RESUMO
Methamidophos (MET) is a highly toxic organophosphate (OP) pesticide that is widely used in developing countries. MET has male reproductive effects, including decreased fertility. We evaluated MET effects on sperm quality, fertilization and DNA integrity, exploring the sensitivity of different stages of spermatogenesis. Adult male mice received MET (3.75 or 5mg/kg-bw/ip/day/4 days) and were euthanized 1, 28 or 45 days post-treatment (dpt) to evaluate MET's effects on epididymal maturation, meiosis or mitosis, respectively. Spermatozoa were obtained from the cauda epididymis-vas deferens and were evaluated for sperm quality, acrosome reaction (AR; Coomassie staining), mitochondrial membrane potential (by JC-1), DNA damage (comet assay), oxidative damage (malondialdehyde (MDA) production), in vitro fertilization and protein phosphorylation (immunodetection), and erythrocyte acetylcholinesterase (AChE) activity. At 1-dpt, MET inhibited AChE (43-57%) and increased abnormal cells (6%). While at 28- and 45-dpt, sperm motility and viability were significantly reduced with an increasing MET dose, and abnormal morphology increased at 5mg/kg/day/4 days. MDA and mitochondrial activity were not affected at any dose or time. DNA damage (OTM and %DNA) was observed at 5mg/kg/day/4 days in a time-dependent manner, whereas both parameters were altered in cells from mice exposed to 3.75 mg/kg/day/4 days only at 28-dpt. Depending on the time of collection, initial-, spontaneous- and induced-AR were altered at 5mg/kg/day/4 days, and the fertilization capacity also decreased. Sperm phosphorylation (at serine and tyrosine residues) was observed at all time points. Data suggest that meiosis and mitosis are the more sensitive stages of spermatogenesis for MET reproductive toxicity compared to epididymal maturation.
Assuntos
Replicação do DNA/efeitos dos fármacos , Inseticidas/toxicidade , Compostos Organotiofosforados/toxicidade , Espermatogênese/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Acetilcolinesterase/metabolismo , Reação Acrossômica/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Ensaio Cometa , Feminino , Fertilização/efeitos dos fármacos , Técnicas In Vitro , Infertilidade Masculina/induzido quimicamente , Infertilidade Masculina/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Oócitos/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Reprodução/efeitos dos fármacosRESUMO
Paternal germline exposure to organophosphorous pesticides (OP) has been associated with reproductive failures and adverse effects in the offspring. Methyl-parathion (Me-Pa), a worldwide-used OP, has reproductive adverse effects and is genotoxic to sperm, possibly via oxidative damage. This study investigated the stages of spermatogenesis susceptible to be targeted by Me-Pa exposure that impact on spermatozoa function and their ability to fertilize. Male mice were exposed to Me-Pa (20 mg/kg bw, i.p.) and spermatozoa from epididymis-vas deferens were collected at 7 or 28 days post-treatment (dpt) to assess the effects on maturing spermatozoa and spermatocytes, respectively. Spermatozoa were examined for DNA damage by nick translation (NT-positive cells) and SCSA (%DFI), lipoperoxidation (LPO) by malondialdehyde production, sperm function by spontaneous- and induced-acrosome reactions (AR), mitochondrial membrane potential (MMP) by using the JC-1 fluorochrome, and fertilization ability by an in vitro assay and in vivo mating. Alterations on DNA integrity (%DFI and NT-positive cells) in spermatozoa collected at 7 and 28 dpt, and decreases in sperm quality and induced-AR were observed; reduced MMP and LPO were observed at 7 dpt only. Negative correlations between LPO and sperm alterations were found. Altered sperm functional parameters evaluated either in vitro or in vivo were associated with reduced fertilization rates at both times. These results show that Me-Pa exposure of maturing spermatozoa and spermatocytes affects many sperm functional parameters that result in a decreased fertilizing capacity. Oxidative stress seems to be a likely mechanism of the detrimental effects of Me-Pa exposure in male germ cells.
Assuntos
Reação Acrossômica/efeitos dos fármacos , Inseticidas/toxicidade , Metil Paration/toxicidade , Espermatogênese/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Feminino , Fertilização/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Estresse Oxidativo/efeitos dos fármacos , Exposição Paterna , Distribuição Aleatória , Motilidade dos Espermatozoides/efeitos dos fármacos , Estatísticas não Paramétricas , Fatores de TempoRESUMO
Pesticide exposure, including organophosphorous (OP) insecticides, has been associated with poor semen quality, and paraoxonase (PON1), an enzyme involved in OP deactivation, may have a role on their susceptibility, due to PON1 polymorphisms. Our objective was to evaluate the role of PON1Q192R polymorphism on the susceptibility to OP toxicity on semen quality and DNA integrity in agricultural workers. A cross-sectional study was conducted in farmers with Mayan ascendancy from southeastern Mexico chronically exposed to pesticides; mostly OP. Fifty four agricultural workers (18-55 years old) were included, who provided semen and blood samples. Semen quality was evaluated according to WHO, sperm DNA damage by in situ-nick translation (NT-positive cells), PON1Q192R polymorphism by real-time PCR and serum PON1 activity by using phenylacetate and paraoxon. Two OP exposure indexes were created: at the month of sampling and during 3 months before sampling, representing the exposure to spermatids-spermatozoa and to cells at one spermatogenic cycle, respectively. PON1 192R and 192Q allele frequencies were 0.54 and 0.46, respectively. Significant associations were found between OP exposure at the month of sampling and NT-positive cells and sperm viability in homozygote 192RR subjects, and dose-effect relationships were observed between OP exposure during 3 months before sampling and sperm quality parameters and NT-positive cells in homozygote 192RR farmers. This suggests that cells at all stages of spermatogenesis are target of OP, and that there exists an interaction between OP exposure and PON1Q192R polymorphism on these effects; farmers featuring the 192RR genotype were more susceptible to develop reproductive toxic effects by OP exposure.
Assuntos
Doenças dos Trabalhadores Agrícolas/induzido quimicamente , Doenças dos Trabalhadores Agrícolas/genética , Arildialquilfosfatase/genética , DNA/efeitos dos fármacos , Inseticidas/toxicidade , Compostos Organofosforados/toxicidade , Sêmen/efeitos dos fármacos , Adolescente , Adulto , Doenças dos Trabalhadores Agrícolas/epidemiologia , Consumo de Bebidas Alcoólicas/epidemiologia , Arildialquilfosfatase/metabolismo , Estudos Transversais , Dano ao DNA , Frequência do Gene , Genótipo , Humanos , Marcação In Situ das Extremidades Cortadas , Modelos Lineares , Masculino , México/epidemiologia , Pessoa de Meia-Idade , Exposição Ocupacional/efeitos adversos , Polimorfismo Genético/efeitos dos fármacos , Polimorfismo Genético/genética , Fumar/epidemiologia , Transtornos Relacionados ao Uso de Substâncias/epidemiologiaRESUMO
Organophosphorous (OP) pesticides are considered genotoxic mainly to somatic cells, but results are not conclusive. Few studies have reported OP alterations on sperm chromatin and DNA, and oxidative stress has been related to their toxicity. Sperm cells are very sensitive to oxidative damage which has been associated with reproductive dysfunctions. We evaluated the effects of methyl-parathion (Me-Pa; a widely used OP) on sperm DNA, exploring the sensitive stage(s) of spermatogenesis and the relationship with oxidative stress. Male mice (10-12-weeks old) were administered Me-Pa (3-20 mg/kg bw/i.p.) and euthanized at 7- or 28-days post-treatment. Mature spermatozoa were obtained and evaluated for chromatin structure through SCSA (Sperm Chromatin Structure Assay; DNA Fragmentation Index parameters: Mean DFI and DFI%) and chromomycin-A(3) (CMA(3))-staining, for DNA damage through in situ-nick translation (NT-positive) and for oxidative stress through lipid peroxidation (LPO; malondialdehyde production). At 7-days post-treatment (mature spermatozoa when Me-Pa exposure), dose-dependent alterations in chromatin structure (Mean DFI and CMA(3)-staining) were observed, as well as increased DNA damage, from 2-5-fold in DFI% and NT-positive cells. Chromatin alterations and DNA damage were also observed at 28-days post-treatment (cells at meiosis at the time of exposure); suggesting that the damage induced in spermatocytes was not repaired. Positive correlations were observed between LPO and sperm DNA-related parameters. These data suggest that oxidative stress is related to Me-Pa alterations on sperm DNA integrity and cells at meiosis (28-days post-treatment) and epididymal maturation (7-days post-treatment) are Me-Pa targets. These findings suggest a potential risk of Me-Pa to the offspring after transmission.
Assuntos
Dano ao DNA , Inseticidas/toxicidade , Metil Paration/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Animais não Endogâmicos , Apoptose/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Relação Dose-Resposta a Droga , Marcação In Situ das Extremidades Cortadas , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Testes de Mutagenicidade , Exposição Paterna , Espermatogênese/efeitos dos fármacos , Espermatozoides/metabolismo , Espermatozoides/patologiaRESUMO
Lead (Pb) alters sperm chromatin condensation (CC) and the mechanisms are investigated. During spermatogenesis, protamines replace histones and disulfide bonds formation during epididymal maturation condense the chromatin. We evaluated sperm Pb uptake in testis and epididymis and the effects on CC in mice (0.06% Pb(2+)/16 weeks/drinking water). Spermatozoa from caput epididymis (CP) and cauda epididymis-vas deferens (CE-VD) were obtained and CC was measured by SCSA. Lead levels in spermatozoa from CP were lower than those from CE-VD, and correlated with a decreased CC, while Pb in CE-VD spermatozoa correlated with an increased CC. Lead accumulation into the nucleus was observed and Pb binding to nuclear sulfhydryl groups decreased chromatin decondensation in vitro. Our results suggest that spermatozoa take up Pb during testicular development and epididymal transport and alter CC, depending of the timing of Pb incorporation into the sperm nucleus, which finally may interfere with the chromatin decondensation process after fertilization.
Assuntos
Núcleo Celular/fisiologia , Cromatina/metabolismo , Epididimo/crescimento & desenvolvimento , Intoxicação por Chumbo/patologia , Espermatozoides/fisiologia , Animais , Peso Corporal/efeitos dos fármacos , Núcleo Celular/patologia , Núcleo Celular/ultraestrutura , Cromatina/patologia , Cromatina/ultraestrutura , Cromomicinas , Epididimo/patologia , Feminino , Masculino , Camundongos , Microscopia Eletrônica , Inibidores da Síntese de Ácido Nucleico , Tamanho do Órgão/efeitos dos fármacos , Gravidez , Reprodução/efeitos dos fármacos , Espermatozoides/patologia , Espermatozoides/ultraestrutura , Compostos de Sulfidrila/químicaRESUMO
Human paraoxonase (PON1) plays a role in detoxification of organophosphorus (OP) compounds by hydrolyzing the bioactive oxons, and in reducing oxidative low-density lipoproteins, which may protect against atherosclerosis. Some PON1 polymorphisms have been found to be responsible for variations in catalytic activity and expression and have been associated with susceptibility to OP poisoning and vascular diseases. Both situations are of public health relevance in Mexico. Therefore, the aim of this study was to evaluate PON1 phenotype and the frequencies of polymorphisms PON1 -162, -108, 55, and 192 in a Mexican population. The studied population consisted of unrelated individuals (n = 214) of either gender, 18-52 years old. Serum PON1 activity was assayed using phenylacetate and paraoxon as substrates. PON1 variants, -162, 55, and 192, were determined by real-time PCR using the TaqMan System, and PON1 -108 genotype by PCR-RFLP. We found a wide interindividual variability of PON1 activity with a unimodal distribution; the range of enzymatic activity toward phenylacetate was 84.72 to 422.0 U/mL, and 88.37 to 1645.6 U/L toward paraoxon. All four PON1 polymorphisms showed strong linkage disequilibrium (D% >90). PON1 polymorphisms -108, 55, and 192 were independently associated with arylesterase activity; whereas the activity toward paraoxon was related only with PON1 192 polymorphism, suggesting that this polymorphism is determinant to infer PON1 activity. A better understanding of the phenotype and genotypes of PON1 in Mexican populations will facilitate further epidemiological studies involving PON1 variability in OP poisoning and in the development of atherosclerosis.
Assuntos
Arildialquilfosfatase/genética , Arildialquilfosfatase/metabolismo , Adulto , Alelos , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Feminino , Haplótipos , Humanos , Desequilíbrio de Ligação/genética , Masculino , México/etnologia , Pessoa de Meia-Idade , Paraoxon/sangue , Fenilacetatos/sangue , Polimorfismo Genético/genéticaRESUMO
Organophosphorus (OP) pesticides, widely used in agriculture and pest control, are associated with male reproductive effects, including sperm chromatin alterations, but the mechanisms underlying these effects are unknown. The main toxic action of OP is related to phosphorylation of proteins. Chemical alterations in sperm nuclear proteins (protamines), which pack DNA during the last steps of spermatogenesis, contribute to male reproductive toxicity. Therefore, in the present study, we tested the ability of diazinon (DZN), an OP compound, to alter sperm chromatin by phosphorylating nuclear protamines. Mice were injected with a single dose of DZN (8.12 mg/kg, i.p.), and killed 8 and 15 days after treatment. Quality of sperm from epididymis and vas deferens was evaluated through standard methods and chromatin condensation by flow cytometry (DNA Fragmented Index parameters: DFI and DFI%) and fluorescence microscopy using chromomycin-A(3) (CMA(3)). Increases in DFI (15%), DFI% (4.5-fold), and CMA(3) (2-fold) were observed only at 8 days post-treatment, indicating an alteration in sperm chromatin condensation and DNA damage during late spermatid differentiation. In addition, an increase of phosphorous content (approximately 50%) in protamines, especially in the phosphoserine content (approximately 73%), was found at 8 days post-treatment. Sperm viability, motility, and morphology showed significant alterations at this time. These data strongly suggest that spermatozoa exposed during the late steps of maturation were the targets of DZN exposure. The correlation observed between the phosphorous content in nuclear protamines with DFI%, DFI, and CMA(3) provides evidence that phosphorylation of nuclear protamines is involved in the OP effects on sperm chromatin.
Assuntos
Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Diazinon/efeitos adversos , Espermatozoides/anormalidades , Espermatozoides/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cromatina/química , Cromomicina A3 , DNA/efeitos dos fármacos , DNA/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Diazinon/administração & dosagem , Diazinon/química , Avaliação Pré-Clínica de Medicamentos/métodos , Citometria de Fluxo/métodos , Injeções Intraperitoneais , Substâncias Intercalantes/efeitos adversos , Substâncias Intercalantes/química , Substâncias Intercalantes/metabolismo , Masculino , México , Camundongos , Camundongos Endogâmicos , Microscopia de Fluorescência/métodos , Matriz Nuclear/química , Matriz Nuclear/efeitos dos fármacos , Matriz Nuclear/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina/química , Protaminas/química , Protaminas/efeitos dos fármacos , Protaminas/metabolismo , Motilidade dos Espermatozoides/efeitos dos fármacos , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/química , Fatores de Tempo , Testes de Toxicidade/métodosRESUMO
The ability of chromium (Cr) salts to increase metallothionein (MT) levels in rat liver, kidney and pancreas, and its relationship with the presence of toxic effects are reported here. Rats were injected subcutaneously with 0, 10, 20, 30, 40, or 50 mg K2Cr2O7/kg and sacrificed 24 h later. Total Cr accumulation followed a dose-dependent pattern, levels in kidney being higher than those in liver or pancreas, suggesting different tissue bioavailabilities and accumulation patterns. Cr(IV) administration resulted in a tissue-specific MT induction: pancreas and liver showed five- and 3.5-fold MT increases, respectively; no increase was observed in the kidney. A positive correlation was observed between zinc and MT concentrations in liver, and between total Cr and MT concentrations in pancreas. Serum alpha-amylase activity showed a dose-dependent increase starting from 20 mg/kg, whereas serum glucose levels increased at doses higher than 30 mg/kg. Serum aspartate aminotransferase and alanine aminotransferase activities were increased in a dose-dependent manner, from 20 and 30 mg/kg, respectively. Our results showed that treatment with Cr(VI) can induce MT synthesis in pancreas and suggests a subsequent binding of Cr to MT. Also, pancreas is a target organ for Cr toxicity, and the usefulness of alpha-amylase activity as a sensitive biomarker of Cr toxicity in human exposed populations merits further study.