Treatment of BG-1 Ovarian Cancer Cells Expressing Estrogen Receptors with Lambda-cyhalothrin and Cypermethrin Caused a Partial Estrogenicity Via an Estrogen Receptor-dependent Pathway

Abstract

Synthetic pyrethroids (SPs) are the most common pesticides which are recently used for indoor pest control. The widespread use of SPs has resulted in the increased exposure to wild animals and humans. Recently, some SPs are suspected as endocrine disrupting chemicals (EDCs) and have been assessed for their potential estrogenicity by adopting various analyzing assays. In this study, we examined the estrogenic effects of lambda-cyhalothrin (LC) and cypermethrin (CP), the most commonly used pesticides in Korea, using BG-1 ovarian cancer cells expressing estrogen receptors (ERs). To evaluate the estrogenic activities of two SPs, LC and CP, we employed MTT assay and reverse-transcription polymerase chain reaction (RT-PCR) in LC or CP treated BG-1 ovarian cancer cells. In MTT assay, LC (10(-6) M) and CP (10(-5) M) significantly induced the growth of BG-1 cancer cells. LC or CP-induced cell growth was antagonized by addition of ICI 182,720 (10(-8) M), an ER antagonist, suggesting that this effect appears to be mediated by an ER-dependent manner. Moreover, RT-PCR results showed that transcriptional level of cyclin D1, a cell cycle-regulating gene, was significantly up-regulated by LC and CP, while these effects were reversed by co-treatment of ICI 182,780. However, p21, a cyclin D-ckd-4 inhibitor gene, was not altered by LC or CP. Moreover, ERα expression was not significantly changed by LC and CP, while downregulated by E2. Finally, in xenografted mouse model transplanted with human BG-1 ovarian cancer cells, E2 significantly increased the tumor volume compare to a negative control, but LC did not. Taken together, these results suggest that LC and CP may possess estrogenic potentials by stimulating the growth of BG-1 ovarian cancer cells via partially ER signaling pathway associated with cell cycle as did E2, but this estrogenic effect was not found in in vivo mouse model.

Keywords: Cyperemethrin; Endocrine disruption; Lambda-cyhalothrin; Ovarian cancer; Pyrethroids; Xenograft models.

Fig. 1.. Chemical structure of two synthetic pyrethroid insecticides.

Fig. 2.. Cell proliferation of BG-1 ovarian cancer cells following the treatment with E2 or two-pyrethroids. To evaluate the effect of E2 and two-pesticides on cell viability, BG-1 cells were seeded at 96-well plate with 3,000 cells per well in 200 μL of phenol red-free DMEM with 5% CD–FBS medium. Cells were cultured in phenol red-free DMEM with 0.1% DMSO (control), E2 (10⁻⁸ M), cyperemethrin (10⁻⁵-10⁻⁸ M) and lambda-cyhalothrin (10⁻⁵-10⁻⁸ M) in the present (B,C) or absence (A) of ICI 182,780 (10⁻⁸ M) for 9 days. The cell viability was measured using an MTT assay. Values are the means ± SD. *: Mean values were significantly different from 0.1% DMSO (control), p< 0.05 (Dunnett’s multiple comparison test).

Fig. 3.. Effects of pyrethroids on the expression of cyclinD1 mRNA in BG-1 ovarian cancer cells. BG-1 cells were seed in 6-well plate and treated with 0.1% DMSO, cypermethrin (10⁻⁵ M) and lambda-cyhalothrin (10⁻⁵ M) in the present (B) or absence (A) of ICI 182,780 in time-dependent manner. Cyclin D1 mRNA levels were determined by RT-PCR using GAPDH (a housekeeping gene) as an internal control. Quantification of gene of cyclin D1 was conducted by scanning the densities of bands on agarose gels using Gel Doc 2000. *: Mean values were significantly different from control (0.1% DMSO), p< 0.05 (Dunnett’s multiple comparison test).

Fig. 4.. Effects of pyrethroids on the expression of ERα mRNA in BG-1 ovarian cancer cells. BG-1 cells were seed in 6-well plate and treated with 0.1% DMSO, cypermethrin (10⁻⁵ M) and lambda-cyhalothrin (10⁻⁵ M) in the present (B) or absence (A) of ICI 182,780 in time-dependent manner. ERα mRNA levels were determined by RT-PCR using GAPDH (a housekeeping gene) as an internal control. Quantification of gene of ERα was conducted by scanning the densities of bands on agarose gels using Gel Doc 2000. *: Mean values were significantly different from control (0.1% DMSO), p< 0.05 (Dunnett’s multiple comparison test).

Fig. 5.. Effects of lambda-cyhalothrin on the tumor growth in the absence of endogenous estrogen. The mice were injected intraperitoneally with DMSO, E2 and lambda-cyhalothrinevere 3-4 days (A) and tumor volumes were measured by length × width × height × 0.5236 (mm³) using a vernier calipers every 10 days during the experiment period of 80 days (B). *: Mean values were significantly different from DMSO (control), p< 0.05 (One-way ANOVA).