Butylated hydroxytoluene (BHT) is a synthetic phenolic antioxidant that has been used as an additive for fat- or oil-containing foods. The exposure index value increases with extended usage of the chemical. Further, estimated total amount of BHT could exceed standard regulation, considering dietary intake or another exposure. Although BHT may induce side effects in reproductive systems, adequate research had not yet been performed to confirm them. In this study, we investigated the effects of BHT on mouse Leydig cells (TM3), which are components of testis. Our results indicated that BHT suppressed cellular proliferation and induced cell cycle arrest in TM3 cells. Moreover, BHT hampered cytosolic and mitochondrial calcium homeostasis in TM3 cells. Furthermore, BHT treatment led to endoplasmic reticulum (ER) stress and DNA fragmentation, simultaneously stimulating intrinsic apoptosis signal transduction. To elucidate the mode of action of BHT on Leydig cells, we performed western blot analysis and confirmed the activation of the PI3K/AKT and MAPK pathways. Collectively, our results demonstrated that BHT has toxic effects on mouse Leydig cells via induction of calcium dysregulation and ER-mitochondria dysfunction.

In this study, seven synthetic phenolic antioxidant (SPA) analogues were positively found in urban and rural indoor dust samples collected from Shandong province in China, among which the novel 2,4,6-tri-tert-butylphenol (AO 246), 2,6-di-tert-butyl-4-sec-butylphenol (DTBSBP), 2,4-di-tert-butylphenol (DBP) and 4,4'-butylidenebis (2-(1,1-dimethylethyl)-5- methyl-phenol) (AO 44B25) analogues accounted for 29% of total SPA concentrations (∑SPAs). Urban dust showed significantly higher ∑SPA levels (range: 1.56e3 - 2.03e4 ng/g) compared with those in rural indoor dust (668–4.39e3 ng/g, p < 0.05). 2,6-Di-tert-butyl-4-methylphenol (BHT) was the dominate analogue in the urban indoor dust, which constituted of 74% in ΣSPAs. While, varied composition profiles of SPAs were noticed in rural indoor dust, for instance, AO 246 (46%) and BHT (43%) had similar contributions to ∑SPAs. Three BHT transformation products (TPs) were also detected in most of the urban and rural dust samples (>97%), with individual residue level in the same order: 2,6-di-tert-butyl-1,4-benzoquinone (BHT-Q) > 2,6-di-tert-butyl-4-hydroxy- 4-methyl-2,5-cyclo-hexadienone (BHT-quinol) > 3,5-di-tert-butyl-4-hydroxybenzal-dehyde (BHT-CHO). Geometric mean values of total TP concentrations were 555 ng/g and 131 ng/g for urban and rural indoor dust samples, respectively. A preliminary estimated daily intake calculation at dust ingestion scenario suggested additional concerns might be paid to simultaneous exposure of several SPA analogues and TPs besides current focus on BHT exposure risks.