Quantitative in Vitro to in Vivo Extrapolation (QIVIVE) for Predicting Reduced Anogenital Distance Produced by Anti-Androgenic Pesticides in a Rodent Model for Male Reproductive Disorders.

BACKGROUND: Many pesticides can antagonize the androgen receptor (AR) or inhibit androgen synthesis in vitro but their potential to cause reproductive toxicity related to disruption of androgen action during fetal life is difficult to predict. Currently no approaches for using in vitro data to anticipate such in vivo effects exist. Prioritization schemes that limit unnecessary in vivo testing are urgently needed. OBJECTIVES: The aim was to develop a quantitative in vitro to in vivo extrapolation (QIVIVE) approach for predicting in vivo anti-androgenicity arising from gestational exposures and manifesting as a shortened anogenital distance (AGD) in male rats. METHODS: We built a physiologically based pharmacokinetic (PBK) model to simulate concentrations of chemicals in the fetus resulting from maternal dosing. The predicted fetal levels were compared with analytically determined concentrations, and these were judged against in vitro active concentrations for AR antagonism and androgen synthesis suppression. RESULTS: We first evaluated our model by using in vitro and in vivo anti-androgenic data for procymidone, vinclozolin, and linuron. Our PBK model described the measured fetal concentrations of parent compounds and metabolites quite accurately (within a factor of five). We applied the model to nine current-use pesticides, all with in vitro evidence for anti-androgenicity but missing in vivo data. Seven pesticides (fludioxonil, cyprodinil, dimethomorph, imazalil, quinoxyfen, fenhexamid, o-phenylphenol) were predicted to produce a shortened AGD in male pups, whereas two (λ-cyhalothrin, pyrimethanil) were anticipated to be inactive. We tested these expectations for fludioxonil, cyprodinil, and dimethomorph and observed shortened AGD in male pups after gestational exposure. The measured fetal concentrations agreed well with PBK-modeled predictions. DISCUSSION: Our QIVIVE model newly identified fludioxonil, cyprodinil, and dimethomorph as in vivo anti-androgens. With the examples investigated, our approach shows great promise for predicting in vivo anti-androgenicity (i.e., AGD shortening) for chemicals with in vitro activity and for minimizing unnecessary in vivo testing. https://doi.org/10.1289/EHP6774.

Nonclassical Phenyl Bioisosteres as Effective Replacements in a Series of Novel Open-Source Antimalarials.

The replacement of one chemical motif with another that is broadly similar is a common method in medicinal chemistry to modulate the physical and biological properties of a molecule (i.e., bioisosterism). In recent years, bioisosteres such as cubane and bicyclo[1.1.1]pentane (BCP) have been used as highly effective phenyl mimics. Herein, we show the successful incorporation of a range of phenyl bioisosteres during the open-source optimization of an antimalarial series. Cubane (19) and closo-carborane (23) analogues exhibited improved in vitro potency against Plasmodium falciparum compared to the parent phenyl compound; however, these changes resulted in a reduction in metabolic stability; unusually, enzyme-mediated oxidation was found to take place on the cubane core. A BCP analogue (22) was found to be equipotent to its parent phenyl compound and showed significantly improved metabolic properties. While these results demonstrate the utility of these atypical bioisosteres when used in a medicinal chemistry program, the search to find a suitable bioisostere may well require the preparation of many candidates, in our case, 32 compounds.

Diterpenes with bicyclo[2.2.2]octane moieties from the fungicolous fungus Xylaria longipes HFG1018.

Xylarilongipins A (1) and B (2), two diterpenes each with an unusual cage-like bicyclo[2.2.2]octane moiety, along with their biosynthetic precursor hymatoxin L (3), were isolated from the culture broth of the fungicolous fungus Xylaria longipes HFG1018 inhabiting in the medicinal fungus Fomitopsis betulinus. The structures and absolute configurations of the three compounds were established by extensive spectroscopic analysis and single-crystal X-ray diffraction analysis. Xylarilongipin A (1) displayed moderate inhibitory activity against the cell proliferation of concanavalin A-induced T lymphocytes and lipopolysaccharide-induced B lymphocytes with IC50 values of 13.6 and 22.4 µM, respectively. Additionally, the biosynthetic pathways for compounds 1-3 are discussed. This work not only corroborates the structure of the 9,16-cyclo-(18-nor-)isopimarane skeleton by single-crystal X-ray diffraction analysis for the first time, but also provides new insights into the biosynthetic origin of the unusual diterpene skeletons.

Use of early phenotypic in vivo markers to assess human relevance of an unusual rodent non-genotoxic carcinogen in vitro.

Foci of altered hepatocytes (FAH) are considered putative, pre-neoplastic lesions that can occur spontaneously in aging rodents, but can also be induced by chemicals or drugs. Progression of FAH to hepatocellular neoplasms has been reported repeatedly but increases in foci in rodents do not necessarily lead to tumors in carcinogenicity studies and the relevance for humans often remains unclear. Here we present the case of RG3487, a molecule which induced FAH and, later on, tumors in rats. Because the molecule was negative in genotoxicity assays it was classified as a non-genotoxic carcinogen. In order to assess the potential for liver tumor formation in humans, we analyzed treatment-induced changes in vivo to establish a possible mode of action (MoA). In vivo and in vitro gene expression analysis revealed that nuclear receptor signaling was unlikely to be the relevant MoA and no other known mechanism could be established. We therefore took an approach comparing phenotypic markers, including mRNA changes, proliferation and glycogen accumulation, in vitro using cells of different species to assess the human relevance of this finding. Since the alterations observed in rats were not seen in the liver of mice or dogs in vivo, we could validate the relevance of the cell models chosen by use of hepatocytes from these species in vitro. This ultimately allowed for a cross-species comparison, which suggested that the formation of FAH and liver tumors was rat specific and unlikely to translate to human. Our work showed that phenotypic species comparison in vitro is a useful approach for assessment of the human relevance of pre-clinical findings where no known mechanism can be established.

Essential Oil of Amomum maximum Roxb. and Its Bioactivities against Two Stored-Product Insects.

Amomum maximum Roxb. is a perennial herb distributed in South China and Southeast Asia. The objective of this work was to analyze the chemical constituents and assess insecticidal and repellent activities of the essential oil from Amomum maximum fruits against Tribolium castaneum (Herbst) and Liposcelis bostrychophila (Badonnel). The essential oil was obtained by hydrodistillation and analyzed by gas chromatography-flame ionization detector and gas chromatography-mass spectrometry. The main components of the essential oil were identified to be ß-pinene (23.39%), ß-caryophyllene (16.43%), α-pinene (7.55%), sylvestrene (6.61%) and ç-cadinene (4.19%). It was found that the essential oil of A. maximum fruits possessed contact and fumigant toxicities against T. castaneum adults (LD50 = 29.57 µg/adult and LC(50) = 23.09 mg/L air, respectively) and showed contact toxicity against L. bostrychophila (LD(50) = 67.46 µg/cm(2)). Repellency of the crude oil was also evaluated. After 2 h treatment, the essential oil possessed 100% repellency at 78.63 nL/cm(2) against T. castaneum and 84% repellency at 63.17 nL/cm(2) against L. bostrychophila. The results indicated that the essential oil of A. maximum fruits had the potential to be developed as a natural insecticide and repellent for control of T. castaneum and L. bostrychophila.

Bioactivity of essential oil from Artemisia stolonifera (Maxim.) Komar. and its main compounds against two stored-product insects.

Artemisia stolonifera, a perennial herb, is widely distrbuted in China. The aim of this study was to analyze the essential oil from the aerial parts of Artemisia stolonifera, as well as to evaluate the bioactivity of the oil and its main constituents. The essential oil was analyzed by gas chromatography-flame ionization detector and gas chromatography-mass spectrometry that allowed characterizing 22 compounds. The main components were eucalyptol (32.93%), ß-pinene (8.18%), camphor (6.12%) and terpinen-4-ol (6.11%), and obtained from the essential oil after a further isolation. During the contact toxicity tests, the essential oil (LD50 = 8.60 µg/adult) exhibited stronger toxicity against Tribolium castaneum adults than those isolated constituents, however, camphor and terpinen-4-ol showed 1 and 2 times toxicity against Lasioderma serricorne adults than the essential oil (LD50 = 12.68 µg/adult) with LD50 values of 11.30 and 5.42 µg/adult, respectively. In the fumigant toxicity tests, especially on Tribolium castaneum, the essential oil (LC50 = 1.86 mg/L air) showed almost the same level toxicity as positive control, methyl bromide (LC50 = 1.75 mg/L air). Moreover, the essential oil and its four isolated constituents also exhibited strong repellency against two stored-product insects.

Estimation of in vivo dose of dermally applied chemicals leading to estrogen/androgen receptor-mediated toxicity from in vitro data--Illustration with four reproductive toxicants.

We present a quantitative in vitro-in vivo extrapolation framework enabling the estimation of the external dermal exposure dose from in vitro experimental data relevant to a toxicity pathway of interest. The framework adapts elements of the biological pathway altering dose (BPAD) method [Judson et al. Chem Res Toxicol 2011;24:451] to the case of dermal exposure. Dermal doses of four toxicants equivalent to concentrations characterizing their effect on estrogen receptor α or androgen receptor activity in chemical-activated luciferase expression (CALUX) assays are estimated. The analysis shows that dermal BPADs, calculated from one in vitro concentration, can differ by up to a factor of 55, due to the impact applied dose and dermal exposure scenarios can have on skin permeation kinetics. These features should therefore be taken into account in risk assessment of dermally applied chemicals.

New quaternary ammonium camphor derivatives and their antiviral activity, genotoxic effects and cytotoxicity.

The synthesis and biological evaluation of a novel series of dimeric camphor derivatives are described. The resulting compounds were studied for their antiviral activity, cyto- and genotoxicity. Compounds 3a and 3d in which the quaternary nitrogen atoms are separated by the C5H10 and С9H18 aliphatic chain, exhibited the highest efficiency as an agent inhibiting the reproduction of the influenza virus A(H1N1)pdm09. The cytotoxicity data of compounds 3 and 4 revealed their moderate activity against malignant cell lines; compound 3f had the highest activity for the CEM-13 cells. These results show close agreement with the data of independent studies on toxicity of these compounds, in particular that the toxicity of compounds strongly depends on spacer length.

Ferutinin induces in vitro eryptosis/erythroptosis in human erythrocytes through membrane permeabilization and calcium influx.

Ferutinin, isolated from the root of Ferula hermonis and proposed to be used as an antiosteoporosis phytoestrogen, has death promoting activities in a number of cancer cells. However, the effect of ferutinin on the induction of apoptosis in human red blood cells (RBCs), also known as eryptosis or erythroptosis, remains unclear. Given that ferutinin is a small molecule that can induce apoptosis in the cancer cells by opening the mitochondrial permeability transition pores, we therefore hypothesized that the effect of ferutinin to elicit apoptosis in human RBCs devoid of mitochondria would be minimal. This study tried to determine the in vitro effect of ferutinin on the induction of apoptosis in human RBCs. Eryptosis/erythroptosis after ferutinin treatment was examined for phosphatidylserine (PS) externalization, calcein leakage, and other apoptotic feature events by flow cytometry and confocal microscopy. Contrary to our prediction, ferutinin caused eryptosis/erythroptosis in human RBCs and simultaneously increased caspase-3 activity and the cytosolic free Ca(2+) ion level ([Ca(2+)]i). Yet, Ca(2+) seems not to be the sole mediator in ferutinin-mediated eryptosis/erythroptosis because depletion of the external Ca(2+) could not eliminate the apoptotic effect from ferutinin. Subsequent replenishment of the external Ca(2+) was able to promote PS externalization, caspase-3 activation, and rise of [Ca(2+)]i. Also, ferutinin at high dose (40 µM or above) was able to permeabilize the membrane of RBC ghosts in a way similar to that of digitonin. At low dose, ferutinin activated the P- and L-type Ca(2+) channels as the ferutinin-mediated [Ca(2+)]i rise was suppressed by the P-type (ω-agatoxin IVA) and L-type (verapamil and diltiazem) Ca(2+) channel blockers. Taken together, we report here for the first time that ferutinin induces in vitro apoptosis in human RBCs. Mechanistically, eryptosis/erythroptosis is mediated by membrane permeabilization and upregulation of [Ca(2+)]i with the activation of caspase-3.

Ultraviolet degradation of procymidone--structural characterization by gas chromatography coupled with mass spectrometry and potential toxicity of photoproducts using in silico tests.

RATIONALE: Procymidone is a dicarboximide fungicide mainly used for vineyard protection but also for different crops. The structural elucidation of by-products arising from the UV-visible photodegradation of procymidone has been investigated by gas chromatography coupled with mass spectrometry. The potential toxicities of photoproducts were estimated by in silico tests. METHODS: Aqueous solutions of procymidone were irradiated for up to 90 min in a self-made reactor equipped with a mercury lamp. Analyses were carried out on a gas chromatograph coupled with an ion trap mass spectrometer operated in electron ionization and methanol positive chemical ionization. Multistage collision-induced dissociation (CID) experiments were performed to establish dissociation pathways of ions. Toxicities of byproducts were estimated using the QSAR T.E.S.T. program. RESULTS: Sixteen photoproducts were investigated. Chemical structures were proposed mainly based on the interpretation of multistage CID experiments, but also on their relative retention times and kinetics data. These structures enabled photodegradation pathways to be suggested. Only three photoproducts remain present after 90 min of irradiation. Among them, 3,5-dichloroaniline presents a predicted rat LD50 toxicity about ten times greater than that of procymidone. CONCLUSIONS: 3,5-Dichloroaniline is the only photoproduct reported in previous articles. Eight by-products among the sixteen characterized might be as toxic, if not more, than procymidone itself considering the QSAR-predicted rat LD50.

Persistent developmental toxicity in rat offspring after low dose exposure to a mixture of endocrine disrupting pesticides.

There is growing concern of permanent damage to the endocrine and nervous systems after developmental exposure to endocrine disrupting chemicals. In this study the permanent reproductive and neurobehavioral effects of combined exposure to five endocrine disrupting pesticides, epoxiconazole, mancozeb, prochloraz, tebuconazole and procymidone, were examined. Pregnant and lactating rat dams were dosed with a mixture of the five pesticides at three different doses, or with the individual pesticides at one of two doses. Adverse effects were observed in young and adult male offspring from the group exposed to the highest dose of the mixture. These included reduced prostate and epididymis weights, increased testes weights, altered prostate histopathology, increased density of mammary glands, reduced sperm counts, and decreased spatial learning. As no significant effects were seen following single compound exposure at the doses included in the highest mixture dose, these results indicate cumulative adverse effects of the pesticide mixture.

The sesquiterpene botrydial produced by Botrytis cinerea induces the hypersensitive response on plant tissues and its action is modulated by salicylic acid and jasmonic acid signaling.

Botrytis cinerea, as a necrotrophic fungus, kills host tissues and feeds on the remains. This fungus is able to induce the hypersensitive response (HR) on its hosts, thus taking advantage on the host's defense machinery for generating necrotic tissues. However, the identity of HR effectors produced by B. cinerea is not clear. The aim of this work was to determine whether botrydial, a phytotoxic sesquiterpene produced by B. cinerea, is able to induce the HR on plant hosts, using Arabidopsis thaliana as a model. Botrydial induced the expression of the HR marker HSR3, callose deposition, and the accumulation of reactive oxygen species and phenolic compounds. Botrydial also induced the expression of PR1 and PDF1.2, two pathogenesis-related proteins involved in defense responses regulated by salicylic acid (SA) and jasmonic acid (JA), respectively. A. thaliana and tobacco plants defective in SA signaling were more resistant to botrydial than wild-type plants, as opposed to A. thaliana plants defective in JA signaling, which were more sensitive. It can be concluded that botrydial induces the HR on its hosts and its effects are modulated by host signaling pathways mediated by SA and JA.

An integrated drug-likeness study for bicyclic privileged structures: from physicochemical properties to in vitro ADME properties.

The concept of drug-likeness has been widely applied in combinatorial chemistry as an approach to reduce attrition in drug discovery and development. Meanwhile, bicyclic privileged structures with versatile binding properties have emerged as ideal source of core scaffolds for the design and synthesis of combinatorial libraries. For the purpose of better assisting the design of bicyclic privileged structure-based combinatorial libraries, we conducted an integrated drug-likeness study on compounds of these scaffolds. Distributions of physicochemical properties (PCPs) were analyzed and in silico prediction models were built. Our results showed that there exist much difference between the drug-like ranges (DLRs) of bicyclic privileged structures and that of others, which have significant impact on compound selection. The DLRs for bicyclic privileged structures were defined as 260 ≤ MW ≤ 524; 0.9 ≤ ALogP ≤ 5.4; 2 ≤ Hacc ≤ 8; Hdon ≤ 3; 21.0 ≤ PSA ≤ 128.6; 6.3 ≤ FPSA ≤ 34.2; 1 ≤ RotB ≤ 10; 2 ≤ Nr ≤ 5; 1 ≤ Nc ≤ 7; SA ≤ 4. Two accurate and easy to understand in silico prediction models, Caco-2 permeability model and metabolic stability classification model, had been built to guide drug candidate optimization. In these models, hydrogen-bond donor and rotatable bond showed major impact on the permeability of compounds, while lipophilicity, flexibility, degree of branching and the existence of some functional groups determined the fate of a drug in metabolic process. Suggestions on structural modification toward higher permeability and metabolic stability were given according to the in silico models.

Combined exposure to endocrine disrupting pesticides impairs parturition, causes pup mortality and affects sexual differentiation in rats.

Risk assessment is currently based on the no observed adverse effect levels (NOAELs) for single compounds. Humans are exposed to a mixture of chemicals and recent studies in our laboratory have shown that combined exposure to endocrine disrupters can cause adverse effects on male sexual development, even though the doses of the single compounds are below their individual NOAELs for anti-androgenic effects. Consequently, we have initiated a large project where the purpose is to study mixture effects of endocrine disrupting pesticides at low doses. In the initial range-finding mixture studies, rats were gavaged during gestation and lactation with five doses of a mixture of the fungicides procymidone, mancozeb, epoxyconazole, tebuconazole and prochloraz. The mixture ratio was chosen according to the doses of each individual pesticide that produced no observable effects on pregnancy length and pup survival in our laboratory and the dose levels used ranged from 25 to 100% of this mixture. All dose levels caused increased gestation length and dose levels above 25% caused impaired parturition leading to markedly decreased number of live born offspring and high pup perinatal mortality. The sexual differentiation of the pups was affected at 25% and higher as anogenital distance was affected in both male and female offspring at birth and the male offspring exhibited malformations of the genital tubercle, increased nipple retention, and decreased prostate and epididymis weights at pup day 13. The results show that doses of endocrine disrupting pesticides, which appear to induce no effects on gestation length, parturition and pup mortality when judged on their own, induced marked adverse effects on these endpoints in concert with other pesticides. In addition, the sexual differentiation of the offspring was affected. This as well as the predictability of the combination effects based on dose-additivity modelling will be studied further in a large dose-response study.

Cytotoxicity and genotoxicity in human lung epithelial A549 cells caused by airborne volatile organic compounds emitted from pine wood and oriented strand boards.

Due to the massive reduction of air-change rates in modern, energy-saving houses and dwellings, the contribution of volatile organic compound (VOCs) emissions from wood-based materials to indoor air quality has become increasingly important. To evaluate toxicity of VOC mixtures typically emitted from pine wood and oriented strand boards (OSB) and their main constituents (selected terpenes and aldehydes), cytotoxicity and genotoxicity were investigated in human A549 lung cells. To facilitate exposure directly via gas phase, a 250 L emission chamber was combined with a Vitrocell exposure system. VOC exposure concentrations were measured by GC/MSD. Biological effects were determined after an exposure time of 1h by measuring cytotoxicity (erythrosine B staining) and genotoxicity (comet assay). Neither cytotoxic nor genotoxic effects were observed for VOC mixtures emitted from pine wood or OSB at loading factors of approximately 13 m(2)/m(3) (worst case conditions) of the panels (with maximum VOC levels of about 80 mg/m(3)) in comparison to clean air. While alpha-pinene and Delta(3)-carene did not induce toxic effects even at exposure concentrations of up to 1800 mg/m(3) and 600 mg/m(3), respectively, hexanal showed a cytotoxic effect at 2000 mg/m(3). The alpha,beta-unsaturated aldehydes 2-heptenal and 2-octenal caused genotoxic effects in concentrations exceeding 100mg/m(3) and 40 mg/m(3), respectively. In conclusion, high concentrations of VOCs and VOC mixtures emitted from pine wood and OSB did not lead to adverse effects in A549 human lung cells even at concentrations 10(2) to 10(5)-fold higher than those found in normal indoor air. Attention must be paid to mutagenic and possibly carcinogenic alpha,beta-unsaturated aldehydes.

Mixture effects of endocrine disrupting compounds in vitro.

Four different equi-molar mixtures were investigated for additive endocrine disrupting effects in vitro using the concentration addition model. It was found that additive effects on the same molecular target (the androgen receptor; AR) can be predicted for both mixtures of compounds with effect on the AR (flutamide, procymidone and vinclozolin) and of compounds with and without effects on the AR [finasteride, mono-(2-ethylhexyl) phthalate, prochloraz and vinclozolin]. For a paraben mixture (methyl paraben, ethyl paraben, propyl paraben, butyl paraben and iso-butyl paraben) antagonistic effect on AR could not be predicted under assumption of additivity in our model system. For a mixture containing three azole fungicides (epoxiconazole, propiconazole and tebuconazole), the observed AR antagonistic effects were close to the predicted effect assuming additivity. Azole fungicides are known inhibitors of androgen biosynthesis and in the steroid synthesis assay using H295R cells, the inhibition of testosterone production was close to additive, whereas the inhibition of oestradiol production was over-estimated for the mixture of azole fungicides, when compared with the effect predicted when assuming additivity. Overall these and other studies show that weak endocrine disrupting compounds, like parabens and azole fungicides, give rise to combination effects when they occur in mixtures. These combination effects should be taken into account in regulatory risk assessment not to under-estimate the risks for adverse effects associated with exposure to disrupting chemicals.

Probabilistic cumulative risk assessment of anti-androgenic pesticides in food.

In this paper, we present a cumulative risk assessment of three anti-androgenic pesticides (vinclozolin, procymidone and prochloraz) using the relative potency factor (RPF) approach and an integrated probabilistic risk assessment (IPRA) model. RPFs for each substance were estimated for three reproductive endpoints (ano-genital distance, and weights of the seminal vesicles and the musculus levator ani/bulbocavernosus) in male rat foetuses exposed in utero. The cumulative dietary intake was estimated based on consumption data and residue data from the Netherlands. The IPRA model combines variability in both exposure and sensitivity between individuals into a distribution of individual margins of exposures (IMoEs) and IMoEs of 1 or less indicate a possible concern. The assessment did not result in IMoEs < or = 1. The endpoint 'weight of seminal vesicles' resulted in the lowest IMoEs (0.1th percentile: 198) and the fraction of individuals with IMoEs<1000 was 1.43%. For the two other endpoints, the fractions were slightly lower. Thus, cumulative dietary exposure of Dutch women to vinclozolin, procymidone and prochloraz is not likely to be of concern for the reproductive development of their male foetuses. However, other anti-androgenic substances and exposure routes should also be included in the cumulative assessment to make it more comprehensive.

Rational design, synthesis and characterization of potent, non-peptidic Smac mimics/XIAP inhibitors as proapoptotic agents for cancer therapy.

Novel proapoptotic Smac mimics/IAPs inhibitors have been designed, synthesized and characterized. Computational models and structural studies (crystallography, NMR) have elucidated the SAR of this class of inhibitors, and have permitted further optimization of their properties. In vitro characterization (XIAP BIR3 and linker-BIR2-BIR3 binding, cytotox assays, early ADMET profiling) of the compounds has been performed, identifying one lead for further in vitro and in vivo evaluation.

The essential oil of turpentine and its major volatile fraction (alpha- and beta-pinenes): a review.

This paper provides a summary review of the major biological features concerning the essential oil of turpentine, its origin and use in traditional and modern medicine. More precisely, the safety of this volatile fraction to human health, and the medical, biological and environmental effects of the two major compounds of this fraction (alpha- and beta-pinenes) have been discussed.

Estrogenic activity of procymidone in rainbow trout (Oncorhynchus mykiss) hepatocytes: a possible mechanism of action.

It is known that procymidone modifies sexual differentiation in vivo and in vitro, and that it induces vitellogenin (Vtg) synthesis in primary cultured rainbow trout hepatocytes. The aim of this study was to evaluate the mechanism underlying this latter in vitro estrogenic action. The cells were treated for 24 h with procymidone 150 microM (with 17beta-estradiol [E2] 20 microM as a positive control) combined with an estrogen receptor (ER) antagonist (tamoxifen 20 microM or ICI 182,780 1 microM) or, given the drug toxic action on the production of reactive oxygen species (ROS), a free radical scavenger (alpha-tocopherol 30 microM). The results from ELISA experiments provided evidence that procymidone Vtg-induction is inhibited by ER antagonists and by alpha-tocopherol suggesting that both ER and ROS are involved in this effect. The ROS detection revealed that the treatment with alpha-tocopherol and tamoxifen completely prevented ROS induction by procymidone, that was not inhibited by ICI 182,780. In exploring the mechanism mediating these events and its timing, we found that procymidone induced mitogen-activated protein kinase (MAPK) at 30 and 60 min, and that this effect was blocked by co-treatment with alpha-tocopherol. In summary, the results of the study clearly support the idea that the estrogenic activity of procymidone in primary cultured trout hepatocytes is mediated by ROS production, and that this activity is similar to that of the ligand-independent ER activation involving MAPK.