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.