Zinc Oxide Nanoparticles' Acute and Subacute Oral Toxic Effects on Pancreas in Adult Male Albino Rats

ABSTRACT

Zinc oxide nanoparticles (ZnO‐NPs) are being progressively employed in various industries, including cosmetics, textiles, and sun protection products. Due to their widespread application, concerns have emerged about their potential health hazards, especially through environmental exposure. This study was conducted to assess the acute and subacute toxicological impact of orally administered ZnO‐NPs (30 ± 5 nm) on the pancreas of adult male albino rats. In this study, the size of ZnO‐NPs was determined by using transmission electron microscopy (TEM) as provided by the company. Eighty male albino rats (2.5–3 months old) were allocated into two equal groups to evaluate acute (1 day) and subacute (28 days) ZnO‐NP toxicity (40 rats each). Each was further divided into control (negative and positive), low‐dose (175 mg/kg), and high‐dose (350 mg/kg) subgroups. Blood was collected via cardiac puncture under anesthesia for biochemical analysis, and pancreatic tissues were examined histopathologically and immunohistochemically. Both acute and subacute ZnO‐NP exposure caused a significant rise in blood glucose and serum Zn. Insulin levels increased acutely but decreased with subacute exposure. The subacute group showed a significant increase in oxidative stress markers indicated by elevated malondialdehyde (MDA) compared to controls. Histological examination revealed pancreatic degeneration, whereas increased inducible nitric oxide synthase (iNOS) immunopositivity indicated an inflammatory response, suggesting the involvement of oxidative stress in both groups. Oral exposure to toxic doses of ZnO‐NPs caused oxidative stress and structural damage in pancreatic cells, affecting insulin and glucose levels. These findings highlight the potential health risks of ZnO‐NP exposure, particularly with prolonged use.

Short abstract

This study evaluated the acute and subacute pancreatic toxicity of orally administered zinc oxide nanoparticles (ZnO‐NPs) in adult male rats. ZnO‐NP exposure led to increased serum zinc, oxidative stress, and significant alterations in glucose and insulin levels. Histopathology revealed dose‐ and time‐dependent pancreatic degeneration, whereas iNOS immunoreactivity suggested inflammatory and oxidative injury. Findings highlight the potential health risks of repeated or high‐dose ZnO‐NP exposure.

1. Introduction

Nanotechnology is a rapidly developing field involving the production and application of nanoparticles (NPs) ranging from 1 to 100 nm. Their high surface area and reactive sites confer unique physicochemical properties, making them valuable in medicine, industry, and consumer products (Aref et al. 2023). Among these, zinc oxide nanoparticles (ZnO‐NPs) are widely used due to their chemical stability, catalytic activity, antimicrobial properties, and UV‐blocking ability. Humans are exposed to ZnO‐NPs via ingestion, inhalation, dermal contact, or environmental contamination (Kim et al. 2025).

The nanoscale dimension of ZnO‐NPs enables rapid cellular internalization. ZnO‐NPs can be internalized as free Zn²⁺ ions or intact nanoparticles, transported via paracellular pathways, transcytosis, or M cells, and reach systemic circulation through the portal vein or lymphatics (Singh 2019). In biological fluids, ZnO‐NPs rapidly bind proteins, such as albumin, transferrin, and immunoglobulins, which can alter their hydrodynamic size, cellular uptake, and cytotoxicity (Mandl et al. 2019).

The liver and kidneys play key roles in ZnO‐NP metabolism and excretion, with hepatocytes and Kupffer cells facilitating clearance; oral administration in rats results mainly in fecal excretion, with minimal urinary excretion. Due to their small size and reactivity, ZnO‐NPs exhibit higher cytotoxicity than bulk ZnO or Zn salts, mediated via Zn²⁺ release and oxidative stress, leading to protein, lipid, and nucleic acid damage (Choi et al. 2015).

The pancreas is a crucial gland with endocrine and exocrine functions, regulating glucose homeostasis and nutrient metabolism. Zinc plays an essential role in insulin synthesis, storage, and secretion; dysregulation of Zn homeostasis is linked to diabetes and other metabolic disorders. ZnO‐NPs have been shown to induce oxidative stress by generating reactive oxygen species (ROS), leading to lipid peroxidation, protein oxidation, and DNA damage. Mechanistic studies have demonstrated that ROS production occurs via both particle dissolution releasing Zn²⁺ ions and direct surface‐catalyzed redox reactions on the nanoparticle surface (Chen et al. 2023).

Oral ZnO‐NP exposure has been reported to increase blood glucose and induce histopathological changes in the pancreas. However, the literature lacks consensus regarding ZnO‐NPs toxicity; these discrepancies may be attributed to variations in nanoparticle size, surface coating, exposure route, dose, and duration of exposure across different experimental models (Chen et al. 2023).

Although several studies have investigated ZnO‐NP‐induced pancreatic toxicity, most have focused on either biochemical alterations or histopathological changes separately. This study aims to provide a more comprehensive assessment by combining biochemical analyses, histopathology, and iNOS immunohistochemistry across both acute and subacute exposures.

Accordingly, this study was designed to assess the toxic impact of ZnO‐NPs on the pancreas of adult male albino rats.

2. Materials and Methods

The study employed an experimental design with appropriate control and treatment groups, dividing the rats into acute and subacute exposure groups.

2.1. Materials

2.1.1. Chemicals Used in the Study

ZnO‐NPs were acquired from Nanotech Company, Cairo, Egypt: address: Dreamland, El‐Wahaat Road, 6th October, Giza, Egypt; telephone: (+202) 38581 440‐(+202) 38581 439; fax: (+202) 38581 441.

The ZnO‐NP powders were white, spherical, and had a mean particle size of 30 ± 5 nm. TEM were performed on JEOL JEM‐2100 (the JEM‐2100 is a high‐resolution transmission electron microscope [TEM] manufactured by Japan Electron Optics Laboratory [JEOL]) as provided by the company (Figure 1).

FIGURE 1.

The TEM image of ZnO‐NPs.

2.1.2. Animals

In the present study, 80 adult male albino rats, aged 2.5–3 months and weighing 150–200 g, were utilized in the period from May 2023 to April 2024. The animals were given to the animal house of the Faculty of Medicine, Tanta University, Egypt. They took up residence in cages made of wire mesh for 1 week to allow acclimatization, having unfettered access to potable water and a commercial diet. The rats were kept under a 12:12‐h light/dark cycle at a room temperature of 22°C–24°C. The cages were cleaned daily throughout the duration of the experiment. All animal experiments were conducted in accordance with the ARRIVE guidelines.

2.1.3. Ethical Consideration

The study methodology was authorized by the Ethical Committee of Faculty of Medicine, Tanta University (Approval Code: 36264MD67/4/23; approval date: 09/04/2023). Painless procedures were executed with adequate sedation.

2.2. Methods and Experimental Design

The rats were allocated randomly to two groups: acute and subacute exposure groups (40 rats each; Table 1).

TABLE 1.

Experimental grouping and allocation of rats for acute and subacute exposure.

Exposure group	Subgroup	Description	Number of rats	Treatment
Acute exposure (1 day)	Group I (control)	Standard diet and drinking water.	20	Subgroup Ia: Negative control (10 rats) Subgroup Ib: Positive control (2‐mL saline) (10 rats)
	Group II	ZnO‐NPs, 175 mg/kg (1/20 of LD50), orally via gavage tube.	10	ZnO‐NP treatment
	Group III	ZnO‐NPs, 350 mg/kg (1/10 of LD50), orally via gavage tube.	10	ZnO‐NP treatment
Subacute exposure (28 days)	Group IV (control)	Standard diet and drinking water.	20	Subgroup IVa: Negative control (10 rats) Subgroup IVb: Positive control (2‐mL saline) (10 rats)
	Group V	ZnO‐NPs, 175 mg/kg orally via gavage tube.	10	ZnO‐NP treatment
	Group VI	ZnO‐NPs, 350 mg/kg orally via gavage tube.	10	ZnO‐NP treatment

ZnO‐NPs powder was suspended in sterile saline and homogenized using a bath sonicator for 10 min immediately prior to administration to minimize particle agglomeration. The suspension was freshly prepared before each dosing and delivered orally to rats via a gavage tube at a volume of 2 mL. Animals received a single dose for acute exposure, whereas subacute exposure involved once‐daily administration for 28 consecutive days.

The selected doses of ZnO‐NPs, corresponding to 1/20 and 1/10 of the reported LD50 (Abass et al. 2017), follow a standard approach for toxicological studies in rodents.

All rats in the studied groups underwent the following procedures:

2.2.1. Relative Weight of Pancreas

The body weight of all rats was recorded on the first day prior to chemical administration and subsequently on a daily basis, and the dose was adjusted accordingly.

Finally, the body weight of all rats was determined at the end of the experimental duration—1 day after acute exposure and 28 days after subacute exposure.

The rats were anesthetized by diethyl ether anesthesia. The fur and skin of each experimental rat were gently pinched with forceps and lifted slightly. An incision was then made through the abdominal cavity using surgical scissors. The stomach, located on the left side, was carefully manipulated to avoid tearing, and the pancreas was gently separated from the stomach and duodenum using two forceps. Remaining attachments between the pancreas and thoracic cavity were carefully dissected to fully detach the pancreas along with the adjoining spleen. The spleen was then separated from the pancreas using blunt dissection.

The weight of the pancreas was recorded as the absolute organ weight, whereas the relative pancreas weight was estimated by the following equation:

$$\begin{array}{c}\frac{\text{Relative pancreas weight}=\text{Absolute pancreas weight}\left(\mathrm{gm}\right)\times 100}{\text{Final body weight}\left(\mathrm{gm}\right)}\hfill \end{array}$$

2.2.2. Biochemical Investigations

Four milliliters of blood was obtained via direct cardiac puncture and gathered into nonheparinized tubes for biochemical study. The blood from each rat was then centrifuged at 3000 rpm for 5 min to segregate the serum. The isolated sera were stored at −20°C for biochemical analysis of glucose, Zn and insulin levels, and malondialdehyde (MDA), which was determined via commercially available colorimetric methods (diagnostic kits supplied by Bio‐diagnostics; Dokki, Giza, Egypt) as per the manufacturer's guidelines following the thiobarbituric acid reactive substances method as described by del Rio et al. (2005).

2.2.3. Histopathological Examination

2.2.3.1. Preparation of Specimens for Light Microscopic Examination

The pancreatic tissues from all rats in both the control and treated groups were fixed in 10% formalin overnight, followed by processing for paraffin embedding. The tissues were dehydrated through a graded series of ethanol concentrations and subsequently embedded in paraffin wax to prepare tissue blocks.

2.2.3.2. Hematoxylin and Eosin Staining (H&E)

Consecutive 5‐μm‐thick sections were fixed in formalin, embedded in paraffin, and subsequently stained with H&E for histopathological examination (Bancroft and Gamble 2008).

2.2.3.3. Morphometric Analysis

Diameters and areas of islet of Langerhans in each animal of the different groups were measured by ImageJ analysis software Version 1.43 from the National Institutes of Health in the United States. After determination of the area of each islet, the cells within the islets were counted and divided by the area to identify the density, and then, a special count for the number of Beta cells was also done. Fibrosis scores were determined in an area of 1 mm² using ImageJ analysis software and expressed as a percentage.

2.2.4. Immunohistochemistry

Immunohistochemical reaction was performed via inducible nitric oxide synthase (iNOS) Polyclonal Antibody, which was purchased from Thermo Fisher Scientific, Massachusetts, United States, Catalog Number: PA3‐030A, Polyclonal (Rabbit IgG), 1:200 dilution, Antigen Retrieval Protocol: heat‐induced epitope retrieval (HIER) using 10‐mM sodium citrate buffer (pH 6.0), microwaved for 8–15 min. Consecutive 5‐μm‐thick sections from formalin‐fixed, paraffin‐embedded tissue blocks were prepared. The paraffin sections were processed for detection of inducible nitric oxide synthase (iNOS) (Tripathi and Krishna 2008). For quantitative evaluation, sections from at least five animals in each experimental group were examined using immunohistochemistry. Five fields per section were randomly selected for morphometric measurements using the ImageJ Analyzer Version 1.43 software from the National Institutes of Health in the United States. Each experimental group's INOS sections were tested for mean number INOS positive stained cells.

2.3. Statistical Analysis

All statistical analyses were performed utilizing R, Version 4.4.1 (Team 2016). The distribution of continuous numerical variables used the Shapiro–Wilk test and Q–Q plots. Variables that revealed a deviation from normal distribution were summarized using the median, interquartile range (IQR; expressed as 25th–75th percentiles), and range (minimum and maximum values). Comparisons between the studied groups were done using the Kruskal–Wallis test (for abnormally distributed variables), followed by the Dunn test if significant (with false discovery rate [FDR] adjustment). A p value of 0.05 was utilized for the threshold for statistical significance (Hinkle et al. 2003).

3. Results

Neither mortality nor abnormal clinical or behavioral data were recorded throughout the experimental period.

3.1. Relative Weight of Pancreas

The impact of ZnO‐NPs on the relative weight of pancreas revealed no significant differences in both acute and subacute toxic exposures, in the four studied groups. The effect size for the relative pancreas weight was calculated to quantify the magnitude of differences among the studied groups. The analysis revealed a small effect size (Eta‐squared, η ² = 0.006) (Table 2).

TABLE 2.

Statistical analysis of relative weights of pancreas in the studied groups after acute toxic exposure (1 day) and subacute toxic exposure (28 days) to zinc oxide nanoparticles (ZnO‐NPs).

Variables		Subgroup Ia (negative control), n = 10	Subgroup Ib (positive control), n = 10	Group II (175 mg/kg ZnO‐NPs treated group), n = 10	Group III (350 mg/kg ZnO‐NPs treated group), n = 10	Test statistics (p)
Relative weight of pancreas (g) after acute toxic exposure	Median [IQR]	0.21 [0.20–0.23]	0.20 [0.19–0.23]	0.20 [0.19–0.21]	0.18 [0.17–0.21]	3.893 (0.273 a )
	Range	0.10–0.23	0.17–0.23	0.11–0.27	0.15–0.27

Variables		Subgroup IVa (negative control), n = 10	Subgroup IVb (positive control), n = 10	Group V (175 mg/kg ZnO‐NPs treated group), n = 10	Group VI (350 mg/kg ZnO‐NPs treated group), n = 10
Relative weight of pancreas (g) after subacute toxic exposure	Median [IQR]	0.22 [0.20–0.23]	0.26 [0.22–0.27]	0.22 [0.20–0.24]	0.23 [0.18–0.25]	3.369 (0.338 a )
	Range	0.19–0.25	0.19–0.29	0.17–0.30	0.15–0.31

Note: Data are presented as median [IQR] and range.

Abbreviations: g, gram; IQR, interquartile range (25th–75th percentiles); n, number.

^a Kruskal–Wallis rank sum test.

3.2. Biochemical Investigations

3.2.1. Serum Zn Level

In cases of acute toxic exposure, Groups II and III exhibited significantly higher median serum zinc levels when compared to Subgroups Ia and Ib (p = 0.001 and 0.004 for Group II and < 0.001 and 0.001 for Group III). Similarly, under subacute toxic exposure conditions, Group VI showed a notable elevation in median serum zinc levels relative to Subgroup IVa, Subgroup IVb, and Group V (p < 0.001, 0.001, and 0.021, respectively) (Table 3 and Figure 2A).

TABLE 3.

Statistical analysis of serum zinc level in the studied groups after acute toxic exposure (1 day) and subacute toxic exposure (28 days) to zinc oxide nanoparticles (ZnO‐NPs).

Variables		Subgroup Ia (negative control), n = 10	Subgroup Ib (positive control), n = 10	Group II (175 mg/kg ZnO‐NPs treated group), n = 10	Group III (350 mg/kg ZnO‐NPs treated group), n = 10	Test statistics (one‐way analysis of variance) (ANOVA) (p) a	Pairwise comparison between groups (Dunn test with FDR adjustment)
Zinc level (μg/dL) after acute toxic exposure	Median [IQR]	140.8 [132.0–154.0]	155.6 [143.0–182.0]	260.5 [220.0–310.0]	376.0 [297.0–456.0]	29.287 (< 0.001*)	Ia vs. Ib	Ia vs. II	Ia vs. III	Ib vs. II	Ib vs. III	II vs. III
	Range	127.0–196.0	128.0–196.0	171.5–417.0	201.0–510.0		0.528	0.001*	< 0.001*	0.004*	0.001*	0.455
	Range	Range	95.0–188.0	130.0–181.0	140.0–468.0

Variables		Subgroup IVa (negative control), n = 10	Subgroup IVb (positive control), n = 10	Group V (175 mg/kg ZnO‐NPs treated group), n = 10	Group VI (350 mg/kg ZnO‐NPs treated group), n = 10	Test statistics (one‐way analysis of variance) (ANOVA) (p) a	IVa vs. IVb	IVa vs. V	IVa vs. VI	IVb vs. V	IVb vs. VI	V vs. VI
Zinc level (ug/dl) after subacute toxic exposure	Median [IQR]	149.0 [119.0–164.0]	160.0 [145.0–176.0]	167.0 [152.0–230.0]	350.0 [268.0–404.0]	21.725 (< 0.001*)	0.369	0.097	< 0.001*	0.369	0.001*	0.021*

Note: Data are presented as median [IQR] and range.

Abbreviations: μg/dL, microgram per deciliter; IQR, interquartile range (25th–75th percentiles); vs., compared to.

^a Kruskal–Wallis rank sum test.

^*Significant at p < 0.05.

FIGURE 2.

(A) Serum zinc level in the studied groups after exposure. (B) Malondialdehyde (MDA) level in the studied groups after exposure.

3.2.2. Estimation of Serum MDA

Although a slight increase in MDA was observed following acute exposure, it did not reach statistical significance relative to the control groups (p = 0.098). Whereas in subacute toxic exposure, the median MDA in Groups V and VI was significantly higher than those of Subgroups IVa and IVb (p = 0.019 and 0.007 for Group V; p = 0.015 and 0.007 for Group VI) (Table 4 and Figure 2B).

TABLE 4.

Statistical analysis of malondialdehyde (MDA) level in the studied groups after acute toxic exposure (1 day) and subacute toxic exposure (28 days) to zinc oxide nanoparticles (ZnO‐NPs).

Variables		Subgroup Ia (negative control), n = 10	Subgroup Ib (positive control), n = 10	Group II (175 mg/kg ZnO‐NPs treated group), n = 10	Group III (350 mg/kg ZnO‐NPs treated group), n = 10	Test statistics (one‐way analysis of variance, ANOVA) (p) a	Pairwise comparison between groups
Malondialdehyde (MDA) (nmol/L) after acute toxic exposure	Median [IQR] Range	16.37 [14.58–21.09] 9.97–23.65	19.15 [14.83–22.56] 12.73–30.35	19.88 [16.45–25.47] 13.85–33.78	22.32 [20.39–29.83] 15.66–43.51	6.294 (0.098)
	Range	9.97–23.65	12.73–30.35	13.85–33.78	15.66–43.51

Variables

Subgroup IVa (negative control), n = 10

Subgroup IVb (positive control), n = 10

Group V (175 mg/kg ZnO‐NPs treated group), n = 10

Group VI (350 mg/kg ZnO‐NPs treated group), n = 10

Test statistics (one‐way analysis of variance, ANOVA) (p) a

Malondialdehyde (MDA) (nmol/L) after subacute toxic exposure

Median [IQR]

9.27 [7.69–12.44]

8.46 [8.25–9.32]

18.01 [9.51–20.97]

16.76 [11.91–19.87]

16.82 (< 0.001*)

Dunn test with FDR adjustment

IVa vs. IVb

IVa vs. V

IVa vs. VI

IVb vs. V

IVb vs. VI

V vs. VI

Range

6.50–13.86

6.80–10.24

8.72–22.00

8.01–26.00

0.679

0.019*

0.015*

0.007*

0.007*

0.848

Note: Data are presented as median [IQR] and range.

Abbreviations: IQR, interquartile range (25th–75th percentiles); nmol/L, nanomoles per liter; vs., compared to.

^a Kruskal–Wallis rank sum test.

^*Significant at p < 0.05.

3.2.3. Random Blood Glucose Level

In acute toxic exposure, the median random serum glucose levels in both Groups II and III were significantly elevated than in Subgroup Ia and Ib (p = 0.009 and 0.003 for Group II; p = 0.005 and 0.002 for Group III). In subacute toxic exposure, the values of random blood glucose level in Groups V and VI were significantly higher than those in Subgroups IVa and IVb (p = 0.014 and 0.029 for Group V; p = 0.012 and 0.014 for Group VI) (Table 5 and Figure 3A).

TABLE 5.

Statistical analysis of random blood glucose level in the studied groups after acute toxic exposure (1 day) and subacute toxic exposure (28 days) to zinc oxide nanoparticles (ZnO‐NPs).

Variables		Subgroup Ia (negative control), n = 10	Subgroup Ib (positive control), n = 10	Group II (175 mg/kg ZnO‐NPs treated group), n = 10	Group III (350 mg/kg ZnO‐NPs treated group), n = 10	Test statistics (p)	Pairwise comparison between groups (Dunn test with FDR adjustment)
Random blood glucose (mg/dL) after acute toxic exposure	Median [IQR]	116.5 [104.0–133.0]	119.0 [81.0–128.0]	196.5 [188.0–210.0]	203.5 [183.0–254.0]	20.717 (< 0.001*, a )	Ia vs. Ib	Ia vs. II	Ia vs. III	Ib vs. II	Ib vs. III	II vs. III
	Range	83.0–180.0	68.0–141.0	80.0–330.0	111.0–320.0		0.672	0.009*	0.005*	0.003*	0.002*	0.767

Variables		Subgroup IVa (negative control), n = 10	Subgroup IVb (positive control), n = 10	Group V (175 mg/kg ZnO‐NPs treated group), n = 10	Group VI (350 mg/kg ZnO‐NPs treated group), n = 10	Test statistics (p)	IVa vs. IVb	IVa vs. V	IVa vs. VI	IVb vs. V	IVb vs. VI	V vs. VI
Random blood glucose (mg/dL) after subacute toxic exposure	Median [IQR]	99.5 [84.0–110.0]	105.5 [93.0–116.0]	131.5 [122.0–139.0]	129.5 [118.0–157.0]	15.042 (0.002*, a )	0.731	0.014*	0.012*	0.029*	0.014*	0.731
	Range	77.0–154.0	86.0–128.0	88.0–143.0	106.0–180.0

Note: Data are presented as median [IQR] and range.

Abbreviations: FDR, false discovery rate; IQR, interquartile range (25th–75th percentiles); mg/dL, milligrams (mg) per deciliter; n, number; vs., compared to.

^a Kruskal–Wallis rank sum test.

^*Significant at p < 0.05.

FIGURE 3.

(A) Random blood glucose level in the studied groups after exposure. (B) Insulin levels in the studied groups after exposure.

3.2.4. Serum Insulin Level

In acute toxic exposure, a significant increase in insulin level was detected between Groups II and III relative to those of Subgroup Ia and Ib (p = 0.016 and 0.017 for Group II; p = 0.017 and 0.032 for Group III). In subacute toxic exposure, the serum insulin level declined insignificantly in Group V compared to control Groups IVa and IVb (p = 0.066 and 0.329, respectively). Similarly, it declined insignificantly in Group VI compared to Subgroup IVb (p = 0.066). However, a significant decrease in its level was detected in Group VI compared to Subgroups IVa (p = 0.009) (Table 6 and Figure 3B).

TABLE 6.

Statistical analysis of insulin levels in the studied groups after acute toxic exposure (1 day) and subacute toxic exposure (28 days) to zinc oxide nanoparticles (ZnO‐NPs).

Variables		Subgroup Ia (negative control), n = 10	Subgroup Ib (positive control), n = 10	Group II (175 mg/kg ZnO‐NPs treated group), n = 10	Group III (350 mg/kg ZnO‐NPs treated group), n = 10	Test statistics (p)	Pairwise comparison between groups (Dunn test with FDR adjustment)
Insulin level (uIU/mL) after acute toxic exposure	Median [IQR]	5.95 [5.70–7.00]	6.55 [5.60–7.00]	8.16 [7.12–8.60]	8.10 [7.10–8.90]	14.299 (0.003*, a )	Ia vs. Ib	Ia vs. II	Ia vs. III	Ib vs. II	Ib vs. III	II vs. III
	Range	5.22–8.00	5.20–8.21	6.60–9.30	4.00–9.10		0.752	0.016*	0.017*	0.017*	0.032*	0.752

Variables		Subgroup IVa (negative control), n = 10	Subgroup IVb (positive control), n = 10	Group V (175 mg/kg ZnO‐NPs treated group), n = 10	Group VI (350 mg/kg ZnO‐NPs treated group), n = 10	Test statistics (p)	IVa vs. IVb	IVa vs. V	IVa vs. VI	IVb vs. V	IVb vs. VI	V vs. VI
Insulin level (uIU/ml) after subacute toxic exposure	Median [IQR]	5.65 [4.60–6.00]	4.95 [4.30–5.60]	4.55 [4.20–4.90]	4.00 [3.70–4.80]	11.380 (0.005*, a )	0.329	0.066	0.009*	0.329	0.066	0.329
	Range	4.10–6.20	3.62–7.00	3.00–5.30	3.21–5.20

Note: Data are presented as median (IQR) and range.

Abbreviations: FDR, false discovery rate; IQR, interquartile range (25th–75th percentiles; n, number; uIU/mL, microinternational units per milliliter); vs., compared to.

^a Kruskal–Wallis rank sum test.

^*Significant at p < 0.05.

3.3. Histopathological Examination

3.3.1. H&E Staining

All slides were evaluated blindly.

Histological examination of pancreatic tissue from control animals revealed normal architecture. The pancreas consisted of multiple lobules of varying sizes and shapes. Each lobule contained pancreatic acini (exocrine component) and interspersed Langerhans islets (endocrine component). The islets were composed of clusters of endocrine cells, whereas the acinar cells were single‐layered pyramidal cells with basal, rounded, vesicular nuclei. The cytoplasm of acinar cells exhibited acidophilic staining near the acinar lumen and basophilic staining in regions distal to the lumen (Figure 4).

FIGURE 4.

Photomicrographs of rat pancreas from the control group. (A) Low‐power view (H&E, ×200) showing exocrine acini (arrow), duct (D), connective tissue septa (CT), and Langerhans islets (I) composed of clusters of endocrine cells. (B) High‐power view (H&E, ×400) showing pancreatic acini lined with pyramidal cells with basal rounded vesicular nuclei (arrowhead). Cytoplasm is basophilic away from the acinar lumen (curved arrow) and acidophilic near the lumen (*).

In acute toxic exposure, examinations of Group II showed that mild degenerative changes primarily affected entire pancreatic lobules. They appeared as shrunken irregularly shaped acini and wide areas between lobules. Additionally, the acinar cells displayed cytoplasmic vacuolation with hyperchromatic nuclei, and some areas showed complete loss of tissue replaced by fibrous tissue. In contrast, most Langerhans islets appeared normal (Figures 5A–C).

FIGURE 5.

Photomicrographs of rat pancreas after acute ZnO‐NPs exposure. (A) Group II showing vacuolation of acini (arrow) and widened areas between lobules (star) (H&E, ×200). (B) Group II showing shrunken, irregularly shaped acini (arrow), widened interacinar spaces (curved arrow), and areas of total acinar loss replaced by fibrous tissue (star) (H&E, ×400). (C) Group II acinar cells exhibiting cytoplasmic vacuolation (arrow) and darkly stained nuclei (curved arrow); Langerhans islet appears normal (I) (H&E, ×400). (D) Group III showing small acini (arrowhead) and fibrous tissue (curved arrow) with dilated ducts (D); most Langerhans islets appear normal (I) (H&E, ×200).

Furthermore, examinations of higher doses of ZnO‐NPs in Group III treated rats revealed that the pancreatic acini were apparently small, and fibrous tissue was well demonstrated. Besides, dilated ducts were also noticed. Most Langerhans Islets were apparently normal (Figure 5D).

In subacute toxic exposure, the majority of specimens in Group V were affected, exhibiting focal lesions characterized by varying degrees of cellular degeneration. Most of the acini were shrunken with loss of cytoplasm and decreased basal basophilia, and some of them were totally degenerated. Parts of the Langerhans islets cells were surrounded by inflammatory cellular infiltration, accompanied by dilated and engorged blood vessels, which were also noted (Figure 6A,B).

FIGURE 6.

Photomicrographs of rat pancreas after subacute ZnO‐NPs exposure. (A) Group V showing shrunken acini with loss of cytoplasm and decreased basal basophilia (arrowhead); some acini are totally degenerated (curved arrow) (H&E, ×400). (B) Group V showing part of Langerhans islet cells surrounded by inflammatory cell infiltration (wavy arrow) with dilated congested blood vessels (arrow) (H&E, ×200). (C) Group VI showing widened spaces between pancreatic lobules (arrow), small acini, and areas of total acinar loss (curved arrow) (H&E, ×200). (D) Group VI showing inflammatory cell infiltration (wavy arrow) and complete loss of Langerhans islet cells (arrowhead) (I) (H&E, ×200).

Examination of Group VI revealed extensive destructive changes affecting nearly the entire pancreatic lobules, suggesting pronounced toxicity relative to the other groups. These lesions disrupted the normal pancreatic architecture. The acini were delineated by extensive intervals, likely owing to interstitial edema, resulting in pronounced separation between lobules. Focal areas of acinar atrophy were observed, with some acini completely destroyed, leaving empty spaces. The Langerhans islet cells showed complete area of cell loss, leaving empty spaces with inflammatory cell infiltration between the acini (Figure 6C,D).

3.3.2. Immunohistochemistry

In the acute toxic exposure, iNOS‐immunohistochemically stained sections from the control groups demonstrated no immunoreactivity in both the negative and positive control specimens (Subgroups Ia and Ib) (Figure 7A). The pancreatic sections of Groups II and III revealed that some pancreatic acini showed strong cytoplasmic immunoreaction, whereas other acini showed moderate reaction (Figure 7B,C).

FIGURE 7.

Immunohistochemical photomicrographs of rat pancreas stained for inducible nitric oxide synthase (iNOS). (A) Control groups (Subgroups Ia and Ib) showing normal pancreatic tissue with no immunostaining reaction (3,3‐diaminobenzidine and hematoxylin, ×200). (B) Group II (acute exposure) showing focal areas of strong positive reaction (arrow) and other areas with moderate positive reaction (bifid arrow) (3,3‐diaminobenzidine and hematoxylin, ×200). (C) Group III (acute exposure) showing some acini with strong cytoplasmic reaction (arrow) and others with moderate reaction (bifid arrow) (3,3‐diaminobenzidine and hematoxylin, ×400). (D) Groups V and VI (subacute exposure) showing Langerhans islets (I) with weak positive immunoreaction for iNOS (arrow) (3,3‐diaminobenzidine and hematoxylin, ×200).

In subacute toxic exposure, the iNOS immunohistochemical‐stained sections of the control groups (Subgroups IVa and IVb) were similar to those in the acute exposure group. The pancreatic acini of Groups V and VI showed no immunoreaction, whereas islets of Langerhans showed weak immunoreaction (Figure 7D).

3.3.3. Morphometric Analysis

Fibrosis score was evaluated using morphometric analysis and expressed as percentage area. The results demonstrated a significant increase in fibrosis severity among the studied groups, especially following subacute exposure, as confirmed by Dunn's test with FDR adjustment (p = 0.022). Pancreatic islet density was expressed as the number of islets per mm². The analysis showed a significant decrease in islet density following both acute and subacute exposure, with a more marked reduction in the subacute groups. Statistical significance was confirmed using Dunn's test with FDR adjustment (p = 0.022). Morphometric evaluation of β‐cell number per islet showed a significant exposure‐dependent decline, particularly following subacute exposure, as confirmed by Dunn's test with FDR correction (p = 0.022). iNOS score was evaluated using morphometric analysis and expressed as an immunoreactivity score. The results demonstrated a significant increase in iNOS expression among the studied groups, particularly following acute exposure, as confirmed by Dunn's test with FDR adjustment (p < 0.05). In contrast, the subacute exposure groups (IV–VI) exhibited comparatively lower iNOS scores with wider interindividual variability (Figure 8).

FIGURE 8.

Morphometric and immunohistochemical analysis of pancreatic tissue after ZnO‐NP exposure. (A) fibrosis score; (B) islet density; (C) number of β‐cells per islet; and (D) iNOS expression. Acute (Groups I–III) and subacute (Groups IV–VI) exposure induced increased fibrosis, reduced islet density, decreased β‐cell number, and elevated iNOS scores compared to controls. Statistical analysis was performed using Dunn's test with FDR adjustment; p values are indicated.

4. Discussion

Zn is a vital trace element that plays a role in insulin synthesis, storage, and secretion by the pancreas secretion (Farooq 2019). Alterations in Zn levels, coupled with increased oxidative stress in diabetes mellitus, oxidative stress has been seen, especially in pancreatic β cells. Such imbalances may further aggravate insulin resistance in tissues outside the pancreas, including muscle, adipose tissue, and liver, thereby impairing glycemic control and promoting the development of diabetic complications. Conversely, the development of diabetes can further disrupt systemic Zn homeostasis (Hasanato 2020).

In our study, the impact of ZnO‐NPs on the relative weight of the pancreas in both acute and subacute toxic exposures revealed nonsignificant differences in the four studied groups. Holding true to what was found in this investigation, Seok et al. (2013) found nonsignificant change in the weight of the pancreas when ZnO‐NPs were delivered to the rats at doses of 67.1, 134.2, 268.4, and 536.8 mg/kg/bodyweight orally for a 13‐week period.

In our investigation, we found a significant rise in the serum Zn level in Groups II and III in acute exposure and in Group VI in subacute exposure relative to control groups.

These results were in line with the data of Deore et al. (2021), who found significantly increased blood Zn levels after administration of 100 mg/kg/day ZnO‐NPs (60 nm) by mouth for 28 days in adult albino rats. In addition, Khayal et al. (2021) demonstrated significant increases in serum Zn levels in male rats treated by 85 mg/kg ZnO‐NPs (< 100 nm) orally for 8 weeks. They reported that the oral ZnO‐NPs dissolve in the stomach with the release of zinc ions, which are small and more easily absorbed by biological membranes, and then circulate into the blood.

On the contrary, Shaban et al. (2021) reported that Zn concentration showed a nonsignificant alteration in the treated groups relative to the control following a single administration of 1 mL/rat suspensions of 20‐ and 40‐ppm ZnO‐NPs. They attributed their results to the effect of the dose, the experiment's duration, or both.

In the present research, administration of ZnO‐NPs elicited oxidative stress in rats as evidenced by the high level of serum MDA in treated groups relative to control groups in subacute toxic exposure. Moreover, our results showed a significant increase in iNOS scores in the acute exposure groups compared with controls, indicating a strong inflammatory response shortly after exposure. In contrast, subacute exposure resulted in lower and more variable iNOS expression, suggesting a milder and more sustained response (Bogdan 2001).

ZnO‐NPs can generate excessive ROS due to their high surface reactivity and ability to penetrate tissues and cells, overwhelming endogenous antioxidant defenses such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). When ROS production exceeds the scavenging capacity of these antioxidant systems, polyunsaturated fatty acids in cell membranes undergo peroxidation, leading to the formation and accumulation of MDA, a stable end‐product and widely accepted biomarker of lipid peroxidation. Additionally, higher doses of ZnO‐NPs have been shown to decrease the total antioxidant capacity in tissues, further tipping the balance toward oxidative damage and enhancing lipid peroxidation processes (Mirzaei et al. 2025). This agreed with Mohamed et al. (2022) who illustrated that there was a very highly significant increase in MDA level in ZnO‐NPs groups than in the control group in subacute toxic exposure.

In line with this, Abdallah (2018) documented a significant raise in serum MDA after administration of 422 mg/kg body weight ZnO‐NPs daily orally for 8 weeks. They stated that ZnO‐NPs can adversely disrupt bodily systems through oxidative stress, followed by the generation of ROS. Sewelam and Shehata (2021) found a significantly raised pancreatic MDA level in rats after delivery of a single intraperitoneal injection of ZnO‐NPs (35 nm) in two doses (250 and 700 mg); then, the rats were left without medication for 2 weeks. Zaghloul et al. (2023) reported similar results after oral administration of ZnO‐NPs (20 nm) in doses of 100 and 400 mg/kg bodyweight per day for 28 days. Their explanation for that was oxidative stress, generation of ROS, and its accumulation causing lipid peroxidation.

Nitric oxide (NO), a highly reactive free radical, is produced from the amino acid L‐arginine by the action of a nitric oxide synthase (NOS). There are three main subtypes of NOS in the human body: endothelial NO synthase (eNOS), neuronal NO synthase (nNOS), and inducible NO synthase (iNOS) (DiMagno 2007). Induction of iNOS can generate NO. Once induced, it produces large amounts of NO for hours to days (Huang et al. 2022; Kolasa et al. 2009). NO modulates normal pancreatic exocrine secretion, endocrine insulin release, and microvascular blood flow within the pancreas (DiMagno 2007). Excessive nitric oxide production mediated by iNOS may play a functional role in the systemic and local hemodynamic disturbances, as well as the oxidative tissue damage observed in acute pancreatitis (Al‐Mufti et al. 1998). Moreover, β cells exhibit heightened susceptibility to oxidative stress owing to their relatively low expression of antioxidant enzymes (Sewelam and Shehata 2021).

In the current study, immunohistochemical‐stained sections by iNOS in Groups II and III after acute toxic exposure showed moderate to strong cytoplasmic immunoreaction in some acini. Whereas in subacute toxic exposure, it showed weak immunoreaction in islets of Langerhans. In line with that Sewelam and Shehata (2021) revealed moderate to strong Caspase‐3 (apoptosis marker) immunoreactivity in ZnO‐NPs managed groups relative to the control group. They also used the anti‐insulin monoclonal mouse primary antibody to detect B cells in Langerhans islets, which revealed a marked reduction in insulin immunoreactivity in β cells. This was explained by ZnO‐NPs' ability to cause oxidative stress and subsequently acinar cell necrosis.

The current study revealed that random blood glucose levels were significantly raised in ZnO‐NPs managed groups in both acute and subacute toxic exposures. Serum insulin levels showed a significant raise in Groups II and III relative to control groups in acute toxic exposure. However, a significant reduction in its level was determined in Group VI relative to the control group in subacute toxic exposure.

The mode of action underlying the observed raise in random serum glucose levels in both acute and subacute exposures could be attributed to ZnO‐NPs induced oxidative stress, insulin resistance due to high levels of ROS, and elevated Zn level that might stimulate hepatic glycogenolysis (Omar et al. 2025). Yary et al. (2016) stated that excess Zn can lead to hyperactivity of β cells and increased insulin production, potentially causing insulin resistance through overstimulation and subsequent exhaustion of insulin receptors. Therefore, prolonged elevated Zn levels may be detrimental to β‐cell function.

These results were comparable to Kong et al. (2020), who found a significant increase in blood glucose in managed groups by ZnO‐NPs (50 nm) administered intragastric at doses of 40, 80, 160 mg/kg/bodyweight for 90 days. They explained their findings by the oxidative stress triggered by ZnO‐NPs, which could affect the metabolism of glucose, resulting in increased blood glucose. Furthermore, Hu et al. (2020) examined the impact of oral administration of ZnO‐NPs (35.65 ± 6.63 nm) at doses of 10, 25, 50, and 300 mg/kg bodyweight per day for 12 weeks in rats. They observed a significant increase in plasma glucose, whereas insulin level remained unchanged across the groups. The authors attributed these effects to the oxidative stress caused by ZnO‐NPs, which raised the production of ROS. The high levels of ROS induce an inflammatory response, leading to phosphorylation of the insulin receptor substrate, which in turn causes insulin resistance resulting in a rise of plasma glucose. Additionally, Virgen‐Ortiz et al. (2020) showed a raise in serum glucose levels in rats after delivery of 100 mg/kg/bodyweight of ZnO‐NPs (17 ± 3.6 nm) once by oral and intraperitoneal routes. They attributed their results to the direct effect of Zn ions on hepatic metabolism; elevated Zn levels stimulate hepatocytes to produce more glucose through glycogenolysis.

However, the findings of our study disagreed with Hosseini et al. (2020), who found a significant decline in serum glucose level in ZnO‐NPs managed groups compared control group resulting probably from accumulation of Zn in pancreatic tissue and induction of cellular injury through oxidative stress. Also, a significant decline in blood glucose level was said to have been done by Shaban et al. (2021), following a single injection of 1 mL of ZnO‐NP suspensions containing 20 and 40 ppm (ppm) to rats. They explained this by the ability of Zn to enhance insulin secretion and induce glucose utilization and metabolism.

This study detected a variance in insulin secretion between acute and subacute exposures, which might be related to the alteration of pancreatic response to the increased random blood glucose levels. In acute toxic exposure, pancreatic cells were mildly affected, and thus, the increased random blood glucose levels stimulate the pancreas to secrete more insulin, which may represent a compensatory response of pancreatic β cells to oxidative stress, aimed at maintaining glucose homeostasis. However, in subacute toxic exposure, beta cells of Langerhans underwent degeneration and damage, leading to decreased insulin secretion despite the elevated random blood glucose level.

The current conclusions were further supported by the histological data in the present study, which revealed that in acute toxic exposure, most Langerhans islets appeared morphologically normal in treated groups. The acinar cells displayed cytoplasmic vacuolation and hyperchromatic nuclei and area of total loss replaced by fibrous tissue. On the other hand, subacute toxic exposure revealed that most of the acini were shrunken and some of them were totally degenerated. Part of the Langerhans islets cells was surrounded by inflammatory cellular infiltration, and others exhibited extensive loss, leaving empty areas.

The morphometric findings of the present study demonstrated a significant increase in pancreatic fibrosis accompanied by a marked reduction in islet density and β‐cell number in the exposed groups, notably more pronounced in the subacute exposure groups compared to the acute exposure groups, indicating cumulative pancreatic injury with prolonged exposure.

The concurrent increase in fibrosis and loss of endocrine cellularity suggests disruption of islet architecture and impaired β‐cell integrity, which may underline the observed alterations in insulin secretion. These findings are consistent with previous reports, demonstrating that progressive pancreatic fibrosis and β‐cell loss are closely associated with β‐cell dysfunction and impaired insulin production (Dludla et al. 2023).

Zinc oxide nanoparticles (ZnO‐NPs) exert their toxic effects on pancreatic tissues primarily through the generation of excessive ROS and disruption of cellular redox balance, as their small size and high surface reactivity facilitate cellular uptake and intracellular Zn²⁺ release; this leads to oxidative stress, mitochondrial dysfunction, lipid peroxidation, and DNA damage, which in turn activate stress‐responsive signaling pathways (e.g., MAPKs and JNK) and proinflammatory cascades, culminating in inflammation, apoptosis, and necrosis of both acinar and islet cells, with consequent structural and functional impairment of the pancreas (Chang et al. 2012). The greater toxicity observed following subacute exposure may be attributed to cumulative oxidative stress, insufficient antioxidant defense mechanisms, and sustained release of Zn²⁺ ions, leading to progressive pancreatic injury. Repeated exposure may overwhelm cellular repair mechanisms, thereby exacerbating tissue damage compared with acute exposure (Farahani et al. 2025).

Parallel to our results, Abdallah (2018) reported mild pancreatitis characterized by focal lymphocytic infiltration, ductular hyperplasia, and numerous acinar deaths in the pancreatic tissue after administration of ZnO‐NPs orally at a dose of 422 mg/kg bodyweight daily for 8 weeks. ZnO‐NPs can adversely disrupt bodily systems through oxidative stress, followed by the production of ROS. Furthermore, Sewelam and Shehata (2021) observed vascular congestion, ductal dilation, fibrosis, and inflammatory cell infiltration as indicators of dose‐dependent pancreatic tissue damage and harm caused to cells by ZnO‐NPs. This pancreatic toxicity may result from oxidative stress within the pancreatic tissues, leading to subsequent tissue damage.

In the same manner, Zaghloul et al. (2023) found that ZnO‐NPs treated group showed dose‐dependent pancreatic acinar cell damage. The cells exhibited vacuolations, rarefaction, dilated rough endoplasmic reticulum, nuclear condensation, and mitochondrial degeneration, which were more apparent in high doses.

Although previous studies reported similar findings, discrepancies may arise due to differences in nanoparticle size, administered dose, and route of exposure. These factors should be considered when comparing results across studies. Variations in toxicity outcomes may be influenced by nanoparticle surface charge, agglomeration state, and crystallinity, which were not fully characterized in the present study and represent a limitation.

5. Conclusion

In accordance with the findings of our study, it can be deduced that ZnO‐NPs exert significant harmful histological and immunohistochemical effects on the pancreas. ZnO‐NPs toxicity induced alterations in the pancreas impairing its function and resulting in disturbances in blood glucose and insulin levels. Acute exposure mainly induces transient oxidative and inflammatory stress with functional disturbances in glucose–insulin homeostasis and largely preserved pancreatic structure, whereas subacute exposure leads to cumulative oxidative injury with structural degeneration of pancreatic acini and islets, impaired insulin secretion, and marked histopathological changes. The increased MDA serum level together with positive immunoreaction for iNOS in immunohistochemical stained sections of pancreatic tissue suggest that oxidative stress is fundamentally responsible for toxicity of ZnO‐NPs. Additionally, the observed significant elevation of serum Zn level that may result from the issuance of Zn ions from the NPs could contribute to ZnO‐NPs' toxic effects. Furthermore, the current study demonstrated that ZnO‐NPs toxicity was more evident in subacute toxic exposure than in acute toxic exposure, possibly due to the pancreatic susceptibility to oxidative stress over prolonged durations. Acute toxicity may be attributed to immediate oxidative stress and transient biochemical disturbances, whereas subacute toxicity is likely mediated by sustained oxidative stress, cumulative cellular damage, and progressive pancreatic dysfunction over prolonged exposure durations. Given the widespread application of ZnO‐NPs in industrial, medical, and consumer products, these findings raise concerns regarding potential risks to human health, particularly in occupational settings involving repeated or long‐term exposure. Therefore, further comprehensive studies should distinctly address acute and subacute toxic mechanisms by evaluating different dosages, exposure durations, and nanoparticle sizes, in order to elucidate time‐dependent toxic pathways and inform safer use and regulatory guidelines.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by D.M.E.‐G., R.H.E., M.M.O., and M.M.A. The first draft of the manuscript was written by M.‐A.A.R., and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.