The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Proceedings for TestSmart -- Endocrine Disruptors

Posters

Generation Of Two Novel Cell Lines That Stably Express Har And Firefly Luciferase Genes For Endocrine Screening

K.L. Bobseine*1, W.R. Kelce2, P.C. Hartig*1, and L.E. Gray, Jr.1
1USEPA, NHEERL, Reproductive Toxicology Division, RTP, NC, 2Searle, Reproductive Toxicology Division, Skokie, IL

The goal of this project was to develop stable cell lines which are able to detect AR agonists and antagonists for use in high-throughput in vitro screening assays. Two different cell lines were developed for this purpose, both utilizing the firefly luciferase reporter gene. The first stable line was created using CHO (Chinese Hamster Ovarian) parental cells and two plasmids. The receptor plasmid was created to express hAR and confer zeomicin-resistence. The reporter plasmid was created to express firefly luciferase under the control of the MMTV (mouse mammary tumor virus) promotor and to confer neomicin-resistence. The MMTV promotor contains sequences homologous to the hAR and hGR response elements. The CHO cells were cotransfected using a standard calcium phosphate transfection technique with an hAR-zeomicin plasmid construct and a MMTV-luciferase-neomicin plasmid construct. Cells were grown in selection media containing both gentamicin and zeocin. A second stable line was created using the MDA-MB-453 cells, a human breast cancer line which contains endogenous AR and GR. After transfection with Fugene 6 using the manufacturer's protocol and the MMTV-luciferase neomicin reporter plasmid alone, these cells were grown in selection media with gentamicin only. Resulting colonies from the CHO and MDA transfections were isolated and expanded. Colonies were screened for detectable AR/GR induction with the AR agonist, dihydroxytestosterone (DHT); the GR agonist, dexamethasone; and with the AR antagonist, hydoxyflutamide using a luciferase assay in 96-well plates. Both cell lines respond consistently to 0.1nM DHT induction over almost 100 passages.

(This does not reflect EPA policy.)

Standardization And Validation Of Adenoviral Transduction Of An AR Positive Cell Line With An MMTV-Luc Reporter For Endocrine Screening

P. Hartig, K. Bobseine, M. Cardon, C. Lambright and L. E. Gray, Jr.
USEPA, NHEERL, Reproductive Toxicology Division, RTP, NC

(Powerpoint presentation)

The discovery of xenobiotics which interfere with androgen activity has highlighted the need to assess chemicals for their ability to modulate DHT receptor binding. Previous test systems have used cells transfected with plasmid containing a reporter gene. Here we report the novel use of transduction for gene delivery and assessment of the modulation of DHT induced gene activation. Transduction, the ability of replication defective viruses to deliver biologically competent genes, is a well understood biological process which has been utilized to repair defective genes in humans as well as to express exogenous genes in rodent models. Human breast carcinoma cells (MDA-MB-453) containing endogenous copies of the AR and GR were transduced with replication defective human adenovirus type 5 containing the luciferase reporter gene driven by the AR & GR responsive glucocorticoid-inducible hormone response element found with the mammary tumor virus LTR (Ad/MLUC7). Cells were subcultured in 96 well plates, transduced with virus, exposed to chemicals, incubated 48 hrs, lysed and assayed for luciferase. Luc gene expression was induced in a dose dependent manner by DHT,E2, Dex and high concentrations of OHF, and M2, and inhibited by AR antagonist OHF, OH-DDE, HPTE and M1. E2 induced expression was blocked with OHF, but not the anti-estrogen ICI. Transduction of MDA-MB-453 cells with MMTV Luc are nearly identical to those obtained with cell lines stably expressing MMTL-Luc. In summary, this assay positively identified all chemicals examined and induction was found to be sensitive and displayed a high fold (approx 100 X) induction. Transduction can be rapidly applied to other cell lines and utilized to deliver receptors and reporter genes quickly.

(This does not reflect EPA policy).

Binding Of Steroids And Environmental Chemicals To The Rainbow Trout Androgen Receptor Alpha Expressed In COS Cells

Mary C. Cardon, L. Earl Gray. Jr., Phillip C. Hartig and Vickie S. Wilson
U.S. Environmental Protection Agency, ORD, NHEERL, Reproductive Toxicology Division, Research Triangle Park, NC

Typically, in vitro hazard assessments for the identification of endocrine disrupting compounds (EDCs), including those outlined in the EDSTAC Tier 1 Screening (T1S) protocols, utilize mammalian eceptors. However, evidence exists that fish sex steroid hormone receptors differ from mammalian receptors both structurally and in their binding affinities for some steroids and environmental chemicals. Most of the binding information available to date has been conducted using cytosolic preparations from various tissues. The goal of this study was to conduct competitive binding studies using rainbow trout androgen receptor alpha (rtAR) expressed in transfected COS cells. In this system we can investigate the binding affinities of individual receptors without the potentially confounding effects of other steroid receptors present in cytosolic tissue extracts. Saturation ligand binding and Scatchard analysis using [3H]R1881, a synthetic androgen, revealed a KD of 0.24 nM for the rtAR. In the same system, we found a KD of 2.27 nM for the human AR. Binding studies in competition with [3H]R1881 were conducted using steroids and a selection of environmental chemicals shown to bind mammalian AR. Relative order of binding affinities of natural and synthetic androgens was methyl trienolone > trenbolone >11-ketotestosterone > dihydrotestosterone (DHT) > testosterone > androstenedione. Some of our results differ from reports in the literature. For example, androstenedione was reported to be a very high affinity ligand for the AR by Wells and Van Der Kraak in their system using cytosolic brain extracts when competed with [3H]testosterone. Progesterone bound to rtAR with similar affinity to DHT whereas estradiol bound to the receptor only at high concentrations (>100 nM, 50% inhibition ~ 770 nM). The antiandrogens hydroxyflutamide (OHF), p,p'-DDE, vinclozolin and the vinclozolin metabolites, M1 and M2 were also tested in this system. OHF, a potent mammalian antiandrogen and the vinclozolin metabolite M2, bound to rtAR with similar affinity suggests a 50% inhibition of 350nM and 310 nM, respectively.

Currently we are conducting studies that compare the binding affinities of the human AR to these chemicals in the same assay. Studies such as these will facilitate the identification of EDCs that affect many species and ultimately impact future risk assessment protocols.

[This abstract does not necessarily reflect EPA policy.]

Genomic And Proteomic Basis For Interspecies Extrapolations Based Upon Estrogen And Androgen Receptor Structure And Function Among Animals

V.S. Wilson,1 G.T. Ankley,2 K. Bobseine,1 M. Cardon,1 M.P. Gooding,1,3 L.E. Gray, Jr.,1 L.J. Guillette,5 P. Hartig,1 G. Held,1 J. Korte,2 G.A. LeBlanc,3 P.D. Reynolds,1,4 J. Welch,1 and E.M. Wilson4
1USEPA, ORD, NHEERL, 1Reproductive Toxicology Division, RTP, NC; 2Mid-Continent Ecology Division, Duluth, MN; 3North Carolina State University; 4University of North Carolina Chapel Hill; 5University of Florida at Gainsville

(Powerpoint presentation)

Most in vitro hazard assessments for the screening and identification of endocrine disrupting compounds (EDCs), including those outlined in the EDSP Tier 1 Screening (T1S) protocols, use mammalian steroid hormone receptors. There is uncertainty, however, concerning differences that may exist in the binding affinities of toxicants for steroid receptors from other species. For example, metabolites of the fungicide vinclozolin have been shown to bind the androgen receptor from humans and rats, but the results of binding assays in fish are contradictory. The goal of this work is to conduct an in-depth comparison of androgen (AR) and estrogen receptor (ER) structure and function across selected species. The cDNA sequences for the rainbow trout androgen (rtAR) and estrogen (rtER) receptors were obtained from published sources. The rtAR has been transfected into and the protein expressed in COS cells. Scatchard analysis with [3H]R1881 has been completed and competitive binding assays are currently being conducted in this system. Preliminary data indicates that the binding affinity of rtAR for some chemicals appears to differ with some ligands from that of hAR. The cDNA sequence for the rtER has been subcloned into a Baculovirus expression vector and semi-purified. Competitive binding assays comparing the rtER to the hER are planned. Tissues have been collected and cDNA libraries completed for alligator testis (Alligator mississippenisis), fathead minnow viscera (Pimephales promelas), Northern Leopard frog liver (Rana pipens), mud snail body (Ilyanassa obsoleta), and Daphnia magna. Tissues have yet to be collected for Japanese quail and African reed frog. The ER and AR from the fathead minnow have both been isolated and sequences confirmed. The full coding region of the ER was isolated from the library using the rtER as a probe and full length AR was isolated using a fathead AR fragment supplied by scientists from the US EPA lab in Duluth. The alligator cDNA library is currently being screened for the ER by traditional methods using the rtER as a probe and for the AR by rtPCR with primers designed from the canary AR sequence. The frog library is currently being screened with probes targeting the most highly conserved regions of the AR and work has also begun to screen the mud snail library for any steroid hormone receptor. As receptors are identified and sequenced, future studies will include detailed comparisons of receptor sequences to known mammalian sequence information, development of expressions systems for proteins to study functionality in comparison to mammalian receptors, and development of additional in vitro assays that incorporate receptors from these species. These investigations will help resolve some of the across-species extrapolation issues associated with EDCs and, ultimately, may have a major impact on future risk assessment protocols.

Disclaimer: This abstract does not necessarily reflect USEPA policy.

Comparison Of Androgen Receptor (AR) Message Levels In Accessory Organs Of Male Rats Following Exposure To The Antiandrogens Vinclozolin And Procymidone

C.R. Wood, G.A. Held, J.S. Ostby, C. Lambright, V. Wilson, J. Furr and L.E. Gray Jr.
USEPA, NHEERL, Reproductive Toxicology Division, Research Triangle Park, NC 27711

The Food Quality Protection Act requires that the EPA consider the cumulative effects of pesticides that have a common mechanisms of action. Vinclozolin (V) and procymidone (P) are dicarboximide fungicides which appear to be antiandrogenic. In vitro, P and V act as AR antagonists inhibiting DHT induced transcriptional activation (Wong et al., 1995; Ostby et al., 1999) and they have similar antiandrogenic effects, in vivo (Ostby et al., 1999; Gray et. al., 1999; 1994). In addition, V is known to alter AR-dependent genes in vivo (Kelce et al., 1997). Short term treatment with either flutamide (F) or V increases TRPM-2 (testosterone repressed prostatic messenger) and represses C3 (prostatein subunit C3) mRNA levels (Kelce et al., 1997). Castrated males also display increased TRPM-2 and decreased C3 mRNA levels similar or to a greater extent than F and V males. The main objective of this study was to more thoroughly characterize the molecular mechanisms of toxicity of V and P. The hypothesis of this portion of the study was to determine if adult male rats exposed to P display increased AR mRNA, another androgen dependent gene, similar to V using real time RT-PCR. This method was recently developed in our laboratory in a high-throughput mode.

In this study, adult male Sprague-Dawley rats SD were castrated and given silastic 2.5 cm implants (with or without testosterone, T) in the morning and gavaged according to treatment. The treatment groups are as follows: corn oil vehicle + T implant, V (200 mg/kg/d) + T implant, P (200 mg/kg/d) + T implant and corn oil vehicle with an empty implant (no T). Rats were necropsied at 16-20 hours, 4 days, and 7 days after the dosing (using IACUC approved methods). Blood was collected for testosterone RIA and body and organs were weighed. Organ weights included liver, adrenals and kidneys, seminal vesicles, with and without fluids (SV), ventral prostate (VP), and levator ani/bulbocavernosus muscles (LABC). A small piece of the VP was saved for AR and TRPM-2 immunohistochemistry. The VP, SV and LABC were homogenized in Tri Reagent immediately after harvesting and RNA was extracted and stored in -80°C until assayed. mRNA was quantitated using real time RT-PCR using a BioRad iCycler. A dual labeled (fluorescein, TAMARA) hydrolysis probe was used to detect the AR gene. Interassay variability was controlled for using a cDNA standard curve on each plate and by sample grouping. mRNA was measured by OD260 and 100ng of each RNA sample was amplified and compared to the standard curve to determine relative copy number. Results from the real time RT-PCR of the SV mRNA show that V and P treatment increased AR mRNA levels to the same degree. But, these AR mRNA levels were slightly lower than that seen in the castrated (no T) treated group. This assay displayed extremely low variability with an intra-assay coefficient of variation (CV) of 3.5% and an interassay CV was 8.7%. These results show that the real time RT-PCR is a reproducible assay with low variability and has the potential to be used as high throughput screen in a 96 well format. They also demonstrate that V and P induce similar effects on SV AR mRNA levels, providing further evidence that these two fungicides act via a common mechanism of toxicity. After AR levels are measured in the VP and LABC, TRPM-2 and C3 mRNA levels also will be examined in these tissues. TRPM2 levels should increase in the VP after exposure to P or V as in castrated males, while C3 should decrease after V or P treatment. This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.

Scientific And Technological Support On In Vivo Mammalian Assays For The Environmental Protection Agency's Endocrine Disrupter Screening And Testing Program

J. Ostby, L.E. Gray, J. Furr, V. Wilson, C. Lambright, C. Wolf, T. Stoker, S. Laws, J. Goldman, R. Cooper
USEPA, NHEERL, Reproductive Toxicology Division, RTP, NC

(Powerpoint presentation)

In 1996, the USEPA was given a mandate to develop an endocrine screening and testing program. In 1998, the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) proposed a Tiered Screening and Testing Strategy for chemicals that act as or antagonize estrogens or androgens, or alter thyroid (EAT) or hypothalamic-pituitary-gonadal (HPG) function. Our role in the EDSTAC-EDSP process is presented in this poster. We have developed novel in vivo assays, conducted optimization experiments, provided on-site training and detailed standard operating procedures, planned standardization and validation experiments for contract laboratories and the OECD, analyzed data from these studies, and drafted reports and publications on these assays. We also have proposed strategies and experimental designs to reduce animal use (Gray et al., in press). In 1998 we drafted several of the in vivo protocols for EDSTAC including the pubertal male and female assays, the Hershberger assay, the uterotropic assay using intact juvenile or adult ovariectomized female rats and an alternative mammalian reproduction test. Many of the in vivo assays have been used in our laboratories for decades for research purposes since the 1970s. Although the EDSTAC document presented screening and testing protocols, research and scientific expertise are needed in this area and the law mandates that these assays be validated for interlaboratory use. In some cases newer, potentially more useful assays have been developed since this document was published. For these reasons, we are conducting research and providing scientific expertise to the Agency and the OECD on the development, standardization and validation of in vitro and in vivo assays for this program.

In the early 1980s the pubertal male and female rat assays for screening chemicals for reproductive effects were developed to assess the effects of di-n-butyl phthalate, vinclozolin, p,p' DDE, linuron and the estrogenic pesticides chlordecone and methoxychlor (Gray et al., 1988 a,b, 1989; Gray and Ostby, 1998; Monosson et al., 1999). We recently published in-depth, background review papers on the pubertal male and female assays (Goldman et al., 2001; Stoker et al., 2001). A similar manuscript for the Hershberger Assay is in preparation. We also are actively involved in helping the Agency develop an assay for T2 Testing which uses fewer animals than a standard multigenerational test but contains several key sensitive endpoints not in the current multigenerational test (Gray et al., 1988; ; Zenick et al., 1991; Gray et al., 1999; 2001). In 1999, we were designated as the lead laboratory by OECD and the Agency for the Hershberger assay. The EB has conducted several studies with weakly active chemicals to define the sensitivity of the assay and optimize the protocol. In Feb 2000, a draft protocol, and a strategy for standardization and validation of this assay was developed for the OECD. In Phase 1 of the evaluation of the Hershberger assay 16 laboratories generated reproducible dose-response data for testosterone propionate. A similar Hershberger study has been completed using the antiandrogen flutamide. In Phase 2, chemicals with varying potencies and mechanisms of action will be evaluated in multiple laboratories. This presentation will discuss the strengths and weaknesses of the proposed screening and testing battery and the assays within, and discuss strategies for standardizing and validating in vivo assays to evaluate their potential value as alternative assays for screening and testing. This abstract does not reflect EPA policy.