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. 2026 Aug 17;40(16):e72202. doi: 10.1096/fj.202600065RR

Adverse Effects of Carbon Black Nanoparticles on Maternal and Fetal Lungs in Pregnant Mice: Insights From a Metabolomics Perspective

Jing Li 1,2, Biling Gao 2, Hao Chang 2, Jingjing Zhan 3, Zhonghua Lu 2, Zehua Xu 2, Yi Lin 2, Jia Li 3,✉, Heqing Shen 2,4,✉
PMCID: PMC13480342  PMID: 42606525

ABSTRACT

Previous studies have linked prenatal exposure to carbon black nanoparticles (CBNPs), a key constituent of air pollution and tobacco smoke, to respiratory abnormalities. However, the distinct impacts of particle size on the maternal‐fetal lung axis remain poorly understood. This study investigated the maternal‐to‐fetal lung toxicological effects and metabolic shifts following exposure to 30 nm (CBNP30) and 120 nm (CBNP120) particles using a high‐dose mechanistic exposure model. In maternal lungs, histological analysis (H&E and Masson staining) revealed severe inflammatory infiltration and significant fibrotic injury for both sizes, with CBNP120 inducing more pronounced damage. Dark‐field hyperspectral imaging confirmed the transplacental transfer and presence of both CBNP sizes within fetal lung tissues. Notably, fetal lungs did not display overt histological injury. However, significant elevations in oxidative stress and inflammatory markers (mRNA and protein) were observed, indicating clear molecular perturbation. Chemical Isotope Labeling LC–MS further identified size‐specific metabolic disruption, with CBNP120 eliciting more extensive changes in the fetal metabolome. Specifically, CBNP30 primarily affected histidine metabolism in both maternal and fetal lungs, whereas CBNP120 disrupted butanoate and arachidonic acid pathways. These metabolic shifts, supported by altered enzyme expression, suggest that profound biochemical disturbances may occur in the fetus even in the absence of observable morphological damage. This study clarifies the distinction between maternal structural injury and fetal molecular dysfunction, providing critical insights for interventions against air pollution‐related neonatal health risks.

Keywords: adverse effect, CBNPs, lung injury, metabolomics, prenatal exposure

Histopathology revealed inflammation and fibrosis in maternal lungs after exposure to 30 and 120 nm CBNPs, with 120 nm causing more pronounced damage. CBNPs could enter the fetal lungs upon maternal exposure and cause oxidative stress, inflammation, and induce distinct metabolic disruptions in fetal lungs. Metabolomics detected by Chemical Isotope Labeling LC–MS showed 120 nm CBNPs induced stronger metabolic disruptions in both maternal and fetal lungs than 30 nm, particularly impacting the fetus.

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1. Introduction

Black carbon (BC) comes from the incomplete combustion of fossil fuels, biomass, and tobacco smoke. It forms ultrafine carbon black nanoparticles (CBNPs). These particles penetrate deep into the lungs [1, 2, 3, 4, 5]. Pregnancy is a window of heightened susceptibility to xenobiotics. Maternal exposure to fine and ultrafine particulate matter causes adverse birth outcomes. It also impairs offspring respiratory function [6]. Prenatal PM2.5 and tobacco smoke exposure independently affect childhood respiratory health [7, 8]. This raises specific concern about the health risks of CBNPs on maternal and fetal lungs.

CBNPs can trigger pulmonary inflammation and alveolar injury [9, 10]. CBNPs can also penetrate the pulmonary epithelium, relocate into the cardiovascular system, and reach other tissues or organs [11, 12]. Notably, carbon‐related particles have been found to pass through the placenta [13] and transfer into the organs of the human fetus, including the fetal lungs [14]. Several studies have suggested that prenatal exposure to air pollution may cause lung damage in offspring [15]. Exposure to air pollution during pregnancy contributes to the development of asthma and increases the risk of childhood asthma [16, 17]. Most studies have mainly addressed the dose‐dependent effects of airborne particles (including CB particles) [18, 19]. But few studies focused on their size‐related effects [20], particularly in the case of CBNPs. Therefore, we wonder whether different sizes of CBNPs will influence the normal physiological state of the maternal lungs and even cause adverse effects on fetal lungs.

Metabolites are biologically sensitive indicators that reflect the toxicological state of an organism. Additionally, they play a significant role in fetal lung development [21, 22]. Previous studies have investigated the metabolic interference of CBNPs in rats' serum [5] and A549 cells (a lung adenocarcinoma cell line) [23] using liquid chromatography‐mass spectrometry (LC–MS). Limited studies have focused on the metabolic changes caused by particulate matter in maternal or fetal lungs. Traditional non‐targeted LC–MS theoretically enables comprehensive metabolite coverage. However, standard sample preparation often lacks sufficient sensitivity. These limitations in sensitivity and quantitative accuracy significantly reduce actual metabolite coverage in practice. Chemical Isotope Labeling (CIL) LC–MS has emerged as a high‐resolution method for detecting metabolites [24]. CIL LC–MS enhanced accuracy in quantification and comprehensive coverage of chemical group‐based submetabolomes [25], including the hydroxyl, amine/phenol, carboxyl, and carbonyl submetabolomes [26]. Hence, the metabolic disturbance in the maternal lung and fetal lung in response to CBNP exposure needs to be understood by a more accurate method.

The study was to determine whether 30 and 120 nm CBNPs reach fetal lungs and cause structural or metabolic injury. Intratracheal instillation serves as a high‐dose mechanistic tool. It does not replicate typical ambient inhalation during pregnancy. We compare metabolic alterations in maternal and fetal lungs to uncover underlying changes. These findings should advance understanding of particulate matter and tobacco toxicity in the maternal and fetal lung, and underscore the importance of controlling ambient particle exposure during pregnancy.

2. Materials and Methods

2.1. Animal

All C57BL/6 mice were utilized in accordance with the guidelines of Xiamen University Institutional Committee for the Care and Use of Laboratory Animals (XMULAC20200188). Nine‐week‐old healthy female C57BL/6 mice were purchased from the SLAC Laboratory Animal Center (Shanghai, China) and held under specific pathogen‐free conditions at Xiamen University Laboratory Animal Center (Xiamen, China). The environment was maintained at consistent conditions of room temperature (22°C ± 2°C), humidity (55% ± 5%), and a 12:12 h light–dark cycle. Mice had ad libitum access to food and water. Following acclimation for a week, two female mice and one male mouse were housed in the same cage overnight for mating. In the following morning, the female exhibiting either a vaginal plug or the presence of sperm on the vaginal smear was identified as pregnant with gestational day 0.5 (GD0.5).

2.2. CBNPs and Exposure

The particle size of CBNPs (Orion, Germany) used to treat the mice has two scales: 30 and 120 nm, respectively. The characterization of CBNPs was reported in a previous study [27]. On GD0.5, pregnant mice were randomly assigned to one of the following groups which are 0.9% saline group (Control), 200 μg, 30 nm CBNPs group (CBNP30), and 200 μg, 120 nm CBNPs group (CBNP120), respectively (Table S1). Mice were anesthetized with isoflurane by inhalation and their heads and necks were backward exposed to the airway, then 40 μL of CBNPs suspension (5 mg/mL) or 0.9% saline infused into the trachea of the mice at GD3.5, GD6.5, GD9.5, GD12.5 and GD15.5, respectively [28, 29, 30]. Then mice were sacrificed on GD18.5. Carbon black nanoparticles (CBNPs) are frequently used as a surrogate for the black carbon core of PM2.5. The maximum ambient concentration of black carbon is 3 μg/m3 [31]. Healthy adults inhale and exhale approximately 10 L of air per minute, yielding a total daily ventilation volume of 14.4 m3 (10 L/min × 60 min/h × 24 h). Because roughly 90% of inhaled air is exhaled during normal breathing, the estimated daily retained dose of CBNPs is 4.32 μg (3 μg/m3 × 14.4 m3/day × 10%). To account for interspecies variation (factor of 10) and intraspecies variation (factor of 10), we applied a cumulative uncertainty factor of 100 [32]. The dose was therefore calculated as 4.32 μg/day × 3 days × 100 = 1296 μg. The final exposure dose was set at one‐sixth of this maximal value. The exposure interval used in this study during pregnancy referred to previous studies on toxicogenomic effects in dams and offspring after CB prenatal exposure [33], the effects of PM2.5 prenatal exposure on the lungs of offspring rats [34], as well as in vivo exposure experiments in mice on the promoting effect of CBNPs produced by tobacco combustion on lung cancer development [3]. The intratracheal instillation protocol represents an exposure model primarily designed to probe underlying toxicological mechanisms rather than to strictly mimic typical continuous ambient inhalation.

2.3. Hyperspectral Imaging (HSI)

Dark‐field HSI is an innovative technique employed for the visualization and tracking of nanoparticles. The methodology applied in this study was based on established protocols from previous research [35]. Fetal lungs separated from pregnant mice were immersed in fixed liquid (Servicebio, China) for 24 h. Following paraffin embedding, tissues were cut into 5 μm sections and then subjected to a series of dehydration steps using gradient alcohol and xylene. The visualization and spatial localization of 30 and 120 nm CBNPs in fetal lung tissues were performed using a CytoViva dark‐field hyperspectral microscopy system equipped with a pixelFly CCD camera. Image capture and spectral data processing were conducted using ENVI 4.8 software. To ensure high specificity and minimize false positives from endogenous biological structures such as collagen fibers or hemoglobin, a rigorous spectral library protocol was established. Initially, a spectral library was acquired from pure CBNP suspensions using the Region of Interest (ROI) function. To isolate nanoparticle‐specific data, we filtered the spectral library of the CBNP‐exposed fetal lungs against control images, effectively removing background scattering from the biological matrix. Subsequently, we compared the spectral library obtained from the exposed group of fetal lungs with the spectral library of CBNPs. If the lungs of the fetal mice have the same spectrum, it is considered that there is CBNPs deposition.

2.4. Hematoxylin–Eosin (H&E) Staining

The reagents were obtained from Servicebio, China. The tissues were fixed in 4% paraformaldehyde overnight and embedded in paraffin. Five micrometer section of tissue slides was dewaxed and dehydrated. Next, slices were put into the hematoxylin dye solution for 3–5 min, washed with tap water, differentiated with the solution, washed again with tap water, returned the blue solution to blue, rinsed with running water. Then, slices were put into 95% alcohol and dehydrated for 1 min, then dyed in eosin solution for 15 s. Finally, slices were dehydrated and sealed with neutral gum. For each slice, we selected five fields of view for pathological scoring, and the scoring was based on previous literature [36].

2.5. Masson Staining

The reagents were obtained from Servicebio, China. The prepared paraffin sections (using the same method as for H&E staining) were dewaxed and rinsed in pure water for a few seconds. The sections were then immersed in Masson's A solution overnight and subsequently washed with tap water. Masson's B and C solutions were mixed in equal proportions and used to stain the sections for 1 min, washed with tap water, differentiated in the differentiator for a few seconds, and another rinsed in tap water. The sections were then stained with Masson's D solution for 6 min, washed with tap water, and immersed in Masson's E solution for 1 min. After allowing them to drain slightly, the sections were immediately submerged in Masson's F solution for 30 s. The sections were differentiated in 1% acetic acid, dehydrated in two changes of anhydrous ethanol, cleared in xylene (5 min each), and finally mounted with neutral balsam. This method stains muscle fibers, fibrin, and red blood cells in red, collagen fibers in blue, and cell nuclei in purple‐blue.

2.6. RNA Extraction and Polymerase Chain Reaction

TRIzol (ThermoFisher, USA) was utilized to extract RNA. RNA was qualified by a high purity with OD260/280 in the range of 1.8 ~ 2.0, and integrity was determined by agarose gel electrophoresis. RNA was quantified using a plate reader (Gene5, Biotek). 1000 ng RNA was reverse transcribed into cDNA by using NovoScriptPlus All‐in‐one 1st Strand cDNA Synthesis SuperMix (Novoprotein, China). The real‐time polymerase chain reaction was performed on a fluorescence quantitative PCR instrument, real‐time PCR system (Q towere3G, Germany), with the NovoStartSYBR qPCR SuperMix Plus (Novoprotein, No.: E096) for 40 cycles. Actb was used as an internal control for mRNA. Primers are shown in Table S2.

2.7. Western Blot

Homogenates of tissues were centrifuged at 12 000 rpm for 30 min under 4°C. Subsequently, the supernatants were determined by the BCA Protein Assay Kit (Beyotime, China). 5× loading buffer (Solarbio, China) was added to each sample, followed by heating at 100°C for 10 min. The samples were loaded for electrophoresis. Following electrophoresis, proteins were transferred onto membranes using the wet transfer method. After the transfer, the membrane was blocked with skimmed milk to prevent non‐specific binding. The membrane was then incubated overnight at 4°C with primary antibodies targeting the proteins of interest HMGB1 (Beyotime, AF0180), TXNIP (Santa Cruz, sc‐271237), ACSL4 (Santa Cruz, sc‐271800), GAPDH (Proteintech, 10494‐1‐AP). Following this, the membrane was incubated with secondary antibodies for 1 h, and detection was performed using an enhanced chemiluminescence (ECL) substrate (Abclonal, China) to visualize the protein bands.

2.8. Metabolomics Profiling

All liquid chromatography mass spectrometry (LC–MS) analyses were carried out using Agilent 1290 LC linked to Agilent 6545 Q‐TOF Mass Spectrometer. The column used was Agilent eclipse plus reversed‐phase C18 column (150 × 2.1 mm, 1.8 μm particle size) and the column oven temperature was 40°C. Mobile phase A was 0.1% (v/v) formic acid in water and mobile phase B was 0.1% (v/v) formic acid in acetonitrile. The gradient setting was: t = 0 min, 25% B; t = 10 min, 99% B; t = 13 min, 99% B; t = 15 min, 99% B; t = 15.1 min, 25% B; t = 18 min, 25% B. The flow rate was 400 μL/min. Mass spectral acquisition rate was 1 Hz, with an m/z range from 220 to 1000. In Chemical Isotope Labeling LC–MS (CIL LC–MS), four isotope labeled metabolites were detected in the form of peak pairs. Metabolite peak pair extraction, sample‐wise alignment, and missing value retrieval were conducted by using IsoMS Pro (www.novamt.com). Peak pairs where Mean (sample)/Mean (blank) was less than or equal to 4.0 at a significance level of 0.05 were filtered out. Only metabolites that were present in at least 80% of samples of each group were retained to remove unstable information. After filtering, the data were normalized by Ratio of Total Useful Signal. Metabolite identification was accomplished through a three‐tier approach as previously reported [26]. The high‐accuracy and high‐reliability metabolites that were searched and identified by the CIL library (Tier 1) and the Linked Identity (LI) library (Tier 2) were used for subsequent analysis. All metabolites, which were classified one‐to‐one with reference to the classification of Metabolon superpathways, were classified as amino acids and derivatives, carbohydrates, cofactors and vitamins, energy, lipid, nucleic acids, peptides, and others.

2.9. Statistical Analysis

The statistical analysis was carried out in R stats package (version 4.3.0; Vienna, Austria). Multivariate statistical data analysis was performed with SIMCA‐P (version 14.1; Umetrics, Umeå, Sweden). The quality of the partial least‐squares discriminant analysis (PLS‐DA) models was evaluated by R2Y, Q2Y, and 200 times permutation test. In this study, metabolites that satisfy both p < 0.05 and log2fold change (log2FC) > log2(1.5) or log2FC < log2(2/3), and variable importance in projection (VIP) > 1 were considered as differential metabolites. The metabolic correlation network analysis was based on the inter‐ and intra‐organ correlations of the differential metabolites present in the maternal lung and fetal lung (|r| > 0.6, p < 0.01). Visualization of the network was performed using Cytoscape software [37]. Degree of the node is defined as the number of connections it has to other nodes. The top 15 metabolites with the greatest degrees are considered as the hub metabolites. Pathway enrichment analysis was computed by MBROLE 3 (http://csbg.cnb.csic.es/mbrole3/analysis.php) [38] and classified by metabolic pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG). The rich ratio was calculated by dividing the number of differential metabolites by the total number of metabolites profiled in the enrichment pathway. The metabolic pathways were visualized with iPath (https://pathways.embl.de/). For visualizing the upset plot, heatmap, volcano plot, and enrichment pathway, we utilized the ggupset package (version 0.3.0), ComplexHeatmap package (version 2.16.0), and ggplot2 package (version 3.4.2). For qPCR and western blot data, results were presented as the mean ± SD. Comparisons between the two groups were performed using one‐way ANOVA followed by the LSD test. Statistical analysis was performed by SPSS 19.0. The data were considered significant when a value of p < 0.05. All the p‐values used in the text are unadjusted.

3. Results and Discussion

3.1. CBNPs Caused Histopathological Changes in the Maternal Lungs and Reached the Fetal Lungs

Firstly, histopathological changes in the maternal and feral lungs were examined upon CBNPs exposure. H&E staining of the maternal lung (Figure 1A) revealed significant structural changes and localized accumulation of CBNPs in the CBNPs exposure groups compared to the control group. In the CBNPs exposure groups, both sizes of CBNPs deposited in the maternal lungs destroyed alveoli, thickened the alveolar septum, and some of the alveolar cavities became solid with inflammatory cell infiltration. Masson's trichrome staining showed significant collagen deposition in CBNP30 and CBNP120 groups. There are no significant differences in collagen deposition between the two particle size groups (Figure S1). However, HE staining showed CBNP120 exposure induced more severe structural damage than CBNP30. CBNP120 thickened alveolar septa and disorganized cellular distribution. These changes are indicative of extensive inflammatory infiltration. Similarly, the maternal pulmonary toxic effects (e.g., inflammation, oxidative stress, and genotoxicity) of CBNPs have been fully demonstrated [9, 39].

FIGURE 1.

FIGURE 1

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Histopathology of maternal lung and fetal lung upon CBNPs exposure during pregnancy in mice. (A) Representative histopathological images of the maternal lung (n = 4); (B) Dark‐field hyperspectral image of fetal lung (n = 4); (C) Representative histopathological images of fetal lung (n = 4). The arrows indicate: black arrow, CBNPs in pulmonary alveoli or bronchiole; green arrow, macrophages with CBNPs; yellow arrow, alveolar walls and pulmonary mesenchyme thickened; blue arrow, alveolar fusion or collapse; red arrow, collagen fiber deposition.

Masson staining showed collagen fiber deposition and pathological changes of fibrosis in the maternal lung after exposure to both particle sizes of CBNPs (Figure 1A). A previous study also reported that carbon black nanoparticles could induce pulmonary fibrosis [40, 41]. Taken together, the fiber deposition hinted that fibrosis also participated in the process of CBNPs‐induced damage to the maternal lungs.

Dark‐field hyperspectral imaging (Figure 1B) demonstrated that both sizes of CBNPs were transferred to the fetal lung after exposure during pregnant mice. Our previous study showed that CBNPs could enter the mice's placenta [27]. Despite detectable nanoparticle deposition, H&E and Masson staining revealed no significant histopathological alterations in fetal lung tissue relative to controls (Figure 1C). These results indicate that transplacental CBNP transfer occurred without structural injury to the fetal lung. Prenatal lung development is reported to be sensitive to PM2.5 toxicity [42, 43]. We therefore examined whether transplacental CBNP transfer, which occurred without structural injury to the fetal lung, elicited subcellular alterations. CBNP30 additionally decreased Sod2 and Gpx1 expression (Figure S2A). SOD, catalase, and GPx constitute the primary enzymatic defense against superoxide radicals and hydrogen peroxide [44, 45]. The downregulation of these antioxidant enzymes indicates that transplacental CBNP exposure disrupts redox homeostasis in the fetal lung, despite the absence of overt histopathological damage.

We next examined oxidative stress and inflammation‐related proteins in fetal lungs. Western blot analysis showed elevated HMGB1, TXNIP, and ACSL4 protein levels following both CBNP30 and CBNP120 exposure (Figure S2B,C). HMGB1, a mediator of lung inflammation and tissue damage, has been reported to increase in offspring lungs after gestational PM2.5 exposure [34]. TXNIP regulates antioxidant activity [46], and can induce airway inflammation through NF‐κB activation [47]. ACSL4 promotes lipid peroxidation and serves as a marker of oxidative damage [48]. The upregulation of these proteins indicates that transplacental CBNP exposure activates inflammatory and oxidative stress pathways in the fetal lung, despite the absence of structural injury.

These results confirm that CBNP exposure caused overt injury in maternal lungs. In fetal lungs, the absence of histopathological changes was accompanied by detectable alterations in oxidative stress and inflammation markers at the mRNA and protein levels, indicating that transplacental CBNPs transfer elicited subcellular perturbations without structural damage.

3.2. CBNPs Exposure During Pregnancy Disturbed Metabolites in the Maternal Lungs and Fetal Lungs

Using CIL LC–MS, we profiled endogenous metabolite alterations in maternal and fetal lungs. PCA clearly separated the two tissue types, indicating distinct baseline metabolite compositions (Figure 3A). Within each tissue, PLS‐DA further distinguished metabolite profiles across control, CBNP30, and CBNP120 groups (Figure 3B), suggesting size‐specific metabolic disturbances. To reassure model validity, key performance parameters were assessed. PLS‐DA model metrics indicated adequate fit (maternal lung: R2Y = 0.78; fetal lung: R2Y = 0.77) but limited predictive reliability (Q 2 = 0.20 and 0.16, respectively) (Figure S3). The modest Q 2 values, together with PCA capturing 77.9% of variance, suggest substantial inter‐sample variability and potential noise within the dataset. These findings indicate that while size‐related metabolic separation is detectable, the global metabolic structure should be interpreted with caution given the inherent biological heterogeneity of lung tissue. The three‐tier approach was used to identify the metabolites [49]. After the removal of isomers, 816 and 811 metabolites were identified in the maternal lung and fetal lung, respectively (Figure 2C,D). 648 metabolites were identified in both the maternal lung and fetal lung. 163 metabolites were present only in the fetal lung, and 168 were present only in the maternal lung. Most of the metabolites belonged to the class of amino acids and derivatives (Figure 2C,D). The Manhattan plot showed that CBNP120 caused more apparent disruption of metabolites both in the maternal and fetal lungs. Furthermore, distinct metabolite profiles emerged between the CBNP30 and CBNP120 groups, with the fetal lung exhibiting the most significant divergence (Figure 2E). These observations indicate that CBNPs‐induced metabolic perturbations differed between maternal and fetal lungs, and that particle size further shaped the metabolic signature within each tissue type.

FIGURE 3.

FIGURE 3

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Differential metabolites were identified after CBNPs exposure during pregnancy in the maternal lung and fetal lung of mice. (A) Differential metabolites between CBNP30 group and the control group. (B) Differential metabolites between CBNP120 group and the control group.

FIGURE 2.

FIGURE 2

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CBNPs exposure during pregnancy disturbed metabolites in maternal lung and fetal lung of mice. (A) PCA score plot; (B) PLS‐DA score plot; (C, D) Classification of metabolites identified in maternal lung and fetal lung. (E) Differential metabolites identified in the maternal and fetal lung (n = 4–5).

3.3. Differential Metabolites Identified in the Maternal Lungs and Fetal Lungs of Mice Upon CBNPs Exposure During Pregnancy

CBNP‐induced metabolic alterations in maternal and fetal lungs are shown in Figure 3. The divergent patterns between the two tissues (Figure 3A) suggest distinct modes of response, likely reflecting differences in tissue maturity or metabolic capacity. In maternal lungs, CBNP30 exposure altered histamine (a proinflammatory mediator) [50], aminomalonic acid (indicative of oxidative stress) [51], and 7′‐carboxy‐alpha‐tocotrienol (a variant of vitamin E functioning as an antioxidant) [52], pointing to disturbance of inflammatory and redox pathways. These metabolic alterations are consistent with the histopathological findings in maternal lungs. They also align with elevated oxidative stress markers (HMGB1, TXNIP, ACSL4) in fetal lungs (Figure S2). Together, these findings suggest that CBNP exposure perturbs redox‐associated metabolism in both tissues, albeit with distinct metabolic profiles.

CBNP120 exposure altered several metabolites linked to cellular and metabolic stress. 1,4‐diaminobutane and N8‐acetyl‐spermidine, associated with cell proliferation and differentiation [53, 54], were perturbed alongside increased acetylcarnitine, a marker of injury [55]. 2‐hydroxybutyric acid, elevated in conditions involving lipid oxidation [56, 57], and AMP, a neutrophilic inflammation indicator [58], increased in maternal lung. Amino acid metabolism was broadly affected, with histidine pathway alterations consistent with oxidative stress responses [59, 60], and butanoate metabolism changes suggesting shifts in energy metabolism. These findings point to CBNP120‐induced metabolic stress in both maternal and fetal compartments, though the specific pathways affected differed by tissue.

Amino acids and lipids were the primary differential metabolites between CBNP30 and CBNP120 groups in fetal lungs, whereas maternal lungs showed no clear size‐specific metabolic separation. This suggests that fetal lungs may be more sensitive to particle size in terms of metabolic response. Among the lipid alterations, 9(S)‐HOT, 13(S)‐HPOT, and 9(10)‐EpOME were mapped to linoleic acid and alpha‐linolenic acid metabolism. Linoleic acid is a precursor for arachidonic acid, which generates pro‐inflammatory eicosanoids including prostaglandins. Elevated linoleic acid metabolism has been associated with increased inflammatory potential [61]. The observed changes in fatty acid metabolism, together with altered ACSL4 protein levels [62], are consistent with CBNP‐induced lipid peroxidation and redox imbalance in both maternal and fetal compartments.

Table S3 summarizes tissue‐specific and shared differential metabolites. Three metabolites, 7′‐carboxy‐alpha‐tocotrienol, 3‐hydroxy‐2‐methylbutanoic acid, and 3‐hydroxyisobutyric acid were altered in both maternal and fetal lungs following CBNP120 exposure. 3‐Hydroxyisobutyric acid has been associated with oxidative stress [63], suggesting that CBNP120 exposure perturbs redox‐related metabolism in both tissues.

CBNPs exposure altered maternal and fetal lung metabolites in patterns consistent with oxidative stress and inflammatory responses, with the profile of disturbance differing by particle size. CBNP120 elicited more pronounced metabolic shifts than CBNP30, particularly within lipid metabolism pathways in both maternal and fetal lungs.

3.4. Pathway Enrichment of Differential Metabolites in Maternal and Fetal Lungs Upon CBNPs Exposure During Pregnancy in Mice

KEGG enrichment analysis identified pathway alterations associated with CBNP exposure. In maternal lungs, histidine and vitamin B6 metabolism were enriched following both CBNP30 and CBNP120 exposure (Figure 4), consistent with inflammatory responses [64, 65]. CBNP120 additionally altered branched‐chain amino acid metabolism and arachidonic acid metabolism [66]. Across all exposure groups, disrupted pathways spanned nucleotide, cofactor, lipid, amino acid, energy, and carbohydrate metabolism. In fetal lungs, histidine metabolism was shared with maternal tissue after CBNP30 exposure, whereas butanoate and arachidonic acid metabolism were altered after CBNP120 exposure (Table S4). These findings suggest partial overlap in pathway responses between maternal and fetal compartments, with particle size influencing the specific metabolic profile in each tissue.

FIGURE 4.

FIGURE 4

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Pathway enrichment of differential metabolites in the maternal and fetal lung upon CBNPs exposure during pregnancy in mice. (A) KEGG analysis in differential metabolites of the CBNP30 group compared to the control group; (B) KEGG analysis in differential metabolites of the CBNP120 group compared to the control group; (C) KEGG analysis in differential metabolites of the maternal lung; (D) KEGG analysis in differential metabolites of the fetal lung.

In fetal lungs, CBNP30 exposure enriched histidine metabolism, which has been associated with inflammatory responses [65]. Elevated eicosanoid precursors including prostaglandins J2 and C2 suggested metabolic changes consistent with inflammation prior to detectable structural alterations [67]. Both particle sizes affected arachidonic acid metabolism, though the broader metabolic impact was more pronounced in fetal than maternal lungs, encompassing glutathione metabolism, the TCA cycle, and linoleic acid metabolism. Amino acids such as glycine and tryptophan have been noted for their role in reducing oxidative stress, inhibiting apoptosis, and modulating immune responses [68], and energy metabolism perturbations reflected by TCA cycle and glyoxylate/dicarboxylate pathway alterations [69]. Disturbance in AA metabolism was linked to inflammatory responses [70]. The exhaustion of glutathione‐related precursors identified in our CIL LC–MS profiles offers a metabolic basis for the downregulated Gpx1 and Sod1 mRNA levels observed in fetal tissues [71]. Alterations in arachidonic acid and linoleic acid derivatives are of particular relevance given their reported involvement in pregnancy outcomes [72].

CBNP exposure elicited distinct, particle size‐related metabolic alterations in maternal and fetal lungs. Maternal tissue showed enrichment in histidine and vitamin B6 metabolism with both particle sizes, and additional branched‐chain amino acid and arachidonic acid pathway changes with CBNP120. The fetal lungs are enriched in fewer pathways. These subcellular metabolic shifts in fetal tissue aligned with changes in oxidative stress markers, despite absent structural injury.

3.5. Metabolic Correlation Network of Differential Metabolites in Maternal and Fetal Lungs Upon CBNPs Exposure During Pregnancy in Mice

Metabolic network analysis revealed a substantially larger perturbation network for CBNP120 (101 nodes, 668 edges) than for CBNP30 (29 nodes, 44 edges). Amino acids and their derivatives constituted the primary metabolite class in both networks, pointing to a shared metabolic target across particle sizes (Figures 5 and 6).

FIGURE 5.

FIGURE 5

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Maternal‐fetal lung correlation network of the differential metabolites upon CBNP30 exposure during pregnancy in mice. (A) Network of differential metabolites; (B) hub metabolites in maternal and fetal lung upon CBNP30 exposure; (C) hub metabolites pathway enrichment upon CBNP30 exposure.

FIGURE 6.

FIGURE 6

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Maternal‐fetal lung correlation network of the differential metabolites upon CBNP120 exposure during pregnancy in mice. (A) Network of differential metabolites; (B) hub metabolites in maternal and fetal lung upon CBNP120 exposure; (C) hub metabolites pathway enrichment upon CBNP120 exposure.

Network hub analysis identified distinct central metabolites in maternal and fetal lungs following CBNP30 exposure. In maternal tissue, 11‐deoxycortisol was a primary hub, correlating strongly with histamine (Figure 5B), consistent with steroid‐related stress responses [73, 74, 75]. In fetal lung, L‐isoglutamine, prostaglandin J2, and nordeoxycholic acid served as hub metabolites. Complete hub lists and correlation matrices are provided in Figures 5 and 6.

To integrate metabolite findings with transcriptional data, we quantified rate‐limiting enzymes from hub‐implicated pathways by qPCR (Figure S4). This narrowed the analysis to pathways supported by concordant metabolite and mRNA changes (Table S5). In the CBNP30 group, histidine metabolism and lysine biosynthesis showed alterations at both levels. In the CBNP120 group, butanoate metabolism and branched‐chain amino acid degradation displayed concordant changes, whereas propanoate metabolism shifted only at the metabolite level. These findings are summarized in Figure 7.

FIGURE 7.

FIGURE 7

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Disturbed metabolic pathways in the fetal lung upon CBNPs exposure during pregnancy in mice. Green dotted circle (histidine metabolism and lysine biosynthesis): CBNP30‐related disturbed metabolites; Blue dotted circle (propanoate metabolism, butanoate metabolism, and valine, leucine and isoleucine degradation): CBNP120‐related disturbed metabolites; Enzyme: MRNA expression of key enzymes in metabolic pathways (blue bold: downregulation; red bold: upregulation; black: unchanged).

A limitation of this study is the use of intratracheal instillation, which delivers a cumulative bolus dose rather than the continuous low‐dose inhalation typical of ambient air pollution or cigarette smoking. The 200 μg exposure, based on maximum exposure, corresponds to high‐exposure scenarios in mice, such as heavy tobacco use or severe industrial settings. These findings therefore reflect acute, high‐dose biological responses and are not directly applicable to quantitative risk estimates for general population exposure.

4. Conclusions

This study examined maternal and fetal pulmonary responses to prenatal exposure to 30 and 120 nm carbon black nanoparticles. Both particle sizes deposited in maternal lung tissue and were associated with inflammatory and fibrotic changes, and both crossed the placental barrier. Maternal lungs exhibited structural damage, whereas fetal lungs showed no histological injury but displayed particle size‐specific metabolic alterations, including shifts in energy metabolism, arachidonic acid metabolism, and redox‐associated pathways. These findings indicate that CBNPs‐induced fetal pulmonary perturbations occur primarily at the metabolic level, with biochemical changes decoupled from tissue architecture.

Author Contributions

Heqing Shen conceived and designed the study, led the research team, and obtained the funding. Heqing Shen and Yi Lin revised the manuscript. Jing Li and Biling Gao were responsible for conducting the entire experimental process, obtaining data, creating plots, and writing the manuscript. Hao Chang analyzed the metabolomic data, contributing valuable scientific research ideas. Jingjing Zhan and Zhonghua Lu assisted with both qualitative and quantitative analyses. Zehua Xu aided in the collection of animal samples. Jia Li contributed ideas during the metabolomics analysis process. Jia Li provided the instrument testing platform and technical support.

Funding

This work was supported by MOST | National Natural Science Foundation of China (NSFC), 22076158, 22576173, 91543113; Natural Science Foundation of Fujian Province (Fujian Natural Science Foundation), 2025J01762; Start‐up funding for high‐level talent research of Fujian Medical University, XRCZX2024028.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Grouping of exposure conditions for pregnant mice and the number included in each group for analysis.

Table S2: Primers used in qRT‐PCR analysis.

Table S3: Tissue‐specific and shared differential metabolites in maternal and fetal lung.

Table S4: Tissue‐specific and shared metabolism pathways in maternal and fetal lung.

Table S5: Concordance between metabolite changes and mRNA expression of key enzymes in perturbed metabolic pathways in fetal lungs following prenatal CBNP exposure.

Figure S1: Pathological score and Masson staining quantitative diagram of maternal lung. Data were expressed as mean ± SD (n = 3 for H&E, n = 4 for Masson staining). Compared with the control group: *p < 0.05, **p < 0.01.

Figure S2: CBNPs exposure during pregnancy caused oxidative stress and inflammation in fetal lungs. (A) Expression of oxidative stress‐related mRNA (n = 3). (B, C) Expression level of HMGB1, TXNIP, ACSL4 protein (n = 4). All data were expressed as mean ± SD. Compared with the control group: *p < 0.05, **p < 0.01.

Figure S3: PLS‐DA score plots and validation plot of the model obtained from 200 permutations in maternal lung (A), and fetal lung (B).

Figure S4: mRNA expression of key enzymes in the metabolic pathways. n = 3. All data were expressed as mean ± SD. Compared with the control group: *p < 0.05.

FSB2-40-e72202-s003.docx (221.7KB, docx)

Dataset S1

FSB2-40-e72202-s002.xlsx (194.9KB, xlsx)

Dataset S2

FSB2-40-e72202-s001.xlsx (210.6KB, xlsx)

Acknowledgments

We thank the Public Technology Service Center at Fujian Medical University and for giving their technical support in this study.

Contributor Information

Jia Li, Email: lisa02252@163.com.

Heqing Shen, Email: hqshen@xmu.edu.cn.

Data Availability Statement

The mass spectrometry metabolomics data have been deposited in the China National Center for Information (https://ngdc.cncb.ac.cn/omix/preview/p7o1y0iA).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1: Grouping of exposure conditions for pregnant mice and the number included in each group for analysis.

Table S2: Primers used in qRT‐PCR analysis.

Table S3: Tissue‐specific and shared differential metabolites in maternal and fetal lung.

Table S4: Tissue‐specific and shared metabolism pathways in maternal and fetal lung.

Table S5: Concordance between metabolite changes and mRNA expression of key enzymes in perturbed metabolic pathways in fetal lungs following prenatal CBNP exposure.

Figure S1: Pathological score and Masson staining quantitative diagram of maternal lung. Data were expressed as mean ± SD (n = 3 for H&E, n = 4 for Masson staining). Compared with the control group: *p < 0.05, **p < 0.01.

Figure S2: CBNPs exposure during pregnancy caused oxidative stress and inflammation in fetal lungs. (A) Expression of oxidative stress‐related mRNA (n = 3). (B, C) Expression level of HMGB1, TXNIP, ACSL4 protein (n = 4). All data were expressed as mean ± SD. Compared with the control group: *p < 0.05, **p < 0.01.

Figure S3: PLS‐DA score plots and validation plot of the model obtained from 200 permutations in maternal lung (A), and fetal lung (B).

Figure S4: mRNA expression of key enzymes in the metabolic pathways. n = 3. All data were expressed as mean ± SD. Compared with the control group: *p < 0.05.

FSB2-40-e72202-s003.docx (221.7KB, docx)

Dataset S1

FSB2-40-e72202-s002.xlsx (194.9KB, xlsx)

Dataset S2

FSB2-40-e72202-s001.xlsx (210.6KB, xlsx)

Data Availability Statement

The mass spectrometry metabolomics data have been deposited in the China National Center for Information (https://ngdc.cncb.ac.cn/omix/preview/p7o1y0iA).

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