Skip to main content
NCBI home page
Japanese Journal of Cancer Research : Gann logoLink to Japanese Journal of Cancer Research : Gann
. 1992 Nov;83(11):1204–1209. doi: 10.1111/j.1349-7006.1992.tb02746.x

Evidence of Direct Generation of Oxygen Free Radicals from Heterocyclic Amines by NADPH/Cytochrome P‐450 Reductase in vitro

Keizo Sato 1,2, Takaaki Akaike 1, Yuichiro Kojima 1, Masayuki Ando 2, Minako Nagao 3, Hiroshi Maeda 1,✉
PMCID: PMC5918710  PMID: 1336493

Abstract

Highly purified cytochrome P‐450 reductase (also called cytochrome c reductase; EC 1.6.2.4.) and NADPH were used to generate superoxide radical (O2) from 11 different heterocyclic amines (HCAs) as identified by electron spin resonance spectroscopy using the spin trapping method with 5,5‐dimethyl‐1‐pyrroline JV‐oxide (DMPO). The signal intensity of DMP0‐OOH(‐O2−) (i.e., the DMPO spin adduct of ‐O2−) was strongest for 2‐amino‐3,4‐dimethy]imidazo[4,5‐f]quinoline (MeIQ). The O2− generation with HCAs decreased in the following order: 2‐amino‐3,8‐dimethyI‐imidazo[4,5‐f] quinoxaline (MeIQx) = 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ) > 2‐amino‐3,4,8‐trimethylimidazo[4,5‐f]quinoxaline (diMeIQx) ≥ other HCAs; O2− generation was lowest with 2‐amino‐3‐methyl‐9H‐pyrido[2,3‐b]indole CH3COOH (MeAαC). By using Lineweaver‐Burk plots, Km values of cytochrome P‐450 reductase for mitomycin C., IQ, and MeIQ were determined to be 1,60 × 10−6 M, 1.97 × 10−5M, and 2.83 × 10−6 M, respectively. The present findings have important implications for carcinogenesis because of the known effect of oxygen radicals on cell proliferation.

Keywords: Heterocyclic amine, NADPH/cytochrome P‐450 reductase Carcinogenesis, ESR spin trapping

Full Text

The Full Text of this article is available as a PDF (341.1 KB).

REFERENCES

  • 1. ) Guengerich , F. P.Roles of cytochrome P‐450 enzymes in chemical carcinogenesis and cancer chemotherapy . Cancer Res. , 48 , 2946 – 2954 ( 1989. ). [PubMed] [Google Scholar]
  • 2. ) Guengerich , F. P.Reactions and significance of cytochrome P‐450 enzymes . Biol. Chem. , 266 , 10019 – 10022 ( 1991. ). [PubMed] [Google Scholar]
  • 3. ) Witz , G.Active oxygen species as factors in multistage carcinogenesis . Proc. Soc. Exp. Biol. Med. , 198 , 675 – 682 ( 1991. ). [DOI] [PubMed] [Google Scholar]
  • 4. ) Harris , C. C.Chemical and physical carcinogenesis: advances and perspectives for the 1990s . Cancer Res. ( Suppl ), 51 , 5023s – 5044s ( 1991. ). [PubMed] [Google Scholar]
  • 5. ) Kasai , H. , Okada , Y. , Nishimura , S. , Rao , M. S. and Reddy , J. K.Formation of 8‐hydroxydeoxyguanosine in liver DNA of rats following long‐term exposure to a peroxisome proliferator . Cancer Res. , 49 , 2603 – 2605 ( 1989. ). [PubMed] [Google Scholar]
  • 6. ) Shigenaga , M. K. , Gimeno , C. J. and Ames , B.Urinary 8‐hydroxy‐2′‐deoxyguanosine as a biological marker of in vivo oxidative DNA damage . Proc. Natl. Acad. Sci. USA , 86 , 9697 – 9701 ( 1989. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. ) Kalyanaraman , B. , Perez‐Reyes , E. and Mason , R. P.Spin‐trapping and direct electron spin resonance investigation of the redox metabolism of quinone anticancer drugs . Biochim. Biophys. Acta , 630 , 119 – 130 ( 1980. ). [DOI] [PubMed] [Google Scholar]
  • 8. ) Bligh , H. F. J. , Bartoszek , A. , Robson , C. N. , Hickson , I. D. , Kasper , C. B. , Beggs , J. D. and Wolf , C. R.Activation of mitomycin C by NADPH: cytochrome P‐450 reductase . Cancer Res. , 50 , 7789 – 7792 ( 1990. ). [PubMed] [Google Scholar]
  • 9. ) Halliwell , B.Review article. How to characterize a biological antioxidant . Free Radical Res. Comm. , 9 , 1 – 32 ( 1990. ). [DOI] [PubMed] [Google Scholar]
  • 10. ) Dillard , C. J. and Tappel , A. L.Lipid peroxidation products in biological tissue . Free Radical Biol. Med. , 7 , 193 – 196 ( 1989. ). [DOI] [PubMed] [Google Scholar]
  • 11. ) Akaike , T. , Sato , K. , Ijiri , S. , Miyamoto , Y. , Kohno , M. , Ando , M. and Maeda , H.Bactericidal activity of alkyl peroxyl radicals generated by heme‐iron‐catalyzed decomposition of organic peroxides . Arch. Biochem. Biophys. , 294 , 55 – 63 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 12. ) Taffe , B. G. , Takahashi , N. , Kensler , T. W. and Mason , R. P.Generation of free radicals from organic hydro‐peroxide tumor promoters in isolated mouse keratinocytes. Formation of alkyl and alkoxyl radicals from tert‐butyl hydroperoxide and cumene hydroperoxide . J. Biol. Chem. , 262 , 12143 – 12149 ( 1987. ). [PubMed] [Google Scholar]
  • 13. ) Maeda , H. , Katsuki , T. , Akaike , T. and Yasutake , R.High correlation between lipid peroxide radical and tumor‐promoter effect: suppression of tumor promotion in the Epstein‐Barr virus/B‐lymphocyte system and scavenging of alkyl peroxide radicals by various vegetable extracts . Jpn. J. Cancer Res. , 83 , 923 – 928 ( 1992. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. ) Ardies , C. M. , Lasker , J. M. , Bloswick , B. P. and Lieber , C. S.Purification of NADPH: cytochrome c (cytochrome P‐450) reductase from hamster liver microsome by detergent extraction and affinity chromatography . Anal. Biochem. , 162 , 39 – 46 ( 1987. ). [DOI] [PubMed] [Google Scholar]
  • 15. ) Kamataki , T. , Komori , M. , Kitamura , R. , Uchida , T. , Fukuta , H. , Matuoka , H. , Baba , H. , Inoue , H. and Yoshikawa , K.Phase I enzymes responsible for the activation promutagens and procarcinogens . Environ. Mutag. Res. Commun. , ( 1992. ) ( in Japanese ), in press .
  • 16. ) Kato , R. and Yamazoe , Y.Metabolic activation and covalent binding to nucleic acids of carcinogenic heterocyclic amines from cooked foods and amino acid pyrolysates . Jpn. J. Cancer Res. , 78 , 297 – 311 ( 1987. ). [PubMed] [Google Scholar]
  • 17. ) Sugimura , T.Studies on environmental chemical carcinogenesis in Japan . Science , 233 , 312 – 318 ( 1986. ). [DOI] [PubMed] [Google Scholar]
  • 18. ) Wakata , A. , Oka , N. , Hiramoto , K. , Yoshioka , A. , Negishi , K. , Wataya , Y. and Hayatsu , H.DNA strand cleavage in vitro by 3‐hydroxyamino‐1‐methyl‐5H‐pyrido‐[4,3‐6]indole, a direct‐acting mutagen formed in the metabolism of carcinogenic 3‐amino‐1‐methyl‐5H‐pyrido[4,3‐6]indole . Cancer Res. , 45 , 5867 – 5871 ( 1988. ). [PubMed] [Google Scholar]
  • 19. ) Hiramoto , K. , Negishi , K. , Namba , T. , Katsu , T. and Hayatsu , H.Superoxide dismutase‐mediated reversible conversion of 3‐hydroxyamino‐ l‐methyl‐5H‐pyrido[4,3‐6]‐indole, the N‐hydroxy derivative of Trp‐P‐2, into its nitroso derivative . Carcinogenesis , 9 , 2003 – 2008 ( 1988. ). [DOI] [PubMed] [Google Scholar]
  • 20. ) Wataya , Y. , Tamane , K. , Hiramoto , K. , Ohtsuka , Y. , Okubata , Y. , Negishi , K. and Hayatsu , H.Generation of intracellular active oxygens in mouse FM3A cells by 3‐hydroxyamino‐1‐methyl‐5H‐pyrido[4,3‐b]indole, the activated Trp‐P‐2 . Jpn. J. Cancer Res. , 79 , 576 – 579 ( 1988. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. ) Yasukochi , Y. and Masters , B. S. S.Some properties of a detergent solubilized NADPH cytochrome c (cytochrome P‐450) reductase purified by biospecific affinity chromatography . J. Biol. Chem. , 251 , 5337 – 5344 ( 1976. ). [PubMed] [Google Scholar]
  • 22. ) Aoyama , T. , Gelboin , H. V. and Gonzalez , F. J.Mutagenic activation of 2‐amino‐3‐methylimidazo[4,5‐f]‐quinoline by complementary DNA‐expressed human liver P‐450 . Cancer Res. , 50 , 2060 – 2063 ( 1990. ). [PubMed] [Google Scholar]
  • 23. ) Ryan , D. E. , Thomas , P. E. and Levin , W.Hepatic microsomal cytochrome P‐450 from rats treated with isosafrole: purification and characterization of four enzymic forms . J. Biol. Chem. , 255 , 7941 – 7955 ( 1980. ). [PubMed] [Google Scholar]
  • 24. ) Berlin , V. and Haseltine , W. A.Reduction of adriamycin to a semiquinone‐free radical by NADPH cytochrome P‐450 reductase produces DNA cleavage in a reaction mediated by molecular oxygen . J. Biol. Chem. , 256 , 4747 – 4756 ( 1981. ). [PubMed] [Google Scholar]
  • 25. ) Canuto , R. A. , Muzio , G. , Biocca , M. E. and Dianzani , M. U.Lipid peroxidation in rat AH‐130 hepatoma cells enriched in vitro with arachidonic acid . Cancer Res. , 51 , 4603 – 4608 ( 1991. ). [PubMed] [Google Scholar]
  • 26. ) Cerutti , P. A.Prooxidant states and tumor promotion . Science , 227 , 375 – 381 ( 1985. ). [DOI] [PubMed] [Google Scholar]

Articles from Japanese Journal of Cancer Research : Gann are provided here courtesy of Wiley

RESOURCES