Validation of the REA bioassay to detect estrogenic activity in the water cycle

Endocrine disrupting compounds (EDCs) with estrogenic potency contaminate water and might eventually cause adverse effects to the aquatic environment. Many estrogenic compounds are not completely removed by wastewater treatment systems and, together with the run-off from agricultural areas, they enter surface waters. Chemical analytical methods to determine these compounds are usually expensive and laborious. Therefore, screening bioassays which are able to detect compounds based on their effects offer a solution for prior selection of samples that need to be chemically analyzed. In this study, the REA (RIKILT yeast Estrogen bioAssay), which has been developed to detect estrogenic compounds in calf urine and animal feed at RIKILT, is validated at the Water Board Laboratory of Waterproef for water samples. According to EC Decision 2002/657, detection capability CCβ, specificity and stability have to be determined for the internal validation of a qualitative screening test. In addition, surface water and effluent samples were analyzed to further demonstrate the applicability of the validated test procedure. Results demonstrate that the REA assay is reproducible and specific for estrogenic compounds in water and meets the criteria as prescribed in EC Decision 2002/657. The assay was sensitive enough to detect estrogenic activity of pollutants in water with a limit of quantification (LOQ) below 1 ng EEQ/L. This means that samples can be compared with preliminary threshold levels for drinking water and surface waters (7 and 1 ng EEQ/L, respectively). The stability of estrogenic activity in water samples is at least 4 weeks, when stored at 4 °C.     

Evaluation of estrogenic activity in animal diets using in vitro assay

A yeast estrogen bioassay (RIKILT REA) was in-house validated for feed on the 5 μg 17β-estradiol-equivalents per kg level according to EC Decision 2002/657/EC. All the performance characteristics met the criteria as defined in the Decision and the REA is able to detect 17β-estradiol in animal feed at a low level of 1.15–2 μg kg⁻¹. Subsequently, the developed and validated procedure was applied to determine the estrogenic activity in 24 feed samples intended for food producing animals, pets and laboratory animals. Two batches of rodent diet Murigran and one dog feed have been presented as a suspect, i.e. gave responses above the determined decision limit (CCα) and detection capability (CCβ). In assessing the performance of the estrogenic activity in these diets evaluated by comparison with the 17β-estradiol calibration curve, 17β-estradiol-equivalence levels of 7.07 μg EEQ kg⁻¹ and 9.54 μg EEQ kg⁻¹ in two batches of rodent diet and 5.3 μg EEQ kg⁻¹ in dog feed have been established. The activities observed in the rodent feed could be explained by chemical analysis, revealing high amounts of genistein, daidzein and trace amounts of zearalenone. In addition, the estrogenic activity in one of rodent feed was above the established CCα, but below the CCβ values established and all other samples showed no estrogenic activity with responses below the CCα value, which corresponds to levels below 2 μg EEQ kg⁻¹.     

Ipso substitution of bisphenol A catalyzed by microsomal cytochrome P450 and enhancement of estrogenic activity

Bisphenol A (BPA), an industrial chemical with estrogenic activity, was investigated as a substrate for the ipso-metabolism catalyzed by microsomal cytochrome P450 (P450). BPA was expected to be transformed to a quinol via an ipso-addition reaction; however, hydroquinone (HQ) was detected as a metabolite via an ipso-substitution reaction. Isopropenylphenol (IPP) and hydroxycumyl alcohol (HCA) were also produced as eliminated metabolites by C-C bond scission via ipso-substitution. Incorporation of the ¹⁸O atom to HCA from H₂¹⁸O suggested the presence of a carbocation intermediate. Bulkiness of p-substituted group of BPA and/or stability of the eliminated carbocation intermediate may cause ipso-substitution of BPA. CYP3A4 and CYP3A5 showed higher activity for ipso-substitution. CYP2D6*1 also showed the activity; however, the other 9 isozymes did not. IPP showed ER-binding activity in the same degree of BPA. Furthermore, the ER-binding activity of HCA was about a hundred times greater than that of BPA. These results suggested that this new metabolic pathway contributes to the activation of the estrogenic activity of BPA.     

Estrogenic effects of 17 beta-aminoestrogens assessed in uteri of rats and mice

Administration of exogenous estrogens has been associated with an increase of thromboembolic events. The 17β-aminoestrogens produce anticoagulant effects contrasting with the procoagulant effects of the natural occurring estradiol in rodents. This work compares the estrogenic effects induced by 17β-aminoestrogens prolame, butolame, pentolame, and estradiol in vivo models. Dose–response curves were performed using immature CD1 mice and Wistar rats. The animals were injected with estradiol or 17β-aminoestrogens (0.01 to 1000 μg/kg), or vehicle. The uterine wet and dry weights were determined. The 17β-aminoestrogens increased uterine weight in a dose-dependent manner. The uterotrophic effect produced by estradiol induced lower ED₅₀ (6.5 and 4 μg/kg) and higher E_max values (+523–350%) in mice as compared with those from the rat, indicating more susceptibility of the mice model. The 17β-aminoestrogens are partial estrogenic agonists with a relative uterotrophic effect of estradiol (100%) from 9–86%. Only the ED₅₀ values of 17β-aminoestrogens in CD1 mice showed a direct correlation to the length of the amine group substitution in C-17 since their efficacy and potency were in the order: prolame>butolame>pentolame.     

Estrogenic and anti-androgenic activities of 4-nitrophenol in diesel exhaust particles

A 4-nitrophenol (PNP) isolated from diesel exhaust particles (DEP) has been identified as a vasodilator. PNP is also a known degradation product of the insecticide parathion. We used uterotrophic and Hershberger assays to study the estrogenic and anti-androgenic activities of PNP in-vivo. In ovariectomized immature female rats injected subcutaneously with 1, 10, or 100 mg/kg PNP daily for 7 days, significant (P<0.05) increases in uterine weight were seen in only those receiving 10 or 100 mg/kg PNP. Furthermore, in castrated immature male rats implanted with a silastic tube (length, 5 mm) containing crystalline testosterone and injected subcutaneously with 0.01, 0.1, or 1 mg/kg PNP daily for 5 days, those receiving the doses of 0.1 mg/kg showed significant (P<0.05) weight decreases in seminal vesicles, ventral prostate, levator ani plus bulbocavernosus muscles, and glans penis. Plasma FSH and LH levels did not change in female rats but were significantly (P<0.05) increased in male rats treated with 0.1 mg/kg PNP. These results clearly demonstrated that PNP has estrogenic and anti-androgenic activities in-vivo. Our results therefore suggest that diesel exhaust emissions and the degradation of parathion can lead to accumulation of PNP in air, water, and soil and thus could have serious deleterious effects on wildlife and human health.     

In-vitro antiproliferative activity of benzopyranone derivatives in comparison with standard chemotherapeutic drugs

The cytotoxic activities of five new benzopyranone derivatives containing basic amino side chain are described. Their cytotoxicities against ER(+) MCF‐7 and ER(–) MDA‐MB‐231 human breast cancer cell lines, and Ishikawa human endometrial cell line were determined after 72 h drug exposure employing CellTiter‐Glo assay at concentrations ranging from 0.01–1.0 × 10⁵ nM. The antiproliferative activities of these compounds were compared to tamoxifen (TAM), 4‐hydroxytamoxifen (4‐OHT, active metabolite of tamoxifen), and raloxifene (RAL). In‐vitro results indicated that compounds 9 , 10 , 12 , and 13 were more potent than TAM against the human breast cancer cell lines with IC₅₀ < 20 µM. The in‐silico structure–activity relationships of these compounds and their binding mode within the estrogen receptor (ER) binding site using AutoDock vina are discussed.     

The environmental chemical tributyltin chloride (TBT) shows both estrogenic and adipogenic activities in mice which might depend on the exposure dose

Exposure during early development to chemicals with hormonal action may be associated with weight gain during adulthood because of altered body homeostasis. It is known that organotins affect adipose mass when exposure occurs during fetal development, although no knowledge of effects are available for exposures after birth. Here we show that the environmental organotin tributyltin chloride (TBT) exerts adipogenic action when peripubertal and sexually mature mice are exposed to the chemical. The duration and extent of these effects depend on the sex and on the dose of the compound, and the effects are relevant at doses close to the estimated human intake (0.5 μg/kg). At higher doses (50-500 μg/kg), TBT also activated estrogen receptors (ERs) in adipose cells in vitro and in vivo, based on results from acute and longitudinal studies in ERE/luciferase reporter mice. In 3T3-L1 cells (which have no ERs), transiently transfected with the ERE-dependent reporter plus or minus ERα or ERβ, TBT (in a dose range of 1-100 nM) directly targets each ER subtype in a receptor-specific manner through a direct mechanism mediated by ERα in undifferentiated preadipocytic cells and by ERβ in differentiating adipocytes. The ER antagonist ICI-182,780 inhibits this effect. In summary, the results of this work suggest that TBT is adipogenic at all ages and in both sexes and that it might be an ER activator in fat cells. These findings might help to resolve the apparent paradox of an adipogenic chemical being also an estrogen receptor activator by showing that the two apparently opposite actions are separated by the different doses to which the organism is exposed.     

Development of a rapid screening and surveillance for estrogenic chemicals in environment based on recombinant yEGFP yeast cell

A recombinant yEGFP gene yeast strain necessary for the routine screening of estrogen activity in the chemical product of the environment was described. Two plasmids, one containing human estrogen receptor (hER) cDNA fused to the GPD gene promoter and another yEGFP inserted under the control of ERE, were constructed. The use of hER cDNA and the yEGFP reporter in the yeast cell sensor resulted in estrogenic chemical product-dependent light emission of yEGFP without additions owing its advantages: a simple and reagent-free measurement of GFP, and a non-toxic protein characteristic. The time needed for the optimal induction of light emission was 4 h. The maximal fold induction of 8.80 over uninduced levels at the concentration of 10⁻⁵ M of bisphenol A and 0.1 nM sensitivities for four different estrogenic chemicals tested were obtained. Five different chemicals which could not bind to hER did not cause an induction of yEGFP. This bioassay can be performed completely in 96-well plates. Thus, this test system can be used as a rapid screening system for the surveillance of estrogenic chemical products in the environment. The yEGFP assay is sensitive, reproducible, and cheap, which makes it highly suitable to be used as a high throughput system.     

In vitro metabolism of methiocarb and carbaryl in rats,and its effect on their estrogenic and antiandrogenic activities

In this work, we examined the metabolism of the carbamate insecticides methiocarb and carbaryl by rat liver microsomes and plasma, and its effect on their endocrine-disrupting activities. Methiocarb and carbaryl were not enzymatically hydrolyzed by rat liver microsomes, but were hydrolyzed by rat plasma, mainly to methylthio-3,5-xylenol (MX) and 1-naphthol, respectively. When methiocarb was incubated with rat liver microsomes in the presence of NADPH, methiocarb sulfoxide was formed. The hydrolysis product, MX, was also oxidized to the sulfoxide, 3,5-dimethyl-4-(methylsulfinyl)phenol (SP), by rat liver microsomes in the presence of NADPH. These oxidase activities were catalyzed by cytochrome P450 and flavin-containing monooxygenase. Methiocarb and carbaryl both exhibited estrogen receptor α (ERα) and ERβ agonistic activity. MX and 1-naphthol showed similar activities, but methiocarb sulfoxide and SP showed markedly decreased activities. On the other hand, methiocarb and carbaryl exhibited potent antiandrogenic activity in the concentration range of 1 × 10⁻⁶–3 × 10⁻⁵ M. Their hydrolysis products, MX, and 1-naphthol also showed high activity, equivalent to that of flutamide. However, methiocarb sulfoxide and SP showed relatively low activity. Thus, hydrolysis of methiocarb and carbaryl and oxidation of methiocarb to the sulfoxide markedly modified the estrogenic and antiandrogenic activities of methiocarb and carbaryl.     

Evaluation of estrogenic activity of organic biocides using ER-binding and YES assay.

Biocides have been used not only in everyday items such as clothes, kitchenware, daily necessities, and infant utensils, but also in cosmetics and wrapping papers for foodstuffs. Since there is a high possibility of exposure to biocides from such materials, their safety must be assessed adequately using a range of methods. We investigated the estrogenic activity of 20 organic biocides using two in vitro screen assays: estrogen receptor (ER) binding assay and yeast estrogen screen (YES) assay. Twelve of the biocides were positive in the ER-binding assay. Regardless of the presence or absence of rat S9Mix for metabolic activation, 4-chloro-3-methylphenol was positive in both ER-binding and YES assay. 4-Chloro-3,5-dimethylphenol was positive in the ER-binding assay and showed a pseudopositive manner in the YES assay that was observed the dose-independent estrogenic activity at only one dose point. Hiba oil was positive in the ER-binding assay but was positive in the YES assay only in the presence of rat S9Mix. These results suggest that ER-binding and YES assay could be adapted for evaluation of the endocrine-disrupting activity of biocides. The biocides found to be positive in vitro now require assessment by in vivo screening methods.     

Characterization of estrogenic and androgenic activity of phthalates by the XenoScreen YES/YAS in vitro assay

The presented study investigates and compares the estrogenic and androgenic activities of commonly used diesters of phthalic acid (phthalates) using the XenoScreen YES/YAS assay. Phthalates are commonly used plasticizers in polymers dedicated for i.e. food and drug containers. Since phthalates are not chemically bonded to the polymer, they can leach or migrate from the polymer. Therefore, phthalates are identified as contaminants in a variety of consumer products. Investigation of estrogenic and androgenic activities of phthalates (DEP, DBP, BBP, DEHP and DINP) showed no significant effect of tested substances either on hERα or hAR receptors. Phthalates exhibited strong anti-estrogenic (IC₅₀ for BBP = 8.66 μM, IC₅₀ for DEHP = 3.61 μM and IC₅₀ for DINP = 0.065 μM) and anti-androgenic (IC₅₀ for BBP = 5.30 μM, IC₅₀ for DEHP = 2.87 μM and IC₅₀ for DINP = 0.068 μM) activities.     

Male reproductive toxicity of four bisphenol antioxidants in mice and rats and their estrogenic effect

Male mice and rats were fed a diet containing four bisphenol antioxidants, 2,2′-methylenebis(4-ethyl-6-tert-butylphenol) (ME), 2,2′-methylenebis(4-methyl-6-tert-butylphenol) (MM), 4,4′-butylidenebis(3-methyl-6-tert-butylphenol) (BM), or 4,4′-thiobis(3-methyl-6-tert-butylphenol) (TM) at levels of 0.06–0.25% for 2 months. BM and TM decreased epididymal, seminal vesicular, prostate and preputial weights, and injured seminiferous tubules in mice in a dose-dependent fashion. BM and TM also reduced sex accessory organ weights and sperm production capacity in rats, but MM and ME were more toxic to rats than BM and TM. ME and MM did not bind ER_α up to 10⁻³ M, while BM and TM competitively bound ER_α against β-estradiol (E2). Fifty percent inhibitory concentrations (IC₅₀ s) of BM, TM, and bisphenol A (positive control) against E2-binding were 7.3×10⁻⁶ M, 1.8×10⁻⁵ M, and 1.4×10⁻⁵ M, respectively. When ovariectomized (OVX) mice were sc administered TM at doses of 60 and 300 mg/kg/day for 4 days, or when OVX mice were fed BM in the diet at a level of 0.25% for 2 months, uterine weight was significantly increased. These results suggest that BM and TM are weakly toxic, possibly through an estrogenic mechanism to male reproductive organs in mice as well as rats, while MM and ME may be the direct testicular toxins in rats but not mice. A part of this study was presented at the 74th annual meeting of the Japanese Society for Hygiene held from 24–27 March 2004 in Tokyo, Japan.     

Evaluation of the stereoselective biotransformation of permethrin in human liver microsomes: Contributions of cytochrome P450 monooxygenases to the formation of estrogenic metabolites

Permethrin (PM) is a pyrethroid insecticide that exists as 4 enantiomers. Biotransformation of PM to estrogen receptor agonists (3-phenoxybenzyl alcohol (PBOH) and 3-(4′-hydroxyphenoxy)-benzyl alcohol (3,4 PBOH)) has been shown to be stereoselective in other vertebrate species. This study evaluated the biotransformation of PM enantiomers in human liver microsomes and with recombinant CYP3A4 and CYP2C19. PBOH and 3,4 PBOH were the only metabolites detected from in vitro incubations including each of the 4 enantiomers of PM with 1R-trans PM having the most efficient NADPH-catalyzed biotransformation to both metabolites. Coincubation with the CYP inhibitor ketoconazole and time course experiments with liver microsomes and recombinant CYP2C19 and CYP3A4 indicated CYP-catalyzed stereoselective cleavage of the ester followed by 4-hydoxylation to 3,4′ PBOH. These data indicate potential dispositional differences may occur with PM enantiomers and a shift in putative molecular targets. While cleavage of pyrethroid esters lead to detoxification of the acute neurological effects, formation of the benzyl alcohol and hydroxylated metabolite may lead to estrogenic responses, since each of these metabolites are estrogen receptor ligands.     

Central estrogenic pathways protect against the depressant action of acute nicotine on reflex tachycardia in female rats

We have previously shown that acute exposure of male rats to nicotine preferentially attenuates baroreceptor-mediated control of reflex tachycardia in contrast to no effect on reflex bradycardia. Here, we investigated whether female rats are as sensitive as their male counterparts to the baroreflex depressant effect of nicotine and whether this interaction is modulated by estrogen. Baroreflex curves relating reflex chronotropic responses evoked by i.v. doses (1-16 μg/kg) of phenylephrine (PE) or sodium nitroprusside (SNP), were constructed in conscious freely moving proestrus, ovariectomized (OVX), and estrogen (50 μg/kg/day s.c., 5 days)-replaced OVX (OVXE₂) rats. Slopes of the curves were taken as a measure of baroreflex sensitivity (BRS_PE and BRS_SNP). Nicotine (100 μg/kg i.v.) reduced BRS_SNP in OVX rats but not in proestrus or OVXE₂ rats. The attenuation of reflex tachycardia by nicotine was also evident in diestrus rats, which exhibited plasma estrogen levels similar to those of OVX rats. BRS_PE was not affected by nicotine in all rat preparations. Experiments were then extended to determine whether central estrogenic receptors modulate the nicotine-BRS_SNP interaction. Intracisteral (i.c.) treatment of OVX rats with estrogen sulfate (0.2 μg/rat) abolished the BRS_SNP attenuating effect of i.v. nicotine. This protective effect of estrogen disappeared when OVX rats were pretreated with i.c. ICI 182,780 (50 μg/rat, selective estrogen receptor antagonist). Together, these findings suggest that central neural pools of estrogen receptors underlie the protection offered by E₂ against nicotine-induced baroreceptor dysfunction in female rats.     

Functional characterization of estrogen receptor subtypes, ERalpha and ERbeta, mediating vitellogenin production in the liver of rainbow trout

The estrogen-dependent process of vitellogenesis is a key function on oviparous fish reproduction and it has been widely used as an indicator of xenoestrogen exposure. The two estrogen receptor (ER) subtypes, ERalpha and ERbeta, are often co-expressed in the liver of fish. The relative contribution of each ER subtype to modulate vitellogenin production by hepatocytes was studied using selected compounds known to preferentially interact with specific ER subtypes: propyl-pyrazole-triol (PPT) an ERalpha selective agonist, methyl-piperidino-pyrazole (MPP) an ERalpha selective antagonist, and diarylpropionitrile (DPN) an ERbeta selective agonist. First, the relative binding affinity of the test compounds to estradiol for rainbow trout hepatic nuclear ER was determined using a competitive ligand binding assay. All the test ligands achieved complete displacement of specific [(3)H]-estradiol binding from the nuclear ER extract. This indicates that the test ligands have the potential to modify the ER function in the rainbow trout liver. Secondly, the ability of the test compounds to induce or inhibit vitellogenin production by primary cultures of rainbow trout hepatocytes was studied. Estradiol and DPN were the only compounds that induced a dose-dependent increase on vitellogenin synthesis. The lack of vitellogenin induction by PPT indicates that ERalpha could not have a role on this reproductive process whereas the ability of DPN to induce vitellogenin production supports the participation of ERbeta. In addition, this hypothesis is reinforced by the results obtained from MPP plus estradiol. On one hand, the absence of suppressive activity of MPP in the estradiol-induced vitellogenin production does not support the participation of ERalpha. On the other hand, once blocked ERalpha with MPP, the only manifestation of agonist activity of estradiol would be achieved via ERbeta. In conclusion, the present results indicate that vitellogenin production is mainly mediated through ERbeta, implying, furthermore that compounds which only exhibit ERalpha selectivity are not detected by vitellogenin bioassay.     

Chlordecone, a mixed pregnane X receptor (PXR) and estrogen receptor alpha (ERalpha) agonist, alters cholesterol homeostasis and lipoprotein metabolism in C57BL/6 mice

Chlordecone (CD) is one of many banned organochlorine (OC) insecticides that are widespread persistent organic pollutants. OC insecticides alter lipid homeostasis in rodents at doses that are not neurotoxic or carcinogenic. Pretreatment of mice or rats with CD altered tissue distribution of a subsequent dose of [¹⁴C]CD or [¹⁴C]cholesterol (CH). Nuclear receptors regulate expression of genes important in the homeostasis of CH and other lipids. In this study, we report that CD suppresses in vitro reporter systems for human liver X receptors (LXRs) and activates those for human farnesoid X receptor (FXR), pregnane X receptor (PXR) and estrogen receptor α (ERα) in a concentration-dependent manner (0-50 μM). Consistent with human PXR activation in vitro, three days after a single dose of CD (15 mg/kg) hepatic microsomal CYP3A11 protein increases in C57BL/6 mice. CD decreases hepatic CH ester content without altering total CH concentration. Apolipoprotein A-I (apoA-I) contents of hepatic lipoprotein-rich and microsomal fractions of CD-treated mice are higher than controls. There is a significant reduction in non-high density lipoprotein CH but not apolipoprotein B-48/100 (apoB-48/100) in plasma from CD-treated mice after a 4 h fast. At 14 days after 15 mg CD/kg apoA-I and apoB-100 proteins but not CYP3A11 protein in hepatic microsomes are similar to controls. This work indicates that altered CH homeostasis is a mode of OC insecticide action of relevance after a single dose. This at least partially explains altered CH tissue distribution in CD-pretreated mice.     

A comparison of in vitro and in vivo EDSTAC test battery results for detecting antiandrogenic activity

The androgen receptor (AR) transactivation, binding, and Hershberger assays are being developed for large-scale screening of chemicals for endocrine activity. The goal of this study was to evaluate the correlation between in vitro and in vivo antiandrogenicity assays using a variety of compounds (p,p'-DDE, flutamide (FLUT), spironolactone, procymidone, RU486, methoxychlor (MXC), benzo(a)pyrene (BAP), and selected metabolites). For the AR transactivation assay, AR(+) LNCaP prostate carcinoma cells were transfected with an inducible luciferase reporter construct (pGudLuc7ARE) and exposed for 24 h to test materials (< or = 10 microM) in the presence and absence of 1 nM of the AR agonist R-1881. Each of these materials, including the hydroxlated metabolites of BAP and MXC, produced significant antiandrogenic activity in vitro as evidenced by their inhibition of the response to R-1881. Similarly, in vitro AR binding experiments using the recombinant ligand-binding domain (LBD) of the human AR and fluorescence polarization (FP) methodology yielded IC50s comparable to that of testosterone for RU486 and 9-OH-BAP. Other parent compounds and metabolites exhibited lesser binding affinity. In vivo antiandrogenic activity was evaluated with the Hershberger assay, wherein castrated male CD rats were dosed by gavage for 10 days with (mg/kg per day): MXC (10, 50, 100, and 200), BAP (1, 10, 50, and 100), RU486 (1, 5, 10, and 25), and FLUT (10) in the presence of 0.4 mg/kg per day (sc) of testosterone propionate (TP). Neither BAP nor MXC produced significant decreases in accessory sex tissue (AST) weights relative to TP control. However, 200 MXC resulted in a significant decrease in body weight and 100 BAP significantly increased absolute and relative liver weights. RU486 (25) produced significant decreases in ventral prostate, seminal vesicle, and Cowper's gland weights without affecting body weight. FLUT (10) decreased all AST weights measured. The antiandrogenic activities of the remaining materials (p,p'-DDE, spironolactone, and procymidone) have been demonstrated in previous Hershberger assays. These data indicate the importance of including in vivo results in assessing the endocrine activity of test materials and further stress the importance of a weight of evidence approach in assessing endocrine activity of test materials.     

Combined in utero and juvenile exposure of mice to arsenate and atrazine in drinking water modulates gene expression and clonogenicity of myeloid progenitors

The effects of arsenate (As) and atrazine (Atr) on myeloid progenitors (colony-forming unit-granulocyte/macrophage, CFU-GM) cells derived from bone marrow were studied in male and female mice after combined in utero and juvenile exposure. Female adult mice were treated with arsenate in drinking water during gestation. Then, separate groups of males and females' offspring were exposed for 4 months to atrazine, to additional arsenate or to co-exposure of atrazine and arsenate together in drinking water. In male mice, arsenate and the combined exposure did not modulate the percentage of CFU-GM progenitors, whereas atrazine significantly decreases the clonogenicity of myeloid cells. In females, the percentage of CFU-GM significantly decreased after atrazine exposure did not change with arsenate treatment, but dramatically increased after the combined exposure. The expression of estrogen receptors alpha (ERalpha) and beta (ERbeta) in bone marrow cells was investigated, and an up-regulation of receptor beta was observed in both genders. A gene expression profile was generated using nylon membranes spotted with 1185 cancer-related genes. Results from microarrays indicate that atrazine alone did not stimulate the expression of any of the genes analysed in both male and female. Arsenic induced gene expression modulation only in female. Major significant changes on the gene expression resulted following the co-exposure to arsenic and atrazine in both male and female.