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Comparative Study
. 2007 May;115(5):720-7.
doi: 10.1289/ehp.9758. Epub 2007 Feb 5.

Atrazine-induced aromatase expression is SF-1 dependent: implications for endocrine disruption in wildlife and reproductive cancers in humans

WuQiang Fan  1 , Toshihiko Yanase, Hidetaka Morinaga, Shigeki Gondo, Taijiro Okabe, Masatoshi Nomura, Tomoko Komatsu, Ken-Ichirou Morohashi, Tyrone B Hayes, Ryoichi Takayanagi, Hajime Nawata
Affiliations
Comparative Study

Atrazine-induced aromatase expression is SF-1 dependent: implications for endocrine disruption in wildlife and reproductive cancers in humans

WuQiang Fan et al. Environ Health Perspect. 2007 May.

Abstract

Background: Atrazine is a potent endocrine disruptor that increases aromatase expression in some human cancer cell lines. The mechanism involves the inhibition of phosphodiesterase and subsequent elevation of cAMP.

Methods: We compared steroidogenic factor 1 (SF-1) expression in atrazine responsive and non-responsive cell lines and transfected SF-1 into nonresponsive cell lines to assess SF-1's role in atrazine-induced aromatase. We used a luciferase reporter driven by the SF-1-dependent aromatase promoter (ArPII) to examine activation of this promoter by atrazine and the related simazine. We mutated the SF-1 binding site to confirm the role of SF-1. We also examined effects of 55 other chemicals. Finally, we examined the ability of atrazine and simazine to bind to SF-1 and enhance SF-1 binding to ArPII.

Results: Atrazine-responsive adrenal carcinoma cells (H295R) expressed 54 times more SF-1 than nonresponsive ovarian granulosa KGN cells. Exogenous SF-1 conveyed atrazine-responsiveness to otherwise nonresponsive KGN and NIH/3T3 cells. Atrazine induced binding of SF-1 to chromatin and mutation of the SF-1 binding site in ArPII eliminated SF-1 binding and atrazine-responsiveness in H295R cells. Out of 55 chemicals examined, only atrazine, simazine, and benzopyrene induced luciferase via ArPII. Atrazine bound directly to SF-1, showing that atrazine is a ligand for this "orphan" receptor.

Conclusion: The current findings are consistent with atrazine's endocrine-disrupting effects in fish, amphibians, and reptiles; the induction of mammary and prostate cancer in laboratory rodents; and correlations between atrazine and similar reproductive cancers in humans. This study highlights the importance of atrazine as a risk factor in endocrine disruption in wildlife and reproductive cancers in laboratory rodents and humans.

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Figures

Figure 1
Figure 1
Effects of SF-1 on induction of CYP19 by 10−5 mol/L atrazine. (A) SF-1 expression was significantly higher (54-fold; ANOVA, p < 0.05) in atrazine-responsive H295R cells compared with atrazine-nonresponsive KGN cells. (B) SF-1 protein levels were also higher in H295R cells as determined by Western blot.
Figure 2
Figure 2
Effects of atrazine and simazine (10−5 mol/L each for A and B; as marked for C) of three cell types, measured by RLA. (A) Atrazine and simazine stimulated ArPII in H295R cells without exogenous SF-1 supplementation. (B) ArPII response to atrazine and simazine in NIH-3T3 cells required coexpression of SF1. (C) Atrazine and simazine stimulation of SF-1–mediated ArPII in SF-1–co-transfected NIH-3T3 cells was dose dependent. Both triazines were effective at concentrations as low as 10−7 mol/L (ANOVA, p < 0.05). Bars show mean ± SD; letters above bars indicate statistical groups (ANOVA, p < 0.05).
Figure 3
Figure 3
RLA (mean ± SD) of endocrine disruptors that affect forskolin-enhanced SF-1–mediated ArPII presented as chemicals with no effect on luciferase activity, those with significantly induced luciferase activity, or those with significantly inhibited luciferase activity as determined by ANOVA, followed by Fisher’s protected least-significant-difference post hoc test (p < 0.05). Abbreviations: 2,4-DCP, 2,4-dichlorophenol; 2,4-PA, 2,4-dichlorophenoxy acetic acid; BHC, benzene hexachloride; DBCP, dibromochloropropane; DDD, 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane; DDE, 1,1-dichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl)ethylene; DDT, 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane; DEHP, di-2-(ethyl-hexyl)-phthalate; DOA, dioctyl adipate; NAC, N-acetylcysteine; NIP, dinitrophenyl-phosphorothioate; TMBP, 4-(1,1,3,3- tetramethylbutyl) phenol. All chemicals were examined at ecologically relevant concentrations: 10−5 mol/L for all chemicals, except tributyltin and triphenyltin, which were examined at 10−7 mol/L.
Figure 4
Figure 4
Effects of adeno-SF1 on responsiveness of KGN cells (mean ± SD) to 10−5 mol/L atrazine or 10−5 mol/L simazine. (A) Basal aromatase mRNA (CYP19; relative copies) was significantly increased in cells transfected with adeno-SF-1 relative to controls infected with adeno-LacZ. (B) Aromatase enzymatic activity (fold change) also increased in response to atrazine or simazine in adeno-SF-1 infected KGN cells, but not in the control adeno-LacZ infected cells. Letters above bars indicate statistical groups (ANOVA, p < 0.05).
Figure 5
Figure 5
Effects of atrazine and simazine (10−5 mol/L) on SF-1 binding to ArPII. Atrazine and simazine enhanced SF-1–ArPII interactions in H295R cells as determined by common PCR (28 cycles; A) and as quantified by real-time PCR (B); mutation of the SF-1 binding site on ArPII significantly reduced responsiveness to atrazine. The responsiveness to atrazine and simazine was well preserved when ArPII was reduced to 516 bp (ArPII-516), but responsiveness was lost when the SF-1 binding site was mutatated to ATTTCA (ArPII-516-SF1-M) (C). In (B) and (C), bars show mean ± SD. Letters above bars indicate statistical groups (ANOVA, p < 0.05).
Figure 6
Figure 6
Kinetic analysis of atrazine binding to SF-1 protein as analyzed by SPR [changes in mass concentration are detected as differences in the refractive index and shown in resonance units (RU)]. (A) Sensorgrams of six concentrations of 16PC (the positive control ligand) added to SF-1. (B) Sensorgrams showing atrazine, benzophenone, or p-nitrotoluene (100 μM each) binding to immobilized SF-1 (only atrazine caused a significant response). (C) Sensorgrams of six concentrations of atrazine added to the immobilized SF-1 (note the dose-dependent response). (D) Sensorgrams showing interactions between atrazine and SF-1 on a quartz-crystal microbalance. 16PC caused a clear decrease in frequency, demonstrating binding between the ligand and SF-1; atrazine also substantially decreased the frequency, but ethanol (EtOH) and the negative control p-nitrotoluene did not.
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