6. Preliminary figures for androstenol excretion in urine are given. In the age group 18-45 years they ranged from 19 to 2630 ,ug./24 hr. in men and from 66 to 1010 ,ug./24 hr. in women. This work was carried out partly during the tenure of a personal grant from the Medical Research Council to B. W. L. B., who wishes to express his gratitude. Our gratefiul thanks are due to Dr J. A. Pollock, Miss S. Johnson and Miss J. Bradley for their invaluable technical assistance, to the many people who provided 24 hr. urine specimens, to Organon Laboratories Ltd. for a generous gift of androsterone, and to the Medical Research Council for samples of steroids from their Reference Collection. Finally we are indebted to Dr C. L. Hewett of Organon Laboratories Ltd. for his suggestions on the route for the preparation of androstenol.
1. In neutral aqueous solution N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) yields salts of nitrocyanamide as u.v.-absorbing products. With cysteine, as found independently by Schulz & McCalla (1969), the principal product is 2-nitràminothiazoline-4-carboxylic acid. Both these reactions liberate the methylating species; thiols enhance the rate markedly at neutral pH values. An alternative reaction with thiols gives cystine, presumably via the unstable S-nitrosocysteine. 2. Thiols (glutathione or N-acetylcysteine) in vitro at about the concentration found in mammalian cells enhance the rate of methylation of DNA markedly over that in neutral solution. 3. Treatment of cultured mammalian cells with MNNG results in rapid methylation of nucleic acids, the extent being greater the higher the thiol content of the cells. Rodent embryo cells are more extensively methylated than mouse L-cells of the same thiol content. Cellular thiol concentrations are decreased by MNNG. Proteins are less methylated by MNNG than are nucleic acids. 4. Methylation of cells by dimethyl sulphate does not depend on cellular thiol content and protein is not less methylated than nucleic acids. Methylation by MNNG may therefore be thiol-stimulated in cells. 5. Both in vitro and in cells about 7% of the methylation of DNA by MNNG occurs at the 6-oxygen atom of guanine. The major products 7-methylguanine and 3-methyladenine are given by both MNNG and dimethyl sulphate, but dimethyl sulphate does not yield O(6)-methylguanine. Possible reaction mechanisms to account for this difference between these methylating agents and its possible significance as a determinant of their biological effects are discussed.
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