Assessing the combinatorial cytotoxicity of the exogenous contamination with BDE-209, bisphenol A, and acrylamide via high-content analysis
Graphical abstract
Introduction
Polybrominated diphenyl ethers (PBDEs), bisphenol A (BPA), and acrylamide (ACR) have been widely confirmed as endocrine-disrupting chemicals and are present in various human matrices such as blood, breast milk, and saliva (Gys et al., 2020; Mazdai et al., 2003). Dietary intake is the dominant route of exposure to these three contaminants for humans.
Polybrominated diphenyl ethers (PBDEs) are flame retardants that are widely consumed in various industries, including electronics, manufacturing, furniture, and textiles. The lipophilic and thermal stability of PBDEs makes it easy for them to escape from the products into the environment and gradually migrate along the food chain (Bute et al., 2020). Common foods such as fish products, crops (cereals, pumpkins), dairy products and livestock products (meat, eggs) have recently been reported to be contaminated with PBDEs (Zacs et al., 2021). A survey of PBDEs in the diet of Nigerian adults showed that the concentrations of PBDEs in aquatic products, edible oils and meat products ranged from 54.9 to 748 pg/g and estimated that adults consumed at least 131 pg/kg BW PBDEs per day (Babalola and Adeyi, 2018). Toxicology studies have shown that PBDEs have harmful effects on different human systems and organs, including the liver, thyroid, reproductive system, and nervous system (Wu et al., 2020). With the banning of low-brominated PBDEs, BDE-209 is the only PBDE still allowed to be produced and commercialized. Due to the widespread presence of BDE-209 in food, its toxicological ranking is high relative to other common PBDE congeners in adult diets (Tait et al., 2017). BDE-209-related toxicity results mainly from its disturbance to organelles, such as the triggering of mitochondrial dysfunction, endoplasmic reticulum (ER) stress, or lysosomal destabilization (Hou et al., 2019; Rajput et al., 2021; Zhang et al., 2015).
Bisphenol A (BPA) is a ubiquitous component in food packaging. It is commonly designed to be in direct contact with foodstuffs. Most food containers contain BPA, including many types of water bottles, aluminium cans, plastic packaging, and salad boxes. However, BPA can leach into food and water, especially in high-temperature, acidic or alkaline conditions (Ong et al., 2020). A report on BPA and its analogues in canned meat samples (sausage, meatloaf, and wholemeal) found that 30 samples were all contaminated with bisphenol analogues, and the detected concentrations of 15 samples ranged from 50 to 236 μg/kg (Cunha et al., 2020). In addition, BPA has been widely detected in cereal grains (rice, barley, wheat and oat) (Albero et al., 2020). BPA exhibits carcinogenic effects as well as hepatotoxicity, nephrotoxicity, immune toxicity, neurotoxicity, and genetic toxicity. BPA can induce oxidative stress, lead to ROS accumulation, and impair mitochondrial function and cell viability (H. Wang, Zhao, Huang, Chi, Dong and Fan, 2019). The genetic toxicity of BPA is achieved by inducing DNA strand breaks, oxidative DNA damage, and chromosomal mutations (Ma et al., 2019).
Acrylamide (ACR), another known food contaminant, is produced by the Maillard reaction between carbonyl compounds and amino compounds. It is widely produced in the heat treatment of meat, roasted coffee, potatoes, and grain products. The average exposure to ACR is 0.31–1.1 μg/kg BW/day in the diet of Europeans (“Results on acrylamide levels in food from monitoring years 2007–2009 and Exposure assessment,” 2011), 0.36 μg/kg BW/day for Americans (Abt et al., 2019), and 0.18–0.319 μg/kg BW/day for Koreans (Lee and Kim, 2020). ACR can cause carcinogenicity, genetic toxicity, and reproductive and developmental toxicity (Matoso et al., 2019). ACR exerts toxic effects by inducing oxidative stress and destroying the structure of biological macromolecules such as proteins, lipids, and DNA (Allam et al., 2010).
At present, the safety standards for food contamination are based on the individual toxicity of a certain chemical. However, foods are usually exposed to many kinds of pollution during production, storage and processing, and humans ingest multiple exogenous contaminants (L. Wang, Zheng, Zhang, Zhao, Kang and Wang, 2019). While these contaminants have different molecular structures and main toxicity targets, combining them may exacerbate their overall toxicity due to sharing certain pathways. PBDEs, BPA, and ACR are the most typical and widespread contaminants encountered by food in the environment, in storage, and during processing, respectively. The type and toxicity mechanism of the interactions among these three contaminants are unclear. Based on the above background, we make the hypothesis that the coexistence of BDE-209, BPA, and ACR may produce synergistic toxicity.
Section snippets
Materials and reagents
HepG2 cells (SWEXB10107) were supplied by Shanghai Cell Bank (Chinese Academy of Sciences, China). Foetal Bovine Serum was purchased from ExCell Bio (Cas no. FND500, Shanghai, China). Dimethyl sulfoxide (DMSO, cell culture grade) and DAPI solution were obtained from Solarbio (Cas no. D8371 and C0065, respectively, Beijing, China). A Cell Counting Kit-8 (CCK-8) was obtained from Meilunbio (Cas no. MA0218-1, Dalian, China). Hoechst 33342, JC-1, Fluo-4 and a ROS Assay Kit were obtained from
Individual cytotoxicity of BDE-209, BPA, and ACR alone
To determine the inhibitory effects of BDE-209, BPA, and ACR on HepG2 cells, we performed concentration-response experiments for the three chemicals using CCK-8 assays. As shown in Fig. 1, treatment with any of these three chemicals alone resulted in growth inhibition of HepG2 cells in a time- and concentration-dependent manner. The IC50 values of BDE-209, BPA, and ACR against HepG2 cells were 0.1871 mmol/L, 0.3387 mmol/L, and 14.26 mmol/L at 24 h and 0.0834 mmol/L, 0.148 mmol/L, and
Discussion
In this report, we investigated the joint toxicities of BDE-209, BPA, and ACR. First, the types of interaction of binary and ternary combinations of the three contaminants were studied using mathematical models. Mixtures of BDE-209 + BPA, BDE-209 + ACR, BPA + ACR, and BDE-209 + BPA + ACR showed enhanced toxicity (additive or synergistic effect) to HepG2 cells after 24 h and 48 h. Since the effects of the two time-points were the same, we chose 24 h as the condition for the HCA, and the results
Conclusions
Overall, we demonstrated that BDE-209 was the most cytotoxic exogenous contaminant to HepG2 cells, followed by BPA and ACR. Furthermore, the mixtures of BDE-209 + BPA, BDE-209 + ACR, BPA + ACR, and BDE-209 + BPA + ACR displayed enhanced cytotoxicity (synergistic or additive effects) as evaluated by the Chou-Talalay method. HCA showed that the enhanced toxicity induced by BDE-209, BPA, ACR, and their mixtures originated from the destruction of subcellular structures.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by National Key R&D Program of China,China (2017YFC1600302).
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