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) or of 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 addition of arachidonic acid (AA). Studies with cyclooxygenase and lipoxygenase inhibitors indicated that this metabolite is a lipoxygenase product. The metabolite has been purified by sequential use of 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 reference mixture of 8-HETE and 9-HETE was used for which a complete structural analysis could be performed using one- and two-dimensional 1H-n.m.r. at 500 MHz. The production of 8-HETE has been shown to start with a lag phase of 3 h after TPA treatment and to reach a maximum after 24-48 h. Nonpromoting phorbol esters are 10-fold, the Ca2+-ionophore A 23187 100-fold, less efficient in inducing in vivo the lipoxygenase responsible for 8-HETE production. 3-Day-old neonatal mice cannot be induced to generate 8-HETE in response to TPA, whereas 8-day-old mice show an extremely strong response. Neither primary basal keratinocytes, isolated from TPA-treated mouse epidermis nor TPA-treated epidermal cell lines generate 8-HETE, indicating that the metabolite may not originate from these epithelial cells.
Gschwendt
M
M
Fürstenberger
G
G
Kittstein
W
W
Besemfelder
E
E
Hull
W E
WE
Hagedorn
H
H
Opferkuch
H J
HJ
Marks
F
F
D000818:Animals; D000831:Animals, Newborn; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis*; D008084:Lipoxygenase / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D010703:Phorbol Esters / Q000494:pharmacology*; D012867:Skin / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001095:Arachidonic Acids; D006893:Hydroxyeicosatetraenoic Acids; D010703:Phorbol Esters; D016718:Arachidonic Acid; D000001:Calcimycin; C047628:8-hydroxyeicosatetraenoic acid; D008084:Lipoxygenase; D013755:Tetradecanoylphorbol Acetate