Possible involvement of oxidative stress in potassium bromate-induced genotoxicity in human HepG2 cells
Introduction
Potassium bromate (KBrO3, PB) is an important potential contaminant in hypochlorite, and it is also a by-product of ozone used as disinfectant in drinking water, because ozonation of drinking water containing bromide may lead to the formation of bromate. Moreover, PB is a widely used food additive by acting as a bleaching agent to improve the quality of flour. In addition, PB is used in cold-wave hair lotion. It is also used in the production of fish paste and fermented beverages [1].
PB has been used for bread-baking as a safe food additive since 1914 in America. Normally during the bread-baking process, PB decomposes into a stable compound, potassium bromide (KBr) [2]. However, this reduction is sometimes incomplete and residual PB remains in the bread, making it a source of potential carcinogen to humans. In 1987, the International Agency for Research on Cancer (IARC) has classified PB to be “a possible carcinogen in humans” (group 2B) based on sufficient evidence that PB induces cancer in experimental animals [3]. The studies have shown that PB may induce renal cell tumors, mesotheliomas of the peritoneum and follicular cell tumors of the thyroid in rats and renal cell tumors in mice following long-term oral administration in drinking water [4], [5], [6]. According to experiments aimed at elucidating the mode of carcinogenic action, PB was a complete carcinogen, possessing both initiating and promoting activities for rat renal tumorigenesis [6]. The LD50 p.o. in rats of the PB is 321 mg/kg [7]. To date, more and more countries have banned the use of PB in bread production. In 2004, the World Health Organization (WHO) proposed the provisional guideline value of PB 0.01 mg/L in drinking water [8].
With respect to genotoxicity, a series of positive results have been obtained recently. PB has been considered to be a genotoxic carcinogen based on positive results in the Ames test by Ishidate [9]. PB induced DNA strand breaks in human leukocytes and rat kidney epithelial cells, as measured by the Comet assay, and that it caused high 8-oxo-dG levels in calf thymus DNA [10]. It was reported that PB induced micronucleus formation in male ddY mice and a dose-dependent increase in DNA synthesis in male Wistar rats after intraperitoneal injections [11], [12]. It was shown that PB strongly stimulated the synthesis of oxidative stress-related proteins in mesothelial cells of F344 rat [13]. Mutation in the Vhl gene of kidney cells of F344 rat was found after administration of PB in the drinking water [14]. Sai et al. [15] used glutathione, cysteine, liposome-coated superoxide dismutase and vitamin C in order to determine the preventive effects of these substances on micronucleus formation induced by PB in peripheral blood cells of F344 rat. All these anti-oxidants, except for vitamin C, could diminish the genotoxic effect of PB.
Evaluation of the genotoxicity induced by PB in the metabolically competent HepG2 cells has not been reported so far. The HepG2 cell line was originally derived from a human hepatoblastoma [16]. The morphological characteristics and cell shapes are compatible with those of liver parenchymal cells. HepG2 cell line retains many of the functions of normal liver cells [17]. In the past two decades, methodologies have been developed to enable the detection of genotoxic effects in HepG2 cells. In addition, PB may lead to hepatic injury, hence HepG2 cells were considered to be a suitable system for detecting the genotoxicity of PB in vitro.
The purpose of this study was to assess the genotoxic effects of PB and the underlying mechanisms in HepG2 cells. We used the SCGE assay and the micronucleus test (MNT) to study the genotoxic effects of PB. The SCGE assay, which was used to detect genotoxicity of a wide variety of compounds in HepG2 [18], [19], [20], has been proved a reliable and rapid procedure for the quantification of DNA lesions. MN reflects chromosome breakage and/or chromosome loss [21]. In addition, 8-hydroxydeoxyguanosine (8-OHdG), which is a biomarker for oxidative DNA damage [22], [23], was also measured by immunocytochemical analysis. Since the molecular mechanisms may involve the generation of various reactive oxygen species (ROS), the level of intracellular ROS was measured by use of the 2,7-dichlorofluorescein diacetate (DCFH-DA) assay. ROS include superoxide anion, hydrogen peroxide (H2O2), and hydroxyl radical generated by incomplete reduction of oxygen [24] which are much more reactive than molecular oxygen and can cause severe damage to nucleic acids, cell membranes, and proteins [25]. To clarify whether the PB-induced oxidative stress is via the depletion of GSH, we examined the level of GSH. GSH is a major intracellular antioxidant, which serves as a substrate for GSH peroxidase to degrade H2O2 to H2O and also acts as a free radical scavenger [26]. To further elucidate the mechanisms of oxidative DNA damage, we measured the level of lysosomal membrane stability and mitochondrial membrane potential. Lysosomal disturbance has been recognized as a feature of oxidative stress-induced cell damage [27]. Nakanishi et al. [28] reported that the accumulations of lysosome- and mitochondria-derived ROS are the most important causative factors. For the mechanisms of lysosome-mediated apoptosis, it suggested that lysosomal permeabilization usually precedes mitochondrial dysfunction [29], [30], [27]. The present study for the mechanisms of PB-mediated DNA damage will help further to understand the toxic effects of PB and provide important information on the regulation of PB in drinking water and other relevant products.
Section snippets
Chemicals, materials and mediums
PB (CAS No. 7758-01-2) was purchased from Sigma–Aldrich (Germany: purity ≥99.8%). Dimethylsulphoxide (DMSO), RNAase A, cytochalasin B, cyclophosphamide (CPA), ethidium bromide (EB), DCFH-DA, o-phthalaldehyde (OPT), acridine orange (AO) and Rhodamine 123 were purchased from Sigma (St. Louis, MO, USA). Monoclonal 8-OHdG antibody was bought from JaICA (Fukuroi, Japan). Ultrasensitive streptavidin-peroxidase kit was bought from Maixin-Bio (Fujian, China). Buthionine-(S,R)-sulfoximine (BSO), low
DNA strand breaks induced by PB
As shown in Table 1, PB significantly increased the DNA migration in a dose-dependent manner for 1 h at all tested concentrations (1.56–12.5 mM), compared to the control (P < 0.01). In Table 2, no statistical significance was observed in HepG2 cells exposed to PB at 0–12.5 mM for 40 min.
The protection of HT against the DNA breakage induced by PB
Table 3 shows the effect of HT on PB-induced DNA damage. Table 1 shows that HepG2 cells led to serious DNA damage treated with 12.5 mM PB. In contrast, DNA damage induced by PB was significantly reduced in a
Discussion
The present study was aimed to investigate the genotoxic effects of PB and the possible mechanisms of PB-induced genotoxicity in human HepG2 cells.
In the SCGE assay and MNT, dose-dependent increases of DNA migration and of the MN frequencies were found after treatment with PB at the concentrations 1.56–12.5 mM and 0.12–1 mM which are chosen in similar dose with those used in same studies with CHO and human peripheral lymphocytes cells [40], [41]. The results of SCGE assay and MNT are in agreement
Conflict of interest statement
None declared.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (30771820). We thank Xiaoqing Wei and Haibo Chen at the Central Facility Core of Dalian Medical University for instrumental assistance.
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Potassium bromate-induced oxidative stress, genotoxicity and cytotoxicity in the blood and liver cells of mice
2022, Mutation Research - Genetic Toxicology and Environmental MutagenesisCitation Excerpt :A significant elevation in serum levels of AST, ALT, and MDA and decreasing levels of GSH and SOD were reported in Swiss albino rats posttreatment with KBrO3 [32]. KBrO3 treatment also resulted in elevated levels of ROS and significant depletion of GSH levels in HepG2 cells in vitro [7]. Previously, oxidative stress induced by KBrO3 and its effects on liver tissue have been investigated in adult rats [33].