Comparative toxicological evaluations of novel forms nano-pesticides in liver and lung of albino rats

**Background:** Copper oxide nanoparticles (CuO NPs) are increasingly used in agriculture as fertilizers, fungicides, and pesticides due to their unique physicochemical properties. However, concerns about their biosafety have emerged, as their small size and high surface reactivity can promote cellular uptake, oxidative stress, inflammation, and organ toxicity. While CuO nanospheres (CuONSp) have been studied, the toxicological profile of CuO nanoflowers (CuONF)—a novel morphology—remains unexplored. This study aimed to compare the toxic effects of CuONSp and CuONF on the liver and lungs of albino rats to identify the less toxic form for agricultural application.

**Methods:** CuONSp and CuONF were synthesized via hydrothermal methods. CuONSp was prepared using Cu(NO3)2·3H2O and NaOH at 170°C for 24 h, while CuONF was synthesized using CuCl2 and NaOH with cetyl trimethyl ammonium bromide (CTAB) as a stabilizer at 120°C for 6 h. Nanoparticles were characterized by XRD, SEM, HRTEM, and zeta-sizer. Eighteen Wistar male albino rats (120–150 g) were divided into three groups (n=6): untreated control, CuONSp-treated (50 mg/kg/day), and CuONF-treated (50 mg/kg/day) for 30 days via oral gavage. Liver function (AST, ALT, GGT), oxidative stress markers (MDA, GSH), and TNF-α levels were measured in serum and tissues. Histopathological examination (H&E), immunohistochemical staining for TNF-α, NF-kβ, and p53, and ultrastructural analysis by TEM were performed on liver and lung tissues.

**Key Results:** XRD confirmed monoclinic crystalline CuO structures; crystallite size was 20.6 nm for CuONSp and 117.1 nm for CuONF. HRTEM showed CuONSp size ~9 nm (quantum dot range) and CuONF ~228 nm. Hydrodynamic sizes were 284.7±59.7 d.nm (CuONSp) and 363±82.6 d.nm (CuONF). PDI was 0.43 (CuONSp) vs. 0.64 (CuONF); zeta potential was −50.9±6.5 mV (CuONSp) vs. −47.2±7.6 mV (CuONF). CuONSp significantly elevated serum AST (194.6±6.3 U/L vs. control 142.1±6.2 U/L, P<0.01), ALT (101.8±5.2 U/L vs. 61.7±3.1 U/L, P<0.01), and GGT (3.4±0.18 U/L vs. 2.29±0.1 U/L, P<0.01), while CuONF showed near-normal values (AST: 144.2±5.5 U/L; ALT: 63.2±6.6 U/L; GGT: 2.4±0.1 U/L). MDA levels were markedly higher in CuONSp (97.52±8.4 nmol/100 mg) vs. control (42.07±4.4 nmol/100 mg, P<0.01) and CuONF (54.7±4.6 nmol/100 mg). GSH activity was severely reduced by CuONSp (22.89±1.3 U/g) compared to control (55.34±1.5 U/g, P<0.01) and CuONF (48.1±1.4 U/g). TNF-α levels were highest in CuONSp (88.57±4.6 Pg/ml) vs. control (23.23±1.9 Pg/ml, P<0.01) and CuONF (33.97±3.1 Pg/ml). Histopathology revealed bile duct proliferation, mononuclear infiltration, and pyknotic nuclei in CuONSp-treated livers; lungs showed thickened blood vessels, bronchiolar hyperplasia, collapsed alveoli, and thickened septa. CuONF-treated tissues appeared nearly normal. Immunohistochemistry showed strong positive expression of TNF-α, NF-kβ, and p53 in CuONSp-treated liver and lung, while CuONF showed only mild reactivity. Ultrastructurally, CuONSp caused irregular nuclear envelopes, swollen mitochondria with ill-defined cristae, fat droplets in hepatocytes, and pyknotic pneumocyte type 2 nuclei with empty lamellar bodies and collagen deposition in lungs. CuONF-treated tissues showed near-normal ultrastructure.

**Clinical Implications:** This study demonstrates that CuONSp induces substantially greater hepatotoxicity and pulmonary toxicity than CuONF, driven by its smaller size, higher surface charge, greater stability, and enhanced oxidative stress and inflammatory responses. CuONF, with its larger size and lower reactivity, caused minimal tissue damage and may represent a safer alternative for agricultural nano-pesticide applications. However, further long-term safety and ecotoxicological studies are needed before field application.