The tested CuNPs (cat. no. H1605061), copper microparticles (cat. no. A1711069) and copper ions (CuCl₂·2H₂O, cat. no. F1620012) were obtained from Aladdin Industrial Co., Ltd. The sizes of the CuNPs and copper microparticles were 80 nm and 1 µm, respectively. Western blotting and SDS-PAGE preparation kits were purchased from Chengdu Baihe Technology Co., Ltd. Other molecular biology reagents were purchased from Bio-Rad Laboratories, Inc. Antibodies were purchased from Abcam. All analytical commercial kits were purchased from Nanjing Jiancheng Bioengineering Institute.

The sizes of the CuNPs and copper microparticles were confirmed using a Phenom ProX scanning electron microscope (Phenom Scientific Instruments Co., Ltd.). The CuNPs were dispersed in purified water, shaken and sonicated in an ice bath to avoid aggregation. The distribution of particle sizes in the suspension was characterized by dynamic light scattering studies performed using a Zetasizer Nano ZS (Malvern Panalytical, Ltd.) immediately following sonication.

A total of 60 specific pathogen-free (SPF) male rats (100–120 g, 6 weeks old) were purchased from Chengdu Dossy Biological Technology Co., Ltd. Male rats were chosen as the subjects of the study due to differences in the expression of CYP450 between female and male rats (25,26). Rats were housed in plastic cages under SPF conditions at 25±2°C and 70±10% relative humidity, under a 12-h light/dark cycle. Water and food were provided ad libitum. Copper particles were suspended in 1% hydroxypropyl methylcellulose (HPMC) solution (w/v) (Shanghai Ryon Biological Technology Co., Ltd.) every day prior to use. Following 7 days of acclimatization, rats were randomly divided into a control group, which was administered with 1% HPMC, and five test groups that were administered with different concentrations of copper by gavage for 28 days: i) 200 mg/kg 1 µm copper; ii) 200 mg/kg CuCl₂·2H₂O (Cu²⁺); iii) 50 mg/kg CuNPs (low dose); iv) 100 mg/kg CuNPs (medium dose); v) 200 mg/kg CuNPs (high dose) (n=10 per group). The study was approved by Sichuan Agricultural University (Chengdu, China), and the protocols for animal care and treatment were in accordance with their guidelines for animal experiments (approval no. 20170314). All possible efforts were made to relieve unnecessary suffering of the experimental animals.

On day 28 of the experiment, following an overnight fast, the rats were anesthetized by gas anesthesia with diethyl ether at the rate of 0.2 l/min. Anesthesia was confirmed by righting reflex, and the animals were rapidly taken out of the anesthesia machine and sacrificed by cervical dislocation. Brain tissues were snap-frozen and stored at −80°C for oxidative stress and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses.

Brain microsomes were used to analyze the protein expression of the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) and CYP450 enzymes, which were prepared by differential centrifugation as previously described (27). The tissue was homogenized in a 0.05 mM Tris/KCl buffer (pH 7.4; Boster Biological Technology), centrifuged at 10,000 × g at 4°C for 30 min, and the supernatant was centrifuged at 105,000 × g at 4°C for 60 min. Subsequently, the brain protein settlement was re-suspended with 0.05 mM Tris/KCl buffer (pH 7.4) and stored at −80°C until western blot analyses were performed. The protein content in the brain microsomes was determined using the Bicinchoninic Acid Protein Assay kit (Beyotime Biological Technology Co., Ltd.) with the Thermo Scientific™ Multiskan™ GO Microplate reader (Thermo Fisher Scientific, Inc.).

The levels of total superoxide dismutase (T-SOD), glutathione (GSH), hydroxyl radicals (·OH) and malondialdehyde (MDA) in the rat brain were determined to evaluate oxidative stress and damage. This was performed using commercial assay kits from Nanjing Jiancheng Bioengineering Institute, which were: Total Superoxide Dismutase (T-SOD) assay kit (Hydroxylamine method); Malondialdehyde (MDA) assay kit (TBA method); Reduced glutathione (GSH) assay kit (Spectrophotometric method); and Hydroxyl Free Radical assay kit. All assays were performed according to the manufacturer's instructions.

The expression levels of CYP450 1A2, 2D22, 2E1 and 3A11 in the brain were analyzed using RT-qPCR as previously described (28). Total RNA was extracted using an OMGA total RNA kit II (Omega Bio-Tek, Inc.) and cDNA was synthesized using PrimeScript™ RT reagent kit with gDNA Eraser (Takara Bio, Inc.). The qPCR was performed using iQ SYBR^® Premix Ex Taq™ II (Tli RNaseH Plus; cat. no. RR820A; Takara Bio, Inc.). The qPCR was performed under the following conditions: 45 cycles, each involving 5 sec of denaturation at 95°C, and 40 sec of amplification at 60°C. The housekeeping gene GAPDH was used as an internal control. All primers were designed with Primer premier v 5.0 software (Premier Biosoft International) and commercially produced (BGI Tech Solutions Co., Ltd.; Table I) based on the target gene. Melting curves and PCR efficiency were used as standard quality criteria for each qPCR run. The target gene mRNA expression was normalized to GAPDH expression, and were analyzed using the 2^−ΔΔCq method (29).

The protein levels of CYP450 1A1, CYP450 2C11, CYP450 2D6, CYP450 3A1, CAR and PXR in the brain microsome of rats were estimated using western blot analysis as previously described (30,31). Microsomal proteins (10 µg) were separated by 10% SDS-PAGE and transferred to PVDF membranes (Pall Corporation). The membranes were blocked with skimmed milk (Beijing Solarbio Science & Technology Co., Ltd.) and incubated for 12 h at 4°C with primary antibodies against CYP450 1A1 (cat. no. ab22717; 1:1,000; Abcam), CYP450 2C11 (cat. no. ab3571; 1:1,000; Abcam), CYP450 2D6 (cat. no. 73867S; 1:1,000; Cell Signaling Technology, Inc.), CYP450 3A1 (cat. no. ab22724; 1:1,500; Abcam), PXR (cat. no. ab118336; 1:500; Abcam), CAR (cat. no. ab62590; 1:1,500; Abcam), and β-actin (cat. no. bs-0061R; 1:10,000; Beijing Biosynthesis Biotechnology Co., Ltd.). Following incubation with primary antibody, the blots were incubated with a horseradish peroxidase-labeled secondary antibody for 1 h at room temperature (cat. no. bs-0295G; 1:10,000; Beijing Biosynthesis Biotechnology Co., Ltd.). β-actin was used as an internal loading control. The bands were visualized using enhanced chemiluminescence (ECL Western Blotting Substrate; Beijing Solarbio Science & Technology Co., Ltd.) and densitometric analysis was performed using ImageJ software version 1.48u (National Institutes of Health).

The assays were performed in triplicate. All data were expressed as the mean ± standard deviation. Statistical analysis was performed by one-way ANOVA in SPSS version 19.0 (IBM Corp.), and the least significant difference test was used following comparison of the mean values with the control group. P<0.05 was considered to indicate a statistically significant difference.

Physiochemical characteristics of CuNPs and copper microparticles were evaluated using scanning electron microscopy and a laser particle size analyzer (Fig. 1). CuNPs and copper microparticles exhibited spherical morphology (Fig. 1A and B), and the size distribution is presented in Fig. 1C and D. The most common sizes of the CuNPs and copper microparticles were 80 nm (average size: 82.5±33.4 nm) and 1 µm (average size: 987.4±436.7 nm), respectively.

The levels of oxidative stress markers in the rat brains were determined using commercial assay kits (Fig. 2). The levels of T-SOD were significantly decreased compared with those of the control following all treatments, with the exception of Cu²⁺. GSH content was decreased significantly in the 1 µm, Cu²⁺ and high-dose CuNPs groups compared with that in the control group. The levels of ·OH were increased in the 1 µm, Cu²⁺ and high-dose CuNPs groups compared with that in the control group. The level of MDA was increased in the Cu²⁺ and high-dose CuNPs groups compared with that in the control.

RT-qPCR was performed to determine the mRNA expression levels of different CYP450s (Fig. 3). CYP450 1A1 mRNA expression levels were significantly decreased in the 1 µm and Cu²⁺ groups and significantly increased in the low-dose CuNPs group compared with that in the control group. The mRNA expression levels of CYP 2C11 were significantly decreased in rats treated with high-dose CuNPs compared with that in the control. The mRNA expression levels of CYP450 2D6 were significantly decreased in the Cu²⁺ group, but significantly increased in the low- and medium-dose CuNPs groups compared with that in the control group. The mRNA expression levels of CYP450 3A1 were increased in the 1 µm, Cu²⁺ and low-dose groups, but significantly suppressed in the high-dose CuNPs group compared with that in the control. The mRNA expression levels of CAR were reduced in the low and medium CuNPs groups, and significantly reduced in the high CuNPs groups, whereas PXR mRNA expression levels of were reduced significantly in the medium and high dose CuNPs groups, and significantly increased in the 1 µm, Cu²⁺ and low-dose CuNPs groups compared with that in the control group.

Western blot analysis was performed to determine the protein expression levels of CYP450 enzymes (Fig. 4). Protein expression levels of CYP450 1A1 were decreased significantly in the high-dose CuNPs group compared with that in the control. The levels of CYP450 2C11 were significantly decreased in the medium- and high-dose CuNPs groups, but increased in the 1 µm group compared with that in the control group. The protein expression levels of CYP450 2D6 were suppressed in the medium- and high-dose CuNPs groups compared with that in the control. The activity of CYP450 3A1 was suppressed in all treatment groups compared with that in the control group. The CAR protein expression levels did not change under any treatment, whereas the protein levels of PXR were decreased in the Cu²⁺, and the low-, medium- and high-dose CuNPs groups compared with that in the control.