Assessment Of Bromochloroacetonitrile Formed As A Disinfection Byproduct in an HaCaT Cells

doi:10.21203/rs.3.rs-2172185/v1

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Assessment Of Bromochloroacetonitrile Formed As A Disinfection Byproduct in an HaCaT Cells

https://doi.org/10.21203/rs.3.rs-2172185/v1

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Bromochloroacetonitrile is a disinfection byproduct of water chlorination. We investigated, the cytotoxic effects of bromochloroacetonitrile in human keratinocyte cells. Cells were exposed to 5–80 µM bromochloroacetonitrile for 24 and 48 h. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Lactate Dehydrogenase Leakage assays was used to evaluate cytotoxic effects. The changes in cellular Reactive Oxygen Species was determined. In addition, the effect of bromochloroacetonitrile on wound healing in cell culture was investigated by the scratch test. Concentration and time-dependent cytotoxicity was observed. Increasing concentrations of bromochloroacetonitrile have been observed to induce reactive oxygen species assay production in human keratinocyte cells. It was determined that concentrations of 5, 10 and 20 µM of bromochloroacetonitrile did not have a negative effect on wound healing, but when toxic concentrations of 40, 60 and 80 µM were increased, it had a slowing effect on wound healing. In this study, the effects of bromochloroacetonitrile on human keratinocyte cells were evaluated for the first time in the literature.

Bromochloroacetonitrile

Cytotoxic activity

Human keratinocyte cell line

Wound healing

Water disinfection has been used for a long time to inactivate bacterial populations in water or to control contamination, and the most common and effective method to disinfect water is chlorination (Aamir et al. 2020). There are studies showing that chlorine and disinfection by-products formed after chlorination leave common toxic residues and are insufficient in killing some microorganisms (Li and Mitch 2018). While drinking water disinfection effectively prevents water-borne diseases, an undesirable result is the production of disinfection by-products (Liu et al. 2022).

When naturally occurring organic substances (NOMs) in raw water react with chlorine, various DBPs such as trihalomethanes (THMs), haloacetic acids (HAAs) and halogenated acetonitriles (HANs) etc. are formed (Richardson et al. 2007; Aamir et al. 2020). It is known that more than 700 DBPs have been defined in current sources in the literature (Luo et al. 2020; Richardson and Plewa 2020; Liu et al. 2022).

Although waterborne diseases are controlled by disinfection, DBPs produced show a high risk for human health and attract great attention (Yang et al. 2021). Disinfection by-products have long been a human health concern, and many of them have raised concerns as they are cytotoxic, neurotoxic, mutagenic, genotoxic, carcinogenic, and teratogenic (Stalter et al. 2016; Harman et al. 2017; Yang et al. 2021). Bromochloroacetonitrile belongs to a class of unregulated nitrogen-containing disinfection by-products (N-DBPs), which are haloacetonitriles, and Bromochloroacetonitrile has been determined to be toxic (Muellner et al. 2007).

The skin is exposed to DBPs in the water through bathing and swimming (Trabaris et al. 2012). Although chlorine is the most commonly used disinfectant in swimming pools, excessive use of chlorine also increases DBP formation (Richardson et al. 2010). Exposure to DBPs in drinking water occurs through ingestion, while exposure in pools occurs as inhalation, dermal contact, and accidental ingestion (Kanan and Karanfil 2011; Chowdhury et al. 2014).

The human skin cells are, the interface between the body and the environment, provides the first line of defense against environmental attack, pathogens and xenobiotics. The skin is our first barrier and any lesion needs to heal quickly to reduce the risk of infection (Governa et al. 2019). Incomplete healing process leads to increased infection development, higher inflammation state, reactive oxygen species (ROS) production, pain and a change in the pH of the wound (Gabriele et al. 2019). The HaCaT cell line is a spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin (Ölschläger and Schrader 2009) widely used in scientific research. HaCaT keratinocytes are a preliminary in vitro model for investigating skin toxicity (Pelin et al. 2017). The aim of this study was to evaluate the cytotoxic effects, ROS production and wound healing effect of BCAN in HaCaT cells.

HaCaT cells were obtained from CLS (Cell Line Services, Eppelheim, Germany) cell bank and were grown in Dulbecco's Modified Eagles Medium (DMEM) (Cytiva, Utah, USA) containing 10% Fetal Bovine Serum (Sigma Aldrich, Germany), 1% Penicillin streptomycin (Sigma Aldrich, Germany) and 7.5% NaHCO₃ (AppliChem, Darmstadt) in a CO2 incubator at 37ºC until it reached 70% density.

HaCaT cells were seeded into each well of a 96-well plate (TPP, Techno Plastic Products AG, Germany) at 1x10⁴ cells/well in 100 µl of media with 10% FBS and allowed for 24 h to attach before treatment with BCAN (LGC DR EHRENSTORFER, Germany). 50 mg of BCAN substance was dissolved in methanol (CARLO ERBA, S.A.S) as 100,000 µM. Then, 10,000 µM BCAN master stock was prepared with sterile distilled water. 100 µM of the substance was prepared fresh from the master stock in media with 10% FBS. The doses to be used in the study were prepared from 100 µM substance by diluting. HaCaT cells were treated by six different concentrations (5-10-20-40-60-80 µM) of BCAN for 24 and 48 h, eight replicate wells per each concentration were employed and repeated in triplicate at different intervals. Untreated medium controls (blank) and solvent controls (methanol at a final concentration of 0.1%, v/v) were assayed also in parallel. Cytotoxicity of BCAN was evaluated by the measurement of cell viability using MTT and LDH assays. The MTT assay was performed previously described by Mossmann (1983). Absorbance was measured at 570 nm by using microplate reader (Synergy HTX, BioTEK, USA).

The lactate-dehydrogenase (LDH) assay was carried out using the LDH Cytotoxicity Detection Kit (Roche, Germany). Following the treatment with BCAN concentrations, the supernatant was collected and the assay was achieved in accordance with the exact protocol provided with the kit. The OD values of the plates were read at 492 nm wavelength in a microplate reader (Synergy HTX, BioTEK, USA) to determine the released LDH enzyme. Experiments were carried out 3 times and 8 repetitions. Methanol (methanol at a final concentration of 0.1%, v/v) used as a solvent control.

Biovision kit (K936-250) (Milpitas, CA, USA) was used to determine the amount of reactive oxygen species (ROS) in the cell. Cells were seeded in a black/clear-bottom 96-well plate (Greiner Bio-One GmbH, Germany) at 1x10⁴ cells per well. After waiting for 23 hours for cells to adhere, the medium was removed. According to the kit procedure, 100 µL of ROS assay buffer was added to each well and the cells were washed. Then, the wells were incubated with 100 µL of 1X ROS marker prepared in ROS assay buffer for 40 minutes at 37°C. Then, the 1X ROS marker was removed, freshly prepared BCAN substance 5, 10, 20, 40, 60, 80µM concentrations were added to the wells and the plates were incubated at 37°C for 24 hours. At the end of the incubation period, the change in the amount of ROS in the cell was measured fluorometrically in the microplate reader (Synergy HTX, BioTEK, USA) at 495/529 nm and the results were compared with the control group.

Obtained data from MTT, LDH and ROS assays were evaluated using one-way analysis of variance (ANOVA) follewed by Tukey’s test in statistical package for social sciences software (SPSS). The experimental study results given in the figures were expressed according to the percentages of control as the mean ± standard deviation. Asterisks indicate significant difference from control group by the Tukey test (p < 0,005).

In the scratch wound healing assay, HaCaT cells (5x105) were seeded in six-well cell culture plates (TPP, Techno Plastic Products AG, Germany) and allowed to grow to 70–80% confluence as a monolayer. The resulting monolayer was gently drawn in a straight line from the center of each well with a sterile 200 µl pipette tip. After drawing, the medium was removed and the wells were washed in PBS (Sigma) solution, BCAN substance doses prepared with DMEM medium containing 10% Fetal Bovine Serum, 1% Penicillin streptomycin and 7.5% NaHCO3 were added to each well and cells were added to 24 and grown in a 48 hour CO2 incubator. Scratch wound healing of images was observed under an Olympus IX71 inverted microscope (Tokyo, Japan) and photographed with an Olympus DP72 camera (Tokyo, Japan) at 10X magnification at 0, 24 and 48 hours after scratch wound formation in HaCaT cells. Experiments were carried out in three replicates.

The effect of BCAN on cell viability was investigated by MTT test by applying different concentrations for 24 and 48 hours. According to MTT results, 5, 10, 20, 40, 60, 80 µM concentrations of BCAN were applied to HaCaT cells for 24 and 48 hours. After 24 hours of application, a small amount of 3% decrease in cell viability was observed at 5 µM cell viability compared to the control group. 10 µM caused approximately 12%, 20 µM approximately 14–15%, 40µM 16%, 60 µM 30%, and 80 µM 35% decrease in cell viability. After 48 hours of application, compared to the control group; It has been determined that 5 µM 17%, 10 µM approximately 24–25% 20 µM approximately 36%, 40 µM 50%, 60 µM 56%, and 80 µM concentration approximately 57–58% in cell viability and cause a toxic effect (Fig. 1A).

The amount of LDH released to the external environment by applying different concentrations of BCAN for 24 and 48 hours was investigated by the LDH test. Concentrations of 5, 10, 20, 40, 60, 80 µM were applied to HaCaT cells of BCAN for 24 and 48 hours. As a result of the 24-hour application, LDH increased by 5 µM 3%, 10 µM approximately 5%, 20 µM 8–9%, 40 µM 11%, 60 µM 12%, 80 µM 13% compared to the control group. After 48 hours of application, LDH increase was observed at the rate of 5 µM 5%, 10 µM approximately 16%, 20 µM 23%, 40 µM 26%, 60 µM 27%, 80 µM approximately 29% (Fig. 1B).

The change in the amount of ROS after the application of different 5, 10, 20, 40, 60, 80 µM concentrations of BCAN to the HaCaT cell line for 24 hours was evaluated with the ROS test. An increase in the ROS level was observed at all BCAN concentrations after 24 hours of application to HaCaT cells compared to the control. At 5 µM dose of BCAN, an increase in ROS levels was observed by 6-7.10%, µM 7–8%, 20 µM 8–9%, 40 µM 10%, 60 µM 18–19% compared to the control. At 80µM concentration, 32–33% increase in ROS level was determined (Fig. 1C).

Wound healing was observed after 24 hours in the control and solvent control. BCAN was able to induce significant keratinocyte migration at 5 µM, 10 µM and 20 µM concentrations, and an increased wound healing was observed compared to the control, and it was determined that it did not have a negative effect on wound healing. Scratch wound healing was observed at a decreasing rate with increasing BCAN doses, and it was determined that it had a slowing effect on wound healing when toxic concentrations of 40, 60 and 80 µM were increased (Fig. 2). It is the first attempt to evaluate the wound-healing effect of BCAN in HaCaT cells. It has been observed that 40, 60 and 80 µM concentrations of BCAN slow down skin wound healing in vitro by directly suppressing keratinocyte migration.

It is an extremely important issue to investigate the toxicities of the DBPs formed and determine their effects. In our study, it was aimed to investigate the cytotoxic effects of BCAN in the HAN group of DBPs on the human keratinocyte cell line HaCaT, which is the first barrier of our body in constant contact with water, bathing and swimming. Considering the skin's exposure to DBPs in water, the effect on the healing of a scratched wound on the skin was also investigated.

The epidermis, which consists of approximately 95% keratinocytes, is the first layer of the human skin to come into contact with external stimuli (Gantwerker and Hom 2012). Human skin is one of the main entry routes for external substances due to its large surface area and accessibility to cosmetics or environmental exposure. It was evaluated using the human HaCaT keratinocyte cell line, which constitutes a recognized model to evaluate the toxicological potential of chemicals present on the skin that can cause scratches and wounds and damage in vitro. HaCaT keratinocytes are a preliminary in vitro model for investigating skin toxicity (Pelin et al. 2017). Studies on the cytotoxic and genotoxic effects of disinfection by-products have increased in recent years. In the literature, there are studies on DBPs in different chemical groups.

In this study, the cytotoxic effects of 5, 10, 20, 40, 60 and 80 μM concentrations of BCAN on the HaCaT cell line were evaluated. BCAN substance concentrations were determined by preliminary experiments, and different studies with different cell lines were used as a first approximation value as a result of literature searches. In the study by Wei et al. the cytotoxicity of haloacetonitrile DBPs on the HAN Chinese hamster ovary (CHO) cell line, the lowest BCAN concentration that caused a statistically significant reduction compared to the negative controls was 7.0 μM, inducing cell density in 50% of the negative controls. LC50, BCAN concentration is 8.20 ± 0.51μM (Wei et al. 2020). The toxicological effects of DBPs have been demonstrated in different models and cell lines in studies for various purposes. In this study, which we have chosen in accordance with the purpose of its scope, gives an important originality as it is one of the first studies in the literature to investigate and present the cytotoxic effects of BCAN, one of the DBPs, on the HaCaT cell line.

In conclusion, the cytotoxic effects of BCAN, in the HaCaT cell line were determined by MTT, LDH and ROS tests. As a result of the MTT test, it was determined that the 40 µM concentration of BCAN caused a decrease in the cell number of approximately 50% compared to the control after 48 hours of application. There was an increase in LDH with the increase in BCAN concentration. It has been observed that increasing concentrations of BCAN induce ROS production in HaCaT cells. Considering the skin's exposure to disinfection by-products in water, the effect of BCAN on wound healing was investigated by the scratch test. It has been determined that 5, 10 and 20 µM concentrations of BCAN do not have a negative effect on wound healing, but when toxic concentrations of 40, 60 and 80 µM are increased, it has a slowing effect on wound healing. In this study, the effects of BCAN on HaCaT cells were evaluated for the first time in the literature. It is thought that investigating BCAN with in vitro experiments by applying cytotoxicity and genotoxicity tests using different cell lines will be more enlightening in determining the effects of this disinfection by-product on human health.

Disclosure Statement

No potential conflict of interest was reported by the authors.

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No competing interests reported.

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Assessment Of Bromochloroacetonitrile Formed As A Disinfection Byproduct in an HaCaT Cells

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