Simvastatin decreases steroid production in the H295R cell line and decreases steroids and FSH in female rats

doi:10.1016/j.reprotox.2015.10.005

Reproductive Toxicology

Volume 58, December 2015, Pages 174-183

https://doi.org/10.1016/j.reprotox.2015.10.005 Get rights and content

Highlights

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Simvastatin and its metabolite Simvastatin β-hydroxy acid were investigated in-vitro and in rats.
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Simvastatin and its metabolite decrease progestagen production in-vitro.
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Simvastatin decreased progestagens in gonads, brain and plasma from rats.
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Simvastatin affected the HPG-axis by decreasing plasma FSH.

Abstract

Endocrine modulating effects of Simvastatin (SV) and its metabolite, Simvastatin β-hydroxy acid (SVA), were investigated in H295R cells and in female Sprague-Dawley (SPRD) rats. H295R cells were exposed to SV and SVA concentrations from 0 to 10 μM for 48 h. Four groups of SPRD rats received 0 (CT), 1.3 (L), 5.0 (M), and 20.0 (H) mg SV/kg bw/day for 14 days. 10 Steroids were investigated in H295R growth media, and in tissues and plasma from rats using GC–MS/MS. Plasma LH and FSH were quantified by ELISA. In the H295R assay, SV and SVA particularly decreased progestagens with IC50-values from 0.10–0.13 μM for SV and from 0.019–0.055 μM for SVA. In rats, SV decreased progestagens in ovaries, brain and plasma, and plasma FSH in the M (72.4% decrease) and H group (76.6% decrease). Because progestagens and gonadotropins are major players in fertility, administration of SV might exert negative effects on female reproduction.

Graphical abstract

Introduction

In recent years, there has been an increase in the global incidences of obesity [1]. Obesity may result in increased levels of cholesterol (CHOL). The synthesis of CHOL is finely tuned in the mammalian body, and it has been shown that high levels of CHOL, which is the precursor for all the steroid hormones in the steroidogenesis, prolong the time to pregnancy (TTP) [2]. Statins, such as Simvastatin (SV), are the most frequently used cholesterol-lowering drugs (CLDs) in the treatment of dyslipidemia both in Europe and the US [3], [4], [5]. They exert their effect by inhibiting the 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, which is the enzyme catalyzing the conversion of HMG-CoA to mevalonate in the synthesis of CHOL [6], [7], thus lowering the level of CHOL. SV is a lactone prodrug, which is extensively biotransformed (78–87% [8]), by in-vivo hydrolysis, to obtain pharmacological activity. The conversion takes place intracellularly within hepatocytes [9], [10]. Simvastatin β-hydroxy acid (SVA) is the biologically active metabolite.

Several in-vitro studies performed on ovarian theca-interstitial cells and a human study indicate that statins exert disrupting effects on sexual and endocrine function, partially due to the CHOL lowering properties of the compounds, but also due to alterations of key enzymes involved in the steroidogenesis [11], [12], [13], [14]. However, to the best of our knowledge, SV has not been investigated in the H295R cell assay. The H295R cell line is a human adrenocortical carcinoma cell line used for investigating effects on the steroidogenesis due to the expression of all steroidogenic enzymes and the de novo synthesis of all steroids in the pathway. The H295R cell obviously does not possess the ability to control the steroid secretion by feedback mechanisms exerted by the action of the hypothalamic–pituitary–adrenal (HPA) and hypothalamic–pituitary–gonadal (HPG) axes. Therefore, a rat model was included in the study. The control of the steroid secretion by feedback mechanisms can be studied in such an in-vivo model with the ability to metabolize SV to SVA via the hepatic CYP450 system. The present study therefore aims to investigate the potential endocrine modulating effects of SV and its primary metabolite, SVA, on the H295R steroidogenesis and on the plasma and tissue steroid levels and plasma Follicle-stimulating hormone (FSH) and Luteinizing hormone (LH) levels in female Sprague-Dawley (SPRD) rats during daily exposure to SV for a 14-day period. In total, we investigated 10 steroid hormones, 4 progestagens: pregnenolone (PRE), 17α-hydroxypregnenolone (OH-PRE), progesterone (PRO), 17α-hydroxyprogesterone (OH-PRO); 4 androgens: dihydroepiandrosterone (DHEA), androstenedione (AN), testosterone (TS), dihydrotestosterone (DHT) and 2 estrogens: estrone (E1), 17β-estradiol (βE2).

Section snippets

Chemicals

Simvastatin (Cas No. 79902-63-9, purity: ≥97%, Sigma–Aldrich), Simvastatin Hydroxy Acid Ammonium Salt (Cas No. 139893-43-9, Purity: 98%, Toronto Research Chemicals Inc.) and Simvastatin KRKA 10 mg tablets, which are commercially available on prescription, were investigated in the present study. n-heptane, acetone and methanol (MeOH) were of HPLC grade and obtained from Lab-Scan, Gliwice, Poland. The internal standard (IS) consisted of d7-androstenedione (AN-d7), d4-estrone (E1-d4),

H295R cell viability

The fluorescent signal for the SC corresponded to 100% viability. All the tested concentrations resulted in a viability level, which complied with the criteria set by the OECD guideline (≥80% viability) [15]. 10 μM SV and SVA, which was the highest test concentration, resulted in 103% and 94% viability, compared to the SC, for SV and SVA, respectively. Table 1 shows the basal steroid levels obtained in the present study. These levels are in good accordance with previous studies [20] on basal

Discussion

Both SV and SVA were able to significantly decrease the steroid hormone levels downstream from CHOL in the H295R steroidogenesis, with the most pronounced effects at the progestagen levels. Consequently, SV and its primary metabolite, SVA, affected the H295R steroidogenesis at concentrations in the proximity of human C_max values (Table 3). In the in-vivo study, administration of SV also resulted in effects on the steroid levels, in particular on the progestagen levels in ovaries and brain. The

Conclusion

From the obtained results it is clear that SV exerted endocrine modulating effects both on the H295R steroidogenesis and in female rats. The major effects were observed on the progestagen levels (in particular PRE and PRO) at concentrations resembling human C_max values and high dose treatments. Furthermore, an effect was observed on the HPG axis, with a significantly decreased FSH level in the M and H group without an effect on the LH level. Because PRE, PRO and FSH are major players in

Conflict of interest

The authors declare that they have no conflict of interest.

Transparency document

Acknowledgements

The authors would like to thank Kenneth Munk Pedersen and Susanne Hermansen for laboratory assistance. Also Mathilde Caldara and Heidi Marie Nielsen should be thanked for assistance with the animal study. The authors would also like to thank Novo Nordisk A/S and the Drug Research Academy (DRA) for the scholarship to AG.

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Simvastatin decreases steroid production in the H295R cell line and decreases steroids and FSH in female rats

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Chemicals

H295R cell viability

Discussion

Conclusion

Conflict of interest

Transparency document

Acknowledgements

Int. J. Pharm.

J. Chromatogr. B Anal. Technol. Biomed. Life Sci.

Metabolism

Fertil. Steril.

Toxicol. In Vitro

Anal. Biochem.

Biochim. Biophys. Acta

Am. J. Clin. Nutr.

J. Lipid Res.

Trends Endocrinol. Metab.

Mol. Cell Endocrinol.

Horm. Behav.

Exp. Parasitol.

J. Steroid Biochem.

J. Steroid Biochem. Mol. Biol.

Mol. Cell Endocrinol.

Lipid concentrations and couple fecundity: the life study

J. Clin. Endocrinol. Metab.

Factors associated with statin selection among privately insured commercial and medicare patients

Curr. Med. Res. Opin.

Trends in prescribing and utilization of statins and other lipid lowering drugs across Europe 1997–2003

Br. J. Clin. Pharmacol.

Simvastatin reduces melanoma progression in a murine model

Int. J. Oncol.

Comparison of properties of four inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase

Am. J. Cardiol.

In vitro metabolism of simvastatin in humans [SBT]identification of metabolizing enzymes and effect of the drug on hepatic P450s

Drug Metab. Dispos.

Statin adverse effects: a review of the literature and evidence for a mitochondrial mechanism

Am. J. Cardiovasc. Drugs