15 N Investigation into the Effect of a Pollutant on the Nitrogen Metabolism of Tetrahymena pyriformis as a Model for Environmental Medical Research

A pilot study was performed to examine the potential of stable isotope techniques for monitoring the impact of a harmful substance on the cellular nitrogen metabolism in the ciliate species Tetrahymena pyriformis. After identical cultivation periods of control cells and toluene-exposed cells in a defined culture medium enriched with [guanidino-15N2]l-arginine, a number of nitrogen-containing pools were analyzed: 1) quantity and 15N abundance of ammonia as the end product of nitrogen metabolism in the system; 2) pattern and 15N abundances of the protein-bound amino acids in the cells; 3) pattern and 15N abundances of free amino acids in the cells; and 4) pattern and 15N abundances of the amino acids in the culture medium. In addition to 15N emission spectrometry, a new gas chromatography/combustion interface-isotope ratio mass spectrometry/mass spectrometry analytical system was used. The production and 15N content of ammonia were higher in the toluene-exposed system by 30% and 43%, respectively, indicating higher deamination rates and greater arginine consumption. The toluene-exposed cells exhibited increased 15N abundances of protein-bound amino acids in alanine, aspartic acid, glutamic acid, and tyrosine. Furthermore, structural analyses revealed the presence of N[Omega]-acetylarginine and pyrrolidonecarboxylic acid--compounds that had not previously been detected in Tetrahymena pyriformis. Differences in the 15N-enrichment of free amino acids were also evident. This new effect-monitoring system designed to investigate the impact of a pollutant on protein metabolism by using a stable isotope-labeled cell culture is a powerful tool for environmental medical research.