Elsevier

Chemosphere

Volume 90, Issue 3, January 2013, Pages 1227-1235
Chemosphere

Effects of polycyclic musks HHCB and AHTN on steroidogenesis in H295R cells

https://doi.org/10.1016/j.chemosphere.2012.09.056Get rights and content

Abstract

1,3,4,6,7,8-Hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-(γ)-2-benzopyran (HHCB) and 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene (AHTN) are widely used in personal care products. Previous studies showed that HHCB and AHTN can be found in various environmental matrices and have potential endocrine disrupting effects. However, the effects on adrenocortical function of HHCB and AHTN are not fully understood. This study evaluated the influences of HHCB and AHTN on seven steroid hormones (progesterone, aldosterone, cortisol, 17α-OH-progesterone, androstenedione, 17β-estradiol, and testosterone) and 10 genes involved in steroidogenic pathways (HMGR, StAR, CYP11A1, 3βHSD2, CYP17, CYP21, CYP11B1, CYP11B2, 17βHSD, and CYP19) using the H295R cell line in the absence and presence of 8-Br-cAMP. MC2R transcription on the cell membrane was also examined to further investigate the effects of HHCB and AHTN on adrenal steroidogenesis. The results demonstrated that HHCB and AHTN could inhibit progesterone and cortisol production mainly by the suppression of 3βHSD2 and CYP21. Meanwhile, high concentrations of AHTN can affect the sensitivity of H295R cells to ACTH by disrupting MC2R transcription. Overall, the results indicate that high concentrations of HHCB and AHTN can affect steroidogenesis in vitro using the H295R cell line.

Highlights

► HHCB and AHTN can inhibit progesterone and cortisol production. ► HHCB and AHTN can down-regulate 3βHSD2 and CYP21. ► HHCB and AHTN can up-regulate CYP17, CYP11B1 and CYP11B2. ► AHTN can influence steroidogenesis by affecting MC2R expression.

Introduction

Polycyclic musks (PCMs), predominantly 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-(γ)-2-benzopyran (HHCB) and 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene (AHTN), are widely used as essential fragrance ingredients in cosmetics, perfumes, laundry cleaning detergents and other consumer products. Due to their widespread use, lipophilicity, and persistence, PCMs have been detected in various environmental matrices including freshwater, seawater, sediments, biota, and even humans (Zeng et al., 2008, Hu et al., 2010, Hu et al., 2011, Hutter et al., 2010). The wide distribution of PCMs and their polycyclic nature raise the question whether PCMs have the potential to disturb endocrine functions. There are increasing evidences that PCMs could exert estrogenic, anti-estrogenic, anti-androgenic, and anti-progestagenic activities, and they could also cause reproductive disorder and population decline of the polychaete (Capitella species I) (Seinen et al., 1999, Bitsch et al., 2002, Schreurs et al., 2004, Schreurs et al., 2005a, Schreurs et al., 2005b, Yamauchi et al., 2008, Ramskov et al., 2009). Most previous studies are focused on the reproductive effects, but less attention is paid to other endocrine organs, such as the adrenal gland. Because of high lipid content, copious blood supply and steroidogenic capacity, the adrenal gland is likely to accumulate lipophilic chemicals (Ribelin, 1984). Thus, it is important to assess the adrenocortical toxicity of HHCB and AHTN.

The adrenal gland is one of the most important endocrine organs in human and, unlike the gonads, essential for survival (Sanderson, 2006). All steroidogenic processes take place in the adrenal cortex, which is responsible for production of three major types of steroid hormones: mineralocorticoids, glucocorticoids, and the sex hormones (Li and Wang, 2005). The H295R cell line, derived from an adrenocortical carcinoma, preserves the intact steroidogenic pathways in the normal adrenal cortex and allows the measurement of alterations in gene transcription, enzyme activity and hormone production at the same time, which makes it a unique bioassay model for evaluating the effects of chemicals on steroidogenic pathways and adrenocortical toxicity (Fig. 1) (Gazdar et al., 1990, Johansson et al., 2002, Oskarsson et al., 2006, Hecker and Giesy, 2008). Additionally, H295R cells treated with forskolin (a cAMP inducer) could make the steroidogenic transcript profiles more similar to those in normal human adrenal cells, which further suggests that the H295R cell line is particularly suitable for the risk assessment of adrenocoritical toxicity (Oskarsson et al., 2006). Activation of cAMP is also critical for the investigation of adrenocorticotropic hormone (ACTH)’s effect on the steroidogenic process under biological conditions. ACTH is released from the pituitary gland and stimulates the production of steroid hormones by activating adenylyl cyclase to elevate cAMP after binding to melanocortin 2 receptor (MC2R) on the cell membrane (Sewer and Waterman, 2003, Li and Wang, 2005).

In order to evaluate the effects of the tested PCMs on steroidogenic pathways and adrenocortical toxicity, we exposed H295R cells to HHCB and AHTN at different concentrations in the absence and presence of 1 mM 8-Br-cAMP (a cAMP analog) for 48 h. Production of seven steroid hormones (progesterone, aldosterone, cotisol, 17α-OH-progesterone, androstenedione, testosterone, and 17β-estradiol) and transcription of ten genes encoding the steroidogenic enzymes (HMGR, StAR, CYP11A1, 3βHSD2, CYP17, CYP21, CYP11B1, CYP11B2, 17βHSD, and CYP19) were investigated. MC2R transcription on the cell membrane was also examined to further elucidate the effects of HHCB and AHTN on adrenal steroidogenesis.

Section snippets

Chemicals

Tonalide (AHTN, 98%) was obtained from LGC-Promochem and Galaxolide (HHCB, 95%) was from Beijing Wohai Global Technology Co., Ltd. 8-Bromo-cyclic adenosine monphosphate (8-Br-cAMP) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO, 99.9%) was purchased from Amresco (Shanghai, China).

Cell culture

The H295R human adrenocortical carcinoma cell line was purchased from the American Type Culture Collection (ATCC#CRL-2128, Manassas, VA, USA) and cultured in 75 cm2 petri dish with 12 mL

Effects of HHCB and AHTN on H295R cell viability

Preliminary experiments based on Alamar Blue assay showed that no cytotoxicity was observed at currently tested concentrations (0.25, 2.5, 25 μM) of PCMs (data not shown). This dose range is used for the following experiments to estimate PCMs effects on steroidogenesis without causing nonspecific cytotoxicity.

Effects of HHCB and AHTN on hormone production

In 25 μM HHCB basal treatments without 8-Br-cAMP (Fig. 2, white bars), progesterone and cortisol production were decreased by 39% and 27% of the basal control, respectively. Aldosterone,

Discussion

In current study, progesterone production was found to be inhibited by 25 μM HHCB. While for AHTN, the production of progesterone was significantly inhibited even when AHTN concentration was as low as 0.25 μM. The results above indicated that AHTN exhibits higher anti-progestagenic activity than HHCB, which agrees with previous reports that HHCB and AHTN are antagonists toward progesterone receptor in reporter gene bioassays, and the IC50 value of AHTN was lower than that of HHCB in the PR CALUX

Conclusions

In summary, this study demonstrated that HHCB and AHTN were capable of inhibiting progesterone and cortisol production mainly by the suppression of 3βHSD2 and CYP21. In addition, high concentrations of AHTN could affect the sensitivity of H295R cells to ACTH by disrupting MC2R transcription. However, effective doses used in the in vitro assay are considerably higher than those detected in the human and their biological significance in vivo is therefore uncertain. Further in vivo studies are

Acknowledgements

This study was supported by the National Natural Science Foundation of China (20837003), the National High Technology Research and Development Program (863 Project, 2009AA062407) and Key Projects in the National Science & Technology Pillar Program (2007BAC27B02-1a).

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