Butylated hydroxyanisole isomers induce distinct adipogenesis in 3T3-L1 cells

Highlights

• 3-BHA promoted adipogenic differentiation in 3T3-L1 cells, while 2-BHA had no effect.

• 3-BHA increased lipid deposition in the differentiated adipocytes together with the upregulation of phenotypic biomarkers.

• 3-BHA interfered the early molecular events in 3T3-L1 during the first four differentiation days.

• 3-BHA enhanced adipogenic effect was independent of its direct binding to PPARγ.

• Foodborne BHA exposure could potentially be involved in the increasing incidence of obesity.

Abstract

Butylated hydroxyanisole (BHA) isomers, as the widely used anthropogenic antioxidants in food, have been revealed to induce endocrine disrupting effects, while the mechanism how BHA isomers regulate the lipogenic differentiation remains to be elucidated. Using 3T3-L1 differentiation model, the effects of BHA isomers, including 2-tert-butyl-4-hydroxyanisole (2-BHA), 3-tert-butyl-4-hydroxyanisole (3-BHA) and their mixture (BHA), on adipogenesis were tested. The results showed that 3-BHA and BHA promoted adipocyte differentiation and enhanced the cellular lipid accumulation through the regulation of the transcriptional and protein levels of the adipogenetic biomarkers, while 2-BHA had no effect. The effective window for 3-BHA induced lipogenesis was the first four days during 3T3-L1 differentiation. BHA isomers showed no binding affinities for peroxisome proliferator activated receptor γ (PPARγ). Instead, the upstream of PPARγ signaling pathway, i.e. the phosphorylation of cAMP-response element binding protein (CREB), upregulation of CAAT/enhancer-binding proteins β (C/EBPβ) and elevated cell proliferation during postconfluent mitosis stage were induced by 3-BHA exposure. Altogether, this study revealed the adipogenic effect of 3-BHA through interference with the upstream events of the PPARγ signaling pathway. The authorized usage of BHA as food additives and its occurrence in human sera can potentially contribute to the incidence of obesity, which is of high concern.

Introduction

Butylated hydroxyanisole (BHA), a synthetic monophenolic antioxidant, has two isomers, 2-tert-butyl-4-hydroxyanisole (2-BHA) and 3-tert-butyl-4-hydroxyanisole (3-BHA), according to the site of tert-butyl group on the benzene ring (Figure S1). The commercial product is a mixture, typically consisting of 10% 2-BHA and 90% 3-BHA. Considering its superior properties in preventing lipid oxidation, BHA has been authorized for its usage in food industry since late 1950s to delay deterioration, rancidity and discoloration of oils, fats and lipid-containing foods during processing, packing and storage, thus extending their shelf-lives [1,2]. The wide usage of BHA potentially results in substantial releases into the environment. Subsequently, BHA was detected in various environmental media, like municipal sewage sludge, surface water and commercially farmed fish [[3], [4], [5], [6], [7], [8]]. Due to all sorts of unintended exposures like other reported synthetic phenolic antioxidants (SPAs), BHA contributed to prevalent human burden in population at large [9]. The extensive environmental occurrence of BHA and the potential human exposure suggested the high importance of its biosafety evaluation.

The accumulating data has been gathered for the biological effects of BHA from diverse aspects in recent years. Some studies revealed that BHA exerted beneficial effects, regarding its anti-tumor effects [[10], [11], [12], [13]], potentially being a promise in clinic therapy. Nevertheless, the toxicological studies revealed that BHA was genotoxic [14,15], and could cause adverse effects towards developmental and reproductive systems [[16], [17], [18]]. The studies on the endocrine disrupting effects showed that BHA could exert estrogenic or anti-estrogenic activities [19,20]. Steroidogenesis assay indicated that BHA significantly induced estrogen secretion, thus perturbing the steroid hormone hemostasis in vivo [21]. Considering the fact that endocrine disrupting chemicals (EDCs) can contribute to the etiology of obesity and high fat diet is commonly involved in this pathology [22], whether BHA, a potential endocrine disruptor abundant in lipid-containing foods, might perturb adipogenesis and induce obesity development was worthy of being studied. What’s more, previous studies mainly reported the toxicological effects of BHA mixture, further exploration on the specific differences in two BHA isomer-induced effects was thus needed.

Adipose tissue, a dynamic organ primarily composed of adipocytes, plays a crucial role in lipid metabolism, whole-body insulin sensitivity and systemic energy homeostasis. Its dysfunction can cause health risks, including obesity and the associated metabolic abnormalities, such as, insulin resistance, type 2 diabetes, cardiovascular disease or hypertension [23,24]. A growing body of epidemic evidence has revealed the steadily increasing incidence of obesity or overweight around the world over the last several decades, especially for children [25]. The roles of EDCs have now been recognized in the mediation of adipose physiology and energy metabolism [26,27], besides lifestyle changes. A cluster of EDCs, such as tributyltin (TBT), bisphenol A (BPA) and flame retardants, have been shown to alter adipogenesis and predispose individuals to gain weight, thus being considered as environmental obesogens [[28], [29], [30]]. The adipose tissue is likely a critical target for obesogens [31], and 3T3-L1 cell differentiation model has been well established for the screening of environmental obesogens [32,33]. The enhanced lipid accumulation observed in BHA exposed Crypthecodinium cohnii [34] and its affinity for fatty tissues [35] suggested the possibility of this compound as an obesogen. Due to the lack of evidences showing that BHA isomers might perturb adipogenesis in mamals, testing their effects on 3T3-L1 cell differentiation would be helpful to reveal their potential risks in inducing human obesity from the prevalent food additive uasge.

The present study firstly evaluated the effects of BHA isomers on adipogenesis using 3T3-L1 confluent cell differentiation. The findings on BHA-induced perturbation in adipogenesis would be helpful for guiding the sound control of this chemical’s usage in food industries.