Evaluation of Proinflammatory Cytokines in Arsenic Induced Hepatotoxicity In Vivo

In general, the liver is a critical organ that performs a huge range of functions in the body and maintaining homeostasis (Donne et al., 2020), contributing to almost all biochemical pathways to fight diseases (Ghareeb and Ali, 2023; Taher et al., 2024). So, maintaining liver integrity is essential for the continued health of the body, and guarding against liver damage induction by exposure to various environmental toxicants (Souza-Arroyo et al., 2022; Ghareeb and Ali, 2024; Barouki et al., 2023). Since long time with the development of technology and the increase of consumer factories and their waste, liver damage caused by chemical pollutants has become overwhelming due to continuous exposure, thus increasing the risk of toxicity (Ramadhan and Ghareeb, 2021; Gripshover et al., 2023). Increasingly alarming are the escalating concentrations of environmental pollutants, particularly heavy metals like arsenic (Raju, 2022). They have raised environmental levels in many regions of the world, and numerous natural, industrial, and human factors have been linked to this (Ghareeb et al., 2024). Both people and animals are known to be at risk for major health problems from low concentrations of these heavy metals (Ali et al., 2021). Arsenic (As) and many of its compounds are potent toxins and increasing within the ecosystem due to urbanization and industrialization, and its forms could be either organic or inorganic (Prakash and Verma, 2021). In vivo, as undergoes several complex metabolic transformations, interacting with macro-molecules inside and outside cells, particularly ones that contained vicinal thiols, although the exact method of action differs based on the arsenic’s chemical structure (Renu et al., 2023). The link between exposure to arsenic and several adverse health effects, including hepatotoxic effects, has been shown in many investigations (Li et al., 2023). It is considered a carcinogenic heavy metal that can disrupt DNA synthesis and repair. Its toxic mechanism functions in established pathways through the generation of reactive oxygen species, inactivation of enzymes, and suppression of antioxidant defense (Garkal et al., 2023). In this regard, some experimental studies have confirmed that development of liver toxicity caused by arsenic is related to generate of cytokine regulatory networks composed of some pro-inflammatory cytokines (Dong et al., 2021). Although previous studies have suggested that arsenic induces oxidative stress as well as enhances immune responses, few studies have been conducted to evaluate the possible association between cytokines in the acute phase of toxicity (Islam, 2023). Management of environment pollutants among affected individuals is a formidable challenge (Ghareeb, 2023). This study was designed to evaluate pro-inflammatory cytokines in AS-induced hepatotoxicity in rat model.

In order to carry out this experimental investigation, eighteen mature male Wistar rats weighed between 180–220 g and aged about eight weeks old were acquired from the Animal Resources Centre of Urmia University (ARCUU). The NIH Institute’s officially approved 1978 criteria for the ethical handling of animals were followed. Before beginning the experiment, they were acclimatized for one week to the proper conditions, which included food and water at all times, A period of twelve hours of light and dark, an identical temperature of 25 °C, and constant humidity. Three groups of rats (n = 6/group) were set up as follows: The first group served as control (CON), which was not given any treatment, while the other two groups received arsenic (Sodium Arsenite NaAsO2) at single oral dose (13 mg/kg), this dosage was chosen in accordance with the Ghosh et al. (2010). Then laboratory animals were euthanized and sacrificed on 1 day and 7 days after exposure (referred as AS-1 and AS-7, respectively). Cardiac blood was collected from each rat by designated tubes, followed ten-minute centrifugation at 3000 rpm, and then kept at -80°C pending the analysis. Also, the livers were separated and homogenized.

A total of 0.3 g of liver tissue was dissected and washed with chilled normal saline, following the preparation of the tissue homogenate and a 10,000×g centrifugation, the supernatant was collected. Then, using commercial ELISA kits, the amounts of glutathione-reducing (GSH), total antioxidant capacity (TAC), and malondialdehyde (MDA) in hepatic tissue were measured.

The following liver enzymes were tested in serum: ALT, AST, ALP, and GGT using commercial bio-diagnostic kits according to respective manufacture’s protocol. Applying a typical ELISA protocol, the levels of TNF-α, IL-1β, and IL-6 were determined in serum samples. Samples of serum were gathered, processed, and diluted as needed. As directed by the kit, standards were made by serial dilution. At 450 nm, the absorbance was measured with a micro-plate reader. Concentrations of these cytokines were determined in samples by constructing standard curves from known standards and comparing the absorbance values.

Using the SPSS program (USA), data were statistically processed by one-way test (ANOVA). Tukey post hoc test was subsequently carried out to determine the variations between group means. Data were presented as mean ± standard deviation. The graphs were made with GraphPad Prism. At the 5% level, a result of p<0.05 was set as significant variance.

Results showed a significant reduction in TAC level versus a clear raise in MDA content in the hepatic tissues of AS-1 and AS-7 animals when compared to CON rats according to a time-based manner. However, rats exposed to arsenic led to an increase in antioxidant GSH activity compared to control animals but without significant difference as illustrated in Figure 1.

The effect of arsenic on serological level of liver enzymes of intoxicated rats (AS-1 and AS-7) and their comparison with control rats is displayed in Figure 2. The findings demonstrated that, when compared with the control (CON) rats, arsenic exposure considerably raised (p<0.0001) the serological levels of AST, ALT, ALP, and GGT in arsenic treated rats. On the other hand, it was observed that the rats inebriated hepatic enzymes (ALT, ALP, and GGT) increased gradually over time. Otherwise, AST levels in the AS-1 and AS-7 animals showed a significant difference.

According to results, AS-treated groups showed significantly increased levels of serum cytokines TNF-α, IL-1β, and IL-6 (p<0.0001) in time-related manner on the 7th and 1st day after exposure (respectively) in comparison with the control animals, as presented in Figures 3, 4, 5.

Arsenic is an environmental toxicant that negatively impacts several organ systems in the body, particularly the liver and immune system. In general, arsenic absorption from the body is followed by metabolized to toxic forms, converting to mono-, di-, and methylated pentavalent products. Moreover, it has the property of accumulating in some organs and tissues including the liver

(Karak, 2022; Pánico et al., 2022). The liver is the principal organ for heavy metal intoxication, because the majority of the absorbed exogenous toxicant is trapped in the liver and eventually transferred into other organs (Ghareeb, 2021; Ghareeb and Ali, 2024). In general, accumulation of any toxicant in body depends on route, dose and duration of exposure (Gupta and Gupta, 2020). To evaluate the hepatotoxic effect of arsenic in exposed rats, hepatic oxidative stress indicators were determined. Many pathological conditions are intrinsically linked to oxidative stress and its consequences, including lipid peroxidation, which is determined by malondialdehyde (Cordiano et al., 2023; Mas-Bargues et al., 2021). This is supported by the findings of the current experiment, which show an important rise in its level in liver tissue. It is a reactive aldehyde formed during the peroxidation of polyunsaturated fatty acids (Sutaria et al., 2022). One of the severe effects of oxidative stress is cell death by necrosis or apoptosis, including hepatocytes (Allameh et al., 2023). Total antioxidant capacity, was significantly reduced in the livers of animals exposed to arsenic, which represents the total non-enzymatic antioxidant capacity and includes the synergistic effects of all antioxidants in liver tissue (Malik et al., 2020). Antioxidants neutralize reactive oxygen species, which upset the cellular redox balance, thereby protecting them from oxidative damage. Therefore, when antioxidants level decreases, it causes damage at different molecular levels, increasing the risk of pathological damage such as inflammation (Pisoschi et al., 2021; Ramadhan and Ghareeb, 2022).

Glutathione is the most abundant intracellular thiol and a critical antioxidant in hepatocytes. It detoxifies ROS and free radicals, protecting the liver from oxidative damage (Vašková et al., 2023). It conjugates with toxic compounds through the action of glutathione S-transferase (GST), facilitating their excretion. This is particularly important in detoxifying drugs and other xenobiotics (Georgiou-Siafis and Tsiftsoglou, 2023). Rats administered sodium arsenite had significantly higher serum concentrations of hepatic enzymes than control rats. This demonstrated its capacity to increase cell membrane permeability and promote liver cell destruction, resulting in liver dysfunction and the release of liver enzymes into the bloodstream (Taher and Ghareeb, 2022; Kalas et al., 2021). In addition, results confirmed the ability of sodium arsenite to cause a rapid inflammatory response in rats after 1 and 7 days of exposure by disturbing the levels of serum cytokines in comparison to non-exposed animals. An important rise in cytokine serum levels suggested an inflammatory response to arsenic exposure. The pro-inflammatory cytokines IL-1 and TNF induce inflammation by increasing the expression of adhesive molecules on endothelial cells, consequently causing leukocytes in circulation to bind to the endothelial (Muzamil et al., 2021). Increased levels of IL-6 and TNF-α disrupt the internal and external homeostasis of these molecules, leading probable cellular death, restricting enzymes and mitochondrial activities, and thus accelerating the induction of various stress genes (Chen et al., 2018; Duvigneau et al., 2019). This experiment’s results are in line with previous studies that has proven that AS is an immunosuppressive agent and exposure to it at non-permissible doses has resulted in a variety of cellular and molecular immune-related disorders (Bellamri et al., 2018; Giles and Mann, 2022). It is a potent immunotoxicant that affects the release of cytokines in time and dose-dependent manners (Dangleben et al., 2013). Lipid peroxidation products and pro-inflammatory cytokines were found to positively correlate in arsenic-exposed mice, according to a prior study by Afolabi et al. (2013). Other investigations by Lasram et al. (2014) and Abu-Zeid et al. (2021) found that decrease of antioxidant defense enzymes was linked to apoptosis, immunotoxicity, and liver damage. The ability to activate inflammatory cytokines has been linked to immune-related disorders in humans and rodents exposed to arsenic (Dangleben et al., 2013).

This experimental study confirmed the hepatotoxicity and immunotoxicity of arsenic in a rat model through acute inflammatory response and induction of serum pro-inflammatory cytokines in a time-dependent progression.

We express my appreciation to our colleagues who provided us with a stimulating academic environment.

It represents a new topic that aims to evaluate the levels of serum pro-inflammatory cytokines in acute and subacute hepatotoxicity in vivo.

The research was equally involved in the efforts to complete the experiment.

The authors have declared no conflict of interest.