Neutrophil-derived hydrogen peroxide (H202) is believed to play an important role in the pathogenesis of vascular injury and pulmonary edema. H202 time-and dose-dependently increased the hydraulic conductivity and decreased the selectivity of an endothelial cell monolayer derived from porcine pulmonary arteries. Effects of H202 on endothelial permeability were completely inhibited by adenylate cyclase activation with 10-12 M cholera toxin or 0.1 juM forskolin. 10' M Sp-cAMPS, a cAMP-dependent protein kinase A agonist, was similarly effective. The phosphodiesterase (PDE) inhibitors motapizone (10'-M), rolipram (10-' M), and zardaverine (10-8 M), which specifically inhibit PDE-isoenzymes III, IV, and III/IV potently blocked H202-induced endothelial permeability when combined with 10' M prostaglandin El. Overall cellular cAMP content and inhibition of H202 effects on endothelial permeability were poorly correlated. H202 exposure resulted in a rapid and substantial decrease in endothelial cAMP content. The analysis of the PDE isoenzyme spectrum showed high activities of isoenzymes II, III, and IV in porcine pulmonary endothelial cells. The data suggest that adenylate cyclase activation/PDE inhibition is a powerful approach to block H202-induced increase in endothelial permeability. This concept appears especially valuable when endothelial PDE isoenzyme pattern and PDE inhibitor profile are matched optimally. (J.

Glucuronides of the mycotoxin T-2 toxin and its phase I metabolite HT-2 toxin are important phase II metabolites under in vivo and in vitro conditions. Since standard substances are essential for the direct quantitation of these glucuronides, a method for the enzymatic synthesis of T-2 and HT-2 toxin glucuronides employing liver microsomes was optimized. Structure elucidation by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry revealed that besides T-2 toxin glucuronide and HT-2 toxin 3-glucuronide also the newly identified isomer HT-2 toxin 4-glucuronide was formed. Glucuronidation of T-2 and HT-2 toxin in liver microsomes of rat, mouse, pig, and human was compared and metabolites were analyzed directly by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). A distinct, species specific pattern of glucuronidation of T-2 and HT-2 toxin was observed with interesting interindividual differences. Until recently, glucuronides have frequently been analyzed indirectly by quantitation of the aglycone after enzymatic cleavage of the glucuronides by β-glucuronidase. Therefore, the hydrolysis efficiencies of T-2 and HT-2 toxin glucuronides using β-glucuronidases from Helix pomatia, bovine liver, and Escherichia coli were compared.

The mycotoxin T-2 toxin, produced by various Fusarium species, is a widespread contaminant of grain and grain products. Knowledge about its toxicity and metabolism in the human body is crucial for any risk assessment as T-2 toxin can be detected in processed and unprocessed food samples. Cell culture studies using cells of human origin represent a potent model system to study the metabolic fate of T-2 toxin as well as the cytotoxicity in vitro. In this study the metabolism of T-2 toxin was analyzed in a cell line derived from human colon carcinoma cells (HT-29) and primary human renal proximal tubule epithelial cells (RPTEC) using high-performance liquid chromatography coupled with Fourier transformation mass spectrometry (HPLC-FTMS). Both cell types metabolized T-2 toxin to a variety of compounds. Furthermore, cell cycle analysis in RPTEC proved the apoptotic effect of T-2 toxin and its metabolites HT-2 toxin and neosolaniol in micromolar concentrations.

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