availability of in vitro vitellogenin assay for screening...

Environmental Sciences, 13, 3 (2006) 161–183 T. Iguchi et al. 161

161

MYU Tokyo

ES619

Availability of In Vitro Vitellogenin Assay forScreening of Estrogenic and Anti-Estrogenic

Activities of Environmental Chemicals

Taisen Iguchi*, Fumi Irie1, Hiroshi Urushitani2, Osamu Tooi2,Yukio Kawashima3, Mike Roberts4, Leif Norrgren5 and Thomas H. Hutchinson6

National Institutes of Natural Sciences, National Institute for Basic Biology, Okazaki Institute forIntegrative Bioscience, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan

1Environmental Health and Safety Division, Environmental Health Department, Ministry of theEnvironment, Government of Japan, 1-2-1 Kasumigaseki, Tokyo 100-8975, Japan

2Biotechnology Research Laboratory, Towa Kagaku Co. Ltd., 3-13-26 Kagamiyama,Higashihiroshima-shi, Hiroshima 739-0046, Japan

3Japan NUS Co. Ltd., Loop-X, 3-9-15 Kaigan, Minato-ku, Tokyo 108-0022, Japan4DEFRA Chemicals and GM Policy Division, 4/F4 Ashdown House,

123 Victoria Street, London SW1E 6DE, UK5Swedish University of Agricultural Sciences,

Faculty of Veterinary Medicine and Animal Science,Department of Biomedical Sciences and Veterinary Public Health,

Division of Pathology, Pharmacology and Toxicology, 750 07 Uppsala, Sweden6AstraZeneca Global Safety, Health and Environment, Brixham Environmental Laboratory,

Freshwater Quary, Brixham, Devon TQ5 8BA, UK

(Received January 20, 2006; accepted March 24, 2006)

Key words: vitellogenin, hepatocyte culture, fish, amphibian, avian

Vitellogenin (VTG) protein, VTG mRNA, other egg yolk proteins, vitelline envelopeproteins and their mRNAs are produced in the liver of oviparous species by stimulation ofendogenous estrogen and exogenous estrogenic chemicals. The VTG assay based onenzyme-linked immunosorbent assay (ELISA) has been widely used for many fish speciesto screen estrogenic and anti-estrogenic activities of chemicals and sewage effluents usingimmature fish and/or male fish. In order to reduce the number of fish for screening ofestrogenicity and anti-estrogenicity of chemicals, primary cultured fish hepatocytes can beused. In fact, primary cultured hepatocytes have been successfully used for the detection ofestrogenic and anti-estrogenic activities of environmental chemicals in selected OECD fishspecies, e.g., medaka (Oryzias latipes) and rainbow trout (Oncorhynchys mykiss) together

*E-mail: [email protected]

162 Environmental Sciences, 13, 3 (2006) 161–183 T. Iguchi et al.

with other fish species such as Atlantic salmon (Salmo salar L.), Siberian sturgeon(Acipenser baeri), tilapia (Oreochromis mossambicus), carp (Cyprinus carpio), bream(Abramis brama), Carassius auratus, silver eel (Anguilla anguilla L.), and channel catfish(Ictalurus punctanus). In terms of hepatocyte assays relating to other taxa, these includefrogs such as Xenopus laevis and the common green frog (Rana esculenta), chickens (Gallusdomesticus) and herring gulls (Larus argentatus). VTG mRNA measurement by quantita-tive reverse transcription-polymerase chain reaction has also been successfully applied inthe primary cultured hepatocytes of various species.

1. Introduction

There are a variety of testing methods for assessing hazardous chemicals based on animalmodels.(1) In recent years, there has been growing public pressure to minimize the use ofvertebrates in ecotoxicity testing. It was concluded at the 13th Meeting of the ScientificGroup on Methodologies for the Safety Evaluation of Chemicals that progress towards theobjective of minimizing testing that causes suffering would be served by the followinginitiatives: a clear definition of goals and strategies when undertaking testing procedures;development of alternative assays, including in vitro test systems, based on new technology;development of nondestructive assays for vertebrates (e.g., biomarkers) that do not causesuffering; selection of most appropriate species, strains, and developmental stages fortesting procedures (but no additional species for basic testing); better integrated and moreflexible testing procedures incorporating biomarker responses, ecophysiological concepts,and ecological endpoints.(2) The use of fish cells in ecotoxicology was recommended in theECVAM Workshop.(3)

Vitellogenin (VTG) is a phospholipoglycoprotein precursor of egg yolk protein that isnormally produced by sexually active females of all oviparous species. The production ofVTG is controlled by the interaction of endogenous estrogens with the estrogen receptor(ER). Both field and laboratory studies have shown the values of VTG as a rapidly induciblebiomarker for estrogens and anti-estrogens in both adult and juvenile fish.(4–8) Because malefish maintain the capacity to produce VTG in response to stimulation by ER agonists, theproduction of VTG in males and juvenile fish has been successfully exploited as a specificbiomarker for exposure to estrogenic compounds in a variety of OECD fish species, notablymedaka (Oryzias latipes), zebrafish (Danio rerio) and fathead minnow (Pimephalespromelas).(9–15) Useful features of VTG induction as a biomarker are the specificity forestrogens, sensitivity and magnitude of the resulting response; plasma VTG may increaseby up to a million-fold, from ng per ml to mg per ml concentrations.(16) VTG Assays areavailable for a wide range of fish species.

Enzyme-linked immunosorbent assay (ELISA) provides a relatively sensitive andspecific measurement technique for assessing VTG concentration in plasma or liverhomogenates. ELISA using polyclonal and/or monoclonal VTG antibodies could be auseful tool for assessing VTG production in ecotoxicological tests for the endocrinedisrupting activity of chemicals.(12,17) The validation of a method for quantifying VTG isessential to obtain accurate results in all laboratories that will conduct this assay. One reasonfor implementing a standardized VTG assay is the need to perform life-cycle assays which


would routinely use VTG measurement as an endpoint. Ideally, both a standard antibodyand a standard VTG protein should be broadly available for all laboratories performing life-cycle testing. For screening, as well as in studies of wildlife fish (e.g., roach, trout, flounder),the measurement of VTG should be based on a quantitative, validated method. Conse-quently, information concerning the intra-assay and interassay variability of the methodused in a given laboratory to measure VTG should be available.

ELISA was validated for the measurement of VTG in the cyprinid model species,medaka. Both polyclonal antibody- and monoclonal antibody-based kits showed very highcorrelation between VTG values in unknown samples when standardized VTG protein wasused.(18) This finding was confirmed by five laboratories that participated in the Japanesering test. The use of one gold standard VTG is essential to measure VTG values using ELISAkits in future OECD ring tests to screen for estrogenic chemicals, and should cover the threemost popular OECD species (medaka, zebrafish and fathead minnow).

Measurement of VTG mRNA has recently also been used for quantifying the potency ofestrogens(19–21) and has proved to be equally as effective in this capacity as VTG protein.(22–24)

Partial or full-length VTG cDNAs have been cloned in 20 different species of fish (NCBIdatabase) providing potential for a wide application of the VTG transcript(s) as an estrogenbiomarker.(25) The VTG mRNA transcript is induced rapidly (within a few hours of estrogenexposure) providing the potential to develop its use for very short-term screens. VTGprotein, in contrast to VTG mRNA, can be measured nondestructively in fish, favoring it foruse in monitoring estrogen exposure in wild populations and for studies requiring repetitivesampling from the same individual. Under maximal stimulation, the VTG protein alsoundergoes up to a 1000-fold higher level of induction compared with the VTG mRNAtranscript.(21) In some fish species including rainbow trout, more than one VTG has beenidentified.(26,27) In zebrafish, where the full genome has been sequenced, 9 distinct VTGmRNAs have been identified. Two subtypes of VTG (1 and 2) have been shown to havedifferent sensitivities to weak xenoestrogens in medaka.(19)

In addition to VTG, vitelline envelope proteins (VEPs) have been detected in fish byestrogen exposure.(28,29) These glycoproteins (VEP1, 2 and 3) are egg envelope componentsthat form the chorion of the developing egg. VEPs are normally synthesized in femalesduring oogenesis. VEPs are synthesized following exposure to xenoestrogens in malefish.(28,30) However, VEPs are extremely hydrophobic proteins and difficult to measure usingconventional techniques, whereas VEP mRNA can be measured with relative ease for thecloned sequence. VEP mRNA has been shown to be responsive to estrogen exposure inseveral fish species.(21,30,31) However, in the Artic char (Salvelinus forntinalis), productionof VTG and VEP is not mediated via the same mechanism, as VEP induction can be regulatedby cortisol in addition to estrogens.(32)

In order to reduce the number of fish for screening chemicals using the VTG assay,cultured hepatocytes and liver slices can be used in in vitro VTG assays. For the screeningassay using hepatocytes in culture, the following points need to be clarified: 1) sensitivityof cultured hepatocytes to estrogenic chemicals; 2) method of primary culture; 3) cultureconditions of hepatocytes such as medium, serum, temperature, growth factors; 4) availabil-ity of hepatocyte cell lines.(33) In this report, a VTG ELISA assay and a VTG mRNAexpression study as well as a VEP assay using cultured hepatocytes and liver slices describedin published articles are summarized.


2. Hepatocyte Culture

Primary fish hepatocyte culture has been used for studying metabolic activity(34,35) as wellas mechanisms of chemical toxicity in vitro,(36,37) since the liver is a major site of metabolismand a hormone target site. The significant advantages of hepatocyte cultures over the useof whole fish are the following: 1) the in vitro cultures allow the role of selected parametersto be investigated in a well-defined, controlled environment, and 2) a single fish can yieldmillions of cells, allowing numerous parameters to be investigated without needing tosacrifice large numbers of animals, depending on the fish species. Most prominently usedfor hepatocyte isolation is rainbow trout (Onchorhynchys mykiss). For example, rainbowtrout (no information of sex of donor fish) hepatocytes have been employed to study theinduction of cytochrome CYP1A on exposure to dioxin and related compounds, acting asa ligand of the aryl hydrocarbon receptor (AhR),(38–41) and to study estrogenic effects due tothe in vitro expression of an active estrogen receptor (ER) and the inducibility of typicaldownstream effects such as VTG synthesis.(4,42)

3. Fish

3.1 Rainbow trout (Oncorhynchys mykiss)Hepatocytes were isolated from juvenile female rainbow trout by a two-step perfusion

method, diluted in serum-free Leibovitz’s L-15 medium and seeded in 96-well plates keptin an ambient atmosphere at 15°C.(43) The cells were then cultured for one day in growthmedium containing the vehicle or different concentrations of test chemicals. Half of themedium was changed after two days, and the cells re-exposed for an additional two-dayperiod before the medium was removed and frozen at –80°C. The frozen medium wasthawed and analyzed for VTG using ELISA.(44) Various in vitro screening methodscomprising 1) competitive binding to ERs derived from a human breast cancer cell line,MCF-7 (hER) and rainbow trout (rtER), 2) a proliferation assay with MCF-7 cells (E-SCREEN) and 3) induction of VTG (rtVTG) in isolated rainbow trout hepatocytes.(45) Theresults showed substantial differences in assay sensitivity for potent estrogens such as 17 -estradiol (E2), diethylstilbestrol (DES) and zearalenone (ranking order of sensitivity: E-SCREEN>hER=rtER=rtVTG). 4-n-Nonylphenol (NP) and bisphenol A (BPA) had higherrelative binding affinity to the hER, whereas 4-t-butylphenol and 4-n-butylphenol showedthe highest affinity for rtER. Zearalenone and 4-t-butylphenol showed a higher relativepotency in the E-SCREEN than in the rtVTG assay, whereas alkylphenols and 4-t-buty-nitrobenzene were most potent in the rtVTG assay. These results suggest that interspeciesdifferences are largely due to inter-assay variation of the ER-dependent cellular responses,whereas binding to the ER is similar in humans and rainbow trout.

Rainbow trout hepatocytes were isolated from male fish (sex of fish was not identified)using a two-step collagenase perfusion technique.(46) BSA inclusion in the wash solutionimproved the viability of rainbow trout hepatocytes. Cells were plated at a density of 1×106

viable cells/ml/well of serum-free and phenol red-free culture medium in 24-well platespreviously coated with Matrigel (basement membrane matrix extracted from mouse sar-coma, phenol red-free), which contains laminin, collagen type IV and heparin sulfate


proteoglycans. Matrigel greatly enhanced the attachment of rainbow trout hepatocytes.(47)

Cells were cultured in L-15 or M199, the latter of which was supplemented with L-glutamine, 3.7 mM HEPES, 4.2 mM NaHCO3, and 0.9 mM CaCl2. Both culture mediacontained 1% of a mixture of penicillin (10,000 U) and streptomycin (10 mg/ml). Cells wereincubated in an atmosphere of air at 19°C. Using this culture system, two fluorescentindicator dyes, alamar blue, which indicates the metabolic activity of a cell, and 5-carboxyfluorescein diacetate actoxymethyl ester, which indicates an indirect measure of cellmembrane integrity, have been successfully used for studying the viability of monolayersof primary rainbow trout hepatocytes without affecting gene expression. E2 (10–8 M) alsoinduced VTG mRNA/protein as an indicator of hepatocyte-differentiated function.(46)

Two-year-old male rainbow trout were used as donors for primary hepatocyte cultures.Fish were anesthetized with benzocaine and hepatocytes were isolated as described.(48) ForVTG mRNA determination, primary cultures were harvested after 96 h of exposure. Theestrogenic activities of two municipal sewage treatment plant effluents and Rhine watersampled at Worms were investigated using in situ experiments measuring hepatic VTGexpression from caged rainbow trout, a yeast estrogen screen, an ER luciferase assay withHEK 293 cells, primary rainbow trout hepatocytes and chemical analysis.(49) VTG mRNAexpression in male rainbow trout exposed for two weeks was elevated. All three in vitrobioassays indicated the estrogenic activity of Rhine water. Chemical analyses identified upto 5.6 ng/l E2, 19 ng/l estrone (E1), 1.5 ng/l 17 -ethynylestradiol (EE2) and the sum of fecal-and phyto-steroids, resorcyclic lactones and flavonoids (280–1200 ng/l) in effluents, and 3.9ng/l E2 and 250 ng/l of fecal- and phyto-steroids in Rhine water.

On the basis of the use of hepatocyte cultures, Pelissero et al.(42) reported that EE2 andestradiol-3-sulfate were equipotent with E2 and DES slightly less potent. Estradiol-17 -glucuronide exhibited 10% the potency of E2. Six different phytoestrogens were weaklyestrogenic, possessing approximately 1/1,000 the potency of E2, and they were as potent asthe androgens and progesterone. Tamoxifen reduced the production of VTG induced bythese phytoestrogens, androgens and progesterone. The relative estrogenic activities of 0.1 gof extracts of phytosterol, soy isoflavone, red clover, kudzu and soybean extracts weredetermined using this assay and found to be equivalent to 212, 1, 3.2, 132 and 1025 nM ofE2, respectively.(50)

Primary culture of juvenile rainbow trout (no information on sex) hepatocytes developedby Pesonen and Andersson(38) was used to determine estrogenic effects of PCBs atconcentrations of 0.01–5 M and demonstrated that hydroxyl PCBs were more estrogenicthan PCBs, and 2’,4’,6’-trichloro-4-biphenylol was the most active compound studied.(51)

Freshly isolated hepatocytes(34) from hatchery-reared male rainbow trout used for the invitro screening of the VTG-inducing capability of the effluents showed a dose-responsecurve.(52) Three weeks’ exposure to undiluted effluents caused a pronounced increase inplasma VTG in juvenile rainbow trout. A multigeneration test of zebrafish provided clearevidence of the estrogenicity of one of the effluents, the sex ratio of the offspring to favorfemales. The diluted effluent was sufficient to induce VTG production in exposed malezebrafish. The effective concentrations of the effluents tested were lower in the in vitroscreening test than in the zebrafish multigenerational test. Thus, using the in vitro approachaffords advantages of both practicality and sensitivity for an initial screening of effluents for


estrogenicity, but to ascertain significant effects, the multigenerational in vivo test isrecommended.

The estrogenicity of five brominated flame retardants (BFRs), BDE-47, BDE-99, BDE-205, PBB-153 and technical Firemaster BP-6, was analyzed by an in vitro assay, usingrecombinant yeast cells containing a human ER gene and freshly separated juvenile rainbowtrout (no information about sex of fish used) cultured hepatocytes in the VTG assay.(53) Theyeast ER assay failed to give any response to the chemicals tested, however, hepatocytes inculture showed a clear dose-response curve in the presence of all tested flame retardantsexcept Firemaster BP-6. The BFRs tested induced hepatic ethoxyresorufin-O-deethylase(EROD) activity at low test concentrations, but started to inhibit activity at higher concen-trations. The decreased detoxification capacity of the hepatocytes resulted in a decrease inthe VTG production of cells. Rainbow trout hepatocytes in culture produced a clear andsignificant VTG production in response to the presence of phytosterol mixtures.(54)

Estrogenic activity of procymidone, a dicarboximide fungicide, was evaluated usingVTG synthesis in primary cultured hepatocytes from juvenile rainbow trout as an indica-tor,(55) and it was found that 150 M precimidone and 20 M E2 induced VTG protein.Estrogenicity of organochlorine pesticides (0.0001–100 M) such as o,p’-DDT, dieldrin,aldrin, heptachlor, mirex and DDT, and E2 was assessed in rainbow trout hepatocyte culturesusing VTG as the biomarker.(56) The order of the estrogenicity of chemicals tested was:E2>o,p’-DDT> dieldrin>aldrin>DDT. The lower potency of technical grade DDT relativeto o,p’-DDT could be explained by the fact that this pesticide is a mixture of 18% o,p’-DDTand 77% p,p’-DDT, suggesting that o,p’-DDT is a stronger inducer of VTG than p,p’-DDT.Heptachlor and mirex did not induce VTG.

Flouriot et al.(57) demonstrated that the response to E2 of a monolayer culture ofhepatocytes was weaker than that in vivo and cells progressively lost their specific hormonalresponse between 5 and 10 days. However, hepatocyte aggregates with cell-cell contactswere stabilized for a period of at least one month and the levels of rtER and VTG mRNAinduced by E2 were stable and maintained at a level comparable to in vivo levels; VTGsynthesis and secretion remained fully functional for the duration of the culture. Thehepatocyte aggregates were formed as previously described(57) in DMEM/F12 (Dulbecco’smodified Eagle’s medium, nutrient mixture F12) (1:1) supplemented with 2% Ultroser SF.Five-day-old aggregates (1–2×107 cells per 5 cm diameter dish) were treated for 48 h withtest substances. The estrogenicity of 49 chemicals such as herbicides, fungicides, insecti-cides, PCBs, plasticizers/detergents and phytoestrogens was studied using VTG mRNAinduction in hepatocyte cultures and a yeast system consistently expressing rtER.(58)

Aggregates of rainbow trout hepatocytes (no information on sex) were cultured for 48 h inthe presence of 10–6 M E2 or 10–4 M chemicals. Half of the 49 chemicals tested exhibitedestrogenic activity in the two bioassays. Among the estrogenic compounds, 70% were ableto activate rtER in yeast and hepatocytes with variable induction levels according to thesystem. The estrogenic potencies of E2, -zearalanol, testosterone (T), trenbolone,trenbolone acetate, melengestrol acetate, 17 -estradiol (17 -E2), E1, 17 -epitestosterone,19-nortestosterone, androstendione, zearalanone, -zearalanol, -zearalanol, -zearalenol,

-zearalenol, EE2, DES were analyzed using VTG mRNA induction of rainbow trouthepatocyte cultures.(59) In rainbow trout hepatocyte culture, E2 strongly stimulated VTG


mRNA expression at a concentration of 1 M and the maximum was reached at 10 M. Atthese concentrations, E1, DES, EE2 and -zearalenol exhibited high estrogenic activity andinduced the maximum VTG mRNA response. Zearalenoe, -zearalanol and -zearalanoldisplayed a lower estrogenic activity and a concentration of 10 M was required to achieve60–80% of the maximal VTG mRNA induction obtained with 10 M E2.

The rtER competitive binding assays for 16 chemicals yielded calculable relativebinding affinities (RBA) from 179 to 0.0006% for 13 chemicals and partial or no binding foran additional 3 chemicals. Eleven low to no affinity chemicals (RBA<0.1%) were tested inrainbow trout liver slices to measure the induction of rtER-dependent VTG mRNA in thepresence of chemical passive partitioning and liver xenobiotic metabolism.(60) VTGinduction in slices was observed in a concentration-dependent manner for eight chemicalsthat produced complete displacement curves in binding assays, including the lowest affinitybinder with an RBA of 0.0006%. Two chemicals with only partial binding curves up to theirsolubility limit did not induce VTG. The monohydroxy metabolite of methoxychlor was theonly chemical tested that apparently bound rtER but did not induce VTG mRNA. Theseresults suggest the use of the two assays in combination for interpreting the role ofmetabolism in VTG induction, as well as the sensitivity of the assays for measuringenantiomer selective binding and ER-mediated induction.

-Sitostreol has been shown to possess estrogenic activity and to cause reproductiveeffects in various species. In fish, -sitosterol has been associated with lowered concentra-tions of sex steroid levels. Effects of -sitosterol were studied in rainbow trout.(61) Threeassays, based on different levels of cellular organization, were compared in terms of receptorbinding, and VTG induction in primary cultures of rainbow trout hepatocytes and juvenilerainbow trout following 21 days exposure. The estrogenic properties of -sitostreol wereconfirmed in all three assays. However, the in vivo assay showed additional effects notrelated to the estrogenicity of -sitosterol, demonstrating the benefit of using a variety ofassays based on multiple levels of cellular organization.

3.2 Atlantic salmon (Salmo salar L.)The induction of zona radiata (zr) proteins and VTG was studied in primary hepatocytes

from Atlantic salmon exposed to xenoestrogens and mycotoxins.(62) Cells were treated with1, 5 and 10 mM NP, o,p’-DDT, lindane ( -HCH) and BPA, which induced zr proteins andVTG in an approximate dose-dependent manner. Hepatocytes were also treated withcombinations of xenoestrogens at 1 or 2 M, resulting in elevated zr proteins and VTG,compared with single treatment. The estrogenic activity of 10, 100 or 1,000 nM mycotoxinssuch as zearalenone, -zearalenone and ss-zearalenone, with regard to zonagenesis andvitellogenesis, was determined in this system. All mycotoxins induced zr proteins and VTG,with -zearalenone being the strongest inducer. ICI 182,780 inhibited the induction of zrproteins and VTG by these xenoestrogens and mycotoxins. zr proteins were moreresponsive than VTG to both xenoestrogens and mycotoxins.

Induction of VTG synthesis in primary hepatocytes from Atlantic salmon was optimizedand validated as an estrogenic in vitro bioassay using a sensitive capture VTG ELISA.(44)

After optimization, this assay gave a sensitive and reproducible response to both endogenoussteroids (E2>>E3>E1>17 -E2) and a range of common estrogen mimics (EE2,


DES>>genistein, zearalenone>>BPA, 4-t-octylphenol (OP)> 4-n-NP, o,p’-DDT). How-ever, T and the putative estrogen mimics, namely, dieldrin and toxaphene, were not shownto be estrogenic using this hepatocyte bioassay. Estrogen-induced VTG synthesis wasefficiently inhibited by the anti-estrogen ZM189,154, suggesting that this bioassay may beused for testing both the estrogenic and the anti-estrogenic properties of chemicals.

3.3 Siberian sturgeon (Acipenser baeri)Dietary genistein in rainbow trout and sturgeon in vivo showed that sturgeon were

sensitive to 20 ppm genistein, whereas trout were not. To analyze the origin of thisinterspecies difference in sensitivity, hepatocyte culture was used with VTG production asa biomarker. E2 (1 nM to 10 M), its glucuronide and sulfate derivatives (1 nM to 5 M),EE2 (10 nM to 10 M) and DES (100 nM to 10 M) were tested. The sex of fish used inthis study was not identified in the text. The hepatocyte isolation method was adapted fromPelissero et al.(42) using collagenase perfusion. Phytoestrogens, from the isoflavone family,were tested individually at increasing doses exhibiting dose response curves for concentra-tions from 500 nM to 10 M.(63) The estrogenic potency of the isoflavones ranged differentlybetween the two species in the following order: biochanin A < daizein = formononetin <genistein < equol in trout and biochanin A < genistein < daizein < formononetin < equol insturgeon. In sturgeon, formononetin was the most potent phytoestrogen in vitro, whereasits activity was weakest in vivo. A competitive-binding assay on hepatic ER from sturgeonand rainbow trout was performed to determine the relative affinity of phytoestrogenscompared with E2.(64) For each compound, the competition with 50%-bound labeled E2(DC50) was considered in order to classify phytoestrogens according to their affinity for ER:in rainbow trout, E2 (DC50: 7 nM) > formononetin (260 nM) > genistein (570 nM > equol(5,300 nM) > didzein (9,050 nM) > biochanin A (> 100,000 nM). In sturgeon, E2 (5 nM) >genistein (220 nM) > formononetin (1090 nM) > equol (8,300 nM) > daidzein (83,000 nM)> biochanin A (> 100,000 nM). These results demonstrate that phytoestrogens, mimickingE2, can disturb the endocrine system by competing for ER. The higher sensitivity observedin vivo in the Siberian sturgeon VTG assay, compared with rainbow trout, is not due to ahigher affinity of genistein for the hepatic ER. Thus, the metabolism of phytoestrogen couldbe species-dependent and affect sensitivity.

3.4 Tilapia (Oreochromis mossambicus)After anesthetizing the tilapia with 2-phenoxyethanol, the liver was excised from the

abdominal cavity of adult fish (sex was not specified), transferred onto a watch glass andperfused with Ca2+-free hepatocyte buffer (HB: 136.9 mM NaCl, 5.4 mM KCl, 0.81 mMMgSO4, 0.44 mM KH2PO4, 0.33 mM Na2HPO4, 5.0 mM NaHCO3, ph 7.6) for 10 min at roomtemperature. The flow rate of the peristaltic pump used for perfusion was set at 2.5 ml/min.After clearing blood from the liver, the tissue was digested for 15–20 min at roomtemperature with HB-containing collagenase at a concentration of 0.3 mg/ml. The softenedliver was minced with a razor blade and filtered through 200- and then 50- m nylon meshes.The resulting cell suspension was transferred to a sterilized 50-ml centrifuge tube and thencentrifuged 4 times at 50 X g for 90 s at 10°C with HB containing 1.5 mM CaCl2. After thelast wash, the cell pellet was resuspended in L-15 medium. The isolated hepatocytes were


seeded at a density of 1×106/ml (2.1×105/cm2) in a primary plate and incubated at 25°C underair with saturated humidity. The culture medium was changed every 2 days. E2 dissolvedin ethanol was added to the culture medium after 2 days’ preculture. Culture medium wascollected from each plate and used immediately for the measurement of VTG levels byELISA.(65) Two distinct VTG proteins (VTG210 and VTG140) were purified from the bloodof E2-injected tilapia and an ELISA for each VTG was developed. In hepatocyte culture,VTG210 and VTG140 were both detected at E2 concentrations of 1×10–7 M and 5×10–7 M,respectively. There was a difference in the detection time of VTG210 and VTG140 after E2treatment. The concentration of VTG210 was nearly five to eight times higher than that ofVTG140.(65,66) The effects of 3 types of NP product from different companies on in vitro VTGproduction were compared using tilapia hepatocyte culture.(67) NP 10–3 M caused a cessationor delay of monolayer formation of hepatocytes. No cell death was observed at aconcentration of 10–4 M. The addition of E2 (10–7 M) and 2 types of NP (10–4 M) inducedVTG but not NP. Co-treatment with NP and tamoxifen reduced VTG synthesis. Theseresults suggest that NP has estrogenic potential in primary cultures of tilapia hepatocytes andacts through binding to ER and there is a difference in the induction level of VTG amongdifferent NP products. The involvement of androgens (T, 17 -methyltestosterone, MT and5 -dihydrotestosterone, DHT) in VTG synthesis was investigated using tilapia hepatocyteprimary cultures.(68) DHT (5×10–6M), but not T and MT, increased VTG synthesis in theculture medium of male hepatocyte cultures. Cotreatment with E2 and androgens ofhepatocytes increased VTG concentration in the medium, suggesting that androgens playsome roles in VTG synthesis in hepatocytes. The culture conditions of various media (L-15, Willams’ medium E (WE) and Medium 199) and temperature (23, 28 and 33°C) werecompared using hepatocytes of tilapia in the induction of VTG after E2 treatment.(69) Therate of VTG synthesis in L-15 after E2 treatment was higher than those of VTG synthesisin WE and Medium 199. A concentration of NaHCO3 of 5 mM in L-15 resulted in fastermonolayer formation of the cells and higher VTG synthesis than at 0 and 23 mM. Primaryculture of the hepatocytes at 28°C showed a higher rate of VTG synthesis than at 23 and33°C.

3.5 Carp (Cyprinus carpio) and bream (Abramis brama)An in vitro VTG assay was developed using cultured primary hepatocytes from a

genetically uniform strain of male carp and VTG level was measured by indirect competitiveELISA, using polyclonal antiserum against goldfish VTG cross-related with carp VTG.(70)

Male carp hepatocytes were freshly perfused by a two-step retrograde technique, isolatedand cultured in 96-well plates.(71) A heparinized canula was inserted in a large dorsal hepaticvein associated with the ductus hepaticus. The canula was connected to a peristaltic pumpand a portion of the liver was cleared of blood by perfusion with a Ca2+– and Mg2+– freemedium containing EDTA (0.145 M) NaCl; 5.4 mM KCl; 5 mM EDTA; 1.1 mM KH2PO4;12 mM NaHCO3; 3 mM NaH2PO4; 100 mM HEPES; pH 7.5) at a flow rate of 4.5 ml/min.After approximately 25 min, the perfusion was continued with the same medium withoutEDTA, but containing 0.26 mg/ml collagenase D and 2.5 mM CaCl2. After another 25 min,the perfused liver sections were removed, minced, and sieved through nylon mesh (200 m).Subsequently, the hepatocytes were washed three times with perfusion medium containing


2.5 mM Ca2+ and 2.5% bovine serum albumin without EDTA and collagenase. After a finalcentrifugation at 100 g, the cells were resuspended in culture medium and counted. The yieldwas 1.5 to 4×108 cells. Hepatocytes were cultured in phenol red-free DMEM/F12 medium,supplemented with 14.3 mM NaHCO3, HEPES (final concentration: 20 mM), 50 mg/lgentamycin, 1 M insulin, 10 M hydrocortisone, 2% (v/v) Ultroser-SF (steroid-free)serum, and 2 mg/l the protease-inhibitor aprotinin at pH 7.4. Cells were seeded in 96-welltissue culture plates at 1×106 cells/ml, 0.18 ml/well and maintained in air at 24°C.Hepatocytes were cultured for 5 days. One day after culture, hepatocytes attached to thewells and formed a monolayer of 50–60% confluency. At this time, the medium wasremoved and replaced with fresh medium containing test compounds or solvents only.Hepatocytes were exposed for 4 days and the medium was changed after 2 days exposure.VTG was dose-dependently induced by E2 in hepatocytes of both sexes. E2 had the lowestconcentration observed effect for VTG induction of 2 nM, an EC50 between 50 and 150 nM,and a maximum response at 2 m. BPA induced VTG in hepatocytes of both sexes at 50 and100 M. TCDD caused a reduction of E2-induced VTG synthesis in female fish hepatocytesat concentrations < 0.1 nM, making it approximately 10,000-fold more potent thantamoxifen. Carp hepatocytes were also sensitive to the induction of CYP1A activity,measured as EROD. Depending on the exposure time, 18 or 96 h, EROD EC50 values forTCDD were 27 or 6 pM, respectively. The carp hepatocyte assay, using the 96-well plateformat, offers the possibility of screening large numbers of compounds for (anti)estrogenicproperties and AhR agonist properties.

Estrogenic potencies of several xenoestrogens were determined using cultured carphepatocytes from a genetically uniform male carp strain.(72) The order of estrogenic potencywas: methoxychlor > o,p-DDT > chlordecone = BPA = 4-t-pentylphenol. Estrogenicpotencies of these compounds varied from 1×10–3 to 1×10–4 relative to E2. DES had a relativeestrogenic potency of 0.5, whereas dieldrin, -endosulfan, o,p-DDE and toxaphene (tech-nical mixture) did not induce VTG at concentrations up to 100 M.

Neither the triazine herbicides nor their metabolites induced VTG production in malecarp hepatocytes; nor did they antagonize the induction of VTG by 100 nM (EC50) E2.(73)

Freshly isolated carp hepatocytes of a genetically uniform strain of adult male carp(72)

were used. Hepatocytes were first exposed (primary exposure) to 50 or 100 M of eithermethoxychlor or BPA, and different concentrations of E1 (1 or 10 nM) or E2 (0.1 or 1 nM)for 2 days. Hepatocytes were exposed to xenoestrogens (secondary exposure) at both 2 and5 days after isolation and cultured for 8 days. ELISA was used to determine VTG levels after8 days. After primary exposure to 50 and 100 M methoxychlor, VTG production inresponse to E2 increased 4-fold, and VTG production in response to E1 doubled comparedwith VTG production without pretreatment. Primary exposure to 50 and 100 M BPAincreased the maximum VTG production in response to secondary E2 exposure by about 5-and 7-fold, respectively. Primary exposure to BPA (50 and 100 M) followed by secondaryexposure to E1 showed a 4- and 5-fold enhancement in VTG synthesis in comparison to E1exposure alone. The present results indicate that weakly estrogenic environmental chemi-cals such as methoxychlor and BPA can increase the sensitivity of carp hepatocytes toendogenous estrogens with respect to VTG synthesis.(74)

Rankouhi et al.(75) compared the sensitivity of male bream (Abramis brama) to


xenoestrogens with that of carp by measuring the effects of E2, E1, EE2, BPA, NP,methoxychlor and PCB126, PCB118, TCDD and PCDF on VTG synthesis in primaryhepatocytes. Hepatocytes of bream and carp were isolated as described previously.(72) Bycomparing the EC50 values in male bream hepatocytes: EE2 (0.1-0.2 M)<E1 (0.6-0.2 M)<E2 (1.9 M) with those of male carp hepatocytes: EE2 (0.03-0.06 M)<E2 (0.3 M)=E1(0.2-0.3 M), differences in sensitivity and ranking of the estrogenic potency of E2 and E1were identified, indicating interspecies differences. Exposure to BPA, NP, methoxychlor,and halogenated aromatic hydrocarbons did not or only weakly induced VTG. Breamhepatocytes coexposed to E2 and TCDD, PCB126 or PCDF showed a concentration-dependent inhibition of E2-induced VTG production. The IC50 values determined in breamwere as follows: TCDD (0.02–0.09 nM) < PCB126 (0.35–0.1 nM) < PCDF (2.0–0.1 nM) andin carp were as follows: TCDD (0.01 nM) < PCB126 (0.4 nM). PCB118 showed no (anti-)estrogenic response, showing similar sensitivity to these compounds.

The effects of BPA relative to eight BPA-related diphenylalkanes on ER-mediated VTGproduction in hepatocytes from male carp were studied.(76) Of the eight diphenylalkanes,only 4,4’-(hexafluoropropylidene) diphenol (BHF) and 2,2’-bis(4-hydroxy-3-methylphenyl)propane (BPRO) induced VTG., i.e., to a maximum of 3% to 4% (at 100 M) compared with8% for BPA relative to the maximum induction by E2 (1 M). BPA diglycidyl ether(BADGE) was a potent antagonist of VTG production with an IC50 of 5.5 M, virtually 100%inhibition of VTG at 20 M, and an IC50 about one-tenth that of tamoxifen (IC50, 0.6 M).2,2’-Diallyl BPA, 4,4’-(1,4-phenylene-diisopropylidene)bisphenol, BPRO, and BHF hadless inhibitory effects, with IC50 concentrations of 20-70 M, and potencies of 0.03 and0.0009 relative to tamoxifen.

3.6 Goldfish (Carassius auratus)Hepatocytes of Carassius auratus were isolated (no information on sex) and treated

according to the method described by Kim et al.(68) Cells were seeded in MICRO-TESTtissue culture plates at a density of 1×106/ml, 25 ml/well, and cultured in L-15 mediumsupplemented with 5 mM NaHCO3, 1 l/ml penicillin-streptomycin, 10 l/ml polymixin Band 0.25 g/ml fungizone. Cells were incubated at 25°C under air with saturated humidity.After 24 h, E2 was used to induce VTG at 1 nM, 10 nM and 100 nM in the medium. Themedium in each well of the culture plate was changed every 2 days and collected for VTGlevel measurement by ELISA.(77) The detection limit of the ELISA for the C.auratushepatocyte culture ranged from 1 ng/ml to a maximum of 137 ng/ml. VTG levels from theriver next to a sewage treatment works were much higher than those from the feeding streamin Korea. The C. auratus VTG bioassay could be a sensitive and useful tool for thequantification of estrogenic principles in aquatic environments. To determine the sensitivityof the in vitro tests, including a recombinant yeast ER assay, a competitive radio-receptorassay using carp hepatic ER, and assays on VTG induction in cultured hepatocytes ofrainbow trout and carp were compared using E2, EE2, 4-tert-octylphenol (OP) and BPA.(78)

Hepatocytes were isolated following a two-step perfusion technique as described for carpby Segner et al.(71) and for rainbow trout by Scholz et al.(79) Sexually immature or female fishwere used as donor animals. Among the in vitro assays, differences were observed in therelative ranking of the test substances, and in the absolute sensitivity (EC50 values), although


the interassay differences in EC50 values were within one order of magnitude. The in vitroassay tended to overestimate the relative potency of the xenoestrogens.

3.7 Medaka (Orlyzias latipes)Sandwich ELISAs of VTG and lipovitellin, a high-molecular-weight yolk protein, were

developed. The application of ELISAs was tested using primary hepatocyte cultures of adultmedaka (no information on sex).(15) Livers from several fish were placed in a sterile sieve(pore size 100 m), washed in sterile PBS, and incubated with 0.05% (w/v) collagenase for30 min as described by Cao et al.(80) After enzymatic dissociation with collagenase Hdissolved in DMEM/F12 medium, the tissues were pressed with the blunt end of a syringethrough the sieve. The cell lumps were pipetted several times and filtered through a secondsieve (pore size 50 m). The cells were centrifuged for 3 min at 59 g, resuspended in adefined volume of medium and counted using a hemocytometer. The hepatocytes werecultivated in a 24-well plate at a density of approximately 2.5×105 viable cells per well. Themedium (DMEM/F12) contained L-glutamine, 15 mM HEPES, 100 U/ml penicillin, 100 g/ml streptomycin, and 50 g/ml gentamycin. Ultroser SF (2% (v/v)) was prepared andpipetted onto the hepatocytes (1 ml/well). Half of the volume was changed daily. The cellswere cultivated at 25°C in a humidified atmosphere with 5% carbon dioxide. Test chemicalswere added to the primary culture after the first day of culture (i.e., after cell attachment).VTG synthesis in cultured male hepatocytes was induced by 1 and 100 nM EE2. The firstproduction of VTG was detected 6 days after EE2 exposure. In contrast, hepatocytes frommature females treated in the same manner showed VTG expression from the beginning ofcultivation and its synthesis increased and remained high at 100 nM EE2. The induction ofVTG synthesis in male medaka hepatocytes in vitro could be maintained for a least onemonth indicating viable and differentiated liver cells. The hepatocyte cultures of malemedaka in combination with the ELISA were shown to be a suitable tool for detecting andquantifying the estrogenic activity of chemicals.

A comparison of VTG gene expression and protein levels showed that in principle, bothtechniques can be used to study VTG induction in vivo (juvenile and adult males) and in vitro(primary cultures of male medaka hepatocytes). Primary hepatocytes were isolated from 27adult male fish and primary cultures were established as described by Kordes et al.(15) Cellsseeded at 2.5×105 cells/well were exposed to 27 g/l E2 and RNA was isolated from cellsafter 0, 4, and 8 days of culture and subjected to RT-PCR. For the induction of VTG in adultmale medaka, fish were exposed to 10 and 100 ng/l EE2 or 1 and 10 mg/l genistein for 4weeks. For juvenile male medaka, 26 freshly hatched male medaka larvae from a YY x XXcross were exposed for 23 days to 100 ng/l EE2. The detection of mRNA might be sufficientfor short-term in vivo and in vitro exposure studies. For long-term exposure studies thatrequires the detection of weak estrogenic chemicals and precise quantification, ELISA maybe favored.(81)

Huggett et al.(82) investigated the estrogenicity of effluents from municipal wastewaterplants using YES and medaka assays. The in vivo estrogenic activity was nearly 10-foldgreater than that determined by the YES assay. Thus, in vitro assays to screen forestrogenicity may underestimate the estrogenic potential of wastewater.


3.8 Zebrafish (Danio rerio)The estrogenicity of dichlorobenzene was evaluated using YES and zebrafish VTG

assays.(83) The results show that the estrogenic potency of Cl-substituted benzenes is strong,both in vitro and in vivo.

3.9 Sheepshead minnow (Cyprinodon variegates)Folmar et al.(84) compared the sensitivity of YES and MCF7 assays with a 13-day

exposure of sheepshead minnow.(22,85) Five chemicals, including E2, EE2, DES, NP andmethoxychlor, were compared. Based on EC50 values and VTG levels, it was concluded thatthe in vitro tests were at least 1000 times less sensitive than the VTG assay for estimatingthe estrogenic potency of NP and methoxychlor, which suggest that in vitro-based screeningprograms could potentially result in false negative evaluations.

3.10 Channel catfish (Ictalurus punctanus)An in vitro assay has been developed to screen for the estrogenic activity of single

chemicals or complex mixtures using primary hepatocyte cultures from channel catfish andELISA to detect and quantify the production of VTG.(86) The detection limit of the VTGELISA was 15–25 ng/ml medium. Fish (400–500 g body weight) were anesthetized inbuffered deionized water containing 150 ppm 3-aminobenzoic acid ethyl ester (MS-222).The liver was perfused by a peristaltic pump with 0.33 mg collagenase (d/ml) for 20 min atroom temperature (23°C). Diced liver was passed through a sterile sieve to remove allconnective tissue. The hepatocytes were resuspended in HEPES-buffered L-15 containinggentamycin (10 mg/ml) and streptomycin (2.5 mg/ml). Cells at a density of 1×106 cells/mlwere placed in 24-well polystyrene tissue culture plates. Cells were allowed to form amonolayer on the bottom of the wells. After 24 h, the media of all wells were changed withappropriate treatments being introduced. Subsequent media changes occurred at 24- or 48-h intervals over the following 4–6 days. Media removed at these time points were analyzedfor VTG content by ELISA. Using this method, the following compounds were observedto be estrogenic: DES>E2>OP>o,p’-DDT>NP. Tamoxifen and ICI-182,780 significantlyreduced VTG synthesis in cells incubated with E2 or DES.

3.11 Silver eel (Anguilla anguilla L.)A primary culture of isolated liver cells from female silver eels was developed.(87) The

hepatocytes were maintained as monolayers on poly-l-lysine-coated dishes for up to 12 daysin a defined medium alone or supplemented with E2 (from 10–8 to 10–5 M). The amount ofVTG in the cells and secreted into the medium was measured at 2-day intervals using ELISA.After 8 days of culture, when hepatocytes from control eels were used, VTG was undetect-able both in cells and in culture media, even if the culture was performed in the presence of10-5 M E2. However, VTG was detectable both in cells and in the culture media ofhepatocytes from E2-primed (250 g E2) eels. If priming was performed 24 h before theculture, the VTG synthesis significantly increased in the presence of 10–5 M E2 after 10 daysof culture but remained low. When the culture was performed 17 or 21 days after thepriming, the level of VTG was higher than that after the short priming. In hepatocytes from21-day E2-primed eels, the concentration of secreted VTG was 1.5 times higher than that in


controls, in the presence of 10–8 M E2 after 12 days of culture. E2 (10–5 M) increased VTGproduction 2.7-fold over that of controls after 4 days of culture. These results indicate thatcultured hepatocytes retain their functional capacity by synthesizing VTG in the presenceof E2. Using cultured hepatocytes from E2-primed eels, the effects of growth hormone (GH)on VTG synthesis were studied.(88) Purified salmon and homologous eel GH potentiated theVTG response to E2.

4. Amphibians

4.1 African clawed frog (Xenopus laevis)An ELISA was developed for measuring VTG in Xenopus laevis.(89) The working ranges

of the ELISA were 2–1000 ng/ml and the recovery of plasma VTG was more than 90% indilutions of more than 200-fold. The VTG-inducing activity of E2 was measured in wholeanimals and primary cultured hepatocytes. Hepatocytes from adult males were primarycultured in serum-free medium as described by Kawahara et al.(90) The cells were wellmaintained for at least 10 days without marked loss. The cells were precultured for 2–3 daysto allow them to extend to the substratum of the plates, then exposed to various concentra-tions of E2 for 6 days. The culture media harvested were examined by ELISA. More than0.1 nM E2 dose-dependently induced VTG secretion. When the VTG induction levels werenormalized by the use of a concentration ratio of VTG to albumin, the values obtained fromthree independent experiments were mutually comparable irrespective of differences in celldensity and hepatocyte preparation. Immersion of mature male animals in more than 1 nME2 induced a detectable amount of plasma VTG. VTG induction in younger animals wasless potent than in the mature animals but the youngest animals (1.5–3 g body mass) wereusable for the exposure test, irrespective of sex.

A dose-dependent increase in VTG mRNA expression level was observed in adult males.X. laevis was treated intraperitoneally with E2 at 0.05 to 5 mg/kg/day for three consecutivedays, with maximal induction observed in the group receiving 1 mg/kg/day. -Zearalenone,genistein and 4-t-OP also significantly induced VTG mRNA expression, although at higherdoses, indicating the utility of X. laevis as an amphibian model to assess the estrogenicactivity of chemicals.(91)

EROD activity was analyzed in primary hepatocytes of the common green frog Ranaesculenta as a biomarker for cytochrome P4501A induction.(92) Hepatocytes derived frommale and female frogs were exposed to several halogenated aromatic hydrocarbons, such asTCDD, PCDF, PCB126, and PCB-118, and polycyclic aromatic hydrocarbons such asbenzo[a]pyrene (BaP), chrysene, anthracene and pyrene. TCDD and PCDF inducedmaximum EROD activity but PCB-118, anthracene and pyrene did not. Chreysene and BaPresulted in median EC50 of 82-1035 nM. Exposure to TCDD, PCDF and PCB-126 resultedin EC50 values of 0.4 to 8, 0.07 to 0.7, and 3 to 133 nM, respectively, which are in the sameranges as the EC50 values of the primary hepatocytes of birds, but lower than primary rathepatocytes.


5. Birds

5.1 Chicken (Gallus domesticus) and herring gull (Larus argentatus)A bioassay based on the measurement of VTG mRNA in avian embryo hepatocyte

cultures by semiquantitative RT-PCR was developed.(93) Cell cultures were prepared fromboth chicken and herring gull (Larus argentatus) embryos and treated with the E2 analoguemoxestrol or o,p’-DDT. Hepatocyte cultures were prepared from White Leghon chickenembryos (day 20 of incubation) and herring gull embryos (day 26 of incubation) using sterileconditions according to the original method of Fisher and Marks(94) with modifications.VTG mRNA induction was observed for both moxestrol- and o,p’-DDT-treated cultures.Herring gull embryo hepatocyte cultures responded with VTG mRNA induction at moxestrolconcentrations of 1 nM compared with 10 nM in chicken embryo hepatocyte cultures. Bothherring gull and chicken embryo hepatocyte cultures responded with substantial VTGmRNA induction when treated with 10,000 nM o,p’-DDT. These results suggest that thebioassay will be useful for comparing avian embryo hepatocyte culture concentration-response data in terms of intra- and interspecies sensitivities to pharmacological estrogensor environmental contaminants.

A method was developed to measure the estrogenic and anti-estrogenic effects of variouschemicals and organochlorine extracts in chicken embryo primary hepatocyte cultures.(95)

mRNAs for the estrogen-inducible egg yolk proteins, VTG II and very low-densitylipoprotein apoprotein II (apoII), were measured by multiplex quantitative RT-PCR (Q-RT-PCR). After 48 h of exposure, both VTG II and apoII mRNA levels were induced bymoxestrol (1–1,000 nM), E2 (10–1,000 nM), o,p’-DDT (apoII: 1,000 and 10,000 nM, VTGII: 10,000 nM), 4-OP (apoII: 5–50 M, VTG II: 10–50 M), and methoxychlor (apoII: 5–50 M, VTG II: 20 and 50 M). Tamoxifen (100 and 1,000 nM) induced apoII mRNA only,and BPA was not estrogenic. Inhibition of moxestrol-mediated VTG II or apoII mRNAinduction by methoxychlor, o,p’-DDT and tamoxifen indicated that these chemicals werealso anti-estrogenic at concentrations similar to those which caused estrogenic responses.Organochlorine extracts prepared from herring gull embryo yolk sacs obtained from threeGreat Lake sites and one Atlantic coast site (reference site) did not show any estrogenicactivity. However, the same extracts from the three Great Lake sites exhibited anti-estrogenic activity. These results indicate that wild birds may be susceptible to theestrogenic or anti-estrogenic effects of environmental contaminants.

5.2 Japanese quail (Coturnix japonica)There are no publications concerning the VTG assay using hepatocyte culture in

Japanese quail, a key OECD test species for avian toxicology. However, the effects of E2on one-generation reproduction in the Japanese quail have been reported.(96) Pairs of 10-week-old Japanese quail were fed a low-phytoestrogen diet containing E2 at 0 (control), 10,100, and 1000 ppm for 6 weeks. In the E2 100- and 1,000-ppm groups, parental quailexhibited marked toxic changes including high mortality, decreased food consumption,decreased gonad weight, gross and histological toxic changes in reproductive and otherorgans, and reduced reproduction. However, no adverse effects were observed in parentalquail from the E2 10-ppm group. In parental males, serum VTG concentrations were


increased significantly in the E2 10-ppm group, indicating that serum VTG concentrationis one of the highly sensitive endpoints for evaluating estrogenic endocrine activities. In theE2 10-ppm group, various endpoints studied such as number of eggs laid, number of eggswith abnormalities, egg shell strength and thickness, fertility, early and late viabilities ofembryos, normal hatching rate, and clinical signs, mortality, viability, and body weight ofchicks at 14 days of age were not affected. However, histopathology of the chicks in the E210-ppm group revealed distinct morphological changes in the reproductive organs, such ascystic dilatation of seminiferous tubules, increased interstitial cell number in the testis, anddecreased theca cell number in the ovary.

6. Conclusion

In order to screen the estrogenic and anti-estrogenic activities of chemicals, measure-ment of the amount of VTG protein and VTG mRNA produced in the hepatocytes in cultureafter stimulation of estrogen and/or estrogenic chemicals, and chemicals in the presence ofestrogen using ELISA and QRT-PCR has been successfully performed in various oviparousspecies, including various fish species such as rainbow trout (Oncorhynchys mykiss),Atlantic salmon (Salmo salar L.), Siberian sturgeon (Acipenser baeri), tilapia (Oreochromismossambicus), carp (Cyprinus carpio), bream (Abramis brama), goldfish (Carassiusauratus), silver eel (Anguilla anguilla L.), medaka (Oryzias latipes), channel catfish(Ictalurus punctanus), frogs such as Xenopus laevis and the common green frog (Ranaesculenta), and birds such as chicken (Gallus domesticus) and herring gull (Larus argentatus).Methods of hepatocyte culture including hepatocyte isolation by two-step perfusion ofcollagenase, and culture conditions such as media, substrata, and temperature have beentested.

7. Recommendations

A serum-free cell culture system was established in the early 1980s for variousmammalian cells, including those of the mouse mammary gland(97) and mouse reproductivetracts.(98,99) Proliferation and differentiation of these organs are controlled by estrogenicstimulation in vivo. However, primary culture of epithelial cells from these organs did notproliferate in response to estrogen in a serum-free medium or even in the presence ofserum.(98,99) However, the progesterone receptor level was increased by estrogen stimulationin uterine epithelial cells.(100–102) In the fish primary hepatocyte culture, VTG production wasdetected by estrogen and estrogenic chemicals, however, almost no information concerninghepatocyte proliferation in vitro by stimulation of estrogen has been published in theliterature. After initiating fish hepatocyte cultures, VTG protein levels should be normalizedby the number of cells in the culture plate.

The following points should be carefully considered before using hepatocyte culture forscreening of estrogenic and anti-estrogenic activities of chemicals: 1) selection of fishspecies, sex and maturation stage, i.e., immature or mature, and the season of reproductionof fish needs to be considered for the isolation of hepatocytes; multiple laboratories usedmature male rainbow trout and carp, but there has been no information available to date on


the seasonal variation in response to estrogen for VTG production in vitro. 2) The size offish is critical to have a sufficient number of viable hepatocytes per fish followingcollagenase perfusion. Small fish are not suitable for collagenase perfusion and more than20 fish have been used to isolate hepatocytes from medaka. 3) Conditions of hepatocyteculture, such as whether monolayer, aggregates or slices are used, the culture medium,supplements and temperature, culture plates, and substratum all need to be assessed. 4)Culture media should not contain phenol-red and sera to avoid possible contamination ofestrogen and estrogenic chemicals. 5) The number of hepatocytes per culture well andculture period need to be optimized in order to maximize the sensitivity of ELISA and RT-PCR. 6) Selection of VTG detection method is important: ELISA can measure VTG secretedinto the medium and produced in the cells, however, a sensitive ELISA for testing fish isneeded (including intra- and interlaboratory validation data). In contrast to the measurementof VTG, the VTG mRNA assay only detects a brief snapshot of VTG gene expression.Appropriate primer sets of the VTG gene are essential. 7) The sensitivity of hepatocyteculture for the detection of VTG protein and VTG mRNA needs to be validated within andbetween laboratories before use for chemical regulation, according to the principles of theOECD Guidance Document 34 on test method validation. 8) Vitelline envelope proteins andother egg yolk proteins as well as their mRNAs, can be used as biomarkers for detectingestrogenic and anti-estrogenic activities of chemicals. 9) In a reporter gene assay system,cells transfected with the ER gene from fish as well as a reporter gene, can be used as a toolfor detecting the estrogenicity of chemicals. 10) A fish hepatocyte cell line that responds toestrogen and estrogenic chemicals needs to be established. 11) Most importantly, reductionof animal use needs to be implemented using hepatocyte cultures for the screening ofchemicals.

Acknowledgment

This work was supported by a grant from the Ministry of the Environment, Japan.

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