Temephos Decreases Sperm Quality and Fertilization Rate and Is Metabolized in Rat Reproductive Tissues at Low-Dose Exposure.

Temephos is an organophosphorus pesticide used in control campaigns against vectors that transmit diseases, including dengue, a public health concern. The WHO classifies temephos in category III and its safe concentration (low-observable-adverse-effect level) in male rats is 100 mg/kg/day for up to 44 days. Temephos inhibits acetylcholinesterase (AChE) and is metabolized in different tissues, probably by mixed-function oxidases; one of its metabolites is bisphenol S (BPS), which is considered an endocrine disruptor. The aim of this study was to evaluate the effects of temephos on sperm function and its biotransformation in the testis, epididymis, and other tissues to explore its toxicity in rats treated with 100 mg/kg/day/5 or 7 days (gavage). AChE activity was inhibited 70% starting on day 3 and 13 or 41% mortality was observed at 5 or 7 days, respectively. After 7 days, temephos significantly decreased sperm motility (30%) and viability (10%) and increased (10%) lipoperoxidation, and the sperm DNA exhibited no damage. Temephos was distributed and metabolized in all tissues, with the highest levels observed in the adipose tissue and temephos levels were 16-fold higher in the epididymis than in the testis. Notably, BPS was observed in the testis. At 5 days, decreased sperm motility (12.5%) and viability (5.7%) were observed and sperm fertilization decreased (30%). These results suggest that temephos decreases sperm quality and fertilization capacity at recommended safe concentrations and that it is metabolized in male reproductive tissues. This pesticide places the reproductive health of exposed people at risk, suggesting the need to reevaluate its toxicity.

Methyl parathion causes genetic damage in sperm and disrupts the permeability of the blood-testis barrier by an oxidant mechanism in mice.

Methyl parathion (Me-Pa) is an extremely toxic organophosphorus pesticide still used in developing countries. It has been associated with decreased sperm function and fertility and with oxidative and DNA damage. The blood-testis barrier (BTB) is a structure formed by tight junction (TJ) proteins in Sertoli cells and has a critical role in spermatogenesis. We assessed the effect of repeated doses of Me-Pa (3-12 mg/kg/day for 5 days, i.p.) on sperm quality, lipid oxidation, DNA integrity, and BTB permeability in adult male mice and explored oxidation as a mechanism of toxicity. Me-Pa caused dose-dependent effects on sperm quality, lipoperoxidation, and DNA integrity. Testis histology results showed the disruption of spermatogenesis progression and atrophy of seminiferous tubules. The pesticide opened the BTB, as evidenced by the presence of a biotin tracer in the adluminal compartment of the seminiferous tubules. This effect was not observed after 45 days of exposure when a spermatogenic cycle had completed. The coadministration of the antioxidant α-tocopherol (50 mg/kg/day for 5 days, oral) prevented the effects of Me-Pa on sperm quality, DNA and the BTB, indicating the importance of oxidative stress in the damage generated by Me-Pa. As evidenced by immunochemistry, no changes were found in the localization of the TJ proteins of the BTB, although oxidation (carbonylation) of total proteins in testis homogenates was detected. Our results show that Me-Pa disturbs the BTB and that oxidation is involved in the observed toxic effects on sperm cells.

Low concentrations of lead decrease the sperm fertilization ability by altering the acrosome reaction in mice.

Lead (Pb) exposure at high concentrations is associated with poor sperm quality, acrosome alterations, and low fertilization rate. Sperm capacitation and the acrosome reaction (AR) are required for successful fertilization. Actin polymerization is crucial for correct capacitation, and small GTPases, such as RhoA, Rac1, and Cdc42, are involved. This study aimed to evaluate the effects of Pb on sperm fertilization ability, capacitation, AR, and the mechanisms involved in mice exposed to low Pb concentrations. CD1 mice were exposed to 0.01% Pb2+ for 45 days through their drinking water and their spermatozoa were collected from the cauda epididymis-vas deferens to evaluate the following: AR (oAR: initial, sAR: spontaneous, and iAR: induced) using the PNA-FITC assay, sperm capacitation (P-Tyr levels), actin polymerization (phalloidin-TRITC), MDA production (stress oxidative marker), the RhoA, Rac1, and Cdc42 protein levels, and the in vitro fertilization (IVF). After the treatment, the blood Pb (PbB) concentration was 9.4 ±â€¯1.6 µg/dL. Abnormal sperm morphology and the oAR increased (8 and 19%, respectively), whereas the iAR decreased (15%) after a calcium ionophore challenge, and the actin polymerization decreased in the sperm heads (59%) and tails (42%). Rac1 was the only Rho protein to significantly decrease (33%). Spermatozoa from the Pb-treated mice showed a significant reduction in the fertilization rate (19%). Our data suggest that Pb exposure at environmental concentrations (PbB < 10 µg/dL) decreases the acrosome function and affects the sperm fertilization ability; this is probably a consequence of the low Rac1 levels, which did not allow adequate actin polymerization to occur.

Oxidative stress and genetic damage among workers exposed primarily to organophosphate and pyrethroid pesticides.

The indiscriminate use of pesticides in agriculture and public health campaigns has been associated with an increase of oxidative stress and DNA damage, resulting in health outcomes. Some defense mechanisms against free radical-induced oxidative damage include the antioxidant enzyme systems. The aim of this study was to determine the levels of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and the relationship of antioxidant enzyme levels with DNA damage among sprayers (workers) occupationally exposed to pesticides. The determinations of MDA and antioxidant enzymes were performed spectrophotometrically. The genotoxic effects were evaluated using the comet assay. The results showed a marginally significant decrease in SOD and CAT activities in the high exposure group compared to the control group. For MDA, statistically significant differences were found among people working long term vs. those working temporarily (P = 0.02) as sprayers. In the moderate exposure group, a positive correlation was observed between MDA levels and GPx activity. In the high exposure group, a negative correlation was observed between GR and CAT activities, and between MDA levels and GPx activities. Furthermore, in the high exposure group, a positive correlation between DNA damage parameters and MDA levels was observed. The results suggest an important role of antioxidant enzymes for the protection of DNA damage caused by occupational exposure to pesticides.

Increased methylation of repetitive elements and DNA repair genes is associated with higher DNA oxidation in children in an urbanized, industrial environment.

DNA methylation in DNA repair genes participates in the DNA damage regulation. Particulate matter (PM), which has metals and polycyclic aromatic hydrocarbons (PAHs) adsorbed, among others has been linked to adverse health outcomes and may modify DNA methylation. To evaluate PM exposure impact on repetitive elements and gene-specific DNA methylation and DNA damage, we conducted a cross-sectional study in 150 schoolchildren (7-10 years old) from an urbanized, industrial area of the metropolitan area of Mexico City (MAMC), which frequently exhibits PM concentrations above safety standards. Methylation (5mC) of long interspersed nuclear element-1 (LINE1) and DNA repair gene (OGG1, APEX, and PARP1) was assessed by pyrosequencing in peripheral mononuclear cells, DNA damage by comet assay and DNA oxidation by 8-OHdG content. PAH and metal contents in PM10 (≤10µm aerodynamic diameter) were determined by HPLC-MS and ICP-AES, respectively. Multiple regression analysis between DNA methylation, DNA damage, and PM10 exposure showed that PM10 was significantly associated with oxidative DNA damage; a 1% increase in 5mC at all CpG sites in PARP1 promoter was associated with a 35% increase in 8-OHdG, while a 1% increase at 1, 2, and 3 CpG sites resulted in 38, 9, and 56% increments, respectively. An increase of 10pg/m3 in benzo[b]fluoranthene content of PM10 was associated with a 6% increase in LINE1 methylation. Acenaphthene, indene [1,2,3-cd] pyrene, and pyrene concentrations correlated with higher dinucleotide methylation in OGG1, APEX and PARP1 genes, respectively. Vanadium concentration correlated with increased methylation at selected APEX and PARP1 CpG sites. DNA repair gene methylation was significantly correlated with DNA damage and with specific PM10-associated PAHs and Vanadium. Data suggest that exposure to PM and its components are associated with differences in DNA methylation of repair genes in children, which may contribute to DNA damage.

Comparative effect of technical and commercial formulations of methamidophos on sperm quality and DNA integrity in mice.

Methamidophos (MET), widely used in developing countries, is a highly neurotoxic organophosphate pesticide that has been associated with male reproductive alterations. Commercial formulations of pesticides used by agricultural workers and urban sprayers are responsible for thousands of intoxications in developing countries and may not have the same effects as active pure ingredients. Therefore, we compared effects of MET technical (METt) and commercial (METc) grades on sperm quality and DNA integrity. Male mice were injected (intraperitoneal, i.p.) with METt or METc (3.75, 5, and 7 mg/kg bw/day/4 days) and sacrificed 24 h post-treatment. Sperm cells collected from epididymis-vas deferens were evaluated for quality parameters, DNA damage by the comet assay, and lipoperoxidation by malondialdehyde (MDA) production. Erythrocyte acetylcholinesterase (AChE) activity was evaluated by acetylthiocholine inhibition as an index of overall toxicity. A dose-dependent AChE inhibition was observed with both formulations. Sperm quality was decreased after treatment with both MET compounds, but the commercial formulation showed stronger effects; a similar profile was observed with the DNA damage, being METc more genotoxic. None MET formulation increased MDA, suggesting no peroxidative damage involved. In summary, the commercial formulation of MET was more reprotoxic and genotoxic than the active pure ingredient, highlighting that commercial formulations must be considered for more appropriate risk assessment of pesticide exposures.

The role of paraoxonase polymorphisms in the induction of micronucleus in paraoxon-treated human lymphocytes.

Human paraoxonase-1 (PON1) is a high-density lipoprotein-associated enzyme that has a role in the detoxification of organophosphorus compounds by hydrolyzing the bioactive oxons. PON1 polymorphims are responsible, at least in part, for the variation in the catalytic activity and expression of the enzyme and have been associated with susceptibility to organophosphorus pesticide toxicity, mainly neurotoxicity. The aim of this study was to determine whether paraoxon induced micronuclei and to examine the role of PON1 polymorphism in paraoxon's genotoxic potential. First, dose finding cytogenetic experiments were performed on lymphocyte cultures from three donors and a range of paraoxon concentration (1-25 microM) were tested. In a second set of experiments, 5 microM paraoxon was added to blood cultures of 11 donors with two different PON1 haplotypes (PON T(-108)M(55)Q(192) with low activity and haplotype PON C(-108)L(55) R(192) with high activity, referred to as PON1QQ and as PON1 RR, respectively). Because PON1 is present in blood, the effect of adding 5 microM paraoxon and 70 microl of autologous plasma to lymphocyte cultures also was examined. Paraoxon had no effect on cell viability, but caused a significant dose-dependent increase in MN frequency. The basal MN frequencies were similar on QQ and RR genotypes. A significant difference was observed in the MN frequency only in lymphocytes from individuals with the QQ genotype treated with 5 microM paraoxon and the autologous plasma did not modify these effects. The results obtained in this study suggest that PON1 genotype might have an important role in the identification of individuals at risk for cancer development due to occupational exposure to pesticides.

Sperm chromatin alteration and DNA damage by methyl-parathion, chlorpyrifos and diazinon and their oxon metabolites in human spermatozoa.

Extensive use of organophosphorous pesticides (OP) by young men represents a public health problem. Toxicity of OP mainly results in neurotoxicity due to their oxygen analogues (oxons), formed during the OP oxidative activation. OP alter semen quality and sperm chromatin and DNA at different stages of spermatogenesis. Oxons are more toxic than the parent compounds; however, their toxicity to spermatogenic cells has not been reported. We evaluated sperm DNA damage by several OP compounds and their oxons in human spermatozoa from healthy volunteers incubated with 50-750 microM of methyl-parathion (MePA), methyl-paraoxon (MePO), chlorpyrifos (CPF), chlorpyrifos-oxon (CPO), diazinon (DZN) or diazoxon (DZO). All concentrations were not cytotoxic (evaluated by eosin-Y exclusion), except 750 microM MePO. Oxons were 15% to 10 times more toxic to sperm DNA (evaluated by the SCSA parameter, %DFI) than their corresponding parent compounds, at the following order: MePO>CPO=MePA>CPF>DZO>DZN, suggesting that oxon metabolites participate in OP sperm genotoxicity.