Integrated mRNA- and miRNA-sequencing analyses unveil the underlying mechanism of tobacco pollutant-induced developmental toxicity in zebrafish embryos

doi:10.21203/rs.3.rs-3728517/v1

Download PDF

Research Article

Integrated mRNA- and miRNA-sequencing analyses unveil the underlying mechanism of tobacco pollutant-induced developmental toxicity in zebrafish embryos

https://doi.org/10.21203/rs.3.rs-3728517/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 08 Mar, 2024

Read the published version in Journal of Translational Medicine →

You are reading this latest preprint version

Tobacco pollutants are prevalent in the environment, leading to inadvertent exposure of pregnant females. Although there are studies examining the toxic effects of these pollutants on the development, they are not yet able to fully elucidate the potential underlying mechanisms. Therefore, in this study, we aimed to investigate the developmental toxicity induced by cigarette smoke extract (CSE) at concentrations of 0.25%, 1%, and 2.5% using a zebrafish embryo toxicity test and integrated transcriptomic analysis of microRNA (miRNA) and messenger RNA (mRNA). The findings revealed that CSE caused developmental toxicity, including increased mortality and decreased incubation rate, in a dose-dependent manner. Moreover, CSE induced malformations and apoptosis, specifically in the head and heart of zebrafish larvae. mRNA- and miRNA-sequencing analyses were used to compare changes in the expression of genes and miRNAs in zebrafish larvae. The results of bioinformatics analysis indicate that the mechanism underlying CSE-induced developmental toxicity was associated with genetic material damage, apoptosis disorder, as well as lipid metabolism disturbance. Morever, the enrichment analysis and RT-qPCR analysis results shown ctsba gene play crucial function in embryo developmental apoptosis, whereas fads2 gene mainly regulated lipid metabolic toxicity. The results of this study improve the current understanding of CSE-induced developmental toxicity in zebrafish embryos and contribute insights for the formulation of novel preventive strategies against tobacco pollutants during early embryonic development.

cigarette smoke extract

RNA-seq

DNA damage repair

cell apoptosis

lipid metabolism

Tobacco consumption remains prevalent, fueled by the growth of the nicotine-addicted population and smokers in low- and middle-income countries [1]. Combustible or non-combustible tobacco products, such as conventional cigarettes and e-cigarettes, produce smoke and aerosol components, respectively. Gas components produced by tobacco, including nicotine, nitrosamines, carbonyls, aromatic volatile organic compounds, and trace metals, share similarities and can pose risks to human health [2, 3]. Furthermore, tobacco smoke, even when dissipated, continuously accumulates on object surfaces, dust, and air, creating potential exposure routes through inhalation, ingestion, and skin transfer [4]. Tobacco pollutants can also permeate nonsmoking areas through shared spaces [5]. Even long after tobacco smoke dissipating, tobacco pollutants remain at high levels, with a potential for an increase in concentrations [6]. Therefore, the universality and persistence of tobacco pollutants lead to inadvertent exposure of individuals within their living environments.

As age increases and fertility rates decrease, pregnant females prioritize preventing pregnancy-related risks to develop healthy children. Research indicates that tobacco pollutant exposure remains a significant factor in embryonic dysplasia during pregnancy [7]. This exposure increases the incidence of adverse embryonic developmental events such as low birth weight, head and facial malformations, heart defects, and embryonic death [8–10]. The zebrafish pattern is often used to evaluate the correlation between toxic material exposure and embryonic development toxicity. Therefore, the zebrafish pattern is particularly suitable for studying tobacco pollutant-induced developmental toxicity because of its low cost and direct observability of embryonic development processes [11, 12]. While some studies on tobacco pollutant exposure have focused on the developmental toxicity induced by a single component of a tobacco pollutant mixture such as nicotine [13, 14], tobacco smoke is a mixture of over 9,500 chemical components [15]. Therefore, pertinent studies exclusively examining a single component fall short of representing the global effects of tobacco pollutant exposure on embryos [16]. Moreover, although studies using tobacco pollutant mixture have been conducted, most have focused on visible embryonic developmental toxicity, such as malformations, with a limited exploration into the specific molecular mechanisms involved in these processes [17, 18]. Therefore, studying possible underlying molecular mechanisms and the overall effects of tobacco smoke exposure on embryonic development can address the limitations of existing studies and help in the formulation of new and effective preventive measures during pregnancy.

High-throughput sequencing technologies, such as messenger RNA (mRNA) or microRNA (miRNA) sequencing, provide effective tools for studying global transcriptomic changes and mechanisms in zebrafish embryo-larvae in response to harmful external stimuli [19, 20]. Recently, changes in the mRNA transcriptome during developmental toxicity in zebrafish have been described [21, 22]. miRNA, which controls the expression of mRNAs by inhibiting translation or directly grading transcription processes, plays a fundamental role in response to symbiotic stress, which is essential for toxicology research and is gradually gaining attention from researchers [23, 24]. However, the change in miRNA and mRNA expression profiles in response to tobacco smoke stress during the early embryonic development of zebrafish has not yet been described. Therefore, conducting a detailed comparative transcriptome analysis of miRNA and mRNA expression in zebrafish embryos exposed to varying concentrations of tobacco is essential. This endeavor will contribute to a deeper understanding of the underlying mechanism of tobacco pollutant-induced developmental toxicity.

To better understand the effects of tobacco exposure on zebrafish embryonic development in this study, zebrafish embryos were treated with different concentrations of cigarette smoke extract (CSE) to assess developmental toxicity. Sequencing technology was used to compare the expression profiles of miRNAs and mRNAs in zebrafish larvae, followed by comprehensive analysis. Subsequently, the expression of crucial genes was determined using quantitative real-time PCR (RT-qPCR). The combination of the results of embryotoxicity tests and bioinformatics analyses will improve the understanding of the potential mechanisms underlying developmental toxicity induced by cigarette smoke and provide a theoretical basis for formulating effective measures against tobacco pollutants during pregnancy.

2.1 Chemicals and reagents

Following a previously described protocol[25], CSE was freshly prepared from filtered cigarettes within 30 min of use. The acquired CSE suspension was yellowish with an optical density of 0.506 ± 0.008 at 405 nm. The CSE was adjusted to a pH value of 7.4 and filtered using a 0.22 µm filter. The final product was considered to have a concentration of 100% and was diluted to the desired concentrations for further experiments. TRIzol reagent was purchased from Tiangen (Nanjing, China). The PrimeScript RT reagent Kit with gDNA Eraser was purchased from Thermo Scientific (MD, USA), the SYBR Green I kit was purchased from Takara Bio Inc. (Shiga, Japan), and the other reagents used in this study were purchased from Beyotime Biotechnology (Jiangsu, China).

2.2 Zebrafish maintenance and CSE exposure

Adult zebrafish (wild-type AB strain, Danio rerio) were used in this study. Zebrafish were maintained under a standard temperature of 28 ± 1 ℃, pH 7.2 ± 2.0, with a lighting cycle of 14 h of light and 10 h of darkness daily. The zebrafish were fed hatched brine shrimp and dry flake food (Nutreco Skretting) twice daily. To obtain fresh embryos, healthy and sexually mature zebrafish were placed in a feeding box at female-to-male ratios of 1:1 or 1:2 for mating and spawning. Animal experiments were approved by the Animal Ethics Committee of Shantou University Medical College (Approval No.: SUMC2022-062).

Two hours post-fertilization (hpf), normally developed embryos were selected and randomly distributed in a 10 cm petri dish and treated with corresponding concentrations of CSE (0, 0.25%, 1%, and 2.5%). CSE exposure concentrations were chosen according to the lethal concentration 50 and effective concentration 50 values of embryonic zebrafish. After screening for death and deformity, the embryos were placed in 96-well plates (50 embryos per group) at 48 hpf for subsequent CSE exposure experiments up to 7 days post-fertilization (dpf). All experiments were repeated thrice.

2.3 Effects of CSE on the survival and developmental status of zebrafish

The number of dead zebrafish embryos at different concentrations was recorded every 24 h using a stereomicroscope (SZX7, Olympus, Tokyo, Japan). The heart rate (beats/min) of 10 larvae from each group was measured using a stereomicroscope at 96 hpf and recorded using a stopwatch for 1 min. The number of hatched and deformed embryos was recorded to calculate the percentage of larval survival, larvae hatched, and larval malformation (all at 96 hpf).

The developmental conditions of ten randomly selected zebrafish for each group were observed at 72 hpf and 7 dpf. The larvae were anesthetized using tricaine, and morphological changes were photographed and recorded using a stereomicroscope. This encompassed observations of developmental indicators, including morphological changes in the head, presence of pericardial edema, delayed yolk sac absorption, spinal curvature, uneven distribution of pigmentation, development of swim bladders, and shortening of body length.

2.4 Acridine orange staining

Acridine orange (AO) staining of 96-hpf larvae was performed to detect cell apoptosis. Briefly, larvae were washed with phosphate buffer saline (PBS) twice, incubated with 5 mg/mL AO at 28 ℃ in the dark for 20 min, and washed thoroughly with PBS thrice, as described previously. Images of the zebrafish embryos were captured using an inverted fluorescence microscope (Zeiss, USA) and processed using ImageJ software (Ver. 1.52a; NIH, Bethesda, MD, USA).

2.5 Global transcriptome sequencing (mRNA- and miRNA-seq) analysis

At 7 dpf, the larvae were sacrificed using an overdose of a buffered anesthetic solution of tricaine methanesulfonate (0.03% MS-222; Sigma-Aldrich, USA). Larvae (15–20/pool) were collected, flash-frozen in liquid nitrogen, and stored at − 80 ℃ before molecular analyses. TRIzol reagent was used to isolate total RNA from each sample. High-throughput full transcriptome and miRNA sequencing analyses were performed at Applied Protein Technology Co., Ltd. (Shanghai, China).

2.6 Integrated bioinformatics analyses of the sequencing results

The expression value of miRNA- and mRNA-seq in reads per million and fragments per kilobase of exon model per million mapped fragment, respectively, were acquired after cleansing, mapping, and quantitative analysis of the raw sequencing results, which were completed by Applied Protein Technology Co., Ltd., China. The R packages DESeq and edgeR were used for differential expression analysis of mRNAs and miRNAs between the control and CSE-exposed treatments, respectively. Differentially expressed genes (DEGs) and miRNAs (DEMs) were identified using an adjusted p-value (padj) or a false discovery rate < 0.05 and |log2(fold change)| > 1. Subsequently, DEGs of each treatment were uploaded to STRING (http://string-db.org/) to determine their interaction, which was visualized using Cytoscape software. The hub genes in the protein-protein interaction network with the highest Molecular Complex Detection score were obtained using the Cytoscape plug-in CytoHubba. Finally, DEM target genes were predicted from the miRWalk database (http://mirwalk.umm.uni-heidelberg.de).

Functional enrichment analyses of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and GeneOntology (GO) were conducted for three datasets with different concentrations of CSE-regulated DEGs, and hub genes. KEGG and GO enrichment results with a p-value of < 0.05 were considered statistically significant. Networks were established to reveal the relationship between enriched genes and the significant functional enrichment results of the DEGs. In addition, gene set enrichment analysis (GSEA) was conducted for three datasets with different concentrations of CSE-regulated genes and DEM target genes. The screening conditions for GSEA were as follows: |normalized enrichment score (NES)| > 1, padj < 0.25, and p-value < 0.05. To explore the potential relationship between miRNAs and mRNAs, we first crossed the significant GSEA-KEGG results of DEM target genes and all CSE-regulated genes to obtain the candidate KEGG pathway and constructed miRNA–mRNA–pathway regulatory networks with miRNA as a decoy, mRNA as a center, and pathway as a target. These networks were visualized using Cytoscape software. We used the intersection of significant GO terms between miRNA and mRNA levels to demonstrate the GO results using upset and dot plots.

2.7 RT-qPCR for verification

Total RNA was extracted using TRIzol reagent and subjected to complementary DNA (cDNA) synthesis using the RevertAid First Strand cDNA Synthesis Kit. RT-qPCR was performed using SYBR Green I according to the manufacturer’s instructions. The primer sequences are listed in Supplementary Table 1.

2.8 Statistical analysis

Data are expressed as mean ± standard deviation obtained from experiments with three replicates of different CSE concentrations. One-way analysis of variance with Dunnett's post-hoc test was used to analyze the effects of CSE exposure on developmental toxicity. Differences were considered statistically significant at p < 0.05. SPSS software (version 27.0; SPSS, Chicago, IL, USA) was used for statistical analyses.

3.1 Embryonic development toxicity of CSE

The treatment groups exhibited lower hatching and survival rates and higher mortality rates than the control group, specifically in the 2.5% CSE treatment group at 96 hpf (Fig. 1A-C). The results were significant in a dose-dependent manner in zebrafish embryos following exposure to CSE (0.25%, 1%, and 2.5%) compared to those in the control group. In addition, the heart rates in the 0.25%, 1%, and 2.5% CSE groups were higher than those in the control group at 96 hpf (Fig. 1F).

Typical malformations, such as morphological changes in the head, presence of pericardial edema, delayed yolk sac absorption, spinal curvature, uneven distribution of pigmentation, absence of swim bladders, and shortening of body length at 72 hpf and 7 dpf, are shown in Supplementary Fig. S1.

3.2 Effects of CSE exposure on larvae injury and cell apoptosis

AO staining assay was conducted to determine the occurrence of cell apoptosis following CSE exposure. At 72 hpf, no distinct apoptotic cells were observed in the control group. However, in the CSE-exposed treatment groups, apoptotic cells primarily occurred in the head, heart, and surrounding tissues, indicating notable concentration dependence (Fig. 1D). The bax/bcl-2 genes serve as markers of apoptosis, and an imbalance in their expression plays an important role in apoptosis. To further investigate the mechanisms underlying CSE-induced apoptosis, we assessed the expression level of bax/bcl-2. CSE exposure significantly decreased the expression of bcl-2 mRNA (anti-apoptotic gene) but increased that of bax (pro-apoptotic gene) in larval zebrafish (Fig. 1G). These results indicate a strong correlation between cell apoptosis and the injury observed in zebrafish larvae following CSE exposure.

3.3 Changes in the transcriptome profile of miRNA and mRNA following CSE exposure

The number of DEMs initially increased and then decreased as CSE concentration increased. However, the number of DEGs initially decreased and then increased (Fig. 2A, B). miRNA and mRNA transcription were more affected in the experimental groups than in the control group. Exposure to 2.5% CSE yielded a greater effect on gene expression than exposure to the other two concentrations, although 1% CSE exerted a greater effect on miRNA expression. In total, 402 DEMs were identified, among which 57.7% were upregulated and 42.3% were downregulated (Fig. 2C). Simultaneously, 490 DEGs were identified, among which 81.0% were upregulated and 19.2% were downregulated (Fig. 2D). The volcano plot (Supplementary Fig. S2) provides a visual representation of the miRNAs and mRNAs in each concentration group, highlighting the top 10 DEMs or DEGs with significant differences.

3.4 Enrichment analysis of DEGs

To elucidate the mechanism underlying CSE-induced damage, we conducted a functional analysis of DEGs at various doses. KEGG enrichment analysis showed that the 2.5% CSE treatment group was enriched with more pathways for upregulated DEGs and fewer for downregulated DEGs than the other two groups. Specifically, the 2.5% CSE treatment group showed significant upregulation of pathways related to cellular processes and the immune system. Only two pathways—"Phototransduction" and "Biosynthesis of unsaturated fatty acids"—were downregulated. Lipid metabolism-related pathways were primarily downregulated in the 0.25% and 1% CSE treatment groups, which were similar. Notably, alpha-Linolenic acid metabolism involved in all three experimental groups (Fig. 3A, B). To further elucidate the primary functions of these pathways, we employed gene-term network graphs to demonstrated the complex relationships between terms in corresponding exposure to different concentrations of CSE and identified key genes associated with changes in CSE concentration (Fig. 3C, D).

The GO results revealed notable similarities in the up-regulated terms between the 1% and 2.5% CSE treatment groups. Meanwhile, the 0.25% and 1% CSE treatment groups exhibited clear parallelism in the down-regulated terms. Specifically, among the up-regulated outcomes, processes and activities related to potassium ion transport were significantly enriched in the 0.25% CSE treatment group. Nevertheless, as the concentration increased, the 2.5% CSE treatment group was mostly associated with components involved in cell transport, namely vesicle and lysosome. Both the 1% and 2.5% CSE treatment group had comparable enrichment results in response to biological stimuli (Fig. 4A). In the down-regulated outcomes, serine-related enzymes and metallocarboxypeptidase activity were commonly observed in all experimental groups. The 0.25% CSE treatment group primarily exhibited involvement in nucleotide synthesis, lipid transport, and photoreceptors components. Notably, lipid transport processes, such as secretion and transport of arachidonic acid, were also down-regulated in the 1% CSE treatment group, while photoreceptors components were enriched in downregulated DEGs in the 2.5% CSE treatment group. Additionally, downregulated visual conduction was particularly evident in the 2.5% CSE treatment group (Fig. 4B). Interestingly, all three experimental groups demonstrated a greater negative regulation of the damage response but with down-regulated responses to chemical stimuli (Fig. 4). Similarly, we constructed a gene-GO term network diagram which visibly demonstrated how certain genes, through their regulatory functions, establish connections between distinct biological processes, cellular components, and molecular functions (Fig. 5). This visual representation offers a comprehensive overview of the complex interactions influenced by CSE treatment at different concentrations.

To further explore the underlying mechanism through which CSE affects coordinated embryonic development, we performed GSEA for all genes. Down-regulated pathways related to DNA damage repair were significant in all treatment groups, further consummating the KEGG and GO functional enrichment results (Fig. 6). In addition, upregulated GSEA results were almost consistent with upregulated functional enrichment results such as lysosome and apoptosis in the 2.5% CSE treatment group .

3.5 Enrichment analysis of DEM target genes

We initially identified the DEM target genes and subsequently conducted GSEA analyses of the genes to evaluate the association between responsive miRNAs and mRNAs. To identify the most valuable pathways, we first utilized a Venn diagram to illustrate the overlap between the significantly enriched GSEA-KEGG results in the miRNA and mRNA groups (Fig. 7A, B, C). These significant intersected KEGG terms were elucidated using miRNA-mRNA-pathway regulatory networks. The number of significantly overlapped pathways, DEMs, and its target genes steadily declined as the concentration of CSE exposure rose, whereas the number of DEGs increased. Furthermore, the experimental groups eventually saw a rise in the prevalence of up-regulated pathways, but the down-regulated pathways, largely associated to DNA repair, decreased with an increase in CSE concentration. Notably, apoptosis and lysosome were two pathways that were significantly up-regulated over the whole bioinformatics analysis. However, the lipid metabolism manner was only significantly activated in the 2.5% CSE concentration group in this case, whereas in the prior bioinformatics study, it had been strongly correlated with the 0.25% and 1% CSE concentration groups (Fig. 7D, E, F). The noteworthy GO results from the intersection are equally remarkable. As illustrated by the upset plot, the number of GO terms at the intersection gradually decreased with concentration increased (Fig. 8A, B, C). Furthermore, at the miRNA and mRNA levels, the important pathways with high NES rankings remained largely consistent (Fig. 8D, E, F). The significant enrichment outcomes obtained from the intersection at both the miRNA and mRNA levels hold paramount importance.

3.6 Validation of significant DEG expression using RT-qPCR

The RT-qPCR validation analysis revealed that the expression levels of klf2a, socs3a, ddit4, fkbp5, ahsg2, fads2, and ctsba were consistent with the changes observed in the mRNA-seq transcriptome across the three concentration groups (Fig. 9A). Additionally, the expression levels of the selected genes, determined through qPCR, had a significant positive correlation with the expression levels of genes obtained from the mRNA-seq data across all three CSE concentration groups (0.25% CSE group: R = 0.72, p = 0.07; 1% CSE group: R = 0.97, p = 0.00027; 2.5% CSE group: R = 0.9, p = 0.0052) (Fig. 9B).

As environmentally persistent and inevitable pollutants, tobacco smoke and its residues have been proven to be significant environmental contaminants, posing potential health hazards for pregnant females [3, 7]. However, the precise mechanism through which tobacco pollutants affect embryonic development is yet to be fully elucidated. Therefore, this study was undertaken to elucidate the possible underlying mechanisms of CSE-induced developmental toxicity in zebrafish embryos through the combination of developmental indicators, apoptosis, and bioinformatics analysis.

Our results regarding CSE-induced embryotoxicity and apoptosis indicated a dose-response trend similar to that observed in other animals, such as mice and earthworms [26–28]. As a single component in the CSE, nicotine and polycyclic aromatic hydrocarbons are considered teratogenic components of craniofacial malformations and yolk sac edema, respectively, suggesting their importance in tobacco pollutant-induced teratogenesis [13, 29]. The AO staining results indicated the head and heart as the primary target organs affected by CSE exposure. Two target organs showed abnormal shape and significant apoptosis, hinting that their abnormal development might be closely related to cell death events. Notably, in contrast to the decrease in heart rate caused by electronic cigarettes, CSE induced an increase in heart rate, possibly due to compensatory events related to CSE-induced cardiac injury [30]. However, these findings alone do not fully explain the complexities underlying CSE-induced embryonic developmental toxicity. To better understand these mechanisms, we assessed the dysregulation related to genetic damage repair, apoptosis, and lipid metabolism, taking into account the impact of miRNA and mRNA levels, as well as the influence of varying CSE concentrations.

Numerous harmful components in CSE, including nicotine and benzo[a]pyrene, target cellular DNA and damage it [31–33]. In response to this harm, cells launch a sequence of repair processes aimed at mending the damaged DNA and shielding the cell from further harm [34–36]. However, the GSEA enrichment analysis of both the miRNA and gene levels revealed multiple downregulations associated with DNA repair processes, including base excision repair, mismatch repair, and nucleotide excision repair. These findings are consistent with earlier research showing that components of cigarette smoke inhibit DNA repair [33–35]. Among them, as oxidative stress increases, the base excision repair capacity, which plays a crucial part in preventing DNA oxidative damage from being exposed to cigarette smoke, decreases, aggravating DNA damage [37]. The preservation of cellular life activities and genomic stability may be impacted by the delayed damage repair caused by malfunctioning of these repair mechanisms [35, 36]. The effects of cigarette smoke on genetic material can have detrimental effects, particularly during embryonic development. For embryos to develop and expand normally, precise gene regulation is necessary [38, 39]. Therefore, in addition to damaging genetic material, cigarette smoke also reduces the body's capacity for repair, which could be a contributor in embryonic growth delays, deformities, or even death. Furthermore, in line with Park et al.'s findings, GSEA analysis revealed that RNA metabolic processes such ribosome manufacturing, transcription, and translation displayed dynamic alterations akin to those of DNA damage repair [40]. RNA metabolism is critical in DNA damage repair, and its disruption can reduce the effectiveness of restoring damaged genetic material. These results uncover intricate regulatory mechanisms by which CSE affects genetic material damage and repair simultaneously, identifying genotoxicity as an essential step in CSE-induced developmental process.

Apoptosis, a useful process by which the body gets rid of damaged cells and encourages proper development, can be triggered by responding to the genetic material damage [41, 42]. However, we discovered that the improper activation of apoptosis grew progressively with higher CSE concentration in the experimental and bioinformatics data, which is consistent with the facts that CSE chemicals greatly heighten cell death and harm the potential for embryonic development [43–45]. Therefore, cell apoptosis is now considered to be a major progress impairing embryonic development rather than merely a mechanism for correcting genetic material damage. Early embryonic development depends on cell death, which functions as a fine sculpting tool [46]. Previous studies on toxicant-induced developmental defectshave underscored excessive embryonic cell death as a pivotal precursor to structural abnormalities [47]. With a focus on a number of genes encoding lysosomal cysteine proteases including ctsba, ctsc, and ctsk, additional investigation of gene-term networks showed a strong association between apoptosis and lysosome. Enzymes involved in cell processes such as apoptosis, tissue remodeling, and cell proliferation are encoded by these genes [48, 49]. Specifically, cathepsin B, a distinct dipeptidyl peptidase that is the predominant execution protease in programmed cell death, is encoded by ctsba [50, 51]. Similar to our findings, other studies have demonstrated that components of cigarette smoke can significantly raise cathepsin B levels at both the RNA and protein levels [52, 53]. Furthermore, it has been demonstrated that poor-quality embryos exhibit higher cathepsin B activity and mRNA expression than good-quality embryos, while inhibiting cathepsin B can reduce apoptosis levels and enhance embryonic developing capacities [54–56]. Despite the paucity of study on the ctsba gene, Yvette et al. have conclusively shown that it plays a crucial role in morphogenesis and that the deletion of this gene causes a problem in the embryonic development of split top embryos [57]. These results demonstrate the possible crucial function of the ctsba gene in controlling embryo developmental apoptosis, as well as providing a greater knowledge of the harmful consequences of apoptotic abnormalities in embryo development.

Recent research has provided a fresh perspective on the impact of toxicant exposure on lipid metabolism and its deleterious consequences on embryo development, thereby providing further insights into the manner in which CSE impacts the process [58, 59]. While we currently lack direct evidence, the KEGG enrichment analysis of DEG in different concentration groups revealed that pathways associated with lipid metabolism were downregulated, suggesting that exposure to CSE may disrupt the lipid metabolism of embryos. Lipids supply energy and generate signaling molecules such as lysophosphatidic acid and prostaglandins for embryonic development; however, abnormalities in lipid content and metabolites can cause dysplasias and defects [60, 61]. As the gene-term network shows, implicated genes changed from pla2g1b to fads2, accompanied by a rise in CSE concentration, even though alpha-Linolenic acid metabolism was enriched in all treatment groups. Therefore, it is reasonable to initiate further investigation into the critical role of fads2 in the metabolic damage caused by CSE during embryonic development. The process of fatty acid production in embryo cells may be interfered with by the down-regulation of fads2 gene expression, which could also impact signaling transduction pathways as well as the structure and function of cell membranes [62–64]. This disturbance in lipid metabolism may hinder embryos from developing normally, resulting in morphological abnormalities and detrimental effects on tissue differentiation [63, 65, 66]. In conclusion, it is important to take into account the possible metabolic toxicity of CSE to embryonic development as well as fads2 direct effect on the metabolism of lipids in developing embryos.

Treating embryos with a CSE containing multiple components could refine the study of mechanisms underlying the harmful effects of tobacco contaminants [17]. This study utilized a combination of morphological and bioinformatic techniques to investigate the impact of CSE on early embryonic development from various angles. Our findings revealed the genetic toxicity, cytotoxicity, and metabolic toxicity respectively caused by the damaged genetic material, disordered apoptosis, and lipid metabolism disorders in embryos exposed to CSE. This research contributes significant toxicological data towards a comprehensive understanding of CSE-induced developmental toxicity, albeit requiring further experimental validation.

In conclusion, our extensive investigation demonstrated that the exposure of zebrafish embryos to CSE resulted in developmental toxicity, particularly affecting the head and heart. Based on the comprehensive analysis of miRNA and mRNA transcriptomics, we have discovered a close correlation between embryonic developmental toxicity and genetic material damage, apoptosis disorder, as well as lipid metabolism disturbance. These findings provide valuable insights into the potential mechanisms by which CSE exposure induces embryonic developmental toxicity in zebrafish.

CSE, cigarette smoke extract

miRNA, microRNA

mRNA, messenger RNA

RT-qPCR, quantitative real-time PCR

hpf, hour post-fertilization

dpf, day post-fertilization

AO staining, Acridine orange staining

PBS, phosphate buffer saline

Padj, adjusted p-value

DEGs, differentially expressed genes

DEMs, differentially expressed miRNAs

KEGG, Kyoto Encyclopedia of Genes and Genomes

GO, Gene Ontology

GSEA, Gene Set Enrichment Analysis

Ethics approval and consent to participate

The animal study was approved by the Ethics Committee of Shantou University Medical College.

Consent for publication

Not applicable.

Availability of data and material

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Competing interests

The authors have no conflicts of interest to disclose.

Funding

The National Natural Science Foundation of China (82002068 and 82272281); Medical Scientific Research Foundation of Guangdong Province (A2022192 and B2023166); The Project of Traditional Chinese Medicine Bureau of Guangdong Province (20232082); Shantou Science and Technology project (220811205271404) supported this research.

Author Contributions

Jiasheng Chen and Yuxin Lin designed the research, wrote the first draft of the manuscript and contributed equally to this work. Deyi Geng and Wanxian Chen performed the experiments and explained the data together. Han Rui, Liang Yuan and Hao Li assisted in the study design and revised the manuscript. Shijie Tang performed the statistical analysis. Shukai Zheng and Xiaoping Zhong supervised the project and revised the paper. All authors have read and agreed to the final version of the manuscript.

Acknowledgment

The authors sincerely thank all participants in the study.

Le Foll B, Piper ME, Fowler CD, Tonstad S, Bierut L, Lu L, et al. Tobacco and nicotine use. Nat Rev Dis Primers. 2022;8:19.
Li L, Lin Y, Xia T, Zhu Y. Effects of Electronic Cigarettes on Indoor Air Quality and Health. Annu Rev Public Health. 2020;41:363–80.
Soleimani F, Dobaradaran S, De-la-Torre GE, Schmidt TC, Saeedi R. Content of toxic components of cigarette, cigarette smoke vs cigarette butts: A comprehensive systematic review. Sci Total Environ. 2022;813:152667.
Matt GE, Quintana PJE, Destaillats H, Gundel LA, Sleiman M, Singer BC, et al. Thirdhand tobacco smoke: emerging evidence and arguments for a multidisciplinary research agenda. Environ Health Perspect. 2011;119:1218–26.
Matt GE, Quintana PJE, Fortmann AL, Zakarian JM, Galaviz VE, Chatfield DA, et al. Thirdhand smoke and exposure in California hotels: non-smoking rooms fail to protect non-smoking hotel guests from tobacco smoke exposure. Tob Control. 2014;23:264–72.
Whitlatch A, Schick S. Thirdhand Smoke at Philip Morris. Nicotine Tob Res. 2019;21:1680–8.
Havard A, Chandran JJ, Oei JL. Tobacco use during pregnancy. Addiction. 2022;117:1801–10.
Deng C, Pu J, Deng Y, Xie L, Yu L, Liu L, et al. Association between maternal smoke exposure and congenital heart defects from a case-control study in China. Sci Rep. 2022;12:14973.
Kharrazi M, DeLorenze GN, Kaufman FL, Eskenazi B, Bernert JT, Graham S, et al. Environmental tobacco smoke and pregnancy outcome. Epidemiology. 2004;15:660–70.
Sabbagh HJ, Baghlaf KK, Jamalellail HMH, Bakhuraybah AS, AlGhamdi SM, Alharbi OA, et al. Environmental tobacco smoke exposure and non-syndromic orofacial cleft: Systematic review and meta-analysis. Tob Induc Dis. 2023;21:76.
Dai Y-J, Jia Y-F, Chen N, Bian W-P, Li Q-K, Ma Y-B, et al. Zebrafish as a model system to study toxicology. Environ Toxicol Chem. 2014;33:11–7.
Veldman MB, Lin S. Zebrafish as a developmental model organism for pediatric research. Pediatr Res. 2008;64:470–6.
Borrego-Soto G, Eberhart JK. Embryonic Nicotine Exposure Disrupts Adult Social Behavior and Craniofacial Development in Zebrafish. Toxics. 2022;10:612.
Merino C, Casado M, Piña B, Vinaixa M, Ramírez N. Toxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in early development: A wide-scope metabolomics assay in zebrafish embryos. J Hazard Mater. 2022;429:127746.
Li Y, Hecht SS. Carcinogenic components of tobacco and tobacco smoke: A 2022 update. Food Chem Toxicol. 2022;165:113179.
Massarsky A, Jayasundara N, Bailey JM, Oliveri AN, Levin ED, Prasad GL, et al. Teratogenic, bioenergetic, and behavioral effects of exposure to total particulate matter on early development of zebrafish (Danio rerio) are not mimicked by nicotine. Neurotoxicol Teratol. 2015;51:77–88.
Karmach O, Madrid JV, Dasgupta S, Volz DC, Zur Nieden NI. Embryonic Exposure to Cigarette Smoke Extract Impedes Skeletal Development and Evokes Craniofacial Defects in Zebrafish. Int J Mol Sci. 2022;23:9904.
Massarsky A, Jayasundara N, Glazer L, Levin ED, Prasad GL, Di Giulio RT. Outcomes of developmental exposure to total particulate matter from cigarette smoke in zebrafish (Danio rerio). Neurotoxicology. 2018;68:101–14.
Johnston IA, Lee H-T, Macqueen DJ, Paranthaman K, Kawashima C, Anwar A, et al. Embryonic temperature affects muscle fibre recruitment in adult zebrafish: genome-wide changes in gene and microRNA expression associated with the transition from hyperplastic to hypertrophic growth phenotypes. J Exp Biol. 2009;212:1781–93.
Xuan R, Qiu W, Zhou Y, Magnuson JT, Luo S, Greer JB, et al. Parental transfer of an antibiotic mixture induces cardiotoxicity in early life-stage zebrafish: A cross-generational study. Sci Total Environ. 2022;849:157726.
Mattingly CJ, Hampton TH, Brothers KM, Griffin NE, Planchart A. Perturbation of defense pathways by low-dose arsenic exposure in zebrafish embryos. Environ Health Perspect. 2009;117:981–7.
Zhou W, Li X, Wang Y, Wang J, Zhang J, Wei H, et al. Physiological and transcriptomic changes of zebrafish (Danio rerio) embryos-larvae in response to 2-MIB exposure. J Hazard Mater. 2021;416:126142.
Yokoi T, Nakajima M. Toxicological implications of modulation of gene expression by microRNAs. Toxicol Sci. 2011;123:1–14.
Zhang L, Li Y-Y, Zeng H-C, Wei J, Wan Y-J, Chen J, et al. MicroRNA expression changes during zebrafish development induced by perfluorooctane sulfonate. J Appl Toxicol. 2011;31:210–22.
Culpitt SV, Rogers DF, Shah P, De Matos C, Russell REK, Donnelly LE, et al. Impaired inhibition by dexamethasone of cytokine release by alveolar macrophages from patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2003;167:24–31.
Liu H, Liu Z, Meng L, Fu X, Hou Y. Toxic effects of 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal on the reproduction of female mice. Ecotoxicol Environ Saf. 2019;183:109544.
Xi Y, Diao L, Wang Z, Jin Z, Wang Y, Liu W, et al. Toxicity of leachate from smoked cigarette butts to terrestrial animals: A case study on the earthworm Eisenia fetida. Science of The Total Environment. 2023;898:165531.
Zheng S, Huang W, Liu C, Xiao J, Wu R, Wang X, et al. Behavioral change and transcriptomics reveal the effects of 2, 2’, 4, 4’-tetrabromodiphenyl ether exposure on neurodevelopmental toxicity to zebrafish (Danio rerio) in early life stage. Sci Total Environ. 2021;752:141783.
Knecht AL, Goodale BC, Truong L, Simonich MT, Swanson AJ, Matzke MM, et al. Comparative developmental toxicity of environmentally relevant oxygenated PAHs. Toxicol Appl Pharmacol. 2013;271:266–75.
Chang YS, Park SM, Rah YC, Han EJ, Koun SI, Chang J, et al. In vivo assessment of the toxicity of electronic cigarettes to zebrafish (Danio rerio) embryos, following gestational exposure, in terms of mortality, developmental toxicity, and hair cell damage: Toxicity of E-cigs to zebrafish embryos. Hum Exp Toxicol. 2021;40:148–57.
Zenzes M. Immunodetection of benzo[a]pyrene adducts in ovarian cells of women exposed to cigarette smoke. Molecular Human Reproduction. 1998;4:159–65.
Huang J, Okuka M, Lu W, Tsibris JCM, McLean MP, Keefe DL, et al. Telomere shortening and DNA damage of embryonic stem cells induced by cigarette smoke. Reproductive Toxicology. 2013;35:89–95.
Tang M, Lee H-W, Weng M, Wang H-T, Hu Y, Chen L-C, et al. DNA damage, DNA repair and carcinogenicity: Tobacco smoke versus electronic cigarette aerosol. Mutation Research/Reviews in Mutation Research. 2022;789:108409.
Ciccia A, Elledge SJ. The DNA Damage Response: Making It Safe to Play with Knives. Molecular Cell. 2010;40:179–204.
Carusillo A, Mussolino C. DNA Damage: From Threat to Treatment. Cells. 2020;9:1665.
Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature. 2009;461:1071–8.
Cui J, Zhao W, Xu X, Yang M, Ren Y, Zhang Z. DNA polymerase beta is involved in the protection against the cytotoxicity and genotoxicity of cigarette smoke. Environmental Toxicology and Pharmacology. 2012;34:370–80.
Wang Q, Gosik K, Xing S, Jiang L, Sun L, Chinchilli VM, et al. Epigenetic game theory: How to compute the epigenetic control of maternal-to-zygotic transition. Physics of Life Reviews. 2017;20:126–37.
Dejosez M, Ura H, Brandt VL, Zwaka TP. Safeguards for Cell Cooperation in Mouse Embryogenesis Shown by Genome-Wide Cheater Screen. Science. 2013;341:1511–4.
Park H-R, Vallarino J, O’Sullivan M, Wirth C, Panganiban RA, Webb G, et al. Electronic cigarette smoke reduces ribosomal protein gene expression to impair protein synthesis in primary human airway epithelial cells. Sci Rep. 2021;11:17517.
Enoch T, Norbury C. Cellular responses to DNA damage: cell-cycle checkpoints, apoptosis and the roles of p53 and ATM. Trends in Biochemical Sciences. 1995;20:426–30.
Sancar A, Lindsey-Boltz LA, Ünsal-Kaçmaz K, Linn S. Molecular Mechanisms of Mammalian DNA Repair and the DNA Damage Checkpoints. Annu Rev Biochem. 2004;73:39–85.
Yin C, Cai H, Yang D, Jian Y, Zhang J, Li Z, et al. Cigarette smoke induced neural tube defects by down-regulating noggin expression. Birth Defects Research. 2021;113:5–13.
Detmar J, Rabaglino T, Taniuchi Y, Oh J, Acton BM, Benito A, et al. Embryonic loss due to exposure to polycyclic aromatic hydrocarbons is mediated by Bax. Apoptosis. 2006;11:1413–25.
Huang J, Okuka M, McLean M, Keefe DL, Liu L. Effects of cigarette smoke on fertilization and embryo development in vivo. Fertility and Sterility. 2009;92:1456–65.
Compagnucci C, Martinus K, Griffin J, Depew MJ. Programmed Cell Death Not as Sledgehammer but as Chisel: Apoptosis in Normal and Abnormal Craniofacial Patterning and Development. Front Cell Dev Biol. 2021;9:717404.
Brill A, Torchinsky A, Carp H, Toder V. The role of apoptosis in normal and abnormal embryonic development. J Assist Reprod Genet. 1999;16:512–9.
Stoka V, Turk B, Turk V. Lysosomal Cysteine Proteases: Structural Features and their Role in Apoptosis. IUBMB Life. 2005;57:347–53.
Reiser J, Adair B, Reinheckel T. Specialized roles for cysteine cathepsins in health and disease. J Clin Invest. 2010;120:3421–31.
Mort JS, Buttle DJ. Cathepsin B. The International Journal of Biochemistry & Cell Biology. 1997;29:715–20.
Xie Z, Zhao M, Yan C, Kong W, Lan F, Narengaowa, et al. Cathepsin B in programmed cell death machinery: mechanisms of execution and regulatory pathways. Cell Death Dis. 2023;14:255.
Nagaraj NS, Zacharias W. Cigarette smoke condensate increases cathepsin-mediated invasiveness of oral carcinoma cells. Toxicology Letters. 2007;170:134–45.
Luo S, Jiang L, Li Q, Sun X, Liu T, Pei F, et al. Acrolein-induced autophagy–dependent apoptosis via activation of the lysosomal–mitochondrial pathway in EAhy926 cells. Toxicology in Vitro. 2018;52:146–53.
Kim S-H, Zhao M-H, Liang S, Cui X-S, Kim N-H. Inhibition of cathepsin B activity reduces apoptosis by preventing cytochrome c release from mitochondria in porcine parthenotes. Journal of Reproduction and Development. 2015;61:261–8.
Li J, Maeji M, Balboula AZ, Aboelenain M, Fujii T, Moriyasu S, et al. Dynamic status of lysosomal cathepsin in bovine oocytes and preimplantation embryos. J Reprod Dev. 2020;66:9–17.
Balboula AZ, Yamanaka K, Sakatani M, Hegab AO, Zaabel SM, Takahashi M. Intracellular cathepsin B activity is inversely correlated with the quality and developmental competence of bovine preimplantation embryos. Molecular Reproduction Devel. 2010;77:1031–9.
Langdon YG, Fuentes R, Zhang H, Abrams EW, Marlow FL, Mullins MC. Split top: A maternal cathepsin B that regulates dorsoventral patterning and morphogenesis. Development. 2016;dev.128900.
Kögel T, Bjorøy Ø, Toto B, Bienfait AM, Sanden M. Micro- and nanoplastic toxicity on aquatic life: Determining factors. Sci Total Environ. 2020;709:136050.
Szilagyi JT, Avula V, Fry RC. Perfluoroalkyl Substances (PFAS) and Their Effects on the Placenta, Pregnancy, and Child Development: a Potential Mechanistic Role for Placental Peroxisome Proliferator-Activated Receptors (PPARs). Curr Environ Health Rep. 2020;7:222–30.
Prates EG, Nunes JT, Pereira RM. A role of lipid metabolism during cumulus-oocyte complex maturation: impact of lipid modulators to improve embryo production. Mediators Inflamm. 2014;2014:692067.
Ye Q, Zeng X, Cai S, Qiao S, Zeng X. Mechanisms of lipid metabolism in uterine receptivity and embryo development. Trends Endocrinol Metab. 2021;32:1015–30.
Park WJ, Kothapalli KSD, Lawrence P, Brenna JT. FADS2 Function Loss at the Cancer Hotspot 11q13 Locus Diverts Lipid Signaling Precursor Synthesis to Unusual Eicosanoid Fatty Acids. PLoS One. 2011;6:e28186.
Bláhová Z, Franěk R, Let M, Bláha M, Pšenička M, Mráz J. Partial fads2 Gene Knockout Diverts LC-PUFA Biosynthesis via an Alternative Δ8 Pathway with an Impact on the Reproduction of Female Zebrafish (Danio rerio). Genes (Basel). 2022;13:700.
Fadhlaoui M, Pierron F, Couture P. Temperature and metal exposure affect membrane fatty acid composition and transcription of desaturases and elongases in fathead minnow muscle and brain. Ecotoxicology and Environmental Safety. 2018;148:632–43.
De La Cruz-Alvarado FJ, Álvarez-González CA, Llera-Herrera R, Monroig Ó, Kabeya N, Rodríguez-Morales S, et al. Expression of long-chain polyunsaturated fatty acids biosynthesis genes during the early life-cycle stages of the tropical gar Atractosteus tropicus. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 2021;256:110628.
Bobiński R, Mazurek U, Zmarzly N, Ulman-Włodarz I, Dutka M, Pizon M, et al. Placental expression of Fatty Acid Desaturases 1, 2 and 3 in selected pregnancy pathologies. Acta Biochimica Polonica. 2023;70:137–44.

Download PDF

Journal Publication

published 08 Mar, 2024

Read the published version in Journal of Translational Medicine →

Reviewers agreed at journal
10 Feb, 2024
Reviewers invited by journal
21 Dec, 2023
Editor assigned by journal
07 Dec, 2023
First submitted to journal
06 Dec, 2023

You are reading this latest preprint version

Integrated mRNA- and miRNA-sequencing analyses unveil the underlying mechanism of tobacco pollutant-induced developmental toxicity in zebrafish embryos

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Chemicals and reagents

2.2 Zebrafish maintenance and CSE exposure

2.3 Effects of CSE on the survival and developmental status of zebrafish

2.4 Acridine orange staining

2.5 Global transcriptome sequencing (mRNA- and miRNA-seq) analysis

2.6 Integrated bioinformatics analyses of the sequencing results

2.7 RT-qPCR for verification

2.8 Statistical analysis

3. Results

3.1 Embryonic development toxicity of CSE

3.2 Effects of CSE exposure on larvae injury and cell apoptosis

3.3 Changes in the transcriptome profile of miRNA and mRNA following CSE exposure

3.4 Enrichment analysis of DEGs

3.5 Enrichment analysis of DEM target genes

3.6 Validation of significant DEG expression using RT-qPCR

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1