© 1991 Oxford University Press
research-article |
Toxicokinetics of tumour promoters of mouse skin. II. Metabolism of the tumour promoter 12-O-tetradecanoylphorbol-13-acetate in mouse skin and biological activities of metabolites
Insutitute of Biochemistry, German Cancer Research Center Im Neuenheimer Feld 280. D-6900 Heidelberg, FRG
The metabolism of the phorbol diester [20-3H]12-O-tetradecanoylphorbol-13-acetate ([3H]TPA) was studied in the back skin of NMRI mice after topical administration of a single tumour-promoting dose, dp. Up to 72 h after admrrustration most of the radioactivity recovered from the skin surface, and from the epidermis and dermis of the treated skin area was unchanged TPA, as determined by silica gel HPLC of extracts obtained from these skin fractions. The major TPA metabolite was less polar than TPA and chromatographed in the range of bngchain TPA-20-acylates. At 72 h, it accounted for about 25,5 and 30% of total radioactivity extracted from skin surface, epidermis and dermis respectively. Of metabolites more polar than TPA, phorbol-13-acetate (PA) by far predominated over 12-O-tetradecanoylphorbol (TP) in both the epidermis and dermis, and by 72 h its relative amount was 3.9 and 2.4% in these skin fractions. Both phorbol monoesters, PA and TP, were not detected in skin surface extracts. In addition to metabolites, various autoxidation products of TPA were present in small amounts in the extracts from each of the skin fractions. The TPA-20-acylate fraction of metabolites isolated from the extracts of skin fractions at 24 h was separated further into the individual metabolites by the combined use of argentation and reversed-phase HPLC. These individual metabolites were identifled by co-chromatography with authentic reference compounds. They were TPA-20-acylates carrying saturated fatty acids (16–26 carbon atoms), cis-mono-unsaturated fatty acids (16–24 carbon atoms), linoleic acid and arachidonic acid. The conjugation of TPA with long-chain fatty acids is the first example of a new route of xenobiotic metabolism in skin. When tested for irritant activity on the mouse ear, TPA-20-acylates were about one to two orders of magnitude less adive than TPA. Similarly, when TPA-20-tetradecanoate was tested for tumour-promoting activity on the back skin of NMRI mice over a dose range of dp = 50–200 nmol applied twice weekly according to the computer-assisted standard protocol 16, it was found to be of only intermediate potency as compared to the highly potent tumour promoter TPA. The results of the present investigation indicate that metabolic conjugation of TPA with longchain fatty acids to yield TPA-20-acylates is another pathway of metabolic deactivation of TPA, thus supporting the hypothesis that TPA itself is the ultimate tumour promoter in mouse skin.