(169bd) Exploring the Effects of Tributyltin Exposure on Adipocytes: An Integrated Transcriptomics and Metabolomics Study to Decode Molecular Response Linked to Metabolic Syndrome

Endocrine disrupting chemicals (EDCs) are compounds that are known to disrupt physiological processes, such as hormonal regulation, which can in turn lead to adverse health outcomes in humans. EDCs comprise a significant number of environmental pollutants that are indiscriminately released into the environment every year, and most of which have endocrine-disrupting potential. Metabolic syndrome (MetS) is a cluster of seemingly vague medical conditions frequently occurring together and includes obesity, insulin resistance, dyslipidemia, and hypertension, and which have been associated with increased risk of cardiovascular disease and type 2 diabetes. Moreover, MetS is increasingly thought to be attributed, at least in part, to exposure to EDCs through inhalation, ingestion, or absorption from various exposures within the environment. Adipose tissue consists of various cell types such as adipocytes, pre-adipocytes, stromal vascular cells, extracellular matrix components, and blood vessels, and is a highly complex and dynamic tissue that plays a vital role in metabolic regulation. Adipocytes are the primary cell type that compose adipose tissue and is the major repository site for the storage and release of energy in the form of lipids, but also contribute to endocrine function and hormone sensing and response. Certain EDCs have shown the potential to interfere with normal development and function of adipose tissue. To better understand the effects of EDCs on metabolic syndrome, adipocytes were treated to a known lipogenic substance, tributyltin (TBT), and the -omic signature associated with metabolic dysregulation was mapped.

Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocytes were grown to near confluence and then incubated (d0) in serum-free differentiation medium [2 μmol/l rosiglitazone, 25 nmol/l dexamethasone, 0.5 mmol/l methylisobuthylxantine, 0.1 μmol/l cortisol, 0.01 mg/ml transferrin, 0.2 nmol/l triiodothyronine, and 20 nmol/l human insulin]. After 4 days, the medium was changed, and the cells were further cultured in medium supplemented with 0.1 μmol/l cortisol, 0.01 mg/ml transferrin, 0.2 nmol/l triiodothyronine, and 20 nmol/l human insulin. TBT-exposed SGBS preadipocytes were differentiated as specified above; for the initial four days (d0–d4), the differentiation medium was additionally supplemented with TBT (25 nM). Cells were harvested at d10 of differentiation.

Transcriptomic analysis was performed using Agilent microarrays to determine differentially expressed genes (DEGs) between treatment groups. Samples collected for metabolomics were analysed using Agilent 1290 Infinity HPLC LC System coupled to an Agilent 6540 HRMS-QTOF/ LCMS system with Reversed-Phase (RP) and Hydrophilic Interaction (HILIC) Liquid Chromatography columns in positive (PI) and negative ionisation (NI) modes. Data preprocessing, batch correction, and statistical analyses were conducted in R using limma for transcriptomics and xcms, IPO, PMCMRplus, and xMSannotator packages for metabolomics for univariate analyses while for multivariate analyses utilised the mixOmics package. Joint pathway analyses were carried out by importing data from univariate and multivariate analyses, thus leveraging the capabilities of the MetaboAnalystR package to facilitate a better comprehension of multi-omic mechanisms.

The univariate statistical analysis resulted in 1088 differentially expressed genes (DEGs) between TBT-exposed cells compared to controls while 551 and 239 metabolites were statistically significantly differentially detected from HILIC and RP analysis. Overall, the categories of glycerophospholipids and fatty acyls were most commonly detected. Across all analyses, the top three commonly occurring perturbed pathways were glycerophospholipid metabolism, retrograde endocannabinoid signalling, and taurine and hypotaurine metabolism. Glycerophospholipids, a class of lipids fundamental to cellular membranes and signalling, play a pivotal role in various physiological processes, including energy homeostasis, lipid transport, and inflammation, and serve as precursors for signalling molecules that directly modulate metabolic processes like inflammation and insulin sensitivity (Alves et al., 2021; Yoon et. al., 2021). Furthermore, aberrant glycerophospholipid metabolism is implicated in the development of non-alcoholic fatty liver disease (NAFLD), a condition closely linked to metabolic disturbances (Liang & Dai, 2022). Disruption in retrograde endocannabinoid signalling has recently been associated with dyslipidemia, obesity and diabetes by influencing cannabinoid receptors both centrally and peripherally (Lipina et al., 2023). Moreover, the endocannabinoid system in general is suggested to play a crucial role in oxidative/nitrosative stress and inflammatory processes within the liver, and thus, been linked to NAFLD and metabolic dysfunction (Jorgaĉević et al., 2021). Taurine and hypotaurine play vital roles in maintaining normal lipid metabolism and is also involved in antioxidant and bile acid conjugation activities (Du et al., 2020). In addition, hepatic taurine levels could potentially influence hepatic steroid metabolism and other lipid metabolisms (Du et al., 2020). Another study by Miao et al., (2022) found that taurine and hypotaurine supplementation was shown to significantly alter outcomes of liver injury, fat accumulation, and insulin sensitivity in NAFLD mice.

Overall, the integrated omics analysis pipeline demonstrated the complexity of cellular responses to TBT exposure, utilizing both univariate and multivariate analytical methods to deepen our understanding of metabolic mechanisms. This approach not only highlighted individual molecular changes but also revealed their collective impact on cellular pathways. Key findings include the consistent identification of perturbed pathways such as glycerophospholipid metabolism, retrograde endocannabinoid signaling, and taurine and hypotaurine metabolism. These insights lay the groundwork for future mechanistic studies and could guide the development of strategies to mitigate the adverse effects associated with exposures to obesogenic and endocrine disrupting compounds with regards to MetS.

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