Protective Role of Nrf2 in Zinc Oxide Nanoparticles-Induced Lung Inammation in Female Mice and Sexual Dimorphism in Susceptibility

Background Zinc oxide nanoparticles (ZnO-NPs) are used in various products such as rubber, paint, and cosmetics. Our group reported recently that Nrf2 protein provides protection against ZnO-NPs-induced pulmonary inammation in male mice. The present study investigated the effect of Nrf2 deletion on the lung inammatory response in female mice exposed to ZnO-NPs. Twenty-four female Nrf2 −/− mice and the same of female Nrf2 +/+ each into three and each to ZnO-NPs at either or 30 µg/mouse by pharyngeal aspiration. Bronchoalveolar lavage uid (BALF) and were 14 quantify protein of inammatory for inammation histopathologically. The mRNA levels of Nrf2-depedent antioxidant enzymes and proinammatory cytokine in lung tissue were measured. lower susceptibility of females to lung inammation, relative to males, irrespective of Nrf2 deletion, and that enhancement of ZnO-NPs-induced upregulation of HO-1 and TNFα and downregulation of GR by deletion of Nrf2 is specic to female mice. We conclude that Nrf2 provides protection in female mice against increase in BALF eosinophils, probably through down-regulation of proinammatory cytokines/chemokines and upregulation of oxidative stress-related genes. The study also suggests lower susceptibility to lung inammation in female mice relative to their male counterparts and the synergistic effects of sex and exposure to ZnO-NPs on mRNA expression of GR, HO-1 or TNFα. both in vitro and in vivo through different exposure routes. While some studies have described the benecial effects of ZnO-NPs, others have highlighted their toxic effects on different cells and organ systems. Exposure to ZnO-NPs can induce mild but sometimes severe cytotoxicity, inammation, genotoxicity, mutagenicity, neurotoxicity, pulmonary toxicity, cardiac toxicity, test followed by Steel multiple comparison test for neutrophil and eosinophil counts). Simple regression analysis for each genotype and multiple regression analysis in a model with interaction were conducted for the number of total cells, macrophages and lymphocytes, and total protein. Simple ordinal logistic regression analysis for each genotype and multiple ordinal logistic regression analysis in a model with interaction were conducted for neutrophil and eosinophil counts. Since the interaction was statistically insignicant for all of the examined parameters, multiple regression analysis or multiple ordinal logistic regression analysis was conducted in a model without interaction to estimate the separate effects of ZnO-NPs and Nrf2 deletion. 24±10 respectively, while those of male wild type mice were 7.5±3.0 and 1.3±0.3 respectively [18]. Relative expression levels of HO-1 and GR in 0µg ZnO-NP group of HO-1 and GR in 0µg ZnO-NP group of female Nrf2 null mice were 18±8 and 19±9 respectively, while those of male Nrf2 null mice were 5.8±2.5 and 2.1±0.5 respectively [18]. A previous study showed that male mice were more susceptible to acute and chronic pulmonary inammation following single and repeated exposure to nickel nanoparticles than female mice [28]. In contrast, Shvedova et al. reported more severe pulmonary toxicity in female mice exposed to cellulose nanocrystals compared to male mice [29]. Another study showed enhanced susceptibility of female mice to acute and chronic lung inammation induced by multi-walled carbon nanotubes (MWCNTs) [30]. Further studies are needed to explain the above differences in the sex-related susceptibility to toxicity.

hepatotoxicity, nephrotoxicity, intestinal toxicity, and reproductive toxicity [3,[7][8][9]. On the other hand, several other studies have focused on the mechanism(s) of ZnO-NPs-induced toxicities and different molecular mechanisms have been proposed. Among them, the generation of reactive oxygen species (ROS) and oxidative stress state, either directly by ZnO-NPs themselves or secondarily through toxic Zn + ions generated from their dissolution, is considered the main mechanism of ZnO-NPs-induced toxicities. The proposed oxidative stress state initiates several deleterious cellular cascades and signaling pathways involved in the resultant toxicity, including the nuclear factor erythroid 2-related factor 2/antioxidant responsive element (Nrf2/ARE) pathway, which is one of the key endogenous antioxidant stress-protective pathway [3,5,8,[10][11][12][13].
The well-documented sex differences in anatomy and physiology can modify the responses to exogenous agents, and susceptibility, pathophysiology, incidence, course, morbidity, and mortality of several diseases across the lifetime and this is highly apparent in the epidemiology of lung diseases [14,15]. Sex was found to be a key susceptibility candidate to engineered nanomaterials (ENMs)induced lung in ammation, and with the expected occupational exposure to the nanoparticles primarily through inhalation, sexrelated differences in the pulmonary responses to engineered nanomaterials have been the focus of attention recently [16].
The nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor involved in the regulation of various cell processes, including the regulation of the adaptive response and resistance to oxidant stress [17]. Our group reported recently that Nrf2 plays an important role in protection against ZnO-NPs-induced pulmonary cytotoxicity through the prevention of neutrophil migration in male mice [18]. However, it is still unknown whether Nrf2 has the same bene cial effects in female mice. The aim of the present study was to determine the effects of Nrf2 deletion on the ZnO-NPs-related pulmonary in ammatory response in female mice.
2.2 Animals.Nrf2 −/− female mice were generated as described by Itoh et al. [21] and backcrossed six times at the Central Institute for Experimental Animals (Kanagawa, Japan) and then further backcrossed seven times at the Division of Experimental Animals, Nagoya University Graduate School of Medicine (Nagoya, Japan). The genotypes of mice were con rmed by PCR ampli cation of genomic DNA isolated from the tail. PCR ampli cation was carried out using three different primers, 5#-TGGACGGGACTATTGAAGGCTG-3# (Nrf2-sense for both genotypes), 3#-GCCGCCTTTTCAGTAGATGGAGG-5# (Nrf2-antisense for wild-type), and 5#-GCGGATTGACCGTAATGGGATAGG-3# (Nrf2-antisense for LacZ). Another 24 pathogen-free age-matched C57BL/6JJcl female mice (Nrf2 +/+ ) weighing 22-27 g were purchased from CLEA Japan Inc. (Tokyo). All mice were housed and acclimatized in a clean environment for 1 week before the start of exposure experiments. Food and water were provided ad libitum. The animal room was light-and temperature-controlled with a 12-h light-dark cycle (lights on at 9 am and off at 9 pm), room temperature of 23-25°C and relative humidity at 57-60%. One day before the start of the experiment, mice of the two genotype groups were weighed and divided at random into three exposure groups (n=8 each); the control (0 µg ZnO-NPs), low-dose (10 µg ZnO-NPs) and high-dose (30 µg ZnO-NPs) groups. The latter two selected exposure doses are equivalent to 0.5 or 1.5 mg/kg body weight. The lower concentration of 0.5 mg/kg is comparable to deposition of 0.48 mg/kg in adult human lung from inhalation to ZnO for one week at the threshold limit value of 2 mg/m 3 (time-weighted average), as proposed by the American Conference of Governmental Industrial Hygienists (ACGIH), based on the values of 500 mL air/breath, 12 breath/min, 40 h/week [22].
The guide of the Japan Government Laws concerning the protection and control of animals, and the guide of animal experimentation of Nagoya University School of Medicine were followed throughout the experiments. The experiment protocol was approved by Nagoya University Animal Experiment Committee.
2.3 Pharyngeal aspiration of ZnO-NPs. Pharyngeal or oropharyngeal aspiration is proved to be an effective convenient alternative to inhalation exposure for the hazard assessment of nanomaterials [23]. For this purpose, the mouse was rst anesthetized by intraperitoneal injection of pentobarbital, then suspended with a rubber band anchored around the upper incisors and placed on its back on an inclined board. ZnO-NP suspensions were vortexed for 10 seconds rst, then the tongue was gently extended outside the oral cavity using blunt forceps, and 40 µl aliquot of the selected concentration was pipetted into the back of the tongue, which was pulled for 1 minute after pipetting then released. With the tongue protruded, the mouse was unable to swallow, and the liquid trickled down slowly into the lungs. Following release of the tongue, the mouse was gently lifted off the board, placed on its left side, and monitored for recovery from anesthesia.
2.4 Bronchoalveolar lavage (BAL), total and differential cell counts. Fourteen days after exposure, the mice were euthanized by intraperitoneal injection of a lethal dose of pentobarbital. The trachea and lungs of each mouse were exposed and bronchoalveolar lavage was conducted. For this purpose, an 18-gauge needle was inserted into the trachea and both lungs were lavaged by 1 ml of 10% PBS (gentle instillation and aspiration). The instillation and aspiration of PBS was repeated 5 times, making a total volume of 5 ml. The amount of recovered bronchoalveolar lavage uid (BALF) was measured and recorded. The average volume of the retrieved uid was >90% of the instilled; the amounts and recovery rates were not different among the three exposure groups. The collected BALF was kept on ice until centrifuged at 1500 rpm for 5 minutes, and the supernatant was aliquoted into three tubes and kept at -80°C until further analysis. The cell pellets were re-suspended in 1 ml of ACK lysis buffer (for red blood cells lysis) and left for 5 minutes at room temperature. Then 10 ml of 10% PBS were added and the whole volume was re-centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was re-suspended in 1 ml 10% PBS and kept on ice for use to determine the total and differential cell counts. Total cell count was determined using a ChemoMetec Nucleocounter (Allerød, Denmark), while differential cell count was performed under optical microscope on slides prepared by cytospin and stained with May-Grunwald-Giemsa (Merck, Darmstadt, Germany). The BALF cell types included macrophages, neutrophils, lymphocytes and eosinophils. The relative differential counts were presented as percentages of total cells counted in 10 elds of each cytospin smear. The absolute differential count was calculated as the product of the number of the total cell count and the proportion of the relative differential count.
2.5 Measurement of total protein in BALF. Total protein in BALF was measured using a Bio-Rad protein assay kit according to the instructions provided by the manufacturer (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin (BSA) as a standard.
2.6 Histopathological examination of the lung. After completion of BAL, the lungs were removed, washed in saline and the right lung was immediately frozen for further analysis. The left lung was xed in 4% formalin, dehydrated with graded alcohol concentrations, embedded in para n, cut into 3 µm-thick sections, placed on slides, stained with hematoxylin and eosin (H&E) and examined under optical microscope by a pathologist blinded to the exposure. These lung sections were used to determine the degree of lung in ammation. The degree of peribronchial and perivascular in ammation was evaluated on a subjective scale of 0-3, as described previously [24][25][26][27]. A score of 0 represented no detectable in ammation, while score of 1 represented occasional cu ng with in ammatory cells. For score 2, most bronchi or vessels were surrounded by a thin layer (1-5 cells thick) of in ammatory cells. For score 3, most bronchi or vessels were surrounded by a thick layer (>5 cells thick) of in ammatory cells. Total lung in ammation was de ned as the average of the peribronchial and perivascular in ammation scores. Four lung sections per mouse were scored and the in ammation score was expressed as the average value. Tissue slides were examined under an optical microscope (model DM750, Leica Microsystem, Wetzlar, Germany) and images were captured with Leica Application Suite V3 software. 2.7 Quanti cation of total glutathione and oxidized glutathione. The frozen lung tissue samples were homogenized with 5 volumes (w/v) of cold 50mM MES buffer (pH 6.01) containing 1mM EDTA. The protein in each sample was denatured with equal volume of 0.1% metaphosphoric acid (Sigma-Aldrich) and mixed on a vortex mixer. The mixture was allowed to stand at room temperature for 5 min and then centrifuged at 2000 x g for 3 min. The supernatant (95 µl) was kept at -20ºC until used for determination of total glutathione and oxidized glutathione (GSSG). First, 90 µl of supernatant was treated with 4.5 µl of 4M triethanolamine (Sigma-Aldrich) solution and vortexed well before assay. For the analysis of total reduced form of glutathione (GSH), 30 µl TEAM-treated sample was diluted 20-fold with MES buffer (pH 6.0) containing 2mM EDTA. An aliquot (50 µl) of the diluted solution was treated with 150 µl freshly prepared assay cocktail and assayed at 405 nm with a microplate reader (Gen5™ & Gen5 Secure, BioTek® Instruments, Inc.). For GSSG determination, 30 µl of TEAM-treated sample was diluted 10 times with MES buffer before derivatization with 2-vinylpyridine., Two µl of 1M 2-vinylpyridine was added to 200 µl of diluted solution of every sample or GSSG standard in tube, and then the tubes were mixed on a vortex mixer and incubated for 1 h at room temperature. Total GSH and GSSG concentrations were calculated from a standard curve using GSSG (Cayman; 703014) prepared according to the GSH assay kit (Cayman Chemical Company, Ann Arbor, MI; #703002), and normalized versus protein concentration. Total GSH and GSSG were expressed in micromoles of GSH (or GSSG) per milligram of protein.
2.8 Malondialdehyde assay. The malondialdehyde (MDA) assay (Life Science Specialties, LLC; NWK-MDA01) was performed according to the protocol supplied by the manufacturer. A 10% wt/vol homogenate was prepared from lung tissue in cold Assay Buffer (Phosphate buffer, pH 7.0 with EDTA). Absorbance was read at 532 nm using a PowerScan4 microplate reader (DS Pharma Medical Co., Japan) after reaction of the sample with thiobarbituric acid (TBA). Samples were analyzed in duplicate, and MDA level was expressed in micromoles of MDA per milligram of protein.
2.10 Statistical analysis. Data were expressed as mean ± standard deviation. Differences between the control and exposure groups were examined using Dunnett`s multiple comparison method following one-way ANOVA or Steel multiple comparison method following Kruskal Wallis nonparametric test in each genotype. To test a trend with level of exposure to ZnO-NPs, simple regression analysis or simple ordinal logistic regression analysis on the exposure level of ZnO nanoparticles was applied in each genotype separately. Multiple regression analysis or multiple ordinal logistic regression analysis using dummy variables for genotype in full model was applied to examine effect of interaction between genotype and exposure level. When the interaction between genotype and exposure level was not signi cant, multiple regression analysis on exposure level and genotype in a non-interaction model was applied to test the effects of exposure level and genotype.
Statistical analysis was performed using the JMP software version 16 (SAS Institute, Cary, NC) and probability (p) value <0.05 was considered statistically signi cant.

Changes in body and lung weight
There was no signi cant difference in body weight and lung weight between ZnO-NPs exposure groups and the control, both for female wild-type mice and female Nrf2-null mice. The percentage of lung weight to body weight (relative lung weight) was signi cantly different between the ZnO-NPs exposure groups and the control only in wild-type mice (p=0.043, ANOVA), but not in Nrf2-null mice. However, post-ANOVA Dunnett's multiple comparison test did not show signi cant difference between the exposure groups and the control. Simple regression analysis showed signi cant trend with ZnO-NPs exposure level in wild-type mice (Table 1). Multiple regression analysis showed no signi cant interaction between ZnO-NPs exposure level and Nrf2 deletion in body weight, lung weight and relative lung weight. However, multiple regression analysis without interaction showed a signi cant negative effect for Nrf2 on body and lung weights, as well as a positive effect for ZnO-NPs exposure on absolute lung weight and relative lung weight.

Changes in BALF cell count and total protein
Aspiration of ZnO-NPs induced signi cant changes in the absolute numbers of total and individual in ammatory cells in both the wild-type and Nrf2-null mice (ANOVA), with the exception of eosinophils in Nrf2-null mice, with signi cant changes at 10 and 30 mg of ZnO-NPs exposure compared to the non-exposed control group. Exposure to ZnO-NPs dose-dependently increased total cells, macrophages, lymphocytes, neutrophils and eosinophils in BALF in both the wild-type and Nrf2-null mice (simple regression analysis and simple ordinal logistic regression analysis, Table 2). Multiple regression analysis or multiple ordinal regression analysis did not show signi cant interaction between ZnO-NPs exposure level and Nrf2 deletion, and the analysis without interaction showed signi cant harmful effect for ZnO-NPs exposure level on total cells and all types of cells in BALF, and signi cant harmful effect for Nrf2 deletion only on eosinophils in BALF. Further simple regression analysis showed that exposure to ZnO-NPs dose-dependently decreased total protein only in wild-type mice, and multiple regression analysis without interaction showed signi cant harmful effect for ZnO-NPs exposure level on total protein. 3.3 Changes in total in ammation score, peribronchial in ammation score and perivascular in ammation score Exposure to ZnO-NPs increased signi cantly all of the examined scores at 30mg in both the wild-type mice and Nrf2-null mice (Kruskal Wallis nonparametric test followed by Steel multiple comparison test), and simple ordinal logistic regression analysis con rmed the signi cant trend with ZnO-NPs exposure level in both the wild-type and Nrf2-null mice (Table 3). Multiple ordinal logistic regression analysis with the full model did not show signi cant interactions between ZnO-NPs exposure level and Nrf2 deletion for all of the examined scores. Multiple ordinal logistic regression analysis without interaction showed signi cant effects of ZnO-NPs exposure level but no signi cant effect of Nrf2 deletion for all of the examined scores.

Changes in glutathione and malondialdehyde expression levels in lung tissue
The GSSG/GSH ratio was signi cantly affected by the ZnO-NPs exposure level in wild-type mice (p=0.049, ANOVA), but the differences between the two exposed groups and the control were statistically insigni cant (Dunnett's multiple comparison test).
Simple regression analysis showed that ZnO-NPs exposure level had a signi cant effect on glutathione disul de in wild-type mice (Table 4). No signi cant interactions between ZnO-NPs exposure level and Nrf2 deletion were noted in total glutathione, glutathione disul de, GSSG/GSH rate and MDA (multiple regression analysis). On the other hand, Nrf2 deletion modulated signi cantly the levels of total glutathione, whereas ZnO-NPs exposure signi cantly altered glutathione disul de expression level. Table 4 Total glutathione (GSH), oxidized glutathione (GSSG), GSSG/total GSH ratio and malondialdehyde (MDA) in the lung of female mice at 14 days after exposure to zinc oxide nanoparticles by pharyngeal aspiration. 3.5 Changes in expression levels of lung oxidative stress-related genes ( Table 5) There were no signi cant ZnO-NPs dose-related differences in the gene expression levels in Nrf2-null mice with the exception of GR (p<0.0001, ANOVA) and HO-1 (p=0.0036, ANOVA). Further analysis with the Dunnett's multiple comparison test showed that exposure to the two doses of ZnO-NPs was associated with signi cant decreases in GR and signi cant increases in HO-1 in Nrf2-null mice. Furthermore, simple regression analyses con rmed the signi cant trends of GR and HO-1 with exposure level (Table 5).
Multiple regression analysis showed signi cant interaction of ZnO-NPs exposure level with GR and HO-1, but not with other genes. Nrf2 deletion was associated with signi cant overexpression of GcLm and MT-2 and under-expression of CAT, GcLc and NQO1.
3.6 Changes in expression levels of lung proin ammatory cytokines and brosis-related proteins At 30 µg exposure level, ZnO-NPs signi cantly increased the expression levels of KC, MIP-2, IL-6, IL-1β, MCP-1 and MMP2 (ANOVA followed by Dunnett's test) and these changes were con rmed by simple regression analysis in wild-type mice (Table 6). Multiple regression analysis showed no signi cant interaction of ZnO-NPs exposure level with Nrf2 deletion for all the tested cytokines and MMP2. Non-interaction multiple regression analysis showed ZnO-NPs exposure signi cantly altered the expression levels of MMP2 whereas Nrf2 deletion signi cantly affected the levels of KC, MIP-2, IL-6, IL-1β, MCP-1 and TNFα. The present study showed that Nrf2 deletion additively enhanced the effects of exposure to ZnO-NPs on the number of eosinophils in BALF of female mice. This is accompanied by enhancement of ZnO-NPs-induced upregulation of HO-1 and TNFα and ZnO-NPsinduced down-regulation of GR by Nrf2 deletion, in addition to reduction of total glutathione, downregulation of CAT, GcLc and NQO1 mRNA levels and upregulation of KC, MIP-2, IL-6, IL-1β and MCP-1 mRNA levels by Nrf2 deletion. The results indicated that in female mice, Nrf2 inhibits in ltration of eosinophils in the lung, at least in part through upregulation of anti-oxidative stress genes and downregulation of proin ammatory cytokines or chemokines. reported more severe pulmonary toxicity in female mice exposed to cellulose nanocrystals compared to male mice [29]. Another study showed enhanced susceptibility of female mice to acute and chronic lung in ammation induced by multi-walled carbon nanotubes (MWCNTs) [30]. Further studies are needed to explain the above differences in the sex-related susceptibility to toxicity.
Nrf2 deletion enhanced ZnO-NPs-induced upregulation of HO-1 and TNFα and downregulation of GR in female mice, but not in male mice [18]. It is known that HO-1 and GR expression shows sexual dimorphisms and is regulated by estradiol [31][32][33][34][35]. Trauma and hemorrhage are reported to induce a 2-fold increase in hepatic HO-1 expression in proestrus female rats compared to male rats [34]. Treatment with 17β-estradiol upregulate the expression of HO-1 in the liver of mice [33]. The ndings of another in vivo study suggested that increased HO activity and expression in female rats compared to male rats can explain the sexual dimorphism of cardiovascular ischemia during reproductive age [31]. Finally, higher activity of GR from 4 to 24 weeks of age was described in the liver of male rats compared to female rats [32]. The mechanism for the sexual dimorphism in expression of HO-1 or GR remains elusive. In vitro studies have shown that 17β-estradiol can upregulate Nrf2 in nuclear extracts and increase the expression of HO-1, SOD, GST and GCL in hypoxia/reoxygenation model of primary myocardial cells [35] and 17β-estradiol increases Nrf2 activity through activation of the PI3K pathway in MCF-1 breast cancer cells [36]. However, these studies on Nrf2 activation by estradiol cannot explain our results that ZnO-NPs-induced upregulation of HO-1 is enhanced in Nrf2 null mice.
Although HO-1 plays an adverse role in carcinogenesis and neurodegenerative disease, it is known to play a protective role against oxidative injury and other stress conditions [37]. We believe that ZnO-NPs-induced upregulation of HO-1 in female mice only is involved with the observed low susceptibility of female mice to peribronchial in ammation compared to male mice, though further studies are needed to test this hypothesis.
Nrf2 deletion signi cantly increased eosinophil in BALF, while did not signi cantly increase the lung in ammation score in female mice. This is in agreement with the previous study on male Nrf2 null mice and wild type mice, suggesting limitation of semiquanti cation in in ammation scoring [18].
In conclusion, our study demonstrated the protective role of Nrf2 against ZnO-NPs-induced in ltration of eosinophils in lung of female mice, which might be explained by negative regulation of proin ammatory cytokines and chemokines and positive regulation of oxidative stress-related genes by Nrf2. The results also suggested lower susceptibility to lung in ammation in female mice compared with male mice and the synergistic effect of sex and ZnO-NPs exposure on GR, HO-1 or TNFα mRNA expression, although further studies are needed to de ne the relationship between sex-related susceptibility and gene expression.