Butylated hydroxyanisole alters rat 5α-reductase and 3α-hydroxysteroid dehydrogenase: Implications for influences of neurosteroidogenesis

Highlights

• Butylated hydroxyanisole inhibits synthesis of neurosteroids.

• Butylated hydroxyanisole competitively inhibits SRD5A1 and AKR1C14.

• Butylated hydroxyanisole potently inhibits SRD5A1 and AKR1C14.

• Docking shows that butylated hydroxyanisole binds to the AKR1C14 steroid-binding pocket.

Abstract

Butylated hydroxyanisole is a synthetic antioxidant. It may affect the function of the nerve system. The objective of the present study is to investigate the direct effects of butylated hydroxyanisole on rat brain neurosteroidogenic 5α-reductase 1 (SRD5A1), 3α-hydroxysteroid dehydrogenase (AKR1C14), and retinol dehydrogenase 2 (RDH2). Rat SRD5A1, AKR1C14, and RDH2 were cloned and expressed in COS1 cells, and the effects of butylated hydroxyanisole on these enzyme activities were measured. Butylated hydroxyanisole inhibited SRD5A1, AKR1C14, and RDH2 with IC₅₀ values of 4.731 ± 0.079 μM, 5.753 ± 0.073 μM, and over 100 μM, respectively. Butylated hydroxyanisole is a competitive inhibitor for both SRD5A1 and AKR1C14. Docking analysis shows that butylated hydroxyanisole binds to the dihydrotestosterone-binding site of AKR1C14. In conclusion, butylated hydroxyanisole is a potent inhibitor of SRD5A1 and AKR1C14, thus reducing the formation of active neurosteroids.

Introduction

Butylated hydroxyanisole (BHA) is an organic chemical. It is synthesized to be added to foods for their long-term preservation as an antioxidant. Structurally, BHA contains an aromatic cycle. It exists in either of the two isomeric forms or as a mixture of 2- and 3-tert-butyl-4-methoxyphenol (Fig. 1). It has been added to a wide range of foods, including beverages, ice cream, candy, baked goods, edible fats, and oils. BHA is considered by the US FDA to be generally recognized as safe when the antioxidant content does not exceed 0.02% by weight of the food's total fat or oil content [1].

Being an antioxidant, BHA has also some beneficial effects for the nerve system. BHA is able to protect methamphetamine-induced dopaminergic neuronal cell death caused by the neurotoxicity of dopamine quinone formation in mice [2]. BHA is capable of protecting the learning and memory deficits after sub-chronic exposure to benzo(a)pyrene in rats [3]. In vitro addition of BHA can prevent the neuron death in the cultured rat neuron [4]. Besides its anti-oxidative effect, BHA may have other pharmacological activities. In the regards, BHA has been demonstrated to replace the binding of estradiol to the fish estrogen receptor at concentrations up to 1 mM [5]. BHA was capable of stimulating the growth of human breast MCF7 cancer cells at a concentration of 50 μM and had a relative effect about 30% of estradiol value, suggesting an estrogenic activity [6]. An in vivo uterotrophic assay demonstrated that BHA increased uteri organ weight gains in immature female rats after administration of 50–500 mg/kg/day BHA [7], also suggesting its estrogenic activity. In recent years, estrogens have been demonstrated to have neural protective benefits [8], [9].

Besides these pharmacological activities, our previous studies [10], [11] demonstrated that BHA can regulate several other steroidogenic enzymes. BHA inhibited rat 3β-hydroxysteroid dehydrogenase and P450 17α-hydroxylase/20-lyase [10]. It inhibited rat and human 11β-hydroxysteroid dehydrogenase 2 [11]. Increasing evidence also shows that brain is a neurosteroid-producing organ. In the nerve system, there are several steroidogenic enzymes, which are responsible for neurosteroid production. The neurosteroids are subsets of steroids that rapidly change neuronal excitability via binding to ligand-gated ion channels, such as GABA-A receptor [12]. The neurosteroids, 3α-hydroxy-5α-pregnan-20-one (allopregnanolone), 3α,21-dihydroxy-5α-pregnan-20-one (tetrahydrodeoxycorticosterone), and 3α,5α-androstanediol (DIOL), are potent GABA-A receptor agonists [13]. These neurosteroids may regulate various functions in the nerve system. For example, upregulation of allopregnanolone was shown to induce significant analgesia for neuropathic pain [14]. Allopregnanolone had potent anticonvulsant activity in the adult brain of mouse [12]. DIOL is also a positive modulator of GABA-A receptors to protect against the seizures induced by hippocampus kindling [15]. The levels of allopregnanolone, tetrahydrodeoxycorticosterone, and DIOL are regulated by their biosynthetic enzymes, 5α-reductase 1 (SRD5A1) and 3α-hydroxysteroid dehydrogenase (AKR1C14). These two enzymes are extensively present in the brain [16], [17]. SRD5A1 is a microsomal reductase 1, using NADPH as a cofactor to transfer electrons to form dihydroprogesterone, dihydrodeoxycorticosterone, and dihydrotestosterone from progesterone, deoxycorticosterone, and testosterone [18] (Fig. 2). AKR1C14 is a cytosolic NADPH-dependent enzyme [19], which primarily catalyzes the formation of allopregnanolone, tetrahydrodeoxycorticosterone, or DIOL from dihydroprogesterone, dihydrodeoxycorticosterone, or dihydrotestosterone (Fig. 2). AKR1C14 belongs to the aldo-keto reductase family and adds a hydrogen to the 3α-position of many steroids, including these neurosteroids [20]. Besides, neurosteroid levels are also modulated by retinol dehydrogenase 2 (RDH2). RDH2 is a microsomal NAD⁺ dependent enzyme, which catalyzes the opposite direction of AKR1C14. RDH2 belongs to the short-chain dehydrogenase/reductase family and removes a hydrogen from 3α-position of neurosteroids [21]. Many regions of the brain contain RDH2 [22], [23], [24]. In the present study, we investigated the effects of BHA on rat SRD5A1, AKR1C14, and RDH2 enzymes.