The mouse bioassay for the detection of estrogenic activity in rodent diets: III. Stimulation of uterine weight by dextrose, sucrose and corn starch

We have shown previously that mice fed the American Institute of Nutrition (AIN-76A) purified diet experience a significant increase in uterine:body weight (U:BW) ratios when compared to the U:BW ratios of mice fed a closed formula natural ingredient diet (Certified Rodent Chow #5002) for 7 days. The AIN-76A purified diet contains 5% corn oil and 65% carbohydrates with 50% of the carbohydrates coming from sucrose or dextrose and 15% from corn starch. The objective of this study was to determine whether the fat and carbohydrate content contributed to the unexpected uterine growth promoting activity observed in mice fed the AIN-76A diet. Estrogen bioassays were performed using CD-1 mice weaned at 15 days of age and assigned randomly to the negative control diet (Certified Rodent Chow #5002) or to the positive control diet (#5002) containing 4 or 6 ppb DES for comparison or to the test diets. The test diets were prepared by adding sucrose, dextrose, corn starch, corn oil or soybean oil to the #5002 negative control diet at 10% w/w concentration. Uterine:BW ratios were determined at 7 days post-feeding. The uterine weights and the U:BW ratios of mice fed the test diets containing dextrose, corn starch, or corn oil, were increased significantly (P less than 0.05) over those of mice fed the negative control diet. The uterine weights and U:BW ratios of mice fed the test diets containing sucrose or soybean oil also were increased over those of mice fed the negative control diet. These increases in uterine weights and U:BW ratios were similar to the increases in uterine weights and U:BW ratios of mice fed the positive control diet containing 4 ppb DES. It was concluded that the fats and carbohydrates caused preferential increases in uterine weights and in U:BW ratios and may account for the estrogen-like uterine growth promoting activity observed in mice fed the AIN-76A purified diet.     

The mouse bioassay for the detection of estrogenic activity in rodent diets: I. A standardized method for conducting the mouse bioassay

A standardized procedure was developed for conducting the mouse bioassay for detecting estrogenic activity in rodent diets. Studies were conducted with CD-1 mice to determine the appropriate weaning age and length of bioassay period. Uterine growth curves were generated from mice weaned at 15 days of age and fed a negative control diet until 28 days of age. These mice showed slow regular increases in uterine weights from 15 22 days of age followed by rapid uterine growth in some mice from 24 to 28 days of age. Estrogenic bioassays using female mice weaned at 15 days of age and fed the positive control diets containing 4 or 6 ppb diethylstilbestrol (DES) demonstrated significant (P less than 0.05) increases in uterine weight and in uterus to body weight (U:BW) ratios over those of mice fed the negative control diet without DES for 5, 7 or 9 days after weaning. In contrast, mice weaned at 17 days of age showed significant (P less than 0.05) increases in uterine weight and in U:BW ratios only at 5 days after weaning. Six ppb DES was required in the positive control diet to produce a 1.5 fold increase in the U:BW ratio over those of mice fed the negative control diet. It was concluded that mice should be weaned at 15 days of age and that the bioassay period should be terminated at 7 days, when the mice are 22 days old, for best reproducible results. The criteria for a valid bioassay should include the demonstration of a significant statistical increase in the U:BW ratios of mice fed the DES positive diet over those of mice fed the negative control diet.     

Absence of estrogenic activity in a diet that promotes estrous cyclicity in C57BL/6J mice

A breeder diet that shortens estrous cycles in mice has been reported to contain estrogenic substances, based on its ability to increase uterine weight of immature mice. However, the estrogenicity of the diet was inferred from uterine weight gain of immature mice that were intact. The increased uterine weight of mice on the breeder diet could thus have resulted from a precocious pubertal increase of endogenous estrogens induced by the diet rather than estrogenic substances in the diet. We therefore measured the estrogenicity of the breeder diet in ovariectomized animals. C57BL/6J mice were fed the breeder diet or a standard diet for 1 or 4 weeks. The breeder diet failed to increase uterine weights above control values for either treatment interval. Intact mice that were fed the breeder diet had twice the number of cycles of mice fed the standard diet, a confirmation of earlier studies. These results indicate that the breeder diet does not contain biologically significant estrogenic activity, and thus potentiates cyclicity by other means.     

Maternal diet alters exencephaly frequency in SELH/Bc strain mouse embryos

BACKGROUND: The SELH/Bc mouse inbred strain, with a high frequency of nonsyndromic, genetically-multifactorial exencephaly, is a model for human cranial neural tube defects (NTDs). Maternal diet affects risk of human NTDs. METHODS: Exencephaly frequencies in SELH/Bc embryos were compared in 8 studies in which dams were fed alternative commercial Purina diets (5015 and 5001) or semisynthetic diets, and in several studies in which maternal diet was supplemented with a specific nutrient, either in drinking water or food before and during pregnancy, or by intraperitoneal injection on E7 and/or E8. RESULTS: The exencephaly frequency in SELH/Bc embryos was 2- to 8-fold higher when the dams were fed Purina 5015 (averaging 23% exencephaly) or a semisynthetic diet modeled on Purina 5015 (averaging 28%) or NIH-31 standard diet (23%), compared with Purina 5001 (averaging 7%). The exencephaly frequency remained high (41%) on a semisynthetic diet modeled on Purina 5001. The exencephaly frequency was not reduced significantly by maternal supplementation with folic acid, nor with each of zinc, methionine, niacin, brewers' yeast, riboflavin, vitamin B12, or inositol. Nor was it reduced by maternal diets with supplemental methyl donors and cofactors or with reduced fat. CONCLUSIONS: The frequency of exencephaly in SELH/Bc embryos is strongly influenced by a specific unidentified aspect of the commercial ration Purina 5001 that prevents 55-85% of exencephaly in SELH/Bc embryos, when directly compared with an alternative commercial ration Purina 5015 or its semisynthetic mimic. This strong maternal diet effect on NTD frequency may point to novel nutritional approaches to prevention of human NTDs.     

Phytoestrogen content of purified, open- and closed-formula laboratory animal diets.

BACKGROUND AND PURPOSE: Phytoestrogens exert estrogenic effects on the central nervous system, induce estrus, and stimulate growth of the genital tract of female animals. Over 300 plants and plant products, including some used in laboratory animal diets, contain phytoestrogens. Therefore, the source and concentration of phytoestrogens in rodent diets were determined. METHODS: Twelve rodent diets and six major dietary ingredients were assayed for phytoestrogens (daidzein, genistein, formononetin, biochanin A, and coumestrol), using high-performance liquid chromatography. Three rodent diets recently formulated to reduce phytoestrogen content also were assayed. RESULTS: Formononetin, biochanin A, and coumestrol were not detected. Soybean meal was the major source of daidzein and genistein; their concentrations were directly correlated to the percentage of soybean meal in each diet. CONCLUSIONS: High, variable concentrations of daidzein and genistein are present in some rodent diets, and dietary phytoestrogens have the potential to alter results of studies of estrogenicity. Careful attention should be given to diet phytoestrogen content, and their concentration should be reported. A standardized, open-formula diet in which estrogenic substances have been reduced to levels that do not alter results of studies that are influenced by exogenous estrogens is recommended.     

Induction of vitellogenin production in male tilapia (Oreochromis mossambicus) by commercial fish diets

Mozambique tilapia, (Oreochromis mossambicus), are a euryhaline teleost and an important biological model species. Captive male tilapia frequently have high levels of the estrogen-induced yolk precursor protein vitellogenin (Vg), a common indicator of exposure to estrogenic compounds. Sex steroids are found in commercial fish diets, but relatively few studies have examined the relationship between commercial diets and Vg production. In a fasting experiment to ascertain a dietary role in male Vg production, plasma Vg was reduced to negligible levels after 2 weeks of fasting, while no change in estrogen receptor (ER) expression was seen. When male tilapia were fed a squid-based diet that replaced the commercial trout diet, plasma Vg was reduced to undetectable levels over 40 days, concomitant with significant reductions in hepatic expression of Vgs A, B, and C, and ERβ, compared with control fish fed commercial trout diet. Female tilapia fed the squid-based for 20 days had no change in these parameters. When male tilapia were fed a defined, soy-based diet, plasma Vg reduced to 20% of levels in fish given either commercial trout diet or a defined, fishmeal-based diet. Overall, results from these studies suggest that estrogens in a commercial trout diet induce vitellogenin production by increasing expression of Vg, but not ER genes in male tilapia.     

Selecting the appropriate rodent diet for endocrine disruptor research and testing studies

Selecting the optimum diet for endocrine disruptor (ED) research and testing studies in rodents is critical because the diet may determine the sensitivity to detect or properly evaluate an ED compound. Dietary estrogens can profoundly influence many molecular and cellular event actions on estrogen receptors and estrogen-sensitive genes. The source, concentration, relative potency, and significance of dietary estrogens in rodent diets are reviewed, including dietary factors that focus specifically on total metabolizable energy and phytoestrogen content, which potentially affect ED studies in rodents. Research efforts to determine dietary factors in commercially available rodent diets that affect uterotrophic assays and the time of vaginal opening in immature CD-1 mice are summarized. A checklist is provided of important factors to consider when selecting diets for ED research and testing studies in rodents. Specific metabolizable energy levels are recommended for particular bioassays. Discussions include the between-batch variation in content of the phytoestrogens daidzein and genistein, the effects of total metabolizable energy and phytoestrogens on the timing (i.e., acceleration) of vaginal opening, and increased uterine weight in immature CD-1 mice. It is concluded that rodent diets differ significantly in estrogenic activity primarily due to the large variations in phytoestrogen content; therefore animal diets used in all ED studies should ideally be free of endocrine-modulating compounds.     

Effect of estrogenic activity, and phytoestrogen and organochlorine pesticide contents in an experimental fish diet on reproduction and hepatic vitellogenin production in medaka (Oryzias latipes)

Endocrine-disrupting chemicals (EDCs) are giving rise to serious concerns for humans and wildlife. Phytoestrogens, such as daidzein and genistein in plants, and organochlorine pesticides are suspected EDCs, because their chemical structure is similar to that of natural or synthetic estrogens and they have estrogenic activity in vitro and in vivo. We assessed estrogenic activity and dietary phytoestrogen and organochlorine pesticide contents of various fish diets made in the United Kingdom, and compared them with those features of diets made in Japan that were tested in a previous study. Genistein and daidzein were detected in all of the diets. Using an in vitro bioassay, many of these diets had higher activation of estrogen beta-receptors than estrogen alpha-receptors. Organochlorine pesticides such as hexachlorobenzene, beta-benzene hexachloride (BHC), and gamma-BHC were detected in all fish diets. On the basis of these data, we investigated the effect of differing dietary phytoestrogen content in Japanese fish diets on hepatic vitellogenin production and reproduction (fecundity and fertility) in medaka (Oryzias latipes). Assessment of the effects of a 28-day feeding period on reproduction of paired medaka did not indicate significant differences in the number of eggs produced and fertility among all feeding groups. However, hepatic vitellogenin values were significantly higher for male medaka fed diet C (genistein, 58.5 +/- 0.6 microg/g; daidzein, 37.3 +/- 0.2 microg/g) for 28 days compared with those fed diet A (genistein, < 0.8 microg/g; daidzein, < 0.8 microg/g) or diet B (genistein, 1.4 +/- 0.1 microg/g; daidzein, 2.0 +/- 0.1 microg/g). Our findings indicate that fish diets containing high amounts of phytoestrogens, such as diet C, have the potential to induce hepatic vitellogenin production in male medaka, even if reproductive parameters are unaffected. Therefore, some diets, by affecting vitellogenin production in males, may alter estrogenic activity of in vivo tests designed to determine activity of test compounds added to the diet.     

Estrogenicity of the isoflavone metabolite equol on reproductive and non-reproductive organs in mice

Equol, a metabolite of the phytoestrogen daidzein, is present at significant levels in some humans who consume soy and in rodents fed soy-based diets. Equol is estrogenic in vitro, but there have been limited studies of its activity in vivo. We evaluated equol effects on reproductive and non-reproductive endpoints in mice. Ovariectomized age-matched (30-day-old) female C57BL/6 mice were fed phytoestrogen-free diets and given a racemic mixture of equol by daily injections (0, 4, 8, 12, or 20 mg [kg body weight](-1) day(-1)) or in the diet (0, 500, or 1,000 ppm) for 12 days. Mice were killed, and serum concentrations of total and aglycone equol were measured. Total serum equol concentrations ranged from 1.4 to 7.5 microM with increasing doses of injected equol, but uterine weight increased significantly only at 12 and 20 mg (kg body weight)(-1) day(-1). Dietary equol at 500 or 1,000 ppm produced total serum equol concentrations of 5.9 and 8.1 microM, respectively, comparable with those in rodents consuming certain high-soy chows; the proportion of equol present as the free aglycone was much lower with dietary administration than injections, which may be a factor in the greater biological effects induced by injections. Dietary equol did not significantly increase uterine weight. Increasing dietary and injected equol doses caused a dose-dependent increase in vaginal epithelial thickness. Uterine epithelial proliferation was increased by equol injections at 8-20 mg (kg body weight)(-1) day(-1) and 1,000 ppm dietary equol. Neither dietary nor injected equol decreased thymic or adipose weights. In conclusion, equol is a weak estrogen with modest effects on endpoints regulated by estrogen receptor alpha when present at serum levels seen in rodents fed soy-based diets, but quantities present in humans may not be sufficient to induce estrogenic effects, although additive effects of equol with other phytoestrogens may occur.     

Estrogenic activity of zearalenone and zearalanol in the neonatal rat uterus

Fusarium sp. contaminated feedstuffs elicit adverse estrogenic effects in several commercially important animal species via the mycotoxin zearalenone. An estrogenically active synthetic derivative, zearalanol, is used as an anabolic agent in cattle. Since estrogens can irreversibly alter target tissue development, we investigated the estrogenic activity of these compounds in the neonatal rat uterus. Both induced dose-dependent premature uterine growth when injected daily on postnatal days 1-5 (ED50 = 1.3 mg/kg BW). Nuclear estrogen receptor levels dramatically increased 1 hour after either a single injection on day 5 or after five daily injections. In 5-day-old animals, the translocated nuclear receptor was characterized as a single class of binding sites with a dissociation constant (KD) for estradiol (E2) of 1 nM. At 15 days, zearalanol-treated animals showed greater uterine nuclear receptor retention than zearalenone-treated animals. In 5-day-old animals, single mycotoxin doses induced five fold elevations of ornithine decarboxylase (ODC) at 6 hours. Unlike the growth response, ODC dose-response studies showed zearalanol to be about 20-fold more effective than zearalenone. Time course studies revealed that a low dose of zearalenone, but not of zearalanol, resulted in a shift in peak activity from 6 to 8 hours. These data suggest that metabolism of zearalenone may be important in short-term pharmacodynamics. In a competitive binding assay, neither compound competed [3H]E2 from the E2 binding site on alpha-fetoprotein. We conclude that the uterine growth response and ODC induction demonstrate the neonatal estrogenic action of these mycotoxins, apparently mediated via the estrogen receptor. The greater effectiveness of zearalanol in inducing ODC may be related to nuclear retention and/or zearalenone metabolism.     

The antiestrogen LY117018 is estrogenic in the fetal rat

LY117018 (LY) has high affinity for the adult rat uterine estrogen receptor, has little uterotrophic activity, and inhibits many estradiol (E2)-induced responses in the adult or immature uterus. In these studies, LY was injected into day 19 rat fetuses, with and without diethylstilbestrol (DES) or E2, to determine whether it could block the estrogen-induced teratogenesis. LY at 1, 25, or 50 micrograms/fetus failed to decrease the 15-70% incidences of oviduct malformation and cleft phallus induced by DES (2.5 micrograms/fetus) or E2 (50 micrograms/fetus). However, LY alone (1-50 micrograms/fetus) was more potent than E2 in eliciting these same urogenital malformations. LY also failed to compete in vitro for plasma protein-bound 3H-E2, and therefore, like DES, is more available than E2 for uptake into fetal tissues. Thus, in the fetus, unlike the adult, LY was an estrogen agonist, which indicates that the fetus has a very different sensitivity than the adult to estrogenic compounds.     

Survey of estrogenic activity in fish feed by yeast estrogen-screen assay

Fishes have been used as laboratory animal for research of estrogenic endocrine disrupters by many researchers. However, much less attention was paid to the possibility that compounds with estrogenic activity are present in fish diets. In order to examine this possibility, we measured the estrogenic activity in commercial fish feed by in vitro yeast estrogen-screen (YES) assay based on the binding ability of tested compounds to estrogen receptors. Estrogenic activity was detected in all the commercial fish feed examined (0.2-6.2 ng estradiol equivalent/g fish feed), some phytoestrogens (genistein, formononetin, equol and coumestrol; relative activity to estradiol, 8.6 x 10(-6)-1.1 x 10(-4) by giving a value of 1.0 to estradiol) and some androgens (testosterone, 11-ketotestosterone and 5 alpha-dihydrotestosterone; relative activity to estradiol, 3.0 x 10(-6)-1.2 x 10(-4)). Therefore, it is possible that these compounds could affect the results of in vivo estrogen assay, such as vitellogenin production in male fish, especially when fish are fed commercial feed.     

Tannins and saponin: Interaction in herbivore diets

Mice allowed to choose between diets containing tannin or saponin did not experience food intake depression, weight loss or high faeces weight to food weight ratios. Diets containing tannin produced all these effects and saponin diets resulted in weight losses and high faeces to food ratios. Mice provided with diets containing both tannin and saponin in predetermined proportions experienced weight losses similar to, or greater than, those of mice fed diets containing either toxin alone. Urinary glucuronide production by mice provided with a choice of tannin and saponin diets was less than that of mice feeding on diets containing either tannin or saponin alone. Simultaneous consumption of tannin and saponin (in the right proportions) may promote chemical interactions that inhibit the toxins' absorption from the intestinal tract. This type of interaction is likely to have influenced the evolution of herbivore feeding behaviour.     

The impact of commercial rodent diets on the induction of tumours and flat aberrant crypt foci in the intestine of multiple intestinal neoplasia mice

A large variation in spontaneous tumour development in the multiple intestinal neoplasia (Min) mouse model between laboratories has been reported. The composition of the diet might be an important factor. We examined the impact of five commercial rodent diets: the natural ingredient breeding diet Harlan Teklad 2018 (HT), the purified breeding diet AIN93G, the natural ingredient maintenance diet RM1, and the purified maintenance diets AIN93M and AIN76A, on the spontaneous intestinal tumorigenesis in the Min mouse model. The Min mice were fed one of two breeding diets during gestation and until four weeks of age, thereafter one of the three maintenance diets. Min mice bred on the breeding diet HT had significantly higher numbers and incidences of tumours in the colon, but fewer tumours in the small intestine than the breeding diet AIN93G. The maintenance diet RM1 gave a significantly higher number of small intestinal and colonic tumours and precancerous lesions called flat aberrant crypt foci (ACF) compared with the maintenance diets AIN93M and AIN76A. These findings show the importance of defining the type of diet used in experimental intestinal carcinogenesis studies, and that the diet should be taken into consideration when comparing results from different studies with Min mice.     

Evaluation of microbially enhanced soybean meal as an alternative to fishmeal in weaned pig diets

An experimental, microbially enhanced soybean product (MEPRO) was evaluated as a replacement for fishmeal (FM). Assessment of feedstuffs should include estimation of digestibility as well as pig performance and in combination with dietary additives. Digestibility values determined in growing pigs may not apply to nursery pigs; thus, standardized ileal digestibility (SID) of amino acids (AA) in MEPRO and FM were determined using 30±1.6 kg BW ileal-cannulated barrows (n=6) and 9.8±1.2 kg BW barrows (n=37; serial slaughter). Experimental diets included MEPRO, FM and nitrogen free where FM and MEPRO were included as the sole protein source. The SID of AAs was 3% to 5% lower in MEPRO than FM when fed to 30 kg pigs. The SID of arginine and methionine was greater (P<0.05) in MEPRO than FM when fed to 10 kg pigs. The SID of AAs was 12% to 20% lower in FM when fed to 10 v. 30 kg pigs but only 3% to 9% lower in MEPRO. A total of 336 barrows and gilts were weaned at 21 days of age (initial BW=6.1±0.8 kg) and used in a performance trial. Pens of pigs were assigned to one of the six experimental diets (8 pens/diet in two blocks). Treatment diets were fed in Phase I (7 days) and Phase II (14 days) with all pigs fed a common Phase III diet (14 days). Experimental diets included (1) negative control (NEG) containing corn, soybean meal and whey, (2) NEG+acidifier, (3) NEG+FM (POS), (4) POS+acidifier (POS A+), (5) NEG+MEPRO (MEPRO) and (6) MEPRO+acidifier. The FM and MEPRO were included at 7.5% and 5.0% in Phase I and II diets, respectively. Diets were formulated to meet the standard nutrient requirements for weaned pigs. Pig BW and feed disappearance was measured weekly and fecal scores were measured daily for the first 14 days post-weaning as an indicator of post-weaning diarrhea syndrome (PWDS). Performance (BW, daily gain, feed intake and gain : feed) was not significantly different among treatments. Treatment for PWDS occurred on different days in each block. Analysis of fecal score was completed separately by block. Pigs fed the NEG diets had higher (P=0.02) fecal scores than pigs fed the POS diets on days 2 and 3 (block 1) and higher (P<0.05) than pigs fed MEPRO or POS diets and diets with dietary acidifier on days 6 and 3 (block 2). The MEPRO holds promise as an alternative to FM in nursery pig diets.     

Dietary phytoestrogens accelerate the time of vaginal opening in immature CD-1 mice

The purpose of the study reported here was to determine the effects of dietary phytoestrogens on the time of vaginal opening (VO) in immature CD-1 mice, and to correlate it with phytoestrogen and total metabolizable energy (ME) contents of the diet in an effort to determine the most appropriate diets(s) for comparing or evaluating the estrogenic or antiestrogenic activity of endocrine disruptor compounds (EDC). Mice were weaned at postnatal day (PND) 15 and fed the test diets from PND 15 to 30. Vaginal opening was recorded from PND 20 to 30. The phytoestrogen content of the diet was highly predictive (P < 0.0001) of the proportion of mice with VO at PND 24. Total ME content also was significantly (P < 0.01) correlated with time of VO, although this variable was somewhat less predictive than was phytoestrogen content. Time of VO in mice was significantly (P < 0.05) accelerated in mice fed diets high in phytoestrogens, compared with those containing low phytoestrogen content. It was concluded that: dietary daidzein and genistein can significantly (P < 0.01) accelerate the time of VO in CD-1 mice; the advancement in time of VO is more highly correlated with daidzein and genistein contents of the diets than with total ME content; advancement in the time of VO is a sensitive end point for evaluating the estrogenic activity of EDCs, and should be part of the standard protocol for evaluating EDCs. Phytoestrogen-free diet(s) containing the same amount of ME should be used in bioassays that compare the time of VO, or increases in uterine weight as end points for evaluating the estrogenic activity of an EDC.     

Extrapolation of Rodent Studies on Amniotic Fluid Contaminatnts to Human Populations

If endocrine active chemicals (EACs) adversely affect human development, then there must be evidence of effects in animal models at properly scaled levels of exposure during pertinent sensitive periods as derived from quantified exposures of the human fetus. Our recent studies attempt to address both effects and exposures. First Study: Dams were gavaged from Gestation Day (GD) 14 through weaning on Post-Natal Day (PND) 21 with either corn oil alone (unexposed controls) or Low DES (0.5?mg/kg BW); High DES (5.0?mg/kg BW); GEN (15?mg/kg BW); GEN + DES (GEN at 15?mg/kg BW and DES at 0.5?mg/kg BW). No treatments affected duration of gestation, litter size or birth anogenital distance / birth body weights ((bAGD/bBW) or ratios of bAGD/cube root of bBW of pups of either sex. The ratio of weaning AGD to weaning body weight (wAGD/wBW) differed significantly between the control group and each of the estrogenic treatments in both sexes with larger wAGD/wBW values associated with each of the estrogenic treatments. Males exposed to High DES and GEN alone exhibited earlier onset of puberty. Only females in the low DES group showed an earlier onset of puberty. At 50 to 70 days of age, the ratios of male reproductive organ weights/body weight were unaffected by estrogen treatment in all groups except high DES which increased testicle weight and decreased epididymis, seminal vesicle, and prostate weights. Initial vaginal cycle lengths were affected only in the high DES group. Thus low doses of DES and GEN at levels comparable to the upper range of human exposure affect some but not all markers of sexual development. Second Study: Amniotic fluid samples obtained at routine amniocentesis between 15 and 23 weeks of gestation were assayed by gas chromatographic/mass spectrometric (GC/MS) analysis. The first group of amniotic fluid samples (n = 53) from 51 women were analysed for several xenobiotic EACs. Alpha-hexachlorocyclohexane, with a mean (± SD) concentration of 0.15 ± 0.06 (ng/ml), and p,p′-DDE, with a mean (± SD) concentration of 0.21 ± 0.18?ng/ ml, were detected in several specimens. Overall one in three amniotic fluid samples tested positive for at least one xenobiotic EAC. Another group of amniotic fluid samples (n = 62) from 56 women were analysed for phytoestrogenic EACs. The mean (± SD) concentration of daidzein and genistein in amniotic fluid were 1.14 ± 1.04 and 1.37 ± 1.00?ng/ml with maximum levels of 5.52 and 4.86?ng/ml, respectively. Overall, 26 and 34 of the samples had quantifiable levels of daidzein and genistein, respectively. Conclusions: One in three human fetuses were exposed to xenobiotic EACs and two of three human fetuses were exposed to phytoestrogenic EACs in utero. Our demonstrations that EACs have developmental effects in an animal model at levels of exposure that mimic those found in humans in North America during sensitive time-frames sustains concerns about potential adverse health effects of developmental exposures to EACs for the human fetus/neonate.     

Repellent activity of estrogenic compounds toward zoospores of the phytopathogenic fungus Aphanomyces cochlioides

Screening chemical compounds, we found that a xenoestrogen, bisphenol A, showed potent repellent activity against the zoospores of Aphanomyces cochlioides. Based on this finding, we tested a number of androgenic and estrogenic compounds (e.g. testosterone, progesterone, estradiols, diethylstilbestrol, estrone, estriol, pregnenolone, dienestrol etc.) on the motility behavior of A. cochlioides zoospores. Interestingly, most of the estrogenic compounds exhibited potent repellent activity (1 microg/ml or less by the "particle method") toward the motile zoospores of A. cochlioides. We derivatized some of the estrogens and discussed the relationship between the structure of active molecules and their repellent activity. Apparently, aromatization of the A ring with a free hydroxyl group at C-3 position of a steroidal structure is necessary for higher repellent activity. Interestingly, methylation of diethylstilbestrol (DES) yielded completely different activity i.e. both mono- and di-methyl ethers of DES showed attractant activity. Moreover, the attracted zoospores were encysted and then germinated in the presence of di-methyl ether of DES. The potential usefulness of this repellent test is discussed for the detection of estrogenic activity of naturally occurring compounds, and the possible role of phytoestrogens in host/parasite interactions. So far, this will be the first report of repellent activity of estrogenic compounds toward trivial fungal zoospores.     

Sensitivity to alcohol in mice with an altered brain fatty acid composition

Ethanol-induced sleep onset times, sleep times and blood alcohol levels upon awakening were measured in mice fed an essential fatty acid deficient, Purina Chow or unsaturated fat diet for nine months. These values in animals fed the essential fatty acid deficient and Purina Chow diets did not differ, but mice fed the unsaturated fat diet had longer sleep times and lower blood alcohol levels upon awakening than mice fed essential fatty acid deficient or Purina Chow diets. Crude brain mitochondrial fractions isolated from mice fed the essential fatty acid deficient diet had decreased levels of docosahexaenoic [22:6(n-3)] and increased levels of eicosatrienoic [20:3(n-9)], docosatrienoic [22:3(n-9)] and docosapentaenoic [22:5(n-6)] acids compared to mice fed the Purina Chow diet. The unsaturated fat diet decreased 22:6(n-3) and increased 22:5(n-6) compared to the Purina Chow dietary regimen. The longer sleep times and lower blood alcohol levels found in mice fed the unsaturated fat diet probably resulted from an artifact due to the obesity of the mice fed this diet and from the hinderance of obesity to the righting reflex (our measure of ethanol potency). We conclude that the alteration of several polyunsaturated fatty acid components in the brain has little or no influence on the sensitivity of the nervous system to alcohol.     

Cassava is not a goitrogen in mice

To examine the effect of cassava on the thyroid function of mice, we fed fresh cassava root to mice and compared this diet with low iodine diet and Purina. Cassava provided a low iodine intake and increased urine thiocyanate excretion and serum thiocyanate levels. Mice on cassava lost weight. The thyroid glands of mice on cassava were not enlarged, even when normalized for body weight. The 4- and 24-hr thyroid uptakes of mice on cassava were similar to those of mice on low iodine diets. Protein-bound [125I]iodine at 24 hr was high in mice on either the cassava or low iodine diets. The thyroid iodide trap (T/M) was similar in mice on cassava and low iodine diets. When thiocyanate was added in vitro to the incubation medium, T/M was reduced in all groups of mice; under these conditions, thiocyanate caused a dose-related inhibition of T/M. The serum thyroxine (T4) and triiodothyronine (T3) concentrations of mice on cassava were reduced compared with mice on Purina diet. Thyroid T4 and T3 contents of mice on cassava were relatively low compared with mice on Purina diet. Hepatic T3 content and T4 5'-monodeiodination in liver homogenates were reduced in mice on cassava compared with other groups. The data show that cassava does not cause goiter in mice. The thiocyanate formed from ingestation of cassava is insufficient to inhibit thyroid iodide transport or organification of iodide. The cassava diet leads to rapid turnover of hormonal iodine because it is a low iodine diet. It also impairs 5'-monodeiodination of T4 which may be related to nutritional deficiency. These data in mice do not support the concept that cassava per se has goitrogenic action in man.