Genetic analysis of aflatoxin B1 activation in rat hepatoma cells.
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.
Corcos
L
L
URA 152 du CNRS, Département de Biologie Moléculaire, Institut Pasteur, Paris, France.
Rousset
J P
JP
Kiefer
F
F
Wiebel
F J
FJ
Weiss
M C
MC
D016604:Aflatoxin B1; D000348:Aflatoxins / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D001711:Biotransformation; D002273:Carcinogens; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004351:Drug Resistance; D008114:Liver Neoplasms, Experimental; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D014407:Tumor Cells, Cultured
D000348:Aflatoxins; D002273:Carcinogens; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D006899:Mixed Function Oxygenases; C019647:aldrin epoxidase