Mary C Cardon

North Carolina State University, Raleigh, NC, United States

Are you Mary C Cardon?

Claim your profile

Publications (23)76.48 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Gestagen is a collective term for endogenous and synthetic progesterone receptor (PR) ligands. In teleost fishes, 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S) are the predominant progestogens, whereas in other vertebrates the major progestogen is progesterone (P4). Progestins are components of human contraceptives and hormone replacement pharmaceuticals, and with P4, can enter the environment and alter fish and amphibian reproductive health. In this study, our primary objectives were to clone the fathead minnow (FHM) nuclear PR (nPR), to develop an in vitro assay for FHM nPR transactivation, and to screen eight gestagens for their ability to transactivate FHM nPR. We also investigated the ability of these gestagens to transactivate FHM androgen receptor (AR). Fish progestogens activated FHM nPR, with DHP being more potent than 20β-S. The progestin drospirenone and P4 transactivated the FHM nPR, whereas five progestins and P4 transactivated FHM AR, all at environmentally-relevant concentrations. Progestins are designed to activate human PR, but older generation progestins have unwanted androgenic side effects in humans. In FHMs, several progestins proved to be strong agonists of AR. Here, we present the first mechanistic evidence that environmental gestagens can activate FHM nPR and AR, suggesting that gestagens may affect phenotype through nPR- and AR-mediated pathways.
    Environmental Science and Technology 06/2014; · 5.48 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Widespread environmental contamination by bisphenol A (BPA) has created the need to fully define its potential toxic mechanisms of action (MOA) to properly assess human health and ecological risks from exposure. Although long recognized as an estrogen receptor (ER) agonist, some data suggest that BPA may also behave as an androgen receptor (AR) antagonist. However, direct evidence of this activity is deficient. To address this knowledge gap, we employed a metabolomic approach using in vivo exposures of fathead minnows (FHM; Pimephales promelas ) to BPA either alone or in a binary mixture with 17β-trenbolone (TB), a strong AR agonist. Changes in liver metabolite profiles in female FHM in response to these exposures were determined using high resolution (1)H NMR spectroscopy and multivariate and univariate statistics. Using this approach, we observed clear evidence of the ability of BPA to mitigate the impact of TB, consistent with an antiandrogenic MOA. In addition, a transcriptional activation assay with the FHM AR was used to confirm the AR antagonistic activity of BPA in vitro. The results of these in vivo and in vitro analyses provide strong and direct evidence for ascribing an antiandrogenic MOA to BPA in vertebrates.
    Environmental Science & Technology 07/2012; 46(17):9673-80. · 5.48 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The captive southern white rhinoceros (SWR; Ceratotherium simum simum) population serves as an important genetic reservoir critical to the conservation of this vulnerable species. Unfortunately, captive populations are declining due to the poor reproductive success of captive-born females. Captive female SWR exhibit reproductive problems suggested to result from continual ovarian follicular activity and prolonged exposure to endogenous estrogen. However, we investigated the potential role of exogenous dietary phytoestrogens in the reproductive failure of SWR by cloning and characterizing in vitro phytoestrogen binding and activation of recombinant SWR estrogen receptors (ESR). We compared those characteristics with recombinant greater one-horned rhinoceros (GOHR; Rhinoceros unicornis) ESR, a species that receives similar captive diets yet reproduces relatively well. Our results indicate that phytoestrogens bind rhino ESR in a manner similar to other vertebrate species, but there are no differences found in phytoestrogen binding affinity of SWR ESR compared with GOHR ESR. However, species-specific differences in ESR activation by phytoestrogens were detected. The phytoestrogen coumestrol stimulated greater maximal activation of SWR ESR1 than GOHR ESR1. SWR ESR2 were also more sensitive to phytoestrogens and were activated to a greater extent by both coumestrol and daidzein. The concentrations in which significant differences in ESR activation occurred (10(-7) to 10(-5) m) are consistent with circulating concentrations measured in other vertebrate species. Taken together, these findings suggest that phytoestrogens potentially pose a risk to the reproductive health of captive SWR. However, additional studies are needed to further clarify the physiological role of dietary phytoestrogens in the reduced fertility of this species.
    Endocrinology 03/2012; 153(3):1444-52. · 4.72 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Reproductive abnormalities in alligators exposed to contaminants in Lake Apopka, Florida, USA represent a clear example of endocrine disruption in wildlife. Several of these contaminants that are not able to bind to mammalian estrogen receptors (such as atrazine and cyanazine) have previously been reported to bind to the alligator estrogen receptor from oviductal tissue. Binding of known Lake Apopka contaminants to full length estrogen receptors alpha from human (hERalpha) and alligator (aERalpha) was assessed in a side-by-side comparison within the same assay system. Baculovirus-expressed recombinant hERalpha and aERalpha were used in a competitive binding assay. Atrazine and cyanazine were not able to bind to either receptor. p,p'-Dicofol was able to bind to aERalpha with a concentration inhibiting 50% of binding (IC50) of 4 microM, while only partially displacing 17beta-estradiol (E2) from hERalpha and yielding a projected IC50 of 45 microM. Chemicals that only partially displaced E2 from either receptor, including some dichlorodiphenyltrichloroethane (DDT) metabolites and trans-nonachlor, appeared to have higher affinity for aERalpha than hERalpha. p,p'-Dicofol-mediated transcriptional activation through aERalpha and hERalpha was assessed to further explore the preferential binding of p,p'-dicofol to aERalpha over hERalpha. p,p'-Dicofol was able to stimulate transcriptional activation in a similar manner with both receptors. However, the in vitro results obtained with p,p'-dicofol were not reflected in an in vivo mammalian model, where Kelthane (mixed o,p'- and p,p'-dicofol isomers) did not elicit estrogenic effects. In conclusion, although there was no evidence of exclusively species-specific estrogen receptor binders, some xenoestrogens, especially p,p'-dicofol, had a higher affinity for aERalpha than for hERalpha.
    Environmental Toxicology and Chemistry 09/2010; 29(9):2064-71. · 2.83 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Vinclozolin and iprodione are dicarboximide fungicides that display antiandrogenic effects in the male rat, which suggests that a mixture would lead to cumulative effects on androgen-sensitive end points. Iprodione is a steroid synthesis inhibitor, but androgen receptor antagonist activity, which is displayed by vinclozolin, has not been fully evaluated. Here, we demonstrate that iprodione binds to the human androgen receptor (IC(50) = 86.0 microM), reduces androgen-dependent gene expression, and reduces androgen-sensitive tissue weights in castrated male rats (Hershberger assay). Since vinclozolin and iprodione affect common targets in the pubertal male rat, we tested the hypothesis that a mixture would have cumulative antiandrogenic effects. An iprodione dose, that does not significantly affect androgen-dependent morphological end points, was combined with vinclozolin doses (2 x 5 factorial design). Sprague-Dawley rats were dosed by gavage with vinclozolin at 0, 10, 30, 60, and 100 mg/kg/day with and without 50 mg iprodione/kg/day from postnatal day (PND) 23 to 55-57 (n = 8 per group). The age at puberty (preputial separation [PPS]), organ weights, serum hormones, and ex vivo testis steroid hormone production were measured. Vinclozolin delayed PPS, reduced androgen-sensitive organ weights, and increased serum testosterone. The addition of iprodione enhanced the vinclozolin inhibition of PPS (PND 47.5 vs.49.1; two-way ANOVA: iprodione main effect p = 0.0002). The dose response for several reproductive and nonreproductive organ weights was affected in a cumulative manner. In contrast, iprodione antagonized the vinclozolin-induced increase in serum testosterone. These results demonstrate that these fungicides interact on common targets in a tissue-specific manner when coadministered to the pubertal male rat.
    Toxicological Sciences 07/2009; 111(1):179-88. · 4.48 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mammalian receptors and assay systems are generally used for in vitro screening of endocrine-disrupting chemicals with the assumption that minor differences in amino acid sequences among species do not translate into significant differences in receptor function. Objectives of the present study were to evaluate the performance of two different in vitro assay systems (a whole cell and a cell-free competitive binding assay) in assessing whether binding of chemicals differs significantly between full-length recombinant estrogen receptors from fathead minnows (fhERalpha) and those from humans (hERalpha). It was confirmed that 17beta-estradiol displays a reduction in binding to fhERalpha at an elevated temperature (37 degrees C), as has been reported with other piscine estrogen receptors. Several of the chemicals (17beta-estradiol, ethinylestradiol, alpha-zearalanol, fulvestrant, dibutyl phthalate, benzyl butyl phthalate, and cadmium chloride) displayed higher affinity for fhERalpha than for hERalpha in the whole cell assay, while only dibutyl phthalate had a higher affinity for fhERalpha than for hERalpha in the cell-free assay. Both assays were effective in identifying strong binders, weak binders, and nonbinders to the two receptors. However, the cell-free assay provided a less complicated and more efficient binding platform and is, therefore, recommended over the whole cell binding assay. In conclusion, no strong evidence showed species-specific binding among the chemicals tested.
    Environmental Toxicology and Chemistry 06/2009; 28(10):2175-81. · 2.83 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In the current study, we developed a new system using full-length recombinant baculovirus-expressed estrogen receptors which allows for direct comparison of binding across species. Estrogen receptors representing five vertebrate classes were compared: human estrogen receptor alpha (hERα), quail estrogen receptor alpha (qERα), alligator estrogen receptor alpha (aERα), salamander estrogen receptor alpha (sERα), and fathead minnow estrogen receptor alpha (fhERα). Saturation binding analyses indicated 17β-estradiol (E2) dissociation constants (Kd) were 0.22 ± 0.02 nM for hERα, 0.28 ± 0.04 nM for sERα, 0.44 ± 0.04 nM for aERα, 0.58 ± 0.10 nM for qERα, and 0.58 ± 0.05 nM for fhERα. Binding specificity to each of the receptors was evaluated using E2, dihydrotestosterone (DHT), corticosterone (C), and ethinylestradiol (EE). E2 and EE were strong binders in all species with IC50's ranging from 0.65 nM with hERα to 1.01 nM with sERα for E2 and from 0.68 nM with sERα to 1.20 nM with qERα for EE. DHT was a weak binder with IC50's ranging from 3.3 μM with hERα to 39 μM with fhERα, and C did not bind any of the receptors at concentrations up to 100 μM. This system provides a convenient in vitro approach for directly comparing chemical binding to estrogen receptors across multiple species without the need to sacrifice animals.
    Toxicology Letters 01/2009; · 3.36 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The issue as to whether natural and man-made chemicals interfere with endocrine function has raised concerns. This interference could be biologically significant even at very low doses if the chemicals interact deleteriously with hormone receptors at low concentrations. Therefore, the United States Environmental Protection Agency (USEPA) Office of Coordination and Policy (OSCP) requested that a nonhuman mammalian androgen receptor binding assay be developed for possible use in their Endocrine Disruptor Screening Program (EDSP). Ideally, this assay would be high throughput, not use animals as a source of receptor protein, easily deployed throughout the scientific community, utilize reagents available to both the public and private sector, and have the potential for future automation. We developed a highly modified 96-well plate assay which meets these criteria. It employs a baculovirus expressed recombinant primate androgen receptor which is publically available and exploits the unique ability of some mammalian androgen receptors to remain biologically active after guanidine hydrochloride (GdnHCl) solubilization. This GdnHCl treated receptor remains soluble and requires no additional purification prior to use. We provide a very detailed description of the assay protocol itself, and similarly detailed method for producing and solubilizing the receptor.
    Toxicology Letters 10/2008; 181(2):126-31. · 3.36 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Alteration of gene expression can result in numerous pathologies, including proliferative diseases, functional deficits, and developmental defects. Recent studies have suggested that changes in the expression domains of Homeotic (Hox) genes during development are associated with the induction of chemically induced developmental anomalies. Before meaningful data on the alteration of gene expression can be obtained, the heterogeneity of normal expressions within a species must be evaluated. In situ hybridization is a powerful technique that can detect perturbations in the spatial and/or temporal expression of genes. The hybridization protocol described here utilizes 33P end-labeled, single-stranded DNA oligomeric nucleotides. Some of the technical advantages to this approach are that (1) DNA probes are not sensitive to RNase, (2) the probes are based on published sequences and are chemically synthesized, (3) probes are end-labeled utilizing a terminal transferase reaction, producing probes of high specific activity and uniform length, (4) expression patterns of spliced genes can be easily evaluated by synthesizing probes specific to different areas of the gene relative to the splice site, (5) probes do not require the use of strong reducing agents, and (6) the technique can be used on tissue embedded in paraffin and avoids the use of frozen sections. The utility of this approach is demonstrated in this paper by the analysis of expression of Hoxa-7 during normal development of the CD-I mouse embryo.
    Toxicology mechanisms and methods 09/2008; 6(1):13-22. · 1.55 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The potential effect of receptor-mediated endocrine modulators across species is of increasing concern. In attempts to address these concerns, we are developing androgen and estrogen receptor binding assays using recombinant hormone receptors from a number of species across different vertebrate classes. The United States Environmental Protection Agency (USEPA) Office of Science Coordination and Policy (OSCP) requested that we develop a nonhuman mammalian receptor-binding assay for possible use in their Endocrine Disruptor Screening Program (EDSP). Since the chimpanzee androgen receptor is very similar to that of humans and thus possesses properties which could be exploited in future endocrine studies, we synthesized and expressed this gene in eukaryotic expression plasmids, baculovirus expression vectors and replication deficient adenovirus. In all ligand-binding and transcriptional activation assays tested, the chimpanzee receptor performed essentially identically to the human receptor. This suggests that the chimpanzee gene could substitute for the human gene in endocrine screening assays.
    Toxicology Letters 12/2007; 174(1-3):89-97. · 3.36 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Typically, in vitro hazard assessments for the identification of endocrine-disrupting compounds (EDCs), including those outlined in the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Tier 1 Screening protocols, utilize mammalian receptors. Evidence, however, exists that fish sex steroid hormone receptors differ from mammalian receptors both structurally and in their binding affinities for some steroids and environmental chemicals. Most of the binding studies to date have been conducted using cytosolic preparations from various tissues. In the present study, we compare competitive binding of a set of compounds to full-length recombinant rainbow trout androgen receptor alpha (rtAR), fathead minnow androgen receptor (fhAR), and human androgen receptor (hAR), each expressed in COS cells. Saturation binding and subsequent Scatchard analysis using [3H]R1881, a high-affinity synthetic androgen, revealed an equilibrium dissociation constant (Kd) of 0.11 nM for the rtAR, 1.8 nM for the fhAR, and 0.84 nM for the hAR. Compounds, including endogenous and synthetic steroids, known mammalian antiandrogens, and environmental compounds, were tested for competitive binding to each of the three receptors. Overall, agreement existed across receptors as to binding versus nonbinding for all compounds tested in this study. Minor differences, however, were found in the relative order of binding of the compounds to the individual receptors. Studies such as these will facilitate the identification of EDCs that may differentially affect specific species and aid in the development and support of future risk assessment protocols.
    Environmental Toxicology and Chemistry 10/2007; 26(9):1793-802. · 2.83 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The present study evaluated both diesel fuel exhaust and biomass (wood) burn extracts for androgen receptor-mediated activity using MDA-kb2 cells, which contain an androgen-responsive promoter-luciferase reporter gene construct. This assay and analytical fractionization of the samples were used as tools to separate active from inactive fractions, with the goal of identifying the specific compounds responsible for the activity. A significant androgenic response was detected from the diesel emission. High-performance liquid chromatographic fractionation of the sample indicated that significant androgenic activity was retained in three fractions. 4-Hydroxybiphenyl was identified from the most active fraction using gas chromatography/mass spectroscopy. This purified compound was then tested at doses from 1 nM to 100 microM. 4-Hydroxybiphenol exhibited antagonist activity at low concentrations and agonist activity at high concentrations. A competitive-binding assay confirmed binding to the androgen receptor, with a median inhibitory concentration for radioligand binding of approximately 370 nM. Significant androgenic activity also was detected in the wood burn samples, but we were unable to identify the specific chemicals responsible for this endocrine activity. The present study demonstrates that in vitro bioassays can serve as sensitive bioanalytical tools to aid in characterization of complex environmental mixtures.
    Environmental Toxicology and Chemistry 09/2006; 25(8):2123-31. · 2.83 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Many chemicals that adversely affect reproduction and/or development do so through multiple pathways within the reproductive tract and hypothalamic-pituitary-gonadal axis. Notable in this regard are fungicides, such as prochloraz or fenarimol, which in mammals have the potential to impact endocrine function through inhibition of CYP enzymes involved in steroid metabolism, as well as through antagonism of the androgen receptor(s). The objective of our studies was to assess the effects of prochloraz and fenarimol on reproductive endocrine function in a model small fish species, the fathead minnow (Pimephales promelas), using both in vitro and in vivo assays. The two fungicides inhibited in vitro CYP19 aromatase activity in brain and ovarian homogenates from the fish, with prochloraz exhibiting a greater potency than fenarimol. Prochloraz and fenarimol also bound competitively to the cloned fathead minnow androgen receptor expressed in COS-1 cells. The two fungicides significantly reduced fecundity of the fish in a 21-day reproduction assay at water concentrations of 0.1 (prochloraz) and 1.0 (fenarimol) mg/l. The in vivo effects of prochloraz on plasma steroid (17beta-estradiol, testosterone, 11-ketotestosterone) and vitellogenin (an estrogen-responsive protein) concentrations, as well as on gonadal histopathology, were consistent with inhibition of steroidogenesis. Fenarimol also affected several aspects of endocrine function in vivo; however, the suite of observed effects did not reflect either aromatase inhibition or androgen receptor antagonism. These studies contribute to a better mechanistic understanding of the extrapolation of effects of endocrine-disrupting chemicals across vertebrate classes.
    Toxicological Sciences 09/2005; 86(2):300-8. · 4.48 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In this study, we characterized the effects of flutamide, a model mammalian androgen receptor (AR) antagonist, on endocrine function in the fathead minnow (Pimephales promelas), a small fish species that is widely used for testing endocrine-disrupting chemicals (EDCs). Binding assays with whole cells transiently transfected with cloned fathead minnow AR indicated that flutamide binds competitively to the receptor. However, as is true in mammalian systems, a 2-hydroxylated metabolite of flutamide binds to the AR with a much higher affinity than the parent chemical. Mixture experiments with flutamide and the androgen 17beta-trenbolone demonstrated that the anti-androgen effectively blocked trenbolone-induced masculinization (nuptial tubercle production) of female fathead minnows, indicating antagonism of an AR receptor-mediated response in vivo. Conversely, reductions in vitellogenin in trenbolone-exposed females were not blocked by flutamide, suggesting that the vitellogenin response is not directly mediated through the AR. The results of these studies provide data demonstrating the validity of using the fathead minnow as a model species for detecting EDCs that exert toxicity through interactions with the AR.
    Environmental Science and Technology 01/2005; 38(23):6322-7. · 5.48 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In vitro screening assays designed to identify hormone mimics or antagonists typically use mammalian (rat, human) estrogen (ER) and androgen receptors (AR). Although we know that the amino acid sequences of steroid receptors in nonmammalian vertebrates are not identical to the mammalian receptors, a great deal of uncertainty exists as to whether these differences affect interactions of potential endocrine-disrupting chemicals (EDC) with the receptors. This leads to substantial uncertainty with respect to the utility of mammalian-based screening assays to predict possible effects of EDCs in nonmammalian wildlife. This paper describes preparation of a cDNA library from a small fish model commonly used in ecological risk assessments, the fathead minnow (Pimphales promelas). The cDNA library was subsequently used to isolate and sequence both AR and ERalpha. In addition, the fathead minnow (fh)AR was expressed and characterized with respect to function using saturation and competitive binding assays in COS monkey kidney cells. Saturation experiments along with subsequent Scatchard analysis determined that the Kd of the fhAR for the potent synthetic androgen R1881 was 1.8 nM, which is comparable to that for the human AR in the same assay system. In COS whole cell competitive binding assays, potent androgens such as dihydrotestosterone and 11-ketotestosterone were also shown to be high affinity ligands for the fhAR. We also report affinity of the receptor for a number of environmental contaminants including the AR agonists androstenedione and 17a- and 17beta-trenbolone;AR antagonists such as p,p'-DDE, linuron, and vinclozolin; and the ER agonist 17beta-estradiol. Future plans include comparison of binding affinities of the fhAR to those of the human AR, also expressed in COS cells, using a range of EDCs.
    Environmental Science and Technology 01/2005; 38(23):6314-21. · 5.48 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Trenbolone acetate is a synthetic steroid that is extensively used in the United States as a growth promoter in beef cattle. The acetate is administered to livestock via slow-release implants; some is converted by the animal to 17-beta-trenbolone, a relatively potent androgen receptor agonist in mammalian systems. Recent studies indicate that excreted 17-beta-trenbolone is comparatively stable in animal waste, suggesting the potential for exposure to aquatic animals via direct discharge, runoff, or both. However, little is known concerning the toxicity of trenbolone to fish. Our goal was to assess the effects of 17-beta-trenbolone on reproductive endocrinology of the fathead minnow (Pimephales promelas). An in vitro competitive binding study with the fathead minnow androgen receptor demonstrated that 17-beta-trenbolone had a higher affinity for the receptor than that of the endogenous ligand, testosterone. Male and female fish were exposed for 21 d to nominal (target) concentrations of 17-beta-trenbolone ranging from 0.005 to 50 microg/L. Fecundity of the fish was significantly reduced by exposure to measured test concentrations > or = 0.027 microg/ L. The 17-beta-trenbolone was clearly androgenic in vivo at these concentrations, as evidenced by the de novo production in females of dorsal (nuptial) tubercles, structures normally present only on the heads of mature males. Plasma steroid (testosterone and beta-estradiol) and vitellogenin concentrations in the females all were significantly reduced by exposure to 17-beta-trenbolone. The 17-beta-trenbolone also altered reproductive physiology of male fathead minnows, albeit at concentrations much higher than those producing effects in females. Males exposed to 17-beta-trenbolone at 41 microg/L (measured) exhibited decreased plasma concentrations of 11-ketotestosterone and increased concentrations of beta-estradiol and vitellogenin. Overall, our studies indicate that 17-beta-trenbolone is a potent androgen and reproductive toxicant in fish. Given the widespread use of trenbolone acetate as a growth promoter, and relative stability of its metabolites in animal wastes, further studies are warranted to assess potential ecological risk.
    Environmental Toxicology and Chemistry 07/2003; 22(6):1350-60. · 2.83 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Retinoic acid (RA) alters the developmental fate of the axial skeletal anlagen. "Anteriorizations" or "posteriorizations," the assumption of characteristics of embryonic areas normally anterior or posterior to the affected tissues, are correlated with altered embryonal expression domains of Hox genes after in utero RA treatment. These "homeotic" changes have been hypothesized to result from alterations of a "Hox cod" which imparts positional identity in the axial skeleton. To investigate whether such developmental alterations were specific to RA, or were a more general response to xenobiotic exposure, CD-1 pregnant mice were exposed to RA, valproic acid (VA), or bromoxynil (Br) during organogenesis. Additionally, the expression domains of two Hox genes, Hoxa7 and Hoxa10, were examined in gestation day (GD) 12.5 embryos obtained from control, RA, VA, or Br, treated gravid dams exposed on GD 6, 7, or 8. The anterior expression boundary of Hoxa7 is at the level of the C7/T1 vertebrae and that of Hoxa10 is at L6/S1. Compound-induced changes in the incidence of skeletal variants were observed. These included supernumerary cervical ribs (CSNR) lateral to C7, 8 vertebrosternal ribs, supernumerary lumbar ribs (LSNR) lateral to L1, extra presacral vertebrae, and the induction of vertebral and/or rib malformations. RA and VA administration on GD 6 caused posteriorization in the cervico-thoracic region (CSNR) while GD 8 exposure to any of the three compounds resulted in anteriorizations in the thoraco-lumbar area (LSNR and an increase in the number of presacral vertebrae). These effects occurred across regions of the axial skeleton. Analysis of gene expression demonstrated changes in the anterior boundaries of Hoxa7 expression domains in embryos treated on GD 6 and 8 with RA. VA and Br did not induce any statistically significant alterations in Hoxa7 and none of the compounds caused alterations in Hoxa10 expression domains. The studies indicate that RA GD 6 treatment-induced Hoxa7 shifts were rostral (posteriorization) while the RA-induced GD 8 anterior expression boundary shift was caudal (anteriorization), correlating with the axial skeletal changes noted. These data suggest that xenobiotic compounds such as VA and Br may induce similar axial skeletal changes by affecting different components of the developmental processes involved in the patterning of the axial skeleton.
    Journal of Biochemical and Molecular Toxicology 02/2003; 17(6):346-56. · 1.32 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Trenbolone acetate is a synthetic steroid that is extensively used in the United States as a growth promoter in beef cattle. The acetate is administered to livestock via slow-release implants; some is converted by the animal to 17-β-trenbolone, a relatively potent androgen receptor agonist in mammalian systems. Recent studies indicate that excreted 17-β-trenbolone is comparatively stable in animal waste, suggesting the potential for exposure to aquatic animals via direct discharge, runoff, or both. However, little is known concerning the toxicity of trenbolone to fish. Our goal was to assess the effects of 17-β-trenbolone on reproductive endocrinology of the fathead minnow (Pimephales promelas). An in vitro competitive binding study with the fathead minnow androgen receptor demonstrated that 17-β-trenbolone had a higher affinity for the receptor than that of the endogenous ligand, testosterone. Male and female fish were exposed for 21 d to nominal (target) concentrations of 17-β-trenbolone ranging from 0.005 to 50 μg/L. Fecundity of the fish was significantly reduced by exposure to measured test concentrations ≥ 0.027 μg/ L. The 17-β-trenbolone was clearly androgenic in vivo at these concentrations, as evidenced by the de novo production in females of dorsal (nuptial) tubercles, structures normally present only on the heads of mature males. Plasma steroid (testosterone and β-estradiol) and vitellogenin concentrations in the females all were significantly reduced by exposure to 17-β-trenbolone. The 17-β-trenbolone also altered reproductive physiology of male fathead minnows, albeit at concentrations much higher than those producing effects in females. Males exposed to 17-β-trenbolone at 41 μg/L (measured) exhibited decreased plasma concentrations of 11-ketotestosterone and increased concentrations of β-estradiol and vitellogenin. Overall, our studies indicate that 17-β-trenbolone is a potent androgen and reproductive toxicant in fish. Given the widespread use of trenbolone acetate as a growth promoter, and relative stability of its metabolites in animal wastes, further studies are warranted to assess potential ecological risk.
    Environmental Toxicology and Chemistry 01/2003; 22(6):1350-1360. · 2.83 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The discovery of xenobiotics that interfere with androgen activity has highlighted the need to assess chemicals for their ability to modulate dihydrotestosterone (DHT)-receptor binding. Previous test systems have used cells transfected with plasmid containing a reporter gene. Here we report the use of transduction for gene delivery and assessment of the modulation of DHT-induced gene activation. Transduction, the ability of replication-defective viruses to deliver biologically competent genes, is a well understood biological process, which has been utilized to repair defective genes in humans as well as to express exogenous genes in rodent models. Human breast carcinoma cells (MDA-MB-453) containing endogenous copies of the androgen (hAR) and glucocorticoid (GR) receptors were transduced with replication-defective human adenovirus type 5 containing the luciferase (Luc) reporter gene driven by the AR- and GR-responsive glucocorticoid-inducible hormone response element found with the mammary tumor virus LTR (Ad/MLUC7). In a second set of experiments, CV-1 cells were transduced as above with MMTV-luc and also hAR. Cells were subcultured in 96-well plates, transduced with virus, exposed to chemicals, incubated for 48 h, lysed, and assayed for luciferase. Luc gene expression was induced in a dose-dependent manner by DHT, estradiol, and dexamethasone (MDA only) and inhibited by AR antagonist hydroxyflutamide (OHF), hydroxy-DDE, HPTE (2,2-bis(p-hydroxyphenyl)-1,1, 1-trichloroethane), a methoxychlor metabolite, and M1 and M2 (vinclozolin metabolites). The transduced cells responded to AR agonists and antagonists as predicted from our other studies, with a very robust and reproducible response. Over all replicates, 0.1 nM DHT induced luc expression by about 45-fold in CV-1 cells (intra-assay CV = 20%) and 1micromolar OHF inhibited DHT by about 80%. In the transduced MDA cells, 0.1 nM DHT induced luc by about 24-fold (intra-assay CV = 33%), which was inhibited by OHF by about 85%. DHT-induced luciferase activity peaked in both cell lines between 1 and 100 nM, displaying about 64- and 115-fold maximal induction in the CV-1 and MDA 453 cells, respectively. For agonists, a two-fold induction of luc over media control was statistically significant. For AR antagonists, a 25-30% inhibition of DHT-induced luc expression was typically statistically significant. Comparing the two assays, the transduced CV-1 cells were slightly more sensitive to AR-mediated responses, but the transduced MDA 453 cells were more responsive to GR agonists. In summary, these assays correctly identified the endocrine activity of all chemicals examined and displayed sensitivity with a relatively low variability and a high-fold induction over background. Adenovirus transduction for EDC screening has the potential to be employed in a high-throughput mode, and could easily be applied to other cell lines and utilized to deliver other receptors and reporter genes.
    Toxicological Sciences 04/2002; 66(1):82-90. · 4.48 Impact Factor
  • Phillip C. Hartig, Mary C. Cardon
    [Show abstract] [Hide abstract]
    ABSTRACT: Recombinant baculoviruses have been used to produce foreign proteins and have the potential to be safe, efficacious insecticides. Isolation of recombinant virus is usually by plaque phenotype. Typical recombination rates are less than 1%, thus requiring time consuming inspection of hundreds of individual plaques. We describe a method of generating recombinants which requires less time than current protocols and frequently produces recombinants at rates exceeding 30%. This protocol employs liposome-mediated transfection, reduced post-transfection incubation times, linearized parental virus which produces occlusion positive plaques in clones of the parental genotype, and colorimetric detection of recombinants. This protocol allows the initial, and frequently the final, isolation of recombinants in 7 days.
    Journal of Virological Methods 08/1992; 38(1):61-70. · 1.88 Impact Factor

Publication Stats

435 Citations
76.48 Total Impact Points

Institutions

  • 2009–2010
    • North Carolina State University
      • Department of Molecular Biomedical Sciences
      Raleigh, NC, United States
  • 2005–2006
    • United States Environmental Protection Agency
      • • Office of Research and Development
      • • Mid-Continent Ecology Division
      Washington, D. C., DC, United States