Regulation of the Tpo, Tg, Duox2, Pds, and Mct8 genes involved in the synthesis of thyroid hormones after subchronic exposure to sodium nitrate in female Wistar rats.

Nitrates are natural compounds present in soil and water; however, the intense use of fertilizers has increased their presence in groundwater with deleterious effects on human health. There is evidence of nitrates acting as endocrine disruptors; however, the underlying molecular mechanisms have not been fully described. Here, we investigated the effect of subchronic exposure to different concentrations of sodium nitrate in female Wistar rats, evaluating thyroid hormonal parameters, such as Nis transporter (Na+ /I- symporter, Slc5a5) and Tsh-R receptor protein expression, as well as transcription of the Tpo (thyroperoxidase), Tg (tiroglobulin), Duox2 (dual oxidase 2), Pds (pendrin), and Mct8 (Mct8 transporter, Slc16a2) genes. Hematological and histochemical changes in the liver and thyroid were also explored. Significant differences were found in platelet and leukocyte counts; although a significant increase in the weight of the thyroid gland was observed, no differences were found in the levels of the hormones Tsh, T3, and T4, but a modulation of the mRNA expression of the Tg, Tpo, Duox2, Mct8, and Pds genes was observed. Morphological changes were also found in liver and thyroid tissue according to the exposure doses. In conclusion, subchronic exposure to sodium nitrate induces leukocytosis consistent with an inflammatory response and upregulation of Sod2 in the liver and increases the expression of genes involved in the synthesis of thyroid hormones, keeping thyroid hormone levels stable. Histological changes in the thyroid gland suggest a goitrogenic effect.

Development of a method for the determination of 8-iso-PGF2α in sheep and goat plasma using solid-phase microextraction and ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Isoprostane 8-iso-PGF2α is a biomarker of lipid peroxidation in cell membranes. The method developed to measure plasma total levels (esterified + free) of 8-iso-PGF2α must be reproducible and be able to reduce the use of solvents in solid phase extraction. It should be useful to evaluate oxidative stress due to the excess of free radicals that are generated by some disorder or disease. METHODS: The method was developed using solid-phase microextraction with Oasis®MAX µElution plates and ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS). Electrospray ionization was performed in the negative mode (ESI-); the multiple reaction monitoring mode (MRM) was used. The development of the method included the optimization of the chromatographic conditions to achieve the separation of PGF2α and 8-iso-PGF2α as well as the optimization of the microextraction conditions of the analyte of interest in ovine and goat plasma. RESULTS: The developed method was validated with a calibration curve of plasma samples fortified with standards at five concentration levels in the range 49-639 pg/mL. The average recovery was 89% with a standard deviation of 10.73%. The inter-day precision was evaluated, obtaining a coefficient of variance (CV) less than 15%. The limit of quantification was 20 pg/mL and the limit of detection was 10 pg/mL. 8-iso-PGF2a was determined in the plasma of 14 sheep and 20 goats of 5 months of age and 6 goats of 24 months of age. The concentrations found were 50-300 pg/mL. CONCLUSIONS: The method developed is precise, accurate and reliable with low reagent consumption compared with conventional solid-phase extraction. The analysis time was decreased because, with the use of the microextraction plate, the step of the evaporation and reconstitution of the analyte was avoided. The method is applicable to quantify the plasma total levels (esterified + free) of 8-iso-PGF2α.