Effects of Perinatal Nitrate Exposure on Peripheral Organs' Inflammatory Markers

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2024-06-18

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Royal Melbourne Institute of Technology (RMIT) University

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Introduction: Nitrate exposure through drinking water has been linked to various health issues, including cancers and developmental disorders. However, the effect on the immune response in peripheral organs during development, remains unclear. Aim: This study investigates the effects of perinatal nitrate exposure on the expression of IL-6 and TNF-α in the liver. To undertake this work, first, an effective tissue homogenisation method was established, and we evaluated reference gene (Gapdh, 18SrRNA, Actb) stability in liver and lung tissues. Hypothesis: Perinatal nitrate exposure increases the expression of IL-6 and TNF-α. Materials and Methods: Swiss White mice were exposed to a nitrate concentration of 320 mg/L in the drinking water before and during pregnancy and through to tissue collections. We cryogenically homogenised the tissues with a mortar and pestle, followed by RNA extraction: 17 liver (six control, 11 nitrate-exposed) and eight lung (three control, five nitrate-exposed) at P60. Tissue recovery, RNA yield and quality were assessed. Quantitative real-time PCR measured the expression levels of reference and inflammatory genes; analysed with a two-way ANOVA. Results: There was a higher tissue recovery and RNA yield in liver compared to lung. RNA quality was within the ideal A260/A280 ratio for all samples, and ideal A260/A230 for 82.4% of samples. Reference genes stability was confirmed and Gapdh had the lowest standard deviation. No significant differences in IL-6 and TNF-α expression were found between nitrate-exposed and control groups. Discussion: Nitrate exposure did not impact the expression of IL-6 and TNF-α in the liver. The tissue preparation methods developed are adaptable for further applications. Future research should explore additional inflammatory markers, other tissues, and histopathological evaluations to understand nitrate’s systemic effects.

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Nitrate Exposure Perinatal Development Inflammatory Markers IL-6 TNF-α Liver Inflammation Swiss White Mice RNA Yield and Quality Tissue Homogenization Reference Genes Gene Expression Immune Response Nitrite Supplementation Biochemical Analysis

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