A systematic review on fluoride-induced epigenetic toxicity in mammals.

Fluoride, one of the global groundwater contaminants, is ubiquitous in our day-to-day life from various natural and anthropogenic sources. Numerous in vitro, in vivo, and epidemiological studies are conducted to understand the effect of fluoride on biological systems. A low concentration of fluoride is reported to increase oral health, whereas chronic exposure to higher concentrations causes fluoride toxicity (fluorosis). It includes dental fluorosis, skeletal fluorosis, and fluoride toxicity in soft tissues. The mechanism of fluoride toxicity has been reviewed extensively. However, epigenetic regulation in fluoride toxicity has not been reviewed. This systematic review summarizes the current knowledge regarding fluoride-induced epigenetic toxicity in the in vitro, in vivo, and epidemiological studies in mammalian systems. We examined four databases for the association between epigenetics and fluoride exposure. Out of 932 articles (as of 31 March 2022), 39 met our inclusion criteria. Most of the studies focused on different genes, and overall, preliminary evidence for epigenetic regulation of fluoride toxicity was identified. We further highlight the need for epigenome studies rather than candidate genes and provide recommendations for future research. Our results indicate a correlation between fluoride exposure and epigenetic processes. Further studies are warranted to elucidate and confirm the mechanism of epigenetic alterations mediated fluoride toxicity.

Integrated in silico analysis of transcriptomic alterations in nanoparticle toxicity across human and mouse models.

Nanoparticles, due to their exceptional physicochemical properties are used in our day-to-day environment. They are currently not regulated which might lead to increased levels in the biological systems causing adverse effects. However, the overall mechanism behind nanotoxicity remains elusive. Previously, we analysed the transcriptome datasets of copper oxide nanoparticles using in silico tools and identified IL-17, chemokine signaling pathway, and cytokine-cytokine receptor interaction as the key pathways altered. Based on the findings, we hypothesized a common pathway could be involved in transition metal oxide nanoparticles toxicity irrespective of the variables. Further, there could be unique transcriptome changes between metal oxide nanoparticles and other nanoparticles. To accomplish this, the overall transcriptome datasets of nanoparticles consisting of microarray and RNA-Seq were obtained. >90 studies for 17 different nanoparticles, performed in humans, rats, and mice were assessed. After initial screening, 24 mouse studies (with 196 datasets) and 34 human studies (with 200 datasets) were used for further analyses. The common genes that are dysregulated upon NPs exposure were identified for human and mouse datasets separately. Further, an overrepresentation functional enrichment analysis was performed. The common genes, their gene ontology, gene-gene, and protein-protein interactions were assessed. The overall results suggest that IL-17 and its related pathways might be commonly altered in nanoparticle exposure with lung as one of the major organs affected.

Acute copper oxide nanoparticles exposure alters zebrafish larval microbiome.

Copper oxide nanoparticles (CuO NPs) are being used in healthcare industries due to its antimicrobial properties. The increased consumption of NPs could lead to the rise of these NPs in the environment affecting the biological systems. Altered microbiome has been correlated to disease pathology in humans as well as xenobiotic toxicity in experimental animal models. However, CuO NPs-induced microbiome alterations in vertebrates have not been reported so far. In this study, for the first time, zebrafish larvae at 96 hpf (hours post fertilization) were exposed to CuO NPs for 24 h at 10, 20, and 40 ppm. After exposure, the control and treated larvae were subjected to 16S rRNA amplicon sequencing followed by relative taxa abundance, alpha and beta diversity analysis, single factor analysis, LEfSe, Deseq2, and functional profiling. No significant alteration was detected in the microbial richness and diversity, however, specific taxa constituting the core microbiome such as phylum Proteobacteria were significantly increased and Bacterioidetes and Firmicutes were decreased in the treated groups, indicating a core microbiota dysbiosis. Further, the family Lachnospiraceae, and genus Syntrophomonas involved in butyrate production and the metabolism of lipids and glucose were significantly altered. In addition, the opportunistic pathogens belonging to order Flavobacteriales were increased in CuO NPs treated groups. Moreover, the taxa involved in host immune response (Shewanella, Delftia, and Bosea) were found to be enriched in CuO NPs exposed larvae. These results indicate that CuO NPs exposure causes alteration in the core microbiota, which could cause colitis or inflammatory bowel disease.

Identification of key genes and signalling pathways in clear cell renal cell carcinoma: An integrated bioinformatics approach.

BACKGROUND: Clear cell Renal Cell Carcinoma (ccRCC) is one of the most prevalent types of kidney cancer. Unravelling the genes responsible for driving cellular changes and the transformation of cells in ccRCC pathogenesis is a complex process. OBJECTIVE: In this study, twelve microarray ccRCC datasets were chosen from the gene expression omnibus (GEO) database and subjected to integrated analysis. METHODS: Through GEO2R analysis, 179 common differentially expressed genes (DEGs) were identified among the datasets. The common DEGs were subjected to functional enrichment analysis using ToppFun followed by construction of protein-protein interaction network (PPIN) using Cytoscape. Clusters within the DEGs PPIN were identified using the Molecular Complex Detection (MCODE) Cytoscape plugin. To identify the hub genes, the centrality parameters degree, betweenness, and closeness scores were calculated for each DEGs in the PPIN. Additionally, Gene Expression Profiling Interactive Analysis (GEPIA) was utilized to validate the relative expression levels of hub genes in the normal and ccRCC tissues. RESULTS: The common DEGs were highly enriched in Hypoxia-inducible factor (HIF) signalling and metabolic reprogramming pathways. VEGFA, CAV1, LOX, CCND1, PLG, EGF, SLC2A1, and ENO2 were identified as hub genes. CONCLUSION: Among 8 hub genes, only the expression levels of VEGFA, LOX, CCND1, and EGF showed a unique expression pattern exclusively in ccRCC on compared to other type of cancers.

Angiogenesis Assay for Live and Fixed Zebrafish Embryos/Larvae.

Angiogenesis is the process of new blood vessel formation from preexisting vasculature. It is an integral component in normal embryonic development and tissue repair. Dysregulation of angiogenesis might lead to tissue ischemia (resulting from reduced blood vessel formation) or major diseases such as cancer (abnormal vascular growth). This makes angiogenesis an excellent area of research for cancer therapeutics, and various animal models including zebrafish are used to study blood vessel development. As most of the techniques used to study angiogenesis are complex and cumbersome, in this chapter, we provide two simple assays to study angiogenesis with live and fixed zebrafish embryos/larvae.

Toxicogenomic analysis of physiologically important metals: An integrated in silico approach.

Biologically important metals regulate cellular homeostasis in living systems. Anthropogenic exposure to these metals can cause adverse effects, including an increased incidence of diseases in humans such as cancer, lung, and cardiovascular defects. However, the effects of metals and the common genes/signaling pathways involved in metal toxicity have not been elucidated. Hence, the present study used toxicogenomic data mining with the comparative toxicogenomics database to explore the impact of these metals. The metals were categorized into transition, alkali, and alkaline earth. The common genes were identified and subjected to functional enrichment analysis. Further, gene-gene and protein-protein interactions were assessed. Also, the top ten transcription factors and miRNAs that regulate the genes were identified. The phenotypes and diseases that have increased incidence upon alterations of these genes were detected. Overall, we were able to identify IL1B and SOD2 as the common genes, along with the AGE-RAGE signaling pathway in diabetic complications as the common pathway altered. Enriched genes and pathways specific to each metal category were also found. Further, we identified heart failure as the major disease that could have increase in the incidence upon these metals' exposure. In conclusion, exposure to essential metals might cause adverse effects via inflammation and oxidative stress.

Acute exposure to tenorite nanoparticles induces phenotypic and behavior alterations in zebrafish larvae.

Tenorite or copper oxide nanoparticles (CuO NPs) are extensively used in biomedical fields due to their unique physicochemical properties. Increased usage of these NPs leads to release in the environment, affecting varied ecosystems and the biota within them, including humans. The effect of these NPs can be evaluated with zebrafish, an excellent complementary model for nanotoxicity studies. Previous reports focusing on CuO NPs-induced teratogenicity in zebrafish development have not elucidated the phenotypical changes in detail. In most of the studies, embryos at 3 hpf with a protective chorion layer were exposed to CuO NPs, and their effect on the overall developmental process is studied. Hence, in this study, we focused on the effect of acute exposure to CuO NPs (96-120 hpf) and its impact on zebrafish larvae. Larvae were exposed to commercially available CuO NPs (<50 nm) at various concentrations to obtain the LC50 value (52.556 ppm). Based on the LC50, three groups (10, 20, and 40 ppm) were taken for further analysis. Upon treatment, bradycardia, and impaired swim bladder (reduced/absence of inflation) were found in the treated groups along with alterations in the erythrocyte levels. Also, the angles and distance between the cartilages varied in the treated larvae affecting their craniofacial structures. There was a significant behavior change, as evidenced by the reduced touch escape response and locomotion (speed, distance, time mobile, time frozen, and absolute turn angle). Further, the acetylcholinesterase activity was reduced. Overall, our results suggest that acute exposure to CuO NPs elicits morphological defects in zebrafish larvae.

Garcinia gummi-gutta: Phytochemicals and pharmacological applications.

Garcinia gummi-gutta, also known as Garcinia cambogia, is a member of the Guttiferae family. Garcinia is a polygamous genus consisting 200 species of trees and shrubs. It is found in different zones of the planet including Asia's tropical regions. In India alone, around 30 species have been discovered. They are widely used as a flavoring agent to garnish fish curry in southern India, particularly in Kerala and Karnataka. The fruit rind of G. gummi-gutta has traditionally been used to treat gastrointestinal problems, diarrhea, and ulcers. South Indian people have been utilizing it traditionally as evidenced by its ethnobotanical properties. In vivo and in vitro effects of the crude fruit extract showed anti-inflammatory, anti-cancer, anthelmintic, anti-microbial, and antioxidant activities. G. gummi-gutta fruit rind is medicinally significant and is frequently used in ayurvedic and traditional medicine for many diseases. Various secondary metabolites such as organic acids-hydroxycitric acid (HCA), flavonoids, terpenes, polysaccharides and polyisoprenylated benzophenones-garcinol, xanthochymol, guttiferone, benzophenone, xanthone, biflavonoids, alkaloids, tannins, phenols, and saponins isolated from the G. gummi-gutta have diverse pharmacological activities. This review provides a summary of G. gummi-gutta, including its biological activities, phytochemistry, and ethnobotanical applications.

Fluoride Induced Neurobehavioral Impairments in Experimental Animals: a Brief Review.

Fluoride is one of the major toxicants in the environment and is often found in drinking water at higher concentrations. Living organisms including humans exposed to high fluoride levels are found to develop mild-to-severe detrimental pathological conditions called fluorosis. Fluoride can cross the hematoencephalic barrier and settle in various brain regions. This accumulation affects the structure and function of both the central and peripheral nervous systems. The neural ultrastructure damages are reflected in metabolic and cognitive activities. Hindrances in synaptic plasticity and signal transmission, early neuronal apoptosis, functional alterations of the intercellular signaling pathway components, improper protein synthesis, dyshomeostasis of the transcriptional and neurotrophic factors, oxidative stress, and inflammatory responses are accounted for the fluoride neurotoxicity. Fluoride causes a decline in brain functions that directly influence the overall quality of life in both humans and animals. Animal studies are widely used to explore the etiology of fluoride-induced neurotoxicity. A good number of these studies support a positive correlation between fluoride intake and toxicity phenotypes closely associated with neurotoxicity. However, the experimental dosages highly surpass the normal environmental concentrations and are difficult to compare with human exposures. The treatment procedures are highly dependent on the dosage, duration of exposure, sex, and age of specimens among other factors which make it difficult to arrive at general conclusions. Our review aims to explore fluoride-induced neuronal damage along with associated histopathological, behavioral, and cognitive effects in experimental models. Furthermore, the correlation of various molecular mechanisms upon fluoride intoxication and associated neurobehavioral deficits has been discussed. Since there is no well-established mechanism to prevent fluorosis, phytochemical-based alleviation of its characteristic indications has been proposed as a possible remedial measure.

Evaluation of Maghemite Nanoparticles-Induced Developmental Toxicity and Oxidative Stress in Zebrafish Embryos/Larvae.

Maghemite nanoparticles ([Formula: see text] NPs) have a wide array of applications in various industries including biomedical field. There is an absence of legislation globally for the regulation of the production, use, and disposal of such NPs as they are eventually dumped into the environment where these NPs might affect the living systems. This study evaluates the effect of the [Formula: see text] NP-induced developmental toxicity in zebrafish embryos/larvae. The commercially available Fe2O3 NPs were purchased, and zebrafish embryos toxicity test was done by exposing embryos to various concentrations of [Formula: see text] NPs at 1 hpf and analyzed at 96 hpf. Based on the LC50 value (60.17 ppm), the sub-lethal concentrations of 40 and 60 ppm were used for further experiments. Hatching, lethality, developmental malformations, and heartbeat rate were measured in the control and treated embryos/larvae. The ionic Fe content in the media, and the larvae was quantified using ICP-MS and AAS. The biomolecular alterations in the control and treated groups were analyzed using FT-IR. The Fe ions present in the larvae were visualized using SEM-EDXS. In situ detection of AChE and apoptotic bodies was done using staining techniques. Biochemical markers (total protein content, AChE, and Na+ K+-ATPase) along with oxidants and antioxidants were assessed. A significant decrease in the heartbeat rate and hatching delay was observed in the treated groups affecting the developmental processes. Teratogenic analysis showed increased developmental deformity incidence in treated groups in a dose-dependent manner. The accumulation of Fe was evidenced from the ICP-MS, AAS, and SEM-EDXS. Alterations in AChE and Na+ K+-ATPase activity were observed along with an increment in the oxidants level with a concomitant decrease in antioxidant enzymes. These results show [Formula: see text] NP exposure leads to developmental malformation and results in the alteration of redox homeostasis.

Integrated in silico analysis for the identification of key genes and signaling pathways in copper oxide nanoparticles toxicity.

Copper oxide nanoparticles (CuO-NPs) are used in various industrial and commercial products due to their enhanced physicochemical properties. The vast consumption increases their exposure in the environment, thereby affecting the ecosystem. Even with the rise in research towards understanding their toxicity, the major signaling cascades and key genes involved in CuO-NPs remain elusive due to the various attributes involved (size, shape, charge, coating in terms of nanoparticles, and dose, duration, and species used in the experiment). The focus of the study is to identify the key signaling cascades and genes involved in CuO-NPs toxicity irrespective of these attributes. CuO-NPs related microarray expression profiles were screened from GEO database and were subjected to toxicogenomic analysis to elucidate the toxicity mechanism. In silico tools were used to obtain the DEGs, followed by GO and KEGG functional enrichment analysis. The identified DEGs were then analyzed to determine major signaling pathways and key genes. Module and centrality parameter analysis was performed to identify the key genes. Further, the miRNAs and transcription factors involved in regulating the genes were predicted, and their interactive pathways were constructed. A total of 44 DEGs were commonly present in all the analysed datasets and all of them were downregulated. GO analysis reveals that most of the genes were enriched in functions related to cell division and chemotaxis. Cell-cycle, chemokine, cytokine-cytokine receptor interaction, and p53 signaling pathways were the key pathways with Cdk1 as the major biomarker altered irrespective of the variables (dosage, duration, species used, and surface coating). Overall, our integrated toxicogenomic analysis reveal that Cdk1 regulated cell cycle and cytokine-cytokine signaling cascades might be responsible for CuO-NPs toxicity. These findings will help us in understanding the mechanisms involved in NPs toxicity.

Mitigation of arsenic induced developmental cardiotoxicity by ferulic acid in zebrafish.

We investigated whether ferulic acid (FA), a nutraceutical could mitigate the arsenic (As) induced cardiotoxicity. Zebrafish larvae (60 and 72 h post-fertilization [hpf]) were used to study the effect of FA on As at different time points (24 and 48 h after exposure). The FA exposure was given as pre-treatment (60 hpf) and simultaneous treatment (72 hpf) to translate the results for As contaminated areas. To accomplish this, the lethality assay was done, and based on the results, the dosage for As (1 mM) and FA (30 µM) was fixed. The FA intervention (30 µM) as 12 h pre-treatment (60 hpf) and simultaneous treatment along with As (72 hpf) decreased the As content in zebrafish larvae as evidenced by inductively coupled plasma-mass spectrometry. As exposure showed congenital deformities especially cardiac malformations in zebrafish larvae after 24 and 48 h. These teratogenic effects induced by As were reduced by FA supplementation in both groups. Also, o-dianisidine staining demonstrated that As treated larvae encountered abnormal cardiac function with reduced blood circulation, while FA supplementation reversed these effects. The acetylcholinesterase activity, a biomarker of As-induced cardiotoxicity was also found to be decreased in As group, which was rescued by FA. The modulation in the expression of the genes involved in cardiogenesis (nkx2.5, bmp2b, gata4, gata5, myh6, myl7, and tnnt2) further confirmed the ameliorative effect of FA on As induced malformations.

Chronic exposure to copper oxide nanoparticles causes muscle toxicity in adult zebrafish.

Repeated deposition of copper oxide nanoparticles (CuO-NPs) into aquatic systems makes them a global threat since the NPs accumulate in various organs of the fish particularly skeletal muscle. In the present study, adult zebrafish were exposed to different concentrations of CuO-NPs (1 and 3 mg/L) for a period of 30 days. The status of functional markers (acetylcholinesterase, creatine kinase-MB, and lactate dehydrogenase) and oxidative stress markers (oxidants and antioxidants) were analyzed. The histological changes in muscle were studied followed by the immunohistochemistry expression for catalase. Further, the expression of myoD, myogenin, pax7, ß-actin, and desmin was examined by semi-quantitative reverse transcriptase polymerase chain reaction. The results indicated that chronic exposure to CuO-NPs causes muscular damage as evidenced by elevated levels of functional markers. There was a significant increase in the oxidants with reduction in the antioxidant levels, implying that the antioxidant enzymes were unable to scavenge the free radicals induced by the CuO-NPs. The histopathological analysis showed degeneration and atrophy in the treated groups confirming muscle damage. The immunohistochemical catalase expression in the muscle was reduced in the treated groups further supporting the evidence that the antioxidant has suffered a decline. The altered gene expression indicates skeletal muscle damage due to the CuO-NPs exposure. Overall, the data suggest that chronic exposure to CuO-NPs caused muscular toxicity which may lead to muscle degeneration in adult zebrafish.

MicroRNAs and Xenobiotic Toxicity: An Overview.

The advent of new technologies has paved the rise of various chemicals that are being employed in industrial as well as consumer products. This leads to the accumulation of these xenobiotic compounds in the environment where they pose a serious threat to both target and non-target species. miRNAs are one of the key epigenetic mechanisms that have been associated with toxicity by modulating the gene expression post-transcriptionally. Here, we provide a comprehensive view on miRNA biogenesis, their mechanism of action and, their possible role in xenobiotic toxicity. Further, we review the recent in vitro and in vivo studies involved in xenobiotic exposure induced miRNA alterations and the mRNA-miRNA interactions. Finally, we address the challenges associated with the miRNAs in toxicological studies.

Role of epigenetics in zebrafish development.

The role of epigenetics in development has garnered attention in recent years due to their ability to modulate the embryonic developmental gene expression in response to the environmental cues. The epigenetic mechanisms - DNA methylation, histone modification, and non-coding RNAs have a unique impact on vertebrate development. Zebrafish, a model vertebrate organism is being used widely in developmental studies due to their high fecundability and rapid organogenesis. With increased studies on various aspects of epigenetics in development, this review gives a glimpse of the major epigenetic modifications and their role in zebrafish development. In this review, the basic mechanism behind each modification followed by their status in zebrafish has been reviewed. Further, recent advancements in the epigenetic aspect of zebrafish development have been discussed.