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On the biochemical mechanism of tumorigenesis in mouse skin

VIII. Isolation and characterization of epidermal microsomes and properties of their arylhydrocarbon monooxygenase and epoxide hydr(at)ase

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Summary

  1. 1.

    Epidermis of the dorsal region of mice may be obtained by the scraping procedure described. Homogenization of epidermal scrapings followed by differential centrifugation allows to pellet a microsomal fraction which was characterized chemically, morphologically, and enzymatically. Strain and age of mice used are identical with that employed in the two stage experiment of mouse skin carcinogenesis.

  2. 2.

    As compared to homogenate the specific activities of arylhydrocarbon monooxygenase (AHM) and epoxide hydr(at)ase (EH) in that fraction are increased: AHM about 2–4 times and EH about 6–8 times.

  3. 3.

    Important characteristics of the epidermal arylhydrocarbon monooxygenase reaction with benozo[a]pyrene as substrate are: a fluorescence spectrum of the polar metabolites highly similar to that of the polar metabolites of incubations with liver microsomes as well as with that of standard 3-hydroxybenzo[a]pyrene; linearity of product formation with incubation times and protein concentrations used; a pH-optimum of 7.7–8.2; saturation of enzyme by the concentrations of substrate and cosubstrates employed; synergistic effect of NADPH and NADH; no influence by additional mg2+ ions; an apparent K m value of approximately 2×10−5 molar; inhibition of the reaction by SKF 525-A and 7,8-benzoflavone.

  4. 4.

    Important characteristics of the epidermal epoxide hydr(at)ase reaction with styrene oxide as substrate are: linearity of product formation with incubation times and protein concentrations used; a pH-optimum of 8–9; saturation of enzyme by the substrate concentration employed; an apparent Km value of approximately 2×10−4 molar.

  5. 5.

    These data unambiguously demonstrate the presence of both enzymes in the dorsal epidermis of mice on which the initiation is carried out in our two stage experiment. Moreover, from the influence of the selective inhibitors SKF 525-A and 7,8-benzoflavone on the activity of epidermal arylhydrocarbon monooxygenase in vitro it may be concluded that in epidermis this enzyme contains both main groups of cytochromes, the P-450 as well as the P-448.

Zusammenfassung

  1. 1.

    Epidermis der Rückenregion von Mäusen kann durch die beschriebene Abschabemethode gewonnen werden. Homogenisierung dieser Epidermispräparation gefolgt von differentieller Zentrifugation erlaubt das Abscheiden einer mikrosomalen Fraktion, die chemisch, morphologisch und enzymatisch charakterisiert wurde. Stamm und Alter der hierbei benutzten Mäuse entsprechen den im Zwei-Stufen-Experiment der Karzinogenese an der Mäusehaut verwendeten.

  2. 2.

    Im Vergleich zum Homogenat sind die spezifischen Aktivitäten von Arylkohlenwasserstoff-Monooxygenase (AHM) und Epoxidhydr(at)ase (EH) in dieser Fraktion erhöht: AHM um etwa das 2–4fache und EH um etwa das 6–8 fache.

  3. 3.

    Wichtige Charakteristika der epidermalen Arylkohlenwasserstoff-Monooxygenase-Reaktion mit Benz[a]pyren als Substrat sind: ein Fluoreszenz-Spektrum der polaren Metabolite, das mit jenem der polaren Metabolite aus Ansätzen mit Lebermikrosomen und dem der Bezugssubstanz 3-Hydroxybenz[a]pyren sehr ähnlich ist; Linearität der Produktbildung bei den angewandten Inkubationszeiten und Proteinkonzentrationen; ein pH-Optimum von 7,7–8,2; Sättigung des Enzyms durch die angewandten Konzentrationen an Substrat und Cosubstrat; Synergismus von NADPH und NADH; kein Einfluß zusätzlicher mg2+ -Ionen; ein meßbarer K m-Wert von schätzungsweise 2×10−5M; Hemmung der Reaktion durch SKF 525-A und 7,8-Benzoflavon.

  4. 4.

    Wichtige Charakteristika der epidermalen Epoxidhydr(at)asen-Reaktion mit Styrolepoxid als Substrat sind: Linearität der Produktbildung bei den angewandten Inkubationszeiten und Proteinkonzentrationen; ein pH-Optimum von 8–9; Sättigung des Enzyms durch die angewandte Substratkonzentration; ein meßbarer K m-Wert von schätzungsweise 2×10−4M.

  5. 5.

    Diese Daten beweisen eindeutig, daß beide Enzyme in der Epidermis der Rückenregion von Mäusen vorhanden sind, an denen im Zwei-Stufen-Experiment die Initiation erfolgt. Darüber hinaus kann auf Grund der Wirkung der selektiven Inhibitoren SKF-525-A und 7,8-Benzoflavon auf die Aktivität epidermaler Arylkohlenwasserstoff-Monooxygenase in vitro geschlossen werden, daß dieses Enzym in der Epidermis beide Hauptgruppen von Cytochromen, sowohl P-450 als auch P-448, enthält.

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Abbreviations

PAH:

polycyclic aromatic hydrocarbon(s)

DMBA:

7,12-dimethylbenz[a]-anthracene

BP:

benzo[a]pyrene

3OHBP:

3-hydroxybenzo[a]pyrene

OHBP:

hydroxylated benzo[a]pyrenes (polar metabolites)

BF:

7,8-benzoflavone

SKF:

2-diethylaminoethyl-2,2-diphenylvalerat. HCl (SKF 525-A)

PB:

phenobarbital

NADPH:

nicotine amide adenine dinucleotide phosphate, red.

NADH:

nicotine amide adenine dinucleotide, red.

TCA:

trichloroacetic acid

AHM:

arylhydrocarbon monooxygenase

EH:

epoxide hydr(at)ase

glc6Pase:

glucose-6-phosphatase

s:

standard deviation

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  1. Deutsches Krebsforschungszentrum Heidelberg, Im Neuenheimer Feld 280, D-6900, Heidelberg, Federal Republic of Germany

    W. G. Pyerin &  E. Hecker

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Pyerin, W.G., Hecker, E. On the biochemical mechanism of tumorigenesis in mouse skin. Z. Krebsforsch. 90, 259–279 (1977). https://doi.org/10.1007/BF00284300

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