APPLICATION OF WHOLE-CELL NMR TECHNIQUES TO STUDY THE INTERACTION OF ARSENIC COMPOUNDS WITH CATHARANTHUS-ROSEUS CELL-SUSPENSION CULTURES

H-1 spin-echo NMR spectroscopy of intact cells of C. roseus facilitate s monitoring changes inside the cells on treatment with arsenicals. Th is in situ detection method is non-invasive and nondestructive in comp arison to other available biochemical methods. Short term uptake of th e arsinicals, methylarsinate MMA and dimethylarsenate DMA, by C. roseu s cells that have reached stationary phase in 1-B5 medium, is followed by using NMR spectroscopy, and in particular, the Carr-Purcell-Meiboo m-Gill pulse sequence. An increase in the peak height of the methylars enic resonance over a period of 11 h is indicative of uptake of each a rsenical. However, there is no evidence of any biotransformation produ cts in the H-1 NMR spectra. The accumulation site of DMA is probably t he vacuole as is seen from the change in the chemical shift of DMA as it moves into a compartment of lower pH. Biochemical changes associate d with the presence of arsenicals are evident in the H-1 NMR spectra o f C. roseus cells isolated at different stages in the growth cycle. Al though uptake has been demonstrated by other analytical techniques, th e resonances corresponding to both MMA and DMA are not observed in the H-1 NMR spectra of cells growing in media containing each arsenical. The association of these arsenicals with large biomolecules in the cel l may account for these absences. In this event, the spin-spin relaxat ion time of the arsenic species would shorten and the signals would no t be seen in the spin-echo NMR spectrum. In cells growing in the prese nce of MMA, a new resonance is observed at a chemical shift position 2 .2 ppm after 15 days of growth. The shift in position of the resonance , from 1.75 ppm expected at physiological pH, may indicate an altered environment around the arsenic species such as high intracellular acid ity.