência da 5alfa-RD2 com sexo social masculino. Nas pacientes com sexo social feminino o desejo de ter filhos foi maior naquelas com DDS 46, XY por defeitos na produção de testosterona do que naquelas com deficiência da 5alfa-RD2 (p < 0,05), enquanto que o desejo de ter filhos foi maior nos homens com deficiência 5alfa-RD2 (p > 0,05). O número de indivíduos casados foi significativamente maior no grupo dos pacientes com DDS 46, XY por defeitos na produção de testosterona do que no grupo dos pacientes com DDS 46, XY por deficiência da 5alfa-RD2 (p = 0,003). Em conclusão, nossos resultados indicam uma possível influência da exposição aos andrógenos durante a vida pré-natal no desenvolvimento psicológico e social, bem como em aspectos da vida sexual dos pacientes adultos com DDS 46, XY / The aim of this study was to evaluate the influence of exposure to normal or reduced levels of testosterone during intra-uterine life in psychological, social, and sexual development of patients with DSD 46, XY. Patients and methods: This is a retrospective study. The 53 participants were patients with DSD 46, XY due to defects in production of testosterone or deficiency of 5alfa-RD2, all of them with ambiguous genitalia and female sex assignment at birth. These patients were divided into two groups: Group 1 (G1) - patients with DSD 46, XY, due to a defect in the production of testosterone, consisting of 29 people, 8 with deficiency of 17beta-HSD3, 7 with Leydig cell hypoplasia, 7 with partial gonadal dysgenesis, 6 with 17alfa-hydroxylase deficiency ,1 with 3beta-HSD2 deficiency, Group 2 (G2) - consisting of 24 patients with deficiency of 5alfa-RD2. We used a questionnaire with 32 questions covering aspects of psychological, social and sexual development of these patients. Results: A significant difference was found in the following aspects of psychological, social and sexual development of these patients: higher incidence of masturbation, erotic fantasies and desire for children in patients with deficiency of 5alfa-RD2 with male social sex. In patients with female social sex, the desire to have children was higher in those with DSD 46, XY by defects in the production of testosterone than in those with deficiency of 5alfa-RD2 (p < 0.05), while the desire to have children in men was higher in those with 5alfa-RD2 (p > 0.05). The number of married individuals was significantly higher in the group of patients with DSD 46, XY by defects in the production of testosterone than in the group of patients with DSD 46, XY by deficiency of 5alfa-RD2 (p = 0.003). In conclusion, our results indicate a possible influence of exposure to androgens during prenatal life in psychological and social development, as well as in aspects of sexual life of adult patients with DSD 46, XY

O objetivo deste estudo foi avaliar a influência da exposição a níveis normais ou reduzidos de testosterona durante a vida intrauterina no desenvolvimento psicológico, social e sexual dos pacientes com DDS 46, XY. Pacientes e métodos: Trata-se de um estudo retrospectivo. Os 53 participantes são pacientes portadores de DSD 46,XY devido a defeitos de produção de testosterona ou deficiência da 5alfa-RD2, todos eles com genitália ambígua que resultou na atribuição do sexo feminino ao nascimento. Os pacientes foram divididos em dois grupos: Grupo 1 (G1) - pacientes com DDS 46, XY, devido a defeito na produção de testosterona, constituído por 29 pacientes, 8 deles com deficiência de 17beta-HSD3, 7 com hipoplasia das células de Leydig, 7 com disgenesia gonadal parcial, 6 com deficiência 17alfa-hidroxilase e 1 com deficiência 3beta-HSD2; Grupo 2 (G2) - constituído por 24 pacientes com deficiência de 5alfa-RD2. Foi utilizado um questionário com 32 perguntas abrangendo aspectos do desenvolvimento psicológico, social e sexual destes pacientes. Resultados: Foi encontrada uma diferença significativa nos seguintes aspectos do desenvolvimento psicológico, social e sexual dos participantes do estudo: maior incidência de masturbação, fantasias eróticas e desejo de ter filhos em pacientes com deficiência da 5alfa-RD2 com sexo social masculino. Nas pacientes com sexo social feminino o desejo de ter filhos foi maior naquelas com DDS 46, XY por defeitos na produção de testosterona do que naquelas com deficiência da 5alfa-RD2 (p < 0,05), enquanto que o desejo de ter filhos foi maior nos homens com deficiência 5alfa-RD2 (p > 0,05). O número de indivíduos casados foi significativamente maior no grupo dos pacientes com DDS 46, XY por defeitos na produção de testosterona do que no grupo dos pacientes com DDS 46, XY por deficiência da 5alfa-RD2 (p = 0,003). Em conclusão, nossos resultados indicam uma possível influência da exposição aos andrógenos durante a vida pré-natal no desenvolvimento psicológico e social, bem como em aspectos da vida sexual dos pacientes adultos com DDS 46, XY / The aim of this study was to evaluate the influence of exposure to normal or reduced levels of testosterone during intra-uterine life in psychological, social, and sexual development of patients with DSD 46, XY. Patients and methods: This is a retrospective study. The 53 participants were patients with DSD 46, XY due to defects in production of testosterone or deficiency of 5alfa-RD2, all of them with ambiguous genitalia and female sex assignment at birth. These patients were divided into two groups: Group 1 (G1) - patients with DSD 46, XY, due to a defect in the production of testosterone, consisting of 29 people, 8 with deficiency of 17beta-HSD3, 7 with Leydig cell hypoplasia, 7 with partial gonadal dysgenesis, 6 with 17alfa-hydroxylase deficiency ,1 with 3beta-HSD2 deficiency, Group 2 (G2) - consisting of 24 patients with deficiency of 5alfa-RD2. We used a questionnaire with 32 questions covering aspects of psychological, social and sexual development of these patients. Results: A significant difference was found in the following aspects of psychological, social and sexual development of these patients: higher incidence of masturbation, erotic fantasies and desire for children in patients with deficiency of 5alfa-RD2 with male social sex. In patients with female social sex, the desire to have children was higher in those with DSD 46, XY by defects in the production of testosterone than in those with deficiency of 5alfa-RD2 (p < 0.05), while the desire to have children in men was higher in those with 5alfa-RD2 (p > 0.05). The number of married individuals was significantly higher in the group of patients with DSD 46, XY by defects in the production of testosterone than in the group of patients with DSD 46, XY by deficiency of 5alfa-RD2 (p = 0.003). In conclusion, our results indicate a possible influence of exposure to androgens during prenatal life in psychological and social development, as well as in aspects of sexual life of adult patients with DSD 46, XY