Generation of the arachidonic acid metabolite 8-HETE by extracts of mouse skin treated with phorbol ester in vivo; identification by 1H-n.m.r. and GC-MS spectroscopy

After a single in vivo application of 20 nmol of the ‘complete’tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) orof the ‘incomplete’ promoter 12-O-retinoylphorbol-13-acetate(RPA) to the back skin of mice, a major arachidonic acid metabolite(AAMX) was found in cell-free epidermal preparations after additionof arachidonic acid (AA). Studies with cyclooxygenase and lipoxygenaseinhibitors indicated that this metabolite is a lipoxygenaseproduct. The metabolite has been purified by sequential useof t.l.c. and h.p.l.c. and identified as 8-hydroxy-5Z, 9E, 11Z,14Z-eicosatetraenoic acid (8-HETE) by means of GC-MS and ¹H-n.m.r.spectroscopy. To assist in the characterization of AAMX, a referencemixture of 8-HETE and 9-HETE was used for which a complete structuralanalysis could be performed using one- and twodimensional ¹H-n.m.r.at 500 MHz. The production of 8-HETE has been shown to startwith a lag phase of 3 h after TPA treatment and to reach a maximumafter 24–48 h. Nonpromoting phorbol esters are 10-fold,the Ca²⁺-ionophore A 23187 100-fold, less efficient in inducingin vivo the lipoxygenase responsible for 8-HETE production.3-Day-old neonatal mice cannot be induced to generate 8-HETEin response to TPA, whereas 8-day-old mice show an extremelystrong response. Neither primary basal keratinocytes, isolatedfrom TPA-treated mouse epidermis nor TPA-treated epidermal celllines generate 8-HETE, indicating that the metabolite may notoriginate from these epithelial cells.