Investigation of Biomarkers in Sub-Acute Hepatorenal Toxicity against Bulk and Zinc Oxide Nanoparticles Exposed Mice

Research Article

Austin J Pharmacol Ther. 2022; 10(1).1162.

Investigation of Biomarkers in Sub-Acute Hepatorenal Toxicity against Bulk and Zinc Oxide Nanoparticles Exposed Mice

Monika Sudhakar Deore¹, Jasleen Kaur¹ and Saba Naqvi1,2*

1Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER-R), India

2Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research (NIPER-R), India

*Corresponding author: Saba Naqvi, National Institute of Pharmaceutical Education and Research- Raebareli, Lucknow (UP) - 226002, India; Email: saba. [email protected]; [email protected]; ORCID: 0000-0001-6591-790X

Received: July 15, 2022; Accepted: August 10, 2022; Published: August 17, 2022


Zinc oxide nanoparticles have been utilized and produced at a very large scale due to their wide range of applications. Nevertheless, its toxicity is one of the concerns addressed by various researchers. Widely used methods of measuring plasma zinc have poor sensitivity and impaired specificity. Currently, there is no specific biomarker for determination of excess zinc inside our body is explored well. Furthermore, it is vital to know the toxic effects of zinc oxide nanoparticles and their bulk counterparts at early exposure level on human health due to day to day increased use of zinc oxide nanoparticles in various applications. In current study we investigate the kidney and liver as the primary target organs for toxicity and their oxidative stress parameters. Twenty-four male mice were divided into three groups(n=8). Control (group I) served as vehicle control; group II:50mg/kg ZnO (bulk) and group III:ZnO NPs (50mg/kg nano). The mice were sacrificed after 14 days exposure, liver and kidney tissue toxicity biomarkers were performed. Our results demonstrate increased levels of GPx, metallothionein, GST, and decrease level of ceruloplasmin, GSH:GSSG ratio in bulk ZnO administered animals. The acute kidney toxicity was further confirmed by increased levels of their biomarkers i.e. Kim-1 and Clusterin. The levels of serum cytokines and caspase were also analyzed. Hence, this study investigated the comparative effect of early exposure of zinc oxide nanoparticles (nZnO) and its bulk form and we conclude that at 50mg/kg b.wt dose, ZnO NPs is comparatively safe to bulk ZnO.

Keywords: Zinc oxide nanoparticles; Bulk zinc oxide comparison; Clusterin, KIM-1; Ceruloplasmin; Metallothionein; GSH; GSSG ratio; Glutathione peroxidase


Zinc oxide nanoparticles(ZnO NPs) are already known for a wide variety of applications because of their enhanced and exceptional properties over their bulk form. ZnO NPs are released into the aquatic system through domestic and industrial wastewater [1]. Researchers have investigated the toxicity of ZnO NPs over the years and confirmed the toxicity in both in-vitro and in-vivo depending on various parameters such as size, dose, and duration. The studies conducted so far have shown toxicity to most of the organs including the liver, kidney, brain, and spleen, etc. However, these studies were limited because of no much comparison with its bulk form and others. Also, no single study confirmed the detailed mechanisms and pathways involved in toxicity in addition to its investigating interlink of this pathology. Studies conducted so far showed the oxidative stress mechanism involved in ZnO NPs toxicity [2]. Thus the generated ROS further causes oxidative DNA damage, cytotoxicity, genotoxicity, etc [3]. ZnO NP is known to be more toxic than other metal oxide nanoparticles due to its ability to shed Zn2+ upon particle dissolution [3,4]. Analysis of the oxidative stress enzyme such as GPx and GST activity upon exposure to ZnO NPs is considered one of the parameters of their toxicity [5]. These are other oxidative stress biomarkers involved in redox cycling. Oxidative stress requires rapid detoxification before it impairs other cellular processes. Early biomarker assessment is essential to tackle any kind of toxicity [6-8]. ZnO NP is exposed to humans as well as animals mainly via dermal, inhalation, and oral routes [9].

Currently, there is a lack of satisfying biomarkers for the assessment of zinc status. However, plasma zinc measurement is the most widely and accepted biomarker of zinc status; but, it has poor sensitivity and impaired specificity. Zinc has been used as a nutritional supplement in various health conditions [10]. Nevertheless, an unusually high zinc intake might cause side effects such as fever, coughing, fatigue, stomach pain, and many other problems including cancer [11-13]. Therefore, the utility of reliable biomarkers in assessing zinc status is essential for the detection of potentially toxic intakes [8,10]. Excess zinc causes copper deficiency which is further affects iron metabolism, causes oxidative stress, weakens the immune system, and decreases the cognitive ability in Alzheimer’s disease [13,14].

Glutathione Peroxidase-1 (GPx-1) is an intracellular antioxidant enzyme function like catalase, it also detoxifies hydrogen peroxide generated by catabolism of superoxide anions to water to limit its harmful effects [15,16]. GPx-1 expression is unique in the regulation of oxidative stress as it contains selenocysteine and is involved during translation hence in the development and prevention of many common and complex diseases [17]. Also, it modulates cell responses such as apoptosis or inflammation to drug toxicity, ischemiareperfusion injury, etc [16].

The cytosolic Glutathione S-Transferase (GST) is an important phase II biotransformation enzyme that catalyzes a nucleophilic attack by glutathione sulfur atom [18]. GST enzymes are involved in the detoxification reactions by conjugation to glutathione. GSTs have served as ideal early biomarkers of organ damage applicable to both human and animal models. Also, they are regiospecifically located in the liver and kidney [19]. GSTs are phase 2 conjugation enzymes protecting the cells from oxidative stress [20]. GST plays a role in preventing oxidative damage by conjugating breakdown products of lipid peroxides to GSH and thus generating less toxic and more hydrophilic molecules [15]. Monitoring of hepatic function is done by liver enzymes according to the genetic polymorphism in early-phase treatment. So, GST is known to be a sensitive biomarker compared to liver enzymes. The specificity of GSTs is due to their large hepatic distribution, high cytosolic concentration, and short plasma half-life [20]. GST is a potential biomarker that can give early warning in nZnO toxicity [21]. Hence, the GSH: GSSG ratio is also a potential biomarker of oxidative stress shows the redox status of the cells. The imbalance in this ratio is indicative of oxidative stress [22].

Ceruloplasmin, a ferroxidase is an alpha-globulin that is involved in both copper and iron homeostasis. It plays an important role in iron transport by oxidizing ferrous iron to the ferric form thus promoting the loading onto transferrin. Oxidizing ferrous to ferric iron also has an antioxidant function [23]. It was reported that high zinc level blocks intestinal absorption of copper [24]. Ceruloplasmin (Cp) is a known biomarker assessed in copper metabolism [25]. In current study, we have evaluated the ceruloplasmin level in ZnO NPs exposed groups, ceruloplasmin catalyzes redox reactions. The lower level indicates the cupper deficiency and also indicates the hypoproteinemic state [26]. The low copper level is also indicated by anemia, and neutropenia [27]. Copper is required for physiological processes such as hemoglobin synthesis, iron oxidation, antioxidant defense peptide amidation, etc. [28].

Metallothionein (MTs) are cysteine-rich low molecular weight proteins that play a role in metal homeostasis [29]. Their levels are known to be overexpressed in response to high metal concentrations. Metal from the GSH–metal complexes involved in metal metabolism is further transferred to MT apoproteins. MT has thus shown its usefulness in environmental monitoring even in complex environments where interference of other xenobiotics can be found. [15]. It detoxifies the heavy metal from the body upon exposure to heavy metal [30]. Metallothionein has also been investigated along with ceruloplasmin levels in zinc exposed rats [31].

Previous studies on renal toxicity showed the mechanisms such as changes in liver enzymes, oxidative stress, inflammation, DNA damage and apoptosis etc. [32]. Among many methods used for the assessment of kidney function, the most widely used method is the measurement of serum creatinine, glomerular function rate (GFR) and urea. But, measuring GFR is time consuming and tedious method [33]. Moreover, serum creatinine level and glomerular filtration rate are detectable when kidney function becomes half. Although Acute Kidney Injury (AKI) progresses towards Chronic Kidney Disease (CKD) as a long-term consequence, the exact pathogenesis of AKI to CKD is largely unknown [34]. Delay in CKD diagnosis cause deterioration of nephron function which furthermore causes endstage renal disease and at that point patients require dialysis or kidney transplant [35].

KIM-1 is one type I transmembrane protein barely expressed in normal kidneys. Increased KIM-1 level is a novel sensitive biomarker for evaluating early kidney damage as compared to traditional biomarkers such as creatinine. Thus, this suggests acute kidney injury particularly in proximal tubular epithelial cells [36-38]. It may play an important role in early tubular epithelial cell damage by modulating damage and repair mechanisms [39]. Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) confirmed KIM-1 as a highly sensitive and specific biomarker [39]. Clusterin is also a newly identified glycoprotein biomarker that shows association with tubulointerstitial renal lesions. Similar to KIM-1 it also serves as the early biomarker of tubular injury [39].

Our study aimed at investigating the early biomarker in target organ like kidney and liver of bulk and nano ZnO exposed mice. This would help in comparing the early exposure toxicity of bulk zinc oxide and ZnO NPs and further helps in designing its prevention and treatment methodology at an early stage.

Materials and Methods

Drugs and chemicals

Zinc oxide-nanopowder and ZnO were purchased from Sigma Aldrich Co., St Louis, Missouri, USA. Enzyme-linked immunosorbent assay (ELISA) kits for analysis of KIM-1 (Cat # ELK-1165), Clusterin (Cat # ELK-1310) were procured from ELK Biotechnology (Hubei, P.R.C).All other chemicals used in the study were of analytical grade.


Twenty-four male albino mice (weighing: 25-30 g) were procured from CSIR-Central Drug Research Institute, Lucknow, India. The mice were kept in the animal house facility of the National Institute of Pharmaceutical Education and Research (NIPER), Raebareli which is approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The animal experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC), NIPER-Raebareli. Standard laboratory animal feed (purchased from altromin, Im Seelenkamp Lage, Germany) and water (Aquapure) were provided ad libitum. The metal contents of the animal feed (in mg/kg) were (Al: 79.37, Cl: 3,484.07, Fe: 192.51, F: 2.80, I: 1.66, Co: 0.34, Cu: 12.81, Mg: 95.06, Mo: 1.10, S: 1,141.22, Se: 0.25, Zn: 95.18). Animals were acclimatized to the experimental conditions for one week before the start of the experiment. All the animal experimental procedures were performed as per the guidelines specified by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Characterization of ZnO NPs

The size and morphology of ZnO NPs were analyzed by transmission electron microscopy (TEM, JEM-200CX) at the CSIRIITR, Lucknow, India. One drop of the ZnO NPs was taken put onto the carbon-coated copper grid and left for 10 min. The excess amount of sample was removed carefully and then negative staining was done using 2% Phosphotungstic Acid (PTA). The samples were air-dried for 15 min and viewed under the TEM.

The hydrodynamic size and surface charge of the nanoparticles were determined using the Malvern Zetasizer Nano-ZS instrument at the National Institute of Pharmaceutical Education and Research (NIPER), Raebareli, Lucknow, India.

Dose Preparation

The ZnO NPs suspension was made using deionized water to minimize reactive oxygen species generation during sonication. Then, the suspension was sonicated for 20 min in a bath sonicator. Bulk ZnO powder was also mixed in deionized water and vortexed before administration to the animals.

Experimental Design

Twenty-four (24) male mice were divided into three groups of 8 each and were administered with normal water, Zinc oxide bulk, and nanoparticles (50 mg/kg, orally through gavage) for 14 days.

Group 1: Normal control

Group 2: Zinc oxide nanoparticles (50mg/kg, p.o.)

Group 3: Bulk Zinc oxide (50mg/kg, p.o.)

Daily body weight, food intake, and water intake were recorded. After 14 days of treatment, blood was collected from retro orbital plexus of animals and then sacrificed.

Functional Observational Battery (FOB): Functional observational battery (FOB) assessments were recorded for 6 animals/groups after completion of exposure. Testing was performed by trained personnel. The FOB was performed in a separate animal room. Animals were observed for the parameters listed in Table 1, which are based on previously developed protocols [40-43].