Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish

In ecotoxicology, in vitro assays with fish cells are currently applied for mechanistic studies, bioanalytical purposes and toxicity screening. This paper discusses the potential of cytotoxicity assays with fish cells to reduce, refine or replace acute lethality tests using fish. Basal cytotoxicity data obtained with fish cell lines or fish primary cell cultures show a reasonable to good correlation with lethality data from acute toxicity tests, with the exception of compounds that exert a specific mode of toxic action. Basal cytotoxicity data from fish cell lines also correlate well with cytotoxicity data from mammalian cell lines. However, both the piscine and mammalian in vitro assays are clearly less sensitive than the fish test. Therefore, in vivo LC50 values (concentrations of the test compounds that are lethal to 50% of the fish in the experiment within 96 hours) currently cannot be predicted from in vitro values. This in vitro-in vivo difference in sensitivity appears to be true for both fish cell lines and mammalian cell lines. Given the good in vitro-in vivo correlation in toxicity ranking, together with the clear-cut difference in sensitivity, the role of cytotoxicity assays in a tiered alternative testing strategy could be in priority setting in relation to toxic hazard and in the toxicity classification of chemicals and environmental samples.     

Effects of the chemically synthesized flavanone 7-(O-prenyl)naringenin-4'-acetate on the estrogen signaling pathway in vivo and in vitro

The flavanone naringenin is known to possess only weak estrogenic properties, but some of its derivatives such as 8-prenylnaringenin are potent phytoestrogens. The aim of this study was to further clarify structure-function relationships of flavanones regarding their estrogenic or antiestrogenic properties by characterizing the new chemically synthesized naringenin derivative 7-(O-prenyl)naringenin-4'-acetate (7-O-PN). A yeast based reporter gene assay and MVLN cells, a MCF-7-derived cell line that possesses a luciferase reporter gene under the control of a vitellogenin estrogen responsive element, were used to investigate estrogenic actions of 7-O-PN in vitro. Estradiol (E2) has been used as a positive control. Subsequently a 3-day rat uterotrophic assay was performed to test for estrogenic effects. In addition, mRNA expression of estrogen sensitive genes in the uteri of these rats was measured using real time rtPCR. While E2 leads to a strong dose dependent signal in the yeast based reporter gene assay and in MVLN cells, 7-O-PN shows mild E2 antagonistic properties at concentrations 10(-8) and 10(-7)M, E2 agonistic properties at 10(-6) and 10(-5)M in MVLN cells and no effects on the yeast based system. In contrast to E2 treatment, 7-O-PN treatment did not increase uterus wet weight compared to the negative control. These findings are supported by mRNA expression studies of proliferation markers. Additionally, mRNA expression studies of estrogen regulated genes revealed very strong antiestrogenic properties of 7-O-PN regarding regulation of complement C3 expression while some estrogenic effects could be observed on the expression of estrogen receptor beta, clusterin and possibly on progesterone receptor and vascular endothelial growth factor.     

Estrogen- and androgen-sensitive bioassays based on primary cell and tissue slice cultures from three-spined stickleback (Gasterosteus aculeatus)

Endocrine disrupting compounds are chemicals that may interfere with the endocrine system causing severe effects in organisms. The three-spined stickleback (Gasterosteus aculeatus L.) offers a potential for the assessment of endocrine disruption caused by a) estrogenic xenobiotics through the estrogen-dependent protein vitellogenin and b) androgenic xenobiotics through the androgen-dependent protein spiggin. The stickleback is presently the only known fish species with a quantifiable androgen and anti-androgen biomarker endpoint. In the current study, hepatocyte and kidney primary cell cultures and liver and kidney tissue slice cultures were prepared and used for detecting estrogenic or androgenic activity in vitro through the action of hormones or municipal sewage water. The results indicate that stickleback male hepatocyte cultures are suitable in detecting estrogenic activity and stickleback female kidney tissue slice cultures in detecting androgenic activity. The tested sewage water showed high estrogenic activity but no significant androgenic activity. Primary cell and tissue slice cultures isolated from the three-spined stickleback will allow simultaneously screening in vitro for potential estrogenic and androgenic activity of complex samples.     

Differential activation of wild-type and variant forms of estrogen receptor alpha by synthetic and natural estrogenic compounds using a promoter containing three estrogen-responsive elements

Structure-dependent estrogen receptor alpha (ER alpha) agonist and antagonist activities of synthetic and natural estrogenic compounds were investigated in human HepG2, MDA-MB-231 and U2 cancer cell lines. Compounds used in this study include 4'-hydroxytamoxifen, ICI 182,780, bisphenol-A (BPA), 2',4',6'-trichloro-4-biphenylol (3Cl-PCB-OH), 2',3',4',5'-tetrachloro-4-biphenylol (4Cl-PCB-OH), p-t-octylphenol, p-nonylphenol, naringenin, kepone, resveratrol, and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). Cells were transfected with a construct (pERE(3)) containing three tandem estrogen responsive elements (EREs) and either wild-type estrogen receptor alpha (ER-wt) or variants expressing activation function-1 (ER-AF1) or AF-2 (ER-AF2). The ER agonist activities of the synthetic mono and dihydroxy aromatic compounds are comparable in all three-cell lines, whereas the activities of naringenin, kepone and resveratrol are dependent on cell context and expression of wild-type or variant forms of ER alpha. In contrast, the ER antagonist activities for these compounds were highly complex and, with the exception of 3Cl-PCB-OH, all compounds inhibited E2-induced wild-type or variant ER action. Results of this in vitro study suggest that the estrogenic and antiestrogenic activity of structurally diverse synthetic and natural estrogenic compounds is complex, and this is consistent with published data that often give contradictory results for these compounds.     

Prenylation has a compound specific effect on the estrogenicity of naringenin and genistein

A variety of plant derived substances, so-called phytoestrogens (PEs), although structurally not related to steroids, produce effects similar to the mammalian estradiol. However, little is known so far about the structural requirements which determine PE activities. Taking into consideration that prenylation reactions are relatively common in plant secondary metabolism, the activity of a set of three PE derivatives of genistein and naringenin, namely genistein, 8-prenylgenistein (8PG), 6-(1,1-dimethylallyl)genistein (6DMAG), naringenin, 8-prenylnaringenin (8PN) and 6-(1,1-dimethylallyl)naringenin (6DMAN) was compared regarding structure–estrogenicity relationships in three functionally different estrogen receptor assays.Strong estrogenic activities were recorded for 6DMAN and 8PN in all assays used, while the parent compound naringenin showed only very weak estrogenicity.In contrast, in the case of genistein derivatives, only genistein itself exhibited estrogenic activity in a yeast based assay. In MVLN breast cancer cells, a bioluminescent MCF-7-derived cell line, the estrogenic activity of all three genistein derivatives was similar. Studying alkaline phosphatase activity in Ishikawa endometrial cancer cells as an estrogenic response marker revealed a similar pattern of estrogenicity of the genistein derivatives compared to the yeast based assay although a slight estrogenic effect of 6DMAG and 8PG was apparent.In summary, this study demonstrates that prenylation often found in plant secondary metabolism differentially modifies estrogenic properties of PEs depending on the basic structure of the respective PE.     

4-Hydroxytamoxifen-induced cytotoxicity and bisphenol a: Competition for estrogen receptors in human breast cancer cell lines

Summary Increasing concerns over the effects of environmental estrogens on wildlife and humans have highlighted the need for screening systems to assess potentially estrogenic effects of test compounds. As a result, in vitro screening methods such as cell proliferation assays using the estrogen-responsive human breast cancer cell line, MCF-7, have been developed. The present study describes an alternative in vitro approach for the assessment of such xenoestrogens, based on estrogenic rescue of MCF-7 cells from antiestrogen-induced cytotoxicity. This method measures the ability of various estrogenic compounds to compete with a known estrogen-receptor-mediated antihormonal drug, 4-hydroxytamoxifen, using the 1-[4,5-dimethylthiazol-2-yl]-3,5-diphenylformazan (MTT) assay to assess mitochondrial activity. Because 4-hydroxytamoxifen treatment of cells results in a dramatic decrease in mitochondrial dehydrogenase activity which is directly related to their estrogen-receptor content, inhibition of this effect with estrogenic compounds represents an estrogen-receptor interaction, or estrogenic rescue. The estrogenic compounds tested include a weak xenoestrogen, bisphernol A (BPA), and two biological estrogens, 17α- and 17β-estradiol. Competitive inhibition of 4-hydroxytamoxifen-induced cytotoxicity by BPA was compared to that of the biological estrogens. The results indicate that the biological estrogens can successfully compete with the antiestrogen in a dose-dependent manner. In addition, the assay is sensitive enough to detect estrogenic rescue by even the very weak xenoestrogen, BPA, albeit at high BPA concentrations. This simple in vitro method could be used as an alternative or second-line screen for potential xenoestrogens.     

Estrogenic, anti-estrogenic and cytotoxic effects elicited by water from the type localities of the endangered goodeid fish Girardinichthys viviparus

Girardinichthys viviparus is a Mexican endangered endemic fish species living only in Lake Texcoco (LTX), one of two extant type localities for this species. The other type locality is Lake Zumpango (LZ). LTX and LZ are fed by wastewater treated at secondary level that contains several endocrine disrupting chemicals. Our goal was to assess the estrogenic and anti-estrogenic effects elicited in G. viviparus by water from the two type localities and by these same matrices enriched with PCBs in order to understand potential damage due to increased xenobiotic levels. Estrogenic and anti-estrogenic effects were evaluated in vitro by E-screen assay in MCF-7 cells and cytotoxicity by MTT assay. PCBs were quantified in type localities. In vivo vitellogenin (VTG) induction was determined by a hybrid ELISA in adult laboratory-born fish exposed during 1, 2, 4, 8 and 16 days to LTX or LZ water in static exposure systems, and by the same matrices enriched with PCBs. We found PCBs only in LTX, but the water from both type localities elicit estrogenic and anti-estrogenic effects in vitro. Cytotoxicity was not observed in MCF-7 cells exposed to LTX or LZ water. VTG induction was higher with LTX water than with LZ water; also the response of males was greater than in females. In the PCB-enriched matrices, VTG induction in both sexes exposed to LTX water was reduced compared to un-enriched matrices. Thus, the sublethal increases in PCB levels may be hazardous to both sexes since they are linked probably to hepatotoxicity.     

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.     

Two major metabolites of 8-prenylnaringenin are estrogenic in vitro

8-prenylnaringenin (8-PN) and preparations containing 8-prenylnaringenin have been suggested for use in medicinal and cosmetic applications like hormone replacement or bust enhancement. However, the safety of application is still under considerable debate. Recently it has been shown that human liver microsomes are converting 8-prenylnaringenin to 12 metabolites, with (E)-8-(4'-hydroxyisopentenyl)naringenin (8-PN-OH) and (E)-8-(4'-oxoisopentenyl)naringenin (8-PN=O) being among the most abundant. Applying two independent in vitro test systems we demonstrate that these two metabolites of 8-prenylnaringenin are estrogenic in vitro. These results represent an important piece of information towards the discussion of safety of use of preparations containing 8-prenylnaringenin.     

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.     

Sex steroid-induced inhibition of food intake in sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>)

This study was conducted to test the sensitivity to gonadal steroids of the systems regulating food intake in sea bass. Animals were treated with silastic implants containing 17-β-estradiol or testosterone. Self-feeding was recorded for 31 days using computerized demand feeders and unfed-pellet recovery systems. Both steroids strongly decreased self-feeding levels, feed efficiency and specific growth rates. The linear growth of fish treated with testosterone was higher than in 17-β-estradiol treated fish. In the second experiment, fish were treated with lower 17-β-estradiol doses and 11-keto-androstenedione, a precursor of the main fish androgen (11-keto-testosterone). The results demonstrated a dose–response effect of estrogen and no effect of non-aromatizable androgens on food intake or growth performance. The inhibitory effect of testosterone on food intake seems to be mediated by its aromatization to estradiol, while linear growth promotion is mediated by the androgen per se. Data suggest that gonadal steroids may be involved in the seasonal feeding pattern of sea bass. The results demonstrate the sensitivity of the mechanisms regulating food intake to estrogenic compounds and point to the risk of including feed containing estrogenic substances in fish diets as well as the risk involved in exposure to “estrogenic environments”.     

Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies

Ethylenediamine (EDA) was evaluated for potential genotoxic activity using a battery in vitro and in vivo mammalian tests. The tests employed were the Chinese hamster ovary (CHO) gene mutation assay, the sister-chromatid exchange (SCE) test with CHO cells, unscheduled DNA synthesis (UDS) assays with primary rat hepatocytes and a dominant lethal study with Fischer 344 rats. EDA did not produce a positive, dose-related, mutagenic effect in either the CHO mutation assay or in the SCE test when evaluated both with and without the addition of a rat-liver S9 activation system. With hepatocytes, no positive effects of EDA upon UDS values were noted in 2 separate studies using either a scintillation counting procedure or an autoradiographic method to determine UDS activity. In a dominant lethal study, male rats fed for 23 weeks with dietary levels of EDA X 2HCl of 0, 0.05, 0.15 or 0.50 g/kg/day, and mated with 1 virgin female/week for 3 consecutive weeks, showed no dose-related or statistically significant effects upon fertility, total number of implantations/female, or the number of living and dead implants per female; marked effects upon the incidence of dominant lethal mutations were noted in the positive control group injected intraperitoneally with one dose of 0.25 mg/kg triethylenemelamine. We conclude that EDA was not genotoxic in the in vitro and in vivo mammalian test systems employed.     

Proliferation assays for estrogenicity testing with high predictive value for the in vivo uterotrophic effect

Proliferation assays based on human cell lines are the most used in vitro tests to determine estrogenic properties of compounds. Our objective was to characterise to what extent these in vitro tests provide alternatives for the in vivo Allen and Doisy test, a uterotrophic assay in immature or ovariectomised rodents with uterus weight as a crucial read-out parameter. In the present study four different human cell lines derived from three different female estrogen-sensitive tissues, i.e. breast (MCF-7/BOS and T47D), endometrial (ECC-1) and ovarian (BG-1) cells, were characterised by investigating their relative ERα and ERβ amounts, as the ERα/ERβ ratio is a dominant factor determining their estrogen-dependent proliferative responses. All four cell lines clearly expressed the ERα type and a very low but detectable amount of ERβ on both the mRNA and protein level, with the T47D cell line expressing the highest level of the ERβ type. Subsequently, a set of reference compounds representing different modes of estrogen action and estrogenic potency were used to investigate the proliferative response in the four cell lines, to determine which cell line most accurately predicts the effect observed in vivo. All four cell lines revealed a reasonable to good correlation with the in vivo uterotrophic effect, with the correlation being highest for the MCF-7/BOS cell line (R2=0.85). The main differences between the in vivo uterotrophic assay and the in vitro proliferation assays were observed for tamoxifen and testosterone. The proliferative response of the MCF-7/BOS cells to testosterone was partially caused by its conversion to estradiol by aromatase or via androstenedione to estrone. It is concluded that of the four cell lines tested, the best assay to include in an integrated testing strategy for replacement of the in vivo uterotrophic assay is the human MCF-7/BOS breast cancer cell line.     

Tools to evaluate estrogenic potency of dietary phytoestrogens:A consensus paper from the EU Thematic Network "Phytohealth" (QLKI-2002-2453)

Phytoestrogens are naturally occurring plantderived polyphenols with estrogenic potency. They are ubiquitous in diet and therefore, generally consumed. Among Europeans, the diet is rich in multiple putative phytoestrogens including flavonoids, tannins, stilbenoids, and lignans. These compounds have been suggested to provide beneficial effects on multiple menopause-related conditions as well as on development of hormone-dependent cancers, which has increased the interest in products and foods with high phytoestrogen content. However, phytoestrogens may as well have adverse estrogenicity related effects similar to any estrogen. Therefore, the assessment of estrogenic potency of dietary compounds is of critical importance. Due to the complex nature of estrogenicity, no single comprehensive test approach is available. Instead, several in vitro and in vivo assays are applied to evaluate estrogenic potency. In vitro estrogen receptor (ER) binding assays provide information on the ability of the compound to I) interact with ERs, II) bind to estrogen responsive element on promoter of the target gene as ligand-ER complex, and III) interact between the co-activator and ERs in ligand-dependent manner. In addition, transactivation assays in cells screen for ligand-induced ERmediated gene activation. Biochemical in vitro analysis can be used to test for possible effects on protein activities and E-screen assays to measure (anti)proliferative response in estrogen responsive cells. However, for assessment of estrogenicity in organs and tissues, in vivo approaches are essential. In females, the uterotrophic assay is applicable for testing ERa agonistic and antagonistic dietary compounds in immature or adult ovariectomized animals. In addition, mammary gland targeted estrogenicity can be detected as stimulated ductal elongation and altered formation of terminal end buds in immature or peripubertal animals. In males, Hershberger assay in peri-pubertal castrated rats can be used to detect (anti)androgenic/ (anti)estrogenic responses in accessory sex glands and other hormone regulated tissues. In addition to these short-term assays, sub-acute and chronic reproductive toxicity assays as well as two-generation studies can be applied for phytoestrogens to confirm their safety in long-term use. For reliable assessment of estrogenicity of dietary phytoestrogens in vivo, special emphasis should be focused on selection of the basal diet, route and doses of administration, and possible metabolic differences between the species used and humans. In conclusion, further development and standardization of the estrogenicity test methods are needed for better interpretation of both the potential benefits and risks of increasing consumption of phytoestrogens from diets and supplements.     

Intrinsic estrogenicity of some progestagenic drugs.

The intrinsic estrogenic activity of some progestins cannot be properly evaluated by using hormone responsive systems when the chosen end-points are also sensitive to progestagenic activity, usually antagonistic of estrogenic actions. We have therefore applied to the evaluation of some drugs commonly used in contraceptive and hormone replacement formulations a recently developed in vitro method to estimate estrogenic activities, which is based on measurements of the estrogen-stimulated alkaline phosphatase activity in cells of the Ishikawa-Var I human endometrial adenocarcinoma line, a response not influenced by progestins. Whereas progesterone, medroxyprogesterone acetate and danazol were found to be devoid of estrogenic activity in this assay, Org OD-14, norethynodrel, gestrinone (R 2323), norethindrone and dl-norgestrel provoked half maximal increases in alkaline phosphatase activity at concentrations (EC-50) of 7, 14, 140, 200 and 2900 nM, respectively, under conditions in which the corresponding value for estradiol was 8 pM. This intrinsic estrogenic activity can be inhibited by antiestrogens, as verified by reversing the effect of R 2323 with 4-hydroxytamoxifen. Since prostaglandin F2 alpha output by secretory endometrium is increased by estrogens and diminished by progestins, this end-point can serve to evaluate the net effect of drugs with intrinsic estrogenic and progestagenic activities. For instance, R 2323 showed estrogenic activity in this assay whereas Org OD-14 did not. The same in vitro system can be used to evaluate estrogen antagonistic activities of test compounds, using estradiol as the agonist. These in vitro systems are useful in establishing a profile of activities of a drug on a relevant human target tissue, in the screening of synthetic or natural compounds under investigation, and in studies on structure/action relationships.     

CpG oligodeoxynucleotides up-regulate antibacterial systems and induce protection against bacterial challenge in rainbow trout (Oncorhynchus mykiss)

The effects of unmethylated CpG oligodeoxynucleotides (ODN) on the mammalian immune system are relatively well studied but much less is known of their effects on the immune systems of different fish species. Here we show that CpG ODNs significantly enhance the survival of rainbow trout (Oncorhynchus mykiss) following bacterial challenge when used both as stand-alone prophylactic agents, or as adjuvants to a commercially available vaccine. They are also capable of increasing serum lysozyme activity in vivo and stimulating the production of chemoattractant factors for rainbow trout head kidney (HK) leucocytes in vitro.     

On the mutagenicity of nitroimidazoles

Regarding mutagenicity, metronidazole is one of the best-investigated compounds of the nitroimidazoles. This drug is mutagenic on bacteria, especially if base-pair tester strains are used and bacterial nitroreductases are present. The serum levels attained in man after intake of this drug are sufficient to cause mutations in bacteria. Furthermore, interaction with and binding to DNA occurs under anaerobic conditions and sometimes DNA breaks are observed. However, metronidazole does not show mutagenic activity in mammalian cells in vitro; the micronucleus test is negative and chromosome aberrations are only found under anaerobic conditions. With microbial systems the mutagenicity of 47 nitroimidazoles has been investigated. Only 4 compounds were always negative in the applied test systems. Because with base-pair tester strains mutagenicity was assessed, this class of compounds should be regarded as a base-pair mutagen. In fungi, some compounds (e.g. ZK 26173 and azathioprine) are potent mutagens, whilst with most investigated nitroimidazoles only a weak or no mutagenic activity could be detected. Somewhat similar observations have been made in tests with Drosophila melanogaster, a test for gene mutations in mammalian cells, the micronucleus test, cytogenic tests and the dominant lethal test. The reduction products of metronidazole, misonidazole and 1-methyl-2-nitro-5-vinylimidazole, cause DNA damage if the nitro group is reduced in the presence of DNA. Reduction products are formed by microbes in the gut or by mammalian cells under anaerobic conditions. No teratological effect due to metronidazole or most other nitroimidazoles has been observed. Metronidazole is carcinogenic in mice and rats, and dimetridazole in rats. Up to the present, no carcinogenic effects have been observed in man. Azathioprine is probably carcinogenic for man. It is unlikely that the therapeutic applications of the presently used nitroimidazoles, except for azathioprine, will cause an increase in the tumor incidence in man or will cause other genotoxic effects, although such effects cannot be excluded with certainty.     

Selecting appropriate animal models and experimental designs for endocrine disruptor research and testing studies

Evidence that chemicals in the environment may cause developmental and reproductive abnormalities in fish and wildlife by disrupting normal endocrine functions has increased concern about potential adverse human health effects from such chemicals. US laws have now been enacted that require the US Environmental Protection Agency (EPA) to develop and validate a screening program to identify chemicals in food and water with potential endocrine-disrupting activity. EPA subsequently proposed an Endocrine Disruptor Screening Program that uses in vitro and in vivo test systems to identify chemicals that may adversely affect humans and ecologically important animal species. However, the endocrine system can be readily modulated by many experimental factors, including diet and the genetic background of the selected animal strain or stock. It is therefore desirable to minimize or avoid factors that cause or contribute to experimental variation in endocrine disruptor research and testing studies. Standard laboratory animal diets contain high and variable levels of phytoestrogens, which can modulate physiologic and behavioral responses similar to both endogenous estrogen as well as exogenous estrogenic chemicals. Other studies have determined that some commonly used outbred mice and rats are less responsive to estrogenic substances than certain inbred mouse and rat strains for various estrogen-sensitive endpoints. It is therefore critical to select appropriate biological models and diets for endocrine disruptor studies that provide optimal sensitivity and specificity to accomplish the research or testing objectives. An introduction is provided to 11 other papers in this issue that review these and other important laboratory animal experimental design considerations in greater detail, and that review laboratory animal and in vitro models currently being used or evaluated for endocrine disruptor research and testing. Selection of appropriate animal models and experimental design parameters for endocrine disruptor research and testing will minimize confounding experimental variables, increase the likelihood of replicable experimental results, and contribute to more reliable and relevant test systems.