Immature uterotrophic assay is more sensitive than ovariectomized uterotrophic assay for the detection of estrogenicity of p-nonylphenol in Sprague-Dawley rats

Many efforts have been made to develop assays for detecting endocrine disrupters (EDs). Among them, uterotrophic assay has been known efficient for detecting EDs, especially estrogenic compounds. This study was performed to compare the immature uterotrophic assay with an ovariectomized assay using p-nonylphenol (NP), a weakly estrogenic compound. NP was given to either immature or ovariectomized rats subcutaneously or orally (only immature) at doses of 10, 100, and 1000 mg/kg for 3 days. After treatment with NP, the rats were examined for parameters such as uterine weight, uterine weight per body weight ratio, luminal epithelial height of uterus and vagina, diameter of uterine ducts, and number of uterine glands. Both systems were shown to increase uterine weight in a dose-dependent manner. In the immature system (subcutaneous injection), uterine weight, diameter of uterine duct and vaginal luminal epithelial height were significantly increased at 100 mg/kg/day, while in the ovariectomized system these parameters were not significant at the same dose (except for vaginal luminal epithelial height). These results suggest that the immature system (subcutaneous injection) might be most sensitive to detecting a weakly estrogenic compound and that the measurement of vaginal epithelium is a good end-point.     

The estrogenic effects of apigenin,phloretin and myricetin based on uterotrophic assay in immature Wistar albino rats

Chemicals that occur in vegetal food and known as phytoestrogens, because of their structures similarity to estrogen, have benefits on chronic diseases. Despite this, when they are taken at high amounts, they can cause harmful effects on endocrine system of human and animals. In this study, it has been intended to determine the estrogenic potencies of phytoestrogens apigenin, phloretin and myricetin whose affinities for estrogen receptors in vitro. The female rats divided into 17 groups, each containing six rats. There was a negative control group and there were positive control dose groups which contains ethinyl estradiol, ethinyl estradiol + tamoxifen and genistein. The other dose groups which were tested for estrogenic activity contains apigenin, myricetin and phloretin All chemicals have been given to Wistar immature female rats with oral gavage for 3 consecutive days. By using uterotrophic analysis, uterus wet and blotted weights, vaginal opening, uterus length of female rats has been recorded at the end of the experiment. For detect of cell response, luminal epithelium height, gland number and lactoferrin intensity in luminal epithelium of uterus were evaluated. Biochemical analysises in blood were performed.     

Comparative estrogenic effects of p-nonylphenol by 3-day uterotrophic assay and female pubertal onset assay

Nonylphenol (NP) is widely used as a component of detergents, paints, pesticides, and many other formulated products. Several studies have demonstrated that NP is estrogenic in fish, avian, and mammalian cells. NP also competitively inhibits the binding of 17 beta-estradiol (E2) to the estrogen receptor (ER). However, there are relatively few in vivo data related to this issue in mammals. The aim of this study was to investigate the estrogenic activity of NP in animal models. We performed a 3-day uterotrophic assay using immature female rats for comparison with other endpoints of Tier I screening including vaginal opening (VO) in prepubertal intact female rats. For the uterotrophic assay, diethylstilbestrol (DES) (0.2 and 1.0 microg/kg) and p-NP (10, 25, 50, 100, and 200 mg/kg) were administered subcutaneously to immature Sprague-Dawley female rats for 3 consecutive days (postnatal days (PND) 20, 21, and 22). For the female pubertal onset assay, DES (0.2, 1.0, and 5.0 microg/kg) and p-NP (10, 50, and 100 mg/kg) were administered daily by oral gavage from 21 days of age for 20 days. In the uterotrophic assay, statistically significant increases in uterine wet weight were observed at doses of 100 and 200 mg/kg p-NP. DES (0.2 and 1.0 microg/kg) also significantly increased uterine weight compared to the vehicle control. In the female pubertal onset assay, the age of VO was advanced following oral exposure to DES (1.0 and 5.0 microg/kg) and p-NP (50 and 100 mg/kg). Estrous cyclicity was monitored in prepubertal rats from the day of VO to the day of necropsy. Irregular estrous cycles were observed in the groups treated with DES (5.0 microg/kg) and p-NP (50 and 100 mg/kg). High-dose DES (5.0 microg/kg) produced a persistent estrus state, whereas p-NP (50 and 100 mg/kg) increased the number of days in diestrus. Serum thyroxine (T(4)) concentrations were decreased in a dose-dependent manner by DES and p-NP treatment. A significant decrease in serum T(4) level was observed at high-dose DES (5.0 microg/kg) and p-NP (100 mg/kg). Serum TSH level was significantly increased by DES (5.0 microg/kg) treatment. Statistically significant decreases in ovarian weight were observed in female rats treated with DES (5.0 microg/kg) and p-NP (100 mg/kg). Our data demonstrate that p-NP can accelerate the onset of puberty and alter estrous cyclicity in prepubertal female rats at oral doses lower than the subcutaneous doses typically used in the uterotrophic assay. We therefore suggest that the female pubertal onset assay may be used as a sensitive testing method to detect environmental agents with weak estrogenic activity, but requires further research.     

Evaluation of estrogenic and androgenic activity of butylated hydroxyanisole in immature female and castrated rats

We evaluated the estrogenic and androgenic activity of butylated hydroxyanisole (BHA) using immature rat uterotrophic assay and Hershberger assay. To investigate (anti-) estrogenicity, BHA alone or with 17beta-estradiol was administered to 20-days-old immature female rats for three consecutive days. Absolute and relative uterine weights were significantly decreased by BHA (50, 100, 250, 500 mg/kg) alone and 17beta-estradiol-stimulated weights of uterine and vagina were also decreased by BHA (500 mg/kg), while uterine epithelial cell height was not affected. In Hershberger assay, BHA alone or with testosterone propionate (TP) was administered daily to 51-days-old castrated male rats for 10 days. BHA alone or with testosterone propionate (TP) caused no significant effect on androgen-dependent accessory sex organ weights; seminal vesicle/coagulative glands, glans penis, Cowper's gland, ventral prostate gland and levator ani plus bulbocarvernosus muscle. Although, the relative weight of ventral prostate gland was increased by the co-treatment of BHA 250 mg/kg with TP 0.4 mg/kg compared to that of TP alone, the relative and absolute weights of other androgen-dependent organs and absolute and formalin-fixed ventral prostate gland weight showed no changes. Our studies suggest that BHA have anti-estrogenic activity for the decrease of uterine weight in immature female rat but have negligible effect on the androgenic activity in castrated male rats.     

Potential estrogenic and antiestrogenic activity of the cyclic siloxane octamethylcyclotetrasiloxane (D4) and the linear siloxane hexamethyldisiloxane (HMDS) in immature rats using the uterotrophic assay.

The cyclic siloxane octamethylcyclotetrasiloxane (D4) and the linear siloxane hexamethyldisiloxane (HMDS) have numerous industrial and consumer applications and thus have the potential for human exposure. The present study was undertaken to examine potential estrogenic and antiestrogenic activities of D4 and HMDS. To address potential differences in sensitivity between rat strains the study used both Sprague-Dawley (SD) and Fischer 344 (F-344) rats. Estrogenicity of the test compounds was determined by measuring absolute and relative uterine weights in immature rats and by monitoring uterine epithelial cell height. In order to place the data obtained for D4 into perspective relative to strong and weak estrogenic compounds, the response produced by D4 at 0, 10, 50, 100, 250, 500, and 1000 mg/kg/day was compared to responses produced by ethinyl estradiol (EE) (1, 3, 10, or 30 microg/kg/day), diethylstilbestrol dipropionate (DES-DP) (0.5, 1.5, 5, 15 microg/kg/day), and coumestrol (CE) (10, 35, 75, 150 mg/kg/day). Antiestrogenic effects were evaluated by co-administering D4 (500 mg/kg/day) with EE at 1, 3, 10, and 30 microg /kg/day. All compounds were administered in sesame oil at a volume of 5 mL/kg by oral gavage. Beginning on postnatal day 18 (SD) or 21 (F-344) each pup (12 per group) received a single dose of test compound once a day for 4 consecutive days. The pups were euthanized the morning after the last treatment and their uteri removed, weighed, and processed for histological examination. EE and DES-DP produced a significant dose-dependent increase in absolute and relative uterine weights and uterine cell height. The maximum increase in uterine weight following EE exposure was approximately 350% relative to controls in both strains. The weak phytoestrogen CE also produced a dose-related increase in absolute and relative uterine weight and epithelial cell height, but the response occurred over a much higher range of doses. At the highest dose of CE, uterine weight was increased approximately 230% relative to controls. Following exposure to D4, absolute and relative uterine weights and uterine epithelial cell height were statistically significantly increased in both strains of rats at doses above 100 mg/kg/day. In terms of uterine weight, D4 was approximately 0.6 million times less potent than EE or DES-DP in SD pups and 3.8 million times less potent than EE or DES-DP in F-344 pups. The maximal increase in uterine weight, relative to controls, produced by D4 at 1000 mg/kg/day was approximately 160% in SD rats, while the maximum increase produced by D4 in F-344 rats was 86%. D4 co-administered over a wide range of EE doses, resulted in a significant reduction in uterine weight compared to EE alone. HMDS was evaluated in SD rats only. The response produced by HMDS (600 and 1200 mg/kg/day) was compared to EE (3 microg/kg/day). Antiestrogenic effects were evaluated by co-administering HMDS (1200 mg/kg/day) with EE at 3 microg/kg/day. HMDS had no measurable effect on uterine weight under the experimental conditions described here. However, HMDS coadministered with EE did produce a small, but statistically significant reduction in uterine weight compared to EE alone. In conclusion, D4 showed weak estrogenic and antiestrogenic activity that was several orders of magnitude less potent than EE, and many times less potent than the weak phytoestrogen CE.     

Assessment of polystyrene extract for estrogenic activity in the rat uterotrophic model and an in vitro recombinant receptor reporter gene assay

The purpose of the study was to determine whether polystyrene used in food-contact applications would elicit an estrogenic response when extracts simulating exaggerated conditions of use were subjected to in vivo and in vitro tests. A sample of polystyrene was subjected to extraction conditions that simulate, or exaggerate, the actual food-contact uses of polystyrene to maximize the amount of low molecular weight polystyrene extractables. The food-simulating solvent and the time and temperature conditions recommended by the Food and Drug Administration (FDA) were selected to maximize the level of extractable components from polystyrene. The extract was examined for its estrogenic response in vivo using the immature rat uterotrophic assay and in vitro using an estrogen receptor (ER)-mediated recombinant receptor reporter gene assay. In vivo, the uterine weights of juvenile female Sprague Dawley rats (10 rats/group) were determined after oral gavage exposure to the extract (two dosage levels: one represents the maximum potential daily human exposure to polystyrene extractables and the other represents one-tenth of the maximum exposure level), vehicle control (sesame oil), or positive control [diethylstilbestrol (DES), at 200 micrograms/kg body weight]. In addition, five treatment groups were dosed by subcutaneous injection of either estradiol (1, 50, and 500 micrograms/kg body weight) or DES (2 and 200 micrograms/kg body weight). Dosing began on postnatal day (pnd) 21 and continued daily through pnd 23. Body weights were collected at study initiation (pnd 21) and at necropsy (pnd 24). Body weights were not different statistically between treatment groups at study initiation or at necropsy. Uterine wet weights and uterine weights relative to body weights were significantly increased (p < 0.05) for estradiol at 50 and 500 micrograms/kg, DES at 2 and 200 micrograms/kg, and DES at 200 micrograms/kg (oral) over vehicle control. The polystyrene extract had no effect on uterine wet weight or uterine weights relative to body weights at either level tested. An in vitro recombinant estrogen receptor/reporter gene assay that involved transiently transfecting MCF-7 human breast cancer cells with the chimeric human ER, Ga14-HEGO, consisting of the yeast Ga14 DNA binding domain linked to the ligand binding domain of the human ER and a Ga14 response element (17mer)-regulated reporter gene (17m5-G-Luc) was employed. Dose-dependent induction of the reporter gene, 17m5-G-Luc, was observed with the positive control, 17 beta-estradiol (E2). Induction of greater than 100-fold was obtained following incubation of transfected MCF-7 cells with 10 nM E2 for 24 hours. No induction of reporter gene activity was observed with the polystyrene extracts dissolved in dimethylsulfoxide (0.01, 0.1 or 0.01 mg/ml) using the same assay conditions. These results indicate that polystyrene extract does not elicit ER-mediated activity using the Ga14-HEGO/17m5-G-Luc recombinant receptor/reporter gene assay. In conclusion, extracts from polystyrene produced no estrogenic response in either the rat uterotrophic assay or the MCF-7 cell assay for estrogen receptor-mediated activity.     

Determination of the oestrogenic (uterotrophic) activity of extracts of 'general purpose polystyrene (GPPS)' using immature female rats

In Japan there is growing concern about the possible adverse effects of consumption of food from styrene containers (mainly those made from polystyrene paper) due to the alleged oestrogenic activity of styrene oligomers (dimers and trimers), which may migrate into the food. To examine the possible oestrogenic activity of styrene dimers and trimers, extracts were made from 'general purpose polystyrene (GPPS)' and administered orally to immature female rats over a 4 day period. Increase of uterus weight (wet and blotted) was used for assessment of possible oestrogenic activity. To establish the sensitivity of the test method, immature rats were treated with diethylstilboestrol (DES), a well-known oestrogenic compound. It was found that treatment of rats with levels of up to 60 microg of styrene dimers and 930 microg of styrene trimers per kilogram body weight per day did not give any statistically significant increase of the uterus weight (wet or blotted), whereas DES caused statistically significant, dose-related increases in uterus weight at levels as low as 0.89 microg kg(-1) body weight day(-1). It was concluded that, compared with the estimated maximum human daily intake of styrene trimers of 1 microg kg(-1) body weight day(-1) from polystyrene food containers, the risk of adverse human health effects with respect to oestrogenicity may be considered negligible.     

Variability in the uterotrophic response assay (an in vivo estrogenic response assay) in untreated control and positive control (DES-DP, 2.5 microG/kg, bid) Wistar and Sprague-Dawley rats

Inclusion of biological outlier values was found to bias the results of rat uterotrophic assays towards false negatives, i.e., not identify uterotrophic effects in treated populations. The present investigation was conducted to identify the background variability in the rat uterotrophic assay and to evaluate the need to exclude biological outlier values in untreated control groups. The Styrene Steering Committee (SSC) of the European Chemical Industry Council (CEFIC) co-sponsored this work with Argus Research Laboratories (Argus). The rat uterotrophic response assay originally was used as a pharmacology screen to identify estrogenic agents. Classically, 5 to 10 immature female rats (18 to 22 days of age) are administered an agent for three or four days. At sacrifice on the following day (21 to 26 days of age), the uterus is removed, weighed and a uterine weight/terminal body weight ratio calculated. This in vivo assay has been adapted for use in identifying the potential estrogenicity of chemicals, generally using 10 immature female rats per group, more closely controlling the ages, and adding one or more positive control groups to demonstrate sensitivity and response of the test system. Statistically significant increases in the positive control group means for absolute and relative uterine weights, as compared with the untreated (or vehicle-treated) means, is generally interpreted as identifying a sensitive test system. The untreated (and/or vehicle-treated) control group is then compared with the various test groups, and statistically significant increases in the mean absolute and relative uterine weights are identified as evidence of estrogenicity of the agent. Although not fully described previously, the inherent biological variability existing in both untreated and treated animals, can confound interpretation of the data, especially when numbers are relatively small. Our laboratories have identified that under controlled GLP-compliant conditions, some Wistar rats [randomly assigned (weight-ordered) to groups of ten at 22 +/- 1 days of age, and sacrificed when 26 +/- 1 days of age] in untreated control groups have high relative uterine weights that skew data distributions such that statistically significant differences are not present between untreated control and positive control groups. Based on these observations, further evaluations of untreated control and positive control (DES-DP, 2.5 micrograms/kg, b.i.d.) populations of three rat strains [Wistar--Chbb:THOM-SPF, Wistar--Crl:(WI)BR and Sprague-Dawley--Crl:CD(SD)IBS BR VAF/Plus "International Genetic Standard"] were made to define when such normal findings should be considered biological outliers, and whether outlier values should be excluded from analyses. Our data indicate that body weight is not always predictive of uterine weight, that relative uterine weight outlier values occur in each of these rat strains, and that statistically significant differences exist between groups of untreated control animals when outlier values are included in analyses. Of 98, 60 and 60 untreated control rats in the three respective strains, 11 (11.2%), 16 (26.7%) and 15 (25.0%) had relative uterine weights > or = 0.150%, and 5 (5.1%), 4 (6.7%) and 9 (15.0%) of these rats had relative uterine weights > or = 0.200%, values within the positive control range. All positive control rats attained relative uterine weights > or = 0.100%. Of 50, 60 and 60 positive control rats in the three respective rat strains, 27 (54%), 47 (78.3%) and 36 (60%) had relative uterine weights > or = 0.200%, 9 (18%), 2 (3.3%) and 7 (11.7%) had relative uterine weights > or = 0.300% and 5 (10%), 1 (1.7%) and 3 (5%) had relative uterine weights > or = 0.400%. The incidences of relative uterine weights > or = 0.300% in the positive control group may indicate the presence of high responders. Histological evaluations of uteri of positive control rats and untreated control rats with relative uterine weights > or = 0.     

Comparison of the reporter gene assay for ER-alpha antagonists with the immature rat uterotrophic assay of 10 chemicals

We performed a reporter gene assay for estrogen receptor (ER)-alpha agonists and antagonists of 10 chemicals that showed both estrogen agonistic and reduced the estrogenic effect of ethinyl estradiol in a rat uterotrophic assay. The chemicals tested by the immature uterotrophic assay were p-(tert-pentyl)phenol, 4,4'-thiobis-phenol, 4,4'-(hexafluoroisopropylidene)diphenol, 2,2-bis(4-hydroxyphenyl)-4-methyl-n-pentane, 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol, 4-(phenylmethyl)phenol, 4,4'-dihydroxybenzophenone, 2,2',4,4'-tetrahydroxybenzophenone, 4-hydroxybenzophenone and 2,4,4'-trihydroxybenzophenone. Although all chemicals examined in this study were positive in the reporter gene assay for ER-alpha agonists, 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol was only positive in the reporter gene assay for ER-alpha antagonists. These findings demonstrate that results of the reporter gene assay for ER-alpha agonists correlated well with those of the uterotrophic assay, but antagonistic change of 9 of 10 chemicals in the uterotrophic assay was not detected by the reporter gene assay for ER-alpha antagonists.     

Validation and application of a reporter gene assay for the determination of estrogenic endocrine disruptor activity in milk

Endocrine disruptors (EDs) are compounds known to interfere with the endocrine system by disturbing the action or pathways of natural hormones which may lead to infertility or cancer.Our diet is considered to be one of the main exposure routes to EDs. Since milk and dairy products are major components of our diet they should be monitored for ED contamination. Most assays developed to date utilise targeted, chromatography based methods which lack information on the biological activity and mixture effects of the monitored compounds.A biological reporter gene assay (RGA) was developed to assess the total estrogen hormonal load in milk. It has been validated according to EU decision 2002/657/EC. Analytes were extracted by liquid–liquid extraction with acetonitrile followed by clean up on a HLB column which yielded good recovery and small matrix effects. The method has been shown to be estrogen specific, repeatable and reproducible, with covariance values below 20%. In conclusion, this method enables the detection of low levels of estrogen hormonal activity in milk with a detection capability of 36 pg g⁻¹ EEQ and has been successfully applied in testing a range of milk samples.     

Potential estrogenic activity of triclosan in the uterus of immature rats and rat pituitary GH3 cells

Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol; TCS) is used as an antimicrobial agent in personal care, pharmaceutical, industrial, and household products. In this study, we established an in vivo model for screening estrogenic activity of TCS in the uteri of immature rats. In addition, we employed temporarily transfected cells with plasmids containing estrogen response element (ERE) and progesterone (P4) response element (PRE) sequences. We found that uterine weight was significantly increased by 17α-ethinylestradiol (EE) as a positive control and TCS at doses of 7.5, 37.4, and 187.5 mg/kg. In addition, the expressions of calbindin-D_9k (CaBP-9k) and complement C3 (C3) were significantly induced by EE and TCS in the uteri of immature rats, indicating that TCS can induce their expression mediated by estrogenic activity. Co-treatment with steroid antagonists ICI 182,780 (ICI) and RU 486 in conjunction with TCS (37.5 mg/kg) reversed TCS-induced uterine weight and CaBP-9k mRNA and protein expression increases in immature rats. Moreover, ERE and PRE luciferase activity was evaluated in GH3 cells following treatment with TCS. Concentrations of TCS at increasing doses (10⁻⁹, 10⁻⁷, and 10⁻⁵ M) resulted in a significant increase in ERE luciferase activity compared to control; however, no difference was observed in PRE luciferase activity following TCS treatment. To confirm that ER signaling is involved in TCS-induced CaBP-9k expression, we treated GH3 cells with the anti-estrogen ICI, which can block TCS-induced up-regulation of CaBP-9k in these cells. Taken together, these results indicate that TCS has an estrogen-like property, which may be mediated through an ER-involved signaling pathway in both in vivo and in vitro models.     

Comparative study of the uterotrophic potency of 14 chemicals in a uterotrophic assay and their receptor-binding affinity

We performed an immature rat uterotrophic assay of 14 chemicals having various receptor-binding affinities in order to assess the relationship between their uterotrophic potency and receptor-binding affinity. The chemicals tested were phthalic acid di-n-hexyl ester, phthalic acid di-n-amyl ester, phthalic acid di-n-propyl ester, 2-ethylhexyl-p-hydroxybenzoate, 4,4'-biphenol, 4,4'-sulfonyldiphenol, 4,4'-dihydroxydiphenylmethane, 2,4-dihydroxybenzophenone, 4,4'-cyclohexylidenebisphenol, 4-t-butylpyrocatechol, clomiphene citrate, 4,4'-(1,3-phenylenediisopropylidene)bisphenol, p-t-butylphenol, and diallylterephthlate. Two of the 14 chemicals, phthalic acid di-n-propyl ester and diallylterephthlate, exhibited no receptor-binding affinity, and the receptor-binding affinity of phthalic acid di-n-hexyl ester and phthalic acid di-n-amyl ester was lower than that of the other chemicals. Ten of the chemicals showed uterotrophic potency, the exceptions being phthalic acid di-n-propyl ester, diallylterephthlate, phthalic acid di-n-hexyl ester, and phthalic acid di-n-amyl ester. The results of the present study demonstrate that the affinity of the chemicals in the receptor-binding assay correlated well with their potency in the uterotrophic assay except for a few chemicals with very low receptor-binding affinity.     

Validation of a photobeam system for assessment of motor activity in rats

To evaluate the validity and sensitivity of test procedures for routine assessment of chemically induced changes in motor activity in rats, studies were carried out with a commercially available photocell cage system. Testing of single, untreated rats showed that motor activity decreased within 10 min to a steady level. Overall activity was the same when testing was repeated at weekly intervals for 6 weeks. In groups of each 10 rats tested immediately after i.p. injection, chlorpromazine HCl (6 or 2 mg/kg) and physostigmine salicylate (0.5 mg/kg) decreased activity. d-Amphetamine sulfate (1.0 mg/kg) caused an increase of virtually all parameters, whereas scopolamine HCl (1.0 mg/kg) and physostigmine salicylate (0.02 mg/kg) led to an increase of particular aspects of motor activity only. Data generated with a mechanical calibrator varied 1-5% between the cages tested. It is concluded that the above procedures of recording of selected parameters of motor activity in groups of 10 rats for periods of up to 20 min would comply with the guidelines recommended by EPA TOSCA.     

Effects of amitraz on cytochrome P450-dependent monooxygenases and estrogenic activity in MCF-7 human breast cancer cells and immature female rats.

This study investigated the ability of amitraz, a formamidine insecticide, to induce cytochrome P450-dependent monooxygenases and to disrupt estrogenic activity in human breast cancer MCF-7 cells and immature female rats. In MCF-7 cells, treatment with 10 microM amitraz for 24 h increased 7-ethoxyresorufin O-deethylase activity in cell homogenate. Treatment of MCF-7 cells with 1 and 10 microM amitraz for 3 h replaced previously bound [(3)H]17beta-estradiol (E(2)) from estrogen receptors. Treatment with 0.1 and 1 microM amitraz for 2 days inhibited [(3)H]thymidine incorporation into the DNA of MCF-7 cells while the inhibition was blocked in cells co-treated with 1 nM E(2) and amitraz. In immature female rats, treatment with 50 mg/kg amitraz intraperitoneally for 3 days increased cytochrome P450 content, 7-ethoxyresorufin, methoxyresorufin and pentoxyresorufin O-dealkylases, and benzo[a]pyrene hydroxylase activities in liver microsomes. The results of immunoblot analysis revealed that amitraz induced liver microsomal CYP1A1/2, 2B1/2B2, and 3A proteins. Treatment with 10 and 25 mg/kg amitraz for 3 days dose-dependently decreased uterine weight and peroxidase activity in immature female rats while the decreases were blocked in rats co-treated with 10 microg/kg E(2) and 10 or 25 mg/kg amitraz. These in vitro and in vivo findings suggest that amitraz induces multiple forms of P450 and exerts weak antiestrogenic activity.     

Critical review and evaluation of the uterotrophic bioassay for the identification of possible estrogen agonists and antagonists: in support of the validation of the OECD uterotrophic protocols for the laboratory rodent. Organisation for Economic Co-operation and Development

A current issue for regulatory agencies is endocrine-related modes of action such as those mediated by the estrogen, androgen, and thyroid nuclear receptors. At the national and international levels, the consensus recommendation for the assessment of such modes of action is a tiered series of in vitro and in vivo protocols. The tiered framework begins with screens for structural alerts and then moves to rapid, mechanistic in vitro screening assays, and then to in vivo screening bioassays. The objective of these screens is to identify substances that may warrant testing for endocrine-mediated adverse effects. The final framework tier as needed is to test these substances in long-term bioassays for adverse endocrine-mediated reproductive and/or developmental effects. The subject of this review, the rodent uterotrophic bioassay, is intended to be a rapid in vivo screening bioassay for possible estrogen agonists and based on the response of the estrogen-sensitive uterus. The central metric of bioassay is a statistically significant increase in the weight of the uterus after 3 consecutive days of test substance administration. The extensive background literature is summarized in this review on the mode of action underlying the bioassay and the uterine response to estrogens. The review includes the bioassay's history of development and how its employment has changed and evolved over time. The review describes two major uterotrophic bioassay versions, the intact, immature female and the mature, ovariectomized female, and the protocol factors likely to influence relevance, reproducibility, and reliability of bioassay. The emphasis of the review is the ability of the uterotrophic bioassay to identify the substances of current interest: weak estrogen agonists with binding affinities relative to the natural 17beta-estradiol in the log 0 to log -3 range. Using selected model substances having RBAs in this target range, the bioassay's performance in a hierarchical, tiered approach is evaluated, including the predictive capability of the uterotrophic bioassay based on available reproductive and developmental testing data. The review concludes that the uterotrophic bioassay is reliable and can identify substances that may act via an estrogen-mode of action, supporting the validity of the uterotrophic bioassay and its regulatory use as an in vivo mechanistic screening bioassay for estrogen agonists and antagonists.     

Weak estrogenic activity of lindane in rats

Administration of lindane (20 mg/kg . d) for 30 d to ovariectomized rats caused no significant change in the weight of the uterus, cervix, or vagina. Histological changes in these organs were comparable to those in ovariectomized control rats. Treatment with estradiol dipropionate alone and in combination with lindane induced a significant increase in the weights of these organs. Microscopically, these organs appeared normal. Lindane induced a significant increase in the glycogen content of the uterus, cervix, and vagina of ovariectomized rats compared to ovariectomized control rats, whereas a two- to fourfold increase in glycogen was observed in ovariectomized rats treated with estradiol dipropionate or estradiol dipropionate plus lindane. There was a significant increase in total erythrocytes and hemoglobin in ovariectomized rats treated with lindane, estradiol dipropionate, or lindane plus estradiol dipropionate. An increase in neutrophils with a corresponding decrease in lymphocytes was also observed in rats treated with lindane or lindane plus estradiol dipropionate.     

A dose-response study on the estrogenic activity of benzophenone-2 on various endpoints in the serum, pituitary and uterus of female rats

The tetrahydroxylated biphenyl-ketone 2,2′,4,4′-tetrahydroxybenzophenone (BP2), one of twelve benzophenone-derived UV-filters, is used in cosmetic products and in packaging materials to protect these products from light induced damage. Recently published studies showed that BP2 exerts estrogenic activity; thus, it is an endocrine active chemical. We present data from a pharmacodynamic dose-response experiment with five dosages of BP2 applied per gavage to adult ovariectomized (ovx) rats for 5 days. Estradiol-valerate (E2) served as a control compound. The uterotrophic assay, proposed by the OECD, was modified to have a broader view on endocrine activity outside the urogenital tract to prevent that undesirable actions in other organs regulated by estrogens are missed. The gene expression levels of marker genes of estrogenic action were measured by semi-quantitative RT-PCR. Metabolic parameters were assessed by determination of the serum concentrations of leptin, cholesterol, high- and low-density lipoproteins, and triglycerides in the serum. Administration of BP2 at dosages of 10–1,000 mg/kg bodyweight led to changes of these parameters comparable to the changes in the E2 group with 0.6 mg/kg bodyweight. For the observed estrogenic activities of BP2, the “no observed adverse effect levels” were determined. Additionally, the data were further analyzed using the benchmark approach. If BP2 is transcutaneously absorbed in the human, the obtained threshold values would suggest refraining from the further use of BP2 as UV-filter in cosmetic products although additional toxicological studies should be conducted to clarify possible adverse effects.     

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.     

Preliminary assessment of the skin sensitizing activity of selected rodent carcinogens using the local lymph node assay

It has been demonstrated previously that there exists an incomplete correlation between the skin sensitizing potential of chemicals and their mutagenic properties as judged by activity in the Salmonella mutation assay. More recently, it has been proposed that there may exist a broader association between carcinogenicity in rodents (including non-genotoxic carcinogenesis) and skin sensitizing activity. To explore further these putative relationships we have here examined the skin sensitizing potential of two non-genotoxic rodent carcinogens which are generally considered not to represent a carcinogenic hazard in humans (limonene and saccharin) and of three genotoxic rodent carcinogens (vinylidene dichloride, ethyl acrylate and bisphenol A diglycidyl ether). For this purpose we have used the local lymph node assay (LLNA), a method for the identification and characterization of skin sensitizing chemicals that has recently been recognized as a stand-alone method for hazard identification purposes. Activity in the LLNA was compared with the results of Salmonella tests conducted previously. This small series of investigations reveals that there exists no general relationship between skin sensitizing potential and rodent carcinogenicity. Furthermore, although a general correlation does exist between mutagenic activity and skin sensitization, this association is not universal and activity in the Salmonella mutation assay does not necessarily imply skin sensitizing potential. Collectively these data suggest that it is inappropriate currently to recommend the use of skin sensitization tests as an adjunct to conventional approaches to the evaluation of potential carcinogenicity.