Multigenerational inheritance of breathing deficits following perinatal exposure to titanium dioxide nanoparticles in the offspring of mice

Background The utilization of titanium dioxide nanoparticles (TIO2NPs) has experienced a significant surge in recent decades, and these particles are now commonly found in various everyday consumer products. Due to their small size, TIO2NPs can penetrate biological barriers and elicit adverse interactions with biological tissues. Notably, exposure of pregnant females to TIO2NPs during the perinatal period has been shown to disrupt the growth of offspring. Furthermore, this exposure induces epigenetic modifications in the DNA of newborns, suggesting the possibility of multigenerational effects. Thus, perinatal exposure to TIO2NPs may induce immediate metabolic impairments in neonates, which could be transmitted to subsequent generations in the long term. Results In this study, we utilized perinatal exposure of female mice to TIO2NPs through voluntary food intake and observed impaired metabolism in newborn male and female F1 offspring. The exposed newborn mice exhibited reduced body weight gain and a slower breathing rate compared to non-exposed animals. Additionally, a higher proportion of exposed F1 newborns experienced apneas. Similar observations were made when the exposure was limited to the postnatal period, highlighting lactation as a critical period for the adverse effects of TIO2NPs on postnatal metabolism. Importantly, the breathing deficits induced by TIO2NPs were transmitted from F1 females to the subsequent F2 generation. Moreover, re-exposure of adult F1 females to TIO2NPs exacerbated the breathing deficits in newborn F2 males. Conclusions Our findings demonstrate that perinatal exposure to TIO2NPs disrupts postnatal body weight gain and respiration in the offspring, and these deficits are transmissible to future generations. Supplementary Information The online version contains supplementary material available at 10.1186/s11671-023-03927-0.


Introduction
The unique physicochemical properties of nanoparticles (NPs), attributed to their small size (below 100 nm), have led to a substantial increase in their production and utilization in various industries.Titanium dioxide nanoparticles (TIO2NPs), for instance, are extensively used in consumer products due to their whitening and photocatalytic properties.These particles can be found in everyday items such as paint, food, sunscreen, pharmaceuticals, toothpaste, cosmetics, and paper [1][2][3].As a result, human exposure to TIO2NPs and their potential toxic effects can occur throughout their lifespan, spanning multiple generations.Over the past decade, numerous studies have raised concerns about the health risks associated with TIO2NPs exposure, particularly during fetal development [4].Recent findings have demonstrated the ability of food-grade TIO2NPs to accumulate and cross the placental barrier in humans [5].Similarly, in mice prenatal exposure to TIO2NPs has been show to damage the placental barrier by altering the proliferation, apoptosis, and vascularization of the placenta [6,7].Moreover, prenatal exposure to TIO2NPs has been linked to pregnancy complications [6,8], and metabolic impairments in both the dam and the offspring [9,10].
Apart from their prenatal toxic effects, TIO2NPs, when administered during lactation, can accumulate in the mammary gland tissue of dams, leading to pathological damages such as oxidative stress.This accumulation can disrupt the blood-milk barrier and subsequently result in inadequate body growth of the offspring [6,[11][12][13].Consequently, when compared to prenatal exposure, perinatal exposure to TIO2NPs (during both gestation and lactation) could be expected to have a more complex impact on the development of the offspring acting both during the gestation and the lactation periods.In a previous study, we demonstrated that prenatal exposure to TIO2NPs altered postnatal breathing in the offspring.Newborns exhibited abnormally elevated breathing frequency, accompanied by impaired respiratory network regulation of breathing frequency [14].However, the consequences of perinatal exposure to TIO2NPs on neonatal respiration, which more closely resembles the exposure experienced by pregnant women, remain unknown.
In this study, we conducted whole-body plethysmography recordings to monitor respiratory frequency and the occurrence of apneas in newborn mice from postnatal day 1 to 11.Compared to non-exposed animals, newborn mice perinatally exposed to TIO2NPs from the first day of gestation until weaning exhibited lower weight, slower breathing rate, and a higher likelihood of experiencing apneas.Similar results were observed when exposure was limited to the lactation period.Furthermore, following perinatal exposure of F0 dams, F1 females were bred to investigate the multigenerational inheritance of breathing alterations in the F2 generation.In the absence of a second perinatal exposure in F1 pregnant females, newborn F2 mice displayed similar breathing deficits as the F1-exposed pups.Moreover, consecutive exposures of F0 and F1 pregnant mice exacerbated the respiratory deficits in newborn F2 males.In conclusion, perinatal exposure to TIO2NPs alters postnatal respiration and body growth of the offspring over multiple generations.

Ethical approval
All procedures were conducted in accordance with the local ethics committee of the University of Bordeaux (APA FIS#11,978-2,017,103,012,063,751) and the European Communities Council Directive (2010/63/EU).Experiments were performed on OF1 mice that were obtained from our laboratory's breeding facility.Newborns had full access to their mother's milk, and the mothers were fed ad libitum with full access to water.Every effort was made to minimize suffering and the number of animals used.

Maternal exposure to TIO2NPs
To generate the F1 offspring, 2 groups of F0 pregnant mice ingested a daily dose of TIO2NPs (100 mg/kg, n = 3; 200 mg/ kg, n = 3) that was mixed with chocolate spread (500 mg/kg), and administered by voluntary food intake, from the first gestational day (determined by the presence of a vaginal plug the morning following the mating night) that was maintained until weaning. 2 other groups of F0 dams received either a daily dose of chocolate spread (500 mg/kg, n = 5) alone or neither of those (n = 3).A 5th group of F0 females (n = 3) was exposed to TIO2NPs (200 mg/kg) during the lactation period (P0-21).
Pregnant mice were weighed daily, as were newborns from birth until the 11th day of life.The litter size was systematically reduced to 12 animals.TIO2NPs were purchased from Sigma-Aldrich (ref.718,467).

Whole body plethysmographic recordings
The breathing rate was measured using a whole-body plethysmograph (Emka Technologies) during a 5-min session [14].Newborns were placed in a 50 ml plethysmography chamber in which the air was renewed continuously (0.8 L/min).A heating lamp was used to maintain a constant (24°C) temperature within the recording chamber.Variation of pressure within the chamber was measured at a sampling rate of 1 kHz using Iox2 software (Emka Technologies).Recordings were analyzed using Spike2 software (Cambridge Electronic Design).Apneas were considered as cessations of breathing that last during at least 3 times the mean breathing period [15].

Physiologically based pharmacokinetic (PBPK) model
To estimate the tissue distribution of TiO 2 NPs, we used a physiologically-based pharmacokinetic model that predicts the absorption, distribution, metabolism, and excretion (ADME) of TiO 2 NPs [16].The model considers the route of exposure (oral), animal model (mice), treatment duration (41 days), daily dose administered (100-200 mg/kg), as well as a set of parameters such as the gastrointestinal absorption rate.

Statistical analysis
Group values were expressed as means ± SD.Differences between means were analyzed using SigmaPlot 11.0 (Systat) and assessed by Chi-square when looking at proportions.Either ANOVA on ranks followed by a Dunn's post hoc test or ANOVA followed by a Tukey's post hoc test was used when comparing two groups of non-parametric or parametric sets of data, respectively.Two-way and three-way MANOVA, as well as Tukey's post hoc tests, were utilized when comparing more than two groups and to assess interactions between independent variables.Differences in mean values for each parameter were taken to be significant at p < 0.05.

Mice gestation is not affected by the administration of TIO2NPs
Two groups of pregnant mice were exposed to daily doses of P25 titanium dioxide nanoparticles (TIO2NPs) at 100 mg/ kg and 200 mg/kg from the first day of gestation until the last day of lactation and weaning of the offspring (Fig. 1A).The European Food Safety Authority estimated that, on average, adults are exposed to 0.3-3.8mg TiO2/kg body weight per day [17].The highest daily dose used in our study was approximatively 50-fold greater than that estimated in humans, however the total amount of TIO2NPs delivered to a pregnant mouse over the ~ 19 day gestation was effectively 500fold less than the estimated quantity taken in by a woman during the entire duration of her pregnancy (~ 280 days).The TIO2NPs used in our study were previously characterized with an average size of 25 nm, a composition of 80% anatase and 20% rutile, and a zeta potential of − 25 mV [14].TIO2NPs were administered as a food additive in a low dose of palatable food (chocolate spread: 500 µg/g) using voluntary food intake.In comparison, a third group (sham group) received a daily dose of chocolate spread alone (500 µg/g), and a fourth group (Control group) received neither (Fig. 1A).Between the first day of gestation (G0) and the last day of lactation (P21), mice exposed to TIO2NPs at 100 mg/kg and 200 mg/ kg ingested a total dose of 180.4 ± 5.9 mg and 353.7 ± 4.6 mg of TIO2NPs, respectively (Fig. 1A, C).We utilized a physiologically based pharmacokinetic (PBPK) model to evaluate the tissue distribution of TIO2NPs in the exposed mothers [16].The model predicted that the intake of TIO2NPs at both concentrations (100 mg/kg and 200 mg/kg) would lead to a rapid increase in the blood levels of TIO2NPs, followed by a plateau (from 0.305 µg/L at G2 to 0.420 µg/L at G14 for a dose of 200 mg/kg).This rise would then be followed by a decrease after the end of the treatment due to the excretion of TIO2NPs in both urine and feces (Figure S1).The model also predicted a gradual increase in the concentration of TIO2NPs in brain tissue during the gestational and lactation periods, from 0.011 µg/g at G2 to 0.202 µg/g at L21 in females exposed to a daily dose of 200 mg/kg (Fig. 1B).A similar increase in TIO2NPs levels was also predicted by the model in various organs such as the kidneys, spleen, stomach, and intestines (Figure S1).Previous studies have reported an accumulation of TIO2 in the placenta of mice exposed to TIO2NPs [6,8].These accumulations coincided with changes in placental morphology, including modifications in weight, size, vascularization, and an ionic imbalance in the maternal serum.These alterations resulted in pregnancy complications, ranging from reduced fetal body weight to a reduced number of viable fetuses [6][7][8]18].
To assess the possibility of abnormal pregnancy outcomes, we conducted daily measurements of the gestational weight gain of the pregnant mice (Fig. 1D).Both exposed and non-exposed females showed similar weight gains during pregnancy and the postnatal period, indicating no apparent effect of TIO2NPs exposure.Additionally, all four groups of animals had similar litter sizes (Fig. 1E) and a similar mortality rate among newborns (Fig. 1F).We also monitored the weight gain and body length of the pups from the first day of life (P0) until the 11th day (P10).At birth, animals from all four groups had similar body weights (ANOVA on ranks, H = 4.182, p = 0.243; Fig. 2A).However, differences were observed between the four groups in the following days.For instance, at P9-10, significant differences were found between the control and sham groups, the TiO2_100 and TiO2_200 groups, and the sham and TiO2_200 groups (ANOVA on ranks, H = 31.380,p < 0.001; Fig. 2A).This was evident when analyzing the cumulative weight gain of the pups between P0 and P10.The exposed pups exhibited significantly lower weight gain compared to the control and sham pups (P0-10: ANOVA on ranks, H = 27.182,p < 0.001; Fig. 2D).We further investigated whether TIO2NPs affected the weight of the pups differently based on their sex.Both exposed females (Fig. 2B) and males (Fig. 2C) showed reduced weight gain compared to the non-exposed pups (Females: P9-10: ANOVA on ranks, H = 17.969, p < 0.001; Males: P9-10: ANOVA on ranks, H = 17.081, p < 0.001).At birth, both TiO2_100 and TiO2_200 animals exhibited shorter body length compared to the control and sham groups (ANOVA on ranks, H = 42.953,p < 0.001; Fig. 2E).However, at P9-10, only the TiO2_200 animals were significantly shorter than the control and sham groups (ANOVA on ranks, H = 44.200,p < 0.001; Fig. 2E-G).Overall, the TiO2_200 animals showed reduced body growth compared to the other groups (P0-10: ANOVA on ranks, H = 34.460,p < 0.001; Fig. 2H).In conclusion, exposure to TIO2NPs, particularly at a dose of 200 mg/kg, alters the body growth of newborn animals during the P0-10 period.Indeed, despite numerous studies on the subject, it remained unclear whether TIO2NPs perinatal exposure affects the growth of neonates.Previous studies have focused the weight gain of newborns following prenatal exposure to TiO 2 , some reporting no significant changes [10,[19][20][21], while others indicated a notable For each group we used the following number of litters: control, n = 3; sham, n = 5; TiO2_100, n = 3; TiO2_200, n = 3 reduction in pups' weight [6,22,23].These discrepancies can be attributed to various factors, such as the use of different animal models (mice vs. rats), variations in the size of TIO2NPs (ranging from 6.5 to 25 nm), differences in the route and dosage of administration (gavage, inhalation, i.v.), and variations in the frequency of administration (single, repeated, or daily).Similarly, comparable observations were made regarding the administration of TIO2NPs during lactation, with some studies demonstrating reduced offspring body weight [12,13], while others showed no effect [11].In this context, our findings provide new insights into the effects of perinatal exposure to TIO2NPs on neonatal growth.In a previous study, we used the same TIO2NPs, with the same number, route, and dosage of administration, but limited the exposure to the gestation period.In a previous study, we observed no changes in the body weight of the pups that were exposed to TIO2NPs through their mother's diet during gestation [14].Conversely, in the present study, we demonstrate a reduced body weight gain in pups exposed to TIO2NPs during lactation (Fig. 2, S2 and Table 1).Consequently, our studies indicate that postnatal, but not prenatal, oral exposure to P25 TIO2NPs alters neonatal body growth, likely through the disruption of breastfeeding.Previous research has shown that the administration of TIO2NPs during the lactation period leads to the accumulation of these particles in the mammary gland, causing pathological damage to the tissue and disruption  of the blood-milk barrier [11][12][13].Notably, the mammary gland exhibited increased oxidative stress, apoptosis rate, and loss of tight junctions.While the milk yield and nutrient quality were not directly affected, the concentration of TIO2NPs and ROS in the dams' milk increased.

Perinatal exposure to TIO2NPs impairs offspring breathing rhythmicity
In the control group, the respiratory frequency of animals increased from 171.07 ± 50.82 at P1-2 to 232.13 ± 47.37 at P5-6 (MANOVA: F = 73.501,p < 0.001, Tukey's test: P1-2 vs. P5-6, p < 0.001), after which it reached a plateau during the following days (MANOVA: F = 73.501,p < 0.001, Tukey's test: P5-6 vs. P9-10, p > 0.05; Fig. 3C).This typical pattern of respiratory development was observed in all groups of animals (MANOVA: F = 73.501,p < 0.001, Fig. 3D).However, neonates from TIO2NPsexposed litters exhibited a slower breathing rate compared to non-exposed animals (MANOVA: F = 73.501,Tukey's test: control vs sham, p > 0.05; control vs TiO2_100, p < 0.001; control vs TiO2_200, p < 0.001; sham vs TiO2_100, p < 0.001; sham vs TiO2_200, p < 0.001; TiO2_100 vs TiO2_200, p > 0.05; Fig. 3B, D).We also observed an interaction between the treatment and the age of the animals (MANOVA: F = 2.034, p = 0.019), indicating a progressive and cumulative-like effect of TIONPs exposure during lactation.When examining sex differences, we observed no differences between males and females (MANOVA: F = 0.336, p = 0.563).Exposed newborn females had a slower breathing frequency compared to sham and control females at both P1-4 and P5-10 (ANOVA: females: P1-4: F = 7.699, p < 0.001; P5-10: F = 11.979,p < 0.001), and a similar difference was also observed between exposed and non-exposed males (ANOVA: males: P1-4: F = 4.712, p = 0.005; P5-10: F = 15.510,p < 0.001; Fig. 3E).Thus, our findings demonstrate that perinatal exposure to TIO2NPs leads to a slower breathing frequency in neonates compared to non-exposed animals, regardless of sex.As discussed in the above section, prenatal exposure to TIO2NPs elicits morphological alterations in the placental barrier and consequently impacts its efficacy, potentially facilitating the transfer of TIO2NPs from the mother to the fetus [6,8,18,23].Other studies notably highlighted an impairment in lung development in the offspring, characterized by inflammatory reactions at the pulmonary level [23,24].In a previous work we demonstrated that prenatal exposure to TIO2NPs similar to the one utilized in this study did not result in any structural modifications of the lungs: the lung/body weight ratio, lung size, and number of lung alveoli were similar between exposed and non-exposed pups [14].Thus, it is likely that our protocol of exposure to TIO2NPs does not significantly alter the lung development of the offspring, although further complementary studies could be conducted to confirm this.

Litters exposed perinatally to TIO2NPs have a higher frequency of apneas
When examining the regularity of breathing in newborn mice, we observed that all groups of animals experienced apneas (Fig. 4A).However, the proportion of animals producing apneas was significantly higher in the TiO2-exposed groups (24.6% for TiO2_100 and 33.5% for TiO2_200) compared to the control and sham groups (11.1% and 8.6%, respectively; Chi-Squared test = 74.923,p < 0.001; Fig. 4B).Between P0 and P10, there was a similar trend of decreasing apnea occurrence in all groups (Fig. 4C).This trend was consistent in both female and male newborns and not specific to a particular sex (females: P1
In comparison to humans, mice have an immature respiratory system at birth, and the presence of apneas has been associated with immature breathing, as observed in preterm infants [28][29][30].Thus, our results suggest a delayed maturation of the respiratory function in exposed pups that could be related to a metabolic distress [6].Notably, the maternal and pup's diet directly influences offspring body weight and breathing [31][32][33].Interestingly, newborn mice subjected to fasting (3-6 h) exhibited breathing instability characterized by decreased breathing frequency and increased occurrence of apneas [33].Despite these important findings, the mechanisms linking impaired neonatal growth and breathing alterations remain unclear.TIO2NPs can directly affect developmental processes such as cell proliferation, apoptosis, neurogenesis, and neuron-glial interactions, all of which are crucial for neural circuit formation [22,[34][35][36].In a previous study, we demonstrated that prenatal exposure to TIO2NPs impairs the development and function of the primary inspiratory center, specifically the pre-Bötzinger complex [14,37].Postnatal exposure to TIO2NPs may also disrupt the functioning of other brainstem nuclei, including the retrotrapezoid nucleus and the parafacial respiratory group (RTN/ pFRG).Mutations in the Phox2b gene, for instance, result in reduced neuronal density in the RTN/pFRG complex and a hypoventilation syndrome in offspring [38].Additionally, it is plausible that TIO2NPs exposure indirectly impairs breathing by dysregulating neurotransmitter systems.Studies in the central nervous system of rats have shown that acute administration of TIO2NPs decreases serotonin levels [39,40], a key neuromodulator in the neural centers that regulate breathing frequency [41,42].Notably, neonates lacking serotoninergic neurons display a higher likelihood of experiencing apneas and severe hypoventilation syndrome [43].Importantly, newborns with serotonin neuron deficiencies also exhibit associated deficits in body growth until the end of the second week of life.
These results demonstrate the long-lasting multigenerational adverse effects of TIO2NPs exposure, highlighting a potential vulnerability of F2 males to re-exposure.Previous studies have indicated that inhalation exposure of pregnant rat to TIO2NPs can lead to persistent alterations in oxidative species production across multiple generations, from F0 dams to F2 offspring [44].These alterations were associated with reduced weight in the F1 and F2 generations and changes in metabolism, including increased glycemia.The perinatal exposure to TIO2NPs induces an early childhood stress response due to its various consequences on F1 newborns, such as reduced weight and breathing rate (Figs.2-4).Notably, metabolic disturbances during the perinatal period have been shown to induce epigenetic modifications with potential implications for subsequent generations [33,45].The increase in oxidative species production triggered by TIO2NPs exposure can disrupt gene expression and DNA methylation [46].The small size and high reactivity of TIO2NPs enable them to induce DNA methylation in cultured cell lines [47][48][49][50][51][52][53], and as they can cross biological barriers, they can be transferred from the mother to the offspring, where they can impact DNA methylation.Indeed, maternal exposure to TIO2NPs can directly cause epigenetic modifications in the DNA of the progeny [21,[54][55][56].These studies have reported genome-wide alterations in DNA methylation in the brains and Furthermore, we observed a notable susceptibility of F2 male pups to perinatal re-exposure to TIO2NPs compared to females.Previous research has reported sex-dependent epigenetic alterations following prenatal TIO2NPs exposure.Specifically, DNA methylation was found to be increased in 614 genes and decreased in 2924 genes in male offspring, while in female offspring, DNA methylation was increased in 6220 genes and decreased in 6477 genes [21].These findings provide a potential explanation for the sex-specific impact of TIO2NPs re-exposure, particularly affecting F2 males (Fig. 8C, D).Although a direct connection between specific epigenetic modifications and respiratory function cannot be established at present, a recent study demonstrated that re-exposure to nicotine in F1 animals, previously exposed during F0 gestation, enhanced the asthmatic phenotype in the F2 generation, primarily in males [57].Once again, these sex-associated phenotypes were linked to sex-specific DNA methylation patterns.

Conclusion
In conclusion, our findings demonstrate that perinatal exposure to TIO2NPs disrupts neonatal respiration throughout multiple generations, thereby establishing a susceptibility to TIO2NPs re-exposure in subsequent pregnancies.We hypothesize that sex-specific epigenetic modifications may underlie the persistent deficits observed across generations, although the precise targets of TIO2NPs remain to be identified.These results raise concerns regarding the transmission of neurophysiological impairments associated with adverse maternal environments, particularly in the context of nanoparticles.

Fig. 1
Fig. 1 Mouse gestation is not affected by TIO2NPs administration.A, Schematic representation of the experimental protocol.B, Evolution of the predicted concentrations of TIO2NPs in the brain and blood of exposed dams during the gestation and lactation periods.C, Cumulative curve illustrating the quantity of TIO2NPs ingested by the dams during the gestation and lactation periods for the mice exposed to TIO2NPs at 100 mg/kg (light blue dots) and 200 mg/kg (blue dots).D, Scatter plot illustrating the evolution of mouse weight during the gestation and lactation.E-F, Bar charts illustrating the mean number of pups per litter (E) and the number of premature deaths (F) for each group of animals.For each group we used the following number of litters: control, n = 3; sham, n = 5; TiO2_100, n = 3; TiO2_200, n = 3

Fig. 6
Fig. 6 Exposure to TIO2NPs during lactation and perinatal period have similar effects on offspring breathing.A, Schematic representation of the experimental protocol.B, Left, Plethysmographic recording at P6 of sham, TiO2_200, and TiO2_200_Lact pups.B, Right, Expanded portion (indicated by dotted lines) of the trace on the left.C, Bar chart illustrating the evolution of the breathing rate of sham (dark grey bars), TiO2_200 (blue bars), and TiO2_200_Lact (blue hatched bars) pups over the P1-10 period.D, Pie chart representation of the number of pups exhibiting apneas during the P1-4 and P5-10 periods.Apneas are represented in black on pie charts.*p < 0.05.For each group we used the following number of pups: sham, n = 55, females = 31, males = 24; TiO2_200, n = 36, females = 19, males = 17; TiO2_200_Lact, n = 36, females = 17, males = 19

Fig. 7
Fig. 7 Transmission of TIO2NPs-induced breathing alterations to F2 sham.A, Schematic representation of the experimental protocol.B, Bar chart illustrating the evolution of the breathing rate of F1 sham (dark grey bars) and F2 sham (purple bars) pups over the P1-10 period.The blue dotted lines indicate the mean values of F1 TiO2_200 animals.C, Bar chart illustrating the breathing rate of females and males for each group of animals during the P1-4 and P5-10 periods.The blue dotted lines indicate the mean values of F1 TiO2_200 animals.D, Proportion of female and male neonates producing apneas for each group over the P1-4 and P5-10 periods.Apneas are indicated in black on pie charts.*p < 0.05.For each group we used the following number of pups: F1 sham, n = 55, females = 31, males = 24; F2 sham, n = 24, females = 12, males = 12

Fig. 8
Fig. 8 Transmission and exacerbation of TIO2NPs-induced breathing alterations by TiO2 re-exposure in F2 females and males, respectively.A, Schematic representation of the experimental protocol.B, Bar chart illustrating the evolution of the breathing rate of F1 TiO2_200 (blue bars) and F2 TiO2_200 (dark blue bars) pups over the P1-10 period.The grey dotted lines indicate the mean values of F1 sham animals.C, Bar chart illustrating the breathing rate of females and males for each group of animals during the P1-4 and P5-10 periods.The grey dotted lines indicate the mean values of F1 sham animals.D, Proportion of female and male neonates producing apneas for each group over the P1-4 and P5-10 periods.Apneas are indicated in black on pie charts.*p < 0.05.For each group we used the following number of pups: F1 TiO2_200, n = 36, females = 19, males = 17; F2 TiO2_200, n = 24, females = 13, males = 11