Elsevier

Fish & Shellfish Immunology

Volume 102, July 2020, Pages 489-498
Fish & Shellfish Immunology

Full length article
Bisphenol A regulates cytochrome P450 1B1 through miR-27b-3p and induces carp lymphocyte oxidative stress leading to apoptosis

https://doi.org/10.1016/j.fsi.2020.05.009Get rights and content

Highlights

  • CYP1B1 is a target gene of miR-27b-3p in fish.

  • BPA exposure alters the expression of miR-27b-3p and CYP1B1 in carp lymphocytes, thereby inducing oxidative stress.

  • BPA exposure induces lymphocyte apoptosis through mitochondrial pathway.

  • Overexpress miR-27b-3p can partially reversed oxidative stress and apoptosis of lymphocytes induced by BPA stimulation.

Abstract

Bisphenol A (BPA) is an industrial raw material widely used in water bottles, medical devices and food packaging, and is now ubiquitous in the environment. However, the effects of BPA on the toxicity of fish lymphocytes and the roles of microRNA (miRNA) in this process remain poorly understood. To explore the mechanism, we exposed carp spleen lymphocytes to BPA of 1, 5 and 10 nM for 24 h. The results showed that BPA induced carp lymphocyte apoptosis. BPA inhibited the expression of miR-27b-3p mRNA, thereby increasing the expression of cytochrome P450 1B1, increasing ROS levels, inhibiting SOD, CAT, GSH-PX activity, GSH content, promoting the accumulation of NOS and MDA. At the same time, BPA activated the mitochondrial apoptosis pathway, inhibited the expression of BCL-2, and promoted the expression of CytC, BAX, Caspase-9 and Caspase-3. Dual luciferase reporter system showed CYP1B1 is the target genes of miR-27b-3p and negatively regulated by it. Overexpression of miR-27b-3p partially reversed oxidative stress and apoptosis of carp spleen lymphocytes induced by BPA stimulation. Taken together, BPA exposure can target up regulate CYP1B1 expression by down regulating miR-27b-3p expression, thus causing oxidative stress and inducing apoptosis of carp spleen lymphocytes through mitochondrial pathway. Our study will provide theoretical basis for immunotoxicology mechanism research and environmental protection of BPA in fish.

Introduction

Bisphenol A (BPA), an industrial raw material, is commonly used in the synthesis of polymer materials, such as polycarbonate plastics and epoxy resins, it plays an crucial role in plastics, food packaging, thermal paper, dental materials, medical devices and many other applications [1]. Studies have shown that BPA acts as an endocrine disruptor with estrogen-like and anti-androgenic effects, causing damage to different tissues and organs, including reproductive system, immune system and neuroendocrine system [2]. However, due to its mass production and wide application, BPA has become ubiquitous in the environment. According to reports, the concentration of BPA in the seawater, suspended particulate matter and biological samples collected from the Pearl River Port in China reached 106 ng/L, 868 ng/g and 41.6 ng/g, respectively [3]. In addition, BPA is still the most important bisphenol substance in wastewater treatment plant, and its detection rate and concentration are very high [4]. It has been found that exposure to BPA analogues may have an impact on human health, especially in childhood obesity and other adverse health conditions [5]. Studies have shown that BPA affects the supporting cell barrier of mammals and fish to disturb reproductive function [6]; BPA is immunotoxic to carp, and the functional changes of lymphocytes and macrophages may be different at low and high doses [7]; long-term exposure to environmentally relevant concentrations of BPA can significantly interfere with the immune response of fish [8].

MicroRNAs (miRNAs) are a class of small non-coding RNAs that serve as key post-transcriptional regulators of gene expression [9]. For example, miR-27b-3p has been reported to have a negative regulatory effect on the expression of cytochrome P450s (CYPs) 1B1 in human cancer cells [10,11]. CYP1B1 is a member of the CYPs superfamily and has been proved to be overexpressed in many tumors and associated with angiogenesis, which can be regulated by the aromatic hydrocarbon receptor to most effectively mediate toxic responses [12,13]. CYPs play key roles in the metabolism of drugs, steroids, fat-soluble vitamins, carcinogens, pesticides and many other types of chemicals [14]. It has been reported that CYP19a is up-regulated in male zebrafish exposed to BPA, and BPA exposure in marine rotifer Brachionus koreanus has a significant regulatory effect on CYPs gene expression [15,16]. In addition, activation of CYPs can induce excessive production of reactive oxygen species (ROS) and promote the emergence of oxidative stress. For example, atrazine can induce CYPs mRNA expression in neutrophils of carp, increase ROS levels, promote oxidative stress and cause apoptosis [17]; CYP1B1 is able to induce ROS production and causing oxidative stress to cause aortic lesions and hypertension in mice [18]. In many cases, when cells undergo irreversible and severe oxidative stress, the cells undergo apoptosis. It has been reported that diethylhexyl phthalate can significantly increase ROS levels and inhibit the expression of antioxidant genes, leading to apoptosis in mouse ovarian cells [19]. BPA can cause oxidative stress, inflammation, apoptosis and mitochondrial dysfunction in the colon and liver of mice [20]. The mitochondrial pathway in apoptosis is associated with cellular stress or development. These factors can change the expression and/or function of BCL-2 family proteins, activate BCL-2 effector protein BAX, increase the mitochondrial outer membrane permeability, release mitochondrial membrane gap proteins including Cytochrome C (CytC). Subsequently, CytC binds with cytoplasmic protein apaf1, oligomerize them into apoptotic bodies and activate Caspase-9 which ultimately activates the executioner caspase endopeptidases Caspase-3 [21]. Many toxic substances induce apoptosis through the mitochondrial pathway. For example, bisphenol C can cause CytC to be released from mitochondria, thereby inducing apoptosis in HepG2 cells [22]. Benzene metabolites can cause hematologic toxicity by reducing miR-133a and then inducing mitochondria-mediated apoptosis [23].

To date, few studies have shown that exposure to BPA can affect the expression of miR-27b-3p. The potential mechanism of BPA toxicity in fish spleen lymphocytes and the roles of miR-27b-3p in this process are not clear. In our experiment, we took the spleen lymphocytes of carp exposed to BPA as the research object to detect the activity or content of antioxidant enzymes and the production of ROS. Meanwhile, we detected the expression levels of miR-27b-3p and CYP1B1 by real-time quantitative PCR (qRT-PCR) and observed the apoptosis of spleen lymphocytes by fluorescence microscopy and flow cytometry. Additionally, apoptosis-related genes and protein expression levels were detected by qRT-PCR and Western blot. The purpose of this study is to explore the manner of cell death in carp spleen lymphocytes caused by BPA exposure and elucidate the roles of miRNAs in it.

Section snippets

Test chemicals

BPA (purity 99%) was purchased from Shanghai Mackon Biochemical. China. BPA stock solution was prepared with DMSO (purity 99.5%). All the working solution of following experiments used the stock solutions, and the DMSO final concentration in each solution was kept at< 0.05%.

Treatment of experimental animals

The carps (mean weight 1000 ± 10 g, mean body length 32 ± 1.49 cm) were purchased from the local fishing ground specialized in freshwater fish breeding, and adapted to the laboratory environment for one week before use. All

BPA exposure induces apoptosis in carp lymphocytes

To identify the death pattern of fish spleen lymphocytes after BPA exposure, lymphocytes were treated with different concentrations (1, 5, and 10 nM) of BPA for 24 h. Hoechst staining showed that apoptotic cells appeared brilliant blue, and normal cells appeared blue. As shown in Fig. 1A, compared with the untreated control group, Hoechst staining in fish spleen lymphocytes treated with different concentrations of BPA was enhanced, and the number of apoptotic cells increased gradually with the

Discussion

BPA is a well-known endocrine disruptor widely existing in the water environment, which involves environmental and human health issues and ecological risks [29]. It has been reported that BPA can accumulate in the viscera, gill, head and muscle of zebrafish after 1 day exposure at 85, 43, 20 and 3 μg/g WW, and can cause neurotoxicity [30]. The stimulation of BPA on adult zebrafish induced the increasing lysozyme activities of the offspring, inhibition of respiratory burst and antioxidant

Credit author statement

All the authors read and approved the final manuscript. The authors declare that they have no conflict of interest.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Acknowledgments

Shiwen Xu provided ideas for the experiment. Shu Li provided experimental supplies. Qingqing Liu completed the experiment and wrote the manuscript. Wei Wang, Yiming Zhang and Yuan Cui completed part of the figures.

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