Hepatotoxicity of nickel nanoparticles in rats 

Nanoparticles (NPs) are materials or structures with a dimension at 1-100 nm range (Nakamura and Watano 2018). Metal NPs are widely used in industry such as chemical catalysts, ceramic capacitors, sensors, hydrogen storage, conductive pains and biological nanomedical applications due to their physicochemical characteristics (Mo et al., 2019).

The nickel nanoparticles (NiNPs) have specific characteristics, such as low melting point and high magnetism, reactivity and surface area (Zhang et al., 2003). The adverse effects of NiNPs to human health have been paid attention by many researchers (Zhao et al., 2009). NiNPs were found to induce neurotoxicity, hepatotoxicity, nephrotoxicity and reproductive toxicity (Patlolla et al., 2019; Abudayyak et al., 2017).

The main mechanism for the cell damage caused by NiNPs is through inducing oxidative stress (Patlolla et al., 2019; Ahmad et al., 2015). Specifically, NiNPs were found to induce DNA damage (Abudayyak et al., 2017), inflam mation, cell degeneration (Razavipour et al., 2015), cell cycle arrest (Ahmad et al., 2015) and cytogenetic alterations (Saquib et al., 2017). The present investigation aimed to focus on the hepatotoxicity of NiNPs in rats using different doses.

NiNPs
 
The powdered 20 nm nickel nanoparticles (NiNPs) procured from Sigma-Aldrich Co.USA were used.
 
Three different doses of NiNPs suspensions were prepared (5, 20, 100 mg/kg body weight). The NiNPs suspensions were ultra-sonicated for 3 h in deionized water, using biologic ultrasonic homogenizer (Model 150VT, BioLogics, Inc., USA), to disperse the NiNPs in a homogenous stable. The NiNPs were vibrated for 2 min, immediately prior to administration in animals.
 
Experimental animals
 
Thirty two male Wistar rats, weighing 200-220 g, kept on commercial pellet feed and water ad libitum were used. The rats were maintained at 21°C±2°C.

The rats were randomly divided into 4 groups. The 1st group represents the control and they were administrated intraperitoneally (i.p.) with normal saline. The groups 2, 3 and 4 (treatment groups) were injected with ultrasonicated NiNPs suspension at 5, 20 and 100 mg/kg body weight respectively. The injections were given once daily for five days a week and it continued for four weeks. All rats were dissected, then liver and blood samples were obtained for histological, biochemical and ultrastructural investigation. The study had approval from Animal Care Ethical Committee protocols in Salahaddin University-College of Science.
 
Biochemical assays
 
Liver tissues were washed in ice-cold normal saline solution, and homogenization was achieved in 20 mM phosphate buffer (pH=7.4) using a glass hand homogenizer. The homogenates were centrifuged at 4000 g at 4°C for 10 min. The supernatants were collected and stored at -80°C until assayed. The level of liver homogenate MDA was determined by thiobarbituric acid (TBA) method using manual commercial reagents kits (Alnahdi et al., 2018).

The liver function measures including ALP, ALT, AST and bilirubin were estimated in blood serum using kits from BIOLABO SA, Maizy, France. Similarly, SOD, GSH, GPX and CAT, kits were estimated using specialized kits and the manufacturers protocols were followed.
 
Histopathology and electron microscopy analysis
 
Liver pieces were fixed in 10% buffered formaldehyde followed by dehydration, clearing and embedding in paraffin. Hematoxylin and eosin were used for routine histology (Tamizhazhagan and Pugazhendy 2017).

For electron microscopic studies, plastic blocks were prepared using liver samples (≤1 mm3) fixed in glutaraldehyde (2.5%) in 0.1M cacodylate buffer followed by 1% osmium tetroxide, dehydrated, cleared and finally embedded in araldite mixture. The ultrathin sections were stained with uranyl acetate and lead citrate and viewed by JEOL JEM 1400 transmission electron microscope.
 
Statistical analysis
 
All the data were expressed by means±standard error (M±SE) and the statistical analysis was achieved by SPSS version 22. One-way analysis (ANOVA) was performed for testing the significant of the treatment followed by Duncan’s multiple range comparison between the groups. P values ≤0.05 were considered to be significant.

NiNPs-induced hepatotoxicity was reflected by the elevated serum bilirubin, ALT, AST and ALP levels (P≤0.05), (Table 1). Increase in bilirubin level and liver function enzymes activity is known to accompany the hepatobiliary damage and leakage of these liver enzymes from hepatocytes (Yaqub et al., 2018; El Shahat et al., 2017). NiNPs accumulation due to increased doses may change the phosphate and ATP metabolism. This change leads to cellular energy depletion and disturbance in the potential of membrane causing hepatocytes necrosis and this consequently may lead to transaminases leakage into the bloodstream (Morsy and Elkon 2014).



The NiNPs treatment caused a significant increase (P≤0.05) of MDA in the liver homogenates of the exposed rats in a dose dependent manner (Fig 1). Probably, this reflected a nanoparticles induced oxidative stress since MDA was considered a marker for lipid peroxidation or oxidative stress and tissue damage (Aitken and Roman 2008). A generation of free radicals and inducing oxidative stress by NiNPs have been recorded previously (Dumala et al., 2018).



On the other hand, a significant decrease (P≤0.05) in SOD, GPX, GSH and CAT level was recorded in NiNPs treated rat liver homogenate compared to the control (Table 2). Several previous studies have shown that the accumulation of NiNPs in tissues may cause significant dose dependent cellular and biochemical changes such as enhancing excessive release of free reactive radicals and altering the endogenous antioxidants, leading to induction of lipid peroxidation (Dumala et al., 2018; Morsy and Elkon 2014).

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The most important histopathological change in NiNPs treated rat liver was the dose dependent increase of the infiltrated inflammatory leucocytes cells in the regions adjacent to the blood vessels especially the portal area (Fig 2). Other histological changes included congestion of blood vessels, fibrosis around the blood vessels, degenerated hepatocytes (with dark condensed nuclei, shrunken cells and hypereosinophilic cytoplasm) and accumulation of lipid droplets in the cytoplasm of some hepatocytes (Fig 2). The normal cellular structure of hepatocyte in control group with normal mitochondrial structure and density is shown in Fig (3A). The histological changes following NiNPs treatment were confirmed by electron microscopy in which lipid accumulated hepatocytes (Fig 3B), apoptotic cells with fragmented nuclei (Fig 3C) and bundles of collagen fibers (Fig 3D) were clearly detected especially in the higher NiNPs doses treated rats.

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NiNPs induced hepatotoxicity may also be the reason for the observed inflammation which may be caused by Kupffer cells activation (Berrahal et al., 2011). Such activation of Kupffer activation may serve as indirect NPs hepatotoxic effect which is associated with apoptosis (Manke et al., 2013). Furthermore, NPs are taken up by Kupffer cells in the liver and then by macrophage in other places (Sadauskas et al., 2007). In the present work, fibrosis was detected near sinusoid lining after injecting the rats with NiNPs (Fig 3D). Kupffer cells are the main sources of TGβ1 production, which caused the stellate cells transformation into myofibroblasts (Kolios et al., 2006).

Thus NiNPs may be considered as a dose dependent inducer of oxidative stress, inflammation and, fatty changes in liver of rats.