Abstract:
Described herein are DNA primer sequences designed for the determination of gene or transcript information from Anuran species, and which may be used in studies for developmental and/or toxicity testing and for environmental toxicology or ecological assessment. Also described herein is a rapid, sensitive, high-throughput assay useful for supporting potential risk assessment across vertebrate clades, and that is also useful for evaluation of complex contaminant mixtures.

Description:
FIELD OF THE INVENTION 
       [0001]    The invention relates to biological assays and the study of animal development, environmental toxicology and ecological assessments. 
       SUMMARY OF THE INVENTION 
       [0002]    The invention describes the DNA primer sequences designed for the determination of gene or transcript information from Anuran species. There are thousands of Anuran species known worldwide and obtaining genetic information from each species for the development of quantitative real time polymerase chain reaction (qPCR) assays is not feasible. Our invention is comprised of one or more specially-designed primer pairs that can be applied to diverse Anuran species spanning over hundreds of million years in evolution, including those species commonly used in laboratory studies for development and/or toxicity testing and sentinel species used for environmental toxicology or ecological assessment. Described herein are optimized conditions for use of these primers in examples of Anuran tissues. Another aspect of the invention is a rapid, sensitive, high-throughput assay useful for supporting potential risk assessment across vertebrate clades, and that is also useful for evaluation of complex contaminant mixtures. 
       BACKGROUND 
       [0003]    Amphibians are used extensively as scientific model organisms, in health and environmental research, as test organisms, and have an undeniable role as sentinel species, as a food source, and in insect control. In contrast to mammals with extensive genomic resources, a particular challenge exists for evaluating gene expression endpoints in amphibian species where clades exhibit evolutionary divergence of over 300 million years (AmphibiaWeb 2012). Of the amphibians, the Anura-representing frogs and toads are the most numerous on the planet representing over 6,000 of the 7,000 known species (AmphibiaWeb 2012). Despite this impressive number, only two closely-related Pipid species,  Xenopus laevis  and  Xenopus tropicalis , have sufficient genomic resources for gene expression studies. Yet many other species including ones that have diverged over 200+ million years ago, serve as important species, regionally and globally. There is a lack of the most minimal genomic information leading to a significant investment in time and resources to even clone a portion of a single gene in order to develop validated gene expression tools for a species of interest. To circumvent this difficulty, we have developed a suite of qPCR-ready primer sets that identify particular genes and/or their transcripts. Each primer pair has been validated to function under stringent criteria in species as diverse as Pipids and Ranids. The use of these primer sets provides a simple, low-cost solution to the issue of cross-species comparison of responses and sensitivities. 
         [0004]    Endocrine disruptors (EDCs) are chemicals, either environmental or man-made, that disturb the endocrine signaling pathways of humans and wildlife. EDC exposure often results in cancer, fertility problems, and other diseases (Vandenberg, Colborn et al. 2012). Therefore, the risk from these exposure effects has led to a great need for sensitive and appropriate methods for indicating deleterious EDC effects. For this reason, there is considerable interest in developing novel diagnostic assays to detect EDCs in the environment as well as in products meant for human consumption or that are used to package food products such as plastic food containers. Over 80,000 chemicals are registered for use in the US (NIEHS 2013) and there is increasing concern regarding their impact since it is now established that EDCs do not follow the classical rule of “the dose makes the poison” (Vandenberg, Colborn et al. 2012). Rather non-monotonic responses and low-dose effects are actually common in studies of natural hormones and EDCs and clear linkages between environmental exposures to EDCs and human diseases/disabilities are becoming evident (Vandenberg, Colborn et al. 2012). EDCs are generally found in low concentrations in the environment, but even minute quantities can have demonstrable impact as hormone disruptors. In fact biological activity can be detected below current analytical detection limits and measurement of EDCs in the context of complex mixtures such as municipal wastewater effluent is not necessarily a good predictor of biological activity (Quanrud and Propper 2010). Given that many aquatic ecosystems contain significant concentrations of environmental contaminants (Kolpin, Furlong et al. 2002), and that many of these compounds and/or their metabolites have been detected in human plasma (National Report on Human Exposure to Environmental Chemicals, Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, http://www.cdc.gov/exposurereport), we have developed a suite of tools for indicating deleterious EDC effects. Such screens can be used as a first level evaluation of exposure health risk for both wildlife species and humans. Most of what we know about EDCs pertains to substances that disrupt estrogen signaling pathways and technologies to detect estrogenic EDCs have been developed (Van Aggelen, Ankley et al. 2010; Hecker and Hollert 2011). 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0005]      FIG. 1  is a cladogram depicting certain major families of the order Anura. 
           [0006]    FIGS.  2 A(1) through 2A(10); 2B(1) through 2B(10) and 2C(1)-2C(10) illustrate the results of the three-tiered quality assurance/control procedure demonstrating activity of the primer sets according to an embodiment of the invention. 
           [0007]      FIG. 3  illustrates the results of a demonstration of thyroid hormone responsiveness of the target gene transcripts in  Rana catesbeiana  according to an embodiment of the invention. 
           [0008]      FIG. 4  is another illustration of the results of a demonstration of thyroid hormone responsiveness of the target gene transcripts in Rana catesbeiana according to an embodiment of the invention. 
           [0009]      FIG. 5  is an illustration of the results of a demonstration of thyroid hormone responsiveness of the target gene transcripts in  Xenopus laevis  according to an embodiment of the invention. 
           [0010]      FIG. 6  illustrates results of tests demonstrating that primer sets according to an embodiment of the invention are usable in a wide range of Anuran species. 
           [0011]      FIG. 7  are illustrations of representative examples of primer set performance on genomic DNA across different Anuran species. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0012]    The thyroid hormones (THs) are crucial for normal growth, development, and metabolism in all vertebrates including humans (Morreale de Escobar, Obregon et al. 2004; Zoeller and Rovett 2004). In fetal and neonatal mammals, environmental contaminants such as perchlorate and polychlorinated biphenyls (PCBs) impact TH action contributing to serious defects in development and organ function including cognitive and motor deficits, abnormal bone growth, cardiac problems, and altered lipid metabolism (Zoeller and Rovett 2004; Bernal 2005; Cheng 2005; Siesser, Cheng et al. 2005; Liu and Brent 2010). The brain is particularly vulnerable (Porterfield 1994; Brucker-Davis 1998; Jones, Thoemke et al. 2005; Zoeller 2005; Zoeller and Crofton 2005) and the impact of mild hypothyroidism during critical developmental phases can have significant societal costs (Miller, Crofton et al. 2009). In amphibians, THs are required for tadpole metamorphosis into a juvenile frog and disruption of TH action results in the inability of the frog to develop normally (Shi 2000). Therefore, modification of TH action at early life history stages can lead to dramatic, deleterious outcomes for both humans and wildlife species. The mechanisms of TH action are common among vertebrates and involve a complex regulatory pathway yet the high degree of sensitivity to TH disruption of the tadpole has been identified as a powerful surrogate for the identification of chemicals and/or contaminants that perturb TH signaling in vertebrates (OECD 2009; Helbing 2012). 
         [0013]    The invention presents an innovative method that relies upon a gene expression readout for detecting TH disrupting activities using Anuran species as model organisms, thereby greatly improving the power of the frog tadpole metamorphosis assay in predictivity and applicability to both laboratory testing and field applications across a range of all frog sentinel species. Moreover, the invention describes validated DNA primer sets for genes that are essential as references or “normalizers.” These normalizers can be used alone or in conjunction with primers used to ascertain TH responsiveness increasing the breadth of utility and application. Furthermore, this set of normalizer genes is not just specific for thyroid function, but will also serve as a strong suite of reference genes for any type of qPCR application utilizing any response genes evaluated for any study using Anuran species. 
         [0014]    The invention comprises one or more DNA primer pairs (Table 1) that have been designed against “normalizer” and/or responsive genes such that the same primer pair representing a given gene can reliably and quantitatively function in Anura regardless of species. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Sequence, qPCR running conditions and charac- 
               
               
                 teristics of the Anuran DNA primer sets 
               
             
          
           
               
                   
                   
                 Gene 
                   
                   
                 Annealing 
                 Thermo- 
                   
               
               
                 999 
                 Gene 
                 Abbre- 
                 Primer 
                 Primer 
                 Tem- 
                 cycle 
                 Ampli- 
               
               
                 Gene 
                 Targets 
                 viation 
                 UP 
                 DOWN 
                 perature 
                 Profile 
                 con 
               
               
                   
               
             
          
           
               
                 TAX 
                 alpha 2 
                 col1a2 
                 TAX1 
                 CTGGTGGTGGA 
                 TAX1 
                 GAGTCTTAAGTC 
                 60 
                 15s, 30s, 
                 182 
               
               
                 1 
                 type 1 
                   
                 up 
                 TATGATGGT 
                 dn 
                 ACGGCAAG 
                   
                 45s 
                   
               
               
                   
                 collagen 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 TAX 
                 cytoplasmic 
                 actb 
                 TAX5 
                 TACAGCTTCACC 
                 TAX5 
                 TCCACATCTGCT 
                 60/64* 
                 15s, 30s, 
                 478 
               
               
                 5 
                 beta- 
                   
                 up 
                 ACCACAG 
                 dn 
                 GGAAGGT 
                   
                 45s 
                   
               
               
                   
                 actin 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 TAX 
                 ribosomal 
                 rps10 
                 TAX7 
                 
                   TTTGCYTGGCGK 
                 
                 TAX7 
                 ARCRGCACTGCG 
                 60 
                 15s, 30s, 
                 213 
               
               
                 7 
                 protein 
                   
                 up 
                 
                   CACTTTT 
                 
                 dn 
                 YCTGTA 
                   
                 45s 
                   
               
               
                   
                 S10 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 TAX 
                 myelin 
                 plp1 
                 TAX8a 
                 TGGCTGARGGATT 
                 TAX8 
                 ACAGCAGAGCAG 
                 60 
                 15s, 30s, 
                 256 
               
               
                 8 
                 proteolipid 
                   
                 up 
                 YTAYACC 
                 dn 
                 GCAAMGA 
                   
                 45s 
                   
               
               
                   
                 protein A 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 TAX 
                 thyroid 
                 thra 
                 TAX9 
                 TGATAAGGCCACA 
                 TAX9 
                 CGGGTGATCT 
                 60 
                 15s, 30s, 
                 141 
               
               
                 9 
                 hormone 
                   
                 up 
                 GGRTACCACTA 
                 dn 
                 TGTCGATRA 
                   
                 45s 
                   
               
               
                   
                 receptor 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 alpha 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 TAX 
                 thryoid 
                 thrb 
                 TAX10 
                 CTATAGAAGAAAA 
                 TAX10 
                 GAAGGCTTCTAA 
                 60 
                 15s, 30s, 
                 237 
               
               
                 10 
                 hormone 
                   
                 up 
                 CAGAGAAAARAGA 
                 dn 
                 GTCCACTTTTCC 
                   
                 45s 
                   
               
               
                   
                 receptor 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 beta 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 TAX 
                 deiodinase 
                 dio2 
                 TAX12a 
                 CCTGGCTCTSTAY 
                 TAX12d 
                 RGCTGATCCRA 
                 62 
                 15s, 30s, 
                 295 
               
               
                 12 
                 type 
                   
                 up 
                 GACTC 
                 dn 
                 ARTTGAC 
                   
                 45s 
                   
               
               
                   
                 II 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 TAX 
                 TH-induced 
                 thibz 
                 TAX15a 
                 ASCTCCRCAGAA 
                 TAX15a 
                 TCACGTACCAG 
                 62 
                 15s, 30s, 
                 354 
               
               
                 15 
                 basic region 
                   
                 up 
                 YCAGCA 
                 dn 
                 GCCAAAA 
                   
                 30s 
                   
               
               
                   
                 leucine  
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 zipper 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 containing 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 TAX 
                 Kruppel- 
                 klf9 
                 TAX16a 
                 CYGCTCAGTGT 
                 TAX16a 
                 ARGGGCCGGTA 
                 62 
                 15s, 30s, 
                 250 
               
               
                 16 
                 like 
                   
                 up 
                 CTGGTGT 
                 dn 
                 CTTGTTT 
                   
                 30s 
                   
               
               
                   
                 factor 9 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 TAX 
                 fibronectin 
                 fn1 
                 TAX18 
                 GTTGCCATGARG 
                 TAX18 
                 CCATTGCCWGT 
                 60 
                 15s, 30s, 
                 373 
               
               
                 18 
                   
                   
                 up 
                 GWGGAC 
                 dn 
                 GCAGATA 
                   
                 45s 
                   
               
               
                   
               
               
                 TAX 
                 matrix 
                 mmp2 
                 TAX20a 
                 TACAACAGCTGC 
                 TAX20a 
                 TCCTTGTCATAGT 
                 62 
                 15s, 30s, 
                 248 
               
               
                 20 
                 metallo- 
                   
                 up 
                 ACTGAT 
                 dn 
                 CTTCTGT 
                   
                 30s 
                   
               
               
                   
                 peptidase 2 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 TAX 
                 ribosomal 
                 rpl8 
                 TAX22 
                 CAGGGGACAGA 
                 TAX22 
                 TGAGCTTTGTT 
                 60 
                 15s, 30s, 
                 270 
               
               
                 22 
                 protein L8 
                   
                 up 
                 GAAAAGGTG 
                 dn 
                 GCCACAG 
                   
                 45s 
                   
               
               
                   
               
               
                 TAX 
                 elongation 
                 eef1a1 
                 TAX23 
                 GCTGCTGGTGTT 
                 TAX23 
                 AGCATGTTGTC 
                 60 
                 15s, 30s, 
                 257 
               
               
                 23 
                 factor 
                   
                 up 
                 GGTGART 
                 dn 
                 ACCRTTCC 
                   
                 45s 
                   
               
               
                   
                 1 alpha 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 TAX 
                 translocase 
                 timm50 
                 TAX27 
                 GCTWCAYCCAGA 
                 TAX27 
                 GCTGYTCCTC 
                 60 
                 15s, 30s, 
                 497 
               
               
                 27 
                 of inner 
                   
                 up 
                 GTGGTCGTT 
                 dn 
                 CTGCTCCA 
                   
                 45s 
                   
               
               
                   
                 mitochondrial 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 membrane 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 50 homolog 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 TAX 
                 ornithine 
                 otc 
                 TAX28 
                 YATGACYGATG 
                 TAX28 
                 CATAWCCCTTT 
                 60 
                 15s, 30s, 
                 272 
               
               
                 28 
                 trans 
                   
                 up 
                 CTGTTCTAG 
                 dn 
                 GGTGTTGC 
                   
                 30s 
                   
               
               
                   
                 carbamylase 
                   
                   
                   
                   
                   
                   
                   
                   
               
             
          
           
               
                 * Xenopus laevis / Rana catesbeiana  annealing temperatures 
               
               
                   
               
             
          
         
       
     
         [0015]    Examples of the use of the invention are to indicate responsiveness to thyroid hormone action or indicators of developmental phases attained. We demonstrate that these primers function well across the evolutionary range of Anurans and also demonstrate the range of tissues in which the primers may be reliably used in the context of assessing mRNA (cDNA) levels or gene levels (genomic DNA). Finally, we also present a specific application of the technology to identifying perturbations in thyroid hormone signaling. 
         [0016]    A great diversity of Anuran species are used in a broad range of disciplines such as developmental and cancer biology, physiology, ecology, molecular biology, biochemistry, from egg to adult stages. Every qPCR-based study requires the use of reference gene transcripts or genes in order to be able to interpret experimental data. The basic assumption is that the reference gene(s) do not vary due to the treatment condition of the experiment and the type of reference or “normalizer” varies depending upon experimental context (Bustin, Beaulieu et al. 2010). Therefore, a selection of normalizer gene candidates is necessary as well as the identification and use of multiple normalizers to enhance the robustness of the data set. Furthermore, this set of normalizer genes is not just specific for thyroid function, but will also serve as a strong suite of reference genes for any type of qPCR application utilizing any response genes evaluated for any study using Anuran species. 
         [0017]    The inventors have identified TH-responsive gene candidates that are common between mammals and frogs that would serve to act as meaningful indicators of TH disruption within the context of an amphibian metamorphosis assay (AMA) (Searcy, Beckstrom-Sternberg et al. 2012). The suggested species for the AMA is  Xenopus laevis  with additional interest in using the related  Silurana  ( Xenopus )  tropicalis  (Mitsui, Fujii et al. 2006). However, several nations are also interested in using native species relevant to their environments such as  Rana pipiens  and  Pseudacris regilla  (Canada),  Rana temporaria  (Europe), and  Rana rugosa  (Japan),  Rhinella  ( Bufo )  marinus  (Latin America, Australia and SE Asia), or the only truly cosmopolitan frog,  Rana catesbeiana  (Bruno 2001; Veldhoen, Skirrow et al. 2006; Oka, Miyahara et al. 2009; Marlatt, Veldhoen et al. 2013). Toxicological evaluations on a variety of species is highly desirable to enable direct comparison between species for sensitivities and evaluation of the impact of different life histories and genome compositions (Relyea and Jones 2009; Helbing 2012). 
         [0018]    The designed primers were subjected to tests of qPCR specificity and suitability within each tissue of interest in BOTH  Xenopus laevis  and  Rana catesbeiana . These two species bracket the most commonly used frog families spanning ˜200 million years of evolution (Sumida, Kato et al. 2004) ( FIG. 1 ). The inventors determined that if the primer pairs work well for both species, then the likelihood that they work for intermediate species is assured. 
         [0019]      FIG. 1  is an anuran cladogram depicting major families of current interest in the scientific and conservation realm.  Xenopus laevis  is a member of the Pipidae whereas  Rana catesbeiana  is a member of the Ranidae. Adapted from AmphibiaWeb: Phylogenetic view of Amphibia [web application] 2012. UC Regents, Berkely, Calif., available: http:amphibiaweb.org (14 May 2012). 
         [0020]    The inventors&#39; rigorous design and verification protocol includes amplicon sequence confirmation, routine melting curve analysis, generation of standard curves for each primer set, and determination of amplification efficiency. All procedures are compliant with the Minimum Information for Publication of qPCR Experiments (MIQE précis) guidelines (Bustin, Beaulieu et al. 2010). qPCR technology presents the most accurate and sensitive genetic technique available that enables high-throughput detection of low abundance mRNA transcripts. Due to its relative robustness and low operating cost, this technology is currently the most conducive to the demands of the regulatory context and the assays developed have immediate translation to regulatory labs. We elected to focus on a SYBR-based detection method because this is the presently least expensive to run (good for the price point of the end user) and more labs have access to the equipment necessary for readout therefore increasing the market potential. 
         [0021]    Our primer quality assurance/control procedure has been standardized and follows a three-tiered quality control and quality assurance (QC/QA) process, as follows. 
         [0022]    Tier 1 QC/QA Total RNA from the target animal species and tissue type is converted to complementary DNA (cDNA) and queried with the putative qPCR primer pairs designed against genes of interest. The function of each primer pair in the qPCR assay is assessed for amplification signal representative of a single targeted cDNA product. Control qPCR reactions containing no cDNA are performed to evaluate background inter-and intra-primer interactions that can contribute to signal noise. Thermocycle conditions are chosen for each gene-specific primer pair that maximizes signal specificity while minimizing background noise. The DNA products generated in the qPCR assay are subsequently confirmed to be the correct predicted length through agarose gel electrophoretic analysis. 
         [0023]    Tier 2 QC/QA The specificity in DNA amplification of each qPCR primer pair is further confirmed through isolation and sequence-dependent assessment of qPCR-generated DNA products that contribute to the SYBR-based signal. Two methods can be used: 1) mapping of restriction endonuclease-derived fragments of qPCR amplified DNA, or 2) DNA sequencing of the product of qPCR. This serves to confirm the specificity of the qPCR SYBR-signal collected and the identity of the gene sequence amplified by each primer pair used in qPCR. 
         [0024]    Tier 3 QC/QA To ensure that the SYBR-based signal data collected during qPCR can be employed in a quantitative manner, similarity in relative primer pair performance must be satisfied using information obtained through qPCR analysis of a dilution series of target cDNA from the tissue of interest. The sensitivity and ability of each gene-specific primer pair to detect linear changes in target cDNA abundance is assessed. Primer pair performance of a test gene is compared to the performance of primer pairs from reference genes to ensure comparable amplification efficiencies. Tissue context is important because we and others have found that some primers, although specific for their gene target, can display reduced performance in certain tissue contexts. 
         [0025]    The results of the three tier QC/QA process for each of the primer sets listed in Table 1 are presented in  FIG. 2 . This demonstrates that the primer sets work to specifically amplify the appropriate amplicon in a variety of contexts. A summary of which tissue types are usable for transcriptomics analyses for each primer set is found in Table 2. In Table 2, the particular tissues of  Rana catesbeiana  and  Xenopus laevis  in which each primer set is usable are denoted as follows:
       T=tail fin   HL=hind limb   I=intestine   H=heart   Li=liver   B=brain   Lu=lung 

       
 
         [0033]    The QC/QA results plus evidence of functionality in  Rana catesbeiana  and  Xenopus laevis  tissues are presented in  FIG. 2 , which is comprised of FIGS.  2 A( 1 ) through FIGS.  2 A( 10 ); FIGS.  2 B( 1 ) through FIGS.  2 B( 10 ) and FIGS.  2 C( 1 ) through FIGS.  2 C( 10 ). In these figures, results of the three tier QC/QA process by primer set on tissues from  Rana catesbeiana  and  Xenopus laevis  are shown. Each of the 30 primer sets is identified at the top of each drawing figure. Quality assurance (QA) of each primer pair is established through a three-tier evaluation procedure that includes:
       Tier 1, a clean cDNA-dependent signal return with low background noise determined through amplification that is associated with the production of a single DNA product for each anuran tissue type as determined through dissociation curves and electrophoretic analyses;   Tier 2, sequence-dependent confirmation of gene-specific targeting through restriction endonuclease mapping or direct DNA sequencing of the isolated amplified DNA product; and   Tier 3, primer pair comparative efficiency assessment across a tissue-specific cDNA dilution series as it related to the qPCR performance of the selected reference primer pair on the same cDNA dilution series. The slope of the interrelated qPCR performance must be ≦0.1 to be considered suitable for that tissue type.       
 
         [0037]    The decision of whether a primer pair would be used to discern a gene transcript as a normalizer or test is highly dependent upon the experimental context and includes considerations of tissue-type, sample time, exposure type, and species examined. 
         [0038]    Examples of normalizer gene candidates that would be used in any qPCR experiment to act as input reference include, but are not limited to: col1a2, actb, fn1, rps10, plp1 , rpl8, eef1a1 and timm50 (Table 1). 
         [0039]    Examples of thyroid hormone-responsive gene candidates in  Rana catesbeiana  ( FIGS. 3 and 4 ) and  Xenopus laevis  ( FIG. 5 ) include, but are not limited to: coil a2, actb, plp1, thra, thrb, dio2, thibz, klf9, fn1, mmp2, and otc. 
         [0040]      FIG. 3  illustrates a demonstration of thyroid hormone responsiveness of the target gene transcripts in  Rana catesbeiana . Fold-change expression data for the indicated gene transcripts as determined using the comparative Ct method (ΔΔCt) using multiple gene transcript normalizers. Data are expressed in box plots as fold-change in expression levels relative to the vehicle control group. Premetamorphic tadpoles were injected with either vehicle control (C) or the indicated concentration of thyroid hormone and the tissues collected after 48 hours. The concentration for T 3  exposures are 0.01, 0.1, 1.0, 10, 25, and 50 nM and 0.05, 0.5, 5.0, 50, 125, and 250 nm for T 4 . The medians are shown as solid black lines within the box and the box indicates the 25 th  and 75 th  percentiles. Whiskers indicated the range. “a”: significantly different from the vehicle control group (p&lt;0.05). “n/a” means not expressed in that tissue. 
         [0041]      FIG. 4  illustrates a demonstration of thyroid hormone responsiveness of the target gene transcripts in  Rana catesbeiana . Fold-change expression data for the indicated gene transcripts as determined using the comparative Ct method (ΔΔCt) using multiple gene transcript normalizers. Data are expressed in box plots as fold-change in expression levels relative to the vehicle control group. Premetamorphic tadpoles were injected with either vehicle control (C) or the indicated concentration of thyroid hormone and the tissues collected after 48 hours. The concentration for T 3  exposures are 0.01, 0.1, 1.0, 10, 25, and 50 nM and 0.05, 0.5, 5.0, 50, 125, and 250 nm for T 4 . The medians are shown as solid black lines within the box and the box indicates the 25 th  and 75 th  percentiles. Whiskers indicated the range. “a”: significantly different from the vehicle control group (p&lt;0.05). “n/a” means not expressed in that tissue. 
         [0042]      FIG. 5  illustrates a demonstration of thyroid hormone responsiveness of the target gene transcripts in  Xenopus laevis  in the tail, brain, liver, and intestine. Fold-change expression data for the indicated gene transcripts as determined using the comparative Ct method (ΔΔCt) using multiple gene transcript normalizers. Data are expressed in box plots as fold-change in expression levels relative to the vehicle control group. Premetamorphic tadpoles were immersed in water containing vehicle control of 10 nM T 3  for 48 hours. The medians are shown as solid black lines within the box and the box indicates the 25 th  and 75 th  percentiles. Whiskers indicate the range. Greyed out regions indicate no detectable specific signal in the indicated tissue (refer to Table 2 for details). “a”: signficantly different from the vehicle control group (p&lt;0.05). 
         [0043]    The primer sets can be used on multiple Anuran species beyond  Rana catesbeiana  and  Xenopus laevis , as is demonstrated in  FIG. 6 .  FIG. 6  illustrates a demonstration that the primer sets are usable in a wide range of anuran specifies. The primer sets in Table 1 were run on cDNA preparations made from a single tissue or mixture of tissues (species-dependent) on the indicated species to demonstrate that the appropriately sized amplicon is produced across a wide range of Anuran species. 
         [0044]    Another use of the primers can be for detection of contaminating genomic DNA in transcriptomic studies or in amplification of genomic DNA ( FIG. 7 ) and their utility in  Rana catesbeiana  and  Xenopus laevis  are summarized in Table 3.  FIG. 7  illustrates representative examples of primer set performance on genomic DNA across different anuran species. Genomic DNA (gDNA) was isolated from skin of  Xenopus laevis  tadpoles and liver of  Rana catesbeiana  adults. For both species, complementary DNA (cDNA) was generated from total RNA isolated from mixed prometamorphic tadpole tissues. Location of the predicted PCR amplicon representative of expressed sequence for each gene target is shown by an arrowhead. MW denotes molecular size standards in kilobase pairs of linear DNA. The results for all of the primer sets are summarized in Table 3. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Characterization of PCR amplified DNA products generated using 
               
               
                 genomic DNA of  Xenopus   laevis  and  Rana   catesbeiana  and the TAXISS primer panel a   
               
               
                 compared to amplicons generated from cDNA. The majority of primer sets amplify a distinctly 
               
               
                 different product or generate no product on genomic DNA from both species. This accentuates 
               
               
                 that, although the genomes of these species are very different, the ability to detect gDNA 
               
               
                 contamination in cDNA preparations is possible. 
               
             
          
           
               
                   
                 
                   Rana 
                   catesbeiana 
                 
                 
                   Xenopus 
                   laevis 
                 
               
             
          
           
               
                 TAXISS 
                 Gene 
                 Similar 
                 Different 
                 No 
                 Similar 
                 Different 
                 No 
               
               
                 Primers 
                 Name 
                 Product 
                 Product 
                 Product 
                 Product 
                 Product 
                 Product 
               
               
                   
               
               
                 TAX1 
                 col1a2 
                 + 
                   
                   
                 + 
                   
                   
               
               
                 TAX5 
                 actb 
                   
                   
                 + 
                   
                 + 
                   
               
               
                 TAX7 
                 rps10 
                   
                 + 
                   
                   
                 + 
                   
               
               
                 TAX8 
                 plp1 
                   
                   
                 + 
                   
                 + 
                   
               
               
                 TAX9 
                 thra 
                   
                   
                 + 
                   
                 + 
                   
               
               
                 TAX10 
                 thrb 
                 + 
                   
                   
                   
                 + 
                   
               
               
                 TAX12 
                 dio2 
                   
                   
                 + 
                   
                 + 
                   
               
               
                 TAX15 
                 thibz 
                 + 
                   
                   
                 + 
                   
                   
               
               
                 TAX16 
                 klf9 
                   
                   
                 + 
                 + 
                   
                   
               
               
                 TAX18 
                 fn1 
                   
                   
                 + 
                   
                   
                 + 
               
               
                 TAX20 
                 mmp2 
                   
                   
                 + 
                   
                 + 
                   
               
               
                 TAX22 
                 rpl8 
                   
                 + 
                   
                   
                 + 
                   
               
               
                 TAX23 
                 eef1a1 
                 + 
                   
                   
                 + 
                   
                   
               
               
                 TAX27 
                 timm50 
                   
                   
                 + 
                   
                   
                 + 
               
               
                 TAX28 
                 otc 
                   
                   
                 + 
                   
                 + 
               
               
                   
               
               
                   a PCR were performed using qPCR-associated reagents and thermoprofile conditions. Genomic DNA and cDNA reactions were carried out concurrently for comparison. The annealing temperature was 60° C. for all reactions. 
               
               
                   b Relative comparison of amplicon band migration on agarose gels comparing amplicons generated from gDNA versus cDNA. 
               
             
          
         
       
     
         [0045]    Each primer pair can be used independently of each other. In one embodiment, the invention comprises use of a primer pair by itself to detect particular genes within an Anuran nucleic acid sample. 
         [0046]    In another embodiment, two or more of any of the primer pairs listed in Table 1 may be used. 
         [0047]    The normalizer gene panel would include two or more of the following primer pairs: col1a2, actb, rps10, plp1, rpl8, eef1a1 and timm50. The end-user would then be able to use evaluation tools readily available to finalize the appropriateness of each reference gene. 
         [0048]    The thyroid hormone responsive gene panel would include two or more of the following primer pairs: colta2, actb, plp1, thra, thrb, dio2, thibz, klf9, fn1, mmp2, and otc. 
         [0049]    The assay of the invention further comprises the following method. Tadpoles are exposed to the compound of interest in water and, at the appropriate time (e.g., within hours to a few days), two standardized portions of the tail are removed. The tail tips are immediately subjected to an apoptosis assay in Phase 1, while the other (second) tail portion is preserved for Phase 2. 
         [0050]    If the results for Phase 1 are negative, then the substance is not thyroid axis active. No further analysis is necessary. If the Phase 1 results are positive, then the second tail portion is analyzed for shifts in the quantity of mRNA transcripts specifically identified for responsiveness in Phase 2. An example of Phase 2 analysis is using quantitative real time polymerase chain reaction (qPCR) for each gene transcript target. 
         [0051]    If the gene transcripts indicative of thyroid hormone (TH) action are not affected, then it can be concluded that the test compound is not thyroid axis active. If the transcripts are affected, then the compound is identified as a likely TH disruptor. 
         [0052]    Accordingly, disclosed herein is a frog tadpole metamorphosis assay method comprising a first phase and a second phase, wherein: the first phase comprises exposing a tadpole to a compound of interest, removing first and second portions of the tadpole&#39;s tail, subjecting the first portion to an apatosis assay, and the second phase comprises subjecting the second portion to an assay to detect shifts in a quantity of pre-determined mRNA transcripts. 
         [0053]    In a more specific embodiment of the method, the first phase further comprises determining whether the compound of interest is thyroid axis active. 
         [0054]    In yet another embodiment of the method, the second phase further comprises subjecting the second portion to qPCR. 
         [0055]    In still yet another embodiment, the the first phase further comprises determining whether the compound of interest is thyroid axis active, and the second phase further comprises subjecting the second portion to qPCR. 
         [0056]    In an embodiment of the invention, disclosed is a metamorphosis assay wherein the first phase comprises exposing a tadpole to a compound of interest, removing first and second portions of the tadpole&#39;s tail, and subjecting the first portion to an apatosis assay, and the second phase comprises subjecting the second portion to qPCR using a primer pair comprising a first primer and a second primer, wherein the primer pair is selected from the group consisting of primer pairs A, B, C, D, E, F, G, H, I, J, K, L, M, N, and O: 
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 Primer 
                 First 
                 Second 
               
               
                 Pair 
                 Primer 
                 Primer 
               
               
                   
               
             
             
               
                 A 
                 CTGGTGGTGGATATGA 
                 GAGTCTTAAGTCACGG 
               
               
                   
                 TGGT 
                 CAAG 
               
               
                   
               
               
                 B 
                 TACAGCTTCACCACCA 
                 TCCACATCTGCTGGAA 
               
               
                   
                 CAG 
                 GGT 
               
               
                   
               
               
                 C 
                 TTTGCYTGGCGKCACT 
                 ARCRGCACTGCGYCT 
               
               
                   
                 TTT 
                 GTA 
               
               
                   
               
               
                 D 
                 TGGCTGARGGATTYTA 
                 ACAGCAGAGCAGGCA 
               
               
                   
                 YACC 
                 AMGA 
               
               
                   
               
               
                 E 
                 TGATAAGGCCACAGGR 
                 CGGGTGATCTTGTCG 
               
               
                   
                 TACCACTA 
                 ATRA 
               
               
                   
               
               
                 F 
                 CTCATAGAAGAAAACA 
                 GAAGGCTTCTAAGTC 
               
               
                   
                 GAGAAAARAGA 
                 CACTTTTCC 
               
               
                   
               
               
                 G 
                 CCTGGCTCTSTAYGA 
                 RGCTGATCCRAARTT 
               
               
                   
                 CTC 
                 GAC 
               
               
                   
               
               
                 H 
                 ASCTCCRCAGAAYCA 
                 TCACGTACCAGGCCA 
               
               
                   
                 GCA 
                 AAA 
               
               
                   
               
               
                 I 
                 CYGCTCAGTGTCTGG 
                 ARGGGCCGGTACTTG 
               
               
                   
                 TGT 
                 TTT 
               
               
                   
               
               
                 J 
                 GTTGCCATGARGGW 
                 CCATTGCCWGTGCA 
               
               
                   
                 GGAC 
                 GATA 
               
               
                   
               
               
                 K 
                 TACAACAGCTGCACT 
                 TCCTTGTCATAGTCT 
               
               
                   
                 GAT 
                 TCTGT 
               
               
                   
               
               
                 L 
                 CAGGGGACAGAGAAA 
                 TGAGCTTTCTTGCC 
               
               
                   
                 AGGTG  
                 ACAG 
               
               
                   
               
               
                 M 
                 GCTGCTGGTGTTGGT 
                 AGCATGTTGTCACCR 
               
               
                   
                 GART 
                 TTCC 
               
               
                   
               
               
                 N 
                 GCTWCAYCCAGAGTG 
                 GCTGYTCCTCCTGCT 
               
               
                   
                 GTCGTT 
                 CCA 
               
               
                   
               
               
                 O 
                 YATGACYGATGCTGT  
                 CATAWCCCTTTGGTG 
               
               
                   
                 TCTAG 
                 TTGC 
               
               
                   
               
             
          
         
       
     
         [0057]    In yet another particular embodiment of the invention, the second phase further comprises subjecting the second portion to qPCR using two or more primer pairs, wherein each pair comprises a first primer and a second primer, and the primer pairs are selected from the group consisting of primer pairs A, B, D, E, F, G, H, I, J, K, and O: 
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 Primer 
                 First 
                 Second 
               
               
                 Pair 
                 Primer 
                 Primer 
               
               
                   
               
             
             
               
                 A 
                 CTGGTGGTGGATATG 
                 GAGTCTTAAGTCACG 
               
               
                   
                 ATGGT 
                 GCAAG 
               
               
                   
               
               
                 B 
                 TACAGCTTCACCACC 
                 TCCACATCTGCTGGA 
               
               
                   
                 ACAG 
                 AGGT 
               
               
                   
               
               
                 D 
                 TGGCTGARGGATTYT 
                 ACAGCAGAGCAGGCA 
               
               
                   
                 AYACC 
                 AMGA 
               
               
                   
               
               
                 E 
                 TGATAAGGCCACAGG 
                 CGGGTGATCTTGTCG 
               
               
                   
                 RTACCACTA 
                 ATRA 
               
               
                   
               
               
                 F 
                 CTCATAGAAGAAAAC 
                 GAAGGCTTCTAAGTC 
               
               
                   
                 AGAGAAAARAGA 
                 CACTTTTCC 
               
               
                   
               
               
                 G 
                 CCTGGCTCTSTAYGA 
                 RGCTGATCCRAARTT 
               
               
                   
                 CTC 
                 GAC 
               
               
                   
               
               
                 H 
                 ASCTCCRCAGAAYCA 
                 TCACGTACCAGGCCA 
               
               
                   
                 GCA 
                 AAA 
               
               
                   
               
               
                 I 
                 CYGCTCAGTGTCTGG 
                 ARGGGCCGGTACTTG 
               
               
                   
                 TGT 
                 TTT 
               
               
                   
               
               
                 J 
                 GTTGCCATGARGGWG 
                 CCATTGCCWGTGCAG 
               
               
                   
                 GAC 
                 ATA 
               
               
                   
               
               
                 K 
                 TACAACAGCTGCACT 
                 TCCTTGTCATAGTCT 
               
               
                   
                 GAT 
                 TCTGT 
               
               
                   
               
               
                 O 
                 YATGACYGATGCTGT 
                 CATAWCCCTTTGGTG 
               
               
                   
                 TCTAG 
                 TTGC 
               
               
                   
               
             
          
         
       
     
         [0058]    The invention further relates to a composition of matter comprising a primer pair selected from the group consisting of primer pairs A, B, C, D, E, F, G, H, I, J, K, L, M, N, and O: 
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 Primer 
                 First 
                 Second 
               
               
                 Pair 
                 Primer 
                 Primer 
               
               
                   
               
             
             
               
                 A 
                 CTGGTGGTGGATATG 
                 GAGTCTTAAGTCAC 
               
               
                   
                 ATGGT 
                 GGCAAG 
               
               
                   
               
               
                 B 
                 TACAGCTTCACCAC 
                 TCCACATCTGCTG 
               
               
                   
                 CACAG 
                 GAAGGT 
               
               
                   
               
               
                 C 
                 TTTGCYTGGCGKC 
                 ARCRGCACTGCGY 
               
               
                   
                 ACTTTT 
                 CTGTA 
               
               
                   
               
               
                 D 
                 TGGCTGARGGATT 
                 ACAGCAGAGCAGGC 
               
               
                   
                 YTAYACC 
                 AAMGA 
               
               
                   
               
               
                 E 
                 TGATAAGGCCACAGG 
                 CGGGTGATCTTGTC 
               
               
                   
                 RTACCACTA 
                 GATRA 
               
               
                   
               
               
                 F 
                 CTCATAGAAGAAAAC 
                 GAAGGCTTCTAAGTC 
               
               
                   
                 AGAGAAAARAGA 
                 CACTTTTCC 
               
               
                   
               
               
                 G 
                 CCTGGCTCTSTAYGA 
                 RGCTGATCCRAART 
               
               
                   
                 CTC 
                 TGAC 
               
               
                   
               
               
                 H 
                 ASCTCCRCAGAAYCA 
                 TCACGTACCAGGCC 
               
               
                   
                 GCA 
                 AAAA 
               
               
                   
               
               
                 I 
                 CYGCTCAGTGTCTG 
                 ARGGGCCGGTACTT 
               
               
                   
                 GTGT 
                 GTTT 
               
               
                   
               
               
                 J 
                 GTTGCCATGARGGW 
                 CCATTGCCWGTGCA 
               
               
                   
                 GGAC 
                 GATA 
               
               
                   
               
               
                 K 
                 TACAACAGCTGCAC 
                 TCCTTGTCATAGTCT 
               
               
                   
                 TGAT 
                 TCTGT 
               
               
                   
               
               
                 L 
                 CAGGGGACAGAGAAA 
                 TGAGCTTTCTTGCC 
               
               
                   
                 AGGTG 
                 ACAG 
               
               
                   
               
               
                 M 
                 GCTGCTGGTGTTGGT 
                 AGCATGTTGTCACCR 
               
               
                   
                 GART 
                 TTCC 
               
               
                   
               
               
                 N 
                 GCTWCAYCCAGAGTGGT 
                 GCTGYTCCTCCTGCT 
               
               
                   
                 CGTT 
                 CCA 
               
               
                   
               
               
                 O 
                 YATGACYGATGCTGTT 
                 CATAWCCCTTTGGT 
               
               
                   
                 CTAG 
                 GTTGC 
               
               
                   
               
             
          
         
       
     
         [0059]    Yet another aspect of the invention relates to a normalizer gene panel comprising two or more primer pairs selected from the group consisting of primer pairs A, B, C, D, L, M, and N: 
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 Primer 
                 First 
                 Second 
               
               
                 Pair 
                 Primer 
                 Primer 
               
               
                   
               
             
             
               
                 A 
                 CTGGTGGTGGATATG 
                 GAGTCTTAAGTCACG 
               
               
                   
                 ATGGT 
                 GCAAG 
               
               
                   
               
               
                 B 
                 TACAGCTTCACCACC 
                 TCCACATCTGCTGGA 
               
               
                   
                 ACAG 
                 AGGT 
               
               
                   
               
               
                 C 
                 TTTGCYTGGCGKCAC 
                 ARCRGCACTGCGYCT 
               
               
                   
                 TTTT 
                 GTA 
               
               
                   
               
               
                 D 
                 TGGCTGARGGATTYT 
                 ACAGCAGAGCAGGCA 
               
               
                   
                 AYACC 
                 AMGA 
               
               
                   
               
               
                 L 
                 CAGGGGACAGAGAAA 
                 TGAGCTTTCTTGCCA 
               
               
                   
                 AGGTG 
                 CAG 
               
               
                   
               
               
                 M 
                 GCTGCTGGTGTTGGT 
                 AGCATGTTGTCACCR 
               
               
                   
                 GART 
                 TTCC 
               
               
                   
               
               
                 N 
                 GCTWCAYCCAGAGTG 
                 GCTGYTCCTCCTGCT 
               
               
                   
                 GTCGTT 
                 CCA 
               
               
                   
               
             
          
         
       
     
         [0060]    Yet another aspect of the invention relates to a thyroid hormone responsive gene panel comprising two or more primer pairs selected from the group consisting of primer pairs A, B, D, E, F, G, H, I, J, K, and O: 
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 Primer 
                 First 
                 Second 
               
               
                 Pair 
                 Primer 
                 Primer 
               
               
                   
               
             
             
               
                 A 
                 CTGGTGGTGGATATG 
                 GAGTCTTAAGTCAC 
               
               
                   
                 ATGGT 
                 GGCAAG 
               
               
                   
               
               
                 B 
                 TACAGCTTCACCACC 
                 TCCACATCTGCTGG 
               
               
                   
                 ACAG 
                 AAGGT 
               
               
                   
               
               
                 D 
                 TGGCTGARGGATTYT 
                 ACAGCAGAGCAGGC 
               
               
                   
                 AYACC 
                 AAMGA 
               
               
                   
               
               
                 E 
                 TGATAAGGCCACAGG 
                 CGGGTGATCTTGTC 
               
               
                   
                 RTACCACTA 
                 GATRA 
               
               
                   
               
               
                 F 
                 CTCATAGAAGAAAAC 
                 GAAGGCTTCTAAGT 
               
               
                   
                 AGAGAAAARAGA 
                 CCACTTTTCC 
               
               
                   
               
               
                 G 
                 CCTGGCTCTSTAYGA 
                 RGCTGATCCRAART 
               
               
                   
                 CTC 
                 TGAC 
               
               
                   
               
               
                 H 
                 ASCTCCRCAGAAYCA 
                 TCACGTACCAGGCC 
               
               
                   
                 GCA 
                 AAAA 
               
               
                   
               
               
                 I 
                 CYGCTCAGTGTCTGG 
                 ARGGGCCGGTACTT 
               
               
                   
                 TGT 
                 GTTT 
               
               
                   
               
               
                 J 
                 GTTGCCATGARGGWG 
                 CCATTGCCWGTGCA 
               
               
                   
                 GAC 
                 GATA 
               
               
                   
               
               
                 K 
                 TACAACAGCTGCACT 
                 TCCTTGTCATAGTC 
               
               
                   
                 GAT 
                 TTCTGT 
               
               
                   
               
               
                 O 
                 YATGACYGATGCTGT 
                 CATAWCCCTTTGGT 
               
               
                   
                 TCTAG 
                 GTTGC 
               
               
                   
               
             
          
         
       
     
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         [0063]    Bernal, J. (2005). “Thyroid hormones and brain development.”  Vitamins and Hormones  71: 95-122. 
         [0064]    Brucker-Davis, F. (1998). “Effects of environmental synthetic chemicals on thyroid function.”  Thyroid  8: 827-856. 
         [0065]    Bruno, J. (2001). Toxicity test using premetamorphic bullfrog tadpoles ( Rana catesbeiana ). North Vancouver, Pacific Environmental Sciences Centre: 1-9. 
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         [0073]    Marlatt, V. L., N. Veldhoen, et al. (2013). “Triclosan exposure alters postembryonic development in a Pacific tree frog ( Pseudacris regilla ) Amphibian Metamorphosis Assay (TREEMA).”  Aquat Toxicol  126: 85-94. 
         [0074]    Miller, M. D., K. M. Crofton, et al. (2009). “Thyroid-disrupting chemicals: interpreting upstream biomarkers of adverse outcomes.”  Environ Health Perspect  117(7): 1033-1041. 
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         [0080]    Porterfield, S. (1994). “Vulnerability of the developing brain to thyroid abnormalities: environmental insults to the thyroid system.”  Environmental Health Perspectives  102: 125-130. 
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         [0083]    Searcy, B. T., S. M. Beckstrom-Sternberg, et al. (2012). “Thyroid hormone-dependent development in  Xenopus laevis : a sensitive screen of thyroid hormone signaling disruption by municipal wastewater treatment plant effluent.”  Gen Comp Endocrinol  176(3): 481-492. 
         [0084]    Shi, Y.-B. (2000). Amphibian Metamorphosis: From morphology to molecular biology. New York, Wiley- Liss. 
         [0085]    Siesser, W. B., S.-Y. Cheng, et al. (2005). “Hyperactivity, impaired learning on a vigilance task, and a differential response to methylphenidate in the TR-PV knock-in mouse.”  Psychopharmacology  181: 653-663. 
         [0086]    Sumida, M., Y. Kato, et al. (2004). “Sequencing and analysis of the internal transcribed spacers (ITSs) and coding regions in the EcoR I fragment of the ribosomal DNA of the Japanese pond frog  Rana nigromaculata .”  Genes &amp; Genetic Systems  79(2): 105-118. 
         [0087]    Van Aggelen, G., G. T. Ankley, et al. (2010). “Integrating omic technologies into aquatic ecological risk assessment and environmental monitoring: hurdles, achievements, and future outlook.”  Environ Health Perspect  118(1): 1-5. 
         [0088]    Vandenberg, L. N., T. Colborn, et al. (2012). “Hormones and endocrine-disrupting chemicals: Low-dose effects and nonmonotonic dose responses.”  Endocr Rev  33: doi: 10.1210/er.2011-1050. 
         [0089]    Veldhoen, N., R. Skirrow, et al. (2006). “The bactericidal agent triclosan modulates thyroid hormone-associated gene expression and disrupts postembryonic anuran development.” Aquatic Toxicology  80: 217-227. 
         [0090]    Zoeller, R. (2005). “Environmental chemicals as thyroid hormone analogues: New studies indicate that thyroid hormone receptors are targets of industrial chemicals.”  Molecular and Cellular Endocrinology  242: 10-15. 
         [0091]    Zoeller, R. and K. Crofton (2005). “Mode of action: developmental thyroid hormone insufficiency-neurological abnormalities resulting from exposure to propylthiouracil.”  Crit Rev Toxicol  35: 771-781. 
         [0092]    Zoeller, R. and J. Rovett (2004). “Timing of thyroid hormone action in the developing brain: clinical observations and experimental findings.”  Journal of Neuroendocrinology  16: 809-818.