The effects of TiO2, ZnO, IONs and Al2O3 metallic nanoparticles on the CYP1A1 and NBN transcripts in rat liver

Abstract

Introduction

Metal oxide nanoparticles are currently used widely in many aspects of human and animal life with broad prospects for biomedical purposes. The present work was carried out to investigate the effects of orally administrated TiO2NPs, ZnONPs, IONs and Al₂O₃NPs on the mRNA expression level of CYP 1A1 and NBN in the rat liver.

Materials and Methods

Four groups of male Albino rats were given their respective treatment orally for 60 days in a dose of 1/20 of the LD50 TiO2NPs (600 mg/Kg b.wt/day), ZnONPs (340 mg/Kg b.wt/day), IONs (200 mg/kg b.wt/day) and Al₂O₃NPs (100 mg/Kg b.wt/day) and a fifth group served as a control group.

Rresults

The mRNA level of CYP 1A1 and NBN showed up-regulation in all the NPs-treated groups relative to the control group. ZnONPs group recorded the highest expression level while the TiO2NPs group showed the lowest expression level transcript. Conclusion:The toxic effects produced by these nanoparticles were more pronounced in the case of zinc oxide, followed by aluminum oxide, iron oxide nanoparticles and titanium dioxide, respectively.

Introduction

Nanotechnology is one of the promising expanding areas of progressing technology due to the unique properties of nanomaterials (chemical, mechanical, optical, magnetic, and biological) which make them anticipated for both commercial and medical applications.¹

Currently, the best probable investigated and applied inorganic nanomaterials are those made of metals such as iron, silver, gold, or titanium. Metal nanoparticles (MNPs) and their oxides are involved in various applications in the medical and industrial fields. MNPs are involved in new drug delivery systems or cancer therapy.²

Interestingly, the extensive use of nanoparticles provoked great concern among health and environmental researchers due to their prospective human and environmental hazards.³

Constant exposure to the MNPs induces toxic effects, manifested by renal, hepatic, and lung injury. Nanoparticles reach different organs even the brain via circulatory, and lymphatic systems due to their minute size.⁴

MNPs-induced hepatotoxicity is a major concern in nanomedicine since the liver is often the first organ that comes into contact with MNPs after their ingestion.⁵

Many reports showed that MNPs are accomplished to produce hepatotoxicity. The toxic effects of MNPs on the liver attracted huge attention in nanomedicine since the liver is considered the first organ that encounters MNPs. MNPs could elicit various harmful cellular effects, including oxidative stress and DNA damage.⁶

Titanium dioxide nanoparticles (TiO2NPs) nanoparticles are produced at high production volumes worldwide and used frequently in cosmetics, toothpaste, sunscreens, food products, paints and drugs.⁷

Zinc oxide nanoparticles (ZnONPs) are extensively used as an active component of food additives, toothpaste, food packaging, sunscreens, and other pharmaceuticals due to their tremendous antibacterial activity and absorption of ultraviolet radiation. However, the exposure to these nanoparticles has been rising progressively and incited more concerns about the extent of their probable toxicity.⁸

During the last few years, iron oxide nanoparticles (IONs) drew attention due to their nano size, high surface area to volume ratios and super-paramagnetism. IONs are implicated in medical imaging, drug delivery and gene therapy. However, their extensive use in many fields was associated with some tissue toxicities mainly in the liver due to induction of oxidative stress and impairment of liver enzymes.⁹

Aluminum and its compounds like aluminium oxide (Al2O3) are common Aluminium oxide nanoparticles (Al₂O₃NPs) that have been used in industrial and biomedical applications. Al2O3-NPs promoted the anticancer activities of immunotherapy.¹⁰ Regrettably, slight studies have reported that Al2O3-NP treatment may drive various dangerous effects, such as genotoxicity, inflammatory response, carcinogenicity, cytotoxicity, and mitochondrial dysfunction.¹¹ This study aimed to investigate the effects of orally administered metal oxide nanoparticles (TiO2NPs, ZnONPs, IONs, and Al2O3NPs) on the mRNA expression level of the CYP 1A1 and NBN gene in the rat liver over a subchronic period.

Materials and methods

Characterization of nanoparticles

The X-ray diffractometer (XRD) was used to assess the density, purity, and size of the NPs using Cu Kα radiation (operated at 20 mA and 40 kV) in the 2θ range of 5–70 at a scanning speed of 5°/min.

Preparation of nanoparticle suspension

TiO(2)NPs, ZnONPs, IONs and Al₂O₃NPs were purchased from the Nanotechnology Department, Faculty of Advanced Basic and Applied Science, Beni Suif University. All nanoparticles were suspended in pH 7 20 mM HEPES buffer containing 1% sodium citrate for oral preparation. The suspension was vortexed just before administration.

Animals

100 adult male rats of Albino strain, weighing 180–200 g were used for this study. The rats will be obtained from the Experimental Animal Care Center, College of Veterinary Medicine, Cairo University. Animals were kept in stainless steel mesh-bottomed cages at a temperature of 22–24 0Crelative humidity of 55 ± 5% and a 12 h light/dark cycle This study was accomplished according to the guidelines approved by the Institutional Animal Ethical Clearance (IAEC).

Dosing and sample collection

Four groups of male rats (n = 20 per group) will daily be administered via oral gavage for 60 days with TiO(2)NPs at a dose of (500 mg/kg b.wt /day) according to HelmyAbdou et al.,⁵ ZnONPs (100 mg/Kg b.wt/day) according to Saman et al,¹² IONs (300 mg/kg b.wt/day)¹³ and Al2O3NPs (70 mg/Kg b.wt/day) according to Park et al.,¹⁴ an additional fifth group of 20 rats received 20 mM HEPES buffer containing 1% sodium citrate which served as a control group.

At the end of the experiment, rats were weighed and anesthetized with alcohol chloroform ether mixture (ACE) mixture in a ratio of 1:2:3 respectively. Blood and tissue samples were collected and stored at −80 °C.

Determination of the CYP 1A1 and NBN transcript levels

The extraction of total RNA from the liver tissue was performed according to the manufacturer protocol of the RNeasyR tissue mini kit (Qiagen). The NanoDrop 1000 Spectrophotometer was used to evaluate the concentration and purity of the extracted total RNA.¹⁵ The first-strand cDNA was generated using the first-strand cDNA synthesis kit (Fermentas, Life Sciences). The quantitative real-time PCR was performed using BioEasy SYBR Green I Real-Time PCR Kit. The program was adjusted to 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C for 45 cycles. Transcript levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).¹⁶ The CYP IA1 gene forward primer; 5′-GCACTCTGGACAAACACCTG-3′ and the reverse primer 5′-ATATCCACCTTCTCGCCTGG-3′; CTTCAGGACAGCAGTGAGGA-3′ and reverse 5′-TCTTTCGAGCATGGTGACCT-3′ for the NBN gene.¹⁷ The PCR specificity was verified by melting curve analysis. All samples were analyzed in duplicate, and relative gene expression was calculated by the 2−ΔΔCt method.¹⁸

Statistical analysis

The statistical analysis employed to identify significant differences between the various groups was one-way analysis of variance (one-way ANOVA). Subsequently, multiple comparisons were conducted using Tukey’s post hoc test. GraphPad Prism version 5.0 software was utilized to perform all statistical analyses. The results were expressed as Mean ± SE (standard error).

Results

Characterization of nano-TiO2

The result of the X-ray diffraction (XRD) showed that the size of the TiO(2)NPs, was 72.1 nm (Fig. 1), the Al₂O₃NPs was 5.24 nm (Fig. 2), the IONs was 34.02 nm (Fig. 3) and the ZnONPs was 78.21 nm (Fig. 4).

Fig. 1.

XRD pattern of Tio₂ Nps.

Fig. 2.

XRD pattern of Zno Nps.

Fig. 3.

XRD pattern of Fe2o3 Nps.

Fig. 4.

XRD pattern of Al₂o₃ Nps.

The transcript level of CYP1A1 and NBN

The quantitative real time PCR results revealed up regulation of both CYP1A1 and nbn genes in all the metallic NPs treated group relative to the negative control group (Table 1).

Table 1.

The relative mRNA expression level of the CYP1A1 and genes.

Genes	Control	TiO2NPs	ZnONPs	Fe2O3NPs	Al2O3NPs
CYP1A1	1 ± 0	2.2 ± 0.07*	3.8 ± 0.61*	2.6 ± 0.03*	2.8 ± 0.1*
nbn	1 ± 0.00	3.3 ± 0.16*	5.9 ± 0.32*	3.3 ± 0.1*	3.77 ± 0.28*

Values are presented as mean + SE. (n = 7 rats/group). The ^* indicates a significant variation relative to the control groups at P < 0.05.

Discussion

Exposure to nanoparticles causes oxidative damage and liver dysfunction.^19,20 Nanoparticles produce injurious influences throughout the production of ROS.²¹ Nanoparticles may directly disrupt both the genomic DNA structure and function by penetrating the nuclear membrane.²²

CYP1A is a member of the P450 superfamily that participates in xenobiotic biodegradation. It has a crucial function in the biotransformation and detoxification of both endogenous and exogenous materials.²³ Overexpression of P450s triggered the endoplasmic reticulum stress response²⁴ which is an essential self-protection mechanism required to stabilize the cellular stress.²⁵ CYP1A is considered a potential environmental biomarker used widely to check the biological consequences of various xenobiotics.²⁶

The present study compares the effect of four different nanoparticles on the liver through estimation of the Cyp 1A1 transcript level which as a member of the phase I cytochrome P450 family, is greatly active in hepatic tissue. It plays a major part in the activation of the heterocyclic aromatic amines.²⁷ Our results showed up-regulation of CYP1A1 in the hepatocytes following exposure to the studied nano metallic particles with various degrees. CYP1A1 is implicated in the metabolism of toxins and protection against xenobiotics,¹⁹ thus it is considered a key marker for the detection of nanoparticle hepatotoxicity.²⁸ Restricted data exist on the regulation of the CYPs by nanoparticles. NPs toxicity induces lipid peroxidation and reduces the cellular antioxidant capacity.²⁹ In the current study, the toxic influences generated by the studied NPs were more distinct in the ZnO NPs, followed by Al₂O₃, Fe₂O₃ and TiO₂, respectively.

The nano-TiO2 hepatotoxicity depends on ROS generation,³⁰ mitochondrial damage and dysregulation of the antioxidant-protecting genes. Our result is in agreement with Li et al³¹ who stated that TiO2 NPs increased the mRNA expression level of CYP1A. Bioactivation of CYP1A is in response to the formation of free radicals and ROS, which initiate lipid peroxidation and protein oxidation leading to hepatocellular membrane damage and hepatocyte toxicity.³² TiO2 NPs induced hepatocyte apoptosis and increased inflammatory reaction.³³ The downregulation of CYP450s leads to drug–drug interactions, while their upregulation minimizes the drug efficacy.⁹

One important gene for DNA repair and maintaining genomic stability is NBN. It is linked to preserving genomic integrity and is important for the cellular response to DNA damage.³⁴ Changes in the expression of the NBN gene may affect the ability to repair DNA and the stability of the genome as a whole.¹⁷ The cellular response to DNA damage caused by the nanoparticles and the activation of DNA repair pathway³⁵ may be shown by the overexpression of NBN. This reaction may be a component of the defense mechanism of the cell to preserve genomic stability when potentially genotoxic substances are present.

Oral administration of TiO2 NPs augmented the ratio of alanine aminotransferase to aspartate aminotransferase, the activity of lactate dehydrogenase as well as the liver weight and induced hepatocyte necrosis. TiO2 NPs are preferentially located in the mitochondria.⁶ The inner mitochondria membrane plays a role in toxicity as it is a major source of ROS which causes damage to lipids, proteins and DNA and leads to loss of cellular function, especially in necrotic and apoptotic cells.³⁶

Similar to our findings, exposure to Fe₂O₃ NPs induced oxidative stress in the rat hepatocytes is mostly correlated to mitochondrial damage and intense production of ROS.³⁷ The IONPs can be metabolized into iron ions in the liver by Kupffer cells and the spleen by macrophages found in the red pulp initiating the cellular responses. When ferritin is overloaded, free iron ions could be released into the cell and react with hydrogen peroxide generated by the mitochondria, microsomes, peroxisomes and cytosolic enzymes and produce the highly reactive hydroxyl radicals and ferric ions (Fe3+) via the Fenton reaction.³⁸

Fe₂O₃ NPs disrupt the mitochondrial membrane potential³⁹ leading to increased ROS production. The CYP1A1 may be involved in detoxification and protection against hepatotoxicity induced by nanoparticles. Further, the ability to affect CYP expression varies according to the type of particles. The up-regulation of CYP1A1 mRNA in response to nanoparticle toxicity may be consequent to the mechanism of cellular protection which is activated for the detoxification of toxins and their deleterious effects among the biological macromolecules.²³

ZnO-NPs are broadly used in paints and many other products. Also, ZnO-NPs depleted the hepatic antioxidants greatly affected the structure and function of hepatocytes.⁴⁰ The ZnO-NPs are characterized by high levels of transparency and chemical reactivity which increase the number of atoms on the surface of the NP and so augment their biological activities.^41,42 The ZnO-NPs induced toxicity is attributed to the Zn ionization to Zn2+ and generation of free radicals on the NPs surface leading to cellular imbalance. ZnOm-NPs nanoparticles were reported to be more genotoxic as compared to TiO₂ nanoparticles.⁴

Aluminum oxide nanoparticles cause toxicity in an animal by entering the food chain. The oral exposure of rats to Al₂O₃Ns has been associated with a genotoxic effect. Oral Al₂O₃NPs administration possibly induced oxidative stress and altered antioxidant status and provoked hepatotoxicity.⁴³ Al2O3 nanoparticles caused deleterious effects on both cellular structures leading to apoptosis and damage to DNA and proteins.⁴⁴ Al2O3NPs may produce their toxic effects through direct interaction with the cellular components, generation of adducts with nucleic acids⁴⁵ and proteins and ROS production and oxidative stress.⁴⁶ Aluminum produced mitochondria-mediated oxidative stress and deregulation of the apoptosis-related genes.²³

Conclusion

The exposure to nanoparticles can cause oxidative damage, liver dysfunction, and various toxic effects. The specific mechanisms and severity of toxicity vary depending on the type of nanoparticles. Understanding the potential adverse effects of nanoparticles is crucial for assessing their safety and developing strategies to mitigate their potential risks in biomedical and industrial applications.

Clinical significance

The current results suggest the contribution of CYP1A1 and NBN upregulation which mostly occurred in response to oxidative stress as one of the main mechanisms elaborated in nanoparticles-induced hepatotoxicity.

Author contributions

All of the authors (W.A.M., M A. I., N. Y. H., A. G. K. and, E. E.) contributed significantly and equally to the research.

Funding

The research receives funds provided by Beni Suef University.

Conflict of interest statement. The authors declare no conflict of interests.

Data availability

All data will be available on request.

Ethical approval

This study was accomplished according to the guidelines approved by the Institutional Animal Ethical Clearance (IAEC).