The toxic and endocrine potential of BPA have been well-studied; however, knowledge regarding its analogue, BPF, is limited, especially in aquatic organisms. BPF is frequently preferred in the manufacturing of industrial and daily products instead of BPA, and its presence has been widely detected in aquatic compartments. Fish, which are an important part of the food chain, are at risk of exposure to environmental chemicals via their surrounding environment, and thus, it is necessary to investigate and elucidate the toxic impacts of BPF and its underlying mechanisms in fish. With this aim, first investigated herein were the antioxidant indicators in primary cultured rainbow trout hepatocytes that had been treated with BPF for 24 h.
The LDH cytotoxicity test demonstrated that BPF exposure affected the cells in a dose-dependent manner and the percentage of cytotoxicity was observed to be 32.55 with the highest concentration of BPF. Several studies of different cell types have reported that BPF-induced cytotoxicity increased with increased concentrations and treatment times. For example, cytotoxicity was increased on a dose-dependent basis in human cell lines (HCLs), such as hepatoma HCL, HepG2; intestinal HCL, LS174T; and renal HCL, ACHN, treated with BPF concentrations ranging from 5 to 100 µM for 24 h [24]. Similarly, BPF-induced cytotoxicity was reported with concentrations of 0–600 µM for 24 h in RWPE-1 cells [16]. Russo et al. reported that mouse embryo fibroblast cells and cancer cells exposed to BPF (0–300 µM for 48 h) resulted in increased cytotoxicity in a dose-dependent manner [25]. Jambor et al. [26] also determined escalated cytotoxic activity in mouse TM3 Leydig cells treated with BPF concentrations of 10–50 µg/mL for 24 h. On the other hand, BPF did not induce cytotoxicity, even at a concentration of 200 µM for 24 h, in the human adrenal carcinoma cell line (H295R), but was found to be cytotoxic at concentrations of 300 and 500 µM [27]. No significant changes in cell viability were observed in the human hepatoma cell line (HepG2) exposed to BPF concentrations of 12.5–100 µmol/L for 24 h [28]. Moreover, Hercog et al. [6] reported that no significant variations were observed in the viability of human hepatoma cells (HepG2) with BPF concentrations of 2.5 and 20 µg/mL for 24 h, while they determined significantly decreased cell viability at the highest BPF concentration for 72 h. Thus, the similar or different findings regarding the cytotoxic impact of BPF on cells from past studies to the current study might have resulted from the cell type used, experimental design, treatment time, or selected assays for the determination of cytotoxicity.
MDA is an end-product of lipid peroxidation and a commonly used assay for monitoring membrane damage or oxidative stress [14]. It has been reported that BPF enhances ROS levels and leads to increased levels of lipid peroxidation in human erythrocytes [15]. In another study, BPF resulted in a high level of MDA contents and finally led to apoptosis in the larvae of zebrafish [7]. Ullah et al. [5] also reported increased levels of lipid peroxidation and ROS in the reproductive tissues of male rats after exposure to BPF. Consistent with these studies, the MDA content was significantly increased in the hepatocytes in the current study, suggesting that BPF is capable of disrupting cell membranes; however, the MDA content remained unchanged or dropped to a level similar to that of the control, even with the highest concentration of BPF. Although the exact mechanism underlying such an effect is unclear, some toxicity studies have reported results similar to those observed in the present study. For example, even though Liu et al. [29] did not observe a significant change in the MDA contents in the cultured hepatocytes of freshwater tilapia (Orechromis niloticus) after 24-h exposure to perfluorooctane sulfonate, an increased ROS level was determined. In another study, MDA levels showed significantly increased levels with lower BPF concentrations, whereas no significant changes were observed with higher concentrations in the liver of carp (Cyprinus carpio) exposed to diethyl phthalate for 2 days [30]. Similar trends in the MDA content were also found in carp [31], and juvenile and adult Daphnia magna exposed to phthalate esters and di‑(2‑ethylhexyl) phthalate [32], respectively. As a result of these observations, two opinions have been put forth. The first was that exposure to a low concentration of the chemical initiated a quick increase in ROS, which damaged the lipids, and then the organisms counteracted a ROS attack via the antioxidant system or by eliminating the damaged lipids [14, 31]. The second was that membrane integrity was disrupted by the harmful chemical, due to the impairment of cellular lipid metabolism and alterations in the plasma phospholipid composition, and finally, the MDA content was decreased [32, 33].
The current study results showed that 24-h exposure of the isolated hepatocytes to BPF affected enzymatic scavengers, including SOD, CAT, GPx, and GST, which were also utilized as biomarkers for a study of environmental toxicity in fish by van der Oost et al. [34]. CAT and SOD act as the first-line of defence against attacks by ROS in cells. Dismutation superoxide anion radicals are catalysed by SOD into hydrogen peroxide and molecular oxygen. In the present study, SOD activities were significantly increased with all of the BPF concentrations when compared to the control group, presumably in response to enhanced cellular superoxide anion radicals. In accordance with the present study, in vitro studies using fish hepatocytes have also reported increased SOD activities after exposure to different types of environmental chemicals, such as BPA [35], perfluorinated organic compounds [29], di(2-ethylhexyl) phthalate [36], and benzo[a]pyrene and nonylphenol [37]. On the other hand, no significant changes were seen in the SOD activity of juvenile common carp liver (Cyprinus carpio) samples after long-term (60 days) waterborne exposure to BPF [38]. Conversely, Gu et al. [7] stated decreased SOD activities in the larvae of zebrafish treated with BPF concentrations of 7–700 µg/L for 3 and 6 days. It can be said that the different findings regarding the SOD activities as a consequence of BPF exposure might have depended on the applied BPF concentration, and design and duration of the experiment. CAT degrades hydrogen peroxide, a hydroxyl radical precursor that results from SOD activity, into water and oxygen and protects unsaturated fatty acids on the cell membrane from peroxidation [14]. In the current study, exposure of hepatocytes to BPF resulted in significantly decreased CAT activity. Similar to these findings, Maćczak et al. found decreased levels of CAT in human erythrocytes treated with BPF for different periods of time (4 h and 24 h) [15]. Decreased CAT activity was also found in juvenile common carp [37], and the larvae of zebrafish after waterborne exposure to BPF [7]. Ullah et al., also reported that CAT activity was inhibited in the testicular tissues of rats exposed to BPF via drinking water [5]. Modesto and Martinez reported that many antioxidant enzymes might be inactivated by an excessive increase in oxidants or the substrate of the enzyme could be an oxidant [39]. Thus, the decreased CAT activity could be explained by accelerated SOD activity as a consequence of the excessive production of superoxide anion radicals, which then lead to higher intracellular hydrogen peroxide generation [40, 41]. In accordance with the results herein, decreased CAT activity with increased SOD activity have been also found in the liver of goldfish (Carassius auratus) exposed to subacute concentrations of nickel [42], and liver samples of Nile tilapia (Oreochromis niloticus) collected from polluted waters [43].
GSH, a tripeptide that contains cysteine, g-glutamine, and glycine, is an antioxidant molecule that directly metabolizes and detoxifies xenobiotics that conjugate directly, in addition to protecting cells from oxidative damage. Both increases and decreases of the molecule are indicative of oxidative stress [44]. GSH levels in this study were observed to be increase in the hepatocytes with concentrations that ranged between 15.63 and 250 µM; however, a significant decrease in the GSH content was determined with 500 µM of BPF. Similar to the results herein, Kose et al. reported elevated GSH levels in RWPE-1 cells that were treated with BPA, as well as its analogues, BPF and BPS, for 24 h [16]. In another study, Maćczak et al. [15] found depleted GSH levels in human erythrocytes exposed to BPF. GSH levels might increase under slight oxidative stress, as an adaptive mechanism to counteract ROS attacks; however, severe oxidative stress could lead to the depletion of the GSH contents as a consequence of the disruption of adaptive mechanisms [45]. Thus, the decrease in the GSH level with 500 µM of BPF was probably due to the severity of the oxidative stress that occurred with such a high concentration.
GPx activity was found to increase remarkably with all of the BPF concentrations, with the exception of 15 µM. GPx is an antioxidant scavenging enzyme that catalyses the degradation of hydroperoxides into hydroxyl compounds, utilizing GSH as a cofactor. The activity of GPx is closely related with its cofactor, GSH, and a reduction of the molecule might cause a decrease in the activity of the enzyme in cells [46]. Thus, a probable increase in the levels of lipid hydroperoxydes and hydroxyperoxides in the hepatocytes after BPF exposure could lead higher GPx activities, in an attempt to cope with the oxidative insult of the BPF. In parallel with the literature knowledge, synchronized increases in GPx activity with GSH levels were observed in the current study; however, an unchanged or decreased level of the enzyme did not occur, even though a significant drop was detected in the GSH level with the highest concentration of BPF. The unaffected levels of GPx activity at this concentration may have resulted because, even if a decrease in the GSH pool occurred with this concentration, the level of GSH was still adequate for the utilization of the enzyme. In support of this explanation, decreases in GSH levels were observed in human erythrocytes with minimal exposure to BPF (4 h), while decreased activities of GPx appeared after 24 h of exposure, depending on the BPF concentration applied [14].
GST is a Phase-II detoxifying enzyme that has a critical role in cellular protection against ROS and toxic xenobiotics. GST catalyses GSH conjugation in reaction to endogenous and exogenous electrophiles [47]. In the study herein, significantly increased GST activity was observed with 125 µM of BPF, while it remained unchanged with the other concentrations. Previous studies performed in both the laboratory and field have displayed that exposure to environmental chemicals that possess endocrine-disrupting potential induced GST activity in the liver of fish; however, unchanged or reduced GST activities were also reported [34, 48, 49]. Due to the lack of research regarding the impact of BPF on liver GST activity, it was not possible to compare the results determined herein. On the other hand, similar to the findings of the current study, in a recent study, GST activity was induced in marine rotifer (Brachionus koreanus) after 24-h exposure to BPF [50].