O-6-METHYLGUANINE-DNA METHYLTRANSFERASE ACTIVITY IN HUMAN BUCCAL MUCOSAL TISSUE AND CELL-CULTURES - COMPLEX-MIXTURES RELATED TO HABITUAL USE OF TOBACCO AND BETEL QUID INHIBIT THE ACTIVITY IN-VITRO

Extracts prepared from tissue specimens of normal, non-tumourous human buccal mucosa, and cultured buccal epithelial cells and fibroblasts, exhibited O-6-methylguanine-DNA methyltransferase (MGMT) activity by c atalysing the repair of the premutagenic O-6-methylguanine lesion in i solated DNA with rates of 0.2 to 0.3 pmol/mg protein, An SV40 T antige n-immortalized buccal epithelial cell line termed SVpgC2a and a buccal squamous carcinoma line termed SqCC/Y1, both of which lack normal tum our suppressor gene p53 function, exhibited about 50 and 10% of the MG MT activity of normal cells, respectively, The normal, experimentally transformed and tumourous buccal cell types showed MGMT mRNA levels wh ich correlated with their respective levels of MGMT activity, Exposure of buccal cell cultures to various organic or water-based extracts of products related to the use of tobacco and betel quid, decreased both cell survival (measured by reduction of tetrazolium dye) and MGMT act ivity (measured subsequently to the exposures in cellular extracts), O rganic extracts of bidi smoke condensate and betel leaf showed higher potency than those of tobacco and snuff, An aqueous snuff extract also decreased both parameters, whereas an aqueous areca nut extract was w ithout effect. The well-established sulph-hydryl-reactive agent Hg2+, a corrosion product of dental amalgam, served as a positive control an d decreased MGMT activity following treatment of cells within a range of 1-10 mu M. Taken together, significant MGMT activities were demonst rated in buccal tissue specimens and in the major buccal mucosal cell types in vitro, Lower than normal MGMT activity in two transformed buc cal epithelial cell lines correlated with decreased MGMT mRNA and lack of functional p53, Finally, in vitro experiments suggested the potent ial inhibition of buccal mucosal MGMT activity by complex mixtures pre sent in the saliva of tobacco and betel nut chewers.