Carbon Black CB-EDA Nanoparticles in Hepatocytes: Changes in the Oxidative Stress Pathway

Background/Aims: Carbon Black (CB) is the most widely produced and commercially used nanocarbon. Growing evidence links nanomaterials to adverse effects, arising from their large surface area capable of interacting with biological systems. Due to the variety of applications and human exposures to nanoparticles, it is important to assess the potential health risk and understand the underlying mechanisms of their toxicity. The present study aims to investigate the cytotoxic effect of the Carbon black nanoparticle covalently linked to ethylenediamine (CB-EDA) on AML-12 cells, a lineage of murine hepatocytes. Methods: For this, the cells were exposed to CB-EDA for 24h, at different concentrations of the nanoparticle (1, 50, 250, 500 and 1000 μg/mL). Effects on cell viability were evaluated using MTT and neutral red dye tests and analysis of changes in cell morphology. Furthermore, the oxidative profile (levels of reactive oxygen species - ROS and nitrogen - RNS) and inflammatory profile (production of IL-6 and TNF-α) were investigated. Results: The results show that CB-EDA nanoparticle causes a reduction in cell viability at concentrations of 250, 500 and 1000 μg/mL after the exposure period. There was a significant increase in the production of ROS, RNS and pro-inflammatory interleukin TNF-α. Conclusion: The data suggest that CB-EDA nanoparticle has a cytotoxic potential in AML-12 hepatocytes, evidenced by the induction of oxidative stress and secretion of inflammatory cytokines, with a consequent decrease in cell viability.


INTRODUCTION
Nanotechnology is a branch of science that encompasses any technology operating on a nanoscale that has practical applications, employing individual atoms and molecules to form functional structures. The field of nanotechnology involves the creation and use of chemical, physical and biological systems with structural characteristics between individual atoms or molecules in submicromic dimensions, and the assimilation of resulting nanostructures into larger systems. The simplest definition of nanotechnology is "nanoscale technology", whose components and structures Corresponding Author: Joice Margareth de Almeida Rodolpho represent significantly improved properties, processes and phenomena due to their nanoscale size [1].
Nanomaterial refers to a material with a crystalline or amorphous particulate structure on a nanoscale. The specificity of the size and structure of nanomaterials makes them have special properties not found in many traditional materials. The development of new nanostructured materials usually leads to a major advance in the field of high technology, of both academic and industry interest [2].
Carbon Black (CB) is among the nanomaterials widely used in various industrial sectors, which is the nanocarbon produced on larger scale and commercially cheaper. The CB are aggregated of spheroidal nanoparticles, typically 10 to 100 nm in diameter and represent a very wide family of materials, with many commercial products available on the market, with a diverse variety of technological specifications and performance requirements. Its applications include the areas of nanotechnology, oil exploration and reservoir characterization, as well as use in medicine, catalysis and environmental remediation [3].
CB has low dispersion in polar solvents and, as a strategy to adjust the interactions of the particle surface with the medium and improve its dispersibility and compatibility, can be covalently bonded to polymers. In this sense, the CB was functionalized with ethylenediamine (EDA) to produce the nanoparticle called CB-EDA [4]. It is believed that the toxicity of nanoparticles is determined by their surface reactivity, influenced by their chemical composition, physical properties and chemical surface. Thus, this modification with EDA can influence the biological nanoparticle activity, due to modified physical-chemical properties, resulting in potential CB-EDA toxicity and altering its biological effects [5].
Growing evidence links nanomaterials to adverse biological outcomes and, due to the variety of applications and potential human exposures to nanoparticles, it is important to assess their toxicity to determine the risk of workers and consumers. It is crucial to understand the underlying mechanisms of its toxicity, as the observation of similar effects after different exposures of nanomaterials does not reflect intracellular processing and interactions in similar organelles. A complete understanding of the mechanisms is necessary not only for predicting potential toxicological impacts, but also for the development of safer applications, modulating the physical-chemical characteristics [6].
Studies evaluating the toxic potential of CB-EDA are still scarce, which makes it extremely important that the performance of this nanoparticle be explored. The tests may include in vitro/in vivo analyses, seeking to understand how oxidative pathways, represented by the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), are related to the synthesis and release of pro-inflammatory molecules [7].
In vitro and in vivo studies have demonstrated the ability of CB nanoparticles to induce toxicity to the respiratory system, but most of them did not address the molecular pathways of toxicity [6].
In view of this, it is necessary to evaluate the cytotoxic effects of the modified nanoparticle CB-EDA in biological models and it is expected that it will present low harmful effects in cell culture models, based on its surface modification and physical-chemical properties. Therefore, this study aims to evaluate the cytotoxic profile of CB-EDA in the AML-12 lineage (murine hepatocytes), in acute exposure for 24 h. For this, the potential cell damage was investigated by oxidative and inflammatory profile analysis in these cells, in addition to the metabolic pathways involved in this process.

MATERIAL AND METHODS Nanoparticles
The CB-EDA nanoparticle was ceded by the petroleum industry Centro de Pesquisa Leopoldo Américo Miguez de Mello CENPES/Petrobrás, Rio de Janeiro, RJ, Brazil. PETROBRAS.

Cell line AML-12
The hepatocytes lineage (AML-12) derived from liver tissue of Mus musculus was acquired in the cell bank of Rio de Janeiro (BCJ) code:0354. The cultivation occurred in bottles containing DMEM F12 (Dulbecco's Modified Eagle's Medium, Sigma) with 10% fetal bovine serum (LGC Biotechnology), at 37 ºC and with 5% CO2. A protocol was established with 3 independent experiments and in triplicates with the following concentrations of the black carbon nanoparticle modified with ethylenediamine (NP CB-EDA): 1, 50, 250, 500 and 1000 μg/mL for 24 hours. The selection criterion was to use concentrations that proved to be more toxic and nontoxic among those initially tested (1, 10, 50, 100, 250, 500, 750 e 1000 µg/mL) for 12,24,48 and 72 hours. the concentrations chosen and hours was selected due to the decrease in cell viability.

Cell Viability by MTT Method
The colorimetric method of Tetrazolium 3 -(4.5dimethyltiltiazol -2 -il) 2.5 -diphenylbromide (MTT) was performed (SIGMA-ALDRICH, USA) (MOSMANN, 1983) to evaluate the integrity of mitochondrial function by measuring the formation of formazan crystals by mitochondrial enzymes. The higher the production of these crystals, the greater the cell viability. In a microplate of 96 flat bottom wells, with treatment for cultivation and with lid (Corning® Costar), 1×10 4 cells per well were inoculated. After 24 hours of support, CB-EDA NP was added. After the exposure times of 24 hours, the wells were washed 2 times with PBS 1X (Saline phosphate buffer: 8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4, 0.2 g KH2PO4 and 1 L distilled water) e 200 μL of MTT solution at 0.5 mg/mL in PBS 1X plus incomplete and phenol-without DMEM F12 medium (1:5) were added. The reaction occurred for 4 hours at 37 ºC and 5% CO2. White controls containing only MTT solution were Corresponding Author: Joice Margareth de Almeida Rodolpho performed. Then, the reagent solution was removed and 100 μL of the DMSO diluent were added per well, followed by the absorption reading at 540 nm in spectrophotometer (Thermo Scientific ™ Multiskan ™ GO Microplate Spectrophotometer). Cell death control (CTRL+) was done with extra 5% enzymatic detergent. The percentage of cytotoxicity occurred by comparing the data obtained with the CTRL group according to the equation.

Viability by Neutral Red Method
The neutral red assay is based on dye accumulation in liposometropic membranes. The more viable cells, the greater the diffusion of the dye through the membrane (REPETTO; DEL PESO; ZURITA, 2008). In a microplate of 96 flat bottom wells, with treatment for cultivation and with lid (Corning® Costar 3527), 1×10 4 cells per well were inoculated. After 24 hours the NP CB-EDA have been added. After the exposure time of 24 hours, the wells were washed 2 times with PBS 1X and 100 μL of dye at 30 μg/mL in DMEM F12 medium without phenol supplemented with 1% of SBF were added. The reaction occurred for 2 hours at 37 ºC and 5% CO2. Then the reagent solution was removed and 200 μL of the Acetic Acid diluent 1% and Ethanol 50% were added per well, followed by the absorption reading at 570 nm in spectrophotometer (Thermo Scientific ™ Multiskan ™ GO Microplate Spectrophotometer).

Cellular Morphology by Optical Microscopy
The cellular morphology of the AML-12 after exposure to nanoparticles was observed by optical microscopy. In microplate of 96 flat bottom wells, with treatment for cultivation and with lid (Corning® Costar 3527), 1×10 4 cells per well were inoculated. After 24 hours of support, CB-EDA NP was added. After the exposure time of 24 hours, the wells were observed in optical microscope Axiovert 40 CFL (Zeiss), with objective lens 10X, whose images were captured with the camera coupled model LOD-3000 (Bio Focus) and analyzed by future WINJOETM version 2.0 software in final resolution of 100X.

Reactive Nitrogen Species (RNS) Production by Griess Reaction
The production of RNS analyzed by means of the Griess reaction, which evaluate the production of nitrite ion (NO2 -) (GREEN et al., 1982;SALTZMAN, 1954 [0.1% n-1-naphtiletilenediamine dicloridra]) for 15 minutes at room temperature. The absorbance was read at 540 nm in spectrophotometer (Thermo Scientific ™ Multiskan ™ GO Microplate Spectrophotometer). The nitrite concentration in the supernatant was quantified from a standard curve with known concentrations of nitrite in mM described in the kit.

Production of Reactive Oxygen Species (ROS)
The production of ROS was measured using the DCFH-DA fluorescent probe (Dichlorodihydrofluorescein Diacetate 2′-7′-Dilorodihydrofluorescein) (Sigma-Aldrich) (WAN, MYUNG, LAU,1993). In a plate of 96 wells, 1x10 4 cells/well were cultivated. After 24 hours for cell support, concentrations of CB-EDA NP were applied. After exposure (24 hours), DCFH-DA probe was diluted in DMEM F12 medium and applied to each well (200μL) for 30 minutes. The wells were washed with PBS 1X and fluorescence emission was read at 485-530 nm in Spectra MAX, i3 plate spectrophotometer ® (Molecular Devices). The values obtained were transformed into percentages using the same formula previously mentioned in the MTT test.

Detection of IL-6 and TNF
For the measurement of IL-6 and TNF cytokines (Kit OptEIATM, BDBiosciences) direct ELISA was performed following the protocol described below. For this procedure, high affinity microtitration plates with 96 wells (Corning® Costar 3590) were also used. Between each step the plates were washed with 300 μL/well of blocking solution (PBS 1X Tween 20 to 0.05%). After sensitization of the plates with 100 μL/well of specific capture antibody in carbonate buffer, there was incubation for 16 hours at 20 ºC. Then, the blocking reaction was performed with 200 μL/10% milk well, followed by incubation for 1 hour at room temperature. It was then performed the application of 50 μL of the samples and the titration curve of the cytokine patterns, with incubation 2 hours also at room temperature. The capture antibody conjugated with the enzyme peroxidase was added in the volume of 100 μL/well under light for 1 hour and 30 minutes. Then 100 μL/well of the TMB substrate was applied and the plates were incubated, still under light, from 15 to 30 minutes approximately. Then, the reaction was blocked with the addition of 50 μL/well of 2N sulfuric acid and the plate reading was performed in the plate spectrophotometer (Thermo Scientific ™ Multiskan ™ GO Microplate Spectrophotometer), at a wavelength of 450 nm. The concentrations were calculated from the titration curve of cytokine patterns and the final concentrations expressed in pg/mL.

Statistical Analysis
The data obtained were expressed in ± SD and analyzed using GraphPad Prism 7.0 (San Diego, California, USA). The entire study was carried out in at least three biological triplicates and three independent experiments. The discrepant data were identified by Grubbs analysis, followed by the Shapiro-Wilk test to verify its parametric or nonparametric nature. For this, Corresponding Author: Joice Margareth de Almeida Rodolpho the ANOVA test (variance analysis) was applied to parametric data and also the Tukey multiple comparation scan (the results were presented in mean and standard deviation). For nonparametric data, the Kruskal-Wallis test and Dunn's multiple comparison post-test were used. Statistical significance was established at p <0.05.

RESULTS
To define the concentrations of NP CB-EDA that would be exposed to AML-12 hepatocytes cells to be evaluated in the study, a preliminary cell viability test was performed using the MTT method. The test consisted of serial dilution of CB-EDA NP in 1, 10, 50, 250, 500, 750 and 1000 μg/mL and exposed to cells for 12, 24, 48 and 72 hours, represented in Figure 1. Data analysis indicated that in 12-hour period there was no significant decrease in the cellular viability of AML-12 hepatocytes due to exposure to CB-EDA NP ( Figure 1A). In 24 hours, there was a significant decrease in concentrations 100, 250, 500, 750 and 1000 μg/mL when compared with the control group -( Figure 1B). In 48 and 72 hours there was a significant decrease in cell viability at the same concentrations 250, 500, 750 and 1000 μg/mL when compared with the control group -( Figure 1C and 1D). These data served as the basis for the construction of EC50 of a graduated dose-response curve, represented by the concentration of the compound for which 50% of the expected effect is observed. In this case, at 12h the EC50 was 255.3; in 24h it was 110; in 48h it was 269.3 and in 72h it was 279.3 ( figure 1E-H). In view of these results, the exposure time chosen to continue the tests was 24 hours, with concentrations of 1, 50, 250, 500 and 1000 μg/mL. Figure 2 shows the results of the cytotoxicity profile by the neutral red dye methodology, at the concentrations and times defined. It can be observed that in 24 hours there was a significant reduction in the number of viable cells in the AML-12 scan at concentrations of 250, 500 and 1000 μg/mL when compared to the control group -. Figure 3 shows, by qualitative data, the cellular morphology of AML-12 hepatocytes after 24 hours of exposure to NP CB-EDA, at different concentrations of 1, 50, 250, 500 and 1000 μg/mL and control group -. It is possible to observe a decrease in size and alteration of cells shape in the groups that were exposed to concentrations of 250, 500 and 1000 μg/mL, when compared to the control group -. The quantification of the production of reactive nitrogen species (RNS) and reactive oxygen species (ROS) for oxidative stress analysis were performed in the AML-12 murine hepatocyte lineage in the 24-hour period, at different concentrations of CB-EDA NP, as observed in Figure 4.
The production of RNS in 1000 μg/mL concentration is observed in Figure 4A and there was a significant increase when compared to the control group -. In ROS production, observed in Figure 4B, there was a significant increase in concentrations of 50, 250, 500 and 1000 μg/mL when compared to the control group -.
The production of IL-6 and TNF cytokines was analyzed after 24 hours of exposure in AML-12 murine hepatocytes to NP CB-EDA, as shown in Figure 5. There was no significant variation in IL-6 production in the exposed groups when compared to the control group -( Figure 5A). In the production of TNF, a significant increase was observed in the group exposed to 1000 μg/mL of NP CB-EDA when compared to the control group -( Figure 5B).

DISCUSSION
In vitro models were developed in cytotoxicity studies to eliminate some variables, to obtain better control over the experimental conditions by researchers. Several studies have evaluated the harmful effects of carbon nanoparticles in different cell types, including macrophages, lymphocytes and fibroblasts [8]. In addition to these cells, hepatocytes are also widely used in cytotoxicity evaluation tests. Primary hepatocytes are differentiated cells capable of reproducing the liver response in vitro and are currently considered a valuable tool to determine the metabolism of compounds and assess the risk of hepatotoxicity. As an alternative to the use of cultured primary hepatocytes, the use of liver cell lines has been established, which facilitates the development of tests [9]. Toxicity studies using hepatocytes are of fundamental importance, since hepatotoxicity can predict conditions such as intoxication, cancer and others, with similar potential results in vivo [10].
When into contact with the AML-12 cell line, CB-EDA nanoparticles trigger a mechanism that results in decreased cell viability at the highest concentrations tested when compared to the control group. These data corroborate other studies that used the ultrathin Carbon Black nanoparticle exposed to murine hepatocytes, whose greatest reductions in cell viability also occurred as nanoparticle concentrations were higher [11,12].
This reduction in cell viability may be correlated with oxidative stress pathways by ROS and RNS production, which, when increased, result in cellular disability of redox control [13]. Reactive oxygen species and reactive nitrogen species are produced in normal hepatocytes and are critical to normal physiological processes including oxidative respiration, growth, regeneration, apoptosis and microsomal defense. When oxidation product levels exceed the capacity of normal antioxidant systems, oxidative stress occurs. This type of stress, in the form of ROS and RNS, can be harmful to all liver cells, including hepatocytes [14].
The main targets of oxidative stress are proteins, lipids and DNA/RNA, and modifications to these molecules may increase the chances of mutagenesis. Overproduction of ROS and RNS, notified for an extended period, can cause damage to cell structure and functions and may induce somatic mutations and preneoplastic and neoplastic transformations. In fact, this process can cause irreversible damage to cells, resulting in cell death by necrotic and apoptotic mechanisms [15]. In response to inflammatory stimuli, the increase in Corresponding Author: Joice Margareth de Almeida Rodolpho RNS in hepatocytes is considered a short-lived cell messenger, which causes its production to be detected early [16].
Niranjan e Thakur (2017), in their studies, address oxidative stress and inflammation resulting from exposure of CB particles in in vivo experiments, highlighting the presence of DNA damage mediated by exacerbated ROS production [17]. The increase in ROS production was discussed by Requardt et al (2019), in in vitro studies based on the cellular exposure of the HepG2 strain to carbon nanotubes [18]. The increase in RNS production is also a mechanism discussed in the literature in the context of nanoparticles, increasingly present in daily life, and detected by authors in studies such as: LA-9 cells exposed to NP CB-EDA [7] and exposed to NP ONCT-TEPA [19].
Several inflammatory stimuli such as excessive ROS/RNS produced in the process of oxidative metabolism, or resulting from chemical compounds, have been reported as initiators of inflammatory process, resulting in synthesis and secretion of pro-inflammatory cytokines. The activation of nuclear-kappa Factor B and the production of tumor necrosis factor alpha (TNF-α) were, for example, documented as playing a critical role in inflammatory process [15]. In this sense, the production of TNF induced by exposure of AML-12 to CB-EDA was investigated, with the expected increase in production of this cytokine in response to inflammatory stimulus exerted in the cell by the nanoparticle.
TNF plays an important role in hepatocytes injury and cell death in various pathophysiological states. The TNFinduced cell death signaling pathway involves binding the linker to the TNF type I receptor, the recruitment of intracellular proteins, and the activation of caspases whose proteolytic activities mediate apoptotic cell death, in addition to independent pathways of caspase and necrosis. Considerable interest focused on the role of antioxidants in regulating hepatocytes sensitization to TNF and induced apoptosis due to the presumed role of oxidative stress in this death [20].
Studies of Jiang et al. (2020) investigated the toxicity of carboxylated carbon in human bronchial epithelial cells (BEAS-2B). Their results showed that the nanoparticle significantly increased the production of IL-6 and TNF-α in cells, in addition to identifying the correlation between ROS generation and the increased production of these inflammatory cytokines induced by carboxylated carbon [21].

CONCLUSION
In view of the studies presented, it is observed that generation of ROS/RNS in induction of oxidative stress, as well as the inflammatory profile of cytokines, are used as good indicators to evaluate the toxicity induced by nanoparticles, which compromises cell viability. According to analyses, NP CB-EDA has a cytotoxicity profile in the AML-12 cell line, evidenced by induction of oxidative stress and secretion of inflammatory cytokines, with consequent decrease in cell viability. Thus, it is suggested that this toxicity be further explored in future studies, emphasizing the importance of elucidating mechanisms involved in cell damage and considering the increasing human and environmental exposure to nanoparticles. Corresponding Author: Joice Margareth de Almeida Rodolpho Cell viability profile (neutral red) in AML-12 murine hepatocytes cells exposed to CB-EDA NP for 24 hours at concentrations 1, 50, 250, 500 and 1000 μg/mL and compared with the Control -group. * p< 0.05, **p<0.01, ***p<0.001 and ****p<0.0001 vs Control in 24 hours. Representative morphology by optical microscopy images with an increase of 100X of the groups exposed to NP CB-EDA at concentrations of 1, 50, 250, 500 and 1000 μg/mL and control groupin 24 hours. Qualitative data.  1, 50, 250, 500 and 1000 μg/mL), compared with the Control-group. * p< 0.05, **p<0.01, ***p<0.001 and ****p<0.0001 vs Control in 24 hours. Figure 5. Cytokine levels in samples of AML-12 cells exposed to NP CB-EDA for 24 hours. Evaluation of IL-6 and TNF levels in the aml-12 cell overn in the control group and different concentrations of NP CB-EDA (1, 50, 250, 500 and 1000 μg/mL) exposed to cells. * p< 0.05, **p<0.01, ***p<0.001 and ****p<0.0001 vs Control in 24 hours