Long recognized as a man-made disruption, artificial light at night (ALAN) has become more prevalent, depriving Earth’s surface of its typical light cycles. The repercussions of change in the light-dark cycle on the environment and human health have been the subject of research [1,2]. Although light is giving ecosystem services to all living beings in the earth for their regulation of cell cycles, development, and growth; longer exposure to artificial CIMlighting has serious consequences [3]. It has been pointed out that longer exposure to ALAN impacts ecosystem functioning and causes strong hormonal responses, which in turn alters physiological processes, daily routines, and other life characteristics [2]. Further, it was also evident that ALAN disrupts invertebrates through changes in their community structure, life cycle, and species interaction, leading to an effect on food web dynamics across ecosystem boundaries, nutrient flow, pollination, and natural control of pest insects [4]. More recently, it has been indicated that ALAN may have contributed to the emergence of COVID-19 [5]. Overall, these investigations demonstrate that ALAN disturbs seasonal or circadian rhythms, endangering every living organism, and eroding biodiversity and wildlife, leading to decreased ecosystem services from the global ecosystem or biosphere.

In the last decade, transcriptomic profiling was performed in a variety of situations, including hypoxia, salt, drought, environmental pollution, and UV radiation [6]. Few transcriptomic studies were carried out to check the repercussions of ALAN in several organisms like birds [7,8], mice [9], coral reefs [10], and common toad tadpoles [11]. The tadpole study revealed that ALAN downregulates the expression of most genes both during the day and at night, and that light at night affects physiological pathways for immunity and lipid metabolism [11]. These studies have significantly enhanced our understanding of the molecular machinery needed for organismal adaptability. Therefore, it was evident that employing a transcriptomic approach to examine the physiological changes through gene expression is one of the best ways to study the consequences of ALAN.

The biology of reproduction in many species, including fish, is influenced by photoperiod and circadian cycles. ALAN also alters the expression of melatonin, a hormone that is largely produced at night, interferes with the synthesis of sex hormones, energy metabolism, antioxidant defences, and immune system components [12,13]. Improper exposure to ALAN has a negative impact on the circadian system, causing short-term effects on sleep and cognition as well as long-term endocrine disruptions that lead to obesity, cardiovascular disease, mood disorders, diabetes, hormonal imbalances during pregnancy, and cancer [14-18]. It has been found that ALAN primarily affects the internal circadian timekeeping system by changing the expression of clock genes [15], as well as other genes whose expression is controlled by photoperiod and involved in processes like reproduction, immunity, and metabolic detoxification [12]. Therefore, a comprehensive study is immediately warranted to analyze the effect of ALAN on a living being.

Zebrafish (Danio rerio) is a reliable vertebrate model for circadian research, originally developed for their utility in developmental biology studies, due to their high fecundity and relatively low cost of maintenance, experimental use of this model has evolved to include a broad range of phenotypes from embryonic to adult stages as well as a wide array of molecular assays [19]. Research on the effects of ALAN on zebrafish reveals multifaceted impacts spanning ovarian physiology, cognitive functions, and transgenerational behavior. In our previous study [15], we uncovered significant chronodisruption in zebrafish ovarian physiology under continuous light exposure, leading to desynchronized clock-associated genes and reduced melatonin levels, ultimately linked to ovarian tumor development. Lucon-Xiccato et al. [20] demonstrated ALAN’s detrimental effects on zebrafish learning abilities, slowing habituation learning, and altering behavioral covariances. Meanwhile, Li et al. [21] highlighted ALAN’s transgenerational impact, showing short-wavelength ALAN’s ability to induce anxiety-like behaviors and affect offspring behavior despite no direct exposure. These findings collectively underscore the profound implications of light pollution on zebrafish reproductive health, cognitive functions, and behavioral traits, urging further attention to mitigate its broader ecological consequences. Moreover, the zebrafish genome was fully sequenced in 2013 and contains homologues of around 70% of human genes, including 80% of those implicated in diseases, which makes zebrafish a reasonable model to study human diseases [22].

This enhances the ability to conduct human comparative transcriptome-based research using zebrafish with proper genomic annotations and identifiers. Previously our group employed a transcriptomic approach for the first time to show the development of thecoma and granulosa cell tumor, induced by a few candidate genes in ALAN-exposed zebrafish or any vertebrate ovary for the first time [15].

This study aims to demonstrate the level of differentially expressed genes (DEGs), the gene and pathway ontology, and diseases, through meticulous bioinformatics analysis of the complete ALAN-exposed ovarian whole transcriptome data.

Our study comprising four transcriptomes of zebrafish ovary exposed to different photic conditions showed that ALAN induces a strong disruption of gene expression starting from one day to a year. Earlier studies indicated that ALAN disrupts sleep and circadian rhythm, raising the risk of cancer, diabetes, cardiovascular disease, obesity, mood disorders, and age-related macular degeneration [18]. A recent study employing the RNA-Seq method to study tadpole larvae exposed to ALAN found that their capacity to deal with immunity is reduced [11]. The impact of ALAN on the transcriptome level has not yet been well investigated in zebrafish, particularly biological rhythms, (controlled transcriptionally) [35]. In this study, the full spectrum of diseases that may develop from exposure to ALAN, is elaborated for the first time (Supplementary Tables 3-5). This helps to predict sustainability of this organism in the ecosystem under rapidly changing environmental parameters. The U-shaped trend in upregulated DEGs suggests an intriguing aspect of light exposure. Initially, LLW shows an acute stress response with high upregulation. At LLM, organisms begin adapting, reducing the number of upregulated genes as they seek homeostasis. By LLY, chronic stress from prolonged exposure increases gene upregulation again, reflecting long-term effects and potential cumulative cellular damage. This pattern highlights the complex, nonlinear nature of biological responses to extended environmental changes, such as continuous light exposure disrupting circadian rhythms and inducing varied stress responses over time. However, the majority of DEGs were found upregulated in all three ALAN exposed conditions in comparison to the normal condition, leading to changes in many physiological pathways like translation, signaling, metabolism, ATP synthesis, methyltransferase, and oxidase activities, causing overall chronodisruption in zebrafish ovary (Figures 3-5 and Supplementary Figure 6). Pathways like RNA degradation, RNA polymerase, signaling, and ABC transporters began enriched in the one-week sample and accelerated over one year of exposure (Figure 4A). The Notch signaling system, which controls cellular differentiation, survival, apoptosis, and proliferation [36], and the Lysine degradation pathway which controls the inborn errors of metabolism [37], were found to be dysregulated in the year-long ALAN exposed samples (Figure 4A). This might have a significant impact on the ability of the organ to function properly. Interestingly these were not the top enriched pathways in the week and month-long ALAN-exposed samples (Figure 4A). The decrease in several genes contributing to oxidative phosphorylation following the year-long exposure to ALAN reveals that the energy needed to maintain metabolism, the physical structure of the cells, and the transportation of molecules and ions are affected and disrupted (Figure 4A). The previous study [38] demonstrated this disruption as the result of the intracellular environment. This was further confirmed by the enrichment of the ATP metabolic process in week and month samples but not in the LLY sample (Figures 4 and 5). Moreover, the activation of the apoptotic signaling pathway and homeostasis in the number of cells in LLM and LLY samples shows that cell death is evident and, hence disrupting the total organ (Figure 5). The GO enrichment provided additional support for the findings of pathway enrichment, indicating a decline in significant biological processes and an increase in significant molecular functions as the ALAN exposure was continued (Figure 5). Furthermore, genes expressed specifically in treated or normal samples demonstrated a disruption in either metabolic pathways or signaling pathways, respectively (Figure 4B). These two processes are bidirectional, and therefore, cells can keep track of the conditions of crucial metabolic pathways and exert feedback control over signal transduction networks [39], allowing the cells to coordinate their metabolic processes with their growth and division. The disruption of these pathways amply demonstrates that the organ is progressing toward a serious diseased condition. In addition, the treated samples showed enrichment in the TGF-beta and GnRH signaling pathways, indicating significant changes in growth and reproduction processes. TGF-beta signaling has complex roles; it can promote growth, inhibit cell multiplication, trigger cell specialization or death, and give rise to various diseases, depending on the situation [40]. Meanwhile, GnRH signaling is crucial for reproductive function, affecting the release of hormones that regulate reproduction (Figure 4B). Our previous findings [27] also support this data, moreover, the effect of ALAN on the brain-pituitary-gonadal axis implies the degree of lethality of this exposure. According to the hub genes pathway enrichment of adherence junction and FoxO signaling system only happened in the upregulated genes of the year-long exposed sample. ALAN’s negative effects were further reduced by preventing cell cycle arrest, stress tolerance, and apoptosis (Supplementary Figure 5). Disruption of the FoxO family of transcription factors results in the sick condition of the ovary because they are essential for the integration of growth factor signalling, oxidative stress, and inflammation [36,41].

The LLY group, being older due to 12 months of continuous light exposure, may likely exhibits age-related gene expression changes. These changes may interact with the effects of ALAN, contributing to the observed dysregulation of genes associated with neoplasms. As zebrafish age, there is a documented disruption in the expression of genes critical for stem cell maintenance and tissue homeostasis, which can lead to increased susceptibility to diseases, including various cancers [42,43]. Though zebrafish are considered as adult from 90 days to 2 years old, and the age of the fish in the present study falls within this range, future research with age-appropriate control might be useful in understanding the exact DEGs [44]. In our study, the significant disruption of various DEGs and pathways in the treated samples indicated the emergence of numerous clinical states, therefore a disease annotation and enrichment analysis using human orthologous data in the entire DisGeNET database was carried out. Previously it was studied that, exposure to ALAN suppresses melatonin production, disrupts circadian rhythms, and is linked to increased risks of hormone-related cancers such as breast, prostate, and colorectal cancers due to its effects on biological pathways like hormone signaling, cell proliferation, DNA repair, and inflammation [45]. Additionally, ALAN is significantly correlated with increased risks of all forms of cancer, including lung, breast, colorectal, and prostate cancers, across 158 countries. This study found positive associations between ALAN exposure and cancer rates, even after adjusting for confounders such as population, electricity consumption, air pollution, and forest coverage [46]. A global study further highlighted higher breast cancer incidence in countries with elevated light pollution levels, suggesting that light pollution might disrupt circadian rhythms and melatonin production, thereby contributing to cancer risk [47]. As anticipated, neoplasms are the most common type of sickness class annotated, followed by the phenotype of pathological states, signs, and symptoms (Figure 7). The most prevalent disorders enriched in all treated samples included malignant prostate cancer (which was previously confirmed by a study [48]), glioblastoma, melanoma, tumor progression, and other cancer-related diseases confirming our previous study [15]. We have taken all significantly dysregulated DEGs in all the contrasts to get a comprehensive picture of the disease enrichment in the treated samples (Figure 9A). We found major classes of neoplasms in the mammary and pancreas, neuroblastoma and glioblastoma in both children and adults, carcinoma, along with intellectual disability, global developmental delay, and seizures. This shows that ALAN’s effects are worse than previously thought. Additionally, genes that were all annotated with less than 5 diseases in the DisGeNET database were chosen to create a signature of genes whose change in expression level may result in these types of diseased states (Figure 9B). It was also noted that, two genes, zgc:56596 and zp2.6, were commonly dysregulated in both the LLW vs. LD and LLM vs. LD contrasts. The zgc:56596 gene is orthologous to the human UBL4A gene which is known to play a role in protein degradation, a critical process for maintaining cellular homeostasis and responding to stress [49,50]. Its dysregulation suggests that ALAN exposure might disrupt protein homeostasis and degradation pathways, contributing to cellular stress responses observed in our study. Similarly, the zp2.6 gene, orthologous to the human ZP4 gene, is involved in reproductive processes, specifically in the structure of the zona pellucida, which surrounds the oocyte and is essential for fertilization [51]. The dysregulation of zp2.6 highlights potential impacts of ALAN on reproductive health, aligning with other findings in our study that suggest reproductive processes are significantly affected by continuous light exposure.

In our transcriptomics analysis of four ALAN exposure scenarios, we identified 777 dysregulated transcripts in the LLW group, 758 in the LLM group, and 802 in the LLY group, compared to the LD sample. The functional enrichment analysis revealed that signaling pathways were more prevalent while ATP-related processes became scarce in the year-long ALAN exposure. Several disease groups that affect the basic functioning of the organism, such as the neural, immune, endocrine, reproductive, digestive, lymphatic, and cardiovascular were recognized by the transcriptomic changes. Major diseases including reproductive problems, global developmental delay, seizures, dwarfism, and cancer (prostate, mammary, lung, kidney, glioblastoma, melanoma, and lymphoma) were found to be triggered by ALAN. It is important to note that while these associations were observed in our study are based on zebrafish models and require validation in human studies to establish direct correlations. Although further studies are required, our study concluded that there is a possibility of ALAN globally affecting the physiology of the organism, initiating various disease-inducing pathways, and may cause numerous diseases by affecting the global gene expression pattern in zebrafish ovary.