ABSTRACT
Prior studies showed that mice deficient in the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in synthesis of the thiol antioxidant glutathione (GSH), have decreased ovarian GSH concentrations, chronic ovarian oxidative stress, poor oocyte quality resulting in early preimplantation embryonic mortality and decreased litter size, and accelerated age-related decline in ovarian follicle numbers. Global deficiency of the catalytic subunit of this enzyme, Gclc, is embryonic lethal. We tested the hypothesis that granulosa cell- or oocyte-specific deletion of Gclc recapitulates the female reproductive phenotype of global Gclm deficiency. We deleted Gclc in granulosa cells or oocytes of growing follicles using Gclc floxed transgenic mice paired with Amhr2-Cre or Zp3-Cre alleles respectively. We discovered that granulosa cell-specific deletion of Gclc in Amhr2Cre;Gclc(f/-) mice recapitulates the decreased litter size observed in Gclm-/- mice, but does not recapitulate the accelerated age-related decline in ovarian follicles observed in Gclm-/- mice. In addition to having lower GSH concentrations in granulosa cells, Amhr2Cre;Gclc(f/-) mice also had decreased GSH concentrations in oocytes. By contrast, oocyte-specific deletion of Gclc in Zp3Cre;Gclc(f/-) mice did not affect litter size or accelerate the age-related decline in follicle numbers, and these mice did not have decreased oocyte GSH concentrations, consistent with transport of GSH between cells via gap junctions. The results suggest that GSH deficiency at earlier stages of follicle development may be required to generate the accelerated follicle depletion phenotype observed in global Gclm null mice.
ABSTRACT
People are widely exposed to polycyclic aromatic hydrocarbons, like benzo[a]pyrene (BaP). Prior studies showed that prenatal exposure to BaP depletes germ cells in ovaries, causing earlier onset of ovarian senescence post-natally; developing testes were affected at higher doses than ovaries. Our primary objective was to determine if prenatal BaP exposure results in transgenerational effects on ovaries and testes. We orally dosed pregnant germ cell-specific EGFP-expressing mice (F0) with 0.033, 0.2, or 2 mg/kg-day BaP or vehicle from embryonic day (E) 6.5-11.5 (F1 offspring) or E6.5-15.5 (F2 and F3). Ovarian germ cells at E13.5 and follicle numbers at postnatal day 21 were significantly decreased in F3 females at all doses of BaP; testicular germ cell numbers were not affected. E13.5 germ cell RNA-sequencing revealed significantly increased expression of male-specific genes in female germ cells across generations and BaP doses. Next, we compared the ovarian effects of 2 mg/kg-day BaP dosing to wild type C57BL/6J F0 dams from E6.5-11.5 or E12.5-17.5. We observed no effects on F3 ovarian follicle numbers with either of the shorter dosing windows. Our results demonstrate that F0 BaP exposure from E6.5-15.5 decreased the number of and partially disrupted transcriptomic sexual identity of female germ cells transgenerationally.
Subject(s)
Ovarian Reserve , Prenatal Exposure Delayed Effects , Pregnancy , Humans , Mice , Male , Female , Animals , Ovary/metabolism , Benzo(a)pyrene/metabolism , Transcriptome , Prenatal Exposure Delayed Effects/metabolism , Mice, Inbred C57BL , Germ CellsABSTRACT
Cannabis use by adolescents is widespread, but its effects on the ovaries remain largely unknown. Δ9-tetrahydrocannabinol (THC) exerts its pharmacological effects by activating, and in some conditions hijacking, cannabinoid receptors (CBRs). We hypothesized that adolescent exposure to THC affects ovarian function in adulthood. Peripubertal female C57BL/6N mice were given THC (5 mg/kg) or its vehicle, once daily by intraperitoneal injection. Some mice received THC from postnatal day (PND) 30-33 and their ovaries were harvested PND34; other mice received THC from PND30-43, and their ovaries were harvested PND70. Adolescent treatment with THC depleted ovarian primordial follicle numbers by 50% at PND70, 4 weeks after the last dose. The treatment produced primordial follicle activation, which persisted until PND70. THC administration also caused DNA damage in primary follicles and increased PUMA protein expression in oocytes of primordial and primary follicles. Both CB1R and CB2R were expressed in oocytes and theca cells of ovarian follicles. Enzymes involved in the formation (N-acylphosphatidylethanolamine phospholipase D) or deactivation (fatty acid amide hydrolase) of the endocannabinoid anandamide were expressed in granulosa cells of ovarian follicles and interstitial cells. Levels of mRNA for CBR1 were significantly increased in ovaries after adolescent THC exposure, and upregulation persisted for at least 4 weeks. Our results support that adolescent exposure to THC may cause aberrant activation of the ovarian endocannabinoid system in female mice, resulting in substantial loss of ovarian reserve in adulthood. Relevance of these findings to women who frequently used cannabis during adolescence warrants investigation.
Subject(s)
Endocannabinoids , Ovarian Reserve , Mice , Female , Animals , Dronabinol/toxicity , Mice, Inbred C57BL , Ovarian FollicleABSTRACT
The endocannabinoid system (ECS) is comprised of a set of lipid-derived messengers (the endocannabinoids, ECBs), proteins that control their production and degradation, and cell-surface cannabinoid (CB) receptors that transduce their actions. ECB molecules such as 2-arachidonoyl-sn-glycerol (2-AG) and anandamide (arachidonoyl ethanolamide) are produced on demand and deactivated through enzymatic actions tightly regulated both temporally and spatially, serving homeostatic roles in order to respond to various challenges to the body. Key components of the ECS are present in the hypothalamus-pituitary-gonadal (HPG) axis, which plays critical roles in the development and regulation of the reproductive system in both males and females. ECB signaling controls the action at each stage of the HPG axis through CB receptors expressed in the hypothalamus, pituitary, and reproductive organs such as the testis and ovary. It regulates the secretion of hypothalamic gonadotropin-releasing hormone (GnRH), pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH), estrogen, testosterone, and affects spermatogenesis in males. Δ9-tetrahydrocannabinol (THC) and other phytocannabinoids from Cannabis sativa affect a variety of physiological processes by altering, or under certain conditions hijacking, the ECB system. Therefore, phytocannabinoids, in particular THC, may modify the homeostasis of the HPG axis by altering CB receptor signaling and cause deficits in reproductive function. While the ability of phytocannabinoids, THC and/or cannabidiol (CBD), to reduce pain and inflammation provides promising opportunities for therapeutic intervention for genitourinary and degenerative disorders, important questions remain regarding their unwanted long-term effects. It is nevertheless clear that the therapeutic potential of modulating the ECS calls for further scientific and clinical investigation.
ABSTRACT
BACKGROUND: Fine particulate matter (PM2.5) exposure accelerates atherosclerosis and contains known ovotoxic chemicals. However, effects of exposure to PM2.5 on the finite ovarian follicle pool have hardly been investigated, nor have interactions between ovarian and cardiovascular effects. We hypothesized that subchronic inhalation exposure to human-relevant concentrations of PM2.5 results in destruction of ovarian follicles via apoptosis induction, as well as accelerated recruitment of primordial follicles into the growing pool. Further, we hypothesized that destruction of ovarian follicles enhances the adverse cardiovascular effects of PM2.5 in females. RESULTS: Hyperlipidemic apolipoprotein E (Apoe) null ovary-intact or ovariectomized female mice and testis-intact male mice were exposed to concentrated ambient PM2.5 or filtered air for 12 weeks, 5 days/week for 4 h/day using a versatile aerosol concentration enrichment system. Primordial, primary, and secondary ovarian follicle numbers were decreased by 45%, 40%, and 17%, respectively, in PM2.5-exposed ovary-intact mice compared to controls (P < 0.05). The percentage of primary follicles with granulosa cells positive for the mitosis marker Ki67 was increased in the ovaries from PM2.5-exposed females versus controls (P < 0.05), consistent with increased recruitment of primordial follicles into the growing pool. Exposure to PM2.5 increased the percentages of primary and secondary follicles with DNA damage, assessed by γH2AX immunostaining (P < 0.05). Exposure to PM2.5 increased the percentages of apoptotic antral follicles, determined by TUNEL and activated caspase 3 immunostaining (P < 0.05). Removal of the ovaries and PM2.5-exposure exacerbated the atherosclerotic effects of hyperlipidemia in females (P < 0.05). While there were statistically significant changes in blood pressure and heart rate variability in PM2.5-compared to Air-exposed gonad-intact males and females and ovariectomized females, the changes were not consistent between exposure years and assessment methods. CONCLUSIONS: These results demonstrate that subchronic PM2.5 exposure depletes the ovarian reserve by increasing recruitment of primordial follicles into the growing pool and increasing apoptosis of growing follicles. Further, PM2.5 exposure and removal of the ovaries each increase atherosclerosis progression in Apoe-/- females. Premature loss of ovarian function is associated with increased risk of osteoporosis, cardiovascular disease and Alzheimer's disease in women. Our results thus support possible links between PM2.5 exposure and other adverse health outcomes in women.
Subject(s)
Ovarian Reserve , Animals , Apolipoproteins , Apolipoproteins E/genetics , Female , Male , Mice , Mice, Knockout , Ovarian Follicle , Particulate Matter/toxicityABSTRACT
Polycyclic aromatic hydrocarbons like benzo[a]pyrene (BaP) are generated during incomplete combustion of organic materials. Prior research has demonstrated that BaP is a prenatal ovarian toxicant and carcinogen. However, the metabolic pathways active in the embryo and its developing gonads and the mechanisms by which prenatal exposure to BaP predisposes to ovarian tumors later in life remain to be fully elucidated. To address these data gaps, we orally dosed pregnant female mice with BaP from embryonic day (E) 6.5 to E11.5 (0, 0.2, or 2 mg/kg/day) for metabolite measurement or E9.5 to E11.5 (0 or 3.33 mg/kg/day) for embryonic gonad RNA sequencing. Embryos were harvested at E13.5 for both experiments. The sum of BaP metabolite concentrations increased significantly with dose in the embryos and placentas, and concentrations were significantly higher in female than male embryos and in embryos than placentas. RNA sequencing revealed that enzymes involved in metabolic activation of BaP are expressed at moderate to high levels in embryonic gonads and that greater transcriptomic changes occurred in the ovaries in response to BaP than in the testes. We identified 490 differentially expressed genes (DEGs) with false discovery rate P-values < 0.05 when comparing BaP-exposed to control ovaries but no statistically significant DEGs between BaP-exposed and control testes. Genes related to monocyte/macrophage recruitment and activity, prolactin family genes, and several keratin genes were among the most upregulated genes in the BaP-exposed ovaries. Results show that developing ovaries are more sensitive than testes to prenatal BaP exposure, which may be related to higher concentrations of BaP metabolites in female embryos.
Subject(s)
Benzo(a)pyrene/metabolism , Gonads/metabolism , Placenta/metabolism , Pregnancy, Animal , Transcriptome , Animals , Chromatography, High Pressure Liquid , Computational Biology , Female , Inflammation , Keratins/metabolism , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Monocytes/metabolism , Ovary/metabolism , Pregnancy , RNA-Seq , Sex Factors , Testis/metabolism , Time FactorsABSTRACT
The effect of thermal cycling on the transformation behavior of a Ti-24Nb-1Mo alloy was investigated by means of electrical resistivity measurement, transmission electron microscopy (TEM), X-ray diffraction (XRD), tensile test and Vickers hardness tests. Electrical resistivity changes were not observed in all alloys. It indicates that thermally induced martensitic transformation does not take place in the alloys. After thermal cycling between 298 K and 77 K, clear X-ray diffraction peaks corresponding to ωath phase, which did not exist before thermal cycling, were observed. Volume fraction of ωath phase increased as increasing the number of thermal cycling. ωath phase formed during thermal cycling increased hardness of the alloy. Although thermally induced martensitic transformation did not occur in the alloys, superelastic deformation behavior was observed in the alloys. The superelastic recovery ratio decreased from 81% to 41% by increasing the number of thermal cycling, which came from the increase in the volume fraction of ωath phase.
ABSTRACT
The tripeptide thiol antioxidant glutathione (GSH) has multiple physiological functions. Female mice lacking the modifier subunit of glutamate cysteine ligase (GCLM), the rate-limiting enzyme in GSH synthesis, have decreased GSH concentrations, ovarian oxidative stress, preimplantation embryonic mortality, and accelerated age-related decline in ovarian follicles. We hypothesized that supplementation with thiol antioxidants, N-acetyl cysteine (NAC), or α-lipoic acid (ALA) will rescue this phenotype. Gclm-/- and Gclm+/+ females received 0 or 80 mM NAC in drinking water from postnatal day (PND) 21-30; follicle growth was induced with equine chorionic gonadotropin (eCG) on PND 27, followed by an ovulatory dose of human CG and mating with a wild type male on PND 29 and zygote harvest 20 h after hCG. N-acetyl cysteine supplementation failed to rescue the low rate of second pronucleus formation in zygotes from Gclm-/- versus Gclm+/+ females. In the second study, Gclm-/- and Gclm+/+ females received diet containing 0, 150, or 600 mg/kg ALA beginning at weaning and were mated with wild type males from 8 to 20 weeks of age. α-Lipoic acid failed to rescue the decreased offspring production of Gclm-/- females. However, 150 mg/kg diet ALA partially rescued the accelerated decline in primordial follicles, as well as the increased recruitment of follicles into the growing pool and the increased percentages of follicles with γH2AX positive oocytes or granulosa cells of Gclm-/- females. We conclude that ovarian oxidative stress is the cause of accelerated primordial follicle decline, while GSH deficiency per se may be responsible for preimplantation embryonic mortality in Gclm-/- females.
Subject(s)
Acetylcysteine/pharmacology , Antioxidants/pharmacology , Glutamate-Cysteine Ligase/metabolism , Glutathione/metabolism , Ovarian Follicle/physiology , Thioctic Acid/pharmacology , Acetylcysteine/administration & dosage , Animals , Antioxidants/administration & dosage , Diet , Dietary Supplements , Estrous Cycle , Female , Genotype , Glutamate-Cysteine Ligase/genetics , Glutathione/deficiency , Glutathione/genetics , Male , Mice , Mice, Knockout , Oocytes , Thioctic Acid/administration & dosageABSTRACT
BACE1 is the rate-limiting enzyme for amyloid-ß peptides (Aß) generation, a key event in the pathogenesis of Alzheimer's disease (AD). By an unknown mechanism, levels of BACE1 and a BACE1 mRNA-stabilizing antisense RNA (BACE1-AS) are elevated in the brains of AD patients, implicating that dysregulation of BACE1 expression plays an important role in AD pathogenesis. We found that nuclear factor erythroid-derived 2-related factor 2 (NRF2/NFE2L2) represses the expression of BACE1 and BACE1-AS through binding to antioxidant response elements (AREs) in their promoters of mouse and human. NRF2-mediated inhibition of BACE1 and BACE1-AS expression is independent of redox regulation. NRF2 activation decreases production of BACE1 and BACE1-AS transcripts and Aß production and ameliorates cognitive deficits in animal models of AD. Depletion of NRF2 increases BACE1 and BACE1-AS expression and Aß production and worsens cognitive deficits. Our findings suggest that activation of NRF2 can prevent a key early pathogenic process in AD.
Subject(s)
Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/metabolism , Cognition Disorders/metabolism , NF-E2-Related Factor 2/metabolism , Alzheimer Disease/pathology , Amyloid Precursor Protein Secretases/genetics , Amyloid beta-Peptides/metabolism , Animals , Aspartic Acid Endopeptidases/genetics , Cognition Disorders/pathology , Disease Models, Animal , Gene Expression Regulation , Humans , Isothiocyanates/pharmacology , Mice , Mice, Transgenic , NF-E2-Related Factor 2/biosynthesis , Promoter Regions, Genetic , Protein Binding , Reactive Oxygen Species/metabolism , Sulfoxides , Transcription, GeneticABSTRACT
Mice lacking the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in glutathione (GSH) synthesis, have decreased tissue GSH. We previously showed that Gclm-/- embryos have increased sensitivity to the prenatal in vivo ovarian toxicity of the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) compared with Gclm+/+ littermates. We also showed that BaP-induced germ cell death in cultured wild type embryonic ovaries is caspase-dependent. Here, we hypothesized that GSH deficiency increases sensitivity of cultured embryonic ovaries to BaP-induced germ cell death. 13.5â¯days post coitum (dpc) embryonic ovaries of all Gclm genotypes were fixed immediately or cultured for 24â¯h in media supplemented with DMSO vehicle or 500â¯ng/ml BaP. The percentage of activated caspase-3 positive germ cells varied significantly among groups. Within each genotype, DMSO and BaP-treated groups had increased germ cell caspase-3 activation compared to uncultured. Gclm+/- ovaries had significantly increased caspase-3 activation with BaP treatment compared to DMSO, and caspase-3 activation increased non-significantly in Gclm-/- ovaries treated with BaP compared to DMSO. There was no statistically significant effect of BaP treatment on germ cell numbers at 24â¯h, consistent with our prior observations in wild type ovaries, but Gclm-/- ovaries in both cultured groups had lower germ cell numbers than Gclm+/+ ovaries. There were no statistically significant BaP-treatment or genotype-related differences among groups in lipid peroxidation and germ cell proliferation. These data indicate that Gclm heterozygous or homozygous deletion sensitizes embryonic ovaries to BaP- and tissue culture-induced germ cell apoptosis.
Subject(s)
Apoptosis/drug effects , Benzo(a)pyrene/toxicity , Embryonic Germ Cells/drug effects , Glutathione/deficiency , Ovary/drug effects , Animals , Cytoprotection , Embryonic Germ Cells/metabolism , Embryonic Germ Cells/pathology , Female , Gestational Age , Glutamate-Cysteine Ligase/deficiency , Glutamate-Cysteine Ligase/genetics , Mice, Inbred C57BL , Mice, Knockout , Ovary/embryology , Ovary/metabolism , Tissue Culture TechniquesABSTRACT
The polycyclic aromatic hydrocarbon pollutant benzo[a]pyrene (BaP) is a known developmental gonadotoxicant. However, the mechanism of BaP-induced germ cell death is unclear. We investigated whether exposure to BaP induces apoptotic germ cell death in the mouse fetal ovary or testis. Mouse fetal gonads were dissected at embryonic day 13.5 days postcoitum (dpc) and fixed immediately or cultured for 6, 24, 48, or 72 h with various concentrations of BaP (1-1000 ng/ml). Germ cells numbers, apoptosis, and proliferation were evaluated by immunostaining. Treatment of fetal ovaries with BaP for 72 h concentration-dependently depleted germ cells. Treatment with BaP elevated the expression of BAX protein at 6 h and activated downstream caspases-9 and -3 at 24 h in a concentration-dependent manner in germ cells of fetal ovaries. As a consequence, ovarian germ cell numbers were significantly and concentration-dependently decreased at 48 h. Pretreatment with z-VAD-fmk, a pan-caspase inhibitor, prior to exposure to 1000 ng/ml BaP prevented BaP-mediated ovarian germ cell death; there were no effects of BaP or z-VAD-fmk on germ cell proliferation. No significant effects of BaP exposure on caspase 3 activation or germ cell numbers were observed in fetal testes after 48 h of culture. Our findings show that BaP exposure increases caspase-dependent and BAX-associated germ cell apoptosis in the mouse fetal ovary, leading to germ cell depletion. In contrast, the cultured 13.5 dpc fetal testis is relatively resistant to BaP-induced germ cell death. This study provides a novel insight into molecular mechanisms by which BaP has direct gonadotoxicity in the mouse fetal ovary.
Subject(s)
Benzo(a)pyrene/toxicity , Ovary/drug effects , Testis/drug effects , Animals , Caspases/metabolism , Cell Proliferation/drug effects , Enzyme Activation , Female , Humans , Male , Mice, Inbred C57BL , Ovary/embryology , Ovary/enzymology , Ovary/metabolism , Pregnancy , Testis/embryology , Testis/enzymology , Testis/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
Polycyclic aromatic hydrocarbons, like benzo[a]pyrene (BaP), are ubiquitous environmental pollutants and potent ovarian toxicants. The transcription factor NRF2 is an important regulator of the cellular response to electrophilic toxicants like BaP and to oxidative stress. NRF2 regulates transcription of genes involved in the detoxification of reactive metabolites of BaP and reactive oxygen species. We therefore hypothesized that Nrf2-/- mice have accelerated ovarian aging and increased sensitivity to the ovarian toxicity of BaP. A single injection of BaP dose-dependently depleted ovarian follicles in Nrf2+/+ and Nrf2-/- mice, but the effects of BaP were not enhanced in the absence of Nrf2. Similarly, Nrf2-/- mice did not have increased ovarian BaP DNA adduct formation compared to Nrf2+/+ mice. Ovarian follicle numbers did not differ between peripubertal Nrf2-/- and Nrf2+/+ mice, but by middle age, Nrf2-/- mice had significantly fewer primordial follicles than Nrf2+/+ mice, consistent with accelerated ovarian aging.
Subject(s)
Benzo(a)pyrene/toxicity , Cellular Senescence/drug effects , Environmental Pollutants/toxicity , Epithelial Cells/drug effects , Gene Deletion , NF-E2-Related Factor 2/deficiency , Ovarian Follicle/drug effects , Ovary/drug effects , Animals , Antioxidants/metabolism , Apoptosis/drug effects , Cell Proliferation/drug effects , DNA Adducts/metabolism , Dose-Response Relationship, Drug , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Genotype , Mice, Inbred C57BL , Mice, Knockout , NF-E2-Related Factor 2/genetics , Ovarian Follicle/metabolism , Ovarian Follicle/pathology , Ovarian Reserve/drug effects , Ovary/metabolism , Ovary/pathology , Oxidative Stress/drug effects , PhenotypeABSTRACT
Glutathione (GSH) is the one of the most abundant intracellular antioxidants. Mice lacking the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH. Our prior work showed that GSH plays antiapoptotic roles in ovarian follicles. We hypothesized that Gclm(-/-) mice have accelerated ovarian aging due to ovarian oxidative stress. We found significantly decreased ovarian GSH concentrations and oxidized GSH/oxidized glutathione redox potential in Gclm(-/-) vs Gclm(+/+) ovaries. Prepubertal Gclm(-/-) and Gclm(+/+) mice had similar numbers of ovarian follicles, and as expected, the total number of ovarian follicles declined with age in both genotypes. However, the rate of decline in follicles was significantly more rapid in Gclm(-/-) mice, and this was driven by accelerated declines in primordial follicles, which constitute the ovarian reserve. We found significantly increased 4-hydroxynonenal immunostaining (oxidative lipid damage marker) and significantly increased nitrotyrosine immunostaining (oxidative protein damage marker) in prepubertal and adult Gclm(-/-) ovaries compared with controls. The percentage of small ovarian follicles with increased granulosa cell proliferation was significantly higher in prepubertal and 2-month-old Gclm(-/-) vs Gclm(+/+) ovaries, indicating accelerated recruitment of primordial follicles into the growing pool. The percentages of growing follicles with apoptotic granulosa cells were increased in young adult ovaries. Our results demonstrate increased ovarian oxidative stress and oxidative damage in young Gclm(-/-) mice, associated with an accelerated decline in ovarian follicles that appears to be mediated by increased recruitment of follicles into the growing pool, followed by apoptosis at later stages of follicular development.
Subject(s)
Aging/physiology , Glutamate-Cysteine Ligase/metabolism , Glutathione/metabolism , Ovary/metabolism , Oxidative Stress , Animals , Apoptosis , Cell Proliferation , Estrous Cycle , Female , Male , Mice, Inbred C57BL , Mice, Transgenic , Ovary/cytologyABSTRACT
A Ti-Ni alloy compositionally graded along the thickness direction in order to obtain a shape change over a wide temperature range, which is beneficial to the actuator for precise position control, was prepared by spark plasma sintering (SPS) after stacking Ti-Ni alloy ribbons in the sequence of Ti-51Ni, Ti-50Ni, Ti-49Ni and Ti-48Ni (at%) followed by annealing. Then, the microstructure and martensitic transformation behavior were investigated by using FE-SEM, DSC and thermal cycling tests under a constant load. The inter-ribbon defects observed after SPS due to insufficient diffusional bonding between the ribbons were eliminated by post-SPS annealing at 1023 K for 36 ks. The compositionally graded sample showed compositional variation of 1.5 at% Ti along the thickness direction (- 120 µm) and a martensitic transformation temperature window as large as 91 K on cooling and 79 K on heating. A recoverable elongation of 0.9% was obtained under a stress of 80 MPa and the deformation rate, which is defined as the ratio of the recoverable elongation to the temperature range where the elongation occurred was 0.015%/K in the compositionally graded sample.
ABSTRACT
Polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (BaP) are ubiquitous environmental pollutants found in tobacco smoke, air pollution, and grilled foods. Prenatal exposure to BaP causes premature reproductive senescence in mice, and other PAHs are transplacental ovarian carcinogens. Glutathione (GSH) is critical for detoxification of the reactive metabolites of PAHs. Therefore, we hypothesized that mice that are genetically deficient in GSH synthesis, due to deletion of the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have increased destruction of oogonia, premature ovarian failure, and ovarian tumorigenesis after transplacental BaP exposure compared with Gclm(+/+) females. Gclm(+/-) female and male mice were mated, and dams were treated with 0, 2, or 10 mg/kg/d BaP in sesame oil by gavage from gestational days 7 to 16. Compared with oil-treated F1 females of the same genotype, Gclm(-/-) prenatally BaP-treated females had significantly greater decrements in offspring production than Gclm(+/+) BaP-treated females. Similarly, we observed significant BaP dose × Gclm genotype interactions on ovarian follicle counts and ovarian tumor multiplicity at 7.5 months of age, with Gclm(-/-) females having greater decrements in follicle numbers and more ovarian tumors in response to prenatal BaP exposure than Gclm(+/+) females. The ovarian tumors were positive for the epithelial marker cytokeratin. Our results show that prenatal exposure of females to BaP causes premature ovarian failure and ovarian tumorigenesis and that embryonic GSH deficiency due to deletion of Gclm increases sensitivity to these transplacental ovarian effects of BaP.
Subject(s)
Benzo(a)pyrene/toxicity , Environmental Pollutants/toxicity , Glutathione/deficiency , Maternal-Fetal Exchange , Ovarian Neoplasms/chemically induced , Primary Ovarian Insufficiency/chemically induced , Animals , Cell Transformation, Neoplastic/metabolism , Estrous Cycle/drug effects , Female , Fertility/drug effects , Glutamate-Cysteine Ligase/genetics , Humans , Mice , Ovarian Follicle/drug effects , PregnancyABSTRACT
Reactive oxygen species (ROS) modulate aging and aging-related diseases. Dietary composition is critical in modulating lifespan. However, how ROS modulate dietary effects on lifespan remains poorly understood. Superoxide dismutase 1 (SOD1) is a major cytosolic enzyme responsible for scavenging superoxides. Here we investigated the role of SOD1 in lifespan modulation by diet in Drosophila. We found that a high sugar-low protein (HS-LP) diet or low-calorie diet with low-sugar content, representing protein restriction, increased lifespan but not resistance to acute oxidative stress in wild-type flies, relative to a standard base diet. A low sugar-high protein diet had an opposite effect. Our genetic analysis indicated that SOD1 overexpression or dfoxo deletion did not alter lifespan patterns of flies responding to diets. However, sod1 reduction blunted lifespan extension by the HS-LP diet but not the low-calorie diet. HS-LP and low-calorie diets both reduced target of rapamycin (TOR) signaling and only the HS-LP diet increased oxidative damage. sod1 knockdown did not affect phosphorylation of S6 kinase, suggesting that SOD1 acts in parallel with or downstream of TOR signaling. Surprisingly, rapamycin decreased lifespan in sod1 mutant but not wild-type males fed the standard, HS-LP, and low-calorie diets, whereas antioxidant N-acetylcysteine only increased lifespan in sod1 mutant males fed the HS-LP diet, when compared to diet-matched controls. Our findings suggest that SOD1 is required for lifespan extension by protein restriction only when dietary sugar is high and support the context-dependent role of ROS in aging and caution the use of rapamycin and antioxidants in aging interventions.
Subject(s)
Dietary Proteins/administration & dosage , Drosophila melanogaster/enzymology , Drosophila melanogaster/physiology , Superoxide Dismutase/metabolism , Age Factors , Animals , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Energy Intake/physiology , Female , Longevity , Male , Oxidative Stress/physiology , RNA, Small Interfering/genetics , Reactive Oxygen Species/metabolism , Signal Transduction , Superoxide Dismutase/genetics , Superoxide Dismutase-1 , TOR Serine-Threonine Kinases/metabolismABSTRACT
Glutathione (GSH) is the most abundant intracellular thiol and an important regulator of cellular redox status. Mice that lack the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH synthesis. Nicotinamide nucleotide transhydrogenase, an inner mitochondrial membrane protein, catalyzes the interconversion of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate; reduced nicotinamide adenine dinucleotide phosphate is required for reduction of GSH disulfide. Previous work supports roles for GSH in preimplantation development. We hypothesized that Gclm-/- mice have increased preimplantation embryonic mortality and that this effect is enhanced by absence of a functioning Nnt gene. Gclm-/- females produced significantly fewer pups per litter than Gclm+/+ littermates. Numbers of oocytes ovulated in a natural estrous cycle or upon superovulation did not differ by genotype. Fewer uterine implantation sites were observed in the Gclm-/- females. Prepubertal Gclm-/- and Gclm+/+ females were superovulated, then mated overnight with a Gclm+/+ male. At 0.5 d postcoitum, Gclm-/- females had significantly lower percentages of zygotes with two pronuclei and higher percentages of zygotes with one pronucleus than Gclm+/+ or Gclm+/- females. At 3.5 d postcoitum, a significantly lower percentage of blastocyst stage embryos was recovered from uteri of Gclm-/- females than Gclm+/+ females. Embryonic development to the blastocyst stage, but not the two-cell stage, was significantly decreased after in vitro fertilization of oocytes from Gclm-/- females compared with Gclm+/+ females. The Nnt mutation did not enhance the effects of Gclm genotype on female fertility. These results demonstrate critical roles for maternal GSH in supporting normal preimplantation development.
Subject(s)
Blastocyst/physiology , Ectogenesis , Glutamate-Cysteine Ligase/physiology , Glutathione/metabolism , Oocytes/metabolism , Protein Subunits/physiology , Animals , Embryo Implantation , Female , Fertilization in Vitro , Glutamate-Cysteine Ligase/genetics , Infertility, Female/genetics , Infertility, Female/metabolism , Infertility, Female/physiopathology , Litter Size , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Mitochondrial Proteins/genetics , NADP Transhydrogenase, AB-Specific , NADP Transhydrogenases/genetics , Protein Subunits/genetics , Sperm-Ovum Interactions , SuperovulationABSTRACT
Oxidative stress has been implicated in various aspects of aging, but the role of oxidative stress in ovarian aging remains unclear. Our previous studies have shown that the initiation of apoptotic cell death in ovarian follicles and granulosa cells by various stimuli is initiated by increased reactive oxygen species. Herein, we tested the hypothesis that ovarian antioxidant defenses decrease and oxidative damage increases with age in mice. Healthy, wild-type C57BL/6 female mice aged 2, 6, 9, or 12 mo from the National Institute on Aging Aged Rodent Colony were killed on the morning of metestrus. Quantitative real-time RT-PCR was used to measure ovarian mRNA levels of antioxidant genes. Immunostaining using antibodies directed against 4-hydroxynonenal (4-HNE), nitrotyrosine (NTY), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) was used to localize oxidative lipid, protein, and DNA damage, respectively, within the ovaries. TUNEL was used to localize apoptosis. Ovarian expression of glutathione peroxidase 1 (Gpx1) increased and expression of glutaredoxin 1 (Glrx1), glutathione S-transferase mu 2 (Gstm2), peroxiredoxin 3 (Prdx3), and thioredoxin 2 (Txn2) decreased in a statistically significant manner with age. Statistically significant increases in 4-HNE, NTY, and 8-OHdG immunostaining in ovarian interstitial cells and follicles were observed with increasing age. Our data suggest that the decrease in mRNA expression of mitochondrial antioxidants Prdx3 and Txn2 as well as cytosolic antioxidants Glrx1 and Gstm2 may be involved in age-related ovarian oxidative damage to lipid, protein, DNA, and other cellular components vital for maintaining ovarian function and fertility.
Subject(s)
Aging/genetics , Aging/metabolism , Ovary/metabolism , Oxidative Stress , Aging/pathology , Animals , Antioxidants/metabolism , Apoptosis , DNA Damage , Estrous Cycle , Female , Gene Expression , Glutaredoxins/genetics , Glutathione Transferase/genetics , Lipid Peroxidation , Mice , Mice, Inbred C57BL , Organ Size , Ovary/pathology , Peroxiredoxin III , Peroxiredoxins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Thioredoxins/geneticsABSTRACT
BACKGROUND/AIMS: Phototherapy using a narrow-band, near-infrared (NIR) light (using a light-emitting diode, LED) is being used to treat certain medical conditions. This narrow-band red light has been shown to stimulate cytochrome c oxidase (CCO) in mitochondria that would stimulate ATP production and has the ability to stimulate wound healing. LED treatment also decreases chemical-induced oxidative stress in tested systems. As renal cystic diseases are known to have evidence of oxidative stress with reduced antioxidant protection, we hypothesized that NIR light therapy might ameliorate the renal pathology in renal cystic disease. METHODS: Wistar-Wpk/Wpk rats with Meckel syndrome (MKS) were treated with light therapy on days 10-18 at which time disease severity was evaluated. Wpk rats were either treated daily for 80 s with narrow-band red light (640-690 nm wavelength) or sham treated. At termination, renal and cerebral pathology was evaluated, and renal expression and activity of enzymes were assessed to evaluate oxidative stress. Blood was collected for blood urea nitrogen (BUN) determination, the left kidney frozen for biochemical evaluation, and the right kidney and head fixed for morphological evaluation. RESULTS: There were no significant effects of LED treatment on body weight (BW) or total kidney weight in non-cystic rats. Total kidney weight was increased and anephric BW was decreased in cystic versus non-cystic controls. LED reduced BW and total kidney weight in cystic rats compared to non-light-treated cystic (control) rats. BUN was already increased almost 6-fold in cystic rats compared to control rats. BUN was further increased almost 2-fold with NIR treatment in both non-cystic and cystic rats compared to cystic and control rats. The hydrocephalus associated with Wpk/Wpk (ventricular volume expressed as total volume and as percent of anephric BW) was also more severe in NIR-treated cystic rats compared to the normal control rats. Renal glutathione peroxidase and catalase (CAT) were reduced in the cystic kidney while superoxide dismutase and CCO were increased. NIR increased CAT and CCO, marginally decreased glutathione S-transferase and slightly decreased glutathione reductase in cystic rats compared to the normal control rats. The detrimental effects of NIR may be related to reduced renal blood flow associated with progression of cystic pathology. Compression by cysts may not allow sufficient oxygen or nutrient supply necessary to support the increased oxidative phosphorylation-associated cellular activity, and the increased demand induced by NIR-increased CCO may have created further oxidative stress. CONCLUSION: LED phototherapy initiated after the onset of symptoms was detrimental to MKS-induced pathology. NIR stimulates CCO thereby increasing the kidney's need for oxygen. We hypothesize that cystic compression of the vasculature impairs oxygen availability and the enhanced CCO activity produces more radicals, which are not sufficiently detoxified by the increased CAT activity.
ABSTRACT
Hyperglycemia causes oxidative damage in tissues prone to complications in diabetes. Low-level light therapy (LLLT) in the red to near infrared range (630-1000nm) has been shown to accelerate diabetic wound healing. To test the hypothesis that LLLT would attenuate oxidative renal damage in Type I diabetic rats, male Wistar rats were made diabetic with streptozotocin (50mg/kg, ip), and then exposed to 670nm light at a dose of 9J/cm(2) once per day for 14weeks. The activity and expression of catalase and the activity of Na K-ATPase increased in kidneys of light-treated diabetic rats, whereas the activity and expression of glutathione peroxidase and the expression of Na K-ATPase were unchanged. LLLT lowered the values of serum BUN, serum creatinine, and BUN/creatinine ratio. In addition, LLLT augmented the activity and expression of cytochrome c oxidase, a primary photoacceptor molecule in the mitochondrial respiratory chain, and reduced the formation of the DNA adduct 8-hydroxy-2'-deoxyguanosine in kidney. LLLT improved renal function and antioxidant defense capabilities in the kidney of Type I diabetic rats. Thus, 670nm LLLT may be broadly applicable to the amelioration of renal complications induced by diabetes that disrupt antioxidant defense mechanisms.