Nanomedicine, the medical application of nanotechnology that deals with particles less than 100 nm in dimensions, has become one of the most promising areas of modern medical research in all related respects [1-4]. Nickel oxide nanoparticles (NiO-NPs) have received a lot of research interest for their huge potential and multiple applications especially in sensors, ceramics, catalysts, photovoltaic materials, paints, cosmetics and others [5,6]. In this context, the environmental burden and the risk of human and animal exposure increase, so it is necessary to understand their toxic effects in the living body [7]. In recent years, evaluations of the potential toxicity and health risks and their management of nanoparticles, including NiO-NPs, have received great attention [8,9].

The nanoparticles can easily enter the body from several entrances, orally, inhalation and skin. These particles easily distribute with the circulatory system, reaching the vital organs [10,11]. Studies have documented that excessive generation of free oxygen radicals (ROS) was the proposed primary mechanism for toxicity induced by NiO-NPs, in addition to stimulation of cell degeneration and inflammatory response [12,13]. Nickel (Ni) has been proven as a chemical element recognized as a carcinogen due to its genotoxicity [14], furthermore the creation of oxidative stress and inflammatory response and among the organs most exposed to Ni are liver and kidneys [15]. Nowadays, there is a growing interest in using raw extracts of medicinal plants to develop traditional alternative medicine as they are valuable sources of phytochemicals that have multiple medicinal properties including antioxidant and tissue damage inhibitor along with low side effects [16,17].Thymus vulgaris (Thyme) is a species of bushy flowering plant with aroma belongs to the Lamiaceae family and is known as a beneficial herbal spice and has antioxidant effects so it is widely cultivated and used by the population [18]. Thyme oil (TO) is a rich source of aromatic bioactive constituents such as thymol and carvacrol and these two essential phenolic constituents are effective antimicrobials as well as immuno-modulatory agents and antioxidant scavengers [19,20]. This experimental study was prepared to assess the ability of thyme oil to improve the hepatic and renal destruction caused by NiO-NPs by examining indices of liver and kidney function as well as oxidative stress in male rats.

Nickel Oxide Nanoparticles (NiO-NPs) and Thyme Oil (TO)

A dispersion of nickel II oxide nanoparticles with a particle size of 3-6 nm (Figure 1) manufactured for research and development purposes was purchased from Nanoshel UK Limited Company. This product has the following characteristics: dissolved in butanol, concentrate 3% by weight, 99.9% purity, milky green, liquid, ph = 6-7. Thyme essential oil manufactured by Allin Exporters (India) was sourced from local markets.

Experiment Design

Thirty-two adult male albino rats (160-195 g) were obtained from the animal house laboratories of the universities of the Ministry of Higher Education and Scientific Research, Kirkuk, Iraq. Animals were housed in cages provided with food and water and acclimated to a standard laboratory environment for 10 days prior to the experiment. Animals were managed ethically according with NIH institutes guidelines officially approved in 1978.They were divided randomly into 4 groups (N = 8/group) and were dosed for 28 consecutive days with nanoparticles and/or thyme oil once a day using an oral gavage (Figure 2), CON group, animals were delivered with deionized water for control; NiO-NPs group, treated with 100 mg/kg body weight [21] of nickel oxide nanoparticles dispersion.

Figure 1: SEM of Nickel II Oxide Dispersion

Figure 2: Diagram Summarizing the Design of the Experiment

TO group rats were dosed with essential oil of thyme at 5 mg/kg [22]. While NiO-NPs +TO group gained like previously mentioned dosages of NiO-NPs and TO groups with an interval of 8 hours between the two doses.

A day after final dose, the anesthetized animals were sacrificed and blood samples were obtained from the puncture of the heart of each rat, placed in designated test tubes and centrifuged after coagulation to separate the serum and stored at a temperature of – 40 ° C until biochemical analyses. The liver and renal organs were collected and homogenized in cold phosphate-buffered saline at −20 °C and centrifuged for about a quarter of an hour and then the supernatants were carefully selected for oxidative stress assessment.

Oxidative Stress Biomarkers

Liver and kidney homogenates were obtained from all experimental animals to assess the activities of oxidative stress markers including malondialdehyde (MDA), glutathione-reducing (GSH) and superoxide dismutase (SOD) by spectrophotometry based on catalog of available commercial companies (Bio-diagnostic).

Liver and Kidney Functions Analysis

Following the standard protocol, alanine and aspartate aminotransferase (ALT and AST), blood urea and creatinine (Cr) serum concentrations were assessed in studied animals according to instructions of private companies (Bio-diagnostic).

Data Analysis

All quantitative results were processed with SPSS (version 25) software program and data were displayed as mean±Std. Deviation. The variation between experimental groups was found by applying one-way analysis of variance (ANOVA) followed by Duncan's test and significance was adopted statistically at p <0.05.

Exposure of experimental animals to NiO-NPs induced a considerable augmentation in of ALT (126.06±10.57), AST (193.80±14.85), Urea (35.76±3.15) and Cr (1.02±0.13) levels when compared to control animals (75.61±8.24; 114.88±10.23; 21.21±1.44; 0.54±0.07 respectively). However, a significant decrease in biomarkers was observed when animals were treated with NiO-NPs plus thyme oil (98.45±7.78; 156.52±16.74; 28.11±2.62; 0.63±0.14, respectively) compared to animals dosed with NiO-NPs only. Otherwise, no important variance in levels of these biomarkers was noted between control and thyme oil animals as displayed in Figure 3.

On the other hand, a significant lower in both of GSH and SOD activities was recorded versus an obvious raise in MDA level in the hepatic and renal homogenous of NiO-NPs animals when compared with the CON animals (P < 0.05). However, dosing with thyme oil post nickel oxide nanoparticles significantly raised the activities of both antioxidants GSH and SOD in the NiO-NPs + TO animals compared to the NiO-NPs animals. Also, an important reduction in MDA content was observed in animals received both NiO-NPs and TO when compared to the NiO-NPs animals (Figure 4).

Figure 3: Serum Levels of Liver and Kidney Function biomarkers: A) ALT, B) AST, C) Urea and D) Cr, for Studied Rats. Uppercase Letters Denote the Statistical Differences (p<0.05) Between the Experimental Groups (n = 8 rats/group).

Figure 4: Levels of Oxidative Stress Markers: A) MDA, B) GSH, C) SOD of Liver and Kidney Tissues for Different Experiment Groups. Data were Presented as the mean±SD and Uppercase Letters (a-d) Indicate Variance Among Experimental Groups (N = 8 rats/group) At Level of p <0.05.

The results of this study demonstrated the destructive ability of NiO-NPs to the liver and kidneys by disturbing the levels of functional indicators of both vital organs in the sera of exposed rats. Dosing experimental rats with NiO-NPs led to markedly higher levels of serum hepatic enzymes ALT and AST compared to control rats. The nanoparticles increased the permeability of the cell membrane and stimulated liver cell damage, which led to liver dysfunction and releasing hepatic enzymes into the blood [23-26]. In general, renal toxicity to chemicals can disrupt excretory task of the kidneys with changes in their cellular structure [27]. Similarity, it has been proven that the kidney is a vital organ in which nanoparticles gather [28-31]. It is worth noting that nickel ions can also cause significant damage to the kidneys, as well as reduce antioxidant activities [32]. According to the results, the role of oxidative stress was observed through the raising MDA levels versus reduction antioxidants concentrations in hepatic and renal homogenates of intoxicated group. This means that NiO-NPs could induce oxidative stress and increased intracellular reactive oxygen species. 

The main proposed mechanism of cell damage induced by Nio-NPs is through stimulation of excess reactive oxygen species (ROS), which are signs of oxidative stress and it was also found to be a major mechanism of cytotoxicity for other nanomaterials [33,34]. This result is in line with another similar study by Ali et al. [35,36], where they found remarkable disturbances in hepatic and renal biomarkers in the sera of male rats dosed with NiO-NPs (100 mg/kg) for twenty-eight days continuously, along with rising in MDA levels and lowering in antioxidants activities in tissue homogenates of both liver and kidney with noting cellular changes histologically [35]. In another experimental study, Iqbal and colleagues [37], exposed male rats to nickel nanoparticles by intraperitoneal injection at various doses for 28 days. They suggested in the results the hepatotoxicity of Ni nanoparticles and their respective dose-dependent toxic pathways, pointing to hepatic impairment by the large production of hepatic serum enzymes in poisoned animals along with the presence of hepatic tissue lesions and clear elevated MDA, CAT and GSH levels in liver homogenates [36]. In a previous study by Abdulqadir and Aziz [38], they found that treated rats with (20 nm) Ni-NPs for 28 consecutive days induced different cellular ultra-structural changes in rat kidneys through various pathways and that the internalization and accumulation of Ni-NPs in the cellular ultrastructure significantly impaired renal function [37]. On the other hand, the results of this experiment showed a clear decrease in the harmful effects of NiO-NPs when the combined doses with TO.

As for NiO-NPs+TO animals, they showed an improvement in hepatic and renal functions, as well as oxidative stress indices when compared to NiO-NPs animals. This denotes to antioxidant power and ability to scavenge free radicals of thymol and carvacrol, the two main phenolic components of T. vulgaris essential oil. Elshopakey et al. [22], found that Thymus vulgaris oil dosed to experimental rats has prospect antioxidant activity and preventative ability upon CCl4-induced hepatotoxicity through regeneration of hepatocytes [22]. In a recent study by Gursul et al. [39], they found that intraperitoneal injection of carvacrol (50 mg/kg) in rats prevented the increase in oxidative stress and liver enzyme levels in serum induced by methanol and raised the levels of antioxidants while mitigating histopathological damage, thus suggesting that carvacrol was beneficial in the treatment of liver damage caused by methanol [38]. In a previous study, Thymus vulgaris extract (500 mg/kg) was shown to have a modulating role on hepatotoxicity in female rats, as the natural antioxidants in thyme enhanced the antioxidant defense system, superior to the oxidative stress caused by dexamethasone [39]. Soliman et al. [40,41] demonstrated that NaNO2-induced liver injury was obviously reduced by administering T. vulgaris extract )0.5 g/kg (to mice orally for 15 days, which counteracted oxidative stress and perturbed biochemical levels as well as restoring tissues to a normal condition [40]. El-Boshy et al. [42], concluded in their experimental study on laboratory rats that T. vulgaris extract (500 mg/kg) reduced lead overload in renal hepatic tissue and this had a potential role in immune modulation, antioxidant activity and a preventative role upon lead toxicity [42]. Recently, it was observed that thyme extract was effective in improving diabetic nephropathy in laboratory mice [43].

This experimental study demonstrated the disturbing effect of NiO-NPs on the biochemical and oxidative stress parameters of the liver and kidneys in a rat model. However, co-dosing with thyme to poisoned animals restored the disturbed levels of the studied indices. Therefore, thyme oil can be considered as a defensive nutritional supplement against possible hepatorenal toxicity of nanoparticles and it is recommended to conduct more experimental research in other body organs to confirm its effectiveness.

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