EVALUATION OF DOSE-DEPENDENT ACTIVITY OF BISPHENOL F ON VIABILITY PARAMETERS AND STEROIDOGENESIS IN H295R CELLS

Increasing concern over bisphenol A (BPA) as an endocrine-disrupting chemical and recent imposition of restriction on the use of BPA paved the way for entry of its analogues in the market. Bisphenol F (BPF) is one of the major analogues of commercial value. Thus, its increasing production and application make it vulnerable to human exposure. The aim of our in vitro study was to assess the potential effect of BPF on H295R cells mitochondrial activity, metabolic activity, membrane integrity, lysosomal function, and testosterone synthesis. Adrenocortical carcinoma cells were cultivated during 24 h in the presence of BPF (0.1, 0.5, 1, 10, 25, 50, 75, 100, 300, 500 μM). Exposure doses of BPF caused a significant decrease of mitochondrial activity starting from 1 μM, we observed a slight increase in mitochondrial activity at the lowest concentration (0.1 μM). Metabolic activity decreased with increasing dose of BPF - from 10 to 500 μM. A significant increase in metabolic activity was observed after cultivation with 0.1 μM BPF and a slight increase was observed after cultivation with 0.5 μM BPF. We observed a slight increase in lysosomal function and membrane integrity after cultivation with 0.1 and 1 μM, although higher exposure doses (25 - 500 μM) caused significant decrease in membrane integrity and lysosomal function. Lowest exposure dose of BPF (0.1 μM) caused a significant increase in testosterone synthesis, on the other hand, higher exposure doses (50 - 500 μM) caused significant decrease of testosterone production. The obtained results confirmed that BPF at higher concentrations caused cytotoxicity and possibly have endocrine-disrupting potential.


INTRODUCTION
In recent years, there is increasing evidence of possible negative effects of bisphenol A (BPA) used in plastics, receipts, food packaging, and other products to human health due to its actions as an endocrine disrupting chemical (EDC) (Rochester, 2013;Rochester and Bolden, 2015). Scientists, regulators, and the general public have raised concerns about the use of BPA, which has prompted industry to seek alternative chemicals such as bisphenol F (BPF). (Vandenberg et al., 2010;Rochester and Bolden, 2015). BPF is industrially used to make epoxy resins and coatings, especially for systems needing increased thickness and durability, such as tank and pipe linings, industrial floors, road and bridge deck toppings, structural adhesives, grouts, coatings, and electrical varnishes (Fiege et al. 2000). BPF epoxy resins are also used for several consumer products such as lacquers, varnishes, liners, adhesives, plastics, water pipes, dental sealants, and food packaging (Office of Environmental Health Hazard Assessment 2012). Cabaton et al. (2006) discovered that after administration of single dose of BPF to pregnant and nonpregnant rats, BPF was absorbed and metabolized, with at least six metabolites identified, (4,4-dihydroxybenzophenone (DHB) and hydroxylated-BPF [BPF-OH]) were main metabolites. BPF residues were measured in the placenta, amniotic fluid and the fetuses. According to Cabaton et al. (2006) and Cabaton et al. (2009), BPF and its metabolites are excreted primarily in the urine (43-54% of administered dose) and to a lesser extent in the feces (15-20%). Ideally, substitutes used to replace a chemical of concern should be inert, or at least far less toxic than the original chemical, but BPF is structural analogue to BPA, thus its effects in physiological systems may be similar. BPA has been identifies as endocrine disruptor based on in vitro and animal laboratory studies (Wetherill et al. 2007, Richter et al. 2007Vandenberg 2014, Rochester andBolden, 2015). Humans are exposed to bisphenol analogues via the same pathways that have been demonstrated for BPA, including oral, dermal, hand tomouth transfer, as well as other mechanisms (Chen et al., 2016). According to the literature, the intake of dietary BPF in the form of contaminated food and water is the main source of exposure. Mainly, exposure to BPA analogues comes from microwaving food in plastic containers made from these materials, from using plastic bowls and cups that are worn out and may be leaching monomers, or even from tap water in areas where bisphenols were used to coat the inside of water pipes (Wu et al., 2017, Liao et al., 2012, Thoene et al., 2020. Exposures to levels of BPA found in environment have been associated with adverse health outcomes in children and adults in more than 75 human studies ( The aim of our in vitro study was to evaluate the potential impact of BPF on mitochondrial activity, metabolic activity, membrane integrity, lysosomal function and synthesis of testosterone by H295R cells.

Cell culture and treatment
The NCI-H295R cells were obtained from the American Type Culture Collections (ATCC CRL-2128; ATCC, Manassas, VA, USA). The cells were cultured in a Good Laboratory Practice (GLP) certified laboratory (National Institute of Chemical Safety, Budapest, Hungary; OGYI/45151-4/2012) according to Increasing concern over bisphenol A (BPA) as an endocrine-disrupting chemical and recent imposition of restriction on the use of BPA paved the way for entry of its analogues in the market. Bisphenol F (BPF) is one of the major analogues of commercial value. Thus, its increasing production and application make it vulnerable to human exposure. The aim of our in vitro study was to assess the potential effect of BPF on H295R cells mitochondrial activity, metabolic activity, membrane integrity, lysosomal function, and testosterone synthesis. Adrenocortical carcinoma cells were cultivated during 24 h in the presence of BPF (0.1, 0.5, 1, 10, 25, 50, 75, 100, 300, 500 μM). Exposure doses of BPF caused a significant decrease of mitochondrial activity starting from 1 μM, we observed a slight increase in mitochondrial activity at the lowest concentration (0.1 μM). Metabolic activity decreased with increasing dose of BPF -from 10 to 500 μM. A significant increase in metabolic activity was observed after cultivation with 0.1 μM BPF and a slight increase was observed after cultivation with 0.5 μM BPF. We observed a slight increase in lysosomal function and membrane integrity after cultivation with 0.1 and 1 μM, although higher exposure doses (25 -500 μM) caused significant decrease in membrane integrity and lysosomal function. Lowest exposure dose of BPF (0.1 μM) caused a significant increase in testosterone synthesis, on the other hand, higher exposure doses (50 -500 μM) caused significant decrease of testosterone production. The obtained results confirmed that BPF at higher concentrations caused cytotoxicity and possibly have endocrine-disrupting potential.
previously established and validated protocols. After the initiation of the H295R culture from the original ATCC batch, the cells were cultured throughout four passages, split and frozen down in liquid nitrogen. The cells used in the scheduled experiments were cultured for a minimum of two additional passages to achieve an optimal hormone production using new H295R batches from frozen stocks. The H295R cells were grown in 25 cm 2 plastic tissue culture flasks (TPP, Trasadingen, Switzerland) in Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham 1:1 mixture (Sigma, St. Louis, MO, USA) supplemented with 1.2 g/L NaHCO3 (Molar Chemicals Halasztelek, Hungary), 12.5 mL/L of BD Nu-Serum (BD Bioscience, Bath, UK) and 5 mL/L of ITSC Premix (BD Bioscience) in a CO2 incubator at 37°C with a 5% CO2 atmosphere. The culture medium was changed 3 times/week, and after obtaining an acceptable cell density, it was removed from the culture flasks. The H295R cells were detached from the bottom of the culture flasks with 0.25% trypsin-EDTA for 3 min (Sigma, St. Louis, MO, USA). The cells were subsequently centrifuged (10 min., 125 x g) and re-suspended in fresh cell culture medium. The cell number was counted using a Burker chamber and adjusted to required cell concentration. The cell suspension was plated into sterile 96-well cell culture plates (6*10 4 cells/100 µL/well) for cytotoxicity and hormone measurements. The cells were incubated for 24 h in a CO2 incubator at 37°C under a humidified atmosphere of 95% air and 5% CO2. To explore the effect of bisphenols, cells were cultured for 24 h in medium containing specific concentrations of BPF (0.1, 0.5, 1, 10, 25, 50, 75, 100, 300, 500 μM) dissolved in dimethyl sulfoxide (Sigma, St. Louis, MO, USA). Control samples were samples without any treatment and negative control were samples treated with dimethyl sulfoxide (Sigma, St. Louis, MO, USA). The specific concentration range of bisphenols was selected according to the results of our pilot range-finding experiments.

Mitochondrial activity assay
In vitro mitochondrial activity of H295R cells exposed to bisphenol F, was determined using the MTT ((3-4,5-dimetyltiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which measures the reduction of a yellow tetrazolium salt to insoluble blue formazan in mitochondria of viable cells (Mosmann, 1983). H295R cells were exposed to different concentrations of BPF. After 24 h of treatment, cells were incubated with MTT tetrazolium salt (Sigma-Aldrich, St. Louis, USA) in CO2 incubator at 37°C under a humidified atmosphere of 95% air and 5% CO2 for 1 h. The supernatants were removed and formed formazan crystals were dissolved by adding isopropanol (p.a. CentralChem, Bratislava, Slovak Republic). Dissolved formazan was measured by an ELISA reader (Multiscan FC, ThermoFisher Scientific, Vantaa, Finland) at 570 nm against 620 nm wavelengths. Cells from three different experiments were analyzed for each treatment. All data were expressed in percentage of control, which was set to 100% (Jambor et al., 2018; Greifová et al., 2020).

Triple assay
For cell viability, three cellular activities were monitored with three indicator dyes: metabolic activity with alamarBlue (ThermoFisher Scientific, USA), plasma membrane integrity with 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM; Sigma-Aldrich, USA), and lysosomal activity with neutral red (NR; Sigma-Aldrich, USA). Schirmer et al., (1997) first identified the use of these 3 dyes in 96-well plates to provide an overview of the cytotoxicity/cytoprotectivity of treatments for cells. With minor modifications, this protocol was followed in this review. With this approach, three cell viability parameters are calculated on the same collection of cells without interference at the same stud&. In the first step, after 24 h of treatment, a solution of almarBlue and CFDA-AM in MEM medium (minimum critical medium eagle; Sigma-Aldrich, St. Louis, USA) was applied to cells seeded in the 96-well plate. The cells were incubated for one hour followed by measurement at individual wavelengths. The cells were then washed with PBS and neutral red dye in MEM medium was added to the cells for 1 h. After incubation cells were washed twice with PBS (phosphate-buffered saline; Sigma-Aldrich, St. Louis, USA) and exposed for another 30 minutes to the lysis buffer followed by measurement at a specific wavelength. The multiple endpoint assay is based on measurements, determined here using a Glomax Multi + Combined Spectro-Fluoro Luminometer (Promega Corp., USA) at respective excitation/emission wavelengths of 525/580-640 nm for alamarBlue, 490/510-570 nm for CFDA-AM and 525/660-720 nm for NR. Cells from three different experiments were analyzed for each treatment. All data were expressed in percentage of control, which was set to 100%.

Assessment of testosterone production
The culture medium was collected, centrifuged (300x g, 4°C) and stored at -20°C until hormone measurement. The testosterone concentration in the samples was assessed by the ELISA method (enzyme-linked immunosorbent assay) using commercial kit (Testosterone, Dialab, Neudorf, Austria) according to the instructional manual. The absorbance was measured at 450 nm by the ELISA reader (Multiscan FC, ThermoFisher Scientific, Vantaa, Finland) at 450 nm (Greifová et al., 2020).

Statistical analysis
Obtained data were statistically analyzed using the GraphPad Prism 8 (GraphPad Software Incorporated, San Diego, California, USA). Descriptive statistical parameters (minimum, maximum, standard error, etc.) were evaluated at first. Oneway analysis of variance (ANOVA) with Dunnett's posttest was used for statistical evaluations. The level of significance was set at ***(P < 0.001), **(P < 0.01) and *(P < 0.05). Each experiment was performed three times independently with cells from different passages (5-10) and expressed in percentage of the control groups, which was set to 100%. Results were presented as means (± SEM).

Mitochondrial activity
We observed a slightly increased mitochondrial activity after cultivation with 0.1 μM of BPF (P > 0.05) and a slightly decreased mitochondrial activity after cultivation with 0.5 μM of BPF (P > 0.05), although administration of higher concentrations (1 -500 μM) caused significantly decreased (P < 0.01) mitochondrial activity when compared to the control group (Tab. 1).

Lysosomal activity
Lysosomal activity measurement in H295R cells treated with BPF for 24 h showed significant changes (P < 0.01) only in experimental groups supplemented with the higher doses of BPF (25 -500 μM), which resulted in the decline of values (Tab. 1).

Membrane integrity
Cell membrane integrity measurement in H295R cells treated with bisphenol F for 24 h showed significant changes (P < 0.001) only in experimental groups treated with the higher doses of BPF (25 -500 μM), which resulted in the decline of values. Cell samples incubated with 0.1 and 0.5 μM of BPF exhibited slightly improved (P > 0.05) level membrane integrity compared with the control group (Tab. 1).

Testosterone production
Assessment of the testosterone production using ELISA revealed that BPF treatment led to significant increase (P < 0.001) in samples cultivated with 0.1 μM. Inversely, significant decrease (P < 0.001) of testosterone production was detected after BPF administration at concentrations 50, 75, 100, 300, and 500 μM (Tab. 1).
Concern over the endocrine-disruptive effects of BPA has resulted in hundreds of laboratory studies. However, a proper human hazard assessment of analogues such as BPF that are believed to have a less harmful toxicity profile is lacking. Although relatively few studies have examined the hormonal actions of BPF (especially in vivo), the in vitro literature indicates that BPF has actions and potencies similar to those of BPA and supports the biological plausibility of its hormonal activity in vivo, which is not surprising because BPF is a structural analogue of BPA and thus mechanisms of action would be expected to be similar (Rochester and Bolden, 2015). Nowadays, many studies prove the endocrine disrupting potential of BPA analogues, BPF also showed other in vitro effects such as cytotoxicity, cellular dysfunction, DNA damage, and chromosomal aberrations ( According to Russo et al. (2018) bisphenols IC50 values confirming their poor acute toxicity. As compared to BPA, bisphenol F was found as the less toxic congener. Their results are partly consistent with the scale of phospholipid affinity showing that toxicity increases at increasing membrane affinity. Therefore, phospholipophilicity determination can be assumed as a useful preliminary tool to select less toxic congeners to surrogate BPA in industrial applications. According to our results, cell metabolic activity, membrane integrity and testosterone production appears to be the most sensitive parameters to the activity of bisphenol F, followed by lysosomal activity membrane integrity and the least sensitive parameter was mitochondrial activity. suggested that BPF had toxic effect on the testes and spermatogenesis (testosterone production was reduced), and also observed that BPF induced oxidative stress in the reproductive tissues of male rats, which is in agreement with our results because significant decrease (P < 0.001) of testosterone production was detected after BPF administration at concentrations 50, 75, 100, 300, and 500 μM. Decreased (P > 0.05) testosterone levels with 50 and 75 μM BPF in H295R cells were also observed in a study by Feng et al., (2016). While the mechanism of action of testesterone decrease was not clear, previous work indicated decreased StAR, CYP11A1 and HSD3B2 expression . Additional research is urgently needed to fill in knowledge gaps and deepen toxicity evaluations, given that the production and applications of bisphenol analogues are on the rise and that many of them have already been present in environmental compartments, foods, and humans.

CONCLUSION
The ban on BPA resulted in its replacement by its analogues, such as BPF. The manufacturing and application of this analogue in the future, is expected to increase. Consequently, we have investigated the potential impact of BPF on mitochondrial activity, metabolic activity, membrane integrity, lysosomal function and synthesis of testosterone by H295R cells. The results of the cytotoxicity evaluation of BPF indicated that a significant level of cytotoxicity was observed at the following tested concentration: 25, 50, 75, 100, 300, and 500 μM. However, its low concentrations led to the improvement of viability parameters (mitochondrial activity, metabolic activity, membrane integrity, and lysosomal activity), as well as testosterone production, which indicates the biphasic, hormetic response of BPF in biological systems.