Pubertal exposure to Bisphenol A increases anxiety-like behavior and decreases acetylcholinesterase activity of hippocampus in adult male mice

The negative effects of Bisphenol A (BPA) on neurodevelopment and behaviors have been well established. Acetylcholinesterase (AChE) is a regulatory enzyme which is involved in anxiety-like behavior. This study investigated behavioral phenotypes and AChE activity in male mice following BPA exposure during puberty. On postnatal day (PND) 35, male mice were exposed to 50 mg BPA/kg diet per day for a period of 35 days. On PND71, a behavioral assay was performed using the elevated plus maze (EPM) and the light/dark test. In addition, AChE activity was measured in the prefrontal cortex, hypothalamus, cerebellum and hippocampus. Results from our behavioral phenotyping indicated that anxiety-like behavior was increased in mice exposed to BPA. AChE activity was significantly decreased in the hippocampus of mice with BPA compared to control mice, whereas no difference was found in the prefrontal cortex, hypothalamus and cerebellum. Our findings showed that pubertal BPA exposure increased anxiety-like behavior, which may be associated with decreased AChE activity of the hippocampus in adult male mice. Further studies are necessary to investigate the cholinergic signaling of the hippocampus in PBE induced anxiety-like behaviors.     

Prenatal ethanol exposure increases susceptibility to depression- and anxiety-like behavior in adult female offspring and its underlying mechanism

Epidemiological investigations have found that maternal alcohol intake increases the risk of mental illness in offspring. Our study investigated changes of depression- and anxiety-like behaviors in adult offspring caused by prenatal ethanol exposure (PEE) and explored the potential mechanism. After Wistar rats were intragastrically administered ethanol at a dose of 4 g/kg·d on the 9–20 t h days of pregnancy, the offspring were given 21 days of chronic unpredictable mild stress (CUMS) starting from the 9th week after birth. Before CUMS, the behavioral results showed that the PEE offspring appeared excited and anxious. After CUMS, the PEE offspring rats were more sensitive to the same intensity of stimulation, and then the behavioral disorders aggravated. In adult offspring from the PEE group, the intercellular space was enlarged in the hippocampus, and there was a loss of pyramidal cells. The expression of brain-derived neurotrophic factor (BDNF) decreased; the mRNA expression of the glucocorticoid receptor and synaptic plasticity-related genes decreased; the apoptosis-related genes expressed disrupted. In order to determine whether hippocampal injury and dysfunction resulted from ethanol directly or indirectly, we performed in vitro study. The outcome was accompanied by disrupted gene expression related to neurogenesis and synaptic plasticity. PEE increases the susceptibility of adult female offspring to depression- and anxiety-like behaviors, and its mechanism may be related to the toxic effects of ethanol, both directly and indirectly. The latter inhibits the hippocampal BDNF pathway, leading to the disruption of hippocampal neurogenesis, apoptosis and decreased synaptic plasticity.     

Sexual behavior decreases pain sensitivity and stimulated endogenous opioids in male rats

In male rats copulation has antinociceptive effects as measured either by shock-induced vocalizations or hindlimb withdrawal to pinch. Prolonged mating reduces the content of endogenous opioids in midbrain but not in hypothalamus or caudate nucleus. Blockage of opiate receptors with the narcotic antagonist naloxone (4 mg/kg) significantly extends the postejaculatory interval. The results indicate that mating is a biological stimulus for the release of endogenous opoids, possibly to (a) prevent intense sexual stimulation from becoming aversive, and (b) increase its reward value.     

Pharmacological manipulation of brain galaninergic system and sexual behavior in male mice

RATIONALE: Available data suggest a complex role for the brain galaninergic system in male sexual behavior; however, the results so far obtained in animals with either galanin or galanin antagonists are conflicting. OBJECTIVE: To define the better influence of galanin on male sexual behavior by studying, in mice, (i) the effect of galanin and of the chimeric galanin peptide M40 on the copulatory performance, and (ii) galanin mRNA levels in hypothalamic arcuate and dorso-medial nuclei. METHODS: For the behavioral testing, only sexually sluggish male mice were used. Galanin mRNA levels were studied in both sexually potent and impotent mice by means of in situ hybridization. Standard behavioral parameters for sexual behavior were recorded or calculated. Synthetic galanin (0.05, 0.1 or 1 microg/mouse) and M40 (5 or 20 microg/mouse) were intracerebroventricularly (ICV) injected, 15 min before the copulatory test. Galanin mRNA levels were evaluated. RESULTS: In sexually sluggish male mice, both galanin (0.1 and 1 microg/mouse ICV) and M40 (20 microg/mouse ICV), significantly increased intromission frequency and ejaculation latency; M40 also improved copulatory efficacy. On the other hand, in the hypothalamic arcuate and dorso-medial nuclei, the levels of galanin mRNA were not significantly different in sexually potent and impotent male mice. CONCLUSIONS: These results show that in sexually sluggish male mice the ICV injection of either galanin or the chimeric analogue M40 greatly prolongs the duration of the copulation; without a reduction of the sexual drive or of the copulatory performance. On the other hand, the hybridization experiments seem to rule out an important physiological role of the brain galaninergic system in the regulation of male sexual behavior, at least in mice.     

Intraventricular 6-hydroxydopamine lowers isolation-induced fighting behavior in male mice

Male mice with high isolation-induced fighting tendencies were administered 200 μg 6-OHDA or vehicle intraventricularly and tested for fighting tendency for up to 10 weeks until sacrifice, and assayed for brain NE levels. A strong correlation was found between NE depletion and reduced fighting tendency after 6-OHDA treatment. The depressed fighting by mice with less than 200 ng NE/g persisted throughout a series of test fights, indicating no recovery in fighting behavior throughout the survival time.     

Prenatal exposure to titanium dioxide nanoparticles increases dopamine levels in the prefrontal cortex and neostriatum of mice

Titanium dioxide (TiO(2)) nanoparticles are widely used in cosmetics, sunscreen and as a photocatalyst. However, little is known about the biological effect of TiO(2) nanoparticles in humans and other animals. Here, we investigated whether prenatal exposure to TiO(2) nanoparticles impacted the central nervous system in mice. We measured the levels of dopamine (DA) and its metabolites in several regions of the brain in mice using high performance liquid chromatography (HPLC). HPLC analysis showed that DA and its metabolites were increased in the prefrontal cortex and the neostriatum following prenatal exposure to TiO(2) nanoparticles. The present study highlights the possibility that maternal exposure to TiO(2) nanoparticles might influence the development of the central dopaminergic system in offspring.     

Developmental exposure to methylmercury alters learning and induces depression-like behavior in male mice.

To investigate the long-term effects of developmental exposure to methylmercury (MeHg), pregnant mice were exposed to at 0.5 mg MeHg/kg/day via drinking water from gestational day 7 until day 7 after delivery. The behavior of offspring was monitored at 5-15 and 26-36 weeks of age using an automated system (IntelliCage) designed for continuous long-term recording of the home cage behavior in social groups and complex analysis of basic activities and learning. In addition, spontaneous locomotion, motor coordination on the accelerating rotarod, spatial learning in Morris water maze, and depression-like behavior in forced swimming test were also studied. The analysis of behavior performed in the IntelliCage without social deprivation occurred to be more sensitive in detecting alterations in activity and learning paradigms. We found normal motor function but decreased exploratory activity in MeHg-exposed male mice, especially at young age. Learning disturbances observed in MeHg-exposed male animals suggest reference memory impairment. Interestingly, the forced swimming test revealed a predisposition to depressive-like behavior in the MeHg-exposed male offspring. This study provides novel evidence that the developmental exposure to MeHg can affect not only cognitive functions but also motivation-driven behaviors.     

Dopamine D5 receptor modulates male and female sexual behavior in mice

Rationale Dopamine exerts its actions through at least five receptor (DAR) isoforms. In female rats, D5 DAR may be involved in expression of sexual behavior. We used a D5 knockout (D5KO) mouse to assess the role of D5 DAR in mouse sexual behavior. Both sexes of D5KO mice are fertile and exhibit only minor disruptions in exploratory locomotion, startle, and prepulse inhibition responses.Objective This study was conducted to characterize the sexual behavior of male and female D5KO mice relative to their WT littermates.Methods Female WT and D5KO littermates were ovariectomized and given a series of sexual behavior tests after treatment with estradiol benzoate (EB) and progesterone (P). Once sexual performance was optimal the dopamine agonist, apomorphine (APO), was substituted for P. Male mice were observed in pair- and trio- sexual behavior tests. To assess whether the D5 DAR is involved in rewarding aspects of sexual behavior, WT and D5KO male mice were tested for conditioned place preference.Results Both WT and D5KO females can display receptivity after treatment with EB and P, but APO was only able to facilitate receptivity in EB-primed WT, not in D5KO, mice. Male D5KO mice display normal masculine sexual behavior in mating tests. In conditioned preference tests, WT males formed a conditioned preference for context associated with either intromissions alone or ejaculation as the unconditioned stimulus. In contrast, D5KO males only showed a place preference when ejaculation was paired with the context.Conclusions In females, the D5 DAR is essential for the actions of dopamine on receptivity. In males, D5 DAR influences rewarding aspects of intromissions. Taken together, the work suggests that the D5 receptor mediates dopamines action on sexual behavior in both sexes, perhaps via a reward pathway.A.E. Kudwa and E. Dominguez-Salazar are co-authors.     

Acute inhalation of combustion smoke triggers neuroinflammation and persistent anxiety-like behavior in the mouse

Context: Acute inhalation of combustion smoke triggers neurologic sequelae in survivors. Due to the challenges posed by heterogeneity of smoke exposures in humans, mechanistic links between acute smoke inhalation and neuropathologic sequelae have not been systematically investigated.

Methods: Here, using mouse model of acute inhalation of combustion smoke, we studied longitudinal neurobehavioral manifestations of smoke exposures and molecular/cellular changes in the mouse brain.

Results: Immunohistochemical analyses at eight months post-smoke, revealed hippocampal astrogliosis and microgliosis accompanied by reduced myelination. Elevated expression of proinflammatory cytokines was also detected. Longitudinal testing in different neurobehavioral paradigms in the course of post-smoke recovery, revealed lasting anxiety-like behavior. The examined paradigms included the open field exploration/anxiety testing at two, four and six months post-smoke, which detected decreases in total distance traveled and time spent in the central arena in the smoke-exposed compared to sham-control mice, suggestive of dampened exploratory activity and increased anxiety-like behavior. In agreement with reduced open field activity, cued fear conditioning test revealed increased freezing in response to conditioned auditory stimulus in mice after acute smoke inhalation. Similarly, elevated plus maze testing demonstrated lesser presence in open arms of the maze, consistent with anxiety-like behavior, for the post-smoke exposure mice.

Conclusions: Taken together, our data demonstrate for the first time persistent neurobehavioral manifestations of acute inhalation of combustion smoke and provide new insights into long-term progression of events initiated by disrupted brain oxygenation that might contribute to lasting adverse sequelae in survivors of smoke inhalation injuries.     

Evaluation of cytogenotoxicity and oxidative stress parameters in male Swiss mice co-exposed to titanium dioxide and zinc oxide nanoparticles

A number of studies have investigated the adverse toxic effects of titanium dioxide (TiO₂) nanoparticles (NPs) or zinc oxide (ZnO) NPs. Information on the potential genotoxic effects of the interactions of TiO₂ NPs and ZnO NPs in vivo is lacking. Therefore, this study was designed to investigate the cytogenotoxicity of TiO₂ NPs or ZnO NPs alone or their mixtures using the bone marrow micronucleus assay, and mechanism of damage through the evaluation of oxidative stress parameters in the liver and kidney tissues of Swiss mice. Intraperitoneal administration of doses between 9.38 and 150.00 mg/kg of TiO₂ NPs or ZnO NPs or TiO₂ NPs + ZnO NPs was performed for 5 and 10 days, respectively. TiO₂ NPs alone induced a significant (P < 0.05) increase in micronucleated (Mn) polychromatic erythrocytes (PCEs) at the applied doses compared with the negative controls, with a significant difference between 5 and 10 days for TiO₂ NPs alone and TiO₂ NPs + ZnO NPs. Concurrently, TiO₂ NPs alone for 5 days and TiO₂ NPs and TiO₂ NPs + ZnO NPs for 10 days significantly (P < 0.05) decreased the percentage PCE: normochromatic erythrocyte (NCE) indicating cytotoxicity; with a significant difference between the two periods. Significant (P < 0.001) changes in the activities of superoxide dismutase (SOD) and catalase (CAT), and levels of reduced glutathione (GSH) and malondialdehyde (MDA) were observed in the liver and kidney of mice exposed to TiO₂ NPs or ZnO NPs alone or their mixtures. These results suggest that TiO₂ NPs alone was genotoxic; TiO₂ NPs and TiO₂ NPs + ZnO NPs were noticeably cytotoxic while ZnO NPs was not cytogenotoxic. The individual NPs or their mixtures induced oxidative stress.     

Differential effects of modafinil, methamphetamine, and MDMA on agonistic behavior in male mice

The aim of the present study was to compare the behavioral effects of modafinil, an atypical psychostimulatory acting and cognitive-function improving drug, with the effects of the psychostimulants methamphetamine (MET) and MDMA (3,4-methylenedioxymethamphetamine, or “ecstasy”) in a model of mouse agonistic behavior. This model enables the observation of ethologically well-defined sociable, timid, aggressive, and locomotor behavioral acts and postures. Singly-housed male mice (isolates) were separated into 4 groups. The observations were performed in 4 sessions, 1 week apart. In each interaction, singly-housed mice were paired with non-aggressive group-housed partners for 4 min in a neutral environment. The isolates received, in a Latin square design, either a) a vehicle or modafinil at doses 2.0, 10.0, or 50.0 mg/kg; or b) a vehicle or MET at doses 1.0, 5.0, or 10.0 mg/kg; or c) a vehicle or MDMA at doses 2.5, 10.0, or 30.0 mg/kg. The isolates were categorized as timid or aggressive according to their behavior in the control interaction (vehicle pre-treatment). Elements of locomotor, sociable, aggressive, and timid behavior were evaluated (one-way ANOVA).In the aggressive mice, no change in the sum of aggressive behavior was measured following modafinil administration, while both methamphetamine and MDMA produced dose-dependent inhibition of aggression (p < 0.01). The substantial difference in the tested drug effects on agonistic behavior was an increased occurrence of sociable acts (p < 0.01) accompanied by a simultaneous increase of timid acts (p < 0.01) recorded after MDMA, but not after modafinil or methamphetamine administration. In the timid mice, at least some doses of modafinil decreased timidity (p < 0.01) and increased aggression (p < 0.01) with no effect on sociability. Administration of MDMA increased timid activities (p < 0.01). Both MDMA and MET decreased sociability (p < 0.01).     

Opioids and sexual behavior in the male rat

Naloxone in the doses of 4 or 16 mg/kg failed to effect copulatory behavior of testosterone-treated castrated male rats. Morphine 10 mg/kg, administered 60 min before behavioral observation, reduced the proportion of animals displaying sexual behavior. Doses of 2.5 or 5 mg/kg reduced the latency to the second ejaculation, whereas the few animals still copulating after morphine 10 mg/kg showed a reduced latency to the first ejaculation. The same doses of morphine administered 5 min before behavioral observation produced a dose-dependent reduction of mount, intromission and ejaculation percentages. However, those animals that did copulate showed a normal copulatory behavior. D-Ala²-Met⁵ enkephalinamide (DALA) infused into the left cerebral ventricle in a dose of 5 μg 5 or 60 min before tests had no effect. When the peptide was infused 30 sec after the first intromission, the number of intromissions as well as the latency to ejaculation were reduced. Opioids may facilitate ejaculatory mechanisms, perhaps as a consequence of their rewarding properties. Moreover, in animals treated with DALA after the first intromission, the number of intromissions and the latency to ejaculation were similar for the first and second copulatory series, while these parameters were much reduced upon the second ejaculation for control animals. It is possible that liberation of endogenous opioids is the cause of ejaculation-induced facilitation of subsequent sexual behavior.     

Long term reduction of male agonistic behavior in mice following early exposure to ethanol

A system was developed to study the ability of early (pre- and neonatal) ethanol input to induce long lasting neural and behavioral changes. Ethanol was administered to C57B1/10Bg and DBA/1 Bg offspring through their parents who received 10% ethanol as their only liquid supply either before and during pregnancy, or from delivery until 14 days post partum, or during both periods. Thus the offspring received ethanol transplacentally and/or through the mother's milk. The present paper is concerned with the male agonistic behavior at age 50 days of the treated offspring as compared with their pair fed controls. Early ethanol input resulted in a 23% increase in latency to attack in C57 mice and 58% in DBA, as well as a 49% (C57) and 38% (DBA) decrease in time spent fighting. The sensitive period to ethanol effect was apparently postnatal. Prenatal administration had no effect on agonistic behavior. DBA offspring were more aggressive than C57 and the scores of C57 offspring were more variable, thus indicating a lower phenotypic buffering in this strain.     

Administration of a hemoglobin solution decreases hypoxia and increases radiation response in a rat mammary carcinoma

Administration of an ultrapurified bovine hemoglobin solution (8 ml/kg, iv) to female Fisher 344 rats bearing the 13672 mammary carcinoma implanted subcutaneously in the hind leg reduced the hypoxic fraction of the tumor from 49 to 36% with normal air breathing and to 28% with carbogen (95% oxygen/5% carbon dioxide) breathing. When administration of the hemoglobin solution was followed by single dose (10, 20, or 30 Gray) local radiation therapy, the dose modifying factor was 1.5 with air breathing and 2.7 with carbogen breathing. For fractionated radiation therapy, 2, 3, or 4 Gray was delivered locally daily for 5 days. When the hemoglobin solution was administered prior to each radiation fraction and air breathing maintained, there was a 1.8-fold increase in tumor growth delay. When daily hemoglobin solution administration was accompanied by carbogen breathing, there was a 2.6-fold increase in tumor growth delay. Less frquent administration of the hemoglobin solution (alternate days, twice weekly or once weekly) resulted in 1.5- to 1.7-fold increases in tumor growth delay. The greatest increases in tumor oxygenation occurred shortly after administration of the hemoglobin solution; in fact at 12 h post administration of the hemoglobin solution, the tumors appeared to be more hypoxic than prior to administration of the hemoglobin solution. By 24 h post administration of the hemoglobin solution, the oxygen tension profile of the tumors were returning to pretreatment levels. Further investigation of this ultrapurified hemoglobin solution in cancer is warranted. © 1995 Wiley-Liss, Inc.     

Effects of delta 9-THC and castration on behavior and plasma hormone levels in male mice

Gonadectomy resulted in a rapid increase in plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels, but had no consistent effects on plasma prolactin (PRL) and growth hormone (GH) levels. In castrated males, oral administration of THC (50 mg/kg) significantly increased plasma LH levels within hours following surgery and again from 3 to several weeks post-castration, while THC treatment decreased LH levels between 1 day and 2 weeks postcastration. Administration of THC to 12-hour sham castrates significantly increased plasma LH levels. Plasma FSH, PRL and GH levels were either reduced or unchanged by THC. Administration of THC significantly reduced levels of gonadotropins, PRL and GH in intact males. In additional studies, we examined the influence of THC on the negative feedback response of the anterior pituitary to gonadal steroids. In testosterone propionate (TP)-treated castrated males, concomitant administration of THC increased plasma testosterone (T) and LH at 20 min, while plasma FSH levels were elevated after 60 min. In contrast, in intact TP-treated mice, concomitant THC exposure reduced plasma T levels except at 60 min, when plasma LH levels were significantly increased. Testosterone replacement failed to restore copulatory behavior in castrated mice given a single dose (50 mg/kg) of THC. In fact, acute THC administration to these TP-treated castrates resulted in marked sedation, which was not observed in intact mice given the same dose of THC in an earlier study. The present findings indicate that the effects of acute THC treatment on pituitary gonadotropin release is dependent upon the time after castration. Furthermore, THC administration can suppress copulatory behavior even in animals whose peripheral T levels have been maintained. Effects of THC on plasma androgen levels in animals injected with TP suggest that THC can alter the metabolism or target tissue response to gonadal steroids.     

Food‐grade titanium dioxide (E171) by solid or liquid matrix administration induces inflammation,germ cells sloughing in seminiferous tubules and blood‐testis barrier disruption in mice

Food‐grade titanium dioxide labeled as E171 has been approved for human consumption by the Food and Drug Administration (USA) and by the European Union for five decades. However, titanium dioxide has been classified as a possible carcinogen for humans by the International Agency of Research in Cancer raising concerns of its oral intake and the translocation to bloodstream, which could disturb barriers such as the blood‐testis barrier. There is evidence that titanium dioxide by intragastric/intraperitoneal/intravenous administration induced alterations on testosterone levels, testicular function and architecture, but studies of the E171 effects on the testicle structure and blood‐testis barrier are limited. E171 is contained not only in foods in liquid matrix but also in solid ones, which can exert different biological effects. We aimed to compare the effects of E171 consumption in a solid matrix (0.1%, 0.5% and 1% in pellets) and liquid suspension (5 mg/kg body weight) on testis structure, inflammation infiltrate and blood‐testis barrier disruption of male BALB/c mice. Results showed that none of the administration routes had influence on body weight but an increase in germ cell sloughing and the infiltrate of inflammatory cells in seminiferous tubules, together with disruption of the blood‐testis barrier were similar in testis of both groups even if the dose received in mice in liquid matrix was 136 or 260 times lower than the dose reached by oral intake in solid E171 pellets in 0.5% E171 and 1% E171, respectively. This study highlights the attention on matrix food containing E171 and possible adverse effects on testis when E171 is consumed in a liquid matrix.     

Maintenance of sexual behavior in castrate male SW mice using the anti-androgen, cyproterone acetate.

The present study was designed to test the hypothesis that cyproterone acetate (C) might selectively block the actions of dihydrotestosterone (D) and via this action, function as an anti-androgen in male sexual behavior. Sexually experienced male SW mice, a strain previously shown to respond to D following castration, were divided randomly into six groups. Beginning on the day after castration, animals received SC injections for 21 days of either testosterone (T), (D), (C), (T+C), (D+C) or vehicle (V). C was found to significantly reduce seminal vesicle and body weights in all androgen treated groups. There was no evidence to support the contention that C selectively blocks the action of D. To the contrary, in sex tests C maintained palpations, thrust mounts, with intromissions and mounts with ejaculations. Indeed, only animals receiving C alone or in combination with T and D exhibited ejaculations throughout the testing. These results suggest that in the SW mouse, C can work like an androgen in the maintenance of male sexual behavior.     

Effect of daidzein on anxiety, social behavior and spatial learning in male Balb/cJ mice

Daidzein is an abundant isoflavone present in soy. It is unique as it can be further metabolized into equol, a compound with greater estrogenic activity than other isoflavones. The potential benefit of daidzein in the prevention of various cancers and cardiovascular diseases has drawn attention to this molecule and isoflavone consumption is increasing around the world. However, it remains unclear whether daidzein affects locomotor activity, anxiety, social behavior or spatial memory. Here we report the results from a range of tests designed to assess anxiety, social behavior and spatial learning and memory in male Balb/cJ mice following consumption of daidzein for 30 days. We found that daidzein treatment significantly increased locomotor activity in the open field, elevated plus-maze and Morris water-maze tests; resulted in increased amicable behavior, decreased aggression and decreased sexual behavior during social interaction tests; and resulted in an anxiolytic effect amongst treated males. We found no effect of daidzein consumption on the acquisition and retrieval of spatial memory. Our results suggest that long-term consumption of daidzein may produce significant effects on locomotor activity, mood and social behavior without significant effects on learning and memory.     

Inhalation of titanium dioxide induces endoplasmic reticulum stress-mediated autophagy and inflammation in mice

Titanium dioxide (TiO₂) nanoparticles are widely used in cosmetics, sunscreen, electronics, drug delivery systems, and diverse bio-application fields. In the workplace, the primary exposure route for TiO₂ nanoparticles is inhalation through the respiratory system. Because TiO₂ nanoparticles have different physiological properties, in terms of size and bioactivity, their toxic effects in the respiratory system must be determined. In this study, to determine the toxic effect of inhaled TiO₂ nanoparticles in the lung and the underlying mechanism, we used a whole-body chamber inhalation system to expose A/J mice to TiO₂ nanoparticles for 28 days. During the experiments, the inhaled TiO₂ nanoparticles were characterized using a cascade impactor and transmission electron microscopy. After inhalation of the TiO₂ nanoparticles, hyperplasia and inflammation were observed in a TiO₂ dose-dependent manner. To determine the biological mechanism of the toxic response in the lung, we examined endoplasmic reticulum (ER) and mitochondria in lung. The ER and mitochondria were disrupted and dysfunctional in the TiO₂-exposed lung leading to abnormal autophagy. In summary, we assessed the potential risk of TiO₂ nanoparticles in the respiratory system, which contributed to our understanding of the mechanism underlining TiO₂ nanoparticle toxicity in the lung.