Genotoxicity of selected pesticides in the mouse bone-marrow micronucleus test and in the sister-chromatid exchange test with human lymphocytes in vitro

Selected pesticides, (aldicarb, 1,3-dichloropropene, methidathion, parathion, triadimefon, vinclozolin) were tested for their clastogenic and aneugenic activities in the mouse bone-marrow micronucleus (MN) test in vivo and for their sister-chromatid exchange-inducing activities in human lymphocytes in vitro in the presence and absence of an exogenous metabolizing system from rat-liver S9. 1,3-Dichloropropene significantly increased the frequencies of micronucleated polychromatic erythrocytes (PCE) in bone-marrow cells of female mice from 3.3 MN/1000 PCE to 15.3 MN/1000 PCE (187 mg per kg body weight). 1,3-Dichloropropene (100 μM) induced 16.0 SCE/metaphase after 24 h of incubation as compared with the basal rate of 11.2 SCE/metaphase (− S9) and of 15.4 SCE/metaphase as compared with 10.5 SCE/metaphase of the control (+ S9). These values were statistically significantly different from each other. The other pesticides tested did neither increase the rate of micronuclei significantly in polychromatic erythrocytes in male nor in female animals. Aldicarb and methidathion induced a significant increase in SCEs in human lymphocytes in vitro only without the metabolic activating system: aldicarb, 5 μm, 24 h incubation: 15.5 SCE/metaphase; control: 12.6 SCE/metaphase; methidathion, 100 μM, 24 h incubation: 15.8 SCE/metaphase, control: 11.1 SCE/metaphase. Parathion, triadimefon and vinclozolin did not have any SCE-inducing effects.     

Anti-hyperglycemic and antigenotoxic potential of Ulva rigida ethanolic extract in the experimental diabetes mellitus

An increased reactive oxygen species (ROS) and insufficient antioxidant activity is known in diabetes mellitus (DM). Antioxidant compounds in the human foods or supplementary diets can be used to counteract several diseases. The analysis of micronuclei (MN) is a cytogenetic technique used to show chromosomal damage caused by clastogenic affects. The present study was designed to evaluate: (i) the effects of diabetes mellitus on bone marrow MN frequency, (ii) the effect of oral administration of Ulva rigida ethanolic extract (URE) on MN frequency produced by DM, and (iii) some hematological values in normal and streptozotocin-induced diabetic rats. Daily fluid and food consumptions, weekly body weights, blood glucose concentrations and serum insulin levels were also examined in the study groups during the two different administration periods. The blood glucose concentration and MN frequency have been significantly increased in diabetic rats compared with the normal rats (p < 0.0001). Especially, URE-30d group treatment in diabetic rats was significantly decreased blood glucose concentrations and MN frequency. This is the first report on the anti-hyperglycemic, anti-oxidative and genotoxic/antigenotoxic capacity of U. rigida in vivo. Our results suggest that URE shows strong anti-hyperglycemic and antigenotoxic effect on the genotoxicity produced by DM in rats.     

Induction of micronuclei following exposure to methylene di-phenyl diisocyanate: potential genotoxic metabolites.

Methylene di-phenyl diisocyanate (MDI) is used to make polyurethane products. The predominant occupational disease attributed to diisocyanates, including MDI, is asthma; however, the potential for genotoxicity has also been of concern. Diisocyanates are very reactive compounds that can undergo nonenzymatic hydrolysis to form methylenedianiline (MDA), or react under physiological conditions with primary amines to form ureas and/or with thiols to form labile thiol acid esters. MDA is a carcinogen in animals and a suspected carcinogen in humans. Brown Norway rats (BNR) were exposed to either 7 or 113 mg/m(3) MDI aerosol for 1 h/week x3 weeks and sacrificed 1 week later. Micronuclei (MN) formation was assessed from bone marrow polychromatic erythrocytes (PCE). A dose-dependent increase in the frequency of micronucleated polychromatic erythrocytes (MN-PCEs) was noted. In vitro exposure of Chinese hamster lung fibroblasts (V79) to MDA or MDI-thiol conjugates, but not to MDI, significantly increased the frequency of MN. MDI-thiol conjugate-exposed cell cultures did not have detectable levels of MDA. A significant increase in the number of V79 cells in metaphase, as well as the number of cells with precipitants within both the cytoplasm and nuclei, were noted in MDI-glutathione-exposed cultures. The results of this study indicate that MDI aerosol exposure can cause MN formation through either the hydrolysis of MDI to MDA or possibly the formation of thiol conjugates.     

MN9202在Beagle犬肝微粒体酶中的代谢动力学

目的研究MN9202在Beagle犬肝微粒体酶中的代谢。方法差速离心法制备Beagle犬肝微粒体酶，0.4 μmol·L^-1的MN9202与1.0 g·L^-1的肝微粒体酶在37 ℃水浴中孵育30 min，加入0.5 mL碱化液终止反应，然后采用RP-HPLC法测定孵育液中MN9202原形药物的浓度。根据所测浓度与反应速度做Lineweave-Brurk双倒数曲线，推导出药物的米氏常数K_m和最大反应速度V_max,并计算机体内在清除率。同时观察不同浓度和不同种类的人肝微粒体酶(CYP450)抑制剂对MN9202代谢的影响。结果MN9202在Beagle犬肝微粒体酶中的K_m为(22.6±8.0) μmol·L^-1；V_max为(0.54±0.17) μmol·g^-1·min^-1；CL_int为(0.024 2±0.000 9) L·g^-1·min^-1。醋竹桃霉素(Tro)和酮康唑(Ket)能够显著抑制MN9202的代谢；反苯环丙胺(Tra)对MN9202的代谢也有一定的抑制作用，而其他CYP450抑制剂对MN9202的代谢无明显影响。结论CYP3A和CYP2C19参与了MN9202的代谢，人CYP3A和CYP2C19的抑制剂可能使MN9202的代谢受到抑制，造成药物的药效或毒性的增加。     

Spinal effect of a neuropeptide FF analogue on hyperalgesia and morphine-induced analgesia in mononeuropathic and diabetic rats.

1DMe, a neuropeptide FF (NPFF) analogue, has been shown to produce antinociception and to enhance morphine analgesia in rats after intrathecal administration. To determine whether 1DMe could correct hyperalgesia and restore morphine efficacy in mononeuropathic (MN) and diabetic (D) rats we examined the spinal effect of 1DMe in MN and D rats without and after spinal blockade of mu- and delta-opioid receptors with CTOP and naltrindole, respectively. The influence of 1DMe on morphine-induced antinociception was assessed in the two models using isobolographic analysis. Whereas 1DMe intrathecally injected (0.1, 1, 7.5 microg rat(-1)) was ineffective in normal (N) rats, it suppressed mechanical hyperalgesia (decrease in paw pressure-induced vocalisation thresholds) in both MN and D rats. This effect was completely cancelled by CTOP (10 microg rat(-1)) and naltrindole (1 microg rat(-1)) suggesting that it requires the simultaneous availability of mu- and delta-opioid receptors. The combinations of morphine: 1DMe (80.6:19.4% and 99.8:0.2%, in MN and D rats, respectively) followed by isobolographic analysis, showed a superadditive interaction, relative to the antinociceptive effect of single doses, in D rats only. In N rats, the combination of morphine: 1DMe (0.5 mg kg(-1), i.v.: 1 microg rat(-1), i.t., ineffective doses) resulted in a weak short-lasting antinociceptive effect. These results show a different efficacy of 1DMe according to the pain model used, suggesting that the pro-opioid effects of the NPFF in neuropathic pain are only weak, which should contribute to hyperalgesia and to the impaired efficacy of morphine.     

Genetic susceptibility of newborn daughters to oxidative stress

A central question in risk assessment is whether newborns' susceptibility to mutagens is different from that of adults. Therefore we investigated whether genotype and/or the DNA strand break repair phenotype in combination with the MN assay would allow estimation of the relative sensitivity of a newborn as compared to his mother for oxidative DNA damage. We compared the in vitro genetic susceptibility for H2O2 in PBMC of 17 mother-newborn daughter pairs taking into account genotypes for relevant DNA repair (hOGG1, XRCC1, XRCC3, XPD) and folate metabolism (MTHFR) polymorphisms. After in vitro challenge with H2O2 the repair capacity was assessed by the Comet assay and chromosome/genome mutations by the cytokinesis-block MN assay. No statistically significant differences were found between mothers and their newborn daughters either for initial DNA damage or for residual DNA damage. Mothers showed higher background frequencies of MN as compared to their newborn daughters, due to the age factor. This was confirmed by significantly higher frequencies of MN observed in mothers versus newborn daughters for several genotypes. No genotype with a significant effect on DNA repair capacity in newborns was identified. Concerning MN frequencies, however, newborns carrying the variant XRCC3(241) genotype might be at higher risk for the induction of MN by oxidative stress. Multivariate analysis revealed a significant protective effect of maternal antioxidant supplementation during pregnancy against oxidative DNA damage in newborns in terms of MN frequencies. However, these conclusions might not be extrapolable to other types of DNA damage and need confirmation in a study on a larger population.     

The genotoxic and antigenotoxic effects of Salvia fruticosa leaf extract in human blood lymphocytes

Abstract

The aim of this study was to investigate the genotoxic and antigenotoxic effects of Salvia fruticosa (Sf) leaf extract with the absence and presence of S9 mix using sister chromatid exchange (SCE), chromosome aberration (CA) and micronucleus (MN) formation test systems in human peripheral blood lymphocytes (HPBLs) that were treated with 1.5-, 3.0- and 6.0-µL/mL concentrations for 24- and 48-hour treatment periods. The cytotoxicity of Sf leaf extract was also investigated by calculating the mitotic index (MI), proliferation index (PI) and nuclear division index (NDI). In the absence of S9 mix, Sf leaf extract alone increased SCE frequency at the 48-hour treatment period; however, it induced the CA and MN at all concentrations and at all treatment periods. Sf plus MMC (mitomycin C) synergically induced SCE and CA, except the highest concentration of Sf leaf extract and MMC on induction of SCE. In addition, Sf leaf extract induced the effect of MMC on MN frequency for 24 hours, but it significantly decreased the effect of MMC on MN frequency for the 48-hour treatment period. Sf leaf extract showed a cytotoxic effect by decreasing the MI; however, it did not decrease the PI and NDI. In the presence of S9 mix, Sf leaf extract did not increase the SCE, when compared to solvent control, whereas it reduced the effect of cyclophosphamide (Cyp). Sf leaf extract induced the CA and MN, but could not increase the effect of Cyp on CA and MN formation. Sf leaf extract had no cytotoxic effect; however, it induced the cytotoxicity of Cyp.     

Assessment of micronuclei in lymphocytes from workers exposed to vapours and aerosols of bitumen

We investigated the micronucleus frequencies in peripheral blood lymphocytes of 225 mastic asphalt workers (age 17-62 years) and 69 non-bitumen-exposed road construction workers (age 18-64 years) in Germany before and after the working shift. Median shift exposure to vapours and aerosols of bitumen of exposed workers was 3.0 mg/m3. Micronuclei (MN) were determined with a standard method using cytochalasin B. Median MN frequency was 6.0 (interquartile range (IQR) 4.0-8.5) MN/1,000 binucleated lymphocytes (MN/1,000 BNC) in exposed workers and 6.0 (IQR 4.0-8.3) MN/1,000 BNC in non-exposed workers before shift. After shift, we observed 6.5 (IQR 4.4-9.3) MN/1,000 BNC in exposed workers and 6.5 (IQR 4.0-9.0) MN/1,000 BNC in non-exposed workers. Regression models were applied with the log-transformed MN frequency as the dependent variable in order to estimate the effects of exposure to vapours and aerosols of bitumen and of potential confounders. Age was the strongest predictor of MN formation in both exposed workers and referents. Our data suggest that MN formation was not associated with concentration of vapours and aerosols of bitumen during shift at the individual level. Although similar MN frequencies were observed in both groups, the modelling of factors potentially influencing MN frequency revealed a weak group difference in the post-shift model. We conclude that this small difference cannot be judged to be a relevant mutagenic effect of exposure to vapours and aerosols of bitumen, also with regard to the lack of adjustment for multiple testing and the lack of a group effect in the original data.     

In vitro antigenotoxic potential of acitretin in human lymphocytes treated with the antineoplastic alkylating agent ASE (NSC-71964).

Acitretin is widely used in the systemic treatment of severe forms of psoriasis and other skin disorders. ASE, namely 3beta-hydroxy-13alpha-amino-13,17-seco-5alpha-androstan-17-oic-13,17-lactam-p-bis(2-chloro-ethyl)amino phenylacetate (AzaSteroidalEster, NSC-71964), is an alkylating agent with antineoplastic activity and mutagenic properties. The aim of this study was to investigate the possible genotoxic and/or antigenotoxic effects of acitretin in human lymphocyte cultures in vitro, using sister chromatid exchange (SCE) and cytokinesis-blocked micronucleus (CBMN) assays. Micronucleus (MN) analysis was achieved in combination with fluorescence in situ hybridization (FISH), using an alpha-satellite DNA pancentromeric probe. It was found that acitretin alone demonstrated no clastogenic or aneugenic activity. However, simultaneous incubation of lymphocyte cultures with ASE and acitretin resulted in a reduction of ASE-induced SCEs. For MN analysis lymphocytes were treated with ASE and acitretin at 21 and 41 h after culture initiation, corresponding to G1 and G2 phases, respectively, and lasted until cell harvest. Acitretin caused a decrease in ASE-induced MN when treatment of cells started at 41 h, but exerted no effect on them when treatment started at 21 h. These findings suggest that acitretin exerts antigenotoxic effects in human lymphocyte cultures, the expression of which may be related to the cycle phase of the cells upon onset and duration of the treatment, at least as far as MN frequency is concerned.     

Involvement of prohibitin 1 and prohibitin 2 upregulation in cBSA-induced podocyte cytotoxicity

Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults, when not effectively treated. The aim of this study was to discover new targets for the diagnosis and treatment of MN. A reliable mouse model of MN was used by the administration of cationic bovine serum albumin (cBSA). Mice with MN exhibited proteinuria, histopathological changes, and accumulation of immune complexes in the glomerular basement membrane. Label-free proteomics analysis was performed to identify changes in protein expression, and the overexpressed proteins were evaluated. There were 273 proteins that showed significantly different expression in mice with MN, as compared to the controls. String analysis showed that functions related to cellular catabolic processes were downregulated in MN. Among the differentially expressed proteins, prohibitin 1 (PHB1) and prohibitin 2 (PHB2) were upregulated in the kidneys of mice with MN, as demonstrated by immunohistochemistry (IHC), and this upregulation was observed in both the tubular cells and glomeruli. Both shRNA-mediated knockdown of PHB1 or PHB2 inhibited tumor suppressor p53 expression and significantly promoted podocyte proliferation. In addition, both PHB1 and PHB2 were responsible for cBSA-induced cytotoxicity. Microarray analysis further revealed that the upregulation of PHB1 and PHB2 may be due to a blockage of proteasome activity. These data demonstrate that the upregulation of PHB2 is involved in cBSA-mediated podocyte cytotoxicity, which may lead to MN development.     

厚朴酚与和厚朴酚的体内药物分析方法及药代动力学研究近况

厚朴酚与和厚朴酚是中药厚朴的主要药效成分,具有多种药理作用,研究其药代动力学过程,对指导临床用药具有现实意义,而适宜的体内药物分析方法的建立,是成功进行药代动力学研究的基础。分类介绍厚朴酚与和厚朴酚的体内药物分析方法,综述并探讨其药代动力学的影响因素。     

Modelling transdermal drug delivery using microneedles: effect of geometry on drug transport behaviour

Transdermal drug delivery using microneedles (MNs) depends on the rate of drug transport through the viable epidermis. Therefore, minimising the distance between the drug-loaded surface and the microcirculation in the dermis where the drug is absorbed into the body is significant in improving drug delivery efficiency. A quantifiable relationship between MN design parameters and skin diffusion properties is therefore desirable, which is what this study aims to achieve. A framework is presented to quantitatively determine the effects of design parameters on drug diffusion through skin, where the effects of compressive strain on skin due to insertion of MN are considered. The model is then used to analyse scenarios of practical importance. For all scenarios analysed, predicted steady-state flux was found to be lower when effect of MN strain on diffusion coefficient was accounted for. For example, simulations results indicated increasing tip radius from 5 to 20 μm and flux increased from 6.56 × 10(-6) to 7.02 × 10(-6) mol/(m(2) s) for constant diffusion coefficient. However, if the effect of strain on diffusion coefficient is considered, the calculated flux increases from 5.30 × 10(-6) to a peak value of 5.32 × 10(-6) mol/(m(2) s) (at 10 μm) and decreases to 5.29 × 10(-6) mol/(m(2) s). This paper contributes by reporting a framework to relate MN geometry to permeability with inclusion of the possible effects the MN design may pose on the diffusion coefficient.     

Absence of synergistic effects on micronucleus formation after exposure to electromagnetic fields and asbestos fibers in vitro.

Exposure of human amniotic fluid (AFC) cells to horizontally applied magnetic fields (hMF) of 50 Hz and 1 mT generated in a Helmholtz-coil system leads to a significant increase in micronucleus frequency (MN), without affecting cell proliferation. To investigate whether hMF-exposure has an additive or synergistic effect on the genotoxic capacity of asbestos fibers, MN induction was investigated in hMF pre-exposed cells, treated before or after with asbestos (1 microg/cm2). Neither synergistic nor additive effects on MN induction were observed. The results indicate, that under our experimental conditions, exposure to hMF and treatment with asbestos fibers possess genotoxic capability, but no interactive effects, in AFC cells.     

Effects of the new mitotic inhibitor pyrimidinsulfone NY 4137 on human cells in vitro and on colchicine binding to tubulin

The effect of 2-(2-thenyl)sulfonyl-5-bromopyrimidine (NY 4137) on cells of the human line NHIK 3025 was investigated. It was shown that NY 4137 induces cell cycle specific inhibition in metaphase. A dose-dependent prolongation of metaphase was found and the fraction of cells able to escape metaphase arrest declined gradually as the concentration of NY 4137 increased. A total block in metaphase was achieved with 0.016 mM and higher concentrations of NY 4137. Interphase was also prolonged in cells treated with 0.016 mM. Inhibition of valine incorporation by NY 4137 was also found to be dose-dependent. Following a 2-h exposure to 0.2 mM NY 4137 valine incorporation was inhibited by 80–85%. Inhibition of colchicine and vincristine binding to purified tubulin was also investigated. Double reciprocal plot and gel filtration chromatography showed that NY 4137 competitively inhibited colchicine binding to DEAE-cellulose purified tubulin. NY 4137 had no effect on vincristine binding to tubulin. The binding of NY 4137 to tubulin at or near the colchicine binding site may be responsible for the metaphase arrest.     

The evaluation of genotoxic potential of ornidazole, nitroimidazole, in lymphocyte culture of patients with amebiasis

The genotoxicity study of ornidazole (ONZ) was carried out on human lymphocyte chromosomes, using sister chromatid exchange (SCE) and micronucleus (MN). Thirty-two patients with Entemoeba histolitica infection who received 1000 mg/day for 10 days were included in this study. SCE and MN were measured before and after therapy. A statistically significant increase was observed in the SCE (P < 0.001) and MN frequencies (P < 0.001) after ornidazole therapy. It was concluded that ONZ has a potential geno- and cytotoxic effect in human peripheral lymphocyte cultures. For this reason, further, detailed studies are needed to elucidate the ONZ mechanism of genotoxicity and its carcinogenic potential.     

Effect of charge and lipid concentration on in-vivo percutaneous absorption of methyl nicotinate from liposomal vesicles

We have investigated the influence of charge and lipid concentration on the in-vivo percutaneous absorption of a model compound, methyl nicotinate (MN), from liposomal vesicles. MN-loaded liposomes were produced by the reverse-phase evaporation method (REV) using different concentrations of phosphatidyl choline (PC), in association with surfactants such as dioctadecyl dimethyl ammonium bromide (DDAB18) and dicetyl phosphate (DCP), which impart a positive or negative charge to the systems, respectively. The liposomal suspensions were then processed to hydrogels and used to study in-vivo the MN permeation profile. MN was chosen as the model compound since it was capable of causing cutaneous erythema, the intensity and duration of which was proportional to the amount entering the living epidermis over time. The extent of the erythema was monitored by reflectance spectrophotometry, a non-invasive technique. In-vivo findings showed an interesting MN delayed release, which was proportional to the amount of phospholipids in each liposomal formulation. Furthermore, it could be noted that the erythematous effect was more prolonged when MN was delivered from neutral or negatively-charged liposomal forms.     

前列腺素E_2调控MN9D细胞孤儿核受体Nurr1表达的研究

目的观察前列腺素E2(prostaglandin E2,PGE2)对多巴胺(dopamine,DA)合成细胞系MN9D细胞内源性孤儿核受体Nurr1表达的作用,并研究Nurr1表达增高对酪氨酸羟化酶(tyrosine hydroxylase,TH)表达的影响,探讨Nurr1调控DA能神经元发育的机制。方法用100μg.L-1的PGE2作用于MN9D细胞2 h至6 h,观察细胞形态变化,并用免疫细胞化学染色及Western blot方法检测PGE2作用前后MN9D细胞内源性Nurr1及TH表达的变化。结果①100μg.L-1的PGE2作用2、4及6 h,MN9D细胞形态没有明显变化。②加入PGE22、4及6 h,MN9D细胞Nurr1抗体免疫荧光染色强度比未加PGE2作用的正常对照组细胞明显增强。MN9D细胞自身表达TH呈不均一性,PGE2作用2~6h,MN9D细胞中TH阳性染色细胞百分比与正常对照组细胞接近。③Western blot结果显示,PGE2作用6 h,MN9D细胞Nurr1蛋白表达比未加PGE2的正常对照组细胞明显增加(P<0.05),而此时TH蛋白表达与正常对照组细胞接近。结论PGE2可在短时间内迅速明显激活MN9D细胞内源性Nurr1表达,但对TH表达没有影响。TH表达的激活或许还需要除Nurr1以外其它环境或因子的共同参与。     

Automation of the in vitro micronucleus and chromosome aberration assay for the assessment of the genotoxicity of the particulate and gas–vapor phase of cigarette smoke

Abstract

Total particulate matter (TPM) and the gas–vapor phase (GVP) of mainstream smoke from the Reference Cigarette 3R4F were assayed in the cytokinesis-block in vitro micronucleus (MN) assay and the in vitro chromosome aberration (CA) assay, both using V79-4 Chinese hamster lung fibroblasts exposed for up to 24?h. The Metafer image analysis platform was adapted resulting in a fully automated evaluation system of the MN assay for the detection, identification and reporting of cells with micronuclei together with the determination of the cytokinesis-block proliferation index (CBPI) to quantify the treatment-related cytotoxicity. In the CA assay, the same platform was used to identify, map and retrieve metaphases for a subsequent CA evaluation by a trained evaluator. In both the assays, TPM and GVP provoked a significant genotoxic effect: up to 6-fold more micronucleated target cells than in the negative control and up to 10-fold increases in aberrant metaphases. Data variability was lower in the automated version of the MN assay than in the non-automated. It can be estimated that two test substances that differ in their genotoxicity by approximately 30% can statistically be distinguished in the automated MN and CA assays. Time savings, based on man hours, due to the automation were approximately 70% in the MN and 25% in the CA assays. The turn-around time of the evaluation phase could be shortened by 35 and 50%, respectively. Although only cigarette smoke-derived test material has been applied, the technical improvements should be of value for other test substances.     

Cell-cycle analysis and micronuclei frequency reveals G0/G1 blockers as weak micronuclei inducers

Micronuclei (MN) formation is generally attributed to error in DNA synthesis or mitosis, which are represented by the S or G₂/M phase respectively, in the cell-cycle histogram. Interestingly, many of the known anticancer drugs target these cell-cycle phases to elicit cytotoxicity. Here, we attempted to identify whether any correlation exists between the cell-cycle effect and MN induction potential using various treatments. In addition, we tracked down MN in cycling cells to assess its final fate. We treated SiHa cells with various known drugs and correlated their effects on cell-cycle and MN frequency. MN-tracking studies were performed in peripheral mononuclear and siHa cells upon staining with Giemsa and ethidium bromide respectively. We observed MN induction by all the tested drugs irrespective of their basic effect on cell cycle. However, MN induction was more with drugs which interfere with the S or G₂/M than the G₀/G₁ phase. Our results indicate G₀/G₁ blockers to be comparatively safer drugs. Additionally, our results show that expulsion out of cells may be one of the main fates of drug-induced MN.     

Effects of repeated maternal oral exposure to low levels of trichlorfon on development and cytogenetic toxicity in 3-day mouse embryos

Trichlorfon is a widely used broad-spectrum agricultural insecticide. Few studies have evaluated the effects of trichlorfon on developing fetuses, especially at early stages of development after low-level maternal exposures. In this study, we evaluated the direct effects of trichlorfon on preimplantation mouse embryos after 30 days of maternal exposure (2, 10 and 50 mg/kg/day) via drinking water.On gestation day 3 (dg3), blastocysts were collected and evaluated for changes in gross morphology; cell number; the presence of interphase, metaphase, micronuclei (MN) cells and fragmented and pycnotic nuclei. Embryos in the 50 mg/kg/day group had a significantly reduced mean cell number per embryo. Furthermore, there was a significant increase in the frequency of pycnotic nuclei and an absence of metaphase cells in the 50 mg/kg/day treated group. None of the developmental endpoints evaluated were observed in the 2 and 10 mg/kg/day trichlorfon-treated groups. A simultaneous decrease in the cell number and an increase in the frequencies of absent metaphases and pycnotic nuclei indicate that embryonic developmental deficits observed in the 50 mg/kg/day exposure group were associated with cytotoxicity.