Deciphering the toxic effects of organochlorine pesticide, dicofol on human RBCs and lymphocytes.

Organochlorine pesticides have generated growing concern owing to their diverse toxicities. In this connection, we have evaluated toxic potential of an acaricide, dicofol (DCF) and its harmful effects on human RBCs and lymphocytes. DCF caused hemolysis and rupture of human erythrocytes as confirmed by scanning electron microscopy (SEM). Significant increase in protein oxidation, lipid peroxidation, ROS production, methemoglobin formation with enhanced activities of superoxide dismutase and catalase but decreased level of reduced glutathione were observed as a result of DCF exposure to human erythrocytes. SEM showed significant DCF induced alterations in RBCs from normal discoid shape to echinocytes. Similarly, lymphocytes showed membrane damage, formation of membrane blebs and distorted cell morphology. In vitro comet assay indicated a significant DNA fragmentation in human lymphocytes upon DCF exposure. These results strongly suggest that DCF induces oxidative stress in RBCs via generation of ROS and alters the cellular architecture directly and indirectly.

A novel mutation in the thyroglobulin gene that causes goiter and dwarfism in Wistar Hannover GALAS rats.

Outbred stocks of rats have been used extensively in biomedical, pharmaceutical and/or toxicological studies as a model of genetically heterogeneous human populations. One of such stocks is the Wistar Hannover GALAS rat. However, the colony of Wistar Hannover GALAS rat has been suspected of keeping a problematic mutation that manifests two distinct spontaneous abnormalities, goiter and dwarfism, which often confuses study results. We have successfully identified the responsible mutation, a guanine to thymine transversion at the acceptor site (3' end) of intron 6 in the thyroglobulin (Tg) gene (Tgc.749-1G>T), that induces a complete missing of exon 7 from the whole Tg transcript by mating experiments and subsequent molecular analyses. The following observations confirmed that Tgc.749-1G>T/Tgc.749-1G>T homozygotes manifested both dwarfism and goiter, while Tgc.749-1G>T/+ heterozygotes had only a goiter with normal appearance, suggesting that the mutant phenotypes inherit as an autosomal semi-dominant trait. The mutant phenotypes, goiter and dwarfism, mimicked those caused by typical endocrine disrupters attacking the thyroid. Hence a simple and reliable diagnostic methodology has been developed for genomic DNA-based genotyping of animals. The diagnostic methodology reported here would allow users of Wistar Hannover GALAS rats to evaluate their study results precisely by carefully interpreting the data obtained from Tgc.749-1G>T/+ heterozygotes having externally undetectable thyroidal lesions.

Effects of DDT and dicofol on population growth of Brachionus calyciflorus under different algal (Scenedesmus obliquus) densities.

A number of organochlorine pesticides, including DDT and dicofol, used to be important in crop protection and management. Their residues may reach water bodies and eventually affect the non-target organisms such as rotifers. In the present study, we evaluated the effects of DDT (0.05, 0.1, 0.2 and 0.4 mg l(-1)) and dicofol (0.1, 0.2, 0.4 and 0.8 mg l(-1)) on the population growth of rotifer Brachionus calyciflorus under two levels of Scenedesmus obliquus (1.0 x 10(6) and 3.0 x 10(6) cell ml(-1)). Regardless of the food level, DDT was more toxic than dicofol to B. calyciflorus. Under low food level, DDT at 0.1 and 0.2 mg l(-1) decreased the population growth rate (r), and DDT at 0.05-0.4 mg l(-1) decreased the maximum population density (K). Dicofol at 0.4 and 0.8 mg l(-1) decreased r and K, but dicofol at 0.2 mg l(-1) increased K. Under high food level, DDT at 0.05-0.2 mg l(-1) increased K, whereas DDT at 0.4 mg l(-1) as well as dicofol at 0.4 and 0.8 mg l(-1) decreased r and K. Increase in food level increased r exposed to DDT at 0.05-0.2 mg l(-1) as well as dicofol at 0.8 mg l(-1), and Kexposed to DDTat 0.05-0.2 mg l(-1) as well as dicofol at 0.1 and 0.2 mg l(-1). DDT concentration, algal density and their interaction affected r and K of B. calyciflorus. Both dicofol concentration and algal density affected r. Dicofol concentration, algal density and their interaction affected K. Both r and K were suitable endpoints for assessing the effects of DDT and dicofol on the rotifers population dynamics under two algal densities, and the latter was more sensitive.

How can environmental conditions influence dicofol genotoxicity on the edible Asiatic clam, Meretrix meretrix?

Genotoxic effects of dicofol on the edible clam Meretrix meretrix were investigated through a mesocosm experiment. Individuals of M. meretrix, were exposed to environmental concentration (D1 = 50 ng/L) and supra-environmental concentration (D2 = 500 ng/L) of dicofol for 15 days, followed by the same depuration period. DNA damage (i.e., strand breaks and alkali-labile sites) was evaluated at day 1, 7 and 15, during uptake and depuration, using Comet assay (alkaline version) and nuclear abnormalities (NAs) as genotoxicity biomarkers. The protective effects of dicofol against DNA damage induced by ex vivo hydrogen peroxide (H2O2) exposure were also assessed. Comet assay results revealed no significant DNA damages under dicofol exposure, indicating 1) apparent lack of genotoxicity of dicofol to the tested conditions and/or 2) resistance of the animals due to optimal adaptation to stress conditions. Moreover, ex vivo H2O2 exposure showed an increase in the DNA damage in all the treatments without significant differences between them. However, considering only the DNA damage induced by H2O2 during uptake phase, D1 animals had significantly lower DNA damage than those from other treatments, revealing higher protection against a second stressor. NAs data showed a decrease in the % of cells with polymorphic, kidney shape, notched or lobbed nucleus, along the experiment. The combination of these results supports the idea that the clams used in the experiment were probably collected from a stressful environment (in this case Pearl River Delta region) which could have triggered some degree of adaptation to those environmental conditions, explaining the lack of DNA damages and highlighting the importance of organisms' origin and the conditions that they were exposed during their lives.

Exploring the binding mechanism and adverse toxic effects of persistent organic pollutant (dicofol) to human serum albumin: A biophysical, biochemical and computational approach.

The organochlorine pesticide dicofol (DCF), a persistent organic pollutant, is used as acaricide worldwide. Considering its large consumption in the agriculture sector and potential toxic effects such as endocrine disruption, carcinogenicity, and environmental persistence are detrimental to human health. To take an extensive evaluation of its potential toxicity, the current study was aimed to explore the binding mechanism and adverse effect of DCF on human serum albumin (HSA) by using an array of biophysical techniques (UV-visible, fluorescence, 3D fluorescence, and circular dichroism spectroscopy), isothermal titration calorimetric (ITC), computational methods and biochemical approaches. Fluorescence quenching and UV-Visible spectra of the HSA-DCF system confirmed static quenching mechanism and complex formation between HSA and DCF. The thermodynamics results from ITC revealed DCF-HSA interaction was exothermic and spontaneous and involved hydrophobic interactions and hydrogen bonding. The esterase activity of HSA displayed constant Vmax and elevated Km values confirming DCF-HSA competitive interaction. Circular dichroism spectra results revealed structural changes in HSA protein on interaction with DCF. Furthermore, molecular-specific site marker and molecular modelling results affirmed that the binding Site of DCF is Site I of HSA. A significant carbonyl content level in DCF-HSA system suggested protein structure damage. This work is likely to add a better understanding of DCF toxicity in human health and helpful in fortifying the check on food safety.

Decreased sperm motility in rats orally exposed to single or mixed pesticides.

The Brazilian Agency of Sanitary Vigilance (ANVISA) conducted a study that demonstrated the presence of residues of several pesticides in fresh fruits and vegetables that were available for purchase by the general populace. In order to evaluate potential adverse health effects of low-level exposure to agrochemicals, the reproductive toxicity of the pesticides dicofol, dichlorvos, permethrin, endosulfan, and dieldrin was evaluated in rats dosed with these chemicals individually or as mixtures. Sixty male Lewis rats (6 wk old, 200 x g) were randomly allocated to 8 groups: (1) control group, received basal diet; (2) 5 groups designated a to e received the diet containing each pesticide individually, at the respective effective doses: lowest-observed-adverse-effect level (LOAEL) for dieldrin and endosulfan, lowest-observed-effect level (LOEL) for dicofol, and lowest effect level (LEL) for dichlorvos and permethrin, respectively, depending on the published data; (3) effective dose group, which received a mixture of pesticides added to basal diet at the respective doses reported to produce adverse effects; and (4) low dose group, which received a pesticide mixture added to the basal diet, where each pesticide was at its no-observed-effect level (NOEL). After 8 wk of treatment, reproductive parameters were evaluated. Sperm morphology, daily sperm production (DSP), sperm transit time through the epididymis, hormonal levels, and histopathological evaluation of testis and epididymis did not differ significantly among the groups. However, sperm motility was significantly decreased in animals that received a mixture of dieldrin, endosulfan, dicofol, dichlorvos, and permethrin, as well as in the group receiving dicofol alone. Exposure to the individual pesticides endosulfan, dichlorvos, and permethrin did not markedly affect sperm motility. The impairment of sperm motility in the mixture of pesticides at the NOEL level indicates that reproductive effects not seen with individual pesticides may occur in presence of several pesticides due to an additive effect. However, the pesticide mixtures did not appear to affect DSP or spermatogenesis despite reduced sperm motility.

Numerical variations and spontaneous malformations in the early embryos of the Korean salamander, Hynobius leechii, in the farmlands of Korea.

Embryo sacs of the Korean salamander, Hynobius leechii, were collected from nine farmlands in Gyeongsangnam-Do, Korea, in early spring of 2002 and 2004. The variations in the number of embryos within each embryo sac and the mortality and abnormality rates among the embryos were investigated. We also analyzed the patterns of spontaneous embryonic malformations and the residual chemicals in the soil of the habitats using multiple-residue GC/MS. A total of 79,195 embryos were obtained from 1933 embryo sacs. There were regional variations in the length of individual embryo sacs and the number of embryos in each. The longest embryo sac averaged by region measured 20.67 cm ± 3.51 and was obtained from 2-Banseong in 2002. Of the embryos collected, 13.71% either died or stopped developing, and 3.54% of the hatched embryos developed abnormally; the latter were classified according to the patterns of malformation. External gill dysplasia was the most frequent malformation, and caudal dysplasia, abdominal blisters, and dysplasia of the fin were also observed frequently. Histopathological analysis showed neural tube abnormalities, acrania, curved notochords, thyroid teratoma, and various other kinds of endodermal developmental abnormalities. In the analysis of the residual pesticides in the soil, carbofuran, endosulfan-sulfate, and endosulfan-ß were detected in the regions with high mortality and malformation rates. These results indicate that various agricultural chemicals and other unknown factors may cause the aforementioned forms of spontaneous malformations in the embryos of Hynobius leechii.

Selected endocrine disrupting compounds (vinclozolin, flutamide, ketoconazole and dicofol): effects on survival, occurrence of males, growth, molting and reproduction of Daphnia magna.

BACKGROUND, AIM AND SCOPE: Pollution-induced endocrine disruption in vertebrates and invertebrates is a worldwide environmental problem, but relatively little is known about effects of endocrine disrupting compounds (EDCs) in planktonic crustaceans (including Daphnia magna). Aims of the present study were to investigate acute 48 h toxicity and sub-chronic (4-6 days) and chronic (21 days) effects of selected EDCs in D. magna. We have investigated both traditional endpoints as well as other parameters such as sex determination, maturation, molting or embryogenesis in order to evaluate the sensitivity and possible use of these endpoints in ecological risk assessment. MATERIALS AND METHODS: We have studied effects of four model EDCs (vinclozolin, flutamide, ketoconazole and dicofol) on D. magna using (i) an acute 48 h immobilization assay, (ii) a sub-chronic, 4-6 day assay evaluating development and the sex ratio of neonates, and (iii) a chronic, 21 day assay studying number of neonates, sex of neonates, molting frequency, day of maturation and the growth of maternal organisms. RESULTS: Acute EC50 values in the 48 h immobilization test were as follows (mg/L): dicofol 0.2, ketoconazole 1.5, flutamide 2.7, vinclozolin >3. Short-term, 4-6 day assays with sublethal concentrations showed that the sex ratio in Daphnia was modulated by vinclozolin (decreased number of neonate males at 1 mg/L) and dicofol (increase in males at 0.1 mg/L). Flutamide (up to 1 mg/L) had no effect on the sex of neonates, but inhibited embryonic development at certain stages during chronic assay, resulting in abortions. Ketoconazole had no significant effects on the studied processes up to 1 mg/L. DISCUSSION: Sex ratio modulations by some chemicals (vinclozolin and dicofol) corresponded to the known action of these compounds in vertebrates (i.e. anti-androgenicity and anti-oestrogenicity, respectively). Our study revealed that some chemicals known to affect steroid-regulated processes in vertebrates can also affect sublethal endpoints (e.g. embryonic sex determination and/or reproduction) in invertebrates such as D. magna. CONCLUSIONS: A series of model vertebrate endocrine disrupters affected various sub-chronic and chronic parameters in D. magna including several endpoints that have not been previously studied in detail (such as sex determination in neonates, embryogenesis, molting and maturation). Evaluations of traditional reproduction parameters (obtained from the 21 day chronic assay). as well as the results from a rapid, 4-6 day, sub-chronic assay provide complementary information on non-lethal effects of suspected organic endocrine disrupters. RECOMMENDATIONS AND PERSPECTIVES: It seems that there are analogies between vertebrates and invertebrates in toxicity mechanisms and in vivo effects of endocrine disruptors. However, general physiological status of organisms may also indirectly affect endpoints that are traditionally considered 'hormone regulated' (especially at higher effective concentrations as observed in this study) and these factors should be carefully considered. Further research of D. magna physiology and comparative studies with various EDCs will help to understand mechanisms of action as well as ecological risks of EDCs in the environment.

Estrogenic activity of dicofol with the human estrogen receptor: Isomer- and enantiomer-specific implications.

Dicofol is a non-systemic acaricide/miticide currently registered in the US and Canada for use on a wide variety of crops. This agrochemical has been identified as a potential candidate substance for the United Nations Economic Commission for Europe (UN-ECE) Persistent Organic Pollutant (POP) Protocol and implicated as a potential "endocrine disrupting compound". The technical product is usually synthesized from technical DDT and consists of approximately 80% and 20% of p,p'- and o,p'-dicofol isomers. The o,p'-substituted isomer of dicofol is chiral and may have enantiomer-specific activity; however, the stereospecific activity of o,p'-dicofol has not been reported. In this study, we examined the isomer- and enantiomer-specific endocrine disruption potential of dicofol using yeast-based steroid hormone receptor gene transcription assay designed with the human estrogen receptor (hER). Estrogenic activity of (+)-17-beta estradiol (positive control), p,p'-dicofol, racemic o,p'-dicofol [(+/-)-o,p'-dicofol] and the individual o,p'-dicofol enantiomers was measured via quantification of beta-galactosidase. The (+/-)-o,p'- and p,p'-dicofol were weak estrogen mimics (EC(50): 4.2 x 10(-6) and 1.6 x 10(-6)M, respectively) relative to estradiol (3.7 x 10(-10)M). For o,p'-dicofol, the beta-galactosidase induction by (-)-o,p'-dicofol (EC(50): 5.1 x 10(-7)M) was greater than the racemic mixture. However, the (+)-o,p'-dicofol enantiomer was found to have negligible estrogenic activity. These data indicate that dicofol is a weak hER agonist due to activity of the achiral p,p'-isomer and (-)-o,p'-substituted enantiomer and emphasizes the influence of chemical structure and configuration on biological responses to exposure from chiral compounds.

Detection of thyroid system-disrupting chemicals using in vitro and in vivo screening assays in Xenopus laevis.

We developed a thyroid hormone (TH) inducible primary screening assay for the identification and assessment of man-made chemicals that interfere with the TH-signalling pathway within target cells. The assay was developed in a Xenopus laevis cell line that was transduced with a self-inactivating (SIN) lentivirus vector (LV) containing a luciferase gene. The luciferase activation in this cell line was TH-specific: 3,3',5-L-triiodothyronine (T(3)) > 3,3'5-L-triiodothyroacetic acid (Triac) > 3,3',5-D-triiodothyronine (D-T(3)), > L-thyroxine (T(4)) > 3,3',5'-L-triiodothyronine (rT(3)). The application of the ligand-dependent luciferase assay for screening for thyroid system-disrupting chemicals revealed that three phthalates (dicyclohexyl phthalate, n-butylbenzyl phthalate, and di-n-butyl phthalate), two herbicides (ioxynil and pentachlorophenol) and a miticide (dicofol) had 3,3',5-L-triiodothyronine- T(3)- antagonist activity at concentrations ranging from 10(-6) to 10(-5) M. These chemicals also inhibited the expression of the endogenous primary T(3)-response TH nuclear receptor beta (TRbeta) gene. The inhibitory characteristics of these chemicals were similar for both assays performed, although the assay for T(3)-dependent activation of TRbeta gene was more sensitive than the luciferase assay. These results indicate that the luciferase assay was a rapid method with a small intra-assay variation for the primary screening of thyroid system-disrupting chemicals. Of the six chemicals, only n-butylbenzyl phthalate and pentachlorophenol exhibited T(3)-antagonist activity in an in vivo metamorphosis-based assay. It should be noted that chemicals elicited thyroid system-disrupting activity in the luciferase assay did not always interfere with the thyroid system in vivo.

Effects of dichlorodiphenyltrichloroethane and its analogues on rat liver mitochondria.

The effects of dichlorodiphenyltrichloroethane (DDT) and sixteen analogues on respiration, swelling and latent ATPase of rat liver mitochondria were examined systematically. The compounds tested could be divided into four groups: DDT-type, DDE-type, kelthane-type and others by substituent groups on the ethane bridge of bis(p-chlorophenyl) ethane. Most compounds tested were shown to inhibit State 3 respiration. A linear relation was observed between the logarithms of the concentrations giving half-inhibition of State 3 respiration and the logarithms of the partition coefficients of the tested compounds. Four compounds of the kelthane type and chlorobenzilate stimulated State 4 respiration to the level of dinitrophenol-stimulated respiration. The compounds that have a hydroxy group on the ethane bridge-rapidly induced mitochondrial swelling, but DDT-type and DDE-type compounds induced swelling when the suspension contained 0.15 M KCl and 5 mM Tris-HCl. Latent ATPase of mitochondria was stimulated to different maximum levels by each of the tested compounds except DDA. The oligomycin-sensitive ATPase of submitochondria was inhibited by a series of kelthane-type compounds.

Standard curves for nuvacron, malathion, sevin, DDT and kelthane tested against the mosquito Culex pipiens L. and the microcrustacean Daphnia magna Straus.

The fourth instar larvae of Culex pipiens L. and adults of microcrustacean Daphnia magna Straus were reared under standardized conditions and used as bioassay test organisms for the preparation of the standard log concentration--probit (lc--p) regression lines for each of the following pesticides: nuvacron, malathion, sevin, DDT and kelthane. When the above pesticides were tested against the larvae of C. pipiens, the LC50 values (in ppm) ranged as follows: nuvacron 0.0014--0.0019, malathion 0.0027-0.0043, sevin 0.059-0.095, DDT 0.233-0.525, kelthane 0.17-0.24 and a mixture of malathion, DDT and kelthane (3 : 10 : 5) 0.1805-0.2451, based on 24-h readings. The corresponding LC50 values (in ppm) for D. magna were 0.00018-0.00032, 0.000074-0.00013, 0.00063-0.00069, 0.0061-0.0064, 0.071-0.090 and 0.055-0.028 for the aforementioned pesticides. D. magna proved to be more sensitive to all tested pesticides than C. pipiens larvae: it succumbed to concentrations ranging from 0.000032 to 0.004 ppm of nuvacron, from 0.000032 to 0.0016 ppm of malathion, from 0.000032 to 0.02 ppm of sevin, from 0.0008 to 0.1 ppm of DDT, from 0.03 to 0.3 ppm of kelthane and from 0.007 to 0.075 ppm of the mixture. C. pipiens larvae were affected by concentrations ranging from 0.0005 to 0.008 ppm of of nuvacron, from 0.0005 to 0.04 ppm of malathion, from 0.008 to 1.0 ppm of sevin, from 0.05 to 2.5 ppm of DDT, from 0.06 to 0.5 ppm of kelthane and from 0.07 to 0.75 ppm of the mixture.

Effect of dicofol formulation on estrous cycle and follicular dynamics in albino rats.

It is very important to study the effects of pesticides on reproduction. Dicofol, an organochlorine pesticide, was administered orally at doses of 20, 30, 40 and 50 mg/kg body weight to normal virgin rats for 30 days. There was no significant change in the number of estrous cycles and duration of each phase with 20 mg dicofol. However, there was a significant decrease in number of cycles, duration of proestrus and diestrus, and increase in the duration of the estrus phase after dicofol treatment at 30, 40 and 50 mg. Body weight and organ weight of liver, kidney, adrenal, ovary, uterus thyroid and thymus were not significantly changed in all the dicofol treated rats. There was a significant decrease in healthy follicles with concomitant increase in atretic follicles at higher doses of dicofol treatment. Hence, the effect of dicofol was dose dependent.

Possible mechanisms for the anti-implantation action of dicofol in albino rats.

Dicofol, a chlorinated pesticide showing estrogenic activity, was administered orally to virgin pregnant rats at doses of 300, 400, 500, 600, or 700 mg/kg/day for 7 consecutive days to examine its effect on blastocyst implantation. One group of rats received estradiol-17beta (10 microg) for comparison and control animals received similar quantities of olive oil. Autopsy on day 8 revealed that the olive-oil treated rats were pregnant and had a normal number of implantations and a normal duration of diestrus. Treatment with estradiol-17beta completely inhibited implantation, significantly decreased the duration of diestrus with concomitant increase in estrus. Treatment with 300, 400, or 500 mg/kg/d dicofol neither inhibited implantation nor significantly changed diestrus. Treatment with 600 mg dicofol partially inhibited implantation and significantly decreased diestrus with concomitant increase in estrus. Treatment with 700 mg dicofol completely inhibited implantation, and the uterus showed placental scars. This group exhibited a significant decrease in diestrus with concomitant increase in estrus. In all dicofol-treated rats, no significant changes were found in body weight or organ, except for a significant decrease in the weight of the uterus in groups receiving either 700 mg dicofol or estradiol-17beta. The inhibitory effect of dicofol on implantation may be due to an imbalanced estrogen-progesterone ratio, essential for implantation. The pesticide is neither tubal locking nor causes expulsion of the blastocyst from the uterus like estradiol-17beta.

Assessing the potential toxic effect of one persistent organic pollutant: non-covalent interaction of dicofol with the enzyme trypsin.

Because of the widespread concern that persistent organic pollutants (POPs) may be adversely affecting the health of humans, reliable assessing their toxic effects is urgently needed. We selectively study the interaction between dicofol (DCF) and trypsin by steady state and time resolved fluorescence quenching measurements and UV-visible absorption spectroscopy under physiological conditions as well as applying molecular docking method to establish the interaction model. The fluorescence results indicate DCF can spontaneously form a complex with trypsin mainly by hydrogen bond with only one binding site, which had been validated in molecular docking. The conformational change of trypsin was proved by UV-visible absorption and synchronous fluorescence spectroscopy indicating a red shift of carbonyl absorption peak. All the results indicated DCF had potential toxic effects on both the structure and activity of the enzyme trypsin and the effects enhanced with the increasing concentration of DCF.

Dicofol degradation to p,p'-dichlorobenzophenone - a potential antiandrogen.

In the present investigation, the degradation of the acaricide dicofol (also known as kelthane) was investigated with special emphasis on generation of p,p'-dichlorobenzophenone (DCBP) under alkaline conditions as well as induced by UV-light. Dicofol was also incubated in the presence and absence of microsomal preparations to measure potential metabolic formation of DCBP. The results indicate that the degradation of dicofol to DCBP primarily proceeds as an abiotic process via hydroxide ion catalysed elimination of a trichloromethyl anion. The generated anion picks up a proton from the solvent to generate chloroform. Microsomal metabolism does not appear to play a major role in the degradation of dicofol. DCBP is structurally analogous to the antiandrogen p,p'-dichlorodiphenylethene (DDE). We therefore investigated whether DCBP displays antiandrogenic properties. In an in vitro transactivation system utilising transiently transfected African green monkey kidney (COS-7) cells, DCBP showed potent antiandrogenic efficacy. This finding was confirmed by further studies in T47D human mammary carcinoma cells by measuring mRNA and protein expression of androgen dependent genes i.e. TRMP-2 (testosterone-repressed prostate message-2) mRNA and PSA (prostate-specific antigen) protein.

Evaluation of the impact of in-ovo exposure to dicofol on avian reproduction.

To assess the possible impact of the currently used organochlorine insecticide, dicofol, on the development and reproduction of avian species, in ovo exposure experiments to its p,p' and o,p' isomers were performed using Japanese quail (Coturnix japonica) eggs. o,p'-Dicofol (0.3-100 µg/g of egg) and p,p'-dicofol (3-100 µg/g) were injected into the yolk prior to incubation and hatched chicks were raised to adulthood. In ovo treatment with o,p'-dicofol impaired the eggshell-forming ability of female quails after sexual maturity; eggshell strength, mass, and thickness were significantly reduced at minimum dosages of 3, 1, and 0.3 µg/g, respectively. o,p'-Dicofol also caused abnormal development of the right oviduct independently of the dose; even a female exposed at the lowest dose tested (0.3 µg/g) possessed a large right oviduct. Minor but significant mass reductions of both the left oviduct and the testis were observed only at 10 µg/g. In addition, the transcript of a gene encoding cytochrome P450 cholesterol side-chain cleavage in the gonads of male hatchlings was markedly reduced by o,p'-dicofol treatment. p,p'-Dicofol did not have any marked effects on the reproductive systems, although some significant changes in eggshell formation and oviduct morphology were observed. The results indicate that transovarian exposure, especially to o,p'-dicofol, could damage avian reproduction mainly through eggshell thinning.