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The effect of Arsenicals on cell suspension cultures of Catharanthus Roseus (Madagascan periwinkle)

University of British Columbia

Although the biotransformation of simple arsenic compounds to a variety of complex arsenosugars inside marine plants is well documented, the role of terrestrial plants has not been thoroughly investigated. The effect of arsenicals on a terrestrial plant, Catharanthus roseus, is examined by using cell suspension cultures as a model system. The minimum inhibitory concentration of arsenate is low at 5 pg mL1 and it is the most toxic species to cell suspension cultures of C. roseus. Arsenite and methylarsonate (MMA) have a less toxic effect. Dimethylarsinate (DMA) is the least toxic of the arsenic species studied, with normal growth rates observed with up to 50 pg mL1 of arsenic in the medium. Significant incorporation of arsenic by cell suspension cultures of C. roseus is observed when grown in media containing arsenicals. The toxicity to each arsenical seems to be a direct function of uptake. Speciation of arsenic in cell extracts shows that C. roseus cells are capable of both methylation and demethylation of arsenicals. However, there is no evidence for the formation of significant levels of complex organoarsenic species. The biosynthesis of indole alkaloids by C. roseus plants as well as tissue and cell cultures has been well documented. Addition of arsenicals into the alkaloid production medium, APM, changes the pattern of alkaloid accumulation. The effect is dependent on the arsenic species, its concentration, as well as the time of application. Treatment with the arsenicals, arsenate, arsenite, MMA and DMA, during early growth stages, has an inhibitory effect on alkaloid production. Although DMA is the least toxic to growth, it has a drastic effect on alkaloid production. Tryptamine, an early precursor of the indole alkaloids, accumulates in cells treated with DMA indicating that the initial step of condensation of tryptamine with secologanin is inhibited. Treatment with arsenicals during the early stationary phase of culture growth enhances the accumulation of some alkaloids, although some are suppressed. For example, treatment with DMA on day 22 of growth results in increased levels of an unidentified alkaloid (MW 354) whereas production of catharanthine is completely suppressed. 1H 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 noninvasive and nondestructive in comparison to other available biochemical methods. Short term uptake of the arsenicals, MMA and DMA, by C. roseus cells that had reached stationary phase in 1-B5 medium, is followed by using the Carr-Purcell-Meiboom-Gill pulse sequence. An increase in the peak height of the methylarsenic resonance over a period of 11 hours, is indicative of uptake of each arsenical. However, there is no evidence of any biotransformation products in the 1H 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 1H 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 1HNMR spectra of cells growing in media containing each arsenical. The association of these arsenicals with large biomolecules- in the cell may account for the absence of these resonances.

Thesis/Dissertation

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2008-12-16

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http://hdl.handle.net/2429/2961

Chemistry

University of British Columbia

UBCV

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