Metal oxide nanoparticles (MONPs) play a significant role in many areas of life. However, their release into the environment may cause harm to humans and/or ecological systems. Once the animal is exposed to NPs, they can move to various organs and tissues due to their extremely small diameter affecting several biochemical parameters (Morishita et al. 2016; Miyazawa et al. 2021). ZnO NPs is an easily accumulated nanomaterial, this accumulation varies according to the tissues type. It was found that the decreasing in size and the increasing of concentrations of this nanometal lead to increasing its toxicity (Lopes et al. 2014; Chen et al. 2016).
The wide range of toxic nanoparticles depends not only on the size and dose but also on the administration route and timing of exposure to nanoparticles (Lee et al. 2016). It is well known that ZnO NPs are used in food packaging, consumer products as coatings and dermatological uses, there is always a risk of ingestion when they are used (Sharma et al. 2012; Choi et al. 2015). Hence, the oral doses of ZnO NPs administration are recommended for the studied rats.
Lynch et al. (2005) and Adeyemi and Adewumi (2014) reported that the reliable ‘markers’ enzymes of liver damage (ALT, AST, ALP, ACP and LDH) are risen in serum elevated during hepatitis, degeneration, necrosis, and inflammatory conditions. Pasupuleti et al. (2012) and Mansouri et al. (2015) recorded a significant rise up in the ALT and AST levels in ZnO NPs treated groups (50 and 300 mg/kg). Moreover, Kavaz et al. (2021) remarked a considerable alteration in serum level markers (ALT, ALP, and AST) of liver function in rats. In their study, rats are exposed to ZnO NPs concentrations ranging 40 and 80 mg/kg for 4 weeks.
In the present study, ALT, AST, ALP, ACP and LDH enzyme activities were elevated significantly in male albino rats treated with 200 mg/kg for 4 weeks, much more than those treated with 50 mg/kg of ZnO NPs. Several studies showed that the activities of these enzymes in treated liver tissues with ZnO NPs were significantly increased (Wang et al. 2009; Shakeel et al. 2016). The current study detected decreasing in enzyme level activities in rats treated with ZnO NPs plus Vita E doses. In support of these results, Hegazy et al. (2018) reported that the changes in AST and ALT levels were reduced in ZnO NPs plus Vita E group when compared with ZnO NPs groups. Welson et al. (2021) noticed that Vita E lowered serum liver enzymes in side by side to the decrease of liver tissue oxidative stress. This may be because Vita E can normalize concentrations of these enzymes (Al-Rasheed et al. 2014; Welson et al. 2021).
A significant decrease in blood total proteins and an increase in total and direct bilirubin levels in groups treated with ZnO NPs compared to the normal control group are additional indicators of liver tissue injury. (Table 1). On the other hand, Kavaz et al. (2021) reported that male rats treated with nano zinc (80 mg/kg for 28 constitutive days oral administration) showed an increasing in the levels of total protein and bilirubin in blood samples compared with control rats. Newly study revealed that ZnO NPs induced elevation in T. protein and bilirubin levels could be attributed to increasing red blood cell hemolysis beyond the hepatic function capacity (Kavaz et al. 2021). In the current study, the relatively normal values detected were in groups exposed to ZnO NPs plus Vita E. It is confirmed that Vita E is regarded hepatoprotective agents versus ZnO NPs toxicity (Al-Rasheed et al. 2014; Welson et al. 2021). They play an important role in improving immune and metabolic disorders associated with liver damage.
ZnO NPs causing a toxic and destructive disruptions in human health, particularly in the reproductive system and remain a concern. Some reports have demonstrated the reproductive toxicity of ZnO NP (Talebi et al. 2013; Ahmed et al. 2019; El-Morshedi et al. 2018).
Lanzafame et al. (2009) declared the role of oxidative stress for male infertility. This is because free radicals and ROS can damage sperm functions. After ZnO NPs treatment, this stress was recorded in different studied animals (Sharma et al. 2012; Pasupuleti et al. 2012). ZnO NPs are considered a testicular toxicant at dosages ranging from 50 to 350 mg kg in rodents (Talebi et al. 2013; Moridian et al. 2015).
The current study showed that male albino rats exposed to both doses (50 and 200) mg kg of ZnO NPs reduced the levels of luteinizing hormone (LH), free and total testosterone in rats compared with the control group (Table 2). Zhang et al. (2006) mentioned that metal NP including ZnO NPs can traverse the blood-testis barrier (BTB).
Exposure to NP may cause a systemic inflammatory response in the host damaging Leydig cells and lowering testosterone blood levels weaken the BTB’s integrity (Meeker et al. 2007; Lan and Yang, 2012). It is essential to understand that any alteration on BTB could potentially produce a high risk to spermatogenesis (Hong et al. 2015; Leclerc et al. 2015). A marked decrease was recorded in testosterone levels in ZnO NP-treated groups, which correlates with the reduced proliferation and activity of Leydig cells. The same results established by the studies of Talebi et al. (2013), Mozaffari et al. (2015) and Hussein et al. (2016).
According to the obtained results, sperm count and sperm motility in male rats treated with ZnO NPs, in particular at the high dose (200 mg/kg) were reduced considerably (P < 0.01) compared with control rats. ZnO NPs cause a reduction in the number, motility and quality of sperm cells and improve the sperm morphological aberrations, such as double head, double tail, and amorphous head (Hussein et al. 2016; Srivastav et al., 2017; Radhi et al. 2019). Therefore, ZnO NPs could probably cross the BTB producing testicular toxicity affecting reproductive hormones and sperm quality. The present study revealed that male rats exposed to ZnO NPs plus vita E (100 mg/kg) for 4 weeks increased LH, total testosterone and free testosterone, sperm count, and sperm motility compared with rats exposed to ZnO NPs only. Aydilek et al. (2004) mentioned that Vita E is indispensable for reproduction in animals. Previous studies, Karanth et al. (2003) and Li et al. (2016) found that Vita E enhanced reproductive hormones production, including LH, and possesses protecting effects on the testicular injury in mice. Moreover, Yin et al. (2012) indicated that mice treated with Vita E at 100 mg/kg significantly increased the secretion levels of LH and testosterone as well as sperm quality.
The disturbance between ROS generation and the biological response of an animal that detoxifies the mechanism is revealed by oxidative stress. The presence of Zn-NPs resulted in a considerable increase in the levels of several antioxidant enzymes as well as gene expression (Saddick et al. 2017).
The effect of ZnO NPs on the enzyme activity levels was evaluated in this study and proved that the activity levels of MDA were increased, but the activity levels of CAT and GPx were decreased in liver and testis (Table 3). The outcomes agree with Hussein et al. (2016), who documented that ZnO NP affected the CAT and GPx levels that were drastically reduced as lipid peroxidation was considerably increased, as showed by the MDA level in tests tissues.
In line with the present study, Abbasi et al. (2018) and Khadra et al. (2021) reported that doses of more than 100 mg/kg ZnO NPs reduced the GPX and CAT activities and increased MDA levels. Syama et al. (2014) suggested that the release of Zn + + ions and rising ROS generation might cause oxidative stress to liver tissues.
In the current study, GPx and CAT activities showed a significant rise in groups 5 and 6 that were treated with Vita E. The protective effect of this vitamin is owing to its essential role in peroxyl radical scavenging, terminating lipid peroxidation and enhancing the glutathione-antioxidant system (Santos et al. 2016; Xu et al. 2014).
Metallothionein (MT) serves as a zinc pool, and its deficiency caused a noticeable upsurge in zinc accretion in the different tissues (Davis et al., 1998). Male rats exposed to ZnO NPs showed a considerable elevation in Zn and MT contents in the liver and testicular tissues. Wang et al. (2017) reported that ZnO NPs significantly enhanced the MT levels in mice tissues by promoting its genes' expression. Swain et al. (2019) mentioned that the expression of MT was proportional to the intake of dietary zinc, which serves a sequestration function to decrease metal toxicity. Increased MT expression in the liver compared to the testis tissues might be attributed to the better bioavailability of zinc from nano zinc particles sources and related to different physiological activities needed of the tissues of the studied animals, and this is greatly contributing to Zn homeostasis.
ROS can damage genetic materials in cells exposed to different metals. The reaction of ROS with DNA molecules has caused a significant damage that can affect purine, pyrimidine and DNA backbones (Martinez et al., 2003).
DNA damage is useful to evaluate the impact of metal exposure in the environmental matrix and in model research (Demir and Yavuz 2020; Jiang et al. 2020). By quantifying breakage in the DNA chain, comet assay offers a sensitive, rapid and multipurpose technique to evaluate the damages produced by various metals. It is an essential example of cellular genotoxicity. Several studies have assessed the potential genotoxic impacts of ZnO NPs in several tissues using comet assay (Sharma et al. 2012; Al-Rasheed et al. 2014). Current data proved the ZnO NPs-triggered oxidative stress in liver and testes coincided with severe DNA damage assessed by comet assay. In comparison to control group, a remarkable improvement was recorded in the tail length (TL) and DNA % in the tail in livers and testis of rats intoxicated with the high dose of ZnO NPs. This damage was more pronounced in rat livers ingesting a high dose of the NPs than in tests tissues (Figs. 2 and 3). According to Singh et al. (1988), there are two signals indicating the amount of DNA breakage: the length and intensity of the comet tail. A remarkable increase in DNA% and tail length, as in our study, have been recorded by Al-Rasheed et al. (2012), in livers of rats intoxicated with ZnO NPs (600 mg/kg/day) for 5 consecutive days.
In hepatocytes and testes tissues, ZnO NPs caused oxidative DNA damage and ROS-triggered mitochondrial-mediated apoptosis, according to several studies (Ghosh et al. 2016; Deepa et al. 2019). In the same way, liver injury was observed after oral exposure of ZnO NPs (300 mg kg − 1) for 14 successive days (Esmaeillou et al. 2013 and Sharma et al. 2012) Nanomaterials can induce intracellular oxidative stress by distressing the equilibrium between oxidant and antioxidant activities (Shi et al. 2020). The rats supplemented with ZnO NPs plus Vita E declined DNA damage in liver and testis compared to those exposed to ZnO NPs only. The same results reported by Niedernhofer et al. (2003) and Al-Rasheed et al. (2014).
Badgujar et al. (2015 and 2017) declared that Vita E supplemented to rats protects DNA from the attack of free radicals that cause damage to DNA molecules and act by one of the two mechanisms as follows: (a) it inactivates ROS molecules and prevents its binding with DNA structure, or (b) scavenges of peroxyl lipid radicals, which breaks the lipid peroxidation chain reaction that results in DNA-damaged products. Nassar et al. (2017) suggested that the protective role of vita E against ZnO NPs induced toxicity in rats could be attributed to its capability to suppress inflammatory mediators' expression. So, the anti-inflammatory action of Vita E might be associated with the trapping of ROS and mitigation of oxidative damage as well as a decrease in the expression levels of IL-6, TNF-α, and CRP that are induced by ZnO NPs (Krishnamoorthy et al. 2007; Faddah et al. 2012). In consistent with these conclusions, Co-administration of the 100 mg/kg Vita E to ZnO NPs intoxicated doses efficiently protected livers and testes tissues from DNA injury indicated by a reduction in DNA % and tail length than intoxicated rats.
The stress markers and genotoxicity results were substantiated by the histopathological inspection of liver and testicular cells. The liver tissues showed disturbed hepatic lobular architecture with cytoplasmic vacuolization of the hepatocytes, focal necrosis, inflammatory cellular infiltration, and dilated congested central vein. In line with these, Johar et al. (2004) suggested that ZnO NP possibly exert an oxidative stress mechanism on rat tissues, which may induce an inflammatory response. Among others, Schrand et al. (2010) and Babele (2019) found that ZnO NPs increased reactive oxygen species (ROS) within mammalian cells, leading to necrosis.
Studies have shown that necrosis is stimulated by toxicants that hurt the cell organelles (endoplasmic reticulum, mitochondria, and cell nuclei), upsetting their activities (Shukla et al. 2011; Sharma et al. 2012). It also leads to glutathione depletion, decreases in catalase and superoxide dismutase activity (Moos et al. 2010; Sharma et al. 2011).
Regarding the testis, tissue lesions in groups exposed to ZnO NPs showed distorted seminiferous tubules with disorganized germ cells with pyknotic nuclei, sloughing, and many sertoli cells and interstitial vacuolation. These findings agree with those obtained by Mozaffari et al. (2015) and Ahmed et al. (2019), who described rats exposed to ZnO NPs had severe changes in seminiferous tubules, sloughing, and reduction in germinal epithelium and pyknosis of spermatogenic cells. Talebi et al. (2013) indicated that rats who received 300mg/kg ZnO NP showed sloughing or even atrophy of the epithelium of seminiferous tubules in rats who received 300mg/kg oral ZnO NPs/day for 35 days. Johnson (2014) suggested that the sloughing of the germinal epithelium that partially or even connects the entire tubule might be related to the toxic effect on the Sertoli cell cytoskeleton. Moreover, disturbance in physical interactions between Sertoli and other germ cells leads to slough and detachment of the germ cells (Erkanlı Şentürk et al. 2012). Halawa (2010) added that nanoparticles can damage and even disrupt the underlying membranes, germ cells and Sertoli cells leading to further destruction of the damaged cell. These observations are in the same line with the earlier reports of Almansour et al. (2017) and Pinho et al. (2020).
In the current study, a moderate histopathological changes on liver and teste of rats accompanied with potent anti-apoptotic outcome of the co-administration of Vita E with ZnO NPs to rats may lead to down-regulate the rise in liver caspase 3 activity, this is in accordance with the results obtained by Aboul-Soud et al. (2011). Al-Rasheed et al. (2012) concluded that by inhibiting lipid peroxidation, Vita E maintains cell membranes' integrity, function, and flexibility. Also, this vitamin plays an important role in reducing inflammation. Also. Abdallah et al. (2018) remarked that supplementation of Vita E during ZnO NPs exposure light have protective effects against tissue dysfunction. Pearce et al. (2019) and Lazzarino et al. (2019) reported that vitamin E may play a role in enhancing the quality of semen and as a result, improving fertility. This could be happened through improving sperm quality and minimizing DNA disintegration and apoptosis.
Treatment of ZnO NPs intoxicated male albino rats with Vita E, pointedly boosted most of the diverged biochemical, genotoxicity and histopathological alterations and effectively prevented the destructive effect caused by ZnO NPs intoxication, which was established microscopically in the histological structures of liver and testes cells.