Organophosphorus flame retardant TDCPP-induced cytotoxicity and associated mechanisms in normal human skin keratinocytes

Highlights

• Human skin is exposed to TDCPP-containing dust on a daily basis;

• We examined effects of TDCPP on normal human skin keratinocytes (HaCaT);

• TDCPP decreased cell viability, and induced cell cycle arrest and apoptosis;

• TDCPP enhanced the expression of apoptosis related genes Bax and Caspase3;

• TDCPP decreased the expression of cell cycle related genes cyclin D1, CDK2, and CDK6.

Abstract

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP), a widely used organophosphorus flame retardant, has been frequently detected in the environment including indoor dust. Long-term exposure to TDCPP-containing dust may adversely affect human skin, however, little is known about its potential cytotoxicity. In this study, human skin keratinocytes (HaCaT) were employed to study TDCPP-induced cytotoxicity and associated mechanisms. The effects of TDCPP on cell morphology, viability, apoptosis, and cycle, and the mRNA levels of apoptosis (Bcl-2, Bax and Caspase-3) and cell cycle (cyclin D1, CDK2, CDK4 and CDK6) regulatory genes were investigated. The results showed that TDCPP caused a concentration-dependent decrease in cell viability after exposing to TDCPP ≥100 μg/mL for 48 h, with a median lethal concentration of 163 μg/mL (LC₅₀). In addition, TDCPP induced cell apoptosis and arrested cell cycle in the G0/G1 phase at 16 and 160 μg/mL by enhancing Bax and Caspase-3 expression besides inhibiting cyclin D1, CDK2, CDK6 and Bcl-2 expression. Our results showed that TDCPP-induced toxicity in HaCaT cells was probably through cell apoptosis and cell cycle arrest. This study provides information on the toxicity of TDCPP to human skin cells, which may help to reduce its toxicity to human skin.

Introduction

Flame retardants are frequently added to various consumer products to prevent fire flame propagation. In recent years, due to their potential health effects, brominated flame retardants including polybrominated diphenyl ethers (PBDEs) have been phased out (Stapleton et al., 2012; Dishaw et al., 2014). As an alternative, organophosphorus flame retardants (OPFR) have been used in consumer products, including building materials, furniture, foam, plastics, electronics, and baby products (Stapleton et al., 2011; Hoffman et al., 2015). At present, OPFR are widely used, with global consumption reaching ~680,000 tons in 2015 (van der Veen and de Boer, 2012; Wang et al., 2015). In China, their production reached 300,000 tons in 2013, increasing ~15% annually (Ou, 2011).

Because OPFR are added to the materials physically, they can be easily released into the environment by volatilization and abrasion. Among OPFR, tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is one of the most widely used and has been frequently detected in indoor dust with concentrations at 56.1–326 μg/g (Stapleton et al., 2009; Carignan et al., 2013). Moreover, it was also detected in many human samples like breast milk (Sundkvist et al., 2010; Kim et al., 2014), and blood and urine (Butt et al., 2014; Zhang et al., 2018). As such, studies have focused on the potential adverse effects of TDCPP on human health. Abou-Elwafa Abdallah et al. (2015) reported that skin absorption may be a main route for human exposure to OPFR including TDCPP. This is because human skin is the largest body organ of the integumentary system, with a total surface area of ~2 m² in adults (Godin and Touitou, 2007). However, little is known about TDCPP-induced effects on human skin.

Animals and cell models have been widely employed to study TDCPP-induced toxicity. Yin et al. (2019) showed that TDCPP disturbed mouse embryonic development by triggering abnormal DNA methylation and apoptosis. TDCPP at 1–86 μg/mL induced neurotoxicity and cytotoxicity in pheochromocytoma neuronal cells and neuroblastoma cells (Li et al., 2017). Xiang et al. (2017a) reported that, in primary normal human corneal epithelial cells, exposure to ≥2 μg/mL TDCPP induced apoptosis, and deregulated the mRNA expression associated with endoplasmic reticulum stress and mitochondrial apoptotic pathway. However, its toxicity on human skin cells is largely unknown. The outermost cell layer of human skin (epidermis) mainly consists of keratinocytes, which acts as a barrier to protect vascular dermis from exposure to contaminants including OPFR. Cequier et al. (2014) found that dermal absorption contributed ~50% to the intake of TDCPP from indoor dust, suggesting that skin absorption may be an important route for human exposure to TDCPP.

To better understand its toxicity, the effects of TDCPP on human skin cells were evaluated using normal human skin keratinocytes (HaCaT). Changes in cell viability, morphology, apoptosis and cycle arrest were determined after 48 h exposure to TDCPP. Besides, the expression of cell apoptosis and cell cycle regulatory genes at mRNA levels were assessed to explore the underlying molecular mechanisms associated with TDCPP-induced adverse effects.