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 facilitates monitoring changes inside the cells on treatment with arsenicals. This in situ detection method is non-invasive and nondestructive in comparison to other available biochemical methods. Short term uptake of the arsinicals, methylarsinate MMA and dimethylarsenate DMA, by C. roseus cells that have reached stationary phase in 1-B5 medium, is followed by using NMR spectroscopy, and in particular, the Carr-Purcell-Meiboom-Gill pulse sequence. An increase in the peak height of the methylarsenic resonance over a period of 11 h is indicative of uptake of each arsenical. However, there is no evidence of any biotransformation products in the H-1 NMR spectra. The accumulation site of DMA is probably the vacuole as is seen from the change in the chemical shift of DMA as it moves into a compartment of lower pH. Biochemical changes associated with the presence of arsenicals are evident in the H-1 NMR spectra of C. roseus cells isolated at different stages in the growth cycle. Although uptake has been demonstrated by other analytical techniques, the 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 cell may account for these absences. In this event, the spin-spin relaxation time of the arsenic species would shorten and the signals would not be seen in the spin-echo NMR spectrum. In cells growing in the presence 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 acidity.