Summary
We present a strategy to elucidate the rate-limiting steps in activation of carcinogenic compounds by cytochromes P450. The principle was to select Reuber rat hepatoma cells for resistance to a procarcinogen. The hypothesis was that resistant cells should be systematically deficient in the P450 enzyme(s) involved in the activation process. Here we present an example of the use of this approach using aflatoxin B1 (AFB1), a potent hepatocarcinogen, as the selective agent. Parental cells as well as individual and pooled colonies selected for AFB1 resistance from three independent rat hepatoma lines were characterized for their content of 1) mRNA hybridizing to cDNA and/or oligonucleotide probes for cytochromes P450IIB1, P450IIB2 and albumin; and 2) aldrin epoxidase activity. Parental aflatoxin B1-sensitive cells were shown to express P450IIB1 but not P450IIB2. The majority of the aflatoxin B1-resistant clones failed to accumulate cytochrome P450IIB1 mRNA and expressed no or only very low aldrin epoxidase activity. Albumin mRNA levels remained unchanged, demonstrating that loss of expression of cytochrome P450IIB1 was not a consequence of a general dedifferentiation event. A revertant population showing restoration of both cytochrome P450IIB1 mRNA accumulation and aldrin epoxidase activity was fully sensitive to aflatoxin B1. The correlation between expression of cytochrome P450IIB1 and sensitivity to aflatoxin B1 in both parental cells and revertants strongly suggests that cytochrome P450IIB1 is a major contributor to the activation of aflatoxin B1 in rat hepatoma cells. The kind of strategy described here could be applied to other compounds that become cytotoxic for hepatoma cells following activation by cytochromes P450.
Article PDF
Similar content being viewed by others
Abbreviations
- AFB1:
-
aflatoxin B1
- AE:
-
aldrin epoxidase
- AHH:
-
aryl hydrocarbon hydroxylase
- PAH:
-
polycyclic aromatic hydrocarbons
- PB:
-
phenobarbital
References
Adesnik M, Atchison M (1986) Genes for cytochromes P450 and their regulation. CRC Crit Rev Biochem 19:247–306
Adesnik M, Bar-Num S, Maschio F, Zunich M, Lippman A, Bard E (1981) Mechanism of induction of cytochrome P-450 by phenobarbital. J Biol Chem 256:10340–10345
Atchison M, Adesnik M (1986) Gene conversion in a cytochrome P450 gene family. Proc Natl Acad Sci USA 83:2300–2304
Beadle GW, Ephrussi B (1936) The differentiation of eye pigment inDrosophila as studied by transplantation. Genetics 21:225–247
Beadle GW, Tatum EL (1941) Genetic control of biochemical reactions inNeurospora. Proc Natl Acad Sci USA 27:499–506
Bertolotti R (1977) A selective system for hepatoma cells producing gluconeogenic enzymes. Somat Cell Genet 3:365–380
Corcos L, Weiss MC (1988a) Phenobarbital, dexamethasone and benzanthracene induce several cytochrome P450 mRNAs in rat hepatoma cells. FEBS Lett 233:37–40
Corcos L, Weiss MC (1988 b) Efficient one-step selection of hepatoma cell variants of a variety of phenotypes by use of aflatoxin B1. Differentiation 38:134–139
Corcos L, Rechenmann C, Weiss MC, Pompon D (1989) Establishment of mouse and rat hepatoma cell clones showing stable expression of rabbit cytochrome P450 IA2. FEBS Lett 259:175–180
Deschatrette J, Weiss MC (1974) Characterization of differentiated and dedifferentiated clones from a rat hepatoma. Biochimie (Paris) 56:1603–1611
Deschatrette J, Moore EE, Dubois M, Weiss MC (1980) Dedifferentiated variants of a rat hepatoma: reversion analysis. Cell 19:1043–1051
Doehmer J, Dogra S, Friedberg T, Monier S, Adesnik M, Glatt Oesch F (1988) Stable expression of rat cytochrome P-45011131 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1. Proc Natl Acad Sci USA 85:5769–5773
Essigmann JM, Croy RG, Nadzan AM, Busby WF Jr, Reinhold VN, Büchi G, Wogan GN (1977) Structural identification of the major DNA adduct formed by aflatoxin B1 in vitro. Proc Natl Acad Sci USA 74:1870–1874
Faletto MB, Koser PL, Battula N, Townsend GK, Maccubbin AE, Gelboin HV, Gurtoo HL (1988) Cytochrome P3-450 cDNA encodes aflatoxin B1–4 hydroxylase. J Biol Chem 263:12187–12189
Friedberg J, Wayman DJ, Atchison M, Kumar A, Haaparanta T, Raphael C, Adesnik M (1986) Isolation and characterization of cDNA clones for cytochromes P450 immunochemically related to rat hepatic P450 form PB1. Biochemistry 25:6975–6983
Guengerich FP (1988) Roles of cytochrome P450 enzymes in chemical carcinogenesis and cancer chemotherapy. Cancer Res 48:2946–2954
Kao FT, Puck TT (1968) Genetics of somatic mammalian cells VII. Induction and isolation of nutritional mutants in Chinese hamster cells. Proc Natl Acad Sci USA 60:1275–1281
Lambiotte M, Thierry N (1979) Enhancement of aflatoxin 131 cytotoxicity in differentiated rat hepatoma cultures by a prior glucocorticoid treatment of the monolayer. Biochem Biophys Res Commun 89:933–942
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Mannervik B (1985) The isoenzymes of glutathione transferse. Adv Enzymol 57:357–417
Metcalfe SA, Colley PJ, Neal GE (1981) A comparison of the effects of pretreatment with phenobarbitone and 3-methylcholanthrene on the metabolism of aflatoxin 131 by rat liver microsomes and isolated hepatocytes in vitro. Chem Biol Interact 35:145–157
Mizokami K, Nohmi T, Fukuhara M, Takanaka A, Omori Y (1986) Purification and characterization of a form of cytochrome P450 with high specificity for aflatoxin B1 from 3-methylcholanthrene-treated hamster liver. Biochem Biophys Res Commun 139:466–472
Nebert DW, Gelboin HJ (1968) Substrate-inducible microsomal aryl hydrocarbon hydroxylase in mammalian cell culture. J Biol Chem 243:6242–6249
Nebert DW, Gonzalez FJ (1987) P450 genes: structure, evolution, and regulation. Annu Rev Biochem 56:945–993
Nebert DW, Nelson DR, Adesnik M, Coon MJ, Estabrook RW, Gonzalez FJ, Guengerich FP, Gunsalus IC, Johnson EF, Kemper B, Levin W, Phillips IR, Sato R, Waterman MR (1989) The P450 superfamily: updated listing of all genes and recommended nomenclature for the chromosomal loci. DNA 8:1–13
Pyerin W, Taniguchi H (1989) Phosphorylation of hepatic phenobarbital-inducible cytochrome P-450. EMBO J 8:3003–3010
Sargent TD, Wu JR, Salat-Trepat JM, Wallace RB, Reyes AA, Bonner J (1979) The rat serum albumin gene: analysis of cloned sequences. Proc Nail Acad Sci USA 76:3256–3260
Shayiq RM, Avadhani NG (1989) Purification and characterization of a hepatic mitochondrial cytochrome P450 active in aflatoxin B1 metabolism. Biochemistry 28:7546–7554
Shimada T, Guengerich FP (1989) Evidence for P450NF, the nifedipine oxidase, being the principal enzyme active in aflatoxin B1 metabolism. Proc Natl Acad Sci USA 86:462–465
Spiegelman BM, Frank M, Green H (1983) Molecular cloning of mRNA from 3T3 adipocytes. Regulation of mRNA content for glycerophosphate dehydrogenase and other differentiationdependent proteins during adipocyte development. J Biol Chem 258:10083–10089
Wiebel FJ, Wolff T, Lambiotte M (1980) Presence of cytochromes P450- and P448-dependent monooxygenase functions in hepatoma cell lines. Biochem Biophys Res Commun 94:466–472
Wiebel FJ, Park SS, Kiefer F, Gelboin HV (1984) Expression of cytochromes P450 in rat hepatoma cells. Eur J Biochem 145:455–462
Wolff T, Deml E, Wanders H (1979) Aldrin epoxidation, a highly sensitive indicator specific for cytochrome P450-dependent mono-oxygenase activities. Drug Metab Dispos 7:301–305
Author information
Authors and Affiliations
Additional information
Communicated by R. Devoret
Rights and permissions
About this article
Cite this article
Corcos, L., Rousset, JP., Kiefer, F. et al. Genetic analysis of aflatoxin B1 activation in rat hepatoma cells. Mol Gen Genet 222, 291–296 (1990). https://doi.org/10.1007/BF00633831
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00633831