Quantitative bioimaging analysis of gonads in olvas-GFP/ST-II YI Medaka (transgenic Oryzias latipes) exposed to ethinylestradiol

This study investigates the adverse and persistent effects of ethinylestradiol (EE2) on mature gonads of transgenic olvas-GFPIST II-YI medaka (Oryzias latipes). The measurement of gonadal size calculating the GFP-fluorescent area was used as a technique that enabled monitoring gonads in living specimens by GFP fluorescence. First, mature medaka were exposed to EE2 (47.8-522 ng/L) for 4 weeks. The gonads showed a significant reduction of the GFP-fluorescent area and Gonadosomatic Index in males exposed to EE2 at >216 ng/L and females exposed at 522 ng/L. Histologically, males at all treatments exhibited testis-ova and additionally, high connective tissue prevalence at > or =216 ng/L. Next, mature male medaka were exposed to EE2 (43.7-473 ng/L) for 3 weeks and allowed to depurate for 6 weeks, to investigate persistent effects of EE2. Continuous gonad observation showed that GFP began to decline 3 weeks after initial exposure to > or =215 ng/L. After depuration, the gonad's fluorescent areas gradually recovered, with no statistical difference at the end of the depuration period; normal spermatogenesis was present in these individuals. Alterations in GFP fluorescence clearly indicate the condition of the gonad in transgenic medaka and this strain showed a facilitated screening fish model to detect the adverse effects on the gonad by estrogenic chemicals.     

In vivo visual reporter system for detection of estrogen-like substances by transgenic medaka

Detection of endocrine disrupting chemicals, in particular, environmental estrogens with living organisms, has many advantages if compared to chemical analysis. The screening of novel pollutants with meaningful endpoints, the integration of uptake, bioconcentration, and excretion as well as the evaluation of endocrine disrupting effects with respect to toxicity require in vivo biotests for estrogen-like substances (ELSs). Critical disadvantages of whole organism biotests are their low sensitivity and the need for laborious and time-consuming work. To overcome these problems, we have developed a transgenic medaka strain harboring the green fluorescence protein (GFP) gene driven by choriogenin H gene regulatory elements. Choriogenin H is an egg envelope protein induced by estrogens in the liver. With yolk sac larvae of this strain, GFP induction in liver was observed 24 h after onset of aqueous exposure to 0.63 nM 17beta-estradiol (E2), 0.34 nM ethynylestradiol, or 14.8 nM estrone. Furthermore, concentrated sewage treatment effluent induced GFP expression. Comparison of E2 equivalents estimated by GFP-induction in transgenic medaka, a YES assay, and GC/MS showed detection limits in the same order of magnitude. These results indicated that the sensitivity of the transgenic medaka strain was sufficient for application as an alternative model in monitoring environmental water samples for ELSs.     

Global gene expression profiling of androgen disruption in Qurt strain medaka

Androgen disrupting chemicals (ADCs) are endocrine disrupting chemicals (EDCs) that mimic or antagonize the effect of physiological androgens. Microarray-based detection of altered gene expression can be used as a biomarker of EDC exposure. Therefore, the purpose of this study was to identify and compare gene expression profiles of the androgen 11-ketotestosterone (11-KT), the antiandrogen flutamide (FLU), and the antiandrogenic fungicide vinclozolin (VIN), on Qurt medaka (Oryzias latipes). Biologically effective concentrations for 11-KT (100 microg/L), VIN (100 microg/L), and FLU (1000 microg/L) determined in range-finding studies were used for exposures. The oligonucleotide microarray included 9379 probes for EDC-affected genes, medaka cDNAs, sequences from the medaka genome project, and the UniGene database. We found that treatment with FLU, VIN, and 11-KT caused significant (false discovery rate = 0.01) differential expression of at least 87, 82, and 578 genes, respectively. Two sets of responsive genes are associated to vertebrate sex differentiation and growth, and 50 genes were useful in discriminating between ADC classes. The discriminating capacity was confirmed by a remarkable similarity of the antiandrogenic expression profiles of VIN and FLU, which were distinct from the androgenic profile of 11-KT. Gene expression profiles characterized in this study allow for reliable screening of ADC activity.     

Reconstitution studies of pesticides and surfactants exploring the cause of estrogenic activity observed in surface waters of the san francisco bay delta

To evaluate the potential role of endocrine disruption in the decline of pelagic fishes in the San Francisco Bay Delta of California, various surface water samples were collected, extracted, and found to elicit estrogenic activity in laboratory fish. Chemical analysis of the estrogenic samples indicated 2 pesticides (bifenthrin, diuron), 2 alkyphenols (AP), and mixtures of 2 types of alkyphenol polyethoxylates (APEOs). Evaluation of estrogenic activity was further characterized by in vitro bioassays using rainbow trout hepatocytes (Oncorhynchus mykiss) and in vivo studies with Japanese medaka (Oryzias latipes). In the in vitro bioassays, hepatocytes exposed to the pesticides alone or in combination with the AP/APEO mixtures at concentrations observed in surface waters failed to show estrogenic activity (induction of vitelloginin mRNA). In the in vivo bioassays, medaka exposed to individual pesticides or to AP/APEO alone did not have elevated VTG at ambient concentrations. However, when the pesticides were combined with AP/APEOs in the 7-day exposure a significant increase in VTG was observed. Exposure to a 5-fold higher concentration of the AP/APEO mixture alone also significantly induced VTG. In contrast to earlier studies with permethrin, biotransformation of bifenthrin to estrogenic metabolites was not observed in medaka liver microsomes and cytochrome P450 was not induced with AP/APEO treatment. These results showed that mixtures of pesticides with significantly different modes of action and AP/APEOs at environmentally relevant concentrations may be associated with estrogenic activity measured in water extracts and feral fish that have been shown to be in population decline in the San Francisco Bay Delta.     

Partiton-controlled delivery of toxicants: a novel in vivo approach for embryo toxicity testing

In conventional static or semi-static embryo toxicity assays with fish, the nominal concentrations of hydrophobic chemicals are often used to establish the toxic thresholds, which often far exceed the solubility limits of test compounds. Saturators and continuous-flow diluters have been used to provide stable concentrations below solubility but are complex, use large amounts of test substance, and produce large volumes of waste. We present a partition-controlled delivery (PCD) method that maintains the concentrations of chemicals in test solutions at or below solubility limits for extended exposure times. Concentrations are maintained by equilibrium partitioning of test chemicals from a series of poly(dimethylsiloxane) films loaded with a range of concentrations of each chemical. The efficacy of the PCD assay was tested by comparisons with static (no renewal) and semi-static (24-h renewal) embryo-larval toxicity tests. The test species was Japanese medaka (Oryzias latipes) exposed to retene (7-isopropyl-1-methylphenanthrene), a compound causing blue sac disease (BSD) in fish embryos. In the PCD assay, the median effective concentration (EC50) for BSD was 10 microg/L, below retene's solubility of 17 microg/L. In contrast, the nominal EC50 values for the semi-static 24-h and static assays were about 10 (150 microg/L) and 150 times (2500 microg/L) greater than solubility, respectively. The PCD method is a more sensitive and realistic method for assessing toxicity of nonpolar compounds than (semi)-static assays.     

Relative potencies and combination effects of steroidal estrogens in fish

The natural steroids estradiol-17beta (E2) and estrone (E1) and the synthetic steroid ethynylestradiol-17alpha (EE2) have frequently been measured in waters receiving domestic effluents. All of these steroids bind to the estrogen receptor(s) and have been shown to elicit a range of estrogenic responses in fish at environmentally relevant concentrations. At present, however, no relative potency estimates have been derived for either the individual steroidal estrogens or their mixtures in vivo. In this study the estrogenic activity of E2, E1, and EE2, and the combination effects of a mixture of E2 and EE2 (equi-potent fixed-ratio mixture), were assessed using vitellogenin induction in a 14-day in vivo juvenile rainbow trout screening assay. Median effective concentrations, relative to E2, for induction of vitellogenin were determined from the concentration-response curves and the relative estrogenic potencies of each of the test chemicals calculated. Median effective concentrations were between 19 and 26 ng L(-1) for E2, 60 ng L(-1) for E1, and between 0.95 and 1.8 ng L(-1) for EE2, implying that EE2 was approximately 11 to 27 times more potent than E2, while E2 was 2.3 to 3.2 times more potent than E1. The median effective concentration, relative to E2, for the binary mixture of E2 and EE2 was 15 ng L(-1) (comprising 14.4 ng L(-1) E2 and 0.6 ng L(-1) EE2). Using the model of concentration addition it was shown that this activity of the binary mixture could be predicted from the activity of the individual chemicals. The ability of each individual steroid to contribute to the overall effect of a mixture, even at individual no-effect concentrations, combined with the high estrogenic potency of the steroids, particularly the synthetic steroid EE2, emphasizes the need to consider the total estrogenic load of these chemicals in our waterways.     

In vitro and in vivo antiestrogenic effects of polycyclic musks in zebrafish

The polycyclic musks 6-acetyl-1,1,2,4,4,7-hexamethyltetraline (AHTN) and 1,2,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-benzopyran (HHCB) are used as fragrance ingredients in perfumes, soaps, and household cleaning products. They are known to be ubiquitously present in the aquatic environment, and because of their lipophilic nature, they tend to bioaccumulate in aquatic biota. In surface waters, concentrations between 1 ng/L and 5 microg/L have been found, depending mainly on the proportion of sewage effluents in the water. In fish, under normal environmental conditions, concentrations in the microgram per kilogram fresh weight (fw) range are found. In a previous study we showed that AHTN and HHCB exert mainly antiestrogenic effects on the human estrogen receptor alpha (ERalpha) and ERbeta in an in vitro reporter gene assay. In the current study, we assessed the in vitro antiestrogenic effects of both musks on zebrafish ERs. Antagonism was observed on zfERbeta, and more pronounced on the newly cloned zfERgamma. Using a transgenic zebrafish assay, we studied antiestrogenicity of the musks in vivo. Dose-dependent antagonistic effects were observed at concentrations of 0.1 and 1 microM AHTN and HHCB. GC-MS analysis showed that the musks bioaccumulated in the fish, with internal concentrations (15-150 mg/kg fw) which were roughly 600 times higher than the nominal test doses. To our knowledge, this is the first time that environmental contaminants are shown to be antiestrogenic in an in vivo fish assay that focuses solely on ER-mediated effects. This makes the transgenic zebrafish assay a promising tool for the rapid detection of both estrogenic and antiestrogenic effects of chemicals in fish.     

Characterization of toxic effects of sediment-associated organic pollutants using the lambda transgenic medaka

A novel sediment-contact assay using embryos of the transgenic medaka was developed to fully characterize the toxic effects induced by exposure to a mixture of organic pollutants in sediments. Embryos of the lambda transgenic medaka were exposed for 10 days to a clean reference sediment spiked with either the solvent alone, benzo[a]pyrene (B[a]P), or three concentrations (0.3x, 1x, and 2x) of an organic extract (OE) of sediments from the Seine estuary. The 1 x OE-spiked sediment contained concentrations of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls similar to those in field-collected sediment. Exposure to this sediment, but not to the B[a]P-spiked sediment, significantly increased embryo-larval mortality and prevalence of spinal deformities. Mutant frequency at the cII mutation target gene in the liver of 10-week-old medaka was significantly increased following exposure to either B[a]P or the three doses of OE. The predominant OE-induced liver mutations were G:C to T:A transversions, consistent with PAHs being the major contributors to the mutation induction. Liver and gonadal tumors were observed in 35-week-old medaka exposed to either B[a]P (1/25) or to the 1 x OE (1/24). The benefits of medaka as a fish model for toxicological assessment and the benefits of the cII mutation assay for mutation detection combine to provide comprehensive assessment of a wide range of genotoxic and nongenotoxic effects of aquatic pollutants.     

Larval exposure to 4-nonylphenol and 17beta-estradiol affects physiological and behavioral development of seawater adaptation in Atlantic salmon smolts

Population declines of anadromous salmonids are attributed to anthropogenic disturbances including dams, commercial and recreational fisheries, and pollutants, such as estrogenic compounds. Nonylphenol (NP), a xenoestrogen, is widespread in the aquatic environment due to its use in agricultural, industrial, and household products. We exposed Atlantic salmon yolk-sac larvae to waterborne 10 or 100 microg L(-1) NP (NP-L or NP-H, respectively), 2 microg L(-1) 17beta-estradiol (E2), or vehicle, for 21 days to investigate their effects on smolt physiology and behavior 1 year later. NP-H caused approximately 50% mortality during exposure, 30 days after exposure, and 60 days after exposure. Mortality rates of NP-L and E2 fish were not affected until 60 days after treatment, when they were 4-fold greater than those of controls. Treatment with NP-L or E2 as yolk-sac larvae decreased gill sodium-potassium-activated adenosine triphosphatase (Na+,K(+)-ATPase) activity and seawater (SW) tolerance during smolt development, 1 year after exposure. Exposure to NP-L and E2 resulted in a latency to enter SW and reduced preference for SW approximately 2- and 5-fold, respectively. NP-L-exposed fish had 20% lower plasma insulin-like growth factor I (IGF-I) levels and 35% lower plasma triiodothyronine (T3). Plasma growth hormone and thyroxine (T4) were unaffected. Exposure to E2 did not affect plasma levels of IGF-I, GH, T3, or T4. Both treatment groups exhibited increased plasma cortisol and decreased osmoregulatory capacity in response to a handling stressor. These results suggest that early exposure to environmentally relevant concentrations of NP, and other estrogenic compounds, can cause direct and delayed mortalities and that this exposure can have long-term, "organizational" effects on life-history events in salmonids.     

Identification and quantitation of nonylphenol ethoxylates and nonylphenol in fish tissues from Michigan

Nonylphenol (NP) and its lower ethoxylates, nonylphenol monoethoxylate (NPE1) and nonylphenol diethoxylate (NPE2), can be present in aquatic environments at total concentrations of more than 10 microg/L. They are metabolites of nonylphenol polyethoxylates (NPE) and have been found to be weakly estrogenic. To evaluate bioaccumulation potential and identify potential risks posed by these chemicals, concentrations of NP, NPE1, NPE2, and nonylphenol triethoxylate (NPE3) were determined in the tissues of fish inhabiting various waters in Michigan. This method involves extraction of samples using exhaustive steam distillation with concurrent liquid extraction. Concentrations of NP among all sites and species ranged from <3.3 to 29.1 ng/g, ww and varied little among sites. NPE1 was detectable in some samples but at concentrations less than the method detection limit (16.8 ng/g). Concentrations of NPE2 and NPE3 in all samples were less than their respective MDLs of 18.2 and 20.6 ng/g.     

Accounting for differences in estrogenic responses in rainbow trout (Oncorhynchus mykiss: Salmonidae) and roach (Rutilus rutilus: Cyprinidae) exposed to effluents from wastewater treatment works

Effluents from wastewater treatment works (WwTWs) contain estrogenic substances that induce feminizing effects in fish, including vitellogenin (VTG) synthesis and gonadal intersex. Fish vary in their responsiveness to estrogenic effluents, but the physiological basis for these differences are not known. In this study, uptake of estrogen from two WwTW effluents (measured in hydrolyzed bile) and estrogenic response (VTG induction) were compared in a salmonid (rainbow trout, Onchorhynchus mykiss) and a cyprinid fish (roach, Rutilus rutilus). Immature rainbow trout were more responsive than maturing roach to the estrogenic effluents. The more potent of the two estrogenic effluents (containing between 24.3 and 104.1 ng estradiol-17beta equivalents/L [E2eq/L]) resulted in a 700-fold and 240-fold induction of plasma VTG in male and female trout, respectively, but only a 4-fold induction in roach (and in males only). The less potent effluent (varying between 4.1 and 6.8 ng E2eq/L) induced VTG in the trout only, with a 4-fold and 18-fold induction in males and females, respectively. In fish exposed to tap water, the estrogenicity of the hydrolyzed bile was 0.03+/-0.01 ng E2eq/microL (for both sexes in trout), 0.18+/-0.04 ng E2eq/microL in male roach, and 0.88+/-0.15 ng E2eq/microL in female roach. The higher bile content of estrogen in control roach reflected their more advanced sexual status (and thus higher endogenous estrogen) compared with the immature female trout. In trout maintained in effluents, the bile content of estrogen was 100-fold and 30-fold higher than controls at WwTW A and B, respectively. Bioconcentration factors (BCFs) for estrogenic activity in bile were between 16 344 and 46 134 in trout and between 3543 and 60 192 in roach (no gender differences were apparent). There were strong correlations between VTG induction and the estrogenic activity of bile extracts for both trout and roach. The results confirm that estrogenic contaminants bioconcentrate to a high degree in fish bile and that the level (and nature) of this accumulation may accountfor responsiveness to the endocrine disruptive effects of estrogenic effluents. Immature fish were the more appropriate life stage for quantifying estrogen exposure and uptake in bile, as they contain little circulating endogenous oestrogen compared with sexual maturing fish. The nature of the estrogenic contaminants is detailed in an accompanying paper.     

Monitoring of vasculogenesis-inhibiting activities in sewage effluents by using medaka embryos

Environmental samples are known to be contaminated with complex chemicals such as estrogens, polycyclic aromatic hydrocarbons, and retinoids. These contaminants have potentially an adverse impact on survival of aquatic animals, because we found previously that medaka (Oryzias latipes) embryos are defective in the development of blood vessels and bones in the presence of these chemicals. Thus, it is important to test whether sewage effluents contain inhibitory activities against the embryonic development. To examine for such activity, medaka embryos were exposed for 48 h to extracts or freeze-evaporated concentrates of effluent samples collected from different municipal sewage treatment plants. We used the transgenic embryos that are hypersensitive to estrogens due to a high-level expression of estrogen receptor for detecting the total (sum of estrogenic and non-estrogenic) vessel-inhibiting activity. The embryos were specifically defective in blood-vessel formation in most effluent samples, showing the activities ranging from 3 to 30 ng of 17beta-estradiol equiv per liter. Detection limit of 17beta-estradiol was 10 ng per liter. For detection of the non-estrogenic vessel-inhibiting activity, we treated the transgenic embryos in the presence of an antiestrogen, tamoxifen, or used the wild-type embryos. The non-estrogenic activities were found in some (7 out of 18) effluents, ranging from half to all of the total activities. Our findings for the first time demonstrate the utility of the vascular assay for monitoring sewage effluents.     

Temporal variation in the estrogenicity of a sewage treatment plant effluent and its biological significance

Daily variation in the estrogenic activity of effluent released by a modern sewage treatment plant (STP) was measured and its effects on the physiology, behavior, and reproductive success of male fish were evaluated. As measured by an estrogen receptor binding assay, the daily estrogenic activity of this effluent was both high and extremely variable (42 +/- 25.4 [mean +/- SD] ng 17beta-estradiol (E2) equivalents/L; n = 18). Liver VTG mRNA expression in male fathead minnows (FHM) covaried with the binding assay estimates, suggesting that these fluctuations are biologically relevant. Tests which exposed male FHMs to either fluctuating levels of E2, a constant concentration of E2 (time-weighted to reflect average concentrations), or control (no E2) demonstrated that while the estrogenic activity of this effluent was detrimental to male spawning success, the fact that its concentration varied in a daily manner was without additional influence. The variability of the effluent's estrogenicity suggests that studies concerned with the effects of STP effluents should collect multiple daily samples and then test them on an appropriate time-weighted basis.     

Reproductive disruption in fish downstream from an estrogenic wastewater effluent

To assess the impact of an estrogenic wastewater treatment plant (WWTP) effluent on fish reproduction, white suckers (Catostomus commersoni) were collected from immediately upstream and downstream (effluent site) of the city of Boulder, CO, WWTP outfall. Gonadal intersex, altered sex ratios, reduced gonad size, disrupted ovarian and testicular histopathology, and vitellogenin induction consistent with exposure to estrogenic wastewater contaminants were identified in white suckers downstream from the WWTP outfall and not at the upstream site. The sex ratio was female-biased at the effluent site in both the fall of 2003 and the spring of 2004; the frequency of males at the effluent site (17-21%) was half that of the upstream site (36-46%). Intersex white suckers comprised 18-22% of the population at the effluent site. Intersex fish were not found at the upstream site. Chemical analyses determined that the WWTP effluent contained a complex mixture of endocrine-active chemicals, including 17beta-estradiol (E2) 17alpha-ethynylestradiol, alkylphenols, and bisphenol A resulting in an estimated total estrogen equivalence of up to 31 ng E2 L(-1). These results indicate that the reproductive potential of native fishes may be compromised in wastewater-dominated streams.     

Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity

Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably transfected with an ER-mediated luciferase gene construct. In the in vivo assay, transgenic zebrafish are used in which the same luciferase construct has been stably introduced. In both assays, luciferase reporter gene activity can be easily quantified following short-term exposure to chemicals activating endogenous estrogen receptors. The objective of this study was to compare responses by known (xeno)estrogenic compounds in both assays. Exposure to the (xeno)estrogens estradiol (E2), estrone, ethynylestradiol (EE2), o,p'-DDT, nonylphenol (NP), and di(2-ethylhexyl)phthalate (DEHP) revealed that EE2 was the most potent (xeno)estrogen tested and was 100 times more potent than E2 in the transgenic zebrafish assay, whereas in the in vitro ER-CALUX assay, EE2 and E2 were equipotent Although the xenoestrogens o,p'-DDT and NP were full estrogen agonists in the in vitro ER-CALUX assay, only o,p'-DDT demonstrated weak dose-related estrogenic activity in vivo. To determine if differences in reporter gene activity may be explained by differential affinity of (xeno)estrogens to human and zebrafish ERs, full-length sequences of the zebrafish ER subtypes alpha, beta, and gamma were cloned, and transactivation by (xeno)estrogens was compared to human ERalpha and ERbeta. Using transiently transfected recombinant ER and reporter gene constructs, EE2 also showed relatively potent activation of zebrafish ERalpha and ERbeta compared to human ERalpha and ERbeta. Zebrafish ERbeta and ERgamma showed higher transactivation by (xeno)estrogens relative to E2 than human ERbeta.     

Transformation of Escherichia coli K-12 with a high-copy plasmid encoding the green fluorescent protein reduces growth: implications for predictive microbiology

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has been widely used as a biomarker and has potential for use in developing predictive models for growth of pathogens on naturally contaminated food. However, constitutive production of GFP can reduce growth of transformed strains. Consequently, a high-copy plasmid with gfp under the control of a tetracycline-inducible promoter (pTGP) was constructed. The plasmid was first introduced into a tetracycline-resistant strain of Escherichia coli K-12 to propagate it for subsequent transformation of tetracycline-resistant strains of Salmonella. In contrast to transformed E. coli K-12, which only fluoresced in response to tetracycline, transformed Salmonella fluoresced maximally without tetracycline induction of gfp. Although pTGP did not function as intended in Salmonella, growth of parent and GFP E. coli K-12 was compared to test the hypothesis that induction of GFP production reduced growth. Although GFP production was not induced during growth on sterile chicken in the absence of tetracycline, maximum specific growth rate (mumax) of GFP E. coli K-12 was reduced 40 to 50% (P < 0.05) at 10, 25, and 40 degrees C compared with the parent strain. When growth of parent and GFP strains of E. coli K-12 was compared in sterile broth at 40 degrees C, mumax and maximum population density of the GFP strain were reduced (P < 0.05) to the same extent (50 to 60%) in the absence and presence of tetracycline. These results indicated that transformation reduced growth of E. coli K-12 independent of gfp induction. Thus, use of a low-copy plasmid or insertion of gfp into the chromosome may be required to construct valid strains for development of predictive models for growth of pathogens on naturally contaminated food.     

Enantioselectivity in estrogenic potential and uptake of bifenthrin

Despite the fact that the biological processes of chiral compounds are enantioselective, the endocrine disruption activity and uptake of chiral contaminants with respect to enantioselectivity has so far received limited research. In this study, the estrogenic potential and uptake of the enantiomers of a newer pyrethroid insecticide, bifenthrin (BF), were investigated. Significant differences in estrogenic potential were observed between the two enantiomers in the in vitro human breast carcinoma MCF-7 cell proliferation assay (i.e., the E-SCREEN assay) and the in vivo aquatic vertebrate vitellogenin enzyme-linked immunosorbent assay (ELISA). In the E-SCREEN assay, the relative proliferative effect ratios of 1S-cis-BF and 1R-cis-BF were 74.2% and 20.9%, respectively, and the relative proliferative potency ratios were 10% and 1%, respectively. The cell proliferation induced by the two BF enantiomers may be through the classical estrogen response pathway via the estrogen receptor (ER), as the proliferation induced by the enantiomers could be completely blocked when combined with 10-6 mol/L of the ER antagonist ICI 182,780. Measurement of vitellogenin induction in Japanese medaka (Oryzias latipes) showed that, at an exposure level of 10 ng/mL, the response to 1S-cis-BF was about 123 times greater than thattothe Renantiomer. Significant selectivity also occurred in the uptake of BF enantiomers in the liver and other tissues of J. medaka. These results together suggest that assessment of the environmental safety of chiral insecticides should consider enantioselectivity in acute and chronic ecotoxicities such as endocrine disruption.