Chapter 28
Tocopherol Inhibition of NO -Mediated Nitrosation Nitrosamines and Related N-Nitroso Compounds Downloaded from pubs.acs.org by UNIV OF CALIFORNIA SANTA BARBARA on 09/17/18. For personal use only.
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In Vitro and Biological Superiority of γ-Tocopherol R. V. Cooney, A. A. Franke, L. J. Mordan, P. J. Harwood, V. Hatch-Pigott, andL.J. Custer Cancer Research Center of Hawaii, University of Hawaii at Manoa, 1236 Lauhala Street, Honolulu, HI 96813 Nitrogen oxides (NO ) are potentially carcinogenic because of their ability to nitrosate amines. Exposure to ΝΟ can produce mutations directly through deamination of primary amino groups of DNA bases (1,2) or through the formation of stable N-nitrosamines which can act as DNA alkylating agents upon metabolic activation. Many antioxidants, including α-tocopherol, have been shown to prevent nitrosation; however, most assays look only at the immediate effects of an inhibitor on nitrosation of amines and do not take into account slower nitrosation reactions from other reaction products. Organic nitrous esters, formed from nitrosating agents, are known to react with amines to yield N-nitrosamines (3,4) and α-tocopherol has been reported to react with NO to form a nitrous ester (5). This property is in contrast to the perception of α-tocopherol as a potent nitrosation inhibitor. We report, in agreement with previous studies, that α-tocopherol is superior to both γ-tocopherol and δ-tocopherol in preventing nitrosation of morpholine by NO when N-nitrosomorpholine is measured immediately after NO exposure (99.6%, 88.4% and 84.2% inhibition of nitrosation respectively, for 10 mM tocopherol and 1 mM morpholine in dichloromethane exposed to 27.5 ppm N O / N for 5 minutes). In contrast, measurement of Nnitrosomorpholine formation after extended incubation of the reaction mixture described above reveals α-tocopherol to be the least effective of the three (66%, 84.9% and 80.9% for α, γ and δ respectively, 168 hours after exposure). Figure 1 indicates that the difference in chemical reactivity between α-tocopherol and γ-tocopherol with NO probably is due to the formation of an intermediate nitrosating agent from α-tocopherol which reacts with morpholine at 37°C to form an N-nitrosamine. The observed reaction is independent of light and does not require oxygen although nitrosation is slightly enhanced in the presence of 0 . 7-Tocopherol, which lacks a methyl group at C-5, does not form a nitrosating agent in its reaction with N 0 . Exposure of 7-tocopherol to NOo resulted in the formation of a colored compound absorbing at 432 nm (in hexane) which was separated from the parent tocopherol by HPLC. As shown in Figure 2, nitric oxide (NO) formed during 7-tocopherol exposure to N 0 . 7-Tocopherol produced significantly more NO than α-tocopherol suggesting that greater efficiency of reduction of N 0 to NO may, in part, account for the superiority of 7-tocopherol as a nitrosation inhibitor. X
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0097-6156/94/0553-0317$08.00/0 © 1994 American Chemical Society
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NITROSAMINES AND RELATED JV-NTTROSO COMPOUNDS
The C3H 10T1/2 mouse fibroblast transformation assay (7) was used to assess the relative effectiveness of α-tocopherol and 7-tocopherol to inhibit neoplastic transformation during the promotional phase of carcinogenesis. At a concentration of 30 μΜ, -tocopherol reduced the development of transformed foci by 90% while α-tocopherol was only 50% effective (p
