Introduction

Titanium dioxide (TiO2) is a fine, white, crystalline, odorless powder that is considered to exhibit relatively low toxicity (Zhang et al. 2000). According to the National Nanotechnology Initiative of America, nanosized TiO2 particles are among those most widely manufactured on a global scale (Liang et al. 2009). Sources of TiO2 nanoparticles (NPs) relevant for oral exposure comprise mainly cosmetics (sunscreen, lipsticks, skin creams, toothpaste) and food (packaging, storage life sensors, food additives, juice clarifiers) (Weir et al. 2012).

The health and safety aspects of NPs are still in the formative phase, and greater effort is needed to understand how NPs interact with the human body (Yokel and Macphail 2011). In this regard, nanotoxicology and nano-risk have been attracting increased attention from toxicologists and regulatory scientists (Oberdörster et al. 2005), particularly in relation to the unique properties of NPs that may render them potentially more dangerous than their fine-sized counterparts and may cause unexpected adverse health effects following human exposure (Li and Nel 2011). Such manufacturing and consumer use can produce multiple and different sources of release of these materials into the environment, ecosystem, water and food supplies, and other routes of non-voluntary exposure into the human body (Jones and Grainger 2009). It is important, however, to distinguish between crystal forms of TiO2 (i.e., rutile versus anatase) when considering their toxicity. One study, in particular, demonstrated that anatase TiO2 NPs resulted in lactate dehydrogenase leakage and widespread necrosis, whereas rutile NPs resulted in higher levels of reactive oxygen species and consequently apoptosis (Braydich-Stolle et al. 2009). This study also demonstrated that although the literature supports a higher level of toxicity for anatase NPs (Wang et al. 2007; Sugibayashi et al. 2008), there is some evidence that rutile NPs, often assumed to induce less of an effect, can produce signs of toxicity.

Fetuses are known to be more sensitive to environmental toxins than adults (Koren et al. 1998), and it has been suggested that many chemical toxins in air, water, and food can induce pregnancy complications (Wigle et al. 2008). For instance, some studies have shown transplacental transport of nanomaterials in pregnant animals and nanomaterial-induced neurotoxicity in their offspring (Tian et al. 2009; Saunders 2009; Chu et al. 2010; Hougaard et al. 2010).

Considering the relevance of the respiratory system as a route for TiO2 NPs exposure in humans, a number of previous studies (Chen et al. 2006; Fujita et al. 2009; Li et al. 2013) were conducted to study the effects of inhalation of these particles on the respiratory system. However, to our knowledge, this is the first study that investigates the histomorphological alterations in maternal and neonatal pulmonary distal airspaces of Wistar rats after oral administration of TiO2 NPs by mothers.

Materials and methods

Experimental animals

Fifteen adult male and 30 adult female albino Wistar Han rats (150–250 g) were obtained from the Animal Breeding Unit of Assiut University (Egypt) and kept under constant conditions, a 12:12 light/dark cycle, and a room temperature of 28 °C. They were allowed access to a standard rat chow diet and water ad libitum, and food intake and body weight were recorded daily throughout the experimental period. Prior to dosing, animals were acclimated to the environment for 7 days. National Institutes of Health (NIH) guidelines for the care and use of laboratory animals (NIH Publication 85–23 Rev. 1985) were observed.

Mating procedure

Males were individually housed. Two females were placed into a cage with one male overnight (12 h). The following morning, mating was confirmed if a vaginal plug was observed, which served as day 0 of the pregnancy (GD0).

Pregnant rats were divided randomly into two main groups: age-matched controls (n = 15) and TiO2 NP-treated rats (n = 15). The control rats received a balanced standard diet together with the vehicle, phosphate-buffered saline (PBS).

Nanoparticles

Titanium dioxide nanopowder was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA; product 718467, CAS number 13463-67-7). According to the product sheet, the nanopowder was approximately 21 nm (TEM), specific surface area: 35–65 m2/g (BET), density: 4.26 g/ml at 25 °C, molecular weight: 79.87 and purity: 99.5 %. NanoTiO2 (5 g/kg body weight) was suspended into PBS, and then the suspension was ultrasonicated before it was used to treat animals to avoid aggregation and provide an optimum size distribution for dispersed particle agglomerates.

Treatment of animals

The methodology adopted was that of Zhang et al. (2010). Before treatment, animals were fasted overnight. TiO2 nanoparticles, in a stable suspending state, were diluted to 20 mg/ml. Subsequently, the TiO2 suspension was administered to the treated group via oral gavage in doses of 0.1 ml/10 g body weight once daily for 7 consecutive days (GD6-12). The control rats were treated with PBS daily for the same period. Food and water were provided 2 h later.

Prenatal evaluation

Maternal behavioral changes, body weight, and quantity of food and water consumption were evaluated daily from GD0 to GD21. The presence of vaginal bloodstains demonstrated the process of abortion or fetal expulsion.

Tissue sampling and processing

On GD21, after previous anesthesia with carbon monoxide, cesarean sections were performed on all pregnant females. Subsequently, the females were sacrificed, and the lungs were separated and weighed. The rat pups were extracted, counted, and examined for external variation. Finally, the rat pups were carefully dissected, and all lungs were harvested. Pressure control and lung fixation was performed as adopted by Vlahovic et al. (1999). Simply, 2 % glutaraldehyde in 0.085 sodium cacodylate buffer (pH 7.4) was instilled through syringes, which were inserted into the airways of collapsed lobes or lung segments. The fixative was instilled quickly to obtain rapid filling of the lung tissue, but overdistension was avoided through careful observation. The alveolar tissue became rigid within seconds and maintained full inflation even though there was a cut surface on the surgically obtained lung lobes.

Pulmonary ultrastructure analysis

After application of the fixative, the tissue remained in the fixative for 24 h, and the specimens for transmission electron microscope (TEM) were postfixed in 0.085 sodium cacodylate buffered 2 % osmium tetroxide in the same buffer at 4 °C, dehydrated using a series of ethanol concentrations, and embedded in epoxy resin.

Semi-thin sections (0.5–1 μm) were cut using a knife with a liquid-filled boat, removed from the boat with a clean wire loop, and transferred to a drop of water on a clean glass slide. The slides were then placed on a low temperature hot plate. The sections were then stained with toluidine blue and evaluated by a histopathologist unaware of the treatments using light microscopy (U-III Multi-point Sensor System; Nikon, Tokyo, Japan).

Ultrathin sections (50–70 nm thick) were stained with uranyl acetate and lead citrate and examined and photographed using a JEOL JEM 1200 EXII Electron Microscope (Jeol Ltd.) Research Laboratory, Assiut University, Egypt (Glauert and Lewis 1998).

Energy-dispersive X-ray (EDX) analysis

Analysis of the TiO2 in the tissue was conducted using an EDX unit attached to a scanning electron microscope (SEM) using glutaraldehyde-fixed carbon dried tissue samples. EDX uses the X-ray spectrum emitted by a solid sample bombarded with a focused beam of electrons to obtain a localized chemical analysis (Agarwal 1991; Scott and Love 1994).

Morphometric lung analysis

Measurement of the interalveolar wall distance was assessed in the adult and neonatal lungs by measuring the mean linear intercept (MLI) (described later in detail). Additionally, the airspace wall thickness, as an indication of the thickness (width) of the septa, was measured directly used an Image Analyzer (Leica Q Win standard, digital camera CH-9435 DFC 290, Germany). The measuring tool was applied to the middle as well as to the marginal parts of the septa to uniformly compute the mean width of whole septa. Measurements were conducted for each lung on five fields (at 200× magnification; frame area = 786,432.0 μ2) and performed by an investigator who was unaware of the identity of the samples.

Method of intercept or chord length estimation

Mean linear intercept is the mean length of straight-line segments (chords) on random test lines crossing the airspace between two sequential intersections of the alveolar surface with the test line. The chord lines often cross from one alveolus through an alveolar duct to an opposite alveolus, indicating that the MLI characterizes the entire acinar airspace complex (alveoli + ducts) and not just alveoli (Fig. 1) (Knudsen et al. 2010).

Fig. 1
figure 1

A photomicrograph of the control neonatal lung shows the method of direct estimation of MLI length based on intercept distribution. Random linear intercepts were measured on microscopic images: test line segments are localized at left (of known length), followed by a dashed line toward the right. Each time the test line intersects the alveolar wall, the distance (intercept length) to the next wall is measured. Boundaries of measurements within alveolar airspace (A) or alveolar and ductal airspace together (D) are marked by arrows (toluidine blue ×200). This figure is modified from Knudsen et al. (2010)

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Results

Particle characterization

Titanium dioxide nanopowder is hydrophilic, and TEM has shown that it is present mostly as agglomerations of smaller particles that are 114–122 nm in diameter, which indicates that the majority of TiO2 NPs were clustered and aggregated in solution.

Prenatal evaluation of maternal and fetal parameters

Table 1 shows the effects of TiO2 NPs ingestion on pregnant rats during the prenatal period. No mortality or treatment-related evident signs of maternal toxicity, stress or abnormal behavioral changes were observed. None of the pregnant rats exhibited vaginal bleeding or expulsions of products of conception. The food and water intakes, maternal weight gain, and lung weights of the treated rats were not statistically (p > 0.05) different from that of the control group.

Table 1 Maternal effects of TiO2 NP ingestion (5 g/kg body weight) by pregnant rats before and during organogenesis (GD1–GD20)
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Titanium dioxide content analysis

In the adult and fetal experimental groups, the contents of titanium in lung, brain, liver, and kidney tissues of female Wistar rats were measured 9 days after the last dose of TiO2 NPs. The highest amount of titanium was found in the maternal and neonatal lungs (0.82 ± 0.15 and 0.4 ± 0.53 ng/g, respectively) (Fig. 2a, b), which was confirmed by further histological examination of the lungs.

Fig. 2
figure 2

The EDX spectrum of adult (a) and neonatal (b) lungs after ingestion of a single dose of 20 nm TiO2 nanoparticles

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Histological examination of maternal lung tissue

Histological examination of the semi-thin sections of the adult control lung samples revealed normal architecture of the alveolar sacs and alveoli. The wall of the alveoli was lined by a single layer of pneumocytes that were composed of type I and type II pneumocytes. Pneumocytes were separated by thin septa and surrounded by a plexus of capillaries constituting the blood-air barrier (Fig. 3a, a′). TEM examination revealed type I and type II pneumocytes having euchromatic nuclei and multiple mitochondria. The latter cells contained many lamellar inclusions with identical pulmonary surfactant and possessed microvilli protruding into the airway spaces (Fig. 4a, a′).

Fig. 3
figure 3

Micrographs a, a′ show the light microscopic appearance of the control adult lung. Alveolar sacs (AS) are seen open into numerous alveoli (A). A terminal bronchiole is seen lined with ciliated columnar epithelium (E) with observable smooth muscle layer (M). A distinct pulmonary artery branch is found next to the bronchiole (Art). The right panel shows that the wall of alveoli is lined by type I pneumocytes (arrowheads) with flattened nuclei and type II pneumocytes (arrows) with large plump nuclei. A number of capillaries (c) form plexus around the alveoli. Micrographs b, b′ show the light microscopic appearance of TiO2 NP-treated adult lung with apparent thinning of airway septa in many areas (thin arrows). The right panel shows apparent widening of pulmonary interstitium (S) and infiltration of lymphocytes (thick arrows) and interstitial macrophages (M). Multiple dense deposits are present in the interstitial spaces of the alveolar walls (dashed arrows), and many apoptotic cells are observed (arrowheads) [a, b toluidine blue ×200—a′, b′ toluidine blue ×1,000]

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Fig. 4
figure 4

Micrographs a, a′ show the electron microscopic appearance of the control adult lung. The alveolar wall contains type II pneumocytes (P 2 ), recognizable by its lamellar inclusions (L) with an apparent pulmonary surfactant, and the cytoplasm is filled with numerous mitochondria (M). A type I pneumocyte (P 1 ) with an elongated euchromatic nucleus is also observed. The right panel shows type II pneumocytes resting on a basement membrane (BM), and a small proportion of its surface that is directly exposed to the alveolar space exhibits small microvilli (arrowheads) (uranyl acetate and lead citrate, Print Mag: a ×6,000—b ×12,000. Micrographs b, b′ show the electron microscopic appearance of TiO2 NP-treated adult lung with an apoptotic type II pneumocyte (P 2 ) with heterochromatic chromatin, atypical lamellar bodies (dashed arrows) and absent microvilli towards the alveolar space (Sp). The alveolar wall contains granulomatous epithelioid cell (EP), and its cytoplasm is burdened with electron opaque particles (arrows). There is an expansion in the interstitial spaces (IS) and dispersed collagen fibers (arrowheads). The right panel shows a type II pneumocyte (P 2 ) with an atypical empty lamellar body (dashed arrows) and short microvilli (arrowhead). An interstitial macrophage (M) is observed with an irregular nucleus and apical pseudopodia (arrows). An eosinophil (Es) is observed having the characteristic cytoplasmic stripped inclusions (n) (uranyl acetate and lead citrate, Print Mag: ×9,940)

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In contrast, semi-thin sections of the lungs exposed to TiO2 NPs exhibited various histological and morphological changes, including a focal decrease in the alveolar wall thickness, widening of the pulmonary interstitium, and inflammatory cell infiltrates. Additionally, there was an interstitial deposition of multiple dense deposits (Fig. 3b, b′). TEM showed type II pneumocytes with heterochromatic nuclei, atypical lamellar bodies, vacuolated cytoplasm, and short microvilli. There were numerous particle-laden interstitial macrophages with heterochromatic nuclei and apical pseudopodia. Multiple black precipitates were observed in the tissues, and the observed histological changes were noticed to be more obvious in these areas of the black precipitates (Fig. 4b, b′).

Histological examination of neonatal lung tissue

Histological examination of the semi-thin sections of the neonatal lungs from unexposed mothers showed many saccules constituting immature alveoli and many intervening airways representing immature alveolar ducts. Immature alveoli were separated by many buds constituting the primary septa that varied in their thickness (Fig. 5a, a′). TEM examination revealed interalveolar septa with type I and type II pneumocytes surrounded by a circle of capillaries. Type I pneumocytes exhibited common basement membranes with an endothelium of the blood capillaries (Fig. 6a).

Fig. 5
figure 5

Micrographs a, a′ show the light microscopic appearance of neonatal lung from control mothers with smooth outlined channels and regularly shaped airspaces (A). The alveolar septa are uniform in width (arrowheads) and mostly completely formed. The right panel shows saccular walls lined with type I pneumocytes (thin arrows) and type II pneumocytes (thick arrows) that are surrounded by a plexus of blood capillaries (c). The alveolar septa are standardized in width and contain few interstitial cells. Micrographs b, b′ show the light microscopic appearance of neonatal lung from TiO2 NP-treated mothers. There are fewer airways with incomplete (dashed arrows) and clearly thickened septations (arrows). The alveolar septa show congested blood vessels (circles). The right panel shows apparent thickening of the primary septa with the presence of various interstitial cells and congested blood capillaries (c′). There are several macrophages present in the alveolar septa (M) and free within the airway spaces. Macrophages lie adjacent to septal capillaries, (c) and their cytoplasms are burdened with dense-stained particles (arrows) [a, b toluidine blue ×200—a′, b′ toluidine blue ×1,000]

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Fig. 6
figure 6

Micrograph a shows the electron microscopic appearance of neonatal lung from control mother with a type II pneumocyte (P 2 ) inside a circle of the blood-air barrier formed as part of the peripheral cytoplasm of a type I pneumocyte (arrow) and that of the endothelium of the blood capillary (dashed arrow) that share a common basement membrane (arrowhead). Micrographs b, c show the electron microscopic appearance of neonatal lung from TiO2 NP-treated mother with a primary septum with many interstitial cells. There is widening of the interstitial spaces (IS) with electron lucent vacuolated areas (thick arrows). A smaller type II pneumocyte (P 2 ) exhibits a shrunken dark and eccentric nucleus (N). The cytoplasm contains empty lamellar bodies (arrows); some lamellar bodies contain apparent atypical surfactant (inside circle). An interstitial macrophage (M) is observed with long pseudopodus (dashed arrow) and numerous lysosomes (arrowheads). The right panel shows widening of the interstitial spaces (IS) and a type I pneumocyte (P 1 ) with a pinocytotic vesicle (dashed arrow). Type II pneumocytes (P 2 ) appear with a dark apoptotic cytoplasm and few short microvilli (arrows). AN interstitial macrophage (M) is observed with an irregular nucleus and numerous lysosomes (L) (uranyl acetate and lead citrate, Print Mag: a, c, ×7,450; b, ×6,000)

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In comparison, semi-thin sections of neonatal lungs from TiO2 NP-treated mothers revealed poorly developed respiratory saccules with interrupted primary septa and focal thickening of the intervening mesenchyme. There were numerous alveolar macrophages scattered in the alveolar wall and free within the airspaces, and their cytoplasms were burdened with dense-stained particles. There were many interstitial cells, and the interstitial spaces contained multiple dense deposits (Fig. 5b, b′). TEM examination showed a thick saccular wall containing interstitial cells, some of which appeared apoptotic with shrunken nuclei and lipid droplets. Additionally, there were numerous type II pneumocytes having dark cytoplasm, shrunken eccentric nuclei, many apparent atypical lamellar bodies, few short microvilli, and variable-sized vesicles containing electron dense particles. There were many interstitial macrophages with irregular nuclei and numerous lysosomes (Fig. 6b, c).

Morphometric analysis

The measurements obtained from the pulmonary alveoli of adult rats are presented in Fig. 7a. The mean linear intercept was slightly lower in the TiO2 NP-treated rats compared with the control group (mean, 108.8 ± 6.9 vs. 101.6 ± 5.6 μm in the control and treated groups, respectively) with no significant difference between the groups (p = 0.207). However, there was a significant reduction in the thickness of the alveolar walls in the treated rats (mean, 76.9 ± 2.2 vs. 25.1 ± 0.9 μm; p < 0.001 in the control and treated groups, respectively) compared with the control group.

Fig. 7
figure 7

Bars the morphometry of pulmonary acini in pregnant rats (a) and neonatal pups (b). Data are expressed as the mean ± SE. *p < 0.05 when the variables in the control and TiO2 NP-treated groups are compared using Mann–Whitney test

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The measurements obtained from the pulmonary alveoli of neonatal pups are presented in Fig. 7b. The mean linear intercept was nearly similar in both groups (mean, 95.7 ± 5.9 vs. 94.9 ± 6.9 μm in the control and treated groups, respectively) with no significant difference (p = 0.579) between groups. However, there was a significant increase in the thickness of the alveolar walls after administration of TiO2 NPs (mean, 85.5 ± 1.9 vs. 105.4 ± 3.8 μm; p = 0.001 in the control and treated groups, respectively) compared with the control group.

Discussion

Large human populations in the world continue to be exposed to pollutants. TiO2 is one type of widely used nanoparticle that has proven hazardous to humans. Through this study, we established novel data about the deposition of TiO2 NPs in maternal and neonatal lungs after 9 days from its maternal administration by an oral route. The presence of these nanoparticles in lung tissue was accompanied by histomorphometric alterations and neonatal alveolar maldevelopment.

In the present study, we used EDX in conjunction with SEM to analyze the energy levels of the dried lung tissue samples from rat mothers administered TiO2 NPs and established the presence of TiO2 in the tissues. Additionally, TEM examination displayed considerable black aggregates inside different pulmonary components as follows: the alveolar epithelium, alveolar spaces and in the interstitium, which further confirms that this substance accessed the pulmonary interstitium. These data prove the transition of TiO2 NPs from the systemic circulation after intake by mouth to pulmonary tissue. Permeation through the gastrointestinal barriers has been shown previously for micro- and nano-particles. These barriers consist of cellular (epithelium) and acellular parts (dead cells, mucus) (Fröhlich and Roblegg 2012). The absorption is estimated to be approximately 15- to 25-fold higher for nanoparticles (Desai et al. 1996). In line with these data, Jani et al. (1994) showed the distribution of orally administered TiO2 particles in the liver, spleen, and lungs. Other studies have reported the presence of titanium in blood and tissues of mice after intravenous injection of nanoparticles (Sugibayashi et al. 2008). A similar observation was reported by van Ravenzwaay et al. (2009), who compared inhalation with an i.v. route of administration for two different TiO2 particle sizes (20–30 nm or 200 nm) in male Wistar rats for up to 28 days.

Consequently, we studied the histomorphological alterations of these deposits in pulmonary tissues compared with the unexposed lung samples. An examination of unexposed adult lung samples revealed normal architecture of the alveolar sacs and alveoli. The lining pneumocytes contained euchromatic nuclei, numerous mitochondria, and regular lamellar inclusions. Additionally there were few interstitial cells with minimal interstitial spaces. However, after TiO2 NP intake, there was thickening of the pulmonary interstitium, changes of the lamellar inclusion ultrastructure, pneumocytic apoptosis, and the presence of interstitial particle-laden macrophages. However, the infiltration of polymorph nuclear inflammatory cells was more pronounced after ingestion of TiO2 NPs. Enhancement of these local effects was observed at the sites of particle retention. In addition, morphometric analysis showed significant thinning of the alveolar septa with no significant change in the MLI (an indication of the interalveolar wall distance) compared with the control group. From the observed results, it is suggested that nanoparticle-induced alveolar epithelial cell apoptosis might be the cause of abnormal thinning of the alveolar septa and the abovementioned histological changes; namely, epithelial cell apoptosis, interstitial thickening, and abnormal surfactant that can induce partial diffusion block for gas exchange.

Similar histopathological changes were observed in the lungs after intratracheal instillation, intraperitoneal injection or oral instillation of TiO2 particles to animals (Inoue et al. 2008; Liu et al. 2009). These findings of pulmonary inflammation are consistent with other studies showing that intratracheal exposure to nanoparticles (Chen et al. 2006) or inhalation (Borm and Kreyling 2004) can induce pulmonary inflammation. Additionally, pulmonary toxicity caused by TiO2 NP inhalation has been reported (Afaq et al. 1998; Sun et al. 2004). Recent studies have shown that TiO2 NPs produced only transient inflammatory effects that recovered after short-term exposure (Warheit et al. 2007). Even with subacute inhalation of these nanoparticles, the inflammatory response was sustained for only 2–3 weeks (Grassian et al. 2007). Our findings of inflammatory reaction and alveolar epithelial apoptosis are suggested to be involved in TiO2 NP-induced pulmonary toxicity. Because TiO2 NPs are widely used in cosmetics and food additives, these results may have important clinical implications regarding their safety (Weir et al. 2012).

In contrast, some authors have reported contradictory results regarding the measured MLI. Chen et al. (2006) found a slight increase in the MLI and in the airspace area on day 3 that significantly increased at the first week after administration of TiO2 NPs and persisted until the second week. They concluded that TiO2 NPs induced emphysema-like lung injury in mice. The discrepancy between our study and others is most likely attributed to differences in the treatment methods. Nevertheless, all studies did demonstrate that TiO2 NPs at higher doses had serious toxicity to rat lungs.

Interestingly, Bermudez et al. (2004) found that there were significant inter-species differences in pulmonary response after exposure to TiO2 particles. Their analysis of pulmonary parameters, including particle clearance from lungs, inflammation, proliferation of lung epithelial cells, and histopathologic alterations, showed that rats developed a more severe pulmonary inflammatory response than mice or hamsters.

In contrast, we studied the effects of maternal intake of TiO2 NPs on the late-term neonatal lung. TEM sections displayed considerable black aggregates that were observed inside the alveolar epithelium, in the alveolar spaces and in the interstitium. These observations were confirmed by the analysis of data by EDX in conjunction with SEM. The detected TiO2 NPs in the pulmonary tissue of neonatal pups suggests the transmission of these nanoparticles from mothers to their offspring, i.e., the ability of the TiO2 nanoparticles to bypass the placental barrier. This is consistent with previous studies that demonstrated the embryotoxic role of a maternal intravenous injection of TiO2 NPs or subcutaneous injections of anatase TiO2 NPs (Takeda et al. 2009; Shimizu et al. 2009; Takahashi et al. 2010). After subcutaneous injection, TiO2 accumulation was detected in the offspring cerebral cortex and olfactory bulb and numerous olfactory bulb cells and induced an altered expression of genes involved in brain development and cell death and a response to oxidative stress in the newborn pups (Shimizu et al. 2009).

Histomorphological examination of neonatal lungs at this age (21-day gestation) revealed the saccular stage of development, which simulates lungs of preterm human babies at gestational ages between weeks 26 and 36 as documented in early studies (Burri 1991). Light and electron microscopic examination of neonates from control mothers revealed large thick-walled saccules with a smooth outline. Saccules were separated by thick primary interalveolar septa because of the presence of many layers of interstitial cells. This is associated with the observation of capillaries on both sides of the primary septa. Moreover, there were numerous type II pneumocytes with numerous lamellar inclusions and obvious microvilli. These observations were in accordance with previous studies (Adamson 1991).

Histological and morphometric analysis of neonatal lungs after maternal intake of TiO2 NPs reveals variable shaped saccules with a significant increase in the thickness of the primary septa but with no significant change in the MLI compared with the control group. Moreover, there was focal thickening of the mesenchyme, poor septation, abnormal lamellar inclusions, pneumocytic apoptosis, and alveolar macrophage infiltration. TiO2 NPs appeared to have been engulfed by interstitial and free alveolar macrophages. Nonetheless, the more pronounced observation was the infiltration of inflammatory cells. The increase in the septal thickness in pups versus its decrease in the mothers could be because of the presence of many interstitial cells in the primary interalveolar septa, which was confirmed by electron microscopic examination together with the focal deposition of the mesenchyme. However, in the mothers, the observed alveolar septal thinning could be a result of apoptotic changes and epithelial cell injury triggered by TiO2 NP deposition in the pulmonary tissue.

It is suggested that the observed thickening of the mesenchyme between the airspaces together with the poor septation could lead to a partial diffusion block for gas exchange. Additionally, pneumocytic apoptosis of type II pneumocytes (producing pulmonary surfactants) and type I pneumocytes (lining the expanded alveoli) could affect alveolar maturation (Adamson 1991). The observed morphological alterations and pneumocytic apoptosis imply a morphological basis for respiratory failure in the neonatal rats. Ambalavanan et al. (2013) suggested that exposure of the developing lung to TiO2 NPs may lead to ineffective clearance by macrophages and persistent inflammation with effects on lung development; this effect was confirmed in our study. This is consistent with Hussain et al. (2011) who observed that low, intrapulmonary doses of TiO2 or gold NPs could aggravate pulmonary inflammation and airway hyperreactivity in a mouse model of diisocyanate-induced asthma. Furthermore, studies have also found that TiO2 NPs cause genetic damage in mice (Trouiller et al. 2009).

In conclusion, the present results suggest the presence of TiO2 NPs in maternal and neonatal pulmonary tissues after maternal oral intake of these particles, and indicate that the pulmonary response manifests as inflammatory lesions and saccular maldevelopment that may affect the risk of respiratory disorders in later life. Although additional studies are necessary to support these findings, they should be considered for a more accurate risk assessment of TiO2 NP exposure, particularly during a complex biological status, such as pregnancy and the early stage of life.