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Toxicology and Applied Pharmacology
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Toxicology and Applied Pharmacology
Evaluation of a bioluminescent mouse model expressing aromatase PII-promoter-controlled luciferase as a tool for the study of endocrine disrupting chemicals
Patricia Rivest, Patrick J. Devine, J. Thomas Sanderson * INRS-Institut Armand-Frappier, Université du Québec, 531 blv des Prairies, Laval, QC, Canada H7V 1B7
ARTICLE INFO
Article history: Received 8 July 2010 Revised 6 August 2010 Accepted 10 August 2010 Available online 17 August 2010
Keywords:
Bioluminescence Aromatase (CYP19) pII promoter Atrazine Endocrine disruption In vivo imaging
ABSTRACT
Dysfunction of the enzyme aromatase (CYP19) is associated with endocrine pathologies such as osteoporosis, impaired fertility and development of hormone-dependent cancers. Certain endocrine disrupting chemicals affect aromatase expression and activity in vitro, but little is known about their ability to do so in vivo. We evaluated a bioluminescent mouse model (LPTA®)CD-1-Tg(Cyp19-luc)-Xen) expressing luciferase under control of the gonadal aromatase pII promoter as an in vivo screening tool for chemicals that may affect aromatase expression. We studied the effects of forskolin, pregnant mare serum gonadotropin and atrazine in this model (atrazine was previously shown to induced pII-promoter-driven aromatase expression in H295R human adrenocortical carcinoma cells). About 2-4 out of every group of 10 male or female Cyp19-luc mice injected i.p. with 10 mg/kg forskolin had increased gonadal bioluminescence after 3- 5 days compared to controls; the others appeared non-responsive. Similarly, about 4 per group of 9 individual females injected with pregnant mare serum gonadotropin had increased ovarian bioluminescence after 24 h. There was a statistically significant correlation between ovarian bioluminescence and plasma estradiol concentrations (n= 14; p=0.022). Males exposed to a single dose of 100 mg/kg or males and females exposed to 5 daily injections of 30 mg/kg atrazine showed no change in gonadal bioluminescence over a 7 day period, but a significant interaction was found between atrazine (100 mg/kg) and time in female mice (p<0.05; two-way ANOVA). Ex vivo luciferase activity in dissected organs was increased by forskolin in testis, epididymis and ovaries. Atrazine (30 mg/kg/day) increased (30%) luciferase activity significantly in epididymis only. In conclusion, certain individual Cyp19-luc mice are highly responsive to aromatase inducers, suggesting this model, with further optimization, may have potential as an in vivo screening tool for environmental contaminants.
@ 2010 Elsevier Inc. All rights reserved.
Introduction
Bioluminescence-based tools have become increasing available to the scientific community in the form of bioluminescent transgenic mice and cell lines as well as affordable imaging equipment. Transgenic mice transfected genomically with a luciferase gene under the control of various promoters of toxicological interest may offer highly sensitive research tools for the in vivo study of effects of disease states and xenobiotics on important physiological processes. One potential target for environmentally and toxicologically relevant chemicals, such as endocrine disruptors, is the enzyme aromatase (cytochrome P450 19; CYP19), which catalyzes the conversion of androgens to estrogens. Over-expression of aromatase has been associated with diseases such as breast and uterine cancer, and
endometriosis, whereas reduced expression has been implicated in osteoporosis and disruption of estrus and sperm quality (Pasqualini et al., 1996; Bulun, 2000).
Current approaches to the screening of effects of chemicals on steroidogenesis use mostly in vitro models. Human placental micro- somes and various human cell lines and cells in primary culture or co- culture have been used for the investigation of effects of xenobiotics on aromatase activity and/or gene expression, each with its advantages and disadvantages (reviewed in (Sanderson, 2006). Microsomal fractions are limited to the identification of inhibitors and do not account for cytotoxicity. Studies with cell lines have as advantage that they have the potential to identify inducers in addition to inhibitors, and further detailed studies of mechanisms of aromatase induction are possible. Primary cell cultures, although more relevant for mechanistic studies and environmental/species relevance than cancer cell lines, have as disadvantage that they are difficult to obtain, maintain and standardize, and loss of gene expression may occur over time. In vitro studies in human placental microsomes or cell lines have shown that aromatase may be inhibited or induced by various
* Corresponding author. INRS-Institut Armand-Frappier, Édifice 18-PRF-K110, 531 blv des Prairies, Laval, QC, Canada H7V 1B7. Fax: + 1 514 686 5309. E-mail addresses: patricia.rivest@iaf.inrs.ca (P. Rivest), patrick.devine@iaf.inrs.ca
(P.J. Devine), thomas.sanderson@iaf.inrs.ca (J.T. Sanderson).
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environmental chemicals, medications or naturally occurring dietary compounds (Mason et al., 1987; Ibrahim and Abul-Hajj, 1990; Campbell and Kurzer, 1993; Yue and Brodie, 1997; Drenth et al., 1998; Le Bail et al., 1998; Vinggaard et al., 2000; Saarinen et al., 2001; Andersen et al., 2002; Sanderson et al., 2002, 2004; Fernandez-Canton et al., 2003; Ohno et al., 2004; Heneweer et al., 2005; Letcher et al., 2005; Sanderson, 2006; He et al., 2008). For example, various dietary (iso)flavonoids induce (genistein, quercetin) or inhibit (chrysin, apigenin) human aromatase activity (Sanderson et al., 2004) and anti-breast cancer medications, such as fadrozole, letrozole and anastrozole (Miller and Dixon, 2000), as well as various pesticides (Vinggaard et al., 2000; Sanderson et al., 2002), are highly potent inhibitors of its catalytic activity. The pesticide atrazine has been shown to induce the catalytic activity of aromatase and pII promoter- mediated CYP19 gene expression in H295R human adrenocortical cells (Sanderson et al., 2000, 2001, 2002). Atrazine also induced aromatase activity in JEG-3 human placental choriocarcinoma and luteinized ovarian granulosa cells (Sanderson et al., 2001; Holloway et al., 2008). Atrazine has further been associated with disturbances in the balance between androgens and estrogens in environmentally exposed wildlife (Guillette et al., 1994; Crain et al., 1997; Crain et al., 1999; Guillette and Gunderson, 2001); Similarly, an increased estrogen-to- androgen ratio was observed in vivo with rats (Babic-Gojmerac et al., 1989; Laws et al., 2000; Stoker et al., 2000, 2002) and African clawed (Xenopus laevis) frogs (Coady et al., 2005; Hecker et al., 2005),
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although in X. laevis no association with altered aromatase activity could be made.
The commercially available bioluminescent mouse model LPTARCD-1-Tg(Cyp19-luc)-Xen (line 125) contains in its genome a modified firefly luciferase gene (Promega pGL-3) under control of the murine gonad-specific aromatase pII-promoter region. This 400 bp promoter region contains all the regulatory elements required for aromatase expression. We characterized this transgenic mouse model to evaluate its potential as an in vivo screening tool for chemicals that may alter CYP19 gene expression.
Materials and methods
Transgenic mice, treatments and imaging. Two breeding pairs of LPTARCD-1-Tg(Cyp19-luc)-Xen mice (founder line 125; transgenic males; CD-1 wild-type females) were a kind gift from Caliper- LifeSciences, Cranbury, NJ. A colony was maintained by crossing transgenic males with wild-type CD-1 females to produce 8 generations of experimental animals over a two-year period (2008-2009). Mice were housed in a 12-h light/dark cycle at 21 ℃ and treated according to the rules of our animal care facility and the requirements of the Canadian Council on Animal Care (CCAC). At 21-days of age animals were separated by sex, weighed and held 5 mice per cage. Presence of the transgene in the offspring was verified by injecting the mice intraperitoneally (i.p.) with 150 mg/ kg D-luciferin (optimized for in vivo imaging, Caliper-LifeSciences) dissolved in 0.9% saline solution, followed 15 min later by scanning for 60 s under isoflurane anesthesia in an IVIS 100 Imager (Caliper LifeSciences). Males were scanned ventrally (on their backs with belly up) and females dorsally for an optimal view of gonadal bioluminescence. Groups of 5 mice per sex and per treatment were then injected i.p. with either forskolin (10 mg/kg; Sigma-Aldrich, St Louis, MO), atrazine (100 mg/kg; Sigma-Aldrich) or with saline solution containing 5% DMSO (Sigma-Aldrich) as vehicle control and assigned to treatment day 1. A repeated-dose experiment was also performed with atrazine by injecting mice with 30 mg/kg atrazine for 5 days. All animals were scanned daily for assessment of gonadal bioluminescence intensity until day 5 (forskolin) or day 7 (atrazine) after initial treatment. A hormonal stimulation (super- ovulation) experiment (three independent times) was carried out in both sexes by injecting 5 mice per experiment with 5 IU PMSG (pregnant mare serum gonadotropin) or vehicle control (n=2) on day 1 and hCG (human choriogonadotropin) on day 3; these animals were scanned daily until day 5. A further experiment was carried out by injecting mice (n=9) with PMSG, or vehicle control (n=9) and monitoring them for 2 days. In these mice plasma estradiol levels were determined by radioimmunoassay (DSL4800, Diagnostics Systems Laboratories, Webster, TX). Gonadal biolumi- nescence intensities were quantified using Living Image software version 3.0.4 (Caliper-LifeSciences). To ensure consistency and objectivity in quantifying gonadal bioluminescent intensities, a fixed integration area (determined by the mouse with the greatest response) was used to quantify bioluminescence in all mice per experiment. This same integration area was also applied to an area of each mouse where there was no bioluminescence (background). This background was subtracted from the bioluminescence values for each mouse to reduce potential overestimation of gonadal bioluminescence in mice where the integration area was larger than the response. This approach resulted in highly reproducible bioluminescence values.
Luciferase expression ex vivo. Testes, epididymides and liver (neg- ative control) of the males, and ovaries, salivary gland, brain and liver of the females were isolated on day 5 of treatment. Tissues were homogenized and assayed for luciferase activity using the Luciferase Assay System (#E1500; Promega, Madison WI) in the
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presence of a protease inhibitor. After 10 min of incubation on ice, the homogenates were vortexed for 30 s and underwent centrifu- gation for 5 min at 6000 rpm. Supernatants were kept at -80 ℃ until analysis. Protein concentrations of the supernatants were determined by a bicinchoninic acid (BCA) protein assay (Pierce, IL, USA, kit #23225) and equal amounts of protein were added to 96- well opaque white plates, together with 50 uL of luciferin reagent for determination of bioluminescence using a SpectraMax M5 multifunctional plate reader (Molecular Devices, Sunnyvale, CA). Luminescence was monitored using an integration time of 1250 ms.
Statistical analyses. Means were presented with standard deviations (SD). Statistically significant (p<0.05) differences from control were determined by Student t-test or one-way ANOVA with Tukey posteriori test (GraphPad Prism, GraphPad Software v5.03, San Diego CA). Statistically significant effects of treatment compared to control over time were determined by two-way ANOVA with Bonferroni posteriori test (GraphPad Prism). Statistically significant deviations of individual responses from the mean response of the experimental sample population were determined using a z-test for outliers (Zar, 1999).
Results
Localization and age-dependency of luciferase expression in vivo
Cyp19-luc mice had identical weight gain over time to wild- type CD-1 mice (not shown). Aromatase pII-promoter-mediated luciferase expression, as measured by bioluminescence, was restricted to the testes and epididymis in males (Fig. 1) and to the ovaries, brain and salivary glands (Fig. 1) in females. This was
confirmed by injecting a small number of male and female mice with D-luciferin followed 15 min later by euthanasia, rapid dissection and scanning of bioluminescence (not shown). Biolu- minescence almost doubled between 21 and 49 days and stabi- lized between 49 and 120 days of age in the gonads of males (Fig. 2). In the ovaries of females there was a statistically significant increase (40%) in bioluminescence between 49 and 120 days of age, although not between these time points and 21- day old mice (Fig. 2). Basal in vivo luminescence from the gonads was about 50-fold greater in males than females.
Effects of pharmacological aromatase inducers and atrazine on luciferase expression in vivo
Forskolin, a stimulant of adenylate cyclase, given once at 10 mg/ kg, did not increase average gonadal luciferase activity in 21-day old male (n=5) or female (n=5) Cyp19-luc mice (Figs. 3A and B) compared to control (n = 5 per sex) (saline with 5% DMSO), although when individually analyzed several mice appeared to respond with about a two-fold induction (Figs. 3C and D). Out of three experiments, 6/15 male and 5/15 female mice responded to forskolin with a statistically significant increase above controls (p<0.05). Cyp19-luc mice of either sex treated with forskolin at a more mature age (49 and 60 days) did not differ in individual responsiveness from 21-d old mice (not shown). Several, but not all (9/20 over 4 experiments), 21- d old female mice that underwent a treatment protocol to induce super-ovulation responded to PMSG (an FSH substitute) with a statistically significant increase in ovarian bioluminescence after 24 h. This response returned to basal levels 24 h later, once the mice were injected with hCG (Fig. 4A). In males that underwent the same treatment no statistically significant changes in gonadal biolumines- cence were observed compared to control at any time-point (Fig. 4B).
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To examine the response to PMSG alone, female Cyp19-luc mice were scanned before being exposed on day 0, and then scanned again 24 and 48 h later. PMSG caused a statistically significant increase (two- way ANOVA, p<0.01) in mean ovarian bioluminescence in the treated animals compared to controls on day 1 and 2 (only the individual responses are shown in Fig. 5). Control bioluminescence values (mean±SD) on day 0 (n=9), 1 (n=9) and 2 (n=7) were 8.6±0.52×105,9.5±1.1×105 and 6.2±0.71x105 photons/s, re- spectively; bioluminescence values of PMSG-treated mice were 10.1 ±0.61×105,22.7±4.35×105 and 21.0±4.58x105, respectively. Graphical presentation of the ovarian bioluminescent response of individual animals clearly demonstrated that of the females treated with PMSG, 4 out of 9 mice on days 1 and 5 out of 7 on day 2 had a response that was between 2 and 5 times greater than the average control response (Fig. 5A). Mice were sacrificed on the final day and plasma estradiol levels were determined. There was a statistically significant correlation between ovarian bioluminescence and estra- diol levels (p=0.022) (Fig. 5B).
Male and female mice exposed to a single dose of 100 mg/kg or 5 daily doses of 30 mg/kg atrazine did not demonstrate any differences in mean gonadal bioluminescence compared to control (5% DMSO in saline) over a 7-day period (Fig. 6). However, in females exposed to 100 mg/kg atrazine, two-way ANOVA revealed a statistically signif- icant time-dependent increase in bioluminescence of treated, but not control mice (p<0.05), but not quite a significant difference between atrazine-treated and control mice at any given time-point (p=0.071). Dexamethasone had no effect on pII-promoter-mediated biolumines- cence in mice of either sex (not shown).
Ex vivo analysis of luciferase expression after in vivo exposure to pharmacological aromatase inducers and atrazine
Bioluminescence was measured in tissues from Cyp19-luc mice 5 days after start of the treatments described above. Forskolin (10 mg/kg) caused a statistically significant increase in pII promot- er-mediated luciferase activity in testis and epididymis in males, and ovary, but not brain or salivary gland, in females (Fig. 7). Atrazine (30 mg/kg/d for 5 days) caused a statistically significant induction of luciferase activity in epididymis (the main site of luciferase expression in male Cyp19-luc mice), but no effect in any other tissues in either sex (Fig. 8).
Discussion
Patho-toxicological relevance of pII promoter-mediated aromatase expression
Aromatase is regulated in a highly tissue-specific way in humans (Mahendroo et al., 1993; Agarwal et al., 1996; Zhao et al., 1997; Kamat et al., 2002; Bulun et al., 2003). In ovaries and testes the CYP19 aromatase gene is mainly under the control of the gonadal proximal pII promoter, which is cAMP-responsive via activation of the protein kinase A pathway. This pathway is activated by gonadotropins such as FSH and LH via their respective Gs-protein-coupled receptors (Simpson, 2003). In other aromatase-expressing tissues, such as breast adipose, skin fibroblast, brain, and placenta, the pII promoter is normally silent, and other promoters in the intron I regulatory region
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control its expression. In breast adipose tissue, for example, aromatase is regulated by the relatively weak glucocorticoid-responsive I.4 promoter (Bulun et al., 2005), with minimal contribution from the pII promoter (Mahendroo et al., 1993). However, in certain disease states such as breast cancer and endometriosis, a “promoter switch” occurs and the tissues become responsive to hormones and other signaling factors that stimulate aromatase expression via multiple promoters, most notably the highly efficient proximal pII promoter (Harada et al., 1993; Agarwal et al., 1996; Zhou et al., 2001). The sudden contribution of multiple promoters to aromatase expression results in local over- expression of the enzyme and increased in situ synthesis of estrogens, which stimulate hormone-dependent growth of proximal tumor cells and contribute to tumor progression. Certain chemicals have been shown to induce aromatase in human cancer cells in vitro (Sanderson et al., 2000, 2001, 2002, 2004; Heneweer et al., 2004; Morinaga et al., 2004; Canton et al., 2005) and in several studies this has been shown to occur via activation of pII promoter-mediated CYP19 gene expression (Heneweer et al., 2004; Sanderson et al., 2004; Fan et al., 2007a,b). Thus, the inappropriate activation of the aromatase pII promoter is of pathophysiological and toxicological importance; however, the relevance and/or significance of the reported in vitro effects for the in vivo situation are poorly, if at all, understood.
In vivo responsiveness of Cyp19-luc transgenic mice to pharmacological inducers and atrazine
The present study has aimed to evaluate an in vivo bioluminescent transgenic mouse model for its ability to identify compounds that may
induce pII-promoter-mediated expression of Cyp19 gene expression. The (LPTA®)CD-1-Tg(Cyp19-luc)-Xen) bioluminescent mouse model demonstrated physiologically relevant localized Cyp19 pII-promoter regulated luciferase expression in male and female gonadal tissues (Fig. 1). Forskolin, an established potent inducer of pII promoter- mediated aromatase gene expression in vitro by stimulating cAMP generation (Agarwal et al., 1995; Sanderson et al., 2004; Watanabe and Nakajin, 2004), caused only a modest increase of in vivo pII- mediated bioluminescence (Fig. 3) and ex vivo luciferase activity (Fig. 7) in Cyp19-luc mice. We noted in our experiments that out of every 10 mice exposed to forskolin only about 2-4 mice would respond with increased bioluminescence, whereas most mice appeared non-responsive; the mice that did respond, responded well with a statistically significant 2- to 3-fold increase in biolumi- nescence. A similar phenomenon was observed when female mice underwent a super-ovulation protocol; in each experiment, approx- imately 2 out of every 5 mice treated would respond with a marked (>2-fold) increase in bioluminescence 24h after PMSG injection (Fig. 4). When female mice were injected with PMSG alone (avoiding the influence of hCG, which abolishes increased aromatase expression by inducing ovulation and loss of aromatase expressing granulosa cells) and were monitored for 2 days does clearly indicate that there is a subpopulation of female Cyp19-luc mice that is highly responsive (up to 5 fold increased bioluminescence above control) (Fig. 5). Given the fact that atrazine has been shown to be a weaker inducer of aromatase in vitro than forskolin, FSH analogs and/or analogs of their downstream effector cAMP (Sanderson et al., 2000, 2001, 2002), it is not surprising that atrazine exposures in our study showed weaker responses and fewer statistically significant increases in biolumines- cence in vivo (Fig. 6) or ex vivo (Fig. 8) compared to controls in this transgenic mouse model. We decisively used doses of atrazine that were toxicologically relevant, although at the higher end of the spectrum of environmental exposures. The lack of response to dexamethasone which stimulates adipose aromatase I.4 promoter activity (Mahendroo et al., 1993; Zhao et al., 1995; Agarwal et al., 1996) is consistent with the lack of this promoter activity in gonadal tissues (Bulun et al., 2003).
Limitations of the in vivo Cyp-luc bioluminescent mouse model
There are several variables to consider that may affect biolumi- nescent signal when working with bioluminescent mammals, most notably physical factors such as fat content, fur thickness and position of the animal when scanning, which probably contribute to some of the variability we observed. However, the sporadic (individual- dependent) response to the prototype aromatase inducer forskolin observed with our transgenic mice requires a different explanation. One possibility is that the mice differ in responsiveness because they are in different phases of their estrus cycle. However, when the mice underwent a super-ovulation protocol, which synchronizes estrus cycle, a similar frequency of non-responsiveness was observed to treatment with forskolin.
Founder line 125 was created by introducing the transgene construct to CD-1 donor embryos by a microinjection technique (personal communication Caliper-LifeSciences). The genomic envi- ronment of the transgene construct will likely be different from the actual aromatase gene and its upstream pII-promoter region. This may have an influence on the effectiveness of various transcription factors that are required for successful usage of the gonadal Cyp19-pII promoter, such as SF-1/NR5A1 (steroidogenic factor 1/nuclear receptor 5A1) and LRH-1/NR5A2 (liver receptor homologue-1) (Sirianni et al., 2002; Pezzi et al., 2004). Another aspect of the influence of location on the pII-promoter activity is the potential for alterations in methylation state of the newly introduced DNA construct, which can be individual dependent (Demura and Bulun, 2008). The pII promoter region contains two cAMP-responsive
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elements (CREs) that are important for promoter activation (Demura and Bulun, 2008). These CREs contain CpG dinucleotides which are targets for DNA methylation, with methylated sites reducing CREB binding and inhibiting pII-promoter activity, an important epigenetic mechanism used to silence promoter activity of unnecessary or unwanted genes in tissues (Siegfried and Cedar, 1997). Differential responsiveness to cAMP-stimulated aromatase induction has been observed in skin fibroblasts from individual human volunteers (Demura and Bulun, 2008). In the individuals whose fibroblasts were responsive to cAMP stimulation of pII-promoter-derived CYP19 transcripts, four out of six CpG sites were unmethylated, whereas in non-responsive fibroblasts all six positions were methylated. These observed differences in individual degree of hypermethylation may play a role in the selective responsiveness of individual Cyp19-luc mice to forskolin and other inducers of Cyp19 pII-promoter-mediated luciferase expression.
Conclusions and perspectives
We evaluated the (LPTAR)CD-1-Tg(Cyp19-luc)-Xen) biolumines- cent mouse model as an in vivo screening tool for the identification of endocrine disrupting chemicals that may alter pII-promoter-mediated Cyp19 expression. Within the population, certain individuals responded well to known pharmacological inducers of aromatase activity, but about half the mice appeared poorly or non-responsive. The herbicide atrazine, which has been shown to induce pII promoter- mediated aromatase activity in vitro (Heneweer et al., 2004), had a weak, but statistically significant effect on pII promoter-mediated bioluminescence in vivo (females at 100 mg/kg) and increased bioluminescence ex vivo with statistical significance only in the
male epididymis. The Cyp19-luc mouse model may, in the future, be optimized for our intended purpose by breeding highly-responsive individuals to obtain a sub-population that responds well to aromatase-inducing chemicals. Overall, we conclude this mouse model has potential, but that in its current form it is of limited use for our intended purpose. Nevertheless, this model may be useful for other types of reproductive studies involving aromatase expression.
Conflict of interest statement
Funding for this research was from NSERC grants to Patrick Devine and Thomas Sanderson, and Caliper-LifeSciences. The financial contribution from Caliper-LifeSciences was entirely without restric- tions or requirements that would influence experimental design, interpretation of results or any other impairment to freely publishing this study regardless of outcome. There were no competing financial interests for any of the authors.
Acknowledgments
This work was supported financially by Natural Sciences and Engineering Research Council (NSERC) of Canada grants to Thomas Sanderson (313313-2005) and Patrick Devine (288304-2009), a financial contribution from Caliper-LifeSciences, and a Fondation Armand-Frappier graduate student bursary to Patricia Rivest. We thank Stephanie Petrillo and To-Quyen Truong for their technical assistance. Patrick Devine currently works at Novartis, Cambridge, MA (patrick.devine@novartis.com).
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References
Agarwal, V.R., Bulun, S.E., Simpson, E.R., 1995. Quantitative detection of alternatively spliced transcripts of the aromatase cytochrome P450 (CYP19) gene in aromatase- expressing human cells by competitive RT-PCR. Mol. Cell. Probes 9, 453-464.
Agarwal, V.R., Bulun, S.E., Leitch, M., Rohrich, R., Simpson, E.R., 1996. Use of alternative promoters to express the aromatase cytochrome P450 (CYP19) gene in breast adipose tissues of cancer-free and breast cancer patients. J. Clin. Endocrinol. Metab. 81, 3843-3849.
Andersen, H.R., Vinggaard, A.M., Rasmussen, T.H., Gjermandsen, I.M., Bonefeld- Jorgensen, E.C., 2002. Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. Toxicol. Appl. Pharmacol. 179, 1-12. Babic-Gojmerac, T., Kniewald, Z., Kniewald, J., 1989. Testosterone metabolism in neuroendocrine organs in male rats under atrazine and deethylatrazine influence. J. Steroid Biochem. 33, 141-146.
Bulun, S.E., 2000. Aromatase deficiency and estrogen resistance: from molecular genetics to clinic. Semin. Reprod. Med. 18, 31-39.
Bulun, S.E., Sebastian, S., Takayama, K., Suzuki, T., Sasano, H., Shozu, M., 2003. The human CYP19 (aromatase P450) gene: update on physiologic roles and genomic organization of promoters. J. Steroid Biochem. Mol. Biol. 86, 219-224.
Bulun, S.E., Lin, Z., Imir, G., Amin, S., Demura, M., Yilmaz, B., Martin, R., Utsunomiya, H., Thung, S., Gurates, B., Tamura, M., Langoi, D., Deb, S., 2005. Regulation of aromatase expression in estrogen-responsive breast and uterine disease: from bench to treatment. Pharmacol. Rev. 57, 359-383.
Campbell, D.R., Kurzer, M.S., 1993. Flavonoid inhibition of aromatase enzyme activity in human preadipocytes. J. Steroid Biochem. Mol. Biol. 46, 381-388.
Canton, R.F., Sanderson, J.T., Letcher, R.J., Bergman, A., van den Berg, M., 2005. Inhibition and induction of aromatase (CYP19) activity by brominated flame retardants in H295R human adrenocortical carcinoma cells. Toxicol. Sci. 88, 447-455.
Coady, K.K., Murphy, M.B., Villeneuve, D.L., Hecker, M., Jones, P.D., Carr, J.A., Solomon, K. R., Smith, E.E., Van Der Kraak, G., Kendall, R.J., Giesy, J.P., 2005. Effects of atrazine on metamorphosis, growth, laryngeal and gonadal development, aromatase activity, and sex steroid concentrations in Xenopus laevis. Ecotoxicol. Environ. Saf. 62, 160-173.
A
1500
3
*
T
Luminescence
1000
T
T
500
0
Liver (Ctl -)
Testes
Epididymis
Organs
B
150
+0
Luminescence
T
100
T
T
50
0
Liver (Cit -)
Ovary
Salivary gland
Brain
Organs
Control (saline)
Atrazine (30 mg/kg/day)
Crain, D.A., Guillette Jr., L.J., Rooney, A.A., Pickford, D.B., 1997. Alterations in steroidogenesis in alligators (Alligator mississippiensis) exposed naturally and experimentally to environmental contaminants. Environ. Health Perspect. 105, 528-533.
Crain, D.A., Spiteri, I.D., Guillette Jr., L.J., 1999. The functional and structural observations of the neonatal reproductive system of alligators exposed in ovo to atrazine, 2, 4-D, or estradiol. Toxicol. Ind. Health 15, 180-185.
Demura, M., Bulun, S.E., 2008. CpG dinucleotide methylation of the CYP19 I.3/II promoter modulates cAMP-stimulated aromatase activity. Mol. Cell. Endocrinol. 283, 127-132.
Drenth, H .- J., Bouwman, C.A., Seinen, W., Van den Berg, M., 1998. Effects of some persistent halogenated environmental contaminants on aromatase (CYP19) activity in the human choriocarcinoma cell line JEG-3. Toxicol. Appl. Pharmacol. 148, 50-55.
Fan, W., Yanase, T., Morinaga, H., Gondo, S., Okabe, T., Nomura, M., Hayes, T.B., Takayanagi, R., Nawata, H., 2007a. Herbicide atrazine activates SF-1 by direct affinity and concomitant co-activators recruitments to induce aromatase expres- sion via promoter II. Biochem. Biophys. Res. Commun. 355, 1012-1018.
Fan, W., Yanase, T., Morinaga, H., Gondo, S., Okabe, T., Nomura, M., Komatsu, T., Morohashi, K., Hayes, T.B., Takayanagi, R., Nawata, H., 2007b. Atrazine-induced aromatase expression is SF-1 dependent: implications for endocrine disruption in wildlife and reproductive cancers in humans. Environ. Health Perspect. 115, 720-727.
Fernandez-Canton, R., Letcher, R.J., Sanderson, J.T., Bergman, A., van den Berg, M., 2003. Effects of brominated flame retardants on activity of the steroidogenic enzyme aromatase (CYP19) in H295R human adrenocortical carcinoma cells in culture. Organohalogen Compd. 61, 104-107.
Guillette Jr., L.J., Gunderson, M.P., 2001. Alterations in development of reproductive and endocrine systems of wildlife populations exposed to endocrine-disrupting contaminants. Reproduction 122, 857-864.
Guillette Jr., L.J., Gross, T.S., Masson, G.R., Matter, J.M., Percival, H.F., Woodward, A.R., 1994. Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lakes in Florida. Environ. Health Perspect. 102, 680-688.
Harada, N., Utsumi, T., Takagi, Y., 1993. Tissue-specific expression of the human aromatase cytochrome P-450 gene by alternative use of multiple exons 1 and promoters, and switching of tissue-specific exons 1 in carcinogenesis. Proc. Natl Acad. Sci. USA 90, 11312-11316.
He, Y., Murphy, M.B., Yu, R.M., Lam, M.H., Hecker, M., Giesy, J.P., Wu, R.S., Lam, P.K., 2008. Effects of 20 PBDE metabolites on steroidogenesis in the H295R cell line. Toxicol. Lett. 176, 230-238.
Hecker, M., Park, J.W., Murphy, M.B., Jones, P.D., Solomon, K.R., Van Der Kraak, G., Carr, J.A., Smith, E.E., du Preez, L., Kendall, R.J., Giesy, J.P., 2005. Effects of atrazine on CYP19 gene expression and aromatase activity in testes and on plasma sex steroid concentrations of male African clawed frogs (Xenopus laevis). Toxicol. Sci. 86, 273-280.
Heneweer, M., van den Berg, M., Sanderson, J.T., 2004. A comparison of human H295R and rat R2C cell lines as in vitro screening tools for effects on aromatase. Toxicol. Lett. 146, 183-194.
Heneweer, M., van den Berg, M., de Geest, M.C., de Jong, P.C., Bergman, A., Sanderson, J.T., 2005. Inhibition of aromatase activity by methyl sulfonyl PCB metabolites in primary culture of human mammary fibroblasts. Toxicol. Appl. Pharmacol. 202, 50-58.
Holloway, A.C., Anger, D.A., Crankshaw, D.J., Wu, M., Foster, W.G., 2008. Atrazine- induced changes in aromatase activity in estrogen sensitive target tissues. J. Appl. Toxicol. 28, 260-270.
Ibrahim, A.R., Abul-Hajj, Y.J., 1990. Aromatase inhibition by flavonoids. J. Steroid Biochem. Mol. Biol. 37, 257-260.
Kamat, A., Hinshelwood, M.M., Murry, B.A., Mendelson, C.R., 2002. Mechanisms in tissue-specific regulation of estrogen biosynthesis in humans. Trends Endocrinol. Metab. 13, 122-128.
Laws, S.C., Ferrell, J.M., Stoker, T.E., Schmid, J., Cooper, R.L., 2000. The effects of atrazine on female wistar rats: an evaluation of the protocol for assessing pubertal development and thyroid function. Toxicol. Sci. 58, 366-376.
Le Bail, J.C., Laroche, T., Marre-Fournier, F., Habrioux, G., 1998. Aromatase and 17beta- hydroxysteroid dehydrogenase inhibition by flavonoids. Cancer Lett. 133, 101-106.
Letcher, R.J., Sanderson, J.T., Bokkers, A., Giesy, J.P., van den Berg, M., 2005. Effects of bisphenol A-related diphenylalkanes on vitellogenin production in male carp (Cyprinus carpio) hepatocytes and aromatase (CYP19) activity in human H295R adrenocortical carcinoma cells. Toxicol. Appl. Pharmacol. 209, 95-104.
Mahendroo, M.S., Mendelson, C.R., Simpson, E.R., 1993. Tissue-specific and hormonally controlled alternative promoters regulate aromatase cytochrome P450 gene expression in human adipose tissue. J. Biol. Chem. 268, 19463-19470.
Mason, J.I., Carr, B.R., Murry, B.A., 1987. Imidazole antimycotics: selective inhibitors of steroid aromatization and progesterone hydroxylation. Steroids 50, 179-189.
Miller, W.R., Dixon, J.M., 2000. Antiaromatase agents: preclinical data and neoadjuvant therapy. Clin. Breast Cancer 1 (Suppl 1), S9-S14.
Morinaga, H., Yanase, T., Nomura, M., Okabe, T., Goto, K., Harada, N., Nawata, H., 2004. A benzimidazole fungicide, benomyl, and its metabolite, carbendazim, induce aromatase activity in a human ovarian granulose-like tumor cell line (KGN). Endocrinology 145, 1860-1869.
Ohno, K., Araki, N., Yanase, T., Nawata, H., Iida, M., 2004. A novel nonradioactive method for measuring aromatase activity using a human ovarian granulosa-like tumor cell line and an estrone ELISA. Toxicol. Sci. 82, 443-450.
Pasqualini, J.R., Chetrite, G., Blacker, C., Feinstein, M.C., Delalonde, L., Talbi, M., Maloche, C., 1996. Concentrations of estrone, estradiol, and estrone sulfate and evaluation of sulfatase and aromatase activities in pre- and postmenopausal breast cancer patients. J. Clin. Endocrinol. Metab. 81, 1460-1464.
Pezzi, V., Sirianni, R., Chimento, A., Maggiolini, M., Bourguiba, S., Delalande, C., Carreau, S., Ando, S., Simpson, E.R., Clyne, C.D., 2004. Differential expression of steroidogenic
factor-1/adrenal 4 binding protein and liver receptor homolog-1 (LRH-1)/ fetoprotein transcription factor in the rat testis: LRH-1 as a potential regulator of testicular aromatase expression. Endocrinology 145, 2186-2196.
Saarinen, N., Joshi, S.C., Ahotupa, M., Li, X., Ammala, J., Makela, S., Santti, R., 2001. No evidence for the in vivo activity of aromatase-inhibiting flavonoids. J. Steroid Biochem. Mol. Biol. 78, 231-239.
Sanderson, J.T., 2006. The steroid hormone biosynthesis pathway as a target for endocrine-disrupting chemicals. Toxicol. Sci. 94, 3-21.
Sanderson, J.T., Seinen, W., Giesy, J.P., van den Berg, M., 2000. 2-Chloro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity? Toxicol. Sci. 54, 121-127.
Sanderson, J.T., Letcher, R.J., Heneweer, M., Giesy, J.P., van den Berg, M., 2001. Effects of chloro-s-triazine herbicides and metabolites on aromatase activity in various human cell lines and on vitellogenin production in male carp hepatocytes. Environ. Health Perspect. 109, 1027-1031.
Sanderson, J.T., Boerma, J., Lansbergen, G.W., van den Berg, M., 2002. Induction and inhibition of aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. Toxicol. Appl. Pharmacol. 182, 44-54.
Sanderson, J.T., Hordijk, J., Denison, M.S., Springsteel, M.F., Nantz, M.H., Van Den Berg, M., 2004. Induction and Inhibition of aromatase (CYP19) activity by natural and synthetic flavonoid compounds in H295R human adrenocortical carcinoma cells. Toxicol. Sci. 82, 70-79.
Siegfried, Z., Cedar, H., 1997. DNA methylation: a molecular lock. Curr. Biol. 7, R305-R307.
Simpson, E.R., 2003. Sources of estrogen and their importance. J. Steroid Biochem. Mol. Biol. 86, 225-230.
Sirianni, R., Seely, J.B., Attia, G., Stocco, D.M., Carr, B.R., Pezzi, V., Rainey, W.E., 2002. Liver receptor homologue-1 is expressed in human steroidogenic tissues and activates transcription of genes encoding steroidogenic enzymes. J. Endocrinol. 174, R13-R17.
Stoker, T.E., Laws, S.C., Guidici, D.L., Cooper, R.L., 2000. The effect of atrazine on puberty in male wistar rats: an evaluation in the protocol for the assessment of pubertal development and thyroid function. Toxicol. Sci. 58, 50-59.
Stoker, T.E., Guidici, D.L., Laws, S.C., Cooper, R.L., 2002. The effects of atrazine metabolites on puberty and thyroid function in the male Wistar rat. Toxicol. Sci. 67, 198-206.
Vinggaard, A.M., Hnida, C., Breinholt, V., Larsen, J.C., 2000. Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro. Toxicol. In Vitro 14, 227-234.
Watanabe, M., Nakajin, S., 2004. Forskolin up-regulates aromatase (CYP19) activity and gene transcripts in the human adrenocortical carcinoma cell line H295R. J. Endocrinol. 180, 125-133.
Yue, W., Brodie, A.M., 1997. Mechanisms of the actions of aromatase inhibitors 4- hydroxyandrostenedione, fadrozole, and aminoglutethimide on aromatase in JEG-3 cell culture. J. Steroid Biochem. Mol. Biol. 63, 317-328.
Zar, J.H., 1999. Biostatistical Analysis. Prentice-Hall, N.J.
Zhao, Y., Nichols, J.E., Bulun, S.E., Mendelson, C.R., Simpson, E.R., 1995. Aromatase P450 gene expression in human adipose tissue. Role of a Jak/STAT pathway in regulation of the adipose-specific promoter. J. Biol. Chem. 270, 16449-16457.
Zhao, Y., Agarwal, V.R., Mendelson, C.R., Simpson, E.R., 1997. Transcriptional regulation of CYP19 gene (aromatase) expression in adipose stromal cells in primary culture. J. Steroid Biochem. Mol. Biol. 61, 203-210.
Zhou, J., Gurates, B., Yang, S., Sebastian, S., Bulun, S.E., 2001. Malignant breast epithelial cells stimulate aromatase expression via promoter II in human adipose fibroblasts: an epithelial-stromal interaction in breast tumors mediated by CCAAT/enhancer binding protein beta. Cancer Res. 61, 2328-2334.