Detecting hormonally active compounds

FIELD OF THE INVENTION

[0004] The invention relates to the fields of molecular genetics, endocrinology, and toxicology. More particularly, the invention relates to compositions and methods for detecting androgenic/estrogenic agents in the environment and screening candidate agents for androgenic/estrogenic activity.

BACKGROUND OF THE INVENTION

[0005] The last decade saw the emergence of the field of endocrine disruption after it was discovered that a variety of anthropogenic chemicals act as weak estrogens. Through their interaction with estrogen receptors (ERs), these endocrine-disrupting compounds (EDCs) can alter normal expression of gene products and proteins at critical times during development and reproduction. Environmental contamination with EDCs is therefore a serious concern.

[0006] In an effort to detect EDCs in environmental samples, a number of methods have been developed including both in vitro and in vivo assays. Available in vitro assays include those based on hormone receptor-ligand binding, cell proliferation, and reporter gene expression. Although these are relatively inexpensive and amenable to high throughput applications, they provide only limited information about how EDCs affect animals in the environment (see, e.g., Zacharewski T. Environ. Sci. Technol. 31:600-623, 1997; Baker V. A. Toxicol In vitro 15:413-419, 2001). In vivo exposure assays, on the other hand, provide useful information about whole animal responses to EDCs, but can be more cumbersome and expensive than in vitro assays. Moreover, such assays do not provide information about the molecular mechanisms underlying EDC-mediated changes in the animals.

SUMMARY

[0007] The invention is based on the discovery of a large number of sheepshead minnow (SHM) and largemouth bass (LMB) genes that are up-regulated or down-regulated in tissues that have been exposed to an estrogenic or androgenic agent. Thus, whether an environmental sample contains an estrogenic or androgenic agent can be determined by examining a fish (or biological sample obtained from the fish) that was exposed to the sample (e.g., a lake or river) for modulation of expression of these genes. A finding that these genes were modulated in the test fish compared to a control fish not exposed to the sample (or an estrogenic or androgenic agent) indicates that the sample contains an estrogenic or androgenic agent. It was also discovered that different classes of estrogenic or androgenic agents modulated expression of the genes in different patterns depending on the class or mechanism of action of the estrogenic or androgenic agent. Thus, the invention can be used to discern that a particular type of estrogenic or androgenic agent is present in the sample. Based on these discoveries, a screening assay to characterize an unknown molecule's hormonal (e.g., estrogenic or androgenic) activity was developed wherein a fish, fish tissue or fish cell is exposed to a test substance and the effect of the substance on gene expression is compared to known patterns of gene up- or down-regulation. On this basis, the agent can be classified as estrogenic or androgenic and is thus determined to be hormonally active.

[0008] Accordingly, the invention features a method for detecting the presence of an agent having estrogenic or androgenic activity in a sample (e.g., a water sample). The method includes the steps of: (A) providing at least one (e.g., at least 2, 3, 4, 5, 10, 25, 100) fish cell which was exposed to the sample; (B) analyzing the at least one fish cell for expression of at least one gene wholly or partially encoded by a nucleotide sequence of SEQ ID NOs: 1-560; and (C) comparing the expression of the at least one gene in the cell compared to the expression of the at least gene in a control cell not exposed to the sample or an agent having estrogenic or androgenic activity. A difference in the expression of the at least one gene in the at least one fish cell compared to the expression of the at least one gene in the control cell indicates that the sample contains an agent having estrogenic or androgenic activity.

[0009] In the method, the fish cell can be a large mouth bass cell or a sheep's head minnow cell. It can also be one obtained from a fish that had been exposed to the sample.

[0010] Also in the method, the step of analyzing the at least one fish cell for expression of at least one gene (e.g., at least 2, 3, 4, 5, 10, 25, 100) might involve isolating RNA transcripts from the at least one cell, and the step of analyzing the at least one fish cell for expression of at least one gene can include contacting the isolated RNA transcripts or nucleic acids derived therefrom using the isolated RNA transcripts as templates with at least one probe (e.g., at least 2, 3, 4, 5, 10, 25, 100) that hybridizes under stringent hybridization conditions to at least one nucleotide sequence of SEQ ID NOs: 1-560.

[0011] The probe can be immobilized on a substrate such as nylon, nitrocellulose, glass, and plastic. It can be on conjugated with a detectable label. In one variation of the method of the invention, the isolated RNA transcripts or nucleic acids derived therefrom are conjugated with a detectable label.

[0012] The method of the invention might also include analyzing the control cell not exposed to the sample or an agent having estrogenic or androgenic activity for expression of at least one gene wholly or partially encoded by a nucleotide sequence of SEQ ID NOs: 1-560. In this version of the method, the step of analyzing the control cell for expression of at least one gene can include isolating RNA transcripts from the control cell and contacting the isolated RNA transcripts or nucleic acids derived therefrom using the isolated RNA transcripts as templates with at least one probe (e.g., at least 2, 3, 4, 5, 10, 25, 100) that hybridizes under stringent hybridization conditions to at least one nucleotide sequence (e.g., at least 2, 3, 4, 5, 10, 25, 100) of SEQ ID NOs: 1-560. Also in this version of the method, the RNA transcripts or nucleic acids derived therefrom isolated from the at least one fish cell can be conjugated with a first detectable label and the RNA transcripts or nucleic acids derived therefrom isolated from the control cell are conjugated with a second detectable label differing from the first detectable label.

[0013] For example, the method can include isolating RNA transcripts from the at least one fish cell and contacting the RNA transcripts isolated from the at least one fish cell or nucleic acids derived therefrom using the RNA transcripts isolated from the at least one fish cell as templates with at least one molecule that hybridizes under stringent conditions to at least one nucleotide sequence of SEQ ID NOs: 1-560. The at least one probe can be conjugated with a first detectable label and the at least one molecule can be conjugated with a second detectable label differing in chemical structure from the first detectable label. The step of comparing the expression of the at least one nucleic acid in the cell compared to the expression of the at least one nucleic acid in a control cell not exposed to the sample or an agent having estrogenic or androgenic activity may be performed by quantifying the amount of first detectable label associated with the RNA transcripts isolated from the control cell or nucleic acids derived therefrom, and quantifying the amount of second detectable label associated with the RNA transcripts isolated from the at least one fish cell or nucleic acids derived therefrom.

[0014] An additional variation of the method of the invention also includes the steps of contacting the fish with the sample; and isolating the at least one fish cell from the fish contacted with the sample.

[0015] In another aspect, the invention features a method for determining whether an agent has estrogenic, anti-estrogenic, androgenic or anti-androgenic activity. This method includes the steps of: providing at least one fish cell; contacting the at least one fish cell with the agent; analyzing the at least one fish cell for expression of at least one gene wholly or partially encoded by a nucleotide sequence of SEQ ID NOs: 1-560; and comparing the expression of the at least one gene in the cell compared to the expression of the at least one nucleic acid in a control cell not exposed to the sample or an agent having estrogenic or androgenic activity. A difference in the expression of the at least one nucleic acid in the at least one fish cell compared to the expression of the at least one nucleic acid in the control cell indicates that the agent has estrogenic, anti-estrogenic, androgenic, or anti-androgenic activity.

[0016] Yet another aspect of the invention is a substrate having immobilized thereon at least one (e.g., at least 2, 3, 4, 5, 10, 25, 100) nucleic acid comprising a nucleotide sequence of SEQ ID NOs: 1-560 and complements thereof.

[0017] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Commonly understood definitions of molecular biology terms can be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: N.Y., 1991; and Lewin, Genes V, Oxford University Press: New York, 1994. Commonly understood definitions of microbiology can be found in Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 3rd edition, John Wiley & Sons: New York, 2002.

[0018] By the term “gene” is meant a nucleic acid molecule that codes for a particular protein, or in certain cases a functional or structural RNA molecule.

[0019] As used herein, a “nucleic acid” or a “nucleic acid molecule” means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid). A “purified” nucleic acid molecule is one that has been substantially separated or isolated away from other nucleic acid sequences in a cell or organism in which the nucleic acid naturally occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants). The term includes, e.g., a recombinant nucleic acid molecule incorporated into a vector, a plasmid, a virus, or a genome of a prokaryote or eukaryote. Examples of purified nucleic acids include cDNAs, fragments of genomic nucleic acids, nucleic acids produced by polymerase chain reaction (PCR), nucleic acids formed by restriction enzyme treatment of genomic nucleic acids, recombinant nucleic acids, and chemically synthesized nucleic acid molecules. A “recombinant” nucleic acid molecule is one made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.

[0020] As used herein, “protein” or “polypeptide” are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation.

[0021] By the term “estrogenic” is meant acting to produce the effects of an estrogen. An “estrogenic agent” and an “estrogen mimic” is a substance that acts to produce the effects of an estrogen.

[0022] As used herein the term “androgenic” means acting to produce the effects of an androgen. An “androgenic agent” and an “androgen mimic” is a substance that acts to produce the effects of an androgen.

[0023] When referring to hybridization of one nucleic acid to another, “low stringency conditions” means in 10% formamide, 5× Denhardt's solution, 6×SSPE, 0.2% SDS at 42° C., followed by washing in 1×SSPE, 0.2% SDS, at 50° C.; “moderate stringency conditions” means in 50% formamide, 5× Denhardt's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 65° C.; and “high stringency conditions” means in 50% formamide, 5× Denhardt's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C. The phrase “stringent hybridization conditions” means low, moderate, or high stringency conditions.

[0024] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions will control. The particular embodiments discussed below are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]
FIG. 1 is a series of macroarrays demonstrating gene expression profiles from SHM exposed to E2, 17α-ethynyl estradiol (EE2), diethylstilbestrol (DES), para-nonylphenol (pNP), methoxychlor (MXC), endosulfan (ES) or untreated control fish. Three separate fish were used for each treatment.

[0026]
FIG. 2 is two graphs showing quantification of the E2, EE2, DES, pNP, MXC, ES and control arrays for SHM. Panel A is a plot of the mean±SEM intensity values for each of the cDNA clones arranged in order of their expression. Panel B is a plot of the mean intensity values for each of the cDNA clones for E2, EE2, DES, pNP, ES, or MXC divided by the mean intensity values of the respective cDNA clones for untreated control fish. Any clones above the line labeled 1.66 were considered up-regulated genes, any clones below the line labeled 0.42 were considered down-regulated genes, and any clones between these lines were considered constitutive. Genes on the macroarray were designated as constitutive if their intensity values fell within the range of the mean plus one standard deviation of the highest and lowest values of the 11 clones that were used to normalize the data.

[0027]
FIG. 3 is a series of graphs plotting the quantification of the EE2 dose response arrays for SHM. Each graph contains a plot of a gene whose expression levels significantly changed more then 2-fold at one or more of the three EE2 concentrations compared to controls as revealed by one way analysis of variance (P<0.05). AMBP=alpha-1-microglobulin/bikunin precursor protein. The data on both axes are plotted using a log10 scale.

[0028]
FIG. 4 shows arrays on a plot for control and E2-treated fish and the results from the array analysis. Panels A and B are arrays that were hybridized with RNA from control (triethylene glycol (TEG)-treated) and E2-treated SHMs, respectively. Panel C is a plot of the mean intensity value of each cDNA clone on the E2-treated blots (N=3) over the mean intensity value of each cDNA clone on the control (TEG treated) blots (N=2). The black circles in panel C represent the 17 cDNA clones that were identified by DD analysis to be constitutive. Any clones above the dotted gray line labeled 1.27 were considered E2 up-regulated genes, any clones below the dotted gray line labeled 0.83 were considered E2 down-regulated genes, and any clones between the two gray dotted lines were considered constitutive genes. In panel C, a is transferrin, b is vitellogenin (Vtg ) β, c is ZP2, and d is vitellogenin α. There is a break in the graph of panel C from 2 to 10 log (intensity) units.

[0029]
FIG. 5 is two graphs showing gene expression profiles from control and E2-treated male LMB. (A) shows the mean±SEM intensity values for each of the cDNA clones arranged in order of their expression (black circles are E2, gray circles are control); (B) illustrates the mean intensity values for each of the cDNA clones for E2 divided by the mean intensity values of the respective cDNA clones from control fish. Any genes outside of the upper and lower solid gray lines in the figure change by more then two-fold and are considered to be up or down-regulated. Genes that exhibited a significant change in expression at P<0.05 are shown by a double asterisk; whereas genes that exhibited a significant change in expression at P<0.1 are shown by a single asterisk (t-tests). Three separate fish were used for each treatment. Only genes that were found in at least one of the treatments to be at least three standard deviations from the mean of the 12 ribosomal protein (r-protein) genes used to normalize the data (0.98±0.41) are plotted. AR=androgen receptor, ER=estrogen receptor, and NADH=Nicotinamide Adenine Dinucleotide (reduced form).

[0030]
FIG. 6 is two graphs showing gene expression profiles from control and 4-NP-treated male LMB. The order of genes in this figure corresponds to the order in FIG. 5.

[0031]
FIG. 7 is two graphs showing gene expression profiles from control and p, p′-DDE treated male LMB. The order of genes in this figure corresponds to the order in FIG. 5.

[0032]
FIG. 8 is two graphs showing gene expression profiles from control and p, p′-DDE treated female LMB. The order of genes in this figure corresponds to the order in FIG. 5.

[0033]
FIG. 9 is a list of genes whose expression is increased or decreased more than two-fold following exposure of LMB to E2, 4-NP, and p,p′-DDE.

DETAILED DESCRIPTION

[0034] The invention is premised in part on the discovery of nucleic acids (e.g., those of SEQ ID NOs: 1-560) whose expression is modulated in response to estrogenic/androgenic agents in fish such as SHM and LMB. Several of these nucleic acids were not previously characterized. Thus, the invention includes these nucleic acids, variants of these nucleic acids, proteins encoded by these nucleic acids, antibodies against these proteins, as well as other embodiments that can be made by one of skill in the art having knowledge of these sequences. An important application of the discovery is an assay for detecting modulation of expression of these nucleic acids in order to analyze an environmental sample or uncharacterized sample molecule. Detection of such modulation in a biological sample indicates that the sample or molecule exerts a hormonal activity (e.g., estrogenic or androgenic activity) or an anti-hormonal activity (e.g., anti-estrogenic, anti-androgenic activity).

[0035] The below described preferred embodiments illustrate adaptations of these compositions and methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.

Biological Methods

[0036] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Various techniques using PCR are described, e.g., in Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. PCR-primer pairs can be derived from known sequences by known techniques such as using computer programs intended for that purpose (e.g., Primer, Version 0.5, ©1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers, Tetra. Letts. 22:1859-1862, 1981, and Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981. Chemical synthesis of nucleic acids can be performed, for example, on commercial automated oligonucleotide synthesizers.

Novel Fish Genes

[0037] As several new genes were identified and characterized in making the invention, the invention provides several purified nucleic acids from SHM and LMB that are modulated in response to androgenic/estrogenic compounds. SHM nucleic acids of the invention have the nucleotide sequences of SEQ ID NOs: 151-419, while LMB nucleic acids of the invention have the nucleotide sequences of SEQ ID NOs: 1-150, 420-560.

[0038] Various assays described herein include a step of analyzing expression of a SHM or LMB gene modulated in response to an estrogenic or androgenic agent. Thus, polynucleotides that preferentially bind to nucleic acids encoded by the gene (e.g., mRNA, cDNA, DNA complements of cDNA, etc.) are also within the invention. Such polynucleotides can have the exact sequence of all or a portion of SEQ ID NOs: 1-560 or the complements of SEQ ID NOs: 1-560. Because hybridization of two nucleic acids does not generally require 100% complementarity, variants of such polynucleotides are also within the invention. These might include naturally occurring allelic variants of native LMB or SHM nucleic acids or non-naturally occurring variants that show sequence similarity to all or portions of SEQ ID NOs: 1-560 or the complements of SEQ ID NOs: 1-560

[0039] Naturally occurring allelic variants of native LMB or SHM nucleic acids within the invention are nucleic acids isolated from LMB and SHM that have at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) sequence identity with native LMB and SHM nucleic acids, and encode polypeptides having at least one functional activity in common with LMB and SHM polypeptides. Homologs of native LMB and SHM nucleic acids within the invention are nucleic acids isolated from other species (e.g., other fish species) that have at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) sequence identity with native LMB and SHM nucleic acids, and encode polypeptides having at least one functional activity in common with native LMB and SHM polypeptides. Naturally occurring allelic variants of LMB and SHM nucleic acids and homologs of LMB and SHM nucleic acids can be isolated by using a library screen, other assays described herein, or other techniques known in the art. The nucleotide sequence of such homologs and allelic variants can be determined by conventional DNA sequencing methods. Alternatively, public or non-proprietary nucleic acid databases can be searched to identify other nucleic acid molecules (e.g., nucleic acids from other species) having a high percent (e.g., 70, 80, 90% or more) sequence identity to native LMB and SHM nucleic acids.

[0040] Non-naturally occurring LMB and SHM nucleic acids variants are nucleic acids that do not occur in nature (e.g., are made by the hand of man), have at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) sequence identity with native LMB and SHM nucleic acids, and encode polypeptides having at least one functional activity in common with native LMB and SHM polypeptides. Examples of non-naturally occurring LMB and SHM nucleic acids are those that encode a fragment of an LMB or SHM protein, those that hybridize to native LMB and SHM nucleic acids or a complement of native LMB and SHM nucleic acids under stringent conditions, those that share at least 65% sequence identity with native LMB and SHM nucleic acids or a complement of native LMB and SHM nucleic acids, and those that encode an LMB or SHM fusion protein.

[0041] Nucleic acids encoding fragments of LMB and SHM polypeptides within the invention are those that encode, e.g., 2, 5, 10, 25, 50, 100, 150, 200, 250, 300, or more amino acid residues of LMB or SHM polypeptides. Shorter oligonucleotides (e.g., those of 6, 12, 20, 30, 50, 100, 125, 150 or 200 base pairs in length) that encode or hybridize with nucleic acids that encode fragments of LMB or SHM polypeptides can be used as probes, primers, or antisense molecules. Longer polynucleotides (e.g., those of 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 or 1300 base pairs) that encode or hybridize with nucleic acids that encode fragments of LMB or SHM polypeptides can be used in place of native LMB or SHM polynucleotides in applications where it is desired to modulate a functional activity of native LMB or SHM polypeptides. Nucleic acids encoding fragments of LMB or SHM polypeptides can be made by enzymatic digestion (e.g., using a restriction enzyme) or chemical degradation of full length LMB or SHM nucleic acids or variants of LMB or SHM nucleic acids.

[0042] Nucleic acids that hybridize under stringent conditions to the nucleic acid of SEQ ID NOs: 1-560 or the complement of SEQ ID NOs: 1-560 are also within the invention. For example, nucleic acids that hybridize to SEQ ID NOs: 1-560 or the complement of SEQ ID NOs: 1-560 under low stringency conditions, moderate stringency conditions, or high stringency conditions are within the invention. Preferred such nucleic acids are those having a nucleotide sequence that is the complement of all or a portion of SEQ ID NOs: 1-560. Other variants of LMB or SHM nucleic acids within the invention are polynucleotides that share at least 65% (e.g., 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99%) sequence identity to SEQ ID NOs: 1-560 or the complement of SEQ ID NOs: 1-560. Nucleic acids that hybridize under stringent conditions to or share at least 65% sequence identity with SEQ ID NOs: 1-560 or the complement of SEQ ID NOs: 1-560 can be obtained by techniques known in the art such as by making mutations in native LMB or SHM nucleic acids, by isolation from an organism expressing such a nucleic acid (e.g., a fish expressing a variant of native LMB or SHM nucleic acids), or an organism other than a fish expressing a homolog of native LMB or SHM nucleic acids.

[0043] Nucleic acid molecules of the present invention may be in the form of RNA or in the form of DNA (e.g., cDNA, genomic DNA, and synthetic DNA). The DNA may be double-stranded (ds) or single-stranded (ss), and if single-stranded may be the coding (sense) strand or non-coding (anti-sense) strand. The nucleic acid molecules of the present invention may also be polynucleotide analogues such as peptide nucleic acids (PNA). See, e.g. Gambari R., Curr. Pharm. Des. 7:1839-1862, 2001; U.S. Pat. No. 6,395,474; and PCT patent application publication number WO 86/05518. The sequences which encode native LMB and SHM gene products may be identical to the nucleotide sequences shown in SEQ ID NOs:1-560. They may also be different sequences which, as a result of the redundancy or degeneracy of the genetic code, encode the same polypeptides as the polynucleotides of SEQ ID NOs:1-560. Other nucleic acid molecules within the invention are variants of nucleic acids of SEQ ID NOs: 1-560 such as those that encode fragments, analogs and derivatives of native proteins encoded by nucleic acids of SEQ ID NOs: 1-560. Such variants may be, e.g., a naturally occurring allelic variant of native nucleic acids of SEQ ID NOs: 1-560, a homolog of native nucleic acids of SEQ ID NOs:1-560, or a non-naturally occurring variant of native nucleic acids of SEQ ID NOs: 1-560. These variants have a nucleotide sequence that differs from native nucleic acids of SEQ ID NOs: 1-560 in one or more bases. For example, the nucleotide sequence of such variants can feature a deletion, addition, or substitution of one or more nucleotides of native nucleic acids of SEQ ID NOs: 1-560. Nucleic acid insertions are preferably of about 1 to 10 contiguous nucleotides, and deletions are preferably of about 1 to 30 contiguous nucleotides.

Probes and Primers

[0044] Nucleic acids that hybridize under stringent conditions to the nucleic acid sequences of SEQ ID NOs: 1-560 or the complement of the nucleic acid sequences of SEQ ID NOs: 1-560 can be used in the invention. For example, such nucleic acids can be those that hybridize to the nucleic acid sequences of SEQ ID NOs: 1-560 or the complement of the nucleic acid sequences of SEQ ID NOs: 1-560 under low stringency conditions, moderate stringency conditions, or high stringency conditions. Preferred such nucleic acids are those having a nucleotide sequence that is the complement of all or a portion of a nucleic acid sequence of SEQ ID NOs: 1-560. Others that might be used include polynucleotides that share at least 65% (e.g., 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99%) sequence identity to a native nucleic acid sequence of SEQ ID NOs: 1-560 or the complement of a native nucleic acid sequence of SEQ ID NOs: 1-560. Nucleic acids that hybridize under stringent conditions to or share at least 65% sequence identity with the nucleic acid sequences of SEQ ID NOs: 1-560 or the complement of the nucleic acid sequences of SEQ ID NOs: 1-560 can be obtained by techniques known in the art such as by making mutations in a native nucleic acid sequence of SEQ ID NOs: 1-560, or by isolation from an organism expressing such a nucleic acid (e.g., an allelic variant).

[0045] Methods of the invention utilize oligonucleotide probes (i.e., isolated nucleic acid molecules conjugated with a detectable label or reporter molecule, e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme); and oligonucleotide primers (i.e., isolated nucleic acid molecules that can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, e.g., a DNA polymerase). Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the PCR or other conventional nucleic-acid amplification methods.

[0046] PCR primers can be used to amplify the nucleic acid sequences of SEQ ID NOs: 1-560 using known PCR and RT-PCR protocols. Such primers can be designed according to known methods as PCR primer design is generally known in the art. See, e.g., methodology treatises such as Basic Methods in Molecular Biology, 2nd ed., ed. Davis et al., Appleton & Lange, Norwalk, CN, 1994; and Molecular Cloning: A Laboratory Manual, 3rd ed., vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001.

[0047] Probes and primers utilized in methods of the invention are generally 15 nucleotides or more in length, preferably 20 nucleotides or more, more preferably 25 nucleotides, and most preferably 30 nucleotides or more. Preferred probes and primers are those that hybridize to a native nucleic acid sequence of SEQ ID NOs: 1-560 (or cDNA or mRNA) sequence under high stringency conditions, and those that hybridize to homologs of the nucleic acid sequences of SEQ ID NOs: 1-560 under at least moderately stringent conditions. Preferably, probes and primers according to the present invention have complete sequence identity with a native nucleic acid sequence of SEQ ID NOs: 1-560. However, probes differing from this sequence that retain the ability to hybridize to a native nucleic acid sequence of SEQ ID NOs: 1-560 under stringent conditions may be designed by conventional methods and used in the invention. Primers and probes based on the nucleic acid sequences of SEQ ID NOs: 1-560 disclosed herein can be used to confirm (and, if necessary, to correct) the disclosed nucleic acid sequences of SEQ ID NOs: 1-560 by conventional methods, e.g., by re-cloning and sequencing a native nucleic acid sequence of SEQ ID NOs: 1-560 or cDNA corresponding to a native nucleic acid sequence of SEQ ID NOs: 1-560.

Proteins Encoded by Nucleic Acid Sequences of SEQ ID NOs: 1-560

[0048] The invention also provides polypeptides encoded in whole or in part by the nucleic acid sequences of SEQ ID NOs: 1-560. Some polypeptides encoded by the nucleic acids of SEQ ID NOs: 1-560 are expressed at higher levels when the nucleic acids are exposed to hormonal compounds compared to control nucleic acids not exposed to the hormonal compound. Other polypeptides encoded in whole or in part by the nucleic acid sequences of SEQ ID NOs: 1-560 are expressed at lower levels when exposed to hormonal compounds compared to the expression of nucleic acids not exposed to the hormonal compound.

[0049] Variants of native proteins encoded in whole or in part by nucleic acid sequences of SEQ ID NOs: 1-560 such as fragments, analogs and derivatives of native proteins encoded by nucleic acid sequences of SEQ ID NOs: 1-560 may also be used in methods of the invention. Such variants include, e.g., a polypeptide encoded in whole or in part by a naturally occurring allelic variant of a native nucleic acid sequence of SEQ ID NOs: 1-560, a polypeptide encoded by an alternative splice form of a native nucleic acid sequence of SEQ ID NOs: 1-560, a polypeptide encoded in whole or in part by a homolog of a native nucleic acid sequence of SEQ ID NOs: 1-560, and a polypeptide encoded in whole or in part by a non-naturally occurring variant of a native nucleic acid sequence of SEQ ID NOs: 1-560.

[0050] Protein variants encoded by a sequence having homology to a nucleic acid sequence of SEQ ID NOs: 1-560 have a peptide sequence that differs from a native protein encoded in whole or in part by a nucleic acid sequence of SEQ ID NOs: 1-560 in one or more amino acids. The peptide sequence of such variants can feature a deletion, addition, or substitution of one or more amino acids of a native polypeptide encoded in whole or in part by a nucleic acid sequence of SEQ ID NOs: 1-560. Amino acid insertions are preferably of about 1 to 4 contiguous amino acids, and deletions are preferably of about 1 to 10 contiguous amino acids. In some applications, variant proteins substantially maintain a native nucleic acid sequence of SEQ ID NOs: 1-560-encoded protein functional activity. For other applications, variant proteins lack or feature a significant reduction in a nucleic acid sequence of SEQ ID NOs: 1-560-encoded protein functional activity. Where it is desired to retain a functional activity of a native protein encoded in whole or in part by a nucleic acid sequence of SEQ ID NOs: 1-560, preferred protein variants can be made by expressing nucleic acid molecules within the invention that feature silent or conservative changes. Variant proteins with substantial changes in functional activity can be made by expressing nucleic acid molecules within the invention that feature less than conservative changes.

[0051] Nucleic acid sequences of SEQ ID NOs: 1-560-encoded protein fragments corresponding to one or more particular motifs and/or domains or to arbitrary sizes, for example, at least 5, 10, 25, 50, 75, 100, 125, 150, 175, 200, and 250 amino acids in length may be utilized in methods of the present invention. Isolated peptidyl portions of proteins encoded by a nucleic acid sequence of SEQ ID NOs: 1-560 can be obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, a protein encoded by a nucleic acid sequence of SEQ ID NOs: 1-560 used in methods of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can function as either agonists or antagonists of a native nucleic acid sequence of SEQ ID NOs: 1-560-encoded protein.

[0052] Methods of the invention may also involve recombinant forms of the nucleic acid sequences of SEQ ID NOs: 1-560-encoded proteins. Recombinant polypeptides preferred by the present invention, in addition to native nucleic acid sequence of SEQ ID NOs: 1-560-encoded protein, are encoded by a nucleic acid that has at least 85% sequence identity (e.g., 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%) with a native nucleic acid sequence of SEQ ID NOs: 1-560. In a preferred embodiment, variant proteins lack one or more finctional activities of native nucleic acid sequence of SEQ ID NOs: 1-560-encoded protein.

[0053] Protein variants can be generated through various techniques known in the art. For example, protein variants can be made by mutagenesis, such as by introducing discrete point mutation(s), or by truncation. Mutation can give rise to a protein variant having substantially the same, or merely a subset of the functional activity of a native protein encoded in whole or in part by a nucleic acid sequence of SEQ ID NOs: 1-560. Alternatively, antagonistic forms of the protein can be generated which are able to inhibit the function of the naturally occurring form of the protein, such as by competitively binding to another molecule that interacts with a protein encoded in whole or in part by a nucleic acid sequence of SEQ ID NOs: 1-560. In addition, agonistic forms of the protein may be generated that constitutively express one or more nucleic acid sequence of SEQ ID NOs: 1-560-encoded protein functional activities. Other protein variants that can be generated include those that are resistant to proteolytic cleavage, as for example, due to mutations that alter protease target sequences. Whether a change in the amino acid sequence of a peptide results in a protein variant having one or more functional activities of a native nucleic acid sequence of SEQ ID NOs: 1-560-encoded protein can be readily determined by testing the variant for a native nucleic acid sequence of SEQ ID NOs: 1-560-encoded protein functional activity.

Antibodies

[0054] Antibodies that specifically bind nucleic acid sequence of SEQ ID NOs: 1-560-encoded proteins can be used in methods of the invention, for example, in the detection of nucleic acid sequence of SEQ ID NOs: 1-560-encoded protein expression. Antibodies of the invention include polyclonal antibodies and, in addition, monoclonal antibodies, single chain antibodies, Fab fragments, F(ab′)2 fragments, and molecules produced using a Fab expression library. Antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

[0055] Antibodies that specifically recognize and bind to nucleic acid sequence of SEQ ID NOs: 1-560-encoded proteins are useful in methods of the present invention. For example, such antibodies can be used in an immunoassay to monitor the level of the corresponding protein produced by a cell or an animal (e.g., to determine the amount or subcellular location of a nucleic acid sequence of SEQ ID NOs: 1-560-encoded protein). Methods of the invention may also utilize antibodies, for example, in the detection of a nucleic acid sequence of SEQ ID NOs: 1-560-encoded protein in an environmental sample. Antibodies also can be used in a screening assay to measure the effect of a candidate agent on expression or localization of a nucleic acid sequence of SEQ ID NOs: 1-560-encoded protein.

Detecting the Presence of an Agent Having Androgenic/estrogenic Activity

[0056] Within the invention, SEQ ID NOs:1-560 are used in various methods for detecting the presence of estrogenic/androgenic agents (e.g., EDCs) such as E2, EE2, DES, MXC, ES, 4-NP, p-chlorophenyl, and p,p′-DDE in a sample. Examples of other EDCs that may be detected using compositions and methods of the invention include benzenehexachloride, 1,2-dibromoethane, chloroform, dioxins, furans, octachlorostyrene, PBBs, PCBs, PCB, hydroxylated PBDEs, and pentachlorophenol as well as others disclosed in Hormonally Active Agents In The Environment, Ed. by The Committee On Hormonally Active Agents In The Environment Board On Environmental Studies and Toxicology Commission On Life Sciences And National Research Council, National Academy Press, Washington D.C., 1999.

[0057] Methods for detecting the presence of an agent having estrogenic or androgenic activity in a sample involve a first step of providing at least one fish cell which was exposed to the sample. A fish cell of the invention can be a cell from any fish, preferably a cell from a SHM or LMB. A sample can be obtained from a number of sources, including a body of water (e.g., river, lake, stream, canal, estuary, pond, etc.) as well as sediment obtained from a body of water or from a site near or contacting a body of water (e.g., sediment from a lake or river bed). The fish cell exposed to the sample can be a cell taken from a fish that was present in a body of water (or in contact with sediment) from which the sample (i.e., environmental sample) was taken. The fish cell can also be a cell isolated from a provided fish that was contacted with the sample (e.g., taken from a fish that was exposed to a sample in controlled, laboratory conditions). Alternatively, the fish cell can be one that was cultured and exposed to the sample in vitro.

[0058] A second step of this method involves analyzing the at least one fish cell for expression of at least one gene encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-560. A number of methods for analyzing gene expression are described below. A third step of this method involves comparing the expression of the at least one gene in the cell compared to the expression of the at least one gene in a control cell not exposed to the sample or an agent having estrogenic or androgenic activity, wherein a difference in the expression of the at least one gene in the at least one fish cell compared to the expression of the same at least one nucleic acid in the control cell indicates that the sample contains an agent having estrogenic or androgenic activity.

[0059] The step of analyzing the at least one fish cell can include analyzing the cell for expression of at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 100) different genes, each being wholly or partially encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-560. To analyze the cell for expression of at least one gene, RNA transcripts can be isolated from the at least one cell. The isolated RNA transcripts or nucleic acids derived therefrom can be used as templates and contacted with at least one probe that hybridizes under stringent conditions to at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-560. This step can also include contacting the RNA transcripts or nucleic acids derived therefrom with at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150) different probes that each hybridize under stringent conditions to a different nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-560. The at least one probe (or probes) or the isolated RNA transcripts (or nucleic acids derived therefrom) can be conjugated with a detectable label such as a fluorphore or a radioactive molecule or compound. The probe(s) can be immobilized on a substrate (e.g., array) before placed in contact with RNA transcripts isolated from a fish cell or control cell, or can be contacted with the RNA transcripts in solution (e.g., real-time PCR assay) rather than in the presence of a substrate. Examples of substrates that may be used include nylon, nitrocellulose, glass, and plastic.

[0060] In another method of detecting the presence of an agent having estrogenic or androgenic activity in a sample, the control cell not exposed to the sample or an agent having estrogenic or androgenic activity is analyzed for expression of at least one gene encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-560. For example, RNA transcripts can be isolated from the control cell and contacted with the RNA transcripts or nucleic acids derived therefrom using the isolated RNA transcripts as templates with at least one probe that hybridizes under stringent conditions to at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-560. This method can further include isolating RNA transcripts from the at least one fish cell and contacting the RNA transcripts isolated from the at least one fish cell (or nucleic acids derived therefrom) with at least one molecule that hybridizes under stringent conditions to at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-560. In some applications, the at least one probe is conjugated with a first detectable label and the at least one molecule is conjugated with a second detectable label differing in chemical structure from the first detectable label. In other applications, the RNA transcripts (or nucleic acids derived therefrom) isolated from the at least one fish cell are conjugated with a first detectable label and the RNA transcripts isolated from the control cell are conjugated with a second detectable label differing in chemical structure from the first detectable label.

[0061] To compare expression of the at least one nucleic acid in the fish cell compared to the expression of the at least one nucleic acid in a control cell not exposed to the sample or an agent having estrogenic or androgenic activity, both 1) the amount of first detectable label associated with the RNA transcripts isolated from the control cell (or nucleic acids derived therefrom) and 2) the amount of second detectable label associated with the RNA transcripts isolated from the at least one fish cell (or nucleic acids derived therefrom) is quantified.

[0062] In one example of comparing expression of the at least one nucleic acid in the fish cell to the expression of the at least one nucleic acid in the control cell, the labeled RNA transcripts (or nucleic acids derived therefrom) isolated from the at least one fish cell and from the control cell are contacted e.g., on an array as described herein. Hybridization of the differentially labeled transcripts to the nucleic acids is then detected (e.g., using an imaging device such as a phosphor screen or autoradiographic film) and signal intensities are quantitatively analyzed (e.g., using a software program such as Atlaslmage™ 2.01 Clontech, Palo Alto, Calif.).

[0063] Among the traditional methods that can be employed for gene expression analyses are DD RT-PCR, nucleic acid arrays, quantitative PCR (e.g., real-time PCR), in situ hybridization, serial analysis of gene expression (SAGE), and subtractive hybridization. DD RT-PCR, for example, isolates differentially expressed genes using both arbitrary and anchored oligo-dT primers (Liang & Pardee, 1992; Liang et al., 1994; and Genome Analysis: A Laboratory Manual Series 1, ed: B. Birren et al., 1997, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). A typical DD RT-PCR protocol involves several steps including reverse transcription using anchored oligo-dT primers, amplification of cDNA using one anchored and one arbitrary primer, electrophoresis of PCR products, purification of the product of interest, and cloning and sequencing of the product. In one method, DD-RT-PCR is performed with the RNAimage mRNA Differential Display system (GenHunter; Nashville, Tenn.) using one-base anchored oligo-dT primers (Liang et al., 1994) as described previously (Denslow et al., 1999a; and Denslow et al., 2001).

[0064] In vitro quantitation of gene expression can be performed using a number of real-time quantitative PCR assays. Real-time quantitative PCR assays typically involve labeling a target nucleic acid with a first fluorescing dye and labeling a probe with a second fluorescing dye. For example, Multiplex TaqMan® (Applied Biosystems, Foster City, Calif.) assays can be performed using the ABI PRISM® 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.), capable of detecting multiple dyes with distinct emission wavelengths. Some real-time quantitative PCR applications involve the use of fluorescence resonance energy transfer (FRET) between fluorochromes introduced into DNA molecules (e.g., molecular beacon assays). For a review of FRET techniques, see Vet et al., Expert Rev. Mol. Diagn. 2:77-86, 2002.

[0065] A preferred technique for detecting the presence of estrogenic compounds involves the use of nucleic acid arrays. Nucleic acid arrays allow the simultaneous monitoring of expression patterns of multiple genes from the same sample. Arrays are an appropriate tool for rapidly screening large numbers of genes. Examples of nucleic acid arrays include microarrays and macroarrays. Methods involving nucleic acid arrays are reviewed in Ringner et al., Pharmacogenomics 3:403-415, 2002; Epstein et al., Curr. Opin. Biotechnol. 11:36-41, 2000; Granjeaud et al., BioEssays 21:781-790, 1999; Hatakeyama K., Nippon Rinsho 57:465-473, 1999; DNA Microarrays: A Molecular Cloning Manual, ed: D. Bowtell and J. Sambrook, 2002, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and U.S. Pat. No. 6,410,229. The construction and use of nucleic acid arrays containing fish genes is described below.

Arrays

[0066] The nucleic acids (and proteins and antibodies) of the invention are preferably useful for assaying a sample for the presence of a hormonal agent (e.g., an estrogenic, sample in an environmental water sample). In this regard, nucleic acid-based assays are presently preferred. The invention thus provides a substrate having immobilized thereon at least one nucleic acid including a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-560 and complements thereof. A typical substrate having immobilized thereon at least one nucleic acid of the invention is an array of.fish nucleic acids, including nucleic acids (e.g., genes and gene fragments) responsive to androgenic and estrogenic compounds. Arrays containing fish-derived nucleic acids responsive to androgenic and/or estrogenic compounds can be used in a number of applications. For example, the arrays can be used to monitor the presence and distribution of androgenic and estrogenic contaminants in the environment. The arrays can also be used to screen for synthetic or natural agents having androgenic or estrogenic activity. An example of an array provided by the invention is a macroarray containing LMB- or SHM-derived nucleic acids. On a preferred macroarray of the invention, a minimum number of nucleotides of 150 is included for each nucleic acid (e.g., 2, 10, 50, 75, 100, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 200, 250, 300, 350, 400 or more). A portion of the nucleic acids on the macroarray are responsive to estrogenic compounds. A list of nucleic acids that may be contained within a macroarray of the invention is presented in Table III (SHM), Table II (LMB), and Table IV (SHM and LMB).

[0067] To construct a cDNA macroarray, cDNA is first prepared from RNA. Techniques for preparing cDNA from RNA are widely known, and are described in methodology treatises such as Sambrook and Russell supra and Ausubel et al., supra. In one example, cDNA clones (e.g., miniprep cDNAs) derived from DD RT-PCR analysis (as described above) are PCR-amplified using primers specific to the cloning vector (e.g., pGEMT-Easy, Promega, Madison, Wis.). Any suitable thermocycling conditions that result in amplification of the desired product may be used. After completion of the PCR, the products are purified (e.g., in a spin-column, Quiagen, Chatsworth, Calif.) and then concentrated (e.g., in a speed-vac). Aliquots of the PCR products are then resolved electrophoretically (e.g., run on a 1.2% agarose gel containing 0.3 mM ethidium bromide). The resultant gels are analyzed (e.g., digitally imaged using a UVP Bio Doc-It camera, Ultra Violet Laboratory Products, Upland Calif.) and the concentration of each PCR product is determined. Typically, concentrations of PCR products are determined by comparing the intensity of each band to a standard curve derived from a low DNA mass ladder (InVitrogen Corporation, Carlsbad, Calif.).

[0068] Once the PCR products are purified and their concentrations determined, they are then spotted onto a membrane (e.g., nylon membrane). Methods for spotting cDNAs onto membranes are discussed in Diehl et al., NAR 29:E38, 2001; Shieh et al., Biotechniques 32:1360-1362 & 1364-1365, 2002; and Schuchhardt et al., NAR 28:E47, 2000. In one method of spotting the cDNAs onto a membrane, PCR products are denatured, quenched on ice, and robotically spotted onto nylon membranes (Fisher Scientific). In this method, membranes are cross-linked and stored under vacuum at room temperature until the hybridization step. Various controls are also spotted onto the membranes. These controls provide information about cDNA labeling efficiency, blocking at the pre-hybridization step, and non-specific binding. Any genes that are not responsive to estrogen may be used as negative control genes on an array of the invention. Control genes that are not responsive to estrogen include Arabidopsis thaliana cDNA clones, Cot-1 repetitive sequences, polyA sequence (SpotReport 3, Stratagene, LaJolla, Calif.), and a M13 sequence (vector but no cDNA insert). The consistency of the spotting technique may be assessed by spotting on the array multiple cDNA products from the same gene that were amplified in separate PCR reactions.

[0069] For the generation of probes, mRNA from fish exposed to an estrogenic compound (e.g., E2, EE2, DES, pNP, ES, MXC) and mRNA from control fish (i.e., fish not exposed to estrogenic compounds), is extracted and purified. mRNA may be purified by a number of known techniques, including the use of affinity columns (Qiagen, Chatsworth, Calif.). In addition to RNA probes, cDNA probes may also be used. The labeling of nucleotide probes is described in Relogio et al., NAR 30:351, 2002; and Yu et al., Mol. Vis. 8:130-137, 2002. Probes may be labeled using any of a number of techniques, including fluorescence (e.g., Atlas Glass Fluorescent Labeling Kit, Clontech, Palo Alto, Calif.), resonance light scattering (Bao et al., Anal. Chem. 74:1792-1797, 2002), gold nanoparticle labeling (Fritzsche et al., J. Biotechnol. 1:37-46, 2001) and radioactive methods. In one example of radiolabeling RNA probes, DNase-treated total RNA from fish is subjected to random primer labeling with α-33P dATP (Strip-EZ RT, Ambion, Austin, Tex.). RNAs may also be radiolabeled using a kit such as AtlasPure™ RNA Labeling System. Typically, blots are prehybridized for several hours, hybridized overnight with probe-containing solution, and then washed several times.

[0070] To detect hybridization of the probe to nucleotides on an array, membranes are exposed to a suitable imaging device, such as a phosphor screen (Molecular Dynamics, Piscataway, N.J.) or autoradiographic film for an appropriate period of time (e.g.,several hours). Signal intensities may be quantitatively analyzed using a suitable software program, such as Atlaslmage™ 2.01 (Clontech, Palo Alto, Calif.). Blots may also be quantitatively evaluated using a Typhoon 8600 imaging system (Molecular Dynamics). For each nucleotide (e.g., cDNA) clone on an array, the general background of each membrane is subtracted from the average value of the duplicate spots on the membrane. The values are normalized to the average value of several (e.g., 11) nucleotide (e.g., cDNA) clones. Gene array data is analyzed using a suitable statistical analysis. For example, linear regression and one-way analysis of variance, with Tukey post-hoc analysis (SigrnaStat and SigmaPlot, Jandel, Calif.) may be used to analyze the gene array data.

Determining Whether an Agent has Estrogenic, Anti-Estrogenic, Androgenic, or Anti-Androgenic Activity

[0071] In addition to detecting the presence of estrogenic compounds in the environment, nucleic acid arrays containing one or more nucleotide sequences of SEQ ID NOs: 1-560 of the invention may also be used to screen for compounds with estrogenic, anti-estrogenic, androgenic, or anti-androgenic activity. Estrogenic compounds (e.g., estrogen, estrogen mimics) have possible uses in a number of disorders, including the treatment of cardiovascular disease, menopausal symptoms and menopausal osteoporosis. Molecules or compounds with anti-estrogenic activity (e.g., flavonoids) have a number of possible applications, including the treatment of breast cancer. Androgenic agents also have a number of applications, including the treatment of sexual dysfunction, depression and pelvic endometriosis. Androgenic agents are also fed to livestock as growth-inducing agents. For the treatment of prostate enlargement and acne, anti-androgenic agents are useful.

[0072] A method for determining whether an agent has estrogenic, anti-estrogenic, androgenic, or anti-androgenic activity involves several steps. A first step in this method includes providing at least one fish cell. In a second step of the method, the at least one fish cell is contacted with the agent. In a third step, the at least one fish cell is analyzed for expression of at least one gene wholly or partially encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-560. A fourth step of the method includes comparing the expression of the at least one gene in the cell compared to the expression of the at least one nucleic acid in a control cell not exposed to the sample or an agent having estrogenic, anti-estrogenic, androgenic or anti-androgenic activity. In this method, a difference in the expression of the at least one nucleic acid in the at least one fish cell compared to the expression of the at least one nucleic acid in the control cell indicates that the agent has estrogenic, anti-estrogenic, androgenic, or anti-androgenic activity.

[0073] In one embodiment of determining if a test agent increases or decreases expression of a gene responsive to estrogen, cells are first exposed to the test agent in vitro. For example, multiple compounds can be tested simultaneously by plating cells in a multi-well plate (e.g., in a 96 well tissue culture plate) and contacting one test compound per well. RNA from the exposed cells as well as from control cells (i.e., negative control cells not exposed to the test compound and positive control cells exposed to the test compound) is isolated and reverse transcribed to cDNAs. The cDNAs are labeled to generate probes as described above, and contacted with the nucleic acid arrays of the invention. Hybridization of the labeled probes to the nucleic acids of the array (e.g., SEQ ID NOs: 1-560) is analyzed as described above. Alternatively, whole fish can be exposed to the test agents in the water or through the food. This allows for normal metabolic processes to occur within the various tissues of the fish to end up with an agent that has either the same or more or less activity then the parent agent.

EXAMPLES

[0074] The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and are not to be construed as limiting the scope or content of the invention in any way.

Example 1—Expression Profiling of Estrogenic Compounds Using A SHM cDNA Macroarray Methods

[0075] Amplification of cDNA to be spotted on macroarrays: Minipreps of 30 cDNA clones derived from DD RT-PCR analysis (Denslow et al., Gen. Comp. Endocrinol. 121:250-260, 2001; Denslow et al., Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 129:277-282, 2001) were PCR amplified in a 300 μL reaction containing 1×PCR Buffer A (Promega, Madison, Wis.), 2 mM MgCl2 (Promega, Madison, Wis.), 160 μM each deoxynucleotide triphosphate (dNTP) (Statagene, La Jolla, Calif.), 0.4 μM M13 primers (5′-GTT TTC CCA GTC ACG ACG TTG (SEQ ID NO:561) and 5′-GCG GAT AAC AAT TTC ACA CAG GA (SEQ ID NO:562), and 1.25 units Taq polymerase (Promega, Madison, Wis.). The PCR reaction conditions were: 1 cycle at 80° C. (1 min); 1 cycle at 94° C. (2min); 32 cycles at 94° C. (1 min) 57° C. (1 min) 72° C. (2 m); 1 cycle at 72° C. (10 min); and then hold at 4° C. After completion of the PCR reactions the products were purified in a spin-column (Qiagen, Chatsworth, Calif.) and then concentrated in a speed-vac. Aliquots of the PCR products were run on a 1.2% agarose gel containing 0.3 mM ethidium bromide. The gels were digitally imaged using a UVP Bio Doc-It camera (Ultra Violet Laboratory Products, Upland Calif.) and the concentration of each PCR product was determined by comparing the intensity of each band to a standard curve derived from a low DNA mass ladder (Invitrogen Corporation, Carlsbad, Calif.). The PCR products were adjusted to a concentration of 160 ng/μL cDNA template.

[0076] Spotting of the macroarrays: The PCR products were loaded into 96 well plates (Fisher Scientific, Pittsburgh, Pa.), denatured with 3 M NaOH, heated to 65° C. for 10 mins, and then immediately quenched on ice. 20× saline sodium citrate (SSC) (3M NaCl, 0.3M sodium citrate, pH 7.0) containing 0.01 mM bromophenol blue was added to the samples to yield a final concentration of 0.3M NaOH, 6×SSC, and 100 ng/μL cDNA template. The PCR products were robotically spotted (Biomek 2000, Beckman Coulter, Fullerton, Calif.) in duplicate onto 11.5 by 7.6 cm neutral nylon membranes (Fisher Scientific) using 100 nL pins. Membranes were UV cross-linked at 1×105 μJoules (UV Stratalinker 1800, Stratagene, La Jolla, Calif.) and stored under vacuum at room temperature until hybridization.

[0077] Array controls: Various controls were also spotted onto the membranes, which provided information about cDNA labeling efficiency, blocking at the pre-hybridization step, and non-specific binding. These controls included: 3 Arabidopsis thaliana cDNA clones, Cot-1 repetitive sequences, poly A sequence (SpotReport 3, Stratagene), and a M13 sequence (vector but no cDNA insert). The consistency of the technique was evaluated by spotting on the array multiple cDNA products from the same gene that were amplified in separate PCR reactions.

[0078] Sample extraction: Total hepatic messenger ribonucleic acid (mRNA) was extracted using affinity columns (Qiagen, Chatsworth, Calif.) from adult male SHMs treated by aqueous exposure to either 65.14 ng/L of E2, 109 ng/L EE2, 100 ng/L DES, 11.81 μg/L pNP, 590.3 ng/L ES or 5.59 μg/L MXC as described previously (Folmar et al., Aquatic Toxicol. 49:77-88, 2000; Hemmer et al., Environ. Toxicol. Chem. 20:336-343, 2001). Three fish were used per treatment group. Criteria for selection of samples from each compound tested were based on previously generated dose response curves (Folmar et al., Aquat. Toxicol. 49:77-88, 2000; Hemmer et al., Environ. Toxicol. Chem. 20:336-343, 2001) and chosen to give similar levels of expression of Vtg mRNA, a well established estrogenic biomarker (Bowman et al., Gen. Comp. Endocrinol. 120:300-313, 2000; Sumpter and Jobling, Environ. Health Perspect. 103:173-178, 1995). By selecting the concentration and length of exposure to yield similar Vtg mRNA expression levels, differing potencies among the chemicals tested was accounted for. Based on this criterion, length of exposure was four days for EE2 and DES, five days for E2 and pNP, and thirteen days for MXC. ES treatment levels ranging from 68.8 ng/L to 788.33 ng/L failed to induce Vtg mRNA. A treatment of 590.3 ng/L of ES for these analyses was chosen. This level of ES was slightly below the maximum acceptable toxicant concentration (MATC) derived for ES for SHMs (Hansen and Cripe 1991).

[0079] Labeling of RNA and hybridization: Radiolabeled probes were generated by random primer labeling of DNase treated (DNA-free, Ambion, Austin,Tex.) total RNA from male SHM livers with [α-33P] dATP (Strip-EZ RT, Ambion, Austin, Tex.). The blots were prehybridized with ultraArray hybridization buffer (Ambion, Austin, Tex.) at 64° C. for 3 hours. Following prehybridization, each probe was diluted 20-fold with 10 mM disodium ethylenediaminetetraacetate (EDTA), pH 8.0 to yield 1×106 cpm incorporated 33P per mL hybridization solution. The diluted probes were heated to 95° C. for 5 mins, quenched on ice for 1 min, and added directly to the prehybridization buffer. The blots were then hybridized overnight at 64° C. Following hybridization, the blots were washed 4×15 minutes each with low (2×SSC, 0.5% SDS) and high (0.5×SSC and 0.5% SDS) stringency washes (Ambion, Austin, Tex.) at 64° C.

[0080] Detection and normalization: The membranes were exposed to a phosphor screen (Molecular Dynamics, Piscataway, N.J.) at room temperature for 48 hrs. The blots were quantitatively evaluated using a Typhoon 8600 imaging system (Molecular Dynamics, Piscataway, N.J.). For each cDNA clone, the general background of each membrane was subtracted from the average value of the duplicate spots on the membrane. The values were normalized to the average value of 11 cDNA clones. These genes include ribosomal proteins L8, S9, two unique genes that are similar to ribosomal protein S9, and several clones that do not match any sequences in the National Center for Biotechnology Information (NCBI) database. These genes were chosen to normalize the data because they did not fluctuate appreciably (<1.3 fold) on macro arrays from E2-treated and control fish and also were shown to be equally expressed in controls and treated fish by DD analysis data. Gene array data was analyzed using linear regression and one-way analysis of variance, with Tukey post-hoc analysis (SigmaStat and SigmaPlot, Jandel, Calif.).

Results

[0081] As a first step toward using array technology, the variability between the macroarrays was determined. To accomplish this, aliquots of identical RNA samples were hybridized onto two separate membranes. A scatter plot correlating the intensity values for each spot on the two membranes was generated. The data points in the graph cluster along a slope of one for all of the spots, including both the low and highly expressed cDNA clones (R2=0.94). Similar R2 values ranging from 0.88-0.97 were observed in replicate experiments.

[0082] cDNAs corresponding to thirty unique genes were spotted on the macroarrays. These genes were originally isolated by comparing gene expression profiles from control and E2-treated fish by DD RT-PCR. Hepatic MRNA from exposed fish were radiolabeled and individually hybridized to membranes to determine if fish treated with E2, EE2, DES, pNP, MXC, and ES shared similar expression profiles. Three separate fish were used for each treatment. FIG. 1 contains representative membranes from the different treatments and a graphical representation of the data is shown in FIG. 2. FIG. 2A illustrates the mean±SEM intensity values for each of the cDNA clones arranged in order of their expression; FIG. 2B illustrates the mean intensity values for each of the cDNA clones for E2, EE2, DES, pNP, MXC or ES divided by the mean intensity values of the respective cDNA clones from the untreated control fish.

[0083] Several of the genes that were spotted on the array were found to be up or-down regulated in E2-treated fish compared to controls. These genes were identified by comparing their intensity values to constitutive genes after correcting for intra-membrane differences based on the intensity values of 11 cDNA clones used to normalize the data. Genes on the macroarray were designated as constitutive if their fold-induction values fell within the range of the mean plus one standard deviation of the highest and lowest values of the 11 clones. Based on this criteria, any cDNA clones in the macroarray experiments above a ˜1.66-fold induction were designated as up-regulated genes respective to control fish, and any cDNA clones that had a value below ˜0.42 were designated as down-regulated.

[0084] Of the 30 genes used on the array, 6 genes were found to be up-regulated by E2 including Vtg α and β, choriogenin 2 and 3, ER α, and coagulation factor XI. Three genes found to be down-regulated by E2 were transferrin, beta actin, and alpha-1-microglobulin/bikunin precursor protein. The remaining genes did not appear to be differentially regulated by E2 when compared to controls.

[0085] The 9 genes that were up or down-regulated by EE2, DES, pNP, and MXC exposures showed a similar pattern of expression to the E2 treatment. Interestingly, ubiquitin-conjugating enzyme 9 was significantly (P<0.05) up-regulated only in the pNP treatments suggesting its regulation is not mediated through the ER. Eight of the nine genes that were found to be up or down-regulated for E2, EE2, DES, pNP, and MXC did not fluctuate for ES-treated fish, but instead resembled the pattern observed in control fish. The primary exception was ER α, which appeared to be up-regulated for all of the compounds, including ES. An additional gene, 3-hydroxy-3-methylglutaryl CoA reductase, appeared to be slightly down-regulated in fish treated with ES compared to all of the other treatments and the controls.

[0086] To determine if the gene expression profiles on the array could be verified by other techniques that monitor MRNA expression, the expression profiles of several genes on the arrays were compared (Vtg α, choriogenin 2, and transferrin) to their profile by Northern blots and DD RT-PCR. Both Vtg α and choriogenin 2 mRNA levels increase in fish treated with E2, as measured by Northern blots and DD RT-PCR. Transferrin decreases with E2 treatment, as measured by Northern blots and DD RT-PCR.

[0087] To assess whether the arrays could be used as a quantitative tool to measure the expression of multiple genes at varying concentrations of an estrogenic chemical, male SHMs exposed for 4 days to either 24, 109, or 832 ng/L of EE2 were examined (Folmar et al., Aquatic. Toxicol. 49:77-88, 2000; Hemmer et al., Environ. Toxicol. Chem. 20:336-343, 2001). FIG. 3 contains graphical illustrations of genes whose expression levels significantly changed more than 2-fold in one or more of the three EE2 concentrations examined (P<0.05). Vtg α and β, choriogenin 2, choriogenin 3, ER α, and clone ND107-B were found to increase in a concentration dependent manner in the EE2-exposed fish. Three other genes, transferrin, alpha-1-microglobulin/bikunin precursor protein, and beta actin, appeared to decrease in a dose-dependent manner. These results were consistent with the same genes that were up or down-regulated in the E2, DES, pNP, and MXC exposed fish (FIG. 2).

Example 2—Expression Profiling of E2 Using a SHM Array

[0088] A SHM estrogen responsive macroarray was developed to investigate the feasibility of applying array technology in monitoring the environmental distribution of endocrine disrupting compounds that mimic estrogen.

[0089] Total hepatic mRNA was extracted from 5 adult male SHMs treated by aqueous exposure to 100 ng/L of E2 dissolved in triethylene glycol (TEG) for 5 days. Minipreps of 54 cDNA clones derived from DD analysis were PCR amplified using primers specific to the M13 sequence of the cloning vector (pGEMT-Easy, Promega, Madison,Wis.). After the PCR reactions the products were purified in spin-columns (Qiagen, Chatsworth, Calif.) and then concentrated in a speed-vac. The cDNA samples were denatured with NaOH, heated to 65° C. for 10 min, and then immediately quenched on ice. 20×SSC (3M NaCl, 0.3M sodium citrate, pH 7.0) that contained 0.01 mM bromophenol blue was then added to the samples to yield a final concentration of 0.3M NaOH, 6×SSC, and 100 ng/μL cDNA template. The samples were then robotically spotted (Biomek 2000, Beckman Coulter, Fullerton, Calif.) in duplicate onto neutral nylon membranes (Fisher Scientific, Pittsburgh, Pa.) using 100 nL pins. The membranes were UV cross-linked and then stored under vacuum at room temperature until hybridized. Various controls, which provided information about the cDNA labeling efficiency, blocking, and non-specific binding of the arrays, were also spotted onto the membranes. These controls included: 3 Arabidopsis thaliana cDNA clones, Cot-1 repetitive sequences, poly A sequence (SpotReport 3, Stratagene, La Jolla, Calif.), and a M13 sequence (vector but no cDNA insert). Labeling of RNA probes and hybridization of blots was performed as described in Example 1.

[0090] The inter-membrane process variability between macroarrays was determined by hybridizing aliquots of identical RNA samples onto two separate membranes. A scatter plot correlating intensity values between the membranes was generated. The data points in the graph clustered along a slope of one (R2 of 0.95, Sigma Stat, Jandel, Calif.), a result which indicates that there is very little variability between membranes.

[0091] To determine if the gene transcripts found to be up- or down-regulated initially by DD analysis reflect the same induction pattern when spotted onto array membranes, RNA from adult male SHMs aqueously exposed to 100 ng/L of E2 dissolved in TEG were radiolabeled and hybridized to several membranes. FIGS. 4A and 4B contain blots of control (TEG-treated) and E2-treated fish, respectively. FIG. 4C is a plot of the mean intensity values. Genes on the macroarray were designated as constitutive genes if their intensity values fell within the range of the highest (1.27) and lowest (0.83) value of the 17 cDNA clones that were used to normalize the data. Based on this criteria, any cDNA clone in the macroarray experiments that had an intensity value above ˜1.27 was designated an E2 up-regulated gene, and any cDNA clone that had a value below ˜0.83 was designated an E2 down-regulated gene. Of the 54 cDNA clones that were spotted on the array, 15 genes appeared to be up-regulated by E2, 32 clones appeared to be constitutive, and 7 genes appeared to be down-regulated by exposure to E2. All of the highly up-regulated genes, including vitellogenin α and β and the choriogenic protein (ZP2) were also shown to be up-regulated on DD analysis. Interestingly, transferrin, a protein involved in iron transport that was identified to be down-regulated by DD analysis also appears to be down-regulated in response to E2 on the macroarrays.

Example 3—Gene Expression Profiles of LMB Exposed to 4-NP and ICI 182,780 Using a LMB Array

[0092] Experimental Design and Sample Collection: Adult male LMB were purchased from American Sports Fish Hatchery (Montgomery, Ala.) and maintained in fiberglass tanks as previously described (Larkin et al., Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 133:543-557, 2002; Larkin et al., Marine Environ. Res. 54:395-399, 2002; and Larkin et al., Comparative Biochemistry and Physiology 133:543-557, 2002). Array technology as a tool to monitor exposure of fish to xenoestrogens. Marine Environ. Res., 2002; Bowman et al., Mol. Cell. Endocrinol. 196:67-77, 2002). Each fish was injected IP with either 50 mg/kg 4-NP (Fluka, St. Louis, Mo. # 74430), the combination of 50 mg/kg 4-NP and 1.0 mg/kg ICI 182,780 (Tocris Cookson), or vehicle, which consisted of ethanol and DMSO (Sigma, St. Louis, Mo. #5879). Each dose was dissolved in 1 ml of ethanol and then diluted to the appropriate concentration with DMSO. The fish were euthanized by submersion in a water bath containing 50-100 ppm MS-22 48 hours post injection and sacrificed by a sharp blow to the head followed by cervical transaction. The livers were excised and immediately flash frozen in liquid nitrogen. The frozen tissues were stored at −80° C. until RNA was isolated.

[0093] RNA Isolation: Isolation of total RNA from liver tissue was performed with the RNA Stat-60 reagent (Tel-test). Briefly, 30 mg-50 mg of tissue stored in RNA later was homogenized in 0.9 mls STAT 60, choloroform was added, and the mixture was centrifuged at 12,000 g for 15 minutes at 4° C. The extraction process was repeated and the pooled RNA was added to 500 μl isopropanol and allowed to precipitate at −20° C. for at least one hour. Following centrifugation at 12,000 g for 50 minutes, the pellet was washed with 70% ethanol, air dried and resuspended in an appropriate volume (50 μl-120 μl of RNA secure. The samples were treated with RNA secure (Ambion, Austin Tex. #7010) to inactivate contaminating RNases. All isolated RNA was treated with DNase solution (Ambion, Austin, Tex. #1906) following the manufacture's protocol. For all RNA samples, the quantity and quality of total RNA was assessed by spectrophotometric readings at 260 nm and by electrophoresis through a 1% formaldahyde agarose gel stained with ethidium bromide.

[0094] Real-Time PCR: Real time PCR was performed using reagents and a 5700 thermocycler purchased from Applied Biosystems (ABI, Foster City, Calif.). The nucleotide sequences of the primers for the ER subtypes and Vtg 1 are as follows: 5′, GACTACGCCTCCGGCTATCAYTATGG (SEQ ID NO:563) AND 5′CATCAGGTAGATCTCAGGGGGYTCNGCNTC (SEQ ID NO:564). Probes and primers for the ER subtypes and Vtg 1 are described in Bowman et al., Ecotoxicology 8:399-416, 1999; and Bowman et al., Mol. Cell Endocrinol. 196:67-77, 2002. Each real time PCR reaction consisted of 0.01-0.2 μg of reverse transcribed total RNA from liver tissue, 1× universal Taqman master mix (ABI, Foster City, Calif.), and primers and probes in a 25 μl reaction. To generate a standard curve, varying amounts of plasmid containing the specific cDNA inserts for each gene were used as template in the PCR reactions. For each gene, a 6 point standard encompassing a 1×106 fold range of approximately 25-2.5×106 copies of cDNA was constructed. Each sample was run in duplicate and normalized 18s rRNA, also obtained by real-time PCR. Both the intra-assay and inter-assay variability never exceeded 10%. The final data is graphed as the mean and standard error of the relative copies of each ER or Vtg MRNA per μg of total RNA. Statistical differences between the treatments were determined by one way analysis of variance with Dunnets post-hoc analysis.

[0095] Amplification of cDNA to be spotted on the macro arrays: The macroarrays were prepared and printed as previously described (Larkin et al., Marine Environ. Res. 54:395-399, 2002). Briefly, the 132 LMB clones were PCR amplified using primers specific to the M13 sequence of the cloning vector (pGEMT-Easy, Promega, Madison,Wis.). After completion of the PCR reactions the products were purified using MultiScreen PCR plates (Millipore, Bedford, Mass.), concentrated, denatured with NaOH, heated to 65° C. for 10 min, and then immediately quenched on ice. 20×SSC (3M NaCl, 0.3M sodium citrate, pH 7.0) containing 0.01 mM bromophenol blue was added to the samples to yield a final concentration of 0.3M NaOH, 6×SSC, and 100 ng/μL cDNA template. The PCR products were robotically spotted (Biomek 2000, Beckman Coulter, Fullerton, Calif.) in duplicate onto neutral nylon membranes (Fisher Scientific, Pittsburgh, Pa.) using 100 nL pins. Membranes were UV cross-linked and stored under vacuum at room temperature until hybridization.

[0096] Labeling of RNA and hybridization was performed as described in Example 1. The membranes were exposed to a phosphor screen (Molecular Dynamics, Piscataway, N.J.) at room temperature for 48 hours. The blots were quantitatively evaluated using a Typhoon 8600 imaging system (Molecular Dynamics, Piscataway, N.J.). For each cDNA clone, the general background of each membrane was subtracted from the average value of the duplicate spots on the membrane. The values were normalized to the average value of 12 cDNA clones specific to ribosomal genes, which included S2, S3, S8, S15, S16, S27, L4, L5, L8, L13, L21, and L28. Ribosomal genes were chosen to normalize the data because they do not appear to fluctuate appreciably (<1.3 fold) in response to estrogenic compounds. Genes were not included for analysis that had values less than the background value for two out of the three replicates and/or fluctuated more then two fold when aliquots of the same RNA were hybridized to blots printed at the beginning, middle, and the end of the array printing process.

[0097] Measurement of ER and Vtg 1 mRNA by real-time PCR: Real-time PCR is a sensitive assay that can be used to quantitate expression levels of genes. Using this technology, assays were designed to quantitate the expression of 4 genes, estrogen receptors alpha, beta, and gamma, and Vtg 1 in LMB following exposure to 4-NP and 4-NP/ICI 182,780. Using primers and probes specific to each gene it was possible to differentiate between the ER isotypes with no cross reactivity. Exposure of LMB to a single IP injection of 4-NP (50 mg/kg) significantly increased ER α by 80 fold (p <0.05) after 48 hours when compared to controls. During the same time frame, the levels of both ER β and ER γ decreased approximately 1.3-fold and 2.6-fold respectively, however these changes were not statistically significant from controls. When the LMB were exposed to a combination of 4-NP (50 mg/kg) and the anti-estrogen ICI 182,780 (1.0 mg/kg), the levels of ER a increased only 4-fold over controls (p<0.08), suggesting that the anti-estrogen had interfered with the activation process. As with the 4-NP treatment, the expression of ER β and γ decreased (1.9-fold) but the values did not differ significantly from controls.

[0098] Since the Vtg gene is an E2-responsive gene that is under transcriptional control by ERs in the liver, the expression levels of Vtg 1 were also determined by real-time PCR. Exposure to 4-NP increased message levels by approximately 40-fold over controls (p<0.05), however, this induction was not repressed by the addition of ICI 182,780.

[0099] LMB gene array analysis: In order to further characterize the effects of 4-NP alone or in conjunction with ICI 182,780 on hepatic gene regulation in LMB, the expression of 132 genes was examined, many of which are estrogen responsive, by gene arrays. Total hepatic RNA isolated from control and exposed fish was radiolabeled and hybridized to the membranes. Of the 132 genes on the array, only genes that changed by at least 3 standard deviations from the mean of the 12 ribosomal genes that were used to normalize the data are included. These include several that are up or down-regulated by more than 2-fold, a conservative cutoff generally used for array interpretation. The mean and standard error for each gene for control and treated LMB was determined. The fold induction of each gene over controls for both the NP and NP/ICI 182,780 treatments was determined.

[0100] In the 4-NP-treated fish (FIG. 6), 9 genes were up-regulated 2-fold or greater including 4 Vtgs, choriogenin 2, choriogenin 3, aspartic protease, signal peptidase, and one unidentified clone designated 92-1. Two genes were found to be down-regulated by 4-NP including transferrin and clone 50-1. In the case of the mixture of 4-NP and ICI 182,780, some genes that were up-regulated by 4-NP treatment alone were reduced, but not all. In fact, the expression levels of 4 Vtgs, 2 choriogenins, and transferrin were not affected at all; instead they appear to be expressed to the same levels as with the 4-NP alone. Vtg 1, 2, 2a, and 3 were induced approximately 74, 28, 37, and 2-fold over controls respectively. The levels of both choriogenins increased to values approximately 35-fold over controls while aspartic protease was induced 16 fold over controls.

[0101] Genes which were reduced by the mixture and that exhibited at least a 2-fold change in expression included aspartic protease, protein disulfide isomerase, integral membrane protein, methionine sulfoxide reductase, ER γ, glucocorticoid receptor, aldose reductase, ER β, FK506 binding protein, and 21 unidentified clones. All of these genes except for clone 53-1 were down regulated by the addition of ICI 182,780 to the 4-NP.

Example 4—Gene Expression Analysis of LMB Exposed to E2 and p,p′-DDE Using a LMB Array Materials and Methods

[0102] Amplification of cDNA to be spotted on the macro arrays: The 132 clones of LMB genes in pGEM-T Easy plasmids were PCR amplified in a 300 μL reaction containing 1×PCR Buffer A (Promega, Madison,Wis.), 2 mM MgCl2 (Promega, Madison, Wis.), 160 μM each dNTP (Statagene, La Jolla, Calif.), 0.4 μM M13 primers (5′-GTT TTC CCA GTC ACG ACG TTG (SEQ ID NO:?) and 5′-GCG GAT AAC AAT TTC ACA CAG GA (SEQ ID NO:?)), and 1.25 units Taq polyrnerase (Promega, Madison, Wis.). The PCR reaction conditions were 1 cycle at 80° C. (1 min), 1 cycle at 94° C. (2min), 32 cycles at 94° C. (1 min), 57° C. (1 min), and 72° C. (2 min), 1 cycle at 72° C. (10), and then hold at 4° C. After completion of the PCR reactions the products were purified using MultiScreen PCR plates (Millipore, Bedford, Mass.) and then concentrated in a speed-vac. Aliquots of the PCR products were run on a 1.2% agarose gel containing 0.3 mM ethidium bromide. The gels were digitally imaged using a UVP Bio Doc-It camera (Ultra violet Laboratory Products, Upland Calif.) and the concentration of each PCR product was determined by comparing the intensity of the gel band to a standard curve derived from a low DNA mass ladder (Invitrogen Corporation, Carlsbad, Calif.). The PCR products were adjusted to a concentration of 160 ng/μL cDNA template.

[0103] Spotting of the gene arrays and various controls used are described in Example 1. Chemicals, Treatment, and Preparation of the hepatic samples: E2 (# E-8875) and p, p′-DDE (#12,389-7) were obtained from Sigma-Aldrich Corporation (St Louis, Mo.); 4-NP #74430, 85% para isomer) was obtained from Fluka (Milwaukee, Wis.).

[0104] Adult (˜1.5 year old) LMB weighing 300±71 grams were obtained from American Sports Fish Hatchery (Montgomery, Ala.). Fish were acclimated for a minimum of one month in an aerated holding tank prior to treatment. The fish were exposed to ambient light and fed Purina Aquamax 5D05 fish chow (St. Louis, Mo.). Groups of fish received a single IP dose of E2 (2.5 mg/kg), 4-NP (50 mg/kg), or p, p′-DDE (100 mg/kg). E2 and 4-NP were dissolved in 1 mL of 100% ethanol and then diluted to the appropriate concentration with DMSO (Sigma, St. Louis, Mo. # 5879), whereas p, p′-DDE was dissolved directly in DMSO. Control fish received an IP injection of the ethanol/DMSO or DMSO diluent without any chemical. During the experimental period the fish were not fed.

[0105] The fish were euthanized 48 hours after the IP injection by addition of 50-100 parts per million (ppm) of tricaine methanesulfonate (MS-222) to the water followed by a sharp blow to the head and cervical transection. The livers were excised from the fish and immediately flash frozen with liquid nitrogen. Total RNA was extracted from the tissue samples using RNeasy affinity columns (Qiagen, Chatsworth, Calif.).

[0106] Labeling of RNA and hybridization was performed as described in Example 1. Detection and normalization was performed as described in Example 3. Transcript data were analyzed using linear regression and student t-tests (SigmaStat and SigmaPlot, Jandel, Calif.).

Results

[0107] Gene array technology has enabled researchers to analyze hundreds to thousands of genes on a single array. As a first step toward using array technology, the inter membrane variability between the gene arrays was determined. To accomplish this, aliquots of identical RNA samples were hybridized onto two separate membranes. A scatter plot correlating the intensity values for the cDNA clones between the two arrays was generated. The data points in the graph cluster along a slope of one starting with the low to the high expressed cDNA clones (R2 of 0.98). Similar results were observed in a replicate experiment.

[0108] In order to determine the specific expression profile of 132 unique genes in LMB exposed to E2, or to the contaminants 4-NP and p, p′-DDE, hepatic total RNAs from exposed fish were radiolabeled and individually hybridized to separate membranes. Three separate fish were used for each treatment. A graphical representation of this data is shown in FIG. 5. FIG. 5A illustrates the mean±SEM intensity values for each of the cDNA clones arranged in order of their expression; FIG. 5B illustrates the mean intensity values for each of the cDNA clones for E2 divided by the mean intensity values of the respective cDNA clones from control fish. Only genes from any of the treatments (E2, NP or DDE) that were 3 standard deviations from the mean (0.98±0.41) of the 12 r-protein genes that were used as constitutive controls are shown. While there are a number of genes whose expression levels meet this criterion, only genes that exhibit a two-fold or greater change in expression were considered to be differentially regulated. A two-fold cutoff is commonly used by researchers to demarcate up or-down regulated genes for array experiments (Nagahama, Y. Int. J. Dev. Biol. 38:217-229, 1994; Lin and Peter, Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 129:543-550, 2001). Of the 132 genes used on the array, 16 genes were up-regulated 2-fold or greater by E2 including four Vtg genes, choriogenin 2, choriogenin 3, aspartic protease, protein disulfide isomerase, aldose reductase, and 7 unidentified clones designated 23-1, 24-1, 34-1, 92-1, 101-1, 132-2, and 136-1. Two genes were down-regulated two-fold or more by E2 including transferrin and a clone designated 53-1.

[0109] Since the mode of action of p, p′-DDE has not been extensively characterized, the influences of this compound on the expression profiles of the 132 genes arrayed in both male and female fish was examined. In male fish (FIG. 7), four genes were up-regulated by p, p′-DDE including Vtg 1, Vtg 2, choriogenin 2, and choriogenin 3, whereas one gene, clone 47-2 was down-regulated. In female fish (FIG. 8) injected with p, p′-DDE, no genes were identified as up-regulated; however, 17 genes were down-regulated two-fold or greater. These included the four Vtg 's, aspartic protease, transferrin, chemotaxin, choriogenin 2, androgen receptor, and 8 unidentified clones designated 50-1, 53-1, 71-1, 101-1, 107-1, 118-1,120-1, and 128-1.

[0110] Summaries of the genes whose expression increased or decreases more than 2-fold for each exposure are depicted in FIG. 9. Light shading indicates down-regulated genes while dark shading indicates up-regulated genes.

Example 5—Altered Gene Expression in Liver of LMB Exposed To Androgens Methods

[0111] Suppressive subtractive hybridization: Juvenile LMB were treated with a single 50 μl intraperitoneal (IP) injection of either a 2 μM solution of dihydrotestosterone (DHT) or progesterone in DMSO (˜2.5 nmol/g BWT). Fish were euthanized four days later and their livers were removed. Hepatic polyA+ RNA was isolated from these samples and subtractive hybridizations (Clontech, Palo Alto, Calif.) were performed in one direction using DMSO as the driver. The subtracted gene pools were then cloned into pGEM T-Easy (Promega, Madison, Wis.) and sequenced. Clones were identified using tBlastx at the National Center for Biotechnology Information (NCBI).

[0112] Gene arrays: cDNAs obtained from SSH were arrayed as previously described (Larkin et al., Marine Environ. Res. 54:395-399, 2002) and then hybridized with 33P-labeled single-stranded cDNAs isolated from adult male LMB treated with 62.5 μg/g DHT or 20 μg/g 11-ketotestosterone (11-KT) or vehicle (DMSO) (n=5 per treatment). For each cDNA clone, the general background of each membrane was subtracted from the average value of the duplicate spots on the membrane. The values were then normalized to the average value of seven cDNA clones specific to ribosomal genes and the fold change calculated by dividing the mean of each treatment by the mean of the control. Those genes which changed by 2-fold or more were graphed. Significant differences (p<0.05) were determined by ANOVA and secondary testing was done by using Tukey's LSD.

Results

[0113] The results are shown in Table 1. Genes that were the most elevated include Vtg 2, spermidine-spermine N1-acetyltransferase (SSAT), and ZPCs 1 and 4, while the LDL receptor, RXR interacting protein, and Vtg receptor were the most decreased. While the patterns of regulation appeared similar for both androgens, some specific differences did occur. For instance, aspartic protease and glutathione peroxidase III were up- and down-regulated, respectively, by DHT alone. Conversely, a fish homolog to pituitary tumor transforming protein (PTTP) and cystatin were up- and down-regulated, respectively by 11-KT alone. One gene that was up-regulated by both androgens was sSAT. This gene was shown to be unaffected by estradiol in the pig (Green et al., Biol. Reprod. 59:1251-1258, 1998).

1TABLE 1Genes Up/Down Regulated By 11-KT and DHTTREATEDWITH 20CHANGEMG/KGBYLOCGene IDE-scoreDHT11KTD14VTG PRECURSOR1.33E−39upupC13SSAT0upupH7EST SEASONAL 646.3upupF297-8upNCE12EST SEASONAL 884upNCO11RIKEN 1110001M012.61E−05upupB14EST SEASONAL 629.04upupB13SOLUTE CARRIER2.29E−37upupF12EST SEASONAL 561.7upNCG13RHAMNOSE BINDING1.00E−43upNCLECTINJ13TFIIIA1.20E−30upNCH14ZPC10upupD13EST SEASONAL 97.58upupI14ZPC42.58E−25upupB8EST SEASONAL 12upNCI7ASP PROTupNCC14UNNAMED PROTEIN1.48E−36upNCE3ATPASE 63.17E−18upNCF9ATPASE SUBUNIT 65.13E−24upNCM11RETINOL9.07E−38upupDEHYDROGENASEK7ATP SYNTHASE1.35E−08upNCK13ESTP4_H072.16upupO13EST SEASONAL F210.82downdownF5ESTDHT606.92downNCL2ALPHA 1 ANTI-2.80E−27downNCTRYPSIND12RIKEN 2700038C091.50E−04downNCM4EST SEASONAL 551.32downdownB253-1downNCC268-1downNCM5IGF-I7.50E−03downNCD6ESTP4_E066.7downNCG9ESTP4_C041.78downNCI5HAPTOGLOBIN5.19E−28downNCK2ALDOLASE B0downNCC3APOLIPOPROTEIN E1.13E−26downNCK12EST SEASONAL 726.57downdownM2ALPHA TUBULIN2.93E−40downdownG5GLUTATHIONE0downNCPEROXIDASE IIIK5ESTP4_E013.43E+00downNCL124-1downdownL14ER GAMMA 5′ 2FdowndownJ5HEPCIDIN2.49E−23downNCL3COMPLEMENT C33.20E−04downdownM12TFIID (change to1.88E−07downdownliver regerationrelated protein)K11EST SEASONAL 510.39downdownK1GP3 11CdowndownL11RXR INTERACTING1.36E−09downdownPROTM14VTG RCdowndownJ10LDL RC1.62E−32downdownL9EST SEASONAL 90downNCN8EST P4_D08upNCO10PTTP3.12E−22upNCD4EST DHT640.322upNCE14warm water acclim3.18E−12upupL8EST P4_06upNCH10EST SEASONAL 420.011downNCB9EST SEASONAL F17downNCO6EST SEASONAL 110.36downNCO3CYSTATIN8.39E−05downNC

Example 6—LMB And SHM Genes Up/down-regulated in Response to Estrogenic Agents

[0114]

2

TABLE II

LMB Gene Regulation

Differentially

LMB#
Gene ID
expressed by

LMB_COMP FACTOR
Putative complement

Bf/C2
factor Bf/C2

LMB_ABMP
ABMP precursor

LMB GLUT-PEROX III
Glutathione
Dn-reg DHT

peroxidase III

LMB_Srnp D1
Small ribonucleoprotein

D1 polypeptide (16 kD)

LMB_RIBO L6
Ribosomal protein L6

LMB_MYOSIN LIGHT
myosin regulatory

light chain

LMB_ZPC1
ZPC1
up-reg DHT;

11-KT

LMB_CYTO-C OX 1
Cytochrome c oxidase

subunit I

LMB_LECTIN STL2
Rhamnose binding
up-reg DHT

lectin STL2

LMB_EMAP2
Echinoderm microtubule

associated protein

like 2

LMB_ALDOLASE-B
Aldolase b

LMB_RIBO L7A
60S ribosomal

protein L7A

LMB_PROTHROMBIN
Prothrombin precursor

LMB_SSAT
SSAT
up-reg DHT;

11-KT

LMB_COMPLEMENT-
Complement C3 precursor
Dn-reg DHT;

C3

11-KT

LMB_RIBO L7
Ribosomal protein L7

LMB H-ATPASE-
H+-ATPase subunit,

SUBUNIT
oligomycin sensitivity

conferring protein

LMB_RIBO L23A
Ribosomal protein L23a

LMB_ALPHA-TUBULIN
alpha tubulin
Dn-reg 11-kt;

DHT

LMB_RIBO-Sa
40S ribosomal

protein Sa

LMB_VTG
Vitellogenin prcursor

LMB NASCENT-POLYPEP
Nascent polypeptide-

associated complex,

alpha polypeptide

LMB_ApoH
Apoliporotein H

LMB_TBT-BP
TBT-binding protein

LMB_SOL-CAR-
solute carrier family
up-reg DHT;

25A#5
25 alpha member 5
11-KT

LMB_UNNAMED-
Unnamed protein

PROTEIN
product

LMB_FIB-B-SUBUNIT
Fibrinogen B subunit

LMB CIS-RETIN
cis-retinol
up-reg DHT

DEHYDRO
dehydrogenase

LMB_SENES-ASSOC
Putative senscence-

PROTEIN
associated protein

LMB_LDL RC
LDL receptor
Dn-reg DHT;

11-KT

LMB_ABC-TRANS
ABC transporter

LMB_CATHEPSIN B
Cathepsin B

LMB_SERPIN-CP9
Serpin CP9

LMB_TFIIIA
Transcription factor

IIIA (TFIIIA)

LMB_ANTITHROMBIN
Antithrombin III

III

LMB_RIKEN
RIKEN cDNA

1810056020
1810056020

LMB_WEE-I
Wee I tyrosine

kinase

LMB_HAPTOGLOBIN
Haptoglobin
Dn-reg DHT

LMB_APOA-I
APOPLIPOPROTEIN A-I

LMB_ALPHA-1
alpha-1 antitrypsin
Dn-reg DHT

ANTITRYPSIN
homolog precursor

LMB_APOE
Apolipoprotein E

LMB_ZPC4
ZPC4
up-reg DHT;

11-KT

LMB_LECTIN 9
C-type lectin

superfamily 9

LMB_ATPASE 6
ATPase subunit 6
up-reg DHT

LMB_ITI
inter-alpha-trypsin

inhibitor “ITI”

LMB_EIF-3#7
Eukaryotic translation

initiation factor 3

subunit 7

LMB_HEPCIDIN
Hepcidin precursor
dn-reg DHT

LMB_PTTP
Pituitary tumor

transforming protein

LMB_TOXIN-1
Toxin-1

LMB_COAG FACTOR
Coagulation factor

VII
VII

LMB_CDC42-2
cdc 42 isoform 2

LMB_WARM-WATER
Warm water acclimation-
up-reg 11-KT

ACC PROTEIN
related protein

LMB_CYTO-C OX II
Cytochrome c oxidase

subunit II

LMB_L10A
60S ribosomal

protein L10A

LMB_KALLIKREIN
Kallikrein

LMB_DANIO EST
Danio EST

3818635
IMAGE: 3818635

LMB_ALPHA-2-
alpha-2-

MACROGLOB-1
macroglobulin-1

LMB_HAPTOGLOB
Haptoglobin-

RELATED PROT
related protein

LMB_FILAMEN-B
Filamen B

LMB_UBIQUITIN
ubiquitin

LMB_RXR INTERACT
Retinoid X receptor
Dn-reg 11-KT;

PROT
interacting protein
DHT

LMB_MITOCHON-
ATP synthase alpha
up-reg DHT

ATP-SYNTHASE
chain mitochondrial

precursor

LMB_TATA BOX BP
TATA-box binding

protein

LMB_DIFF-REG
Differentially

TROUT PROT-1
regulated trout

protein 1

LMB LIVER-REGEN-
liver regeneration

REL PROT
related protein

LMB_SERPIN-2B
Serpin 2b

LMB_APO-A1
Apolipoprotein

A-I-1 precursor

LMB_M-PHASE
M-phase

PROT 6
phosphoprotein 6

LMB_PROSTAGLAND-
Prostaglandin D

D-SYNTHASE
synthase-like protein

(lipocalin type)

LMB_LYRIC
LYRIC

LMB CYSTATIN-PREC
Cystatin precursor
Dn-reg 11-KT

LMB_RIKEN 2700038
RIKEN cDNA 2700038

LMB_DIAZEPAM-
Membrane associated

BINDING INHIB
diazepam-binding

inhibitor

LMB_IGF-I
IGF-I

LMB_ESTP4_D11
ESTP4_D11

LMB_ESTDHT_6
ESTDHT_6

LMB_ESTDHT_7
ESTDHT_7

LMB_ESTDHT_13
ESTDHT_13

LMB_ESTDHT_50
ESTDHT_50

LMB_ESTDHT_51
ESTDHT_51

LMB_ESTDHT_53
ESTDHT_53

LMB_ESTDHT_60
ESTDHT_60

LMB_ESTDHT_62
ESTDHT_62
up-reg DHT;

11-KT

LMB_ESTDHT_68
ESTDHT_68

LMB_ESTDHT_69
ESTDHT_69

LMB_ESTP4_A02
ESTP4_A02

LMB_ESTP4_B03
ESTP4_B03

LMB_ESTP4_B04
ESTP4_B04

LMB_ESTP4_B07
ESTP4_B07

LMB_ESTP4_B08
ESTP4_B08

LMB_ESTP4_B09
ESTP4_B09

LMB_ESTP4_C03
ESTP4_C03

LMB_ESTP4_C04
ESTP4_C04

LMB_ESTP4_C06
ESTP4_C06

LMB_ESTP4_D04
ESTP4_D04

LMB_ESTP4_D08
ESTP4_D08

LMB_ESTP4_D10
ESTP4_D10

LMB_ESTP4_E01
ESTP4_E01

LMB_ESTP4_E03
ESTP4_E03

LMB_ESTP4_E06
ESTP4_E06

LMB_ESTP4_E08
ESTP4_E08

LMB_ESTP4_E12
ESTP4_E12

LMB_ESTP4_F06
ESTP4_F06

LMB_ESTP4_G06
ESTP4_G06

LMB_ESTP4_G11
ESTP4_G11

LMB_ESTP4_H02
ESTP4_H02

LMB_ESTP4_H04
ESTP4_H04

LMB_ESTP4_H05
ESTP4_H05

LMB_ESTP4_H07
ESTP4_H07

LMB_ESTP4_H08
ESTP4_H08

LMB_EST-
EST-SEASONAL_02

SEASONAL_02

LMB_EST-
EST-SEASONAL_03

SEASONAL_03

LMB_EST-
EST-SEASONAL_04

SEASONAL_04

LMB_EST-
EST-SEASONAL_06

SEASONAL_06

LMB_EST-
EST-SEASONAL_09
up-reg DHT;

SEASONAL_09

11-KT

LMB_EST-
EST-SEASONAL_11
dn-reg 11-KT

SEASONAL_11

LMB_EST-
EST-SEASONAL_12
up-reg DHT

SEASONAL_12

LMB_EST-
EST-SEASONAL-14

SEASONAL-14

LMB_EST-
EST-SEASONAL_16

SEASONAL_16

LMB_EST-
EST-SEASONAL_17

SEASONAL_17

LMB_EST-
EST-SEASONAL_22

SEASONAL_22

LMB_EST-
EST-SEASONAL_51
dn 11-KT;

SEASONAL_51

DHT

LMB_EST-
EST-SEASONAL_52

SEASONAL_52

LMB_EST-
EST-SEASONAL_54

SEASONAL_54

LMB EST-
EST-SEASONAL_55
Dn-reg 11-KT;

SEASONAL_55

DHT

LMB EST-
EST-SEASONAL_56
up-reg DHT;

SEASONAL_56

LMB EST--
EST-SEASONAL_58

SEASONAL_58

LMB EST--
EST-SEASONAL_59

SEASONAL_59

LMB EST--
EST-SEASONAL_61

SEASONAL_61

LMB EST--
EST-SEASONAL_62

SEASONAL_62

LMB EST--
EST-SEASONAL_64
up-reg DHT;

SEASONAL_64

11KT

LMB EST--
EST-SEASONAL_68

SEASONAL_68

LMB EST--
EST-SEASONAL_70

SEASONAL_70

LMB EST--
EST-SEASONAL_71

SEASONAL_71

LMB EST--
EST-SEASONAL_72
Dn-reg DHT;

SEASONAL_72

11KT

LMB EST--
EST-SEASONAL_75

SEASONAL_75

LMB EST--
EST-SEASONAL_77

SEASONAL_77

LMB EST--
EST-SEASONAL_85

SEASONAL_85

LMB EST--
EST-SEASONAL_88
up-reg DHT;

SEASONAL_88

LMB EST--
EST-SEASONAL_90
dn-reg DHT

SEASONAL_90

LMB EST--
EST-SEASONAL_92

SEASONAL_92

LMB EST--
EST-SEASONAL_97

SEASONAL_97

LMB EST--
EST-SEASONAL_F11

SEASONAL_F11

LMB EST--
EST-SEASONAL_F17
Dn-reg 11-KT

SEASONAL_F17

LMB EST--
EST-SEASONAL_F21
Dn-reg DHT

SEASONAL_F21

LMB_ER-ALPHA
ESTROGEN RECEPTOR
up-reg E2;

ALPHA
NP

LMB_ER-BETA
ESTROGEN RECEPTOR

BETA

LMB_ER-GAMMA
ESTROGEN RECEPTOR
Dn-reg 11-KT;

GAMMA
up-reg E2

LMB_STAR
STAR PROTEIN
up-reg cAMP;

dn-reg b-

sitosterol

LMB_SF1
SF1 PROTEIN

FRAGMENT

LMB_ESTP4-E01
LMB_ESTP4-E01
down by DHT

LMB_ESTDHT64
LMB_ESTDHT64
up by 11KT

LMB LIV-REGER-
LMB_LIV-
down by DHT

PROT
REGER-PROT

LMB RIKEN
LMB_RIKEN
up by 11KT;

1110001M01
1110001M01
DHT

LMB EST-
LMB_EST-SEASONALf17
down by 11KT

SEASONALf17

LMB1-3
unknown

LMB2-2
unknown

LMB3-1
unknown

LMB4-1
unknown

LMB5
vitellogenin-2A
Up reg E2;

NP; Dn-reg

DDE(F)

LMB6-1
AMBP protein precursor

LMB7-1
Unknown

LMB8-2
Unknown

LMB9-1
Unknown

LMB10-1
Unknown

LMB11-2
Unknown

LMB12-1
Zebrafish

Oligosaccharyl

transferase integral

membrane protein

LMB13-2
Unknown

LMB14-1
Unknown

LMB15-1
NADH dehydrogenase

subunit 1

LMB16-2
unknown

LMB17-2
Mitochondrial control

region

LMB18-3
unknown

LMB19-1
Insulin like growth

factor

LMB20-1
unknown

LMB21-1
unknown

LMB22-1
unknown

LMB23-1
unknown
Up-reg E2

LMB24-1
Unknown
Up-reg E2,

dn-reg DHT;

11-KT

LMB25-1
Ribosomal porotein

S8

LMB26-1
Transferrin

LMB27-1
unknown

LMB28-2
unknown

LMB29-2
unknown

LMB30-1
unknown

LMB31
choriogenin

LMB32-1
G-box binding factor

(bacteria)

LMB33-1
unknown

LMB34-1
unknown
up-reg E2

LMB35-1
unknown

LMB36-1
hypothetical protein

LMB37-1
unknown

LMB38-1
40S ribosomal

protein S2

LMB39-1
unknown

LMB40-1
alport syndrome

chrom region gene

LMB41-1
ribosomal protein L8

LMB42-1
Gamma fibrinogen

LMB43-1
FK506 binding protein,

immunophillin

LMB44-1
Dynein heavy chain

LMB45-1
vitellogenin A

LMB46-1
unknown

LMB47-2
unknown
Down-reg

DDE(M)

LMB48-1
elongation factor

1 beta

LMB49-1
40S ribosomal

protein S15

LMB50-1
unknown
Down reg NP;

DDE(F)

LMB51-1
unknown

LMB52-1
unknown

LMB53-1
unknown
Down-reg. E2;

DDE (F); DHT

LMB54-2
L4 ribosomal

LMB55-1
L4 ribosomal

LMB56-1
40S ribosomal

LMB57
ADP, ATP translocase

LMB58-1
ribosomal L21

LMB59-1
Unknown

LMB60-1
unknown, AK010552

LMB61-1
unknown

LMB63-1
unknown

LMB64-1
unknown

LMB65-1
unknown

LMB66-1
unknown

LMB67-1
signal peptidase,
Up-reg NP

endopeptidase

LMB68-1
hypothetical protein
Dn-reg DHT

LMB69-2
unknown

LMB70-2
NADH dehydrogenase

subunit 1

LMB71-1
unknown
down-reg

DDE (F)

LMB72-1
unknown

LMB73-1
NADH dehydrogenase

subunit 1

LMB74-3
unknown

LMB75-1
40S ribosomal

LMB76-2
unknown

LMB77-1
unknown

LMB78-1
unknown

LMB79-2
unknown

LMB80-1
unknown

LMB81-1
unknown

LMB82-1
unknown

LMB83
vitellogenin-2
up-reg E2;

NP; DDE(M);

dn-reg DDE(F)

LMB84-1
STAR

LMB85-1
CYP1A

LMB86-1
ribosomal protein

L28

LMB87
vitellogenin-1
up-reg E2;

NP; DDE(M);

dn-reg DDE(F)

LMB88-1
unknown

LMB89-1
glucocorticoid

receptor

LMB90-1
unknown

LMB91-1
unknown

LMB92-1
unknown
up-reg E2;

NP

LMB93-1
estrogen receptor

gamma

LMB94-1
transferrin
Down-reg.

E2; NP;

DDE in F

LMB95-1
CAP-rich Zinc finger

protein

LMB96-1
unknown

LMB97
choriogenin-3
Up-reg E2;

NP; DDE(M);

DHT

LMB98-1
estrogen receptor

beta

LMB99-1
estrogen receptor

alpha

LMB100-1
ribosomal protein L5

LMB101-1
unknown
Up-reg E2, Dn

for DDE(F)

LMB102-1
chemotaxin
down-reg

DDE (F)

LMB103-1
proteosome subunit 9

LMB104-3
60S ribosomal protein

L13

LMB105-1
unknown

LMB107-1
unknown
down-reg

DDE (F)

LMB108-1
choriogenin-2
Up-reg E2; NP;

DDE(M); Dn-

reg DDE (F)

LMB109-1
40S ribosomal

protein S3A

LMB110-1
Methionine sulfoxide

reductase

LMB112-1
cathepsin (Aspartic
up-reg E; NP:

protease)
DHT; Dn-DDE (F)

LMB116-1
aldose reductase
up-reg E2

LMB118-1
apolipoprotein
down-reg

precursor
DDE (F)

LMB120-1
hypothetical protein
donw-reg

DDE (F)

LMB121-1
TBT binding protein

LMB122-1
alpha2-HS

glycoprotein

LMB123-1
Urocanase

LMB128-1
unknown
down-reg

DDE (F)

LMB129-1
unknown

LMB130-1
secreted phosphoprotein

precursor

LMB132-1
integrin beta
up-reg E2

LMB133-1
unknown

LMB134-3
unknown

LMB135
protein disulfide
up-reg E2

isomerase

LMB136-1
protein disulfide
up-reg E2

isomerase like

LMB137-2
unknown

LMB138-1
unknown

LMB139-1
apolipoprotein C2

LMB140-1
unknown

LMB141
vitellogenin-3
up reg E2; NP,

dn DDE (F)

LMB142-1
hypothetical protein

(FLJ10530)

LMB144-1
vitellogenin like

LMB150
androgen receptor
Dn-reg DDE(F)

LMB151
vitellogenin receptor
Dn-reg

DHt-11-KT

[0115]

3

TABLE III

SHM Gene Regulation

Clone ID
Identity
E value
Regulation

SHM IK 7A
40 S ribosomal protein
2.00E−39

(Ictalurus punctatus)

SHM IK 24E
similar to ribosomal protein
4.00E−34

L37a, cytosolic

SHM IK 25C
ribosomal protein L5
5 E−05

SHM IK 5D
60 S ribosomal protein L8
5.00E−26

SHM IKIGF-1
IGF I

SHM IKIGF-2
IGF 2

Female Test (SSH)

ndSHM-FT1-A03
sertotransferrin precursor
1.00E−99

(O. Latipes)

ndSHM-FT1-A09
putative transmembrane
7.88E−08
up-reg- E2

4 superfamily member protein

ndSHM-FT1-A10
unknown

up-reg-E2

ndSHM-FT1-A11
phospholipid hydroperoxide
5.61E−44
dn-reg E2

glutathione peroxidase

ndSHM-FT1-A12
sertotransferrin precursor
3.90E−35
dn-reg E2

(O. Latipes)

ndSHM-FT1-B03
Unknown

ndSHM-FT1-B07
Similar to aldehyde dehydrogenase
0

7 family, member A1

ndSHM-FT1-B10
cytochrome b
0

[Orestias silustani]

ndSHM-FT1-C01
Similar to high mobility group
0

box 1 [Danio rerio]

ndSHM-FT1-C03
perforin 1 (pore forming
2.00E−19
up reg E2

protein) human,,

ndSHM-FT1-C04
Prostaglandin D Synthase
1.01E−05
dn-reg E2

[Xenopus laevis]

ndSHM-FT1-C09
endoplasmic reticulum lumenal
0
dn-reg E2

L-amino acid oxidase

ndSHM-FT1-D06
Unknown

up-reg E2

ndSHM-FT1-D10
unknown

up-reg E2

ndSHM-FT1-D12
unknown

dn-reg E2

ndSHM-FT1-E01
probable complement regulatory
2.79E−09
dn-reg E2

plasma protein SB1 -

ndSHM-FT1-E02
Cytochrome C oxidase subunit II
2.00E−62

ndSHM-FT1-E08
unknown

up-reg E2

ndSHM-FT1-E09
unknown

up-reg E2

ndSHM-FT1-E12
Similar to chitinase, (D. rerio)
1.00E−83
dn-reg E2

ndSHM-FT1-F01
leucine-rich alpha-2-glycoprotein
3.29E−13

[Homo sapiens]

ndSHM-FT1-F06
complement component C3
0
dn-reg E2

[Paralichthys olivaceus]

ndSHM-FT1-F09
solute carrier family 27 (fatty
6.13E−19

acid transporter), member

ndSHM-FT1-F10
beta hemoglobin A
1.00E−42
dn-reg E2

[Seriola quinqueradiata]

ndSHM-FT1-F11
unknown

dn-reg E2

ndSHM-FT1-F12

up reg E2

ndSHM-FT1-G02
unknown

ndSHM-FT1-G04
Unknown
2.15847
up-reg E2

ndSHM-FT1-G08
endoplasmic reticulum lumenal
0

L-amino acid oxidase

ndSHM-FT1-H02
FUGRU complement component
3.00E−16

C9 precursor

ndSHM-FT1-H03
35 kDa serum lectin
1.85E−35

[Xenopus laevis]

ndSHM-FT1-H04
Similar to chitinase, acidid
4.00E−83
dn-reg E2

(D. rerio)

ndSHM-FT1-H06
SPI-2 serine protease inhibitor
1.97E−09

(AA 1-407) [Rattus no

ndSHM-FT1-H07
unknown

up-reg E2

ndSHM-FT1-H10
Unknown

ndSHM-FT1-H11
unknown

up-reg E2

ndSHM-FT1-H12
beta hemoglobin A
1.40E−45
up-reg E2

ndSHM-MC1-A02
Liver basic fatty acid bp
2.00E−43
dn-reg E2

ndSHM-MC1-A03
Polyadenylate-binding protein 1
0

ndSHM-MC1-A04
unknown

up-reg E2

ndSHM-MC1-A05
beta galactosidase/ubiquitin
3.00E−44

fusion protein

ndSHM-MC1-A07
Orla C3 (O. latipes)
9.00E−38

ndSHM-MC1-A09
alpha-2-macroglobulin 2
2.00E−06
dn-reg E2

(C. carpio)

ndSHM-MC1-A11
alpha-1-antitrypsin
1.27E−11
dn-reg E2

[Sphenodon punctatus]

ndSHM-MC1-B01
unknown

dn-reg E2

ndSHM-MC1-B03
cytochrome c oxidase, subunit Va
0

ndSHM-MC1-B04
KIAA0018 protein [Homo sapiens]
9.29E−35
up-reg E2

ndSHM-MC1-B05
Unknown

up-reg E2

ndSHM-MC1-B08
complement component C5-1
5.61E−30

[Cyprinus carpio]

ndSHM-MC1-B10
Serotransferrin precursor >gi|
2.00E−39

1814091|dbj|BAA10901.1|

ndSHM-MC1-B11
fibrinogen, B beta polypeptide
2.80E−45
dn-reg E2

ndSHM-MC1-C02
Similar to fibrinogen, gamma
4.06E−41
up-reg-field

polypeptide [Danio rerio]

dn-reg E2

ndSHM-MC1-C04
4-hydroxy-phenylpyruvate-
0

dioxygenase

ndSHM-MC1-C05
unknown

up-reg E2

ndSHM-MC1-C08
serine proteinase inhibitor
8.08E−39
dn-reg E2

CP9 - common carp

ndSHM-MC1-C10
prothrombin precursor
0

[Takifugu rubripes]

ndSHM-MC1-D01

ndSHM-MC1-D02
ATPase, H+ transporting,
1.47E−25

lysosomal,

ndSHM-MC1-D03
fatty acid binding protein 2,
2.00E−58
up-reg-field

hepatic (Japanese seapearch)

ndSHM-MC1-D04
Proteasome Regulatory Particle,
0

ATPase-like

ndSHM-MC1-D06
expressed sequence AL022852
1.63E−21
up-reg E2

[Mus musculus]

ndSHM-MC1-D10
Scavenger receptor with C/type
5.00E−14
dn-reg E2

lectine type I (Human)

ndSHM-MC1-E01
similar to monocarboxylate
2.73E−17

transporter 6

ndSHM-MC1-E05
elastase 4 precursor
0
up-reg field

[Paralichthys

ndSHM-MC1-E06
Unknown

ndSHM-MC1-E08
pre alpha inhibitor heavy
3.00E−14
dn-reg E2

chain 3 rat

ndSHM-MC1-E10
Unknown

up-reg E2

ndSHM-MC1-E12
unknown

up-reg E2

ndSHM-MC1-F01
similar to charged amino acid
1.83E−11
up-reg E2

rich leucine zipper factor-1

ndSHM-MC1-F02
chemotaxis (O. mykiss)
2.00E−60

ndSHM-MC1-F03
dendritic cell protein
0

[Homo sapiens]

ndSHM-MC1-F06
Chain A, Alcohol Dehydrogenase
0

ndSHM-MC1-F11
17-beta-hydroxysteroid
0
up-reg E2

dehydrogenase type IV

ndSHM-MC1-F12
interferon induced protein 2
2.49E−07
up-reg E2

[Ictalurus punctatus]

ndSHM-MC1-G01
Alcohol dehydrogenase >gi|
0
up-reg E2

482344|

ndSHM-MC1-G02
14 kDa apolipoprotein
1.45E−16
dn-reg E2

[Anguilla japonica]

ndSHM-MC1-G03
serine (or cysteine)
1.14E−29
dn-reg E2

proteinase inhibitor,

clade F

ndSHM-MC1-G04
ribosomal protein XL1a -
0

African clawed frog

ndSHM-MC1-G05
microfibrillar-associated
3.27E−13

protein 4

ndSHM-MC1-G07
apolipoprotein E
4.21E−37

[Scophthalmus maximus]

ndSHM-MC1-G11
aldehyde reductase AFAR2
3.34E−36
up-reg E2

subunit [Rattus norvegicus]

ndSHM-MC1-G12
Similar to RIKEN cDNA
4.21E−12

1300018K11 gene [Homo sapiens]

ndSHM-MC1-H02
unknown

ndSHM-MC1-H03
complement factor B/C2B
8.00E−28

(O. mykiss)

ndSHM-MC1-H04
similar to ribosomal protein
1.07E−23
up-reg field

S25, cytosolic [validated] -

ndSHM-MC1-H06
unnamed protein product
0.000421546
up-reg E2

[Homo sapiens]

ndSHM-MC1-H08
peroxisomal proliferator-
2.00E−08

activated receptor beta1

[Salmo salar]

ndSHM-MC1-H09
Ligand-gated ionic channel
1.93158
up-reg E2

family member

ndSHM-MC1-H10
unknown
3.75692
up-reg E2

ndSHM-MC1-H12
Similar to sperm associated
4.36E−18

antigen 7 [Homo sapiens]

Male Test SSH

ndSHM-MT1-A02
chicken fatty acid binding
3.00E−52
up-reg E2

protein

ndSHM-MT1-A03
warm temperature acclimation
6.00E−40

related 65 kDa protein

(O. latipes)

ndSHM-MT1-A05
Transducin beta/like 2 protein
e−107
up-reg E2

ndSHM-MT1-B09
putative mitochonrial inner
1.00E−34
up-reg E2

membrane protease subunit

(Human)

ndSHM-MT1-C05
unknown

up-reg E2

ndSHM-MT1-C08
vitellogenin I
6.89E−43
up-reg E2

[Cyprinodon variegatus]

ndSHM-MT1-D04
WS beta-transducin repeats
1.87E−05

protein [Homo sapiens]

ndSHM-MT1-D05
mesau serum amyloid A/3
5.00E−25
up-reg E2

protein precursor

ndSHM-MT1-D07
vitellogenin (Sillago japonica)
8.00E−78
up-reg E2

ndSHM-MT1-E02
40 S ribosomal protein S3
E−105

ndSHM-MT1-E03
Similar to transducin (beta)-
0
up-reg E2

like 2 [Xenopus laevis]

ndSHM-MT1-E05
Predicted CDS, seven TM
3.00782
up-reg E2

Receptor S

ndSHM-MT1-F11
Similar to transducin (beta)-
1.42E−16

like 2 [Xenopus laevis]

ndSHM-MT1-G03
60S ribosomal protein
2.40E−38

L10a > g

ndSHM-MT1-H05
Similar to transducin (beta)-
0

like 2 [Xenopus laevis]

METHOXYCHLOR-

CONTROL SSH

ndSHM-MXCc1-
Protein involved in recombination
0.0928205
dn-reg E2

A04
repair, homologous to S. pombe

rad18.

ndSHM-MXCc1-
no hit

A09

ndSHM-MXCc1-
KIAA0096 gene product is
5.62E−12
up-reg E2

A10
related to a protein kinase.

ndSHM-MXCc1-
alpha s HS glycogrotein
1.00E−47

A11
(Platichthys flesus)

ndSHM-MXCc1-
dodecenoyl-Coenzyme A
3.82E−37
up-reg E2

B02
delta isomerase

ndSHM-MXCc1-
cytochrome P450 3A56
0
up-reg field

B03
[Fundulus heteroclitus]

ndSHM-MXCc1-
kallistatin
4.93156
up-reg E2

B04
[Rattus norvegicus]

ndSHM-MXCc1-
Fibrinogen beta chain precursor
5.78E−24
dn-reg E2

B06
[Contains: Fibrinopeptide B]

ndSHM-MXCc1-
Apolipoprotein A/I precursor
5.00E−25
dn-reg E2

B07
(sparus aurata)

ndSHM-MXCc1-
Similar to retinol dehydro-
4.16E−32

B08
genase type III [Danio rerio]

ndSHM-MXCc1-
Beta-2-glycoprotein I precursor
1.92E−11

C04
(Apolipoprotein H) (

ndSHM-MXCc1-
tyrosine kinase [Gallus gallus]
1.31312

C06

ndSHM-MXCc1-
ceruloplasmin [Danio rerio]
0
up-reg

C11

field

ndSHM-MXCc1-
vitellogenin I precursor
4.00E−51
up-reg E2

D03
(Mummichog)

ndSHM-MXCc1-
hypothetical protein
0.826071
dn-reg E2

D04
[Ferroplasma acidarmanus]

ndSHM-MXCc1-
cytochrome c oxidase subunit I
8.28E−35
dn-reg E2

D05
[Engraulis japonicus]

ndSHM-MXCc1-
no hit

up-reg E2

D08

ndSHM-MXCc1-
Immunoglobulin domain-
0.991091
up-reg E2

D10
containing protein family

ndSHM-MXCc1-
hypothetical protein
8.1324

D12
[Plasmodium falciparum 3D7]

ndSHM-MXCc1-
hypothetical protein
0.61028

E01
[Magnetospirillum magnetotacticum]

ndSHM-MXCc1-
unknown protein

up-reg E2

E09

ndSHM-MXCc1-
sorting nexin 11 [Homo sapiens]
0

E11

ndSHM-MXCc1-
vitellogenin B (M. aeglefinus)
6.00E−16
up-reg E2

F01

ndSHM-MXCc1-
warm-temperature-acclimation-
6.25E−25
dn-reg E2

F03
related-protein- [Oryzias latipes]

ndSHM-MXCc1-
UDP-glucose pyrophosphorylase
0

F07
[Gallus gallus]

ndSHM-MXCc1-
interferon-related developmental
6.68E−39
up-reg E2

F10
regulator 1 [Mus musculus]

ndSHM-MXCc1-
unknown protein
0.202018
up-reg E2

G02

ndSHM-MXCc1-
thyroid hormone receptor
0
up-reg E2

G03
interactor 12;

ndSHM-MXCc1-
Putative ribosomal protein L21
0

G04

ndSHM-MXCc1-
putative delata 6-desaturase
0
up-reg E2

G12
[Oncorhynchus masou]

ndSHM-MXCc1-
complement control protein
1.72E−23

H05
factor I-A [Cyprinus carpio]

ndSHM-MXCc1-
ATP synthase 6
3.00E−23

H09

METHOXYCHLOR

TEST SSH

ndSHM-MXCt1-
rat liver regeneration related
1.00E−48

B05
protein

ndSHM-MXCt1-
BH2041 ˜unknown conserved
6.52356
up-reg E2

B08
protein [Bacillus halodurans]

ndSHM-MXCt1-
lysophospholipase (Rat)
1.00E−36
up-reg E2

C02

ndSHM-MXCt1-
Unknown protein for MGC:63946
3.00E−29

C11
(D. rerio)

ndSHM-MXCt1-
unknown

up-reg E2

D09

ndSHM-MXCt1-
unknown

up-reg E2

E04

ndSHM-MXCt1-
CG4198-PA [Drosophila
0.385852

E06

melanogaster
]

ndSHM-MXCt1-
PROBABLE IRON OXIDASE
3.15365

E09
PRECURSOR OXIDOREDUCTASE

PROTEIN

ndSHM-MXCt1-
Vitellogenin I precursor
0
up-reg E2

E12
(VTG I) [Contains: Lipovitellin 1 (

ndSHM-MXCt1-
Unknown

up-reg E2

F11

ndSHM-MXCt1-
Unknown

G03

ndSHM-MXCt1-
miro2 pending protein
4.00E−60

H03

ndSHM-MXCt1-
Group XIII secretory
6.05E−40
up-reg E2

H09
phospholipase A2 precursor

NONYLPHENOL

CONTROL SSH

ndSHM-NPc1-A12
unknown

ndSHM-NPc1-B01
NADH subunit 1
2.80E−45
up-reg field

[Cyprinodon variegatus]

ndSHM-NPc1-B08
Chain A, Complex Of The
1.20E−15
up-reg field

Catalytic Portion Of Human

ndSHM-NPc1-B09
calreticulin [Danio
0

rerio
] >gi|6470259|gb|

ndSHM-NPc1-C04
hypothetical protein APE0566 -
0.667761

ndSHM-NPc1-C06
Vitellogenin II precursor (VTG II)
0
up-reg E2

[Fundulus heteroclitus]

ndSHM-NPc1-C11
translation elongation factor
7.14E−10

1-alpha [Stylonychia mytilus]

ndSHM-NPc1-E01
Vitellogenin I
1.20E−33
up-reg E2

[Cyprinodon variegatus]

ndSHM-NPc1-E06
Unknown

ndSHM-NPc1-E11
Unknown

up-reg E2

ndSHM-NPc1-F01
ubiquitin A-52 residue ribosomal
2.00E−37

protein [Homo sapiens]

ndSHM-NPc1-F05
Vitellogenin A
0.000293022
up-reg E2

[Melanogrammus aeglefinus]

ndSHM-NPc1-F06
Unknown

up-reg E2

ndSHM-NPc1-F07
LFA-3 (delta TM) [Ovis sp.]
0.0763225
up-reg E2

ndSHM-NPc1-F08
CG32659-PA
0.0316684

[Drosophila melanogaster]

ndSHM-NPc1-G02
ribophorin I [Danio rerio]
0

ndSHM-NPc1-G08
KIAA1560 protein [Homo sapiens]
6.27E−38

ndSHM-NPc1-G11
ATP synthase alpha chain,
1.29E−23

mitochondrial precursor

ndSHM-NPc1-H01
similar to Tho2 [Homo sapiens]
2.32887

[Rattus norvegicus]

ndSHM-NPc1-H02
Transporter, truncation
5.24069
up-reg E2

[Streptococcus pneumoniae R6]

ndSHM-NPc1-H03
Hemoglobin beta chain >gi|
1.2944

7439519|pir∥S70614

ndSHM-NPc1-H04
unknown

up-reg E2

ndSHM-NPc1-H05
Cytochrome c >gi|65467|
3.47E−32

pir∥C

ndSHM-NPc1-H08
choriogenin L (O. latipes)
1.00E−70
up-reg E2

NONYLPHENOL

TEST SSH

ndSHM-NPt1-A01
RIFIN [Plasmodium falciparum
1.79528

3D7] >gi|23498329|e

ndSHM-NPt1-A02
P0699H05.18 [Oryza sativa
0.244655

(japonica cultivar-group)]

ndSHM-NPt1-A03
hypothetical aminotransferase
0.421189

[Bradyrhizobium japonicum]

ndSHM-NPt1-A04
unknown

up-reg E2

ndSHM-NPt1-A05
serum amyloid A protein
9.34E−14
up-reg E2

[Holothuria glaberrima]

ndSHM-NPt1-A08
unknwon

up-reg E2

ndSHM-NPt1-A09
DNAse II homolog F09G8.2
0.656008
up-reg E2

[Caenorhabditis elegans]

ndSHM-NPt1-B02
similar to peroxisomal long-
2.30E−17
up-reg E2

chain acyl-coA thioesterase;

peroxisomal long-chain acyl-

coA thioesterase ; putative

protein [Homo sapiens]

ndSHM-NPt1-B03
choriogenin Hminor
1.52E−14
up-reg E2

[Oryzias latipes]

ndSHM-NPt1-B05
tryptophan 2,3 dioxygenase
1.00E−60
up-reg E2

ndSHM-NPt1-B06
ATP synthase 6
2.00E−23

(Pomacentrus trilineatus)

ndSHM-NPt1-B07
unknown

up-reg E2

ndSHM-NPt1-B11
embyonic epidermal lectin
4.00E−42
up-reg E2

(X. laevis)

ndSHM-NPt1-B12
perlecan (heparan sulfate
2.00E−31

proteoggllycan 2

ndSHM-NPt1-C01
immunoglobulin light chain
1.38E−14
up-reg E2

[Seriola quinqueradiata]

ndSHM-NPt1-C03
cytochrome c oxidase subunit
0
up-reg E2

I [Arcos sp. KU-149] >gi|

25006169|dbj|BAC23776.1|

cytochrome c oxidase subunit I

[Arcos sp. KU-149]

ndSHM-NPt1-C05
C9 protein
8.96E−18

[Oncorhynchus mykiss]

ndSHM-NPt1-C06
pentraxin [Cyprinus carpio]
9.55E−15
up-reg E2

ndSHM-NPt1-C09
Very-long-chain acyl-CoA
1.09E−13
up-reg E2

synthetase (Very-long-chain-

fatty-acid-CoA ligase) >gi|

2645721|gb|

AAB87982.1| very-long-

chain acyl-CoA synthetase

[Mus musculus]

ndSHM-NPt1-C12
dihydroorotate dehydrogenase
5.60255
up-reg E2

electron transfer subunit

[Clostridium tetani

E88] >gi|28204415|

gb|AAO36853.1|

dihydroorotate dehydrogenase

electron transfer subunit

[Clostridium tetani E88]

ndSHM-NPt1-D04
hypothetical protein
1.69055

[Plasmodium yoelii yoelii]

ndSHM-NPt1-D05
Deoxyribonuclease II precursor
3.09E−16

(DNase II) (Acid DNase)

(Lysosomal DNase II) >gi|

7513450|pir∥JE0205

deoxyribonuclease II

(EC 3.1.22.1) - pig >gi|

3157444|emb|CAA04717.1|

Deoxyribonuclease II

[Sus scrota] >gi|3309153|gb|

AAC39263.1| deoxyribonuclease

II [Sus scrofa]

ndSHM-NPt1-D07
egg envelope protein winter
4.00E−41
up-reg E2

flounder

ndSHM-NPt1-D07
similar to olfactory receptor
4.09975
dn-reg E2

MOR149-1 [Mus musculus]

ndSHM-NPt1-D09
CG31752-PA [Drosophila
1.73966

melanogaster
] >gi|

22946779|gb|

AAN11014.1|AE003660_32

CG31752-PA

[Drosophila melanogaster]

ndSHM-NPt1-D11
Fibrinogen alpha (Rattus)
5.00E−05
up-reg field

ndSHM-NPt1-E02
heparin cofactor II
0

[Danio rerio]

ndSHM-NPt1-E03
FIFO-type ATP synthase
3.32E−22
up-reg E2

subunit g [Homo sapiens]

ndSHM-NPt1-E06
unknown

ndSHM-NPt1-E07
hypothetica protein XP_215519
5.42E−09

[Rattus norvegicus]

ndSHM-NPt1-E12
6.2 kd protein [Homo
1.75E−21

sapiens
] >gi|12643829

|sp|Q9POU1|

OM07_HUMAN Probable

mitochondrial import

receptor subunit TOM7 homolog

(Translocase of outer membrane

7 kDa subunit homolog)

(Protein AD-014) >gi|

7688665|gb|AAF67473.1|

AF150733_1 AD-014 protein

[Homo sapiens] >gi|12804619

|gb|AAH01732.1|AAH01732

6.2 kd protein [Homo sapiens]

ndSHM-NPt1-F01
Hepatocyte growth factor activator
5.12E−17
up-reg E2

[Rattus norvegicus]

ndSHM-NPt1-F05
Unknown

up-reg E2

ndSHM-NPt1-F07
complement component C9
4.46E−34
up-reg field

[Paralichthys olivaceus]

ndSHM-NPt1-F11
alanine-glyoxylate
3.01E−28

aminotransferase 2

[Homo sapiens] >gi|

17432913|sp|Q9BYV1|

AGT2_HUMAN Alanine-glyoxylate

aminotransferase 2, mito-

chondrial precursor (AGT 2)

(Beta-alanine-pyruvate

aminotransferase) (Beta-

ALAAT II) >gi|12406973|

emb|CAC24841.1| alanine-

glyoxylate aminotransferase 2

[Homo sapiens]

ndSHM-NPt1-G03
KIAA1657 protein [Homo sapiens]
8.65698
up-reg E2

ndSHM-NPt1-G07
Unknown

up-reg E2

ndSHM-NPt1-G08
choriogenin H [Oryzias latipes]
3.54E−09
up-reg E2

ndSHM-NPt1-G11
glucose-6-phosphatase,
7.73E−09
up-reg field

catalytic; Glucose-

6-phosphatase [Rattus

norvegicus
] >gi|567864

|gb|AAA74381.1|

glucose-6-phosphatase

ndSHM-NPt1-G12
Orla C4 [Oryzias latipes]
1.04E−36
up-reg E2

ndSHM-NPt1-H03
N-acetylneuraminate pyruvate
3.50E−17

lyase [Mus musculus] >gi|

12832930|dbj|BAB22314.1|

unnamed protein product

[Mus musculus] >gi|

18490967|gb|AAH22734.1|

RIKEN cDNA 0610033B02 gene

[Mus musculus] >gi|

26353976|dbj|BAC40618.1|

unnamed protein product

[Mus musculus]

ndSHM-NPt1-H04
apolipoprotein B -Atlantic salmon
1.14E−10
up-reg field

(fragment) >gi|854620|

emb|CAA57449.1|

apolipoprotein B [Salmo salar]

ndSHM-NPt1-H11
putative aryl-CoA ligase EncN
0.513537

[Streptomyces maritimus]

MALE/FEMALE

UNSUBTRACTED

SHM-D03
cytochrome P450
3.00E−36
up-reg E2;

(Ictalurus punctatus)

field

SHM-D02
unknown

up-reg E2

SHM-B02
retinol binding protein 4
1.00E−17

(D. rerio)

SHM-B07
ribosomal protein L35 (galus)
2.00E−08

SHM-B06
unknown

up-reg E2

SHM-B12
Similar to 60S riboxomal
3.00E−40

protein L18A (D. rerio)

SHM-C03
ribosomal protein P2
3.00E−21

(I. punctatus)

SHM-C07
C type lectins (O. mykiss)
2.00E−11
dn-reg E2

SHM-E04
similar to 60S ribosomal
2.00E−15

protein L21

SHM-D06
unknown protein for MGC:64127
6.00E−68
up-reg E2

(D. rerio)

SHM-E01
G protein B subunit
2.00E−25

(Ambystoma tigrinum)

SHM-E07
precerebellin like protein
7.00E−27

(O. mykiss)

SHM-A06
AMBP protein precursor
3.00E−30

microglobulin

SHM-E02
Natural killer cel enhancement
8.00E−31

factor (O. mykiss)

SHM-C05
unknown

up-reg field

SHM-B10
Similar to ribosomal protein
1.00E−28

L10 (D. rerio)

SHM-D12
unknown

SHM-C01
unknown

SHM1
Glycosylate reductase
3.00E−14

SHM2-1
vitellogenin alpha (2)
in genbank
up-reg E2; EE2,

DES, NP, MXC

SHM3
vitellogenin beta(1)
in genbank

SHM
Ribosomal protein S8
8.00E−45

SHM26
choriogenin 3

SHM6
Unknown

SHM7-3
choriogenin 2
1.00E−45

SHM29
beta actin
in genbank

SHM9-1
ribosomal protein L8

SHM74-1
3-hydroxy-3-methylglutaryl-
9.00E−51
dn-reg ES

CoA reductase

SHM11
Transferrin

dn-reg E2; EE2,

DES, NP, MXC

SHM13-1
Low molecular mass protein 2
2.00E−12

SHM14
Unknown

SHM22
Unknown

SHM23-1
Ribosomal protein S9 like
6.00E−71

SHM24
Unknown

SHM25
Ribosomal protein S9 like
2.00E−45

SHM39
Unknown

SHM41
Ubiquitin-conjugating enzyme 9
EST match
up-reg NP

(putative)

SHN42-1
Unknown

SHM43
Unknown protein, Acession
4.00E−23

numberAAH10857

SHM48
Unknown

SHM48-2
Unknown

SHM51-3
Unknown

SHM56-2
Unknown

SHM62-2
Hepatic lipase precursor
7.00E−06

SHM72-3
Coagulation Factor XI

up-reg E2; EE2,

DES, NP, MXC

SHM73
Unknown

SHM76-2
Alphal-microglobulin/bikunin
1.00E−11
d-reg E2; EE2,

precursor (AMBP) protein

DES, NP, MXC

Estrogen receptor alpha

up-reg E2; EE2,

DES, NP, MXC, ES

[0116]

4

!SHEEPSHEAD? ? !LARGEMOUTH? MINNOW? ? !BASS GENES? Sequence ID? ? GENES? Sequence ID? ? !LMB#? Gene ID? Number? LMB#? Gene ID? Number

LMB_COMP FACTOR
Putative
1
SHM IK 7A
Liver
151

Bf/C2
complement factor

Bf/C2

LMB_ABMP
ABMP precursor
2
SHM IK 24E
Liver
152

LMB_GLUT-PEROX III
Glutathione
3
SHM IK 25C
Liver
153

peroxidase III

LMB_Smp D1
Small
4
SHM IK 5D
Liver
154

ribonucleoprotein D1

polypeptide (16kD)

LMB_RIBO L6
Ribosomal protein
5
SHM IKIGF-1
Liver
155

L6

LMB_MYOSIN LIGHT
myosin regulatory
6
SHM IKIGF-2
Liver
156

light chain

LMB_ZPC1
ZPC1
7
ndSHM-FT1-A03
Liver
157

LMB_CYTO-C OX 1
Cytochrome c
8
ndSHM-FT1-A09
Liver
158

oxidase subunit I

LMB_LECTIN STL2
Rhamnose binding
9
ndSHM-FT1-A10
Liver
159

lectin STL2

LMB_EMAP2
Echinoderm
10
ndSHM-FT1-A11
Liver
160

microtubule

associated protein

like 2

LMB_ALDOLASE-B
Aldolase b
11
ndSHM-FT1-A12
Liver
161

LMB_RIBO L7A
60S ribosomal
12
ndSHM-FT1-B03
Liver
162

protein L7A

LMB_PROTHROMBIN
Prothrombin
13
ndSHM-FT1-B07
Liver
163

precursor

LMB_SSAT
SSAT
14
ndSHM-FT1-B10
Liver
164

LMB_COMPLEMENT-
Complement C3
15
ndSHM-FT1-C01
Liver
165

C3
precursor

LMB_RIBO L7
Ribosomal protein
16
ndSHM-FT1-C03
Liver
166

L7

LMB_H-ATPASE-
H+-ATPase subunit,
17
ndSHM-FT1-C04
Liver
167

SUBUNIT
oligaomycin

sensitivity conferring

protein

LMB_RIBO L23A
Ribosomal protein
18
ndSHM-FT1-C09
Liver
168

L23a

LMB_ALPHA-TUBULIN
alpha tubulin
19
ndSHM-FT1-D06
Liver
169

LMB_RIBO-Sa
40S ribosomal
20
ndSHM-FT1-D10
Liver
170

protein Sa

LMB_VTG
Vitellogenin prcursor
21
ndSHM-FT1-D12
Liver
171

LMB_NASCENT-
Nascent polypeptide-
22
ndSHM-FT1-E01
Liver
172

POLYPEP
associated complex,

alpha polypeptide

LMB_ApoH
Apoliporotein H
23
ndSHM-FT1-E02
Liver
173

LMB_TBT-BP
TBT-binding protein
24
ndSHM-FT1-E08
Liver
174

LMB_SOL-CAR-25A#5
solute carrier family
25
ndSHM-FT1-E09
Liver
175

25 alpha member 5

LMB_UNNAMED-
Unnamed protein
26
ndSHM-FT1-E12
Liver
176

PROTEIN
product

LMB_FIB-B-SUBUNIT
Fibrinogen B subunit
27
ndSHM-FT1-F01
Liver
177

LMB_CIS-RETIN
cis-retinol
28
ndSHM-FT1-F06
Liver
178

DEHYDRO
dehydrogenase

LMB_SENES-ASSOC
Putative senscence-
29
ndSHM-FT1-F09
Liver
179

PROTEIN
associated protein

LMB_LDL RC
LDL receptor
30
ndSHM-FT1-F10
Liver
180

LMB_ABC-TRANS
ABC transporter
31
ndSHM-FT1-F11
Liver
181

LMB_CATHEPSIN B
Cathepsin B
32
ndSHM-FT1-F12
Liver
182

LMB_SERPIN-CP9
Serpin CP9
33
ndSHM-FT1-G02
Liver
183

LMB_TFIIIA
Transcription factor
34
ndSHM-FT1-G04
Liver
184

IIIA (TFIIIA)

LMB_ANTITHROMBIN
Antithrombin III
35
ndSHM-FT1-G08
Liver
185

III

LMB_RIKEN
RIKEN cDNA
36
ndSHM-FT1-H02
Liver
186

1810056020
1810056020

LMB_WEE-I
Wee I tyrosine
37
ndSHM-FT1-H03
Liver
187

kinase

LMB_HAPTOGLOBIN
Haptoglobin
38
ndSHM-FT1-H04
Liver
188

LMB_APOA-I
APOPLIPOPROTEIN
39
ndSHM-FT1-H06
Liver
189

A-I

LMB_ALPHA-1
alpha -1 antitrypsin
40
ndSHM-FT1-H07
Liver
190

ANTITRYPSIN
homolog precursor

LMB_APOE
Apolipoprotein E
41
ndSHM-FT1-H10
Liver
191

LMB_ZPC4
ZPC4
42
ndSHM-FT1-H11
Liver
192

LMB_LECTIN 9
C-type lectin
43
ndSHM-FT1-H12
Liver
193

superfamily 9

LMB_ATPASE 6
ATPase subunit 6
44
ndSHM-MC1-A02
Liver
194

LMB_ITI
inter-alpha-trypsin
45
ndSHM-MC1-A03
Liver
195

inhibitor “ITI”

LMB_EIF-3#7
Eukaryotic
46
ndSHM-MC1-A04
Liver
196

translation initiation

factor 3 subunit 7

LMB_HEPCIDIN
Hepcidin precursor
47
ndSHM-MC1-A05
Liver
197

LMB_PTTP
Pituitary tumor
48
ndSHM-MC1-A07
Liver
198

transforming protein

LMB_TOXIN-1
Toxin-1
49
ndSHM-MC1-A09
Liver
199

LMB_COAG FACTOR
Coagulation factor
50
ndSHM-MC1-A11
Liver
200

VII
VII

LMB_CDC42-2
cdc 42 isoform 2
51
ndSHM-MC1-B01
Liver
201

LMB_WARM-WATER
Warm water
52
ndSHM-MC1-B03
Liver
202

ACC PROTEIN
acclimation-related

protein

LMB_CYTO-C OX II
Cytochrome c
53
ndSHM-MC1-B04
Liver
203

oxidase subunit II

LMB_L10A
60S ribosomal
54
ndSHM-MC1-B05
Liver
204

protein L10A

LMB_KALLIKREIN
Kallikrein
55
ndSHM-MC1-B08
Liver
205

LMB_DANIO EST
Danio EST
56
ndSHM-MC1-B10
Liver
206

3818635
IMAGE: 3818635

LMB_ALPHA-2-
alpha-2-
57
ndSHM-MC1-B11
Liver
207

MACROGLOB-1
macroglobulin-1

LMB_HAPTOGLOB
Haptoglobin-related
58
ndSHM-MC1-C02
Liver
208

RELATED PROT
protein

LMB_FILAMEN-B
Filamen B
59
ndSHM-MC1-C04
Liver
209

LMB_UBIQUITIN
ubiquitin
60
ndSHM-MC1-C05
Liver
210

LMB_RXR INTERACT
Retinoid X receptor
61
ndSHM-MC1-C08
Liver
211

PROT
interacting protein

LMB_MITOCHON-ATP-
ATP synthase alpha
62
ndSHM-MC1-C10
Liver
212

SYNTHASE
chain mitochondrial

precursor

LMB_TATA BOX BP
TATA-box binding
63
ndSHM-MC1-D01
Liver
213

protein

LMB_DIFF-REG
Diiferentially
64
ndSHM-MC1-D02
Liver
214

TROUT PROT-1
regulated trout

protein 1

LMB_LIVER-REGEN-
liver regeneration
65
ndSHM-MC1-D03
Liver
215

REL PROT
related protein

LMB_SERPIN-2B
Serpin 2b
66
ndSHM-MC1-D04
Liver
216

LMB_APO-A1
Apolipoprotein A-I-1
67
ndSHM-MC1-D06
Liver
217

precursor

LMB_M-PHASE PROT
M-phase
68
ndSHM-MC1-D10
Liver
218

6
phosphoprotein 6

LMB_PROSTAGLAND-
Prostaglandin D
69
ndSHM-MC1-E01
Liver
219

D-SYNTHASE
synthase-like protein

(lipocalin type)

LMB_LYRIC
LYRIC
70
ndSHM-MC1-E05
Liver
220

LMB_CYSTATIN-PREC
Cystatin precursor
71
ndSHM-MC1-E06
Liver
221

LMB_RIKEN 2700038
RIKEN cDNA
72
ndSHM-MC1-E08
Liver
223

2700038

LMB_DIAZEPAM-
Membrane
73
ndSHM-MC1-E10
Liver
224

BINDING INHIB
associated

diazepam-binding

inhibitor

LMB_IGF-I
IGF-I
74
ndSHM-MC1-E12
Liver
225

LMB_ESTP4_D11
ESTP4_D11
75
ndSHM-MC1-F01
Liver
226

LMB_ESTDHT_6
ESTDHT_6
76
ndSHM-MC1-F02
Liver
227

LMB_ESTDHT_7
ESTDHT_7
77
ndSHM-MC1-F03
Liver
228

LMB_ESTDHT_13
ESTDHT_13
78
ndSHM-MC1-F06
Liver
229

LMB_ESTDHT_50
ESTDHT_50
79
ndSHM-MC1-F11
Liver
230

LMB_ESTDHT_51
ESTDHT_51
80
ndSHM-MC1-F12
Liver
231

LMB_ESTDHT_53
ESTDHT_53
81
ndSHM-MC1-G01
Liver
232

LMB_ESTDHT_60
ESTDHT_60
82
ndSHM-MC1-G02
Liver
233

LMB_ESTDHT_62
ESTDHT_62
83
ndSHM-MC1-G03
Liver
234

LMB_ESTDHT_68
ESTDHT_68
84
ndSHM-MC1-G04
Liver
235

LMB_ESTDHT_69
ESTDHT_69
85
ndSHM-MC1-G05
Liver
236

LMB_ESTP4_A02
ESTP4_A02
86
ndSHM-MC1-G07
Liver
237

LMB_ESTP4_B03
ESTP4_B03
87
ndSHM-MC1-G11
Liver
238

LMB_ESTP4_B04
ESTP4_B04
88
ndSHM-MC1-G12
Liver
239

LMB_ESTP4_B07
ESTP4_B07
89
ndSHM-MC1-H02
Liver
240

LMB_ESTP4_B08
ESTP4_B08
90
ndSHM-MC1-H03
Liver
241

LMB_ESTP4_B09
ESTP4_B09
91
ndSHM-MC1-H04
Liver
242

LMB_ESTP4_C03
ESTP4_C03
92
ndSHM-MC1-H06
Liver
243

LMB_ESTP4_C04
ESTP4_C04
93
ndSHM-MC1-H08
Liver
244

LMB_ESTP4_C06
ESTP4_C06
94
ndSHM-MC1-H09
Liver
245

LMB_ESTP4_D04
ESTP4_D04
95
ndSHM-MC1-H10
Liver
246

LMB_ESTP4_D08
ESTP4_D08
96
ndSHM-MC1-H12
Liver
247

LMB_ESTP4_D10
ESTP4_D10
97
ndSHM-MT1-A02
Liver
248

LMB_ESTP4_E01
ESTP4_E01
98
ndSHM-MT1-A03
Liver
248

LMB_ESTP4_E03
ESTP4_E03
99
ndSHM-MT1-A05
Liver
249

LMB_ESTP4_E06
ESTP4_E06
100
ndSHM-MT1-B09
Liver
250

LMB_ESTP4_E08
ESTP4_E08
101
ndSHM-MT1-C05
Liver
251

LMB_ESTP4_E12
ESTP4_E12
102
ndSHM-MT1-C08
Liver
252

LMB_ESTP4_F06
ESTP4_F06
103
ndSHM-MT1-D04
Liver
253

LMB_ESTP4_G06
ESTP4_G06
104
ndSHM-MT1-D05
Liver
254

LMB_ESTP4_G11
ESTP4_G11
105
ndSHM-MT1-D07
Liver
255

LMB_ESTP4_H02
ESTP4_H02
106
ndSHM-MT1-E02
Liver
256

LMB_ESTP4_H04
ESTP4_H04
107
ndSHM-MT1-E03
Liver
257

LMB_ESTP4_H05
ESTP4_H05
108
ndSHM-MT1-E05
Liver
258

LMB_ESTP4_H07
ESTP4_H07
109
ndSHM-MT1-F11
Liver
259

LMB_ESTP4_H08
ESTP4_H08
110
ndSHM-MT1-G03
Liver
260

LMB_EST-
EST-
111
ndSHM-MT1-H05
Liver
261

SEASONAL_02
SEASONAL_02

LMB_EST-
EST-
112
ndSHM-MXCc1-A04
Liver
262

SEASONAL_03
SEASONAL_03

LMB_EST-
EST-
113
ndSHM-MXCc1-A09
Liver
263

SEASONAL_04
SEASONAL_04

LMB_EST-
EST-
114
ndSHM-MXCc1-A10
Liver
264

SEASONAL_06
SEASONAL_06

LMB_EST-
EST-
115
ndSHM-MXCc1-A11
Liver
265

SEASONAL_09
SEASONAL_09

LMB_EST-
EST-
116
ndSHM-MXCc1-B02
Liver
266

SEASONAL_11
SEASONAL_11

LMB_EST-
EST-
117
ndSHM-MXCc1-B03
Liver
267

SEASONAL_12
SEASONAL_12

LMB_EST-SEASONAL-
EST-SEASONAL-14
118
ndSHM-MXCc1-B04
Liver
268

14

LMB_EST-
EST-
119
ndSHM-MXCc1-B06
Liver
269

SEASONAL_16
SEASONAL_16

LMB_EST-
EST-
120
ndSHM-MXCc1-B07
Liver
270

SEASONAL_17
SEASONAL_17

LMB_EST-
EST-
121
ndSHM-MXCc1-B08
Liver
271

SEASONAL_22
SEASONAL_22

LMB_EST-
EST-
122
ndSHM-MXCc1-C04
Liver
272

SEASONAL_51
SEASONAL_51

LMB_EST-
EST-
123
ndSHM-MXCc1-C06
Liver
273

SEASONAL_52
SEASONAL_52

LMB_EST-
EST-
124
ndSHM-MXCc1-C11
Liver
274

SEASONAL_54
SEASONAL_54

LMB_EST-
EST-
125
ndSHM-MXCc1-D03
Liver
275

SEASONAL_55
SEASONAL_55

LMB_EST-
EST-
126
ndSHM-MXCc1-D04
Liver
276

SEASONAL_56
SEASONAL_56

LMB_EST--
EST-
127
ndSHM-MXCc1-D05
Liver
277

SEASONAL_58
SEASONAL_58

LMB_EST--
EST-
128
ndSHM-MXCc1-D08
Liver
278

SEASONAL_59
SEASONAL_59

LMB_EST--
EST-
129
ndSHM-MXCc1-D10
Liver
279

SEASONAL_61
SEASONAL_61

LMB_EST--
EST-
130
ndSHM-MXCc1-D12
Liver
280

SEASONAL_62
SEASONAL_62

LMB_EST--
EST-
131
ndSHM-MXCc1-E01
Liver
281

SEASONAL_64
SEASONAL_64

LMB_EST--
EST-
132
ndSHM-MXCc1-E09
Liver
282

SEASONAL_68
SEASONAL_68

LMB_EST--
EST-
133
ndSHM-MXCc1-E11
Liver
283

SEASONAL_70
SEASONAL_70

LMB_EST--
EST-
134
ndSHM-MXCc1-F01
Liver
284

SEASONAL_71
SEASONAL_71

LMB_EST--
EST-
135
ndSHM-MXCc1-F03
Liver
285

SEASONAL_72
SEASONAL_72

LMB_EST--
EST-
136
ndSHM-MXCc1-F07
Liver
286

SEASONAL_75
SEASONAL_75

LMB_EST--
EST-
137
ndSHM-MXCc1-F10
Liver
287

SEASONAL_77
SEASONAL_77

LMB_EST--
EST-
138
ndSHM-MXCc1-G02
Liver
288

SEASONAL_85
SEASONAL_85

LMB_EST--
EST-
139
ndSHM-MXCc1-G03
Liver
289

SEASONAL_88
SEASONAL_88

LMB_EST--
EST-
140
ndSHM-MXCc1-G04
Liver
290

SEASONAL_90
SEASONAL_90

LMB_EST--
EST-
141
ndSHM-MXCc1-G12
Liver
291

SEASONAL_92
SEASONAL_92

LMB_EST--
EST-
142
ndSHM-MXCc1-H05
Liver
292

SEASONAL_97
SEASONAL_97

LMB_EST--
EST-
143
ndSHM-MXCc1-H09
Liver
293

SEASONAL_F11
SEASONAL_F11

LMB_EST--
EST-
144
ndSHM-MXCt1-B05
Liver
294

SEASONAL_F17
SEASONAL_F17

LMB_EST--
EST-
145
ndSHM-MXCt1-B08
Liver
295

SEASONAL_F21
SEASONAL_F21

LMB_ER-ALPHA
ESTROGEN
146
ndSHM-MXCt1-C02
Liver
296

RECEPTOR ALPHA

LMB_ER-BETA
ESTROGEN
147
ndSHM-MXCt1-C11
Liver
297

RECEPTOR BETA

LMB_ER-GAMMA
ESTROGEN
148
ndSHM-MXCt1-D09
Liver
298

RECEPTOR

GAMMA

LMB_STAR
STAR PROTEIN
149
ndSHM-MXCt1-E04
Liver
299

LMB_SF1
SF1 PROTEIN
150
ndSHM-MXCt1-E06
Liver
300

FRAGMENT

LMB1-3

420
ndSHM-MXCt1-E09
Liver
301

LMB2-2

421
ndSHM-MXCt1-F11
Liver
302

LMB3-1

422
ndSHM-MXCt1-E12
Liver
303

LMB4-1

423
ndSHM-MXCt1-G03
Liver
304

LMB5

424
ndSHM-MXCt1-H03
Liver
305

LMB6-1

425
ndSHM-NPc1-A12
Liver
306

LMB7-1

426
ndSHM-NPc1-B01
Liver
307

LMB8-2

427
ndSHM-NPc1-B08
Liver
308

LMB9-1

428
ndSHM-NPc1-B09
Liver
309

LMB10-1

429
ndSHM-NPc1-C04
Liver
310

LMB11-2

430
ndSHM-NPc1-C06
Liver
311

LMB12-1

431
ndSHM-NPc1-C11
Liver
312

LMB13-2

432
ndSHM-NPc1-E01
Liver
313

LMB14-1

433
ndSHM-NPc1-E06
Liver
314

LMB15-1

434
ndSHM-NPc1-E11
Liver
315

LMB16-2

435
ndSHM-NPc1-F01
Liver
316

LMB17-2

436
ndSHM-NPc1-F05
Liver
316

LMB18-3

437
ndSHM-NPc1-F06
Liver
318

LMB19-1

438
ndSHM-NPc1-F07
Liver
319

LMB20-1

439
ndSHM-NPc1-F08
Liver
320

LMB21-1

440
ndSHM-NPc1-G02
Liver
321

LMB22-1

441
ndSHM-NPc1-G08
Liver
322

LMB23-1

442
ndSHM-NPc1-G11
Liver
323

LMB24-1

443
ndSHM-NPc1-H01
Liver
324

LMB25-1

444
ndSHM-NPc1-H02
Liver
325

LMB26-1

445
ndSHM-NPc1-H03
Liver
326

LMB27-1

446
ndSHM-NPc1-H04
Liver
327

LMB28-2

447
ndSHM-NPc1-H05
Liver
328

LMB29-2

448
ndSHM-NPc1-H08
Liver
329

LMB30-1/Forward

449
ndSHM-NPt1-A01
Liver
330

LMB30-1/Reverse

450

LMB31

451
ndSHM-NPt1-A02
Liver
331

LMB32-1

452
ndSHM-NPt1-A03
Liver
332

LMB33-1/A

453
ndSHM-NPt1-A04
Liver
333

LMB33-1/B

454

LMB34-1

455
ndSHM-NPt1-A05
Liver
334

LMB35-1

456
ndSHM-NPt1-A08
Liver
335

LMB36-1

457
ndSHM-NPt1-A09
Liver
336

LMB37-1/A

458
ndSHM-NPt1-B02
Liver
337

LMB37-1/B

459

LMB38-1

460
ndSHM-NPt1-B03
Liver
338

LMB39-1

461
ndSHM-NPt1-B05
Liver
339

LMB40-1

462
ndSHM-NPt1-B06
Liver
340

LMB41-1

463
ndSHM-NPt1-B07
Liver
341

LMB42-1

464
ndSHM-NPt1-B11
Liver
342

LMB43-1

465
ndSHM-NPt1-B12
Liver
343

LMB44-1

466
ndSHM-NPt1-C01
Liver
344

LMB45-1/Forward

467
ndSHM-NPt1-C03
Liver
345

LMB45-1/Reverse

468

LMB46-1

469
ndSHM-NPt1-C05
Liver
346

LMB47-2

470
ndSHM-NPt1-C06
Liver
347

LMB48-1

471
ndSHM-NPt1-C09
Liver
348

LMB49-1

472
ndSHM-NPt1-C12
Liver
349

LMB50-1

473
ndSHM-NPt1-D04
Liver
350

LMB51-1

474
ndSHM-NPt1-D05
Liver
351

LMB52-1

475
ndSHM-NPt1-D07
Liver
352

LMB53-1

476
ndSHM-NPt1-D07
Liver
353

LMB54-2

477
ndSHM-NPt1-D09
Liver
354

LMB55-1

478
ndSHM-NPt1-D11
Liver
355

LMB56-1

479
ndSHM-NPt1-E02
Liver
356

LMB57

480
ndSHM-NPt1-E03
Liver
357

LMB58-1

481
ndSHM-NPt1-E06
Liver
358

LMB59-1

482
ndSHM-NPt1-E07
Liver
359

LMB60-1

483
ndSHM-NPt1-E12
Liver
360

LMB61-1

484
ndSHM-NPt1-F01
Liver
361

LMB63-1

485
ndSHM-NPt1-F05
Liver
362

LMB64-1

486
ndSHM-NPt1-F07
Liver
363

LMB65-1

487
ndSHM-NPt1-F11
Liver
364

LMB66-1

488
ndSHM-NPt1-G03
Liver
365

LMB67-1

489
ndSHM-NPt1-G07
Liver
366

LMB68-1

490
ndSHM-NPt1-G08
Liver
367

LMB69-2

491
ndSHM-NPt1-G11
Liver
368

LMB70-2

492
ndSHM-NPt1-G12
Liver
369

LMB71-1

493
ndSHM-NPt1-H03
Liver
370

LMB72-1

494
ndSHM-NPt1-H04
Liver
371

LMB73-1

495
ndSHM-NPt1-H11
Liver
372

LMB74-3

496
SHM-D03
Liver
373

LMB75-1

497
SHM-D02
Liver
374

LMB76-2

498
SHM-B02
Liver
375

LMB77-1

499
SHM-B07
Liver
376

LMB78-1

500
SHM-B06
Liver
377

LMB79-2

501
SHM-B12
Liver
378

LMB80-1

502
SHM-C03
Liver
379

LMB81-1

503
SHM-C07
Liver
380

LMB82-1

504
SHM-E04
Liver
381

LMB83

505
SHM-D06
Liver
382

LMB84-1

506
SHM-E01
Liver
383

LMB85-1

507
SHM-E07
Liver
384

LMB86-1

508
SHM-A06
Liver
385

LMB87

509
SHM-E02
Liver
386

LMB88-1

510
SHM-C05
Liver
387

LMB89-1

511
SHM-B10
Liver
388

LMB90-1

512
SHM-D12
Liver
389

LMB91-1

513
SHM-C01
Liver
390

LMB92-1

514
SHM1

391

LMB93-1

515
SHM2-1

392

LMB94-1

516
SHM3

393

LMB95-1

517

LMB96-1

518
SHM26

394

LMB97

519
SHM6

395

LMB98-1

520
SHM7-3

396

LMB99-1

521
SHM29

397

LMB100-1

522
SHM9-1

398

LMB101-1

523
SHM74-1

399

LMB102-1

524
SHM11

400

LMB103-1

525
SHM13-1

401

LMB104-3

526
SHM14

402

LMB105-1

527
SHM-18

403

LMB107-1

528
SHM22

404

LMB108-1

529
SHM23-1

405

LMB109-1

530
SHM24

406

LMB110-1

531
SHM25

407

LMB112-1

532
SHM39

408

LMB116-1

533
SHM41

409

LMB118-1

534
SHN42-1

410

LMB120-1

535
SHM43

411

LMB121-1

536
SHM48

412

LMB122-1

537
SHM48-2

413

LMB123-1

538
SHM51-3

414

LMB128-1

539
SHM56-2

415

LMB129-1

540
SHM62-2

416

LMB130-1

541
SHM72-3

417

LMB132-1

542
SHM73

418

LMB133-1

543
SHM76-2

419

LMB134-3

544

LMB135

545

LMB136-1

546

LMB137-2

546

LMB138-1

547

LMB139-1

549

LMB140-1

550

LMB141

551

LMB142-1

552

LMB144-1

553

LMB150

554

LMB151

555

LMB_ESTP4-E01

556

LMB_ESTDHT64

557

LMB_LIV-REGER-PROT

558

LMB_RIKEN 1110001M01

559

LMB_EST-SEASONALf17

560

Other Embodiments

[0117] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Detecting hormonally active compounds

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Provisional Applications (1)