C. hAR Transactivation Assays Using Stable Cell Lines DESCRIPTION Transcriptional activation assay using cells stably expressing a MMTV luciferase reporter together with the hAR in a mammalian cell line such as a CV 1. DEGREE OF USE Under development, likely available in 6 months, could be automated for high throughput. DURATION 24 hours Final EDSTAC Report Chapter Five Appendices August 1998 23 K  ASSAY STABILITY Unknown to date, should be stable. DOES IT METABOLIZE TOXICANTS These cells can activate some antiandrogenic fungicides. ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED Yes, freezer, incubator, and luminometer, robot optional. ARE SPECIAL SKILLS/ TRAINING NEEDED Do not need to transfect cells, but training required and cell techniques needed. HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY Not yet, but can be easily standardized. SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Should be as sensitive, or more sensitive, than transient transfection assays because all the cells are responsive. ARE THERE KNOWN FALSE POSITIVES Theoretically, cytotoxicity may induce apparent inhibition of DHT induced effects ( this is true for many cell reporter assays of antiandrogenicity), hence, use constituitively active mutant receptor as a cytotoxicity control ( decline in luc indicates cytotoxicity) . OR FALSE NEGATIVES Toxicants requiring activation may not be detected. SPECIFICITY ARE THERE OTHER MECHANISMS TO PRODUCE THIS EFFECT ( FALSE POSITIVES) Cell death. COMMENTS Need controls in this and other similar assays for cell viability and health. Must verify toxicant stability in media.

D. Leydig Cell Culture DESCRIPTION Determination of testosterone production in purified, isolated Leydig cells. DEGREE OF USE Limited to a several/ few research laboratories. DURATION A few days ASSAY STABILITY Variable from lab to lab on degree of purification of Leydig cells and T production per cell. DOES IT METABOLIZE TOXICANTS No ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED Yes, quite a bit. ARE SPECIAL SKILLS/ TRAINING NEEDED Requires skills that are not widely available. HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY No Final EDSTAC Report Chapter Five Appendices August 1998 24 K  SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Very sensitive to SIS, less sensitive, and to insensitive to estrogens. ARE THERE KNOWN FALSE POSITIVES Cell toxicants. OR FALSE NEGATIVES Toxicants that require metabolic activation. SPECIFICITY ARE THERE OTHER MECHANISMS TO PRODUCE THIS EFFECT ( FALSE POSITIVES) Cell membrane and second messenger effects.

VII. Androgen and Anti androgen  In Vivo A. Endocrine Challenge Test ( Fail et al., 1995) DESCRIPTION Repeated observation of serum T, LH and other hormones ( with and without LH or GnRH challenge) in catheterized animal. Clearly the best way to detect altered hormone secretion in vivo. DEGREE OF USE Limited DURATION Few weeks. ASSAY STABILITY Stable, once the animal is catheterized. DOES IT METABOLIZE TOXICANTS Yes ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED Yes ARE SPECIAL SKILLS/ TRAINING NEEDED Yes, quite difficult. HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY Has not been standardized. SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS More sensitive than single determination of serum hormones levels. ARE THERE KNOWN FALSE POSITIVES Stress reduces serum T, increases prolactin and corticosterone, effect that is rarely accounted for in most studies. Hormone levels are also subject to circadian effects. COMMENTS. Excellent, but specialized. Tail bleeds can also be used for repeated sampling from the same animal if only a small amount of serum is needed. This method also is not standardized and requires a great deal of practice. Final EDSTAC Report Chapter Five Appendices August 1998 25 K  B. Super Apical Developmental Toxicity Test DESCRIPTION Expose pregnant/ lactating dam and examine hormone ( AR, ER, SIS, Ah, and T3) sensitive endpoints in progeny up to puberty. DEGREE OF USE Such a protocol has been used at EPA, CIIT, NIEHS for xenoestrogens, environmental antiandrogens, Ah receptor agonists, phthalates and antithyroidal toxicants ( PCBs and PTU). DURATION Relatively long ( two to three months) as compared to other " screens," would need to determine if it is quicker and cheaper to run this as opposed to several more focused, shorterterm assays. ASSAY STABILITY Unknown, but should be as good as any developmental test. DOES IT METABOLIZE TOXICANTS Yes ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED Yes, similar to needs for new multigen tests. ARE SPECIAL SKILLS/ TRAINING NEEDED Multiplicity of in vivo techniques are required that are currently not used in toxicology labs, but they should be coming up to speed to implement new test guidelines. HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY Not standardized as used in different labs with respect to dosing or some of the assessments. Some standardization is now taking place between different labs. SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Antiandrogencity via AR, or SIS. In male progeny, AGD, areolas, nipples, reduced weight of prostate. Higher doses of AR mediated, but not SIS, produce agenesis of prostate, undescended testis and agenesis of epididymis. Altered T production by testis, ex vivo.

Estrogenicity. Neonatal Uterine weight and gland development (?), vaginal opening, age at first estrus, vaginal cornification. Prostate size in male (?), and sperm production ( at high dosage levels). SIS or Antiestrogenicity. Pregnancy loss, delay in deliver by dam, delay in VO and possible altered ovarian hormone production ex vivo.

Antithyroid. Reduced perinatal growth and brain size. Lower serum T4 and possibly T3, elevated TSH. Also detects functional developmental alterations induced by phthalates, TCDD and etc. The following endpoints are considered to be insensitive because they take too long to assess in a screen and/ or have never been detected with a pesticide or toxic substance following developmental exposure ( i. e., for ER mediated: cancer, reduced AGD, hypospadias in male of female, undescended testes or any malformations). ARE THERE KNOWN FALSE POSITIVES. Very Apical Test used to screen for several mechanisms at once. OR FALSE NEGATIVES. Not likely, if designed properly. Final EDSTAC Report Chapter Five Appendices August 1998 26 K  C. Pubertal Development in Male Rodent ( Preputial Separation) DESCRIPTION In vivo determination of age at puberty in male rat. DEGREE OF USE Data base includes drugs and antiandrogens and estrogenic pesticides and toxic substance ( vinclozolin, pp DDE, methoxychlor, phthalates, TCDD, PCBs). Required endpoint in new multigenerational test guidelines. DURATION 20 30 days ASSAY STABILITY Varies from block to block and from LE to SD by less than two days if designed carefully. Delays of two to three days are generally significant with sample sizes of 10 12/ group. DOES IT METABOLIZE TOXICANTS Yes ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED No ARE SPECIAL SKILLS/ TRAINING NEEDED Some practice is required. HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY Has been, is almost as easy as puberty in female rat. SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Slightly less sensitive than are some developmental AR mediated alterations. Also moderately sensitive to xenoestrogens ( for ER, PPS is less sensitive than growth, but much better than spermatogenesis and dosing duration is shorter). ARE THERE KNOWN FALSE POSITIVES Yes, this is an apical assay so anti AR, ER, SIS inhibition, altered hypothalamic pituitary function and Leydig cell toxicants will all delay this developmental landmark. OR FALSE NEGATIVES Unknown SPECIFICITY ARE THERE OTHER MECHANISMS TO PRODUCE THIS EFFECT ( FALSE POSITIVES) Nonspecific, apical test. COMMENTS Noninvasive test that could be coupled with both Hershberger test and biochemical assays.

D. Hershberger Assay ( 1953) DESCRIPTION In vivo measurement of effects of antiandrogenic/ androgenic toxicants of androgen dependent tissues in peripubertal/ adult male rat. Weigh sex accessory glands and levator ani/ bulbocavernosus muscle in T implanted, castrate adult or intact juvenile male rat four to seven days after start of study. Can also measure serum T and DHT ( to discriminate ARmediated from SIS mechanisms and liver effects on metabolism), and biochemical indices ( ODC) and tissue gene expression ( TRPM2, C3). Final EDSTAC Report Chapter Five Appendices August 1998 27 K  DEGREE OF USE Extensive in drug development and recently in toxicology of antiandrogens. DURATION One week. ASSAY STABILITY Stable DOES IT METABOLIZE TOXICANTS Yes ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED Balance, scissors. ARE SPECIAL SKILLS/ TRAINING NEEDED Good necropsy and surgical skills. HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY Has been standardized and validated. SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Responds as expected to moderate dosage levels of antiandrogenic pesticides. ARE THERE KNOWN FALSE POSITIVES Yes, especially if conducted in intact peripubertal male. An apical test in intact animal. More specific in castrate but some endpoints are still affected by other mechanisms. OR FALSE NEGATIVES None known. SPECIFICITY ARE THERE OTHER MECHANISMS TO PRODUCE THIS EFFECT ( FALSE POSITIVES) Yes, prolactin, thyroid hormone, and estrogens affect some organ weights. COMMENTS One of the best short term assays for antiandrogens, along with pubertal development.

VIII. Anti androgen  Synthesis Metabolism A. Testis/ Ovary Culture In Vitro or Ex Vivo DESCRIPTION Determination of testosterone production from testicular tissue from animals treated in vivo ( ex vivo) or using in vitro dosing. Used for EDS, estrogens, antiandrogens, several other testicular toxicants, and substances that inhibit steroidogenesis. In female, minced ovary culture can be used ex vivo from pregnant ( i. e., GD 14 16) or cycling females ( proestrus for estradiol production). DEGREE OF USE A few hundred publications over the last 25 years since the advent of RIAs for testosterone. Used in other vertebrates, as well as mammals. Used by several toxicology laboratories in addition to NHEERL EPA. DURATION One day for in vitro, longer for ex vivo ( duration depends upon dosing). Final EDSTAC Report Chapter Five Appendices August 1998 28 K  ASSAY STABILITY Depends on methodology.

DOES IT METABOLIZE TOXICANTS Yes, for ex vivo, little or no metabolism for in vitro. ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED Incubator, freezer, necropsy equipment. ARE SPECIAL SKILLS/ TRAINING NEEDED Some practice, but a rather simple assay that can be learned by competent technicians in a week or less. Need to be able to conduct RIAs for T, estradiol, and progesterone. HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY Has not been standardized between labs, but could be without too much difficulty. SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Fairly sensitive. ARE THERE KNOWN FALSE POSITIVES Cell toxicants. OR FALSE NEGATIVES If metabolic activation is required then in vitro may not work. COMMENTS. These assays could be used rapidly to screen chemicals for ability to inhibit steroidogenesis.

IX. Thyroid  Intrinsic Activity A. TR Binding Assay DESCRIPTION Determines whether a chemical can alter T3 binding to its nuclear receptor. Assay is performed on isolated nuclei. Nuclei can be isolated from liver of any species. In principal, these results would reveal competition for the binding site or an allosteric effect.

DEGREE OF USE Extensive DURATION Four hours. ASSAY STABILITY Prepared nuclei are stable at  80 ¡ C for long periods. Assay is performed at RT. DOES IT METABOLIZE TOXICANTS No ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED Yes ARE SPECIAL SKILLS/ TRAINING NEEDED Yes, but training period is not extensive. Final EDSTAC Report Chapter Five Appendices August 1998 29 K  HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY Yes SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Very sensitive; can be run over broad dose range. ARE THERE KNOWN FALSE POSITIVES No, though chemicals that bind to TR in vitro may not affect TR signaling in vivo. OR FALSE NEGATIVES Conditions of the assay must be standardize because a variety of factors ( e. g., oxidation) blocks T3 binding to its receptor. In addition, solubility of lipophilic compounds may preclude binding in this in vitro system. Finally, chemicals that do not bind to the TR may still disrupt thyroid function or thyroid hormone action. SPECIFICITY High COMMENTS This assay should be practical to do with tissue ( liver) from any vertebrate.

X. Thyroid  In Vitro A. Whole Cell Binding Assays. Cell lines that express the thyroid hormone receptor can be used as a whole cell binding assay. Generally, 125I T 3 is added to the media and after a short incubation period, nuclei are isolated and counted. The difference between a whole cell assay and other binding assays is that the ligand must be taken up into the cell by stereospecific uptake sites that can be blocked by xenobiotics. Thus, this type of screen could potentially detect a broader array of chemicals that affect thyroid hormone action ( i. e., those that bind to the TR and/ or those that affect cellular uptake).

Cell lines from a number of vertebrates have been described to express the thyroid hormone receptor and, therefore, may be suitable. However, cell lines often exhibit a number of growth/ maintenance characteristics that would make them more or less suitable for large screens, and this type of information would require further research to obtain.

B. Stably Transfected Cell Lines. Cells that express the TR can also be stably transfected to provide a number of reporters that would respond to different kinds of thyroid responsive elements ( TREs). An example would be GH 3 cells which have been used to study the role of thyroid hormone on the regulation of growth hormone gene expression. Because there are a number of TRE motifs, one goal would be to establish a cell line that would allow an easy screen of compounds that might affect the ability of TR to influence the expression of several types of regulatory elements. In principle, this type of assay could be established in cell lines from a variety of vertebrates with the same caveats listed above. Final EDSTAC Report Chapter Five Appendices August 1998 30 K  C. Thyroid Hormone Responsive Cells. Various cell lines change phenotype in response to thyroid hormone. PC12 cells are an example. This is a rat pheochromocytoma cell line which can be induced to become neuronlike in response to NGF. Thyroid hormone can block this change, but it requires transient transfection of the thyroid hormone receptor.

Another example is that of the XLT 15 cell line of Yaoita and Nakajima. These cells can be induced to undergo apoptosis by thyroid hormone. Thus, a screen may be developed to determine whether a compound can influence this induction.

D. Specialized Cells. Various cell lines exhibit unique features that can be recruited for development of a screen. For example, FRTL 5 cells are derived from a rat thyrocarcinoma. Marinovich et al. [ Marinovich 1995; 1153] have reported a clonal line that is stably transfected with the human thyroid peroxidase which can be inhibited by a number of chemicals ( e. g., ethylenethiourea, a metabolite of dithiocarbamate pesticides).

XI. Thyroid In vivo A. Short Term Serum T4 DESCRIPTION Determines whether a chemical can alter circulating levels of thyroxine. Several mechanisms for this, including decreased half life ( displacing from serum carrier proteins, activating liver enzymes), or decreasing synthesis by effects on the thyroid itself. T 4 is more sensitive than T 3 or TSH. T 3 and TSH can be measured if T 4 is affected but there are a number of compounds that affect T 4 without affecting T 3 or TSH. Other indices would be required. DEGREE OF USE Extensive DURATION 24 hours or more ASSAY STABILITY N/ A DOES IT METABOLIZE TOXICANTS Yes ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED Yes, but T 4 kits are widely used clinically. ARE SPECIAL SKILLS/ TRAINING NEEDED Yes, but training period is not extensive. HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY Yes SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Final EDSTAC Report Chapter Five Appendices August 1998 31 K  T 4 is not very sensitive in a short term assay, though others may have more extensive knowledge on this. ARE THERE KNOWN FALSE POSITIVES No OR FALSE NEGATIVES Because the half life of thyroid hormone in blood is long (~ 120h) and because some compounds may interfere with thyroid hormone synthesis ( e. g., iodide uptake), short term exposure may not be indicative of thyroid affects. SPECIFICITY High COMMENTS This could be coupled to a screen for reproductive effects.

B. Long Term Serum T4 DESCRIPTION Determines whether a chemical can alter circulating levels of thyroxine within a longer time course. This could be performed on animals being evaluated for reproductive effects ( see RTP proposal). DEGREE OF USE Extensive DURATION 48 hours or more. ASSAY STABILITY N/ A DOES IT METABOLIZE TOXICANTS Yes ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED Yes, but T 4 kits are widely used clinically. ARE SPECIAL SKILLS/ TRAINING NEEDED Yes, but training period is not extensive. HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY Yes SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Unclear ARE THERE KNOWN FALSE POSITIVES No OR FALSE NEGATIVES None that are known. SPECIFICITY Unknown COMMENTS This assay can be included in an experiment for reproductive effects. Final EDSTAC Report Chapter Five Appendices August 1998 32 K  C. Thyroid Peroxidase DESCRIPTION This description was contributed by Dan Sheehan ( excerpted by TZ). The thyroid peroxidase has a broad substate specificity, for all phenolic chemicals essentially. One group of prototypical ED's is the flavones and isoflavones; they inhibit thyroid peroxidase with IC0' s in the low micromolar range and can act as either competitive inhibitors or as suicide substrate inhibitors which inactivate the peroxidase. Either or both of these actions could lower T3/ T4 production, increase TSH and lead to goiter/ carcinoma. A population of human infants consuming soy infant formula, which has a high isoflavone content, has been identified with Graves disease or Hashimotos thyroiditis, both autoimmune thyroid diseases diagnosed by goiter. The prevalence of soy formula consumption was twice as high in these patients as in controls who had consumed cows milk formula. The assay for inhibition is simpler, quicker, and cheaper than a receptor assay. It is a colorimetric assay of peroxidase activity in the presence of various concentrations of a chemical followed by graphical or computer solutions for IC50' s. It seems likely to me that other chemicals may act via the same mechanism. Also note that the phenolic " A" ring ( or its equivalent) is crucial for estrogen receptor binding. Chemicals with a low ER RBA (?) ( i. e., 1/ 1000 tha of E2) and a phenolic group would be active estrogens in the micromolar range AND would also possibly be active on the peroxidase. Given the co occurence of thyroid and reproductive problems in some geographical areas, this may be due to estrogenic activity of low affinity ER ligands which also have peroxidase inhibiting activity. While this assay does not account for all mechanisms leading to thyroid toxicity, just as an ER RBA assay does not, it is cheap, simple, and quick; and , I think, should be in the in vitro battery for the thyroid. DEGREE OF USE Extensive DURATION ? ASSAY STABILITY N/ A? DOES IT METABOLIZE TOXICANTS Yes ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED Modest ARE SPECIAL SKILLS/ TRAINING NEEDED Yes, but training period is not extensive. HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY Yes SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Unclear ARE THERE KNOWN FALSE POSITIVES No OR FALSE NEGATIVES Compounds may interfere with thyroid hormone action without affecting TPO. SPECIFICITY Final EDSTAC Report Chapter Five Appendices August 1998 33 K  Unknown D. Malic Enzyme DESCRIPTION DEGREE OF USE Extensive DURATION ? ASSAY STABILITY N/ A? DOES IT METABOLIZE TOXICANTS Yes ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED Modest ARE SPECIAL SKILLS/ TRAINING NEEDED Yes, but training period is not extensive. HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY Yes SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Unclear ARE THERE KNOWN FALSE POSITIVES No OR FALSE NEGATIVES Compounds may interfere with thyroid hormone action without affecting TPO. SPECIFICITY Unknown COMMENTS See above E. Mammal Development DESCRIPTION This assay can be performed on animals in which screens for reproductive effects are being evaluated as described in the RTP proposal. Simplest measures would be circulating T 4, and brain weight. DEGREE OF USE Brain weight is not used as an index of thyroid hormone action during development, though brain weight is clearly affected by thyroid hormone action. DURATION ? ASSAY STABILITY N/ A? DOES IT METABOLIZE TOXICANTS Final EDSTAC Report Chapter Five Appendices August 1998 34 K  Yes ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED Modest ARE SPECIAL SKILLS/ TRAINING NEEDED Yes, but training period is not extensive. HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY Unclear SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Unclear ARE THERE KNOWN FALSE POSITIVES Yes, compounds that affect nutritional status or eating. However, these may also influence thyroid hormone levels which are linked to nutritional status. OR FALSE NEGATIVES No? SPECIFICITY Unknown COMMENTS See above.

XII. Three Alternative Overviews The following three overviews were submitted as alternatives to existing overviews and are included here as an addendum. ( submitted by Dr. Soto)

A. MCF7 Proliferation Assay ( E SCREEN) DESCRIPTION Measures proliferation of cells in culture in response to estrogens. DEGREE OF USE Used in various laboratories in the U. S. A., Europe and Japan. Many chemicals studied; several toxicants were discovered to have estrogenic properties using this method. Extensive published data on toxicants with estrogenic activity. DURATION From seeding the cells to harvesting cells= 5 to 7 days. It requires 4 manipulations only: ( 1) seeding, ( 2) changing to test medium 24 hours later, ( 3) single step harvesting ( or staining in situ) and ( 4) counting ( or reading in an ELISA plate reader). ASSAY STABILITY Very stable in the labs that started with a line/ subline of proven sensitivity. Clones reported to have stable activity for over ten years. DOES IT METABOLIZE TOXICANTS? Not thoroughly characterized. However, some proestrogens ( methoxychlor, bisphenol A dimethacrylate, alkylphenol monoethoxylates, non hydroxylated PCBs) were reported to have activity in this assay. Final EDSTAC Report Chapter Five Appendices August 1998 35 K  ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED? Like all assays performed with vertebrate cell cultures, it requires laminar flow hood, CO 2 incubator, cell freezing storage, inverted microscope, cell counting devise, and a detector for the specific end point measured ( it may use an electronic cell counter or an ELISA plate reader if using sulforhodamine B assay or other colorimetric assay). ARE SPECIAL SKILLS/ TRAINING NEEDED? Like all assays using cell culture, it requires the ability of maintaining the culture stocks, periodic freezing after several passages to maintain the cultures in case of infections or other mishaps. The assay itself is very easy to perform.

HAS IT BEEN STANDARDIZED EASILY? It is highly reproducible from assay to assay. It is used in several labs in the USA and Europe. By using responsive cells to begin with, it has provided comparable results when a battery of 20 coded chemicals were tested by several labs ( manuscript in preparation; Project Coordinator: Philip Grandjean, Odense University, Denmark). Competent labs appear to get similar results. Cell of appropriate phenotype can be easily distributed. SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Very sensitive; one of the most sensitive assays ( E2 EC50= 10 15 pM range; maximal activity 100 pM). ARE THERE KNOWN FALSE POSITIVES? In competent labs no false positives were found among growth factors. Testosterone is the only steroid that shows activity at TM concentrations. When assaying for antagonists, endocrinologists have used a two step method to assess whether or not the effect is truly antiestrogenic regardless of whether the end point is inhibition of estrogen induced cell proliferation or inhibition of estrogen induction of a gene product: 1) assess the effect of a range of doses of the test compound together with the minimal estrogen dose needed for maximal induction of the gene product ( inhibition), and 2) assess the effect of increasing doses of estradiol administered together with the dose of toxicant found to induce maximal inhibition ( estradiol rescue). OR FALSE NEGATIVES? False negatives due to toxicity were not found using GLP ( it only takes looking at the cells with the inverted microscope to detect unspecific toxicity). TCDD does have toxic effects both in the presence and absence of estrogens. Various substances that require metabolic activation like alkylphenols polyethoxylates may be missed. SPECIFICITY ARE THERE OTHER MECHANISMS OF ACTION TO PRODUCE THIS EFFECT ( FALSE POSITIVES)? So far, all substances found to be estrogenic with the E SCREEN assay that were also tested using gene activation assays were found to be consistently estrogenic. COMMENTS As with all bioassays, it requires obvious good laboratory practices. In the case of rodent bioassays it is important to work with healthy animals, appropriate light cycles, using feeds that do not contain estrogens, etc. When working with cells in culture, one has to start with a subline that expresses the appropriate phenotype. To maintain the phenotype, serum used for Final EDSTAC Report Chapter Five Appendices August 1998 36 K  cell propagation has to be checked before using it for propagation ( therefore, laboratories stock " good serum" to last for one year). Similarly, stocks have to be frozen periodically, and the charcoal dextran stripped serum ( which can be stored frozen in aliquots for up to one year, should be checked once before use). Recently, it was shown that recombinant serum albumin may be used instead of charcoal dextran stripped serum. The assay can be automated, and different labs use 12, 24 or 96 well plates, depending on whether one uses cell numbers, sulforhodamine B staining or MTT reaction as the end point). Also, cells can be fixed in situ at the end of the experiment and stained days or weeks later.

B. MVLN Assay. Stably Transfected Reporter Gene Assay in Mammalian Cells ( submitted by Dr. Soto) DESCRIPTION The assay utilizes a mammalian cell line ( MCF7 with endogenous ER) that has been stably transfected with an ER specific reporter gene ( Vit luc). DEGREE OF USE Used in various laboratories for pharmaceutical and environmental research. No publications are listed in Medline as yet attesting its use for estrogenic toxicants. DURATION From seeding the cells to harvesting lysate and Luc activity takes two to three days. The following manipulations are required: ( 1) seeding and exposure to charcoal dextran stripped serum containing tamoxifen for 24 48 hours; ( 2) changing the medium to tamoxifen free for 12 hours ( or, treat for 1 2 weeks with charcoal dextran stripped serum,); ( 3) incubate with test substance for 24 or more hours; ( 4) finally harvest cells; ( 5) homogenize them; ( 6) read assay; and ( 7) run a protein assay. ASSAY STABILITY Like the parent cell line MCF7, it should be stable when good laboratory practices are followed. It has been stable for 30 passages. Exposure to tamoxifen renders these cells unable to express estrogen induction of the reported gene. DOES IT METABOLIZE TOXICANTS? Not thoroughly characterized. It may maintain same capabilities as those of the parental cell line. ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED? Like all assays performed with vertebrate cell cultures, it requires laminar flow hood, CO 2 incubator, cell freezing storage, inverted microscope, cell counting devise, and a detector for the specific end point measured ( luminometer to measure Luc activity). ARE SPECIAL SKILLS/ TRAINING NEEDED? Like all assays using cell culture, it requires the ability of maintaining the culture stocks, periodic freezing after several passages to maintain the cultures in case of infections or other mishaps. The assay itself is easy to perform. HAS IT BEEN STANDARDIZED EASILY? No published results are available on the performance of this assay to detect estrogenic toxicants. Stably transfected cells can be easily distributed. SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Final EDSTAC Report Chapter Five Appendices August 1998 37 K  Very sensitive; EC50 for E2 in the 20 pM range; maximal activity of E2 was reported at 1 nM. ARE THERE KNOWN FALSE POSITIVES? Hypothetically, ER may be activated by non estrogenic agents through phosphorylation pathways. When assaying for antagonists, endocrinologists have used a two step method to assess whether or not the effect is truly antiestrogenic regardless of whether the end point is cell proliferation or inhibition of estrogen induction of a gene product: ( 1) assess the effect of a range of doses of the test compound together with the minimal estrogen dose needed for maximal induction of the gene product ( inhibition); and ( 2) assess the effect of increasing doses of estradiol administered together with the dose of toxicant found to induce maximal inhibition ( estradiol rescue). OR FALSE NEGATIVES? Similarly to the parental cell line, false negatives due to toxicity should be excluded by GLP ( it only takes looking at the cells with the inverted microscope to detect unspecific toxicity). It is suspected that like in the parent cell line TCDD may have toxic effects both in the presence and absence of estrogens. Various substances that require metabolic activation like alkylphenols polyethoxylates may be missed. SPECIFICITY ARE THERE OTHER MECHANISMS OF ACTION TO PRODUCE THIS EFFECT ( FALSE POSITIVES)? Hypothetically, ER may be activated by non estrogenic agents through phosphorylation pathways. COMMENTS Specific ER activation. As with all bioassays, it requires obvious good laboratory practices. In the case of rodent bioassays it is important to work with healthy animals, appropriate light cycles, using feeds that do not contain estrogens, etc. When working with cells in culture, one has to start with a subline that expresses the appropriate phenotype. To maintain the phenotype, serum used for cell propagation has to be checked before use ( therefore, laboratories stock " good serum" to last for at least one year). Similarly, cell stocks have to be frozen periodically. The assay can be automated; can be done in 12, 24 or 96 well plates.

C. MCF7 AR1 Assay. MCF7 Cells Stably Transfected With " Wild Type" Androgen Receptor. ( submitted by Dr. Soto) DESCRIPTION The assay utilizes a mammalian cell line ( MCF7) stably transfected AR. These cells proliferate maximally in serumless medium supplemented with insulin and transferrin. Androgens inhibit their proliferation; antiandrogens abolish the inhibitory effect of androgens. DEGREE OF USE This method was just published. DURATION From seeding cells to harvesting them, it takes 5 days. ASSAY STABILITY Like the parent cell line MCF7, it should be stable when good laboratory practices are followed. It has been stable for 4 years. Final EDSTAC Report Chapter Five Appendices August 1998 38 K  DOES IT METABOLIZE TOXICANTS? Not thoroughly characterized. It should keep the same capabilities as those of the parental cell line. ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED? Like all assays performed with vertebrate cell cultures, it requires a laminar flow hood, CO 2 incubator, cell freezing storage, inverted microscope, cell counting devise, and a detector for the specific end point measured ( it may use an electronic cell counter or an ELISA plate reader when using sulforhodamine B assay or other colorimetric assay).

ARE SPECIAL SKILLS/ TRAINING NEEDED? Like all assays using cell culture, it requires the ability of maintaining culture stocks, periodic freezing after several passages to maintain the cultures in case of infections or other mishaps. The assay itself is easy to perform. HAS IT BEEN STANDARDIZED EASILY? It can be standardized easily, since cells express AR constitutively. No published results are available on the performance of this assay to detect toxicants that are androgen agonists or antagonists. Stably transfected cells can be easily distributed. SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Very sensitive; EC50 for DHT in the pM range. ARE THERE KNOWN FALSE POSITIVES? In theory, hyperphysiological doses of glucocorticoids may bind to the AR and this may activate AREs. OR FALSE NEGATIVES? As with the parental cell line, false negatives due to toxicity should be excluded by GLP ( it only takes looking at the cells with the inverted microscope to detect unspecific toxicity). In addition, since androgens inhibit cell proliferation, " rescue" from inhibition by an antiandrogen may help differentiating toxicity from androgenicity. Substances that require metabolic activation may be missed. SPECIFICITY ARE THERE OTHER MECHANISMS OF ACTION TO PRODUCE THIS EFFECT ( FALSE POSITIVES)? Probably hyperphysiological doses of glucocorticoids. COMMENTS Like all bioassays, it require good laboratory practices. In the case of rodent bioassays it is important to work with healthy animals, appropriate light cycles, using feeds that do not contain estrogens, etc. When working with cells in culture, the cell line should express the appropriate phenotype. To maintain the phenotype, serum used for cell propagation has to be checked before use ( therefore, laboratories stock " good serum" lots for at least one year). Similarly, cell stocks have to be frozen periodically. The assay can be automated; can be done in 12, 24 or 96 well plates.

XIII. Overview of Non Mammalian Screens Final EDSTAC Report Chapter Five Appendices August 1998 39 K  A. Procedures Using Birds and Reptiles to Determine Endocrine Disruptive Action The number of procedures available are limited for taxa other than mammals. In part, this is because many procedures used in mammals have just not been tried in other taxa, as well as because they are not appropriate for egg producing vertebrates or any phyla other than Vertebrata. Hence, the procedures below have been divided into two matrices with identical structure as the mammalian matrix, but including both birds and reptiles as subjects. The first matrix contains procedures that have already been used to determine endocrine action of contaminants, and the second contains procedures that could be used, but have not. The procedures in the second matrix need varying amounts of development to be ready for inclusion in a screening and testing program. The amount of development needed is estimated in the " comments" section.

Also, the number and variety of in vitro procedures that have been applied to wildlife is orders of magnitude smaller than used with mammalian material. This may be a particular problem for the design of a comprehensive set of screening procedures, because the screening battery is intended to cull out from a list of chemicals to be tested, all those chemical that have no intrinsic endocrine activity. Hence, the screening battery should be comprehensive for all types of endocrine action ( or as many as possible). Thus the availability of short duration procedures using non mammalian material that could be used as screens would seem crucial as the screening and testing program is intended to be protective of all taxa, not just humans and other mammals. The issue of the homology of steroid hormone and receptor structure across vertebrates classes needs to be explored more fully to help design an adequately comprehensive program, especially for the screening phase. At this date, there is no real choice to be made as there are very few if any procedures using non mammalian material that could be used as a screening tool. This area needs thoughtful development. In addition to development of non mammalian vertebrate in vitro procedures, invertebrates in whole animal " biological activity" tests could be exploited, which by dint of the very short life cycle, of Daphnia for instance, could be economically used in a screening battery.

B. Overview of Endocrine Disruptor Relevant Screens to the Lower Vertebrates and Invertebrates Unlike the many and wide array of screens available for mammalians, few exist for the animals outside that taxonomic group. Some assays do exist which can be employed with differing degrees of specificity and sensitivity. Predominantly, the assays available for these animals will be in vivo and somewhat apical. Although this means these assays will not provide detailed mode of action information, the endpoints will be relevant for interpreting " adverse" effects.

For the non mammalian vertebrates, vitellogenin assays are available and will generally provide suggestive evidence of estrogen related disruptions. However, there is some evidence of thyroid hormone involvement with vitellogenin production which may compromise any conclusive evidence of an estrogen specific action. Nonetheless, compounds which affect a change in normal Final EDSTAC Report Chapter Five Appendices August 1998 40 K  vitellogenin levels should be captured in a screening program for more definitive investigation of the effect and its relevance.

Existing standardized tests for evaluating conventional toxicities are also included here because of the availability of such information for certain compounds ( e. g., pesticides). Information from these tests can be used to screen for suggestive endocrine mediated effects to be flagged for further investigation.

XIV. Amphibian Screens A. Vitellogenin Assay DESCRIPTION An assay which measures the amount of an egg yolk protein precursor in males as an indicator of estrogenic activity. REFERENCES Selcer, " Vitellogenin induction in frogs by immersion in xenobiotic estrogens," Am. Zool., 36, 1996, p. 5. Peterson, G. L., Determination of total protein: Methods of Enzymology, 91, 1993, pp. 95 121. DURATION > 72 hours EQUIPMENT Wet lab, antibody, immunology lab STAFF SKILLS Can be trained COST Modest AVAILABILITY Limited by specificity of antibody, but Heppell et al. efforts at developing a " universal" antibody appear promising SENSITIVITY Good SPECIFICITY Good for estrogen activity, but thyroid hormones may also be involved STANDARDIZATION Not yet, but could be made without undue difficulties RELATEDNESS Primarily estrogen box, more research is needed to ascertain whether thyroid or androgen activity is or is not connected B. Frog Embryo Teratogenesis Assay Xenopus ( FETAX) DESCRIPTION Final EDSTAC Report Chapter Five Appendices August 1998 41 K  A 96 hour whole embryo teratogenesis screening assay. Because the exposure is through primary organogenesis all developmentally important processes are taken into account. REFERENCES Dumont et al., " Frog Embryo Teratogenesis Assay  Xenopus ( FETAX)," Shortterm Bioassays in the Analysis of Complex Environmental Mixtures III, 1983. Plenum, ASTM, Standard guide for conducting the Frog Embryo Teratogenesis Assay  Xenopus ( FETAX), E, 1991, pp. 1439 91. DURATION 96 hours EQUIPMENT General wet lab and microscopy lab STAFF SKILLS Specialized knowledge of amphibian embryology and histology COST Modest AVAILABILITY Fair SENSITIVITY Fair, not fully comprehensive for all estrogenic, androgenic, or thyroid related effects SPECIFICITY Does not distinguish hormonal from non hormonal developmental effects STANDARDIZATION Yes through ASTM RELATEDNESS Apical for some estrogenic, androgenic, and thyroid related effects other C. Amphibian Metamorphosis Assay ( Conceptual) COMMENTS Similar to a FETAX like assay but focused on tadpole metamorphosis, perhaps specifically on tail resorption. Apical for thyroid related effects.

D. Frog In Vivo Screening Assay ( Conceptual) COMMENTS Similar in concept to what is proposed for fishes evaluating endpoints such as gonadosomatic index, secondary sex characteristics, oocyte maturation, plasma steroids, and plasma vitellogenin. Apical for estrogen, androgen, and thyroid related effects.

E. Metamorphosis DESCRIPTION Determines whether a chemical can affect the thyroid hormone dependent process of metamorphosis. DEGREE OF USE Extensive Final EDSTAC Report Chapter Five Appendices August 1998 42 K  DURATION Depends on species available. Xenopus or Rana would require about a week of treatment. Scaphiopus would require less time ( 24 hours), but may not be widely available. ASSAY STABILITY N/ A? DOES IT METABOLIZE TOXICANTS Yes ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED Modest ARE SPECIAL SKILLS/ TRAINING NEEDED Yes, but training period is not extensive HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY Yes SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS yes ARE THERE KNOWN FALSE POSITIVES No OR FALSE NEGATIVES Unclear SPECIFICITY Unknown COMMENTS The ease of compound administration may make this attractive. The use of ± T3/ T4 paradigm would allow for identification of thyroid action disruption. Endpoints ( e. g., hind limb growth rate) are ÒintegratedÓ measures; thus, the screen would detect compounds that affect thyroid hormone action along the entire pathway. There are clearly a number of endpoint which may be more or less sensitive or reliable. Structure of internal organs ( liver, pronephros), and production of urea are two.

XV. Bird Screens A. Avian Egg Injection Assay TEST AND FUNCTION: The effects of steroids ( and EDCs) on development of the reproductive tract and steroid concentrations. Subject are various species of wildlife. Exposure is by egg injection before organogenesis; responses are measured at hatching and include the morphology histology of the reproductive tract of males ( females may be useful also), and plasma steroid concentrations. It can be extended into the breeding age of the affected animal to look check for functional impairment. REFERENCES: Final EDSTAC Report Chapter Five Appendices August 1998 43 K  Fry, D. M., C. K. Toone, S. M. Speich, and R. J. Peard, " Sex ratio skew and breeding patterns of gulls: demographic and toxicological considerations," Studies in Avian Biology 10, 1987, pp. 26 43. Nisbet et al., 1996. Several other studies now in progress using similar methodology with other species. DURATION: Depends on the length of incubation (~ three weeks minimum), and availability of eggs: wild birds usually not easily available unless a breeding colony exists. EQUIPMENT: Incubator, hatchabator, general lab facilities and facilities for RIAs.

STAFF SKILLS: Animal husbandry skills, general dissection and microscope skills, tissue handling and preparation for sectioning, biochemical expertise for RIA work. AVAILABILITY: Wide. SENSITIVITY: Good for estrogens. SPECIFICITY: Estrogenic effects in males: possibly non aromatizable androgen effects in females. STANDARDIZATION: Needs work, especially in preparation and analysis of histological responses. OTHER: The choice of subject species is crucial for operational ease, practicality and cost control. Embryos must be large enough at hatching to get a gonad that is easily handled and that has enough blood to collect for RIA on the plasma. Very similar responses to procedure R2. Compare to A2.

B. Japanese Quail Early Life Stage TEST AND FUNCTION: The effects of steroids ( and EDCs) on development of sexual maturity in Japanese quail, and, if desired, of the reproductive tract and plasma steroid concentrations in siblings. This tests the time to onset of maturity as measured by secondary sex characters ( cloacal or foam gland size) and behavior ( crowing) in males; all are testosterone dependent. Exposure varies, see below. REFERENCES: Ottinger, M. A. and H. J. Brinkley, " Testosterone and sex related behavior and morphology: Relationship during maturation in the adult Japanese quail," Hormones and Behavior 11, 1978, pp. 175 182; and other references. DURATION: ~ 18 days incubation + 60 days to maturity. EQUIPMENT: Avian husbandry, general lab, tape recorder. Final EDSTAC Report Chapter Five Appendices August 1998 44 K  STAFF SKILLS: Husbandry, general lab. AVAILABILITY: Quail are widely available; strains can be different, recommend out bred line. SPECIFICITY: Depending on the timing of exposure it can be made specific to estrogen OR androgens: · if chicks are implanted or injected with EDC, male time to maturity is androgen sensitive; · if eggs are injected, the gonadal development of male offspring is impaired by estrogens ( and possibly non aromatizable androgens in female offspring, although this has not been demonstrated [ matrix 2]); this is similar to procedure A1. Estrogenic effects on time to maturity in males is not known. STANDARDIZATION: This test has been used widely and can be easily standardized further . OTHER: Excellent background information on the endocrinology of Japanese quail; this procedure might be developed into an Androgen antagonist assay in males with co administration of EDC and androgen, and androgen alone as control [ matrix 2].

C. Japanese Quail Androgenicity Screen TEST AND FUNCTION: Foam gland size and crowing behavior in photo regressed adult males to look at the Androgenic effect of EDCs on peripheral targets, using Japanese quail. This procedure uses individuals that are not secreting GNRH, and thus the HPG axis is shut down. REFERENCES: [ Matrix 2] DURATION: Two weeks to regress; expose for two days, responses in five to seven days. EQUIPMENT: Animal husbandry, photoperiod control, general lab. SPECIFICITY: Measures androgenic effects only. STANDARDIZATION: Good potential. RELATEDNESS: Could be made into an Androgen antagonist procedure, with the co administration of testosterone + EDC, and using testosterone treatment alone as a control; may not be very sensitive, and there is likely to be a threshold effect of the administered testosterone, making the test for an antagonist less sensitive. OTHER: Almost ready to go; needs testing with EDCs.

D. Vitellogenin Production in Female Japanese Quail Final EDSTAC Report Chapter Five Appendices August 1998 45 K  TEST AND FUNCTION: Vitellogenenin production in photo regressed adult females to look at the estrogenic effect of EDCs on peripheral targets, using Japanese quail. REFERENCES: [ Matrix 2] DURATION: Two weeks to regress; expose for two days, vitellogenin response in 24 hours. EQUIPMENT: Animal husbandry, photoperiod control, electrophoresis lab. STAFF SKILLS: As above.

SPECIFICITY: Specific for estrogen binding peripherally; could be made into a test for the E antagonist with co administration of E2 and the potential EDC, along with E2 to the controls. STANDARDIZATION: Good potential: husbandry can be made standard, but a common source of antibody is needed; ring testing probably needed for electrophoresis OTHER: Needs more development than A3. Could be used as a procedure for estrogen antagonists with co administration of estrogen and using estrogen alone as a control.

E. Vitellogenin Production in Adult Male Birds: Japanese Quail, Chickens. REFERENCES: Robinson, G. A. and A. M. V. Gibbins, " Induction of vitellogenesis in Japanese quail as a sensitive indicator of the estrogen mimetic effect of a variety of environmental contaminants," Poultry Science, 63, 1984, pp. 1529 1536. ( and other references ) Heppell, S. A., M. D. Denslow, L. C. Folmar and C. V. Sullivan, " Universal assay of vitellogenin as a biomarker of environmental estrogens," Environ. Health Perpsectives, 103 ( Suppl 7), 1996, pp. 9 15. [ see also: Matter, J. M., A. B. Anthony, K. Alonso, and R. L. Dickerson, " TCDD suppression of vitellogenin synthesis in female hens," Abstract. 17th Annual meeting of SETAC, 1996.] DURATION: Eight to ten days. EQUIPMENT: Animal facilities, electrophoresis equipment. STAFF SKILLS: Animal husbandry, electrophoresis, general lab. AVAILABILITY: Depends on antibody: birds widely available. SENSITIVITY: " High", but o, p DDT did not induce vitellogenesis. Final EDSTAC Report Chapter Five Appendices August 1998 46 K  SPECIFICITY: High. OTHER: Looks pretty good; a little shorter duration than A5 ( photo regressed females) but relative sensitivity is unknown. Lots of current work on different species of wildlife that is not published F. Avian " Plaque Assay" TEST AND FUNCTION: Avian " Plaque assay"; slices of brain are prepared in vitro and infused with steroids, peptides or EDCs and GNRH production is measured. Exploits the negative feedback control of steroids on GNRH production.

REFERENCES: Personal communication; Tom Porter's lab at Texas A& M; reprints in the mail for more details. AVAILABILITY: Only this lab uses the technique, as far as can be told. OTHER: This assay should be explored as it is one of the only in vitro assays for birds. Drawbacks include killing birds for brains, independence of different slices from the same brain is questionable, and the expertise rather local, I believe. Needs development.

G. Avian Cell Culture TEST AND FUNCTION: To test the action of steroids and EDCs on the handful of immortalized avian cell lines. In general, steroidogenic endpoints have not been identified, much less evaluated for sensitivity. Further only one avian cell line ( fibroblasts) is spontaneously immortal and not chemically or virally transformed, hence is probably the best for results relevant to an intact animal. Again, steroidogenic responses are have not been identified in these fibroblasts ( are any expected in a fibroblast?). REFERENCES: Personal communication, Doug Foster, U Minn. AVAILABILITY: Patented, but is willing to license for this use. OTHER: Needs development, but promising.

H. Chicken Early Life Stage TEST AND FUNCTION: Coxcomb size, vent sex and gonadal morphology and histology of chickens; EDCs or steroid administered by injecting the eggs before organogenesis; similar to B1 and B2. REFERENCES: Final EDSTAC Report Chapter Five Appendices August 1998 47 K  Elbrecht, A., R. G. and Smith, " Aromatase enzyme activity and sex determination in chickens," Science, 225, 1992, pp. 467 470. Snedecor, J. G., " A study of some effects of sex hormones on the embryonic reproductive system and comb of the white leghorn chick," J. Exptl. Zool., 110, 1949, pp. 205 246.  and others.. DURATION: Long: 23 weeks to maturity, but for morphology of the reproductive tract, can collect material from hatchlings after 21 day incubation. EQUIPMENT: Animal facilities, RIA, tissue handling. STAFF SKILLS: Husbandry, RIA techniques, histological imbedding and slicing. AVAILABILITY: No problem. SPECIFICITY: For Estrogenic activity in males chicks, and for aromatase inhibition in females; aromatizable androgen has no effect ( but DHT, and hence other non aromatizable androgens ?). Needs development for possible A+ procedure. STANDARDIZATION: Easy for coxcomb, subjective assessments of gonadal morphology can be made more quantitative. OTHER: Coxcomb assay is a classic and has been widely used in the past, but it is very time consuming as maturity is reached not until five months; however, differences can be seen in the incompletely mature cock and may be useful. A nice twist exploited by Elbrecht and Smith is to use a cross that produced sexual dimorphism in feather color which was independent of steroid dependent characters, thus revealing genetic sex. In some studies the EDC has been topically applied to the comb directly. Needs development for possible A+ procedure.

I. Cartilage Growth in Chick DESCRIPTION Determines whether a chemical can alter the response of chick cartilage to thyroid hormone. DEGREE OF USE Extensive. ASSAY STABILITY N/ A? DOES IT METABOLIZE TOXICANTS Yes. ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED Modest. ARE SPECIAL SKILLS/ TRAINING NEEDED Final EDSTAC Report Chapter Five Appendices August 1998 48 K  Yes, but training period is not extensive. HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY Yes. SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Unclear. ARE THERE KNOWN FALSE POSITIVES No. OR FALSE NEGATIVES Compounds may interfere with thyroid hormone action without affecting cartilage. SPECIFICITY Unknown.

XVI. Fish Screens A. Vitellogenin Assay DESCRIPTION An assay which measures the amount of an egg yolk protein precursor in males as an indicator of estrogenic activity. REFERENCES Sumpter, J. P., " The purification, radioimmunoassay and plasma levels of vitellogenin from the rainbow trout. Salmo gairdneri.," Proceedings of the Ninth International Symposium on Comparative Endocrinology, Hong Kong University Press, Hong Kong, 1985, pp. 335 357. Peterson, G. L., Determination of total protein: Methods of Enzymology, 99, 1993, pp. 95 121. DURATION > 72 hours. EQUIPMENT Wet lab, antibody, immunology lab. STAFF SKILLS Can be trained. COST Modest. AVAILABILITY limited by specificity of antibody although Heppell et al. are developing a " universal" antibody. SENSITIVITY Good. SPECIFICITY Good for estrogen activity, but thyroid hormones may also be involved. STANDARDIZATION Not yet, but could be made without undue difficulties. Final EDSTAC Report Chapter Five Appendices August 1998 49 K  RELATEDNESS Primarily estrogen box, more research is needed to ascertain whether thyroid or androgen activity is or is not connected.

B. In Vitro Vitellogenin Assay DESCRIPTION A procedure isolating trout hepatocytes, treating them with a xenobiotic, and then measuring the amount of vitellogenin secreted into the culture medium. REFERENCES Pelissero et al., " Vitellogenin synthesis in cultured hepatocytes: an in vitro test for the estrogenic potency of chemicals," J. Steroid Biochemistry and Molecular Biology 44, 1993, pp. 263 272. EQUIPMENT Immunology lab. STAFF SKILLS Moderate, can be trained. AVAILABILITY Fair. SENSITIVITY Good. SPECIFICITY Good for estrogen activity. STANDARDIZATION Practical. RELATEDNESS Applicable to estrogen boxes .

C. In Vivo Screening Assay Refer to Peter Thomas' write up.

D. Early Life Stage Test DESCRIPTION Newly fertilized eggs are exposed to a test chemical through hatching and early development and growth of the juvenile fish. Endpoints measured are hatching success, survival, and growth. REFERENCES OECD 210, EPA 850.1400, ASTM E 1241 92. DURATION 31  72 days. EQUIPMENT Wet lab sufficient for flow through studies. STAFF SKILLS General. Final EDSTAC Report Chapter Five Appendices August 1998 50 K  AVAILABILITY Commercially available. SENSITIVITY Good. SPECIFICITY Poor. STANDARDIZATION Yes. RELATEDNESS Apical for all boxes, but does not differentiate hormonal from non hormonal driven effects and is not fully comprehensive.

E. Embryo and Sac Fry Test DESCRIPTION This is a short term test in which the life stages from the newly fertilized egg to the end of the sac fry stage are exposed. OECD 212. DURATION 8 to 55 days. EQUIPMENT Wet lab. STAFF SKILLS General. AVAILABILITY Commercially available. SENSITIVITY Unknown. SPECIFICITY None. STANDARDIZATION Yes. RELATEDNESS Apical for general fish health.

F. Partial Life Cycle Test REFERENCES Standard Methods for the Examination of Water and Wastewater, APHA, AWWA, and WPCF 810B, 1985, p. 2. DURATION Long term, > 250 days. EQUIPMENT Wet lab. STAFF SKILLS Experienced. Final EDSTAC Report Chapter Five Appendices August 1998 51 K  COST High. AVAILABILITY Commercially available. SENSITIVITY Good. SPECIFICITY Apical, but does not address transgenerational effects. STANDARDIZATION Fair. RELATEDNESS Applicable to all boxes, but more appropriate in definitive testing as a screen it should only be used if the test has already been completed.

G. Full Life Cycle Test REFERENCE Standard Methods for the Examination of Water and Wastewater, APHA, AWWA, and WPCF, 810B, 1985, p. 3. DURATION Long term, > 250 days. EQUIPMENT Wet lab. STAFF SKILLS Experienced. COST High. AVAILABILITY Commercially available. SENSITIVITY Good. SPECIFICITY Apical, but does not address transgenerational effects. STANDARDIZATION Fair. RELATEDNESS Applicable to all boxes, but more appropriate in definitive testing as a screen it should only be used if the test has already been completed.

H. Flounder Metamorphosis DESCRIPTION Determines whether a chemical can affect the thyroid hormone dependent process of metamorphosis. DEGREE OF USE Final EDSTAC Report Chapter Five Appendices August 1998 52 K  Extensive. DURATION Seven days. ASSAY STABILITY N/ A? DOES IT METABOLIZE TOXICANTS Yes. ARE SPECIAL EQUIPMENT/ REQUIREMENTS MANDATED Modest. ARE SPECIAL SKILLS/ TRAINING NEEDED Yes, but training period is not extensive. HAS IT BEEN OR CAN IT BE STANDARDIZED EASILY Yes. SENSITIVITY TO LOW DOSES OR WEAKLY ACTIVE CHEMICALS Yes. ARE THERE KNOWN FALSE POSITIVES No. OR FALSE NEGATIVES Unclear. SPECIFICITY Unknown. COMMENTS The ease of compound administration may make this attractive. The use of ± T3/ T4 paradigm would allow for identification of thyroid action disruption. Endpoints ( e. g., eye migration) are ÒintegratedÓ measures; thus, the screen would detect compounds that affect thyroid hormone action along the entire pathway.

I. In Vitro Steroid Receptor Competition Assay No information submitted.

J. In Vitro Steroid Production Bioassay No information submitted.

K. In Vitro Germinal Vesicle Breakdown ( GVBD) Bioassay No information submitted.

XVII. Invertebrate Screens A. Daphnia Reproduction ( Life Cycle) Test Final EDSTAC Report Chapter Five Appendices August 1998 53 K  DESCRIPTION The objective of this test is to assess the effect of a chemical on the reproductive output of Daphnia magna. Information on growth is also obtained. REFERENCES EPA 850.1300, OECD 211, ASTM E 1193 93. DURATION 21 days. EQUIPMENT Wet lab. STAFF SKILLS Basic. COST Moderate. AVAILABILITY Commercially available. Final EDSTAC Report Chapter Five Appendices August 1998 54 K  SENSITIVITY Unknown. SPECIFICITY Apical for invertebrate endocrine related effects, unknown as far as vertebrates are concerned. STANDARDIZATION Yes. RELATEDNESS B. Mysid Life Cycle Test DESCRIPTION The objective of this test is to assess the survival, growth, and reproduction of mysids through a complete life cycle. REFERENCES EPA 850.1350, ASTM E 1191 90 DURATION 28 days. EQUIPMENT Wet lab, saltwater capability. STAFF SKILLS Basic. COST Moderate. AVAILABILITY Commercially available. SENSITIVITY Unknown. SPECIFICITY Apical for invertebrate endocrine related effects, unknown as far as vertebrates are concerned. STANDARDIZATION Yes. RELATEDNESS N/ A XVIII. Reptilian Screens A. Vitellogenin Production In Adult Male Turtles TEST AND FUNCTION: Final EDSTAC Report Chapter Five Appendices August 1998 55 K  Vitellogenin production in adult male turtles ( Red eared slider, Trachemys scripta) and male frogs ( Xenopus laevis); indicates estrogen receptor binding in the liver and/ or estrogen receptor production. REFERENCES: Palmer, B. D. and S. K. Palmer, " Vitellogenin induction by xenobiotic estrogens in the Redeared turtle and African clawed frog," Environ. Health Perspectives, 103 ( suppl 4), 1995, pp. 19 25. ( and others in the bibliography) DURATION: Injection daily for seven days, plasma collected on day 14. EQUIPMENT: General lab space for animals, equipment for ELISA. STAFF SKILLS: ELISA. COST: Not high; turtles and frogs purchased. AVAILABILITY: Animals easily obtainable. SENSITIVITY: Sliders more sensitive than frogs when comparing DDT exposed to estradiol controls, but frogs equally as sensitive as sliders when comparing the absolute of vitellogenin produced in response to DDT. SPECIFICITY: Estrogen dependant response. STANDARDIZATION: Good potential, depends on common source of antibody . OTHER: Exact same protocol used for amphibians; possibly useful as E procedure with coadministration of estrogen.

B. Sex Determination in Turtles TEST AND FUNCTION: Sex determination in turtles; the red eared slider ( Trachemys scripta) and snapping turtles ( Chelydra serpentina) have be used; responses measured are the morphology of the gonads and genital ducts and the histology of the gonads. REFERENCES: Bergeron, J. M., D. Crews, and J. A. McLaughlin, " PCBs as environmental estrogens: Turtle sex determination as biomarker of environmental contamination," Environ Health Perspectives, 102, 1994, pp. 780 781. Crews, D., J. M. Bergeron, and J. A. McLaughlin, " The role of estrogen in turtle sex determination and the effects of PCBs," Environ Health Perpectives 103 ( suppl 7), 1994, pp. 73 77. Final EDSTAC Report Chapter Five Appendices August 1998 56 K  Wibbles, T. and D. Crews, " Steroid induced sex determination at incubation temperatures producing mixed sex rations in a turtle with TSD," General and Comparative Endocrinology 100, 1995, pp. 53 60. DURATION: Rather long ~ 12 weeks to hatch and collection of samples. EQUIPMENT: General lab facilities. STAFF SKILLS: Tissue handling. AVAILABILITY: Eggs of T. scripta are commercially available. SENSITIVITY: Incubation temperature is crucial and needs some discussion for development of the best conditions for testing; there is greater physiological sensitivity to steroids at incubation temperatures that produce mixed sexes from the clutch, but greater statistical sensitivity when clutch is incubated at male producing temperatures. SPECIFICITY: An estrogen dependant alteration of male gonads. STANDARDIZATION: ? Some subjectivity in the morphological and histological analysis. OTHER: The red eared slider ( Trachemys scripta) has been studied most extensively; are other species more appropriate or available? Again, possibly useful as a E antagonist procedure with co administered estrogen at male temperatures, OR with EDC alone at female temperatures. This needs development, but would be worth the effort as so much of the basics are known in this system. EDSTAC Final Report Chapter Five Appendices August 1998 Appendix L Protocols for Tier 1 Screening Assays Many of the following draft protocols are undergoing internal validation within NHEERL laboratories and until completion of this process they should be considered preliminary. The final protocols will likely differ from what is currently presented.

Table of Contents In Vitro Assays ( presented as examples) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 I. Rat Estrogen Receptor Equilibrium Exchange Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 A. Purpose and Applicability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 B. Safety and Operating Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 C. Equipment and Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 D. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 II. Protocol to Determine Affinity of Compounds for the Rat AR Androgen Receptor by Equilibrium Exchange Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 A. Purpose and Applicability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 B. Safety and Operating Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 C. Equipment and Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 D. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 E. Data Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 F. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 III. MVLN Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 B. Maintenance of Cell Stocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 C. MVLN Assay Setup and Time Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 D. MVLN Assay Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 E. Example Experiment Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 F. Example Stock and Experiment Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 G. Regarding an Estrogen Free Laboratory Environment . . . . . . . . . . . . . . . . . . . . . . . . . . 15 H. Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 I. Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 J. Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 K. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 IV. CV1 Cell Transfections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 V. Procedures to Assess Individual Steroidogenic Enzyme Activities in the Rat . . . . . . . . 20 A. Purpose and Applicability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 EDSTAC Final Report Chapter Five Appendices August 1998 B. Safety and Operating Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 C. Equipment and Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 D. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 E. Data Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 F. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 VI. Research Protocol for Assessment of Steroidogenesis Using In Vitro Testes Culture . 24 A. 50 Mg Pieces ( Rat or Other Species) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 B. Pieces and 1/ 4 Testes ( Rat And Other Species) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 C. Stock Solutions and Media Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 D. Additional Language to Consider In Developing Steroidogenesis Assay Procedure . . . . 28 In Vivo Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 VII. Research Protocol for Assessment of Uterotropic Activity in the Adult Ovariectomized and Juvenile ( 21 Day Old) Female Rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 A. General Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 B. Subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 VIII. Research Protocol for Assessment of Pubertal Development and Thyroid Function in Juvenile ( 21 Day Old) Female Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 A. General Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 B. Subjects  Juvenile Female Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 C. Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 D. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 E. Vaginal Opening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 F. Necropsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 G. Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 H. Data Summarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 IX. Research Protocol for Assessment of ( Anti) Androgenic Activity in the Immature Male Rat: The " Hershberger" Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 A. General Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 B. Subjects  Castrated Immature Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 C. Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 D. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 E. Necropsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 F. Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 G. Data Summarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 X. Fish Gonadal Recrudescence Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 A. Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 B. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 C. Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 D. Principle of the Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 E. Information on the Test Substance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 F. Validity of the Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 EDSTAC Final Report Chapter Five Appendices August 1998 G. Description of the Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 H. Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 I. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 Alternative In Vivo Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 XI. Development of an In Vivo Battery for Identifying Endocrine Modulators in Male Crl: Cd Ò Br Rats Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 B. Study Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 C. Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 D. Statistical Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 E. Safety And Housekeeping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 F. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 XII. Development of an In Vivo Battery for Identifying Endocrine Modulators in Female Crl: Cd Ò Br Rats Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 C. Materials And Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 D. Statistical Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 E. Safety And Housekeeping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 XIII. Research Protocol for Assessment of Pubertal Development and Thyroid Function in Immature ( 33 53 Day Old) Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 A. General Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 B. Subjects  Peripubertal Male Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 C. Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 D. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 E. Preputial Separation ( PPS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 F. Necropsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 G. Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 H. Data Summarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 EDSTAC Final Report Chapter Five Appendices August 1998 1 L  In Vitro Assays ( presented as examples)

I. Rat Estrogen Receptor Equilibrium Exchange Assay A. Purpose and Applicability The purpose of this protocol is to outline a procedure for the quantitation of estrogen receptor number and binding affinity in various adult female rat reproductive tissues. As tissue receptor number is finite, the binding of ligand to the receptor ( i. e., specific binding) is a saturable process. Unsaturable binding of ligand is called nonspecific binding and is due to ligand binding to non receptor proteins etc. Total binding is saturable binding + unsaturable binding. Total and nonspecific binding are determined empirically, while specific binding is calculated as their difference ( i. e., total nonspecific). The assay described below measures the binding of radiolabelled synthetic ligand ( i. e., [ 3H] E2) by cytosolic and/ or nuclear receptor extracts. Total [ 3H] E2 binding is determined by incubating the extracts with increasing concentrations of [ 3H] E2 during which time the labelled ligand binds to the unoccupied receptors in the cytosol extract or exchanges with endogenous hormone bound to the nuclear receptors. The total bound ligand ( i. e., saturable + nonsaturable binding) is separated from free ligand via hydroxylapatite extraction, eluted from the receptor with ethanol and quantified using liquid scintillation counting. Nonspecific binding is determined exactly as above except that a 100 fold molar excess of radioinert E2 is included in each incubation together with the increasing concentrations of [ 3H] E2 ( i. e., binding of [ 3H] E2 in the presence of a 100 fold molar excess of radioinert E2 represents unsaturable binding). Specific binding is calculated as total  nonspecific binding and is analyzed graphically via Scatchard analysis.

B. Safety and Operating Precautions All procedures with radioisotopes should follow the regulations and procedures as described in the Hazardous Agent Protocol ( HAP) and in the Radiation Safety Manual and Protocols.

C. Equipment and Materials 1. Equipment · Corning Stir/ hot Plates · Digital Pipets · Balance · Polytron PT 35/ 10 Tissue Homogenizer · Vacuum Concentrator · Refrigerated General Laboratory Centrifuge · High Speed Refrigerated Centrifuge ( up to 105,000 x g) · pH Meter with Tris Compatible Electrode · Scintillation Counter 2. Chemicals EDSTAC Final Report Chapter Five Appendices August 1998 2 L  · Tris HCL & Tris Base · Glycerol 99%+ · Ethylenediaminetetraacetic acid ( EDTA); Disodium salt · Dithiothreitol ( DTT) · Potassium Chloride · Hydroxylapatite ( BIO RAD) · Scintillation Cocktail ( Flow Scint III) · Ethyl Alcohol, anhydrous · [ 3H] Estradiol & Radioinert Estradiol ( NEN) · Triamcinolone Acetonide · Steroids ( Steraloids  recrystallized) 3. Supplies · 20 ml Polypropylene Scintillation Vials · 12 x 75 mm Borosilicate Glass Test Tubes · 1000 ml graduated cylinders · 500 ml Erlenmeyer flasks · Yellow ( 0 200 ml) pipet tips D. Methods 1. Preparation of TEDG buffer · To make 250ml: · Add to 150ml distilled water: · .303g Tris base · .140g EDTA · 25ml glycerol · Bring volume to 250 ml and stir. · pH to 7.6 · Add .04g DTT before use 2. Preparation of 50 mM TRIS Buffer Add 50.0 ml 1.0 M TRIS to 950 ml ddH 2 O. Store at 4o C. Check pH of the final solution to make sure it is 7.4 at 4o C.

3. Preparation of 60% Hydroxylapatite ( HAP) Slurry Shake BIO RAD HT GEL until all the HAP is in suspension ( i. e., looks like milk). The evening before the receptor extraction, pour 100 mls ( or an appropriate volume) into a 100 ml graduated cylinder, parafilm seal the top and place in the refrigerator for at least 2hours. Pour off the phosphate buffer supernatant, and bring the volume to 100 mls with 50 mM TRIS. Suspend the HAP by parafilm sealing the top of the graduated cylinder and inverting the cylinder several times. Place in the refrigerator overnight. The next morning, repeat the washing steps two times with fresh 50 mM TRIS buffer. After the last wash, add enough 50 EDSTAC Final Report Chapter Five Appendices August 1998 3 L  mM TRIS to make the final solution a 60% slurry ( i. e., if the volume of the settled HAP is 60 ml bring the final volume of the slurry to 100 mls with 50 mM TRIS). Store at 4o C until ready for use in the extraction.

4. Standard Curve Construction for Saturation and Scatchard Analysis The first step is to pipet the radioactive ligand ( i. e., [ 3H] E2) with and without a 100 fold excess of radioinert E2 into each tube so that the final concentrations of [ 3H] E2 are 6,3,1,. 6,. 3,. 1,. 03,. 01 nM in a 300 ml total volume. To accomplish this, label tubes and pipet the following into separate 12 x 75 mm glass test tubes.

Total Binding Tubes Nonspecific Binding Tubes (+)

6 nM= 21 ml of 10 7 [ 3H] E2 ± 21 ml of 10 5 radioinert E2 3 nM= 10.5 ml of 10 7 [ 3H] E2 ± 10.5 ml of 10 5 radioinert E2 1 nM= 35 ml of 10 8 [ 3H] E2 ± 35 ml of 10 6 radioinert E2 .6 nM= 21 ml of 10 8 mM [ 3H] E2 ± 21 ml of 10 6 radioinert E2 .3 nM= 10.5 ml of 10 8 [ 3H] E2 ± 10.5 ml of 10 6 radioinert E2 .1 nM= 35 ml of 10 9 [ 3H] E2 ± 35 ml of 10 7 radioinert E2 .03 nM= 10.5 ml of 10 9 [ 3H] E2 ± 10.5 ml of 10 7 radioinert E2 .01 nM = 3.5 ml of 10 9 [ 3H] E2 ± 3.5 ml of 10 7 radioinert E2 After the tubes have been pipetted as above, place them in the speed vac and dry the tubes according to the instructions in the speed vac SOP. When tubes are dry place them in an icewater bath and cover with aluminum foil until ready to pipet the nuclear or cytosolic extract into each tube for incubation. ( Total tubes have hot only and NSB tubes have hot + cold.)

5. Estrogen Receptor Assay Procedure · Make TEDG buffer and place in an ice water bucket. · Kill the rat and excise uterus. Tissues should be trimmed of fat, weighed and the weights recorded. Place on dry ice. Before assay, place tissues into a homogenizing tube in an icewater bath and add TEDG buffer at 1.0 ml/ 50mg tissue. It is extremely important to keep the tissues/ extracts at 4o C at all times. · Mince tissues with Metzenbaum scissors until all pieces are small 1 2mm cubes. Then homogenize the tissues at 4o C with a Polytron homogenizer using five second bursts of the Polytron. [ Note: place probe of the Polytron in an ice water bath with TEDG buffer to cool it down prior to its use for homogenization] · Transfer homogenates to pre cooled centrifuge tubes, balance, and centrifuge at 105,000 x g for 60 minutes ( i. e., 35,000 rpm using 90 Ti Beckman rotor). The supernatant is the low salt unoccupied cytosolic receptors. · While these tubes are spinning, the tubes containing [ 3H] E2 ± radioinert E2 for the saturation experiment can be pipetted as described in section 4.4 above. Dry these tubes in the speed vac, and place in a rack in an ice water bath as indicated. EDSTAC Final Report Chapter Five Appendices August 1998 4 L  · Pipet 300 ml of the cytosolic extract into each of the total and nonspecific binding tubes containing the [ 3H] E2 and [ 3H] E2 + radioinert E2, respectively. Gently vortex the samples and place them in the refrigerator overnight in rotor covered with foil for 20 hours. · Before leaving for the day, prepare the first wash of the HAP slurry as described in section 4.3 above. Also, label the HAP tubes and the scintillation vials to be used the following day  see underlines below. · The following morning, wash the HAP as described above, dilute with 50 mM TRIS to yield a 60% slurry, and transfer contents to a 100 ml Erlenmeyer flask. Place a stir bar in the flask and place the flask into a beaker containing ice water; stir the HAP slurry by placing the beaker on a magnetic stir plate. · While the HAP slurry is constantly being stirred, pipet 500 ml of the HAP slurry into clean pre labelled 12 x 75 mm glass test tubes. These tubes should be prepared in duplicate ( i. e., two tubes for every incubation tube). Place these tubes in a rack in an ice water bath prior to pipetting the HAP slurry and keep them in the ice water bath for the remainder of the assay. · Take the incubation tubes from the refrigerator and place them in an ice water bath with the HAP tubes. Pipet 100 ml in duplicate from each of the incubation tubes into the appropriate pre labelled tubes containing HAP. Repeat for all tubes. Quickly take each rack from the ice water bath and vortex each rack of tubes using the whole rack vortex unit. Place racks back into the ice water bath and vortex as above every five minutes for 20 minutes. · During the above 20 minute HAP extraction, pipet 30 ml in duplicate from the remaining 100 ml in each incubation tube into pre labelled 20 ml scintillation vials. These tubes will be used to estimate the concentration of total [ 3H] E2 used in the equilibrium assay and will be called the total counts tubes. The extraction tubes containing the remaining 40 ml of incubate can be discarded into the plastic radiation safety barrel together with some sawdust to adsorb the excess liquid. · Centrifuge the HAP tubes for two to three minutes at 4o C and 600 x g ( 1780 rpm in a Beckman GLC refrigerated centrifuge). Place the tubes back into the rack and into the icewater bath. · While the tubes remain in the ice water bath, aspirate the supernatant from each tube using a 9 inch pipet connected to an aspiration apparatus as per the radiation safety protocol. · Add 2 ml of 50 mM TRIS to each tube, vortex and centrifuge at 600 x g as above. Place the tubes into decanting racks in an ice water bath and decant the supernatant TRIS wash into the radiation safety container. Gently tap the tube openings on a clean adsorbent diaper, place the rack back in the ice water bath and add 2 mls of 50 mM TRIS. Repeat the TRIS washing procedure three or four times ( to be determined empirically) keeping the tubes at 4oC at all times. · Following the last wash and decanting, add 2 mls of ethanol to each tube, vortex three times at five minute intervals and centrifuge the tubes at 600 x g for ten minutes. Decant the supernatants into pre labelled 20 ml scintillation vials. Add 14 ml of Optifluor EDSTAC Final Report Chapter Five Appendices August 1998 5 L  scintillation cocktail and count samples using the single label DPM program with quench correction.

II. Protocol to Determine Affinity of Compounds for the Rat AR Androgen Receptor by Equilibrium Exchange Assay A. Purpose and Applicability The purpose of this protocol is to outline a procedure for the quantitation of androgen receptor number and binding affinity in various adult male rat reproductive tissues. As tissue receptor number is finite, the binding of ligand to the receptor ( i. e., specific binding) is a saturable process. Unsaturable binding of ligand is called nonspecific binding and is due to ligand binding to non receptor proteins etc. Total binding is saturable binding + unsaturable binding. Total and nonspecific binding are determined empirically, while specific binding is calculated as their difference ( i. e., total nonspecific). The assay described below measures the binding of radiolabelled synthetic ligand ( i. e., [ 3H] R1881) by cytosolic and/ or nuclear receptor extracts. Total [ 3H] R1881 binding is determined by incubating the extracts with increasing concentrations of [ 3H] R1881 during which time the labelled ligand binds to the unoccupied receptors in the cytosol extract or exchanges with endogenous hormone bound to the nuclear receptors. The total bound ligand ( i. e., saturable + nonsaturable binding) is separated from free ligand via hydroxylapatite extraction, eluted from the receptor with ethanol and quantified using liquid scintillation counting. Nonspecific binding is determined exactly as above except that a 100 fold molar excess of radioinert R1881 is included in each incubation together with the increasing concentrations of [ 3H] R1881 ( i. e., binding of [ 3H] R1881 in the presence of a 100 fold molar excess of radioinert R1881 represents unsaturable binding). Specific binding is calculated as total  nonspecific binding and is analyzed graphically via Scatchard analysis.

B. Safety and Operating Precautions All procedures with radioisotopes should follow the regulations and procedures as described in the Hazardous Agent Protocol ( HAP) and in the Radiation Safety Manual and Protocols.

C. Equipment and Materials 1. Equipment · Corning Stir/ hot Plates · Digital Pipets · Balance · Polytron PT 35/ 10 Tissue Homogenizer · Hewlett Packard HPLC with on line Radiomatic Radiochromatograph · Vacuum Concentrator · Hamilton Syringes ( 50 ml) · Refrigerated General Laboratory Centrifuge · High Speed Refrigerated Centrifuge ( up to 30,000 x g) EDSTAC Final Report Chapter Five Appendices August 1998 6 L  · pH Meter with Tris Compatible Electrode · Scintillation Counter 2. Chemicals · Tris HCL & Tris Base · Phenylmethylsulfonyl Fluoride ( PMSF) · Glycerol 99%+ · Sodium Molybdate · Ethylenediaminetetraacetic acid ( EDTA); Disodium salt · Dithiothreitol ( DTT) · Potassium Chloride · Hydroxylapatite ( BIO RAD) · Scintillation Cocktail ( Flow Scint III) · Ethyl Alcohol, anhydrous · [ 3H] R1881 & Radioinert R1881 ( NEN) · Triamcinolone Acetonide · Steroids ( Steraloids  recrystallized) 3. Supplies · 20 ml Polypropylene Scintillation Vials · 12 x 75 mm Borosilicate Glass Test Tubes · 1000 ml graduated cylinders · 500 ml Erlenmeyer flasks · Yellow ( 0 200 ml) pipet tips D. Methods 1. Preparation of TEDG Stock Solutions · Add 7.444g disodium EDTA to 100 ml ddH 2 O = 200mM. Store at 4o C. Use 750 ml/ 100ml TEDG buffer = 1.5 mM. · Add 1.742 g PMSF to 100 ml ethanol = 100 mM. Store at 4o C. Use 1.00 ml/ 100ml TEDG buffer = 1.0 mM. · Add 2.419 g sodium molybdate to 8.0 ml ddH 2 O in a 10 ml volumetric flask; bring the total volume to 10 mls = 1.0 M. Store at 4o C. Use 100ml/ 100ml TEDG buffer = 1.0 mM. · Add 15.4 mg DTT directly to 100 ml TEDG buffer the morning of the receptor isolation = 1.0 mM. Add 147.24 g Tris HCL + 8.0 g Tris base to 800mls ddH 2 O in a volumetric flask; bring the final volume to 1.0 liter. Refrigerate to 4o C and pH ( using 4oC pH standardizing solutions) the cooled solution to 7.4. Store at 4o C. Use 1.0 ml/ 100 ml TEDG buffer = 10mM. · Add 298.2 g KCL to 600 ml ddH 2 O in a 1000 ml volumetric flask; bring the total volume to 1000 ml = 4.0 M. Store at room temperature. Use 10.0 ml per 100 ml high salt TEDG buffer = 0.4M. EDSTAC Final Report Chapter Five Appendices August 1998 7 L  2. Preparation of Low Salt TEDG Buffer ( pH 7.4) To make 100 mls of low salt TEDG buffer add the following together in this order: · 87.15 ml ddH 2 O · 1.0 ml 1M TRIS · 10.0 ml glycerol · 100 ml 1M sodium molybdate · 750 ml 200mM EDTA · 1.0 ml 100mM PMSF · 15.4 mg DTT Check pH of the final solution to make sure it is 7.4 at 4o C.

3. Preparation of High Salt TEDG Buffer ( pH 7.4) To make 100 mls of high salt TEDG buffer add the following together in this order: · 77.15 ml ddH 2 O · 10 ml 4.0 M KCL · 1.0 ml 1M TRIS · 10.0 ml glycerol · 100 ml 1M sodium molybdate · 750 ml 200mM EDTA · 1.0 ml 100mM PMSF · 15.4 mg DTT Check pH of the final solution to make sure it is 7.4 at 4o C.

4. Preparation of 50 mM TRIS Buffer Add 50.0 ml 1.0 M TRIS to 950 ml ddH 2 O. Store at 4o C. Check pH of the final solution to make sure it is 7.4 at 4o C.

5. Preparation of 60% Hydroxylapatite ( HAP) Slurry Shake BIO RAD HT GEL until all the HAP is in suspension ( i. e., looks like milk). The evening before the receptor extraction, pour 100 mls ( or an appropriate volume) into a 100 ml graduated cylinder, parafilm seal the top and place in the refrigerator for at least two hours. Pour off the phosphate buffer supernatant, and bring the volume to 100mls with 50 mM TRIS. Suspend the HAP by parafilm sealing the top of the graduated cylinder and inverting the cylinder several times. Place in the refrigerator overnight. The next morning, repeat the washing steps two times with fresh 50 mM TRIS buffer. After the last wash, add enough 50 mM TRIS to make the final solution a 60% slurry ( i. e., if the volume of the settled HAP is 60 ml bring the final volume of the slurry to 100 mls with 50 mM TRIS). Store at 4o C until ready for use in the extraction.

6. Preparation of [ 3H 17a Methyl] R1881 Stock Solutions Dilute the original 1.0 mCi/ ml stock of [ 3H 17a methyl] R1881 to 0.1 mM ( i. e., 1 x 10 7 M). This is most easily accomplished by pipeting 1 ml of the stock solution for every specific EDSTAC Final Report Chapter Five Appendices August 1998 8 L  activity unit ( Ci/ mmol) and diluting this to 10.0 mls with ethanol. Thus, if the specific activity of the stock vial is 86 Ci/ mmol, then pipet 86.0 ml into an amber colored vial ( i. e., R1881 is photosensitive) and add 10.0 mls ethanol to the vial; this solution is 1 x 10 7M.

Calculation Check 86 ml x 1.0 mCi/ 1000ml = 86 x 10 3 mCi R1881 = 86 x 10 6 Ci R1881 86 x 10 6 Ci ¸ 86.0 Ci/ mmol = 1 x 10 6 mmol R1881 = 1 x 10 9 moles R1881 1 x 10 9 moles R1881 ¸ .010 liters = 1 x 10 7 moles/ liter = 0.1 mM To prepare the 1 x 10 8M stock simply make a 10 fold dilution of the 1 x 10 7M stock ( i. e., pipet 1.0 ml of the 1 x 10 7 M stock into a clean amber colored vial and add 9 mls ethanol = 0.01 mM).

7. Preparation of 100X Radioinert R1881 Solutions The R1881 comes as a 5.00 mg quantity. Dilute the original stock to 5.0 ml with ethanol = 3.52 mM. Take 56.82 ml and dilute to 20 ml in an amber vial with ethanol = 1 x 10 5 M R1881. This is the 10 mM radioinert R1881 stock. To make the 1.0 mM radioinert R1881 stock, pipet 2 ml of the 10 mM stock into an amber vial and dilute to 20 ml with ethanol = 1 x 10 6M = 1.0 mM radioinert R1881 stock.

8. Standard Curve Construction for Saturation and Scatchard Analysis The first step is to pipet the radioactive ligand ( i. e., [ 3H] R1881) with and without a 100 fold excess of radioinert R1881 into each tube so that the final concentrations of [ 3H] R1881 are 20, 16, 12, 8, 4, 2, 1, 0.5 nM in a 300 ml total volume. To accomplish this, label tubes and pipet the following into separate 12 x 75 mm glass test tubes.

Total Binding Tubes Nonspecific Binding Tubes a) 20nM= 60 ml of 0.1 mM [ 3H] R1881 ± 60 ml of 10 mM radioinert R1881 b) 16nM= 48 ml of 0.1 mM [ 3H] R1881 ± 48 ml of 10 mM radioinert R1881 c) 12nM= 36 ml of 0.1 mM [ 3H] R1881 ± 36 ml of 10 mM radioinert R1881 d) 8nM= 24 ml of 0.1 mM [ 3H] R1881 ± 24 ml of 10 mM radioinert R1881 e) 4nM= 12 ml of 0.1 mM [ 3H] R1881 ± 12 ml of 10 mM radioinert R1881 f) 2 nM= 60 ml of 0.01 mM [ 3H] R1881 ± 60 ml of 1.0 mM radioinert R1881 g) 1 nM= 30 ml of 0.01 mM [ 3H] R1881 ± 30 ml of 1.0 mM radioinert R1881 h) .5 nM= 15 ml of 0.01 mM [ 3H] R1881 ± 15 ml of 1.0 mM radioinert R1881 After the tubes have been pipetted as above, place them in the speed vac and dry the tubes according to the instructions in the speed vac SOP. When tubes are dry place then in an icewater bath and cover with aluminum foil until ready to pipet the nuclear or cytosolic extract into each tube for incubation.

9. Androgen Receptor Assay Procedure · Make low and high salt TEDG buffer and place in an ice water bucket. EDSTAC Final Report Chapter Five Appendices August 1998 9 L  · Kill the rat and excise epididymis, ventral prostate, and/ or seminal vesicles. Tissues should be trimmed of fat, weighed and the weights recorded. As quickly as possible, place the tissues into a homogenizing tube in an ice water bath and add low salt TEDG buffer at 10 ml/ gm tissue. It is extremely important to keep the tissues/ extracts at 4o C at all times. · Mince tissues with Metzenbaum scissors until all pieces are small 1 2mm cubes. Then homogenize the tissues at 4o C with a Polytron homogenizer using five second bursts of the Polytron. [ Note: place probe of the Polytron in an ice water bath with TEDG buffer to cool it down prior to its use for homogenization] · Transfer homogenates to pre cooled centrifuge tubes, balance, and centrifuge at 30,000 x g for 30 minutes ( i. e., 15,262 rpm using JA 17/ JA 21 Beckman rotors). · The supernatant is the low salt unoccupied cytosolic receptors and this material can be assayed directly or frozen at  70o C for future analysis. · Add high salt TEDG at 10 ml/ gm original tissue weight and homogenize the pellet thoroughly at 15 minute intervals for one hour ( i. e., four times). This procedure extracts the occupied androgen receptor from the nucleus. · Balance the tubes and centrifuge at 30,000 x g for 30 minutes at 4o C. While these tubes are spinning, the tubes containing [ 3H] R1881 ± radioinert R1881 for the saturation experiment can be pipetted as described in section 4.8 above. Dry these tubes in the speed vac, and place in a rack in an ice water bath as indicated. · Transfer the supernatant from the nuclear extract to a 16 x 100 mm polypropylene tube and place in an ice water bath. Pipet 300 ml of the cytosolic or nuclear extract into each of the total and nonspecific binding tubes containing the [ 3H] R1881 and [ 3H] R1881 + radioinert R1881, respectively. Gently vortex the samples and place them in the refrigerator overnight for 20 hours. · Before leaving for the day, prepare the first wash of the HAP slurry as described in section 4.5 above. Also, label the HAP tubes and the scintillation vials to be used the following day  see underlines below. · The following morning, wash the HAP as described in section 4.5 above, dilute with 50 mM TRIS to yield a 60% slurry, and transfer contents to a 100 ml Erlenmeyer flask. Place a stir bar in the flask and place the flask into a beaker containing ice water; stir the HAP slurry by placing the beaker on a magnetic stir plate. · While the HAP slurry is constantly being stirred, pipet 500 ml of the HAP slurry into clean pre labelled 12 x 75 mm glass test tubes. These tubes should be prepared in duplicate  i. e., two tubes for every incubation tube. Place these tubes in a rack in an ice water bath prior to pipetting the HAP slurry and keep them in the ice water bath for the remainder of the assay. EDSTAC Final Report Chapter Five Appendices August 1998 10 L  · Take the incubation tubes from the refrigerator and place them in an ice water bath with the HAP tubes. Pipet 100 ml in duplicate from each of the incubation tubes into the appropriate pre labelled tubes containing HAP. Repeat for all tubes. Quickly take each rack from the ice water bath and vortex each rack of tubes using the whole rack vortex unit. Place racks back into the ice water bath and vortex as above every five minutes for 20 minutes. · During the above 20 minute HAP extraction, pipet 30 ml in duplicate from the remaining 100 ml in each incubation tube into pre labelled 20 ml scintillation vials. These tubes will be used to estimate the concentration of total [ 3H] R1881 used in the equilibrium assay and will be called the total counts tubes. The extraction tubes containing the remaining 40 ml of incubate can be discarded into the plastic radiation safety barrel together with some sawdust to adsorb the excess liquid. · Centrifuge the HAP tubes for two to three minutes at 4o C and 600 x g ( 1780 rpm in a Beckman GLC refrigerated centrifuge). Place the tubes back into the rack and into the ice water bath. · While the tubes remain in the ice water bath, aspirate the supernatant from each tube using a nine inch pipet connected to an aspiration apparatus as per the radiation safety protocol. · Add 2 ml of 50 mM TRIS to each tube, vortex and centrifuge at 600 x g as above. Place the tubes into decanting racks in an ice water bath and decant the supernatant TRIS wash into the radiation safety container. Gently tap the tube openings on a clean adsorbent diaper, place the rack back in the ice water bath and add 2 mls of 50 mM TRIS. Repeat the TRIS washing procedure three or four times ( to be determined empirically) keeping the tubes at 4o C at all times. · Following the last wash and decanting, add 2 mls of ethanol to each tube, vortex three times at five minute intervals and centrifuge the tubes at 600 x g for ten minutes. Decant the supernatants into pre labelled 20 ml scintillation vials. Add 14 ml of Optifluor scintillation cocktail and count samples using the single label DPM program with quench correction.

E. Data Processing 1. Free Concentration of [ 3H] R1881 Multiply the DPM in the total counts tubes by 1.8047 x 10 5. This value will yield the free concentration ( i. e., nM) of [ 3H] R1881 initially present in each incubation tube.

Calculation Check X DPM = 4.5045 x 10 13 Ci = 5.4141 x 10 15 mmole = 5.4141 x 10 18 moles 2.22 x 1012 dpm/ Ci 83.2 Ci/ mmole 1000 mmole/ mole 0.0003 liters = 1.8047 x 10 14 moles/ liter = X ( 1.8047 x 10 5) nM 1 x 10 9 moles/ nmole EDSTAC Final Report Chapter Five Appendices August 1998 11 L  2. Calculation of Total, Nonspecific and Specific [ 3H] R1881 Binding · Total binding is calculated by multiplying the DPM from the tubes that contained only radiolabelled R1881 x ( 1.6242 x 10 2). This value will be total binding in fmoles. · Nonspecific binding is calculated by multiplying the DPM from the tubes containing radiolabelled R1881 + 100 fold molar excess of radioinert R1881 x ( 1.6242 x 10 2). This value will be nonspecific binding in fmoles. · Specific binding is calculated by subtracting nonspecific binding from total binding i. e., fmoles total binding  fmoles nonspecific binding = specific binding in fmoles.

Calculation Check To get fmoles multiply the DPM values by 1.6242 x 10 2. This is simply nM x 300, i. e.,

1.8047 x 10 5 nM x 0.0003 liter = 1.6242 x 10 2 fmoles 1 x 10 6 nmoles/ fmole 3. Graphical Presentation of the Data An example of a typical saturation curve and the associated Scatchard analysis is illustrated in Figure 1. Maximal binding capacity ( Bmax) and association/ dissociation constants ( Ka / Kd) can be estimated using a number of commercially available iterative nonlinear regression analysis programs. One of the better programs was developed by Munson and Rodbard and is called LIGAND ( Munson and Rodbard, 1980).

F. References Munson, P. J., and D. Rodbard, Anal. Biochem., 107, 1980, pp. 220 239. Nonneman, D.. J., V. K. Ganjam, W. V. Welshons, and F. S. vom Saal, Biol. Reprod., 47, 1992, pp. 723 729. Segel, I. H., Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems., 1st ed., John Wiley and Sons, Inc., New York, NY, 1975. Tekpetey, F. R., and R. P. Amann, Biol. Reprod., 38, 1988, pp. 1051 1060.

III. MVLN Assay A. Introduction The MVLN estrogen specific transcription assay can be used to characterize estrogenic chemicals ( Pons et al. 1990). The assay utilizes an MCF 7 ( Soule et al. 1973) derivative that has been stably transfected with the Vit Luc reporter gene ( Pons et al. 1990). Thus, the MVLN cell line expresses the endogenous estrogen receptor of MCF 7 and at the same time, contains an exogenous estrogen responsive reporter ( luciferase). Therefore, the estrogen specific EDSTAC Final Report Chapter Five Appendices August 1998 12 L  transcription activity of a test chemical is directly related to the activity of luciferase measured in the lysate of treated MVLN cells. The MVLN assay procedure presented here is a modified version of published methods ( Gagne et al. 1994; Pons et al. 1990). While the protocol herein does utilize the MVLN subclone of MCF 7, all tissue culture materials, such as media and sera, are commercially available. In brief, MVLN cells are seeded into 12 well plates, fed media containing treatment compounds and then two days later, cell lysates are harvested and measured for luciferase activity. When cell counting is called for in this procedure, consult the method " Monolayer Cell Counting with a Coulter Counter" from this laboratory.

B. Maintenance of Cell Stocks The MVLN cell line must be obtained from its source ( Pons et al. 1990). The MVLN clone has been shown to maintain a stable, estrogen responsive phenotype in this laboratory over many passages ( at least 15). Stock cultures should be maintained in 10% fetal bovine sera ( FBS) media under 5% CO 2 in a 37 ° C incubator. Such culture conditions will be " estrogen rich" and tend to favor cells that require estrogen for growth ( MCF 7, MVLN). A regular schedule of passing stocks weekly ( Monday into 8, T 25 flasks at a density of 5 x 105 to 1 x 106 cells/ flask should provide enough cells for seeding stocks as well as experimentals ( 8, 12 well plates) seven days later. MVLN cells may grow slower than other MCF 7 derivitives. In addition, MVLN cells are very sensitive to seeding density. If seeded too light, MVLN cells will grow exceedingly slow and may not thrive. The common pH indicator phenol red has been shown to be estrogenic and therefore should not be used in cell cultures utilized for MVLN assays.

For routine passage, the MVLN cell monolayer is removed with trypsin/ EDTA treatment, diluted to 10 ml with whole media and then dispersed into a suspension ( mostly single cell) through the use of a 10 ml syringe and bent canula. This canula is a 14 gauge, blunt tip, Luer lock needle ( Thomas Scientific) in which 1 cm at the tip is bent 30 ° to 45 ° . The 10 cm length can be used with T 25 flasks while the 15 cm will work with T 25 or T 75 flasks. First, count one duplicate flask. Second, remove media from flask( s) to be passed ( monolayer of cells stays attached). Then, wash each flask three times with Ca++ free HBSS, remove and then add 1 2 ml trypsin for three to five minutes @ 37 ° C. Be sure to disperse the trypsin evenly over the monlayer. After incubation, dilute trypsin to 10 ml with whole media. With the sterile canula syringe, draw the 10 ml of media up into the syringe. Expel the media, with moderate force, through the bent canula, towards the cell monolayer ( almost, but not touching the monolayer surface) with a circular motion covering the cell surface of the flask. Repeat for a total of three cycles making sure that all the monolayer has been removed from the flask ( keep air bubbles to a minimum). After last cycle, leave the cell suspension in the flask. Then, with a 10 ml pipette, rinse down the inside of the flask three times with the cell solution. The cells will not be harmed by this procedure. This method disperses cells better than trypsin treatment alone. Note that since all MVLN cells can express the reporter gene in response to estrogen, it is essential that cells are dispersed evenly before seeding experimental plates.

An aliquot of this concentrated media cell solution should then be diluted with media in a sterile vessel, mixed and used to seed flasks. For precise seeding, it is recommended that the entire EDSTAC Final Report Chapter Five Appendices August 1998 13 L  volume of cells and media to seed all the flasks/ plates desired is mixed in a single vessel. For example, to seed eight flasks with 7 x 105 cells each, make 50 ml of a seeding solution that is 1.16 x 105 cells/ ml, mix well and then add 6 ml to each T 25 flask. Recall that you know how many cells are in each flask since a duplicate flask was counted previously. The goal of this method is to seed all flasks/ plates the same ( high precision). Since all MVLN cells can express the reporter gene in response to estrogen, precision in MVLN assays is completely dependent on uniform seeding of plates. It may be a good idea to practice seeding flasks and then count them the next day.

C. MVLN Assay Setup and Time Sequence With the following exceptions, passing MVLN cells for assays should be done as described above. First, it is essential that cells used to seed experimental plates were withdrawn from estrogen six days prior to passage. A suggested way to withdraw cells from estrogen involves seeding stocks ( eight flasks) in 10% FBS media one week before you plan on seeding experimentals. The day after seeding the stocks, label two flasks as " stock" and replace the media in these with 10% FBS media These stock flasks should be fed 10% FBS media every other day until used to seed eight more stock flasks the following week. The remaining 6 flasks are then withdrawn from estrogen by rinsing three times with sterile PBS ( all flask surfaces) and fed 10% dextran coated charcoal ( DCC) FBS media. The 10% DCC FBS media is almost devoid of estrogens. These " withdrawn" flasks should be rinsed 3X and fed DCC media every other day until used for seeding a MVLN experiment. When seeding MVLN stocks or experiments, one of the duplicate flasks is always counted to determine the cells per flask count for that series ( one of two stocks is counted, one of six withdrawn is counted). Keep in mind that it takes a week to get cells ready for a MVLN experiment.

MVLN experiment 12 well plates are seeded on day  1 ( Monday) with 1 x 105 cells/ well in 10% DCC FBS media. To ensure constant seeding, mix the required cells and media in a sterile bottle and then seed aliquots ( 2 ml) into each well. The seeding mixture bottle should be mixed often during this procedure. The following day ( day 0, Tuesday), the seeded cells are left alone and allowed to firmly attach to the plates.

Two days after seeding ( day 1, Wednesday), cells are fed treatment media ( 2 ml/ well). Treatment media is 5% DCC FBS into which treatments in ethanol carrier have been added. Treatments may be made up in 50 ml polyethylene tubes ( do not use polycarbonate or polystyrene tubes! ( Soto et al. 1991) and should be no more than 0.05% v/ v ethanol carrier solvent. Higher levels of ethanol may have confounding effects on MVLN studies. Treatment carrier solvents such as DMSO and methanol should be avoided since they are toxic to cells and will have confounding effects on MVLN studies. Experimental cells should have new treatment media applied the next day ( day 2, Thursday).

On day 3 ( Friday), treated cell lysate should be harvested for luciferase assay. First remove treatment media from each well. Then, wash each well one time with 2 ml PBS. It is essential that all PBS is removed from each well at this step. Removal of the PBS by aspiration followed EDSTAC Final Report Chapter Five Appendices August 1998 14 L  by a one minute bench top incubation with the plate tipped 45 ° and final aspiration of drained residue is recommended. To lyse cells, add 500 ml lysis buffer to each well and then incubate at room temperature on a rocker for at least 30 minutes. Transfer lysate from wells with micro pipet to micro tubes. Make sure that all cell debris is mixed and removed uniformly between wells. Lysate can be left at room temperature for up to six hours, kept at 4 ° C over night or stored indefinitely at  70 ° C without loss of luciferase activity. Sample tubes should be equilibrated at room temperature, vortexed and then spun down prior to sampling ( 20 to 50 ml) for luciferase assay.

D. MVLN Assay Design Properly designed MVLN assays can be utilized to answer only the following four questions. First, does the test compound stimulate estrogen receptor mediated transcription ( what is the shape of the corresponding dose response curve)? Second, if the test compound stimulates transcription, is this response an estrogen receptor mediated mechanism ( is the compound an estrogen receptor agonist)? Third, can the test compound block the agonist effects of E 2 ( is the compound an antiestrogen)? Last, is the test compound toxic to MVLN cells? Attempts to obtain additional information from the MVLN assay may be misleading. Keep in mind that this assay does not necessarily determine if the test compound binds to the ER.

Each data point of the MVLN assay should be run in duplicate ( minimum) or triplicate during a trial. Then, that same trial should be repeated at least two more times.

E. Example Experiment Setup Blank ( no additions to DCC FBS media) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 2 wells E 2 ( positive controls) 10 12, 10 11, 10 10, 10 9, 10 8, 10 7 M · · · · · · · · · · · · · · · · · · · · · · · · · · 12 wells ICI 182,780 10 6 M ( check of estrogen free conditions) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 2 wells E2 10 10 M + ICI 182,780 10 6 M ( Check of ICI) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 2 wells Test compound A: 10 8, 10 7, 10 6, 10 5 M · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 8 wells Test compound B: etc.

ICI 10 6 M + TC A 10 5 M ( ER mechanism test) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 2 wells ICI 10 6 M + TC B 10 5 M etc.

E2 10 10 M + TC A 10 5 M ( antiestrogen test) · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 2 wells E2 10 10 M + TC B 10 5 M etc.

to a Total of 96 wells Note: Test compound toxicity is determined by comparing Luc activity of test compound treatments with blank activity and/ or test compound with ICI to ICI alone. EDSTAC Final Report Chapter Five Appendices August 1998 15 L  F. Example Stock and Experiment Schedule Day  1 ( Monday) Pass stocks, experimentals plated. Day 0 ( Tuesday) Cells left undisturbed to attach to flasks/ plates.

Day 1 ( Wednesday) Feed stocks ( 2 flasks FBS, 6 flasks withdrawn in DCC FBS).

Feed experimentals treatments. Day 2 ( Thursday) Feed experimentals treatments.

Day 3 ( Friday) Feed/ withdraw stocks.

Experimentals harvested ( morning).

G. Regarding an Estrogen Free Laboratory Environment All glassware, caps, hoses, etc. that may contact media must be free of estrogens. Soap wash and 3X hot rinse followed by rinsing three times with ddH 2 O, air dry, rinse with 95% ethanol ( from glass bottle; cans and plastic may have estrogenic contamination!) followed by rinse three times with ddH 2 O water. Baking of foil covered glassware for 12 hours in a 250 ° C oven will sterilize and remove more organic compounds. Wash and rinse bottle caps as above, dry in low temp oven and autoclave. Alternatively, bottles and caps may be autoclaved together with the risk of contamination by estrogenic condensates. Your cell culture environment should be characterized for estrogen contamination by proliferation assay tests with and without added ICI 182, 780 ( Wakeling and Bowler 1992). If the " estrogen free" cells grow faster (> 10%) than the ICI treated cells, you have estrogen contamination. All experiments conducted in the presence of such contamination are useless since regardless of how they are set up, you are testing combinations of chemicals. We have found plastic vessels and implements to be the major source of estrogen contamination. Polystyrene and polycarbonate seem to be the big problems. Do not use culture flasks with " phenolic" caps. Filter units may add estrogenic substances to media. The Corning bottle top units ( orange) are suspect. Zap Caps seem to add some kind of nonestrogenic mitogen which induces MCF 7 cells to grow at maximum rate, even in the presence of ICI. It is unclear what effect Zap Cap contamination has on MVLN assays. Also, it appears to be relatively easy to extract estrogens from gloves and/ or hands when rinsing items with ethanol. Lastly, ethanol may confound MVLN assays. Ethanol rinsed vessels and implements must be thoroughly dry before use.

H. Media 1. DMEM powder for 10L ( Gibco 13000 096). EDSTAC Final Report Chapter Five Appendices August 1998 16 L  2. gm HEPES ( Gibco 11344 033), media will be 20 mM. 3. ml non essential amino acids ( Gibco 11140 019), media will be 0.1 mM. 4. ml sodium pyruvate ( Gibco 11360 070), media will be 1 mM. 5. gm NaHCO3. 6. ml/ L media of Antibiotic Antimycotic solution ( Gibco 15240 062).

In 3 L tissue culture grade water, add 1, 2, 3 & 4 above. Mix 45 minutes in 4 L beaker. Add 5, mix 15 minutes. pH to 7.3. Dilute to 10 L and mix 15 minutes. Check pH. Filter into 500 ml sterile bottles ( Gelman VacuCap 4622 or Gelman Micro Culture Capsule 12158). Store media at 4 ° C. Add Antibiotic Antimycotic solution to media bottle when used ( made up with sera). To make one bottle of 10% sera media, add 50 ml sera to 500 ml of media.

I. Sera FBS Hyclone Characterized Fetal Bovine Sera ( A 1115 L) DCC FBS Hyclone Charcoal/ Dextran Fetal Bovine Sera ( A 1120 L)

J. Buffers Ca++ Free HBSS Gibco 14185 052 PBS Gibco 14080 055 Lysis Buffer Promega E153A EDSTAC Final Report Chapter Five Appendices August 1998 17 L  K. References Gagne, D., P. Balaguer, E. Demirpence, C Chabret, F. Trousse, J. C. Nicolas, and M. Pons, " Stable luciferase transfected cells for studying steroid receptor biological activity," Journal of Bioluminescence & Chemiluminescence, 9, 1994, pp. 201 209. Pons, M., D. Gagne, J. C. Nicolas, and M. Mehtali, " A new cellular model of response to estrogens: a bioluminescent test to characterize ( anti) estrogen molecules," Biotechniques, 9, 1990, pp. 450 459. Soto, A. M., H. Justicia, J. W. Wray, and C. Sonnenschein, " p Nonyl phenol: an estrogenic xenobiotic released from ` modified' polystyrene," Environmental Health Perspectives, 92, 1991, pp. 167 173. Soule, H. D., J. Vazquez, A. Long, S. Albert, and M. Brennan, " A human cell line from a pleural effusion derived from a breast carcinoma," Journal of the National Cancer Institute, 51, 1973, pp. 1409 1416. Wakeling, A. E., and J. Bowler, " ICI 182,780, a new antioestrogen with clinical potential [ Review]," Journal of Steroid Biochemistry & Molecular Biology, 43, 1992, pp. 173 177.

IV. CV1 Cell Transfections Purpose and Applicability The purpose of this protocol is to outline a procedure for the quantitation of AR mediated alterations of transcriptional activation in CV1 cells.

MONDAY · plate 0.2 x 106 CV1 cells / 6cm Corning TC dish in the following media DMEM  ( high glucose + L glutamine + 25 mM Hepes + NaHCO 3)  Take a 0.5 liter bottle and add · 5 ml of 100X antibiotic/ antimycotic ( frozen aliquots) · 50 ml fetal bovine serum ( frozen 50 ml aliquots) NOTES: · Grow cells up in T 150 culture flasks in 10% FCS / DMEM. · When there are enough cells for the experiment, decant media from the flasks into funnel waste beaker layered with sterile gauze to prevent backsplash. · Wash cells twice with 20 ml of 1X HBSS and decant into waste funnel ( this gets rid of the serum which inactivates trypsin). · Add 1 2 ml of 1X Trypsin ( from 1.0X frozen stock)  roll the trypsin over the cells and place cells in the incubator for five to ten minutes. · After the cells detach ( i. e., round up and float), thump the side of the flask with the palm of your hand and add 10 ml of 10% FCS / DMEM ( the serum in the media inactivates the trypsin). · Transfer the cells to a 50 ml centrifuge tube ( be sure to get all the cells off the plates) and spin the cells down at 1000 rpm ( 250 x g). EDSTAC Final Report Chapter Five Appendices August 1998 18 L  · Aspirate or decant the media and thump pellet loose from the tube  add 10 ml of 10% FCS / DMEM and resuspend the cells by pipeting up and down with a 10 ml pipet. · Count cells on a hemocytometer as follows: · Total cell # in all center squares x 0.01 = # cells ( in millions) / ml · Dilute the cell suspension out to 50,000 cells/ ml and add 4ml of this cell stock solution to each 6 cm plate using a 10 ml pipet. · In general, the following numbers of CV1 cells are obtained: · T 150 culture flask of almost confluent CV1 cells will contain 8 10 x 106 cells · T 75 culture flask of almost confluent CV1 cells will contain 4 5 x 106 cells ** HANDS ARE WASHED WELL WITH SOAP AND WATER ( ESPECIALLY UNDER NAILS) AND BEFORE PLACING HANDS IN THE CULTURE HOOD, HANDS SHOULD BE SPRAYED WITH 70% ETOH. · THE HOOD SURFACES ARE SPRAYED WITH 70% ETOH AND WIPED CLEAN BEFORE USE. · ANYTHING THAT GOES IN THE HOOD THAT IS NOT STERILE IS SPRAYED WITH 70% ETOH INCLUDING MEDIA BOTTLES, TUBE RACKS, PIPETMEN, ETC. CAN ALSO FLAME THE OUTSIDE OF GLASS CULTURE MEDIA BOTTLES TUESDAY · Prepare 2X HBS ( 100 ml) as follows: · 90 ml ddH 2 O · 5.6 ml 5M NaCl ( from sterile stock) · 2.5 ml 2M HEPES ( Na+ salt) · 1.5 ml 0.1M Na 2 HPO 4 **** pH to 7.12 7.13**** , then filter, sterilize and store in the refrigerator.

· Prepare CaPO 4 precipitates ( listed below are the amounts for 14 dish batches) after all components have reached room temperature. Add the following to a sterile 5.0 ml tube in this order · 1.53 ml ddH 2 O · 700 ng pCMVhAR ( P 9; 50 ng/ dish) · 70 ug MMTV LUC ( 5 ug/ dish) ** vortex to mix** · 219 ul 2M CaCl 2 ( from frozen stock made by adding 22.197g CaCl 2 to a 100 ml volumetric flask and bring the volume to 100 mls with ddH2O, sterile filter).

** vortex to mix**

· While vortexing ( setting 2.5) a 1.75 ml aliquot of the 2X HBS solution in a 15 ml screw top conical c fuge tube add the contents of the 5.0 ml tube containing CaCl 2 DNA with a 5.0 ml plastic pipet.

** DROPWISE AND SLOWLY**. EDSTAC Final Report Chapter Five Appendices August 1998 19 L  · Repeat for the remaining tubes then wait 10 minutes for ppt's to form. · At ten minutes add 3.5 ml of media to each tube to stop the precipitation ( the ppt will be stable for at least 30 minutes).... repeat for the other tubes at their ten minute time periods. After all ppts have been stabilized, check the ppts to be sure they are small with individual grains. If particles are linked into chains or lumpy looking do not use it. Dilute the contents of each tube ( now containing 7.0 mls) with 49 mls media and combine all in a sterile flask ( i. e., for multiple 14 dish batches). · Aspirate the media from the CV1 cell dishes and immediately add 4 ml of gently mixed DNA ppt media to each of the dishes in the experiment using a 10 ml glass pipet. · Return dishes to 37 ° C incubator for four to six hours. · Aspirate the precipitate from each dish and wash each dish two times with 4 ml DPBS and add 4 ml 5% DCC FBS DMEM media. · Place all dishes in 37 ° C incubator overnight WEDNESDAY · Make up a 1000X concentrated stock solution for each treatment in ETOH ( i. e., so won't have to sterilize) as follows: 10.0 mM, 1 mM, 0.5 mM, 0.2 mM, 0.05 mM. · Make up 10X DHT working solution by adding 24 ul of 10 uM DHT ( made fresh from frozen 10 mM stock) to a 5.0 ml sterile tube and combine with 2.4 mls of 5% DCC media ( this is enough for 60 dishes). · Take plates out of incubator and add 4 ul of each treatment to appropriately labelled dishes ( in duplicate).

** Add treatments to the side of each dish and let run down into the media rock plates after addition.**

· Immediately add 40 ul of 10X DHT ( to the side of each dish to receive DHT) using the positive displacement pipetting technique. · Move plates back and forth to distribute the treatments evenly in the media. · Return the dishes to the 37 ° C incubator overnight.

** Notes: Final [ ]' s are 1nM DHT and 10 uM, 2 uM, 0.5 uM, 0.2 uM, 0.05 uM treatments.

 2X TBS Buffer ( 500 ml) For 4 liters  8.18 g NaCl  65.44 g  0.23 g KCl  1.84 g  0.147 g CaCl 2  2H 2 O  1.18 g  0.1 g MgCl 2  6 H 2 O  0.80 g  0.128 g Na 2 HPO 4  H 2 O  1.02 g  3.03 g Tris  HCL  24.24 g pH the buffer to 7.4 and filter sterilize into a sterile bottle before use. EDSTAC Final Report Chapter Five Appendices August 1998 20 L  THURSDAY MORNING  Aspirate media from all dishes and replce with 4.0 mls of fresh 5% DCCmedia using a 10 ml pipette. Repeat the above dosing protocol.

AFTERNOON  Harvest cells · Aspirate media from all dishes; wash cells twice with 2 ml of 1X HBSS and aspirate the HBSS from all dishes. · add 0.5 ml cell culture lysis buffer ( at room temperature) to each plate and let rock at room temperature for 20 30 minutes. · using a rubber scraper ( if necessary), scrape the cells loose from the dish and then slowly pipette the lysis buffer and cells up and down.  Need to go slow because one does not want excessive bubbles.

** Can use the same rubber scraper for all plates, if the scraper is rinsed off with ddH 2 0 between dishesand go from high treatment dose to low treatment dose ( i. e., low luciferase to high luciferase).

** Place all of the cell lysate into prelabelled 1.0 ml microcentrifuge tubes and store at  70 ° C until assay ( i. e., Friday morning).

FRIDAY  LUCIFERASE ASSAY · Prepare 20 mls of rxn buffer in a 50 ml centrifuge tube as follows ( 20 mls is enough for 50 samples): · Add 18 mL ddH 2 O to a 50 ml centrifuge tube · Add 500 uL of 1 M Glycylglycine ( make a 50 ml stock by adding 6.605g gly gly ( MW 132.1) to 50 ml ddH 2 O, pH to 7.8, sterile filter and store at 4o C). · Add 300 uL 1M MgCl 2 ( from Sigma also stored at 4o C) · Add 1.0 mL of 100 mM ATP ( make a 50 ml stock by adding 2.755g of ATP ( MW 551.1 g/ mole) to 50 ml ddH 2 O, pH the soln to 7.8, aliquot to 1.0 mL and store at  80 ° C). · Add 200 uL of 50 mg/ ml BSA ( make a 10 ml stock by adding 500 mg BSA to 10 ml ddH 2 O, aliquot to 200 uL and store at  20 ° C). · Place the tube at room temperature ** NOTE: IT IS CRITICAL THE THAT THE pH OF THE RXN BUFFER IS 7.8 AT ROOM TEMPERATURE. IF THE GLY GLY AND ATP SOLUTIONS ARE CAREFULLY pH'ED, THEN THE FINAL MIX WILL BE pH 7.8  CHECK TO BE SURE. **

· Take cell lysates out of the freezer and allow to thaw at room temperature. · Fill brown luminometer bottle with ddH 2 O and connect to automatic injector in the front panel of the luminometer. Make sure a cuvette is in the chamber and flush the luminometer lines with water ( Service 14). Empty the cuvette and replace in the chamber. EDSTAC Final Report Chapter Five Appendices August 1998 21 L  ** NOTE: NEVER PLACE WET CUVETTES IN THE INSTRUMENT AND ALWAYS KEEP A CUVETTE IN THE INSTRUMENT WHEN NOT IN USE. **

· Flush the lines with air ( i. e. disconnect the brown bottle) again using Service 14. · Add the luciferin to brown bottle # 1 and fill the bottle approximately to the shoulder ( should be enough for about 100 samples). Add rxn buffer to the brown bottle # 2 and fill the bottle approximately to the shoulder ( should be enough for about 100 samples). Place a new cuvette in the chamber and flush the lines with the luciferin/ rxn solution. · The 1mM D luciferin stock solution is made and stored as follows: · Add 159.21 mg D luciferin ( K+ salt; MW 318.41 g/ mole) to 500 ml ddH 2 O. Make 10 ml aliquots in 15 ml centrifuge tubes. Wrap tubes with aluminum foil ( i. e., luciferin is light sensitive) and store at  20 ° C.

NOTE: Can also use the Na+ salt of D luciferin but it sometimes turns yellow  its probably still ok but better to use the K+ salt to avoid any potential problems.

· Set up luminometer ( 20 sec read, continuous measurements, raw data, no duplicates) so will get raw data ( i. e., relative light units) for each sample. · Vortex the cell lysate and spin tubes for 30 seconds to isolate cell debris. · Add 50 ul of vortexed cell lysate to a new cuvette, place in the luminometer and press start. While the sample is being read get another tube ready ( this usually works out so that by the time the machine is ready the next sample is also ready to go). · Use a new cuvette for each sample, do not reuse cuvettes. · When finished, exit and press service 13 to back the contents of each line back into the respective brown bottles, decant to stock solution and place the rxn buffer in the refrigerator and the luciferin in  70C freezer. · Wash the brown bottles out with ddH 2 O and place ddH 2 O in each bottle and place back in line. Place an empty cuvette in the machine and flush the lines ( service 14) first with ddH 2 O then with air. Leave the lines dry, shut off the luminometer and always keep an empty cuvette in the sample chamber.