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Genetic analysis of aflatoxin B1 activation in rat hepatoma cells
L Corcos1, J P Rousset, F Kiefer
1URA 152 du CNRS, Département de Biologie Moléculaire, Institut Pasteur, Paris, France.
Abstract:
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.
Insights
This study identifies cytochrome P450IIB1 as a key enzyme in activating the carcinogen aflatoxin B1. Resistant cells lacking this enzyme confirm its crucial role in aflatoxin B1 activation by P450 enzymes.
Area of Science:
- Biochemistry
- Toxicology
- Carcinogenesis
Background:
- Cytochromes P450 (CYPs) are crucial for metabolizing xenobiotics, including procarcinogens.
- Identifying specific CYPs involved in carcinogen activation is vital for understanding toxicity and developing interventions.
Purpose of the Study:
- To develop and apply a novel strategy for identifying rate-limiting P450 enzymes in procarcinogen activation.
- To investigate the role of cytochrome P450IIB1 in the activation of aflatoxin B1 (AFB1).
Main Methods:
- Utilized Reuber rat hepatoma cells selected for resistance to AFB1.
- Assessed mRNA levels for P450IIB1, P450IIB2, and albumin via hybridization.
- Measured aldrin epoxidase activity as a marker for P450IIB1 function.
Main Results:
- Parental cells expressed P450IIB1 but not P450IIB2.
- AFB1-resistant clones predominantly lacked P450IIB1 mRNA and aldrin epoxidase activity.
- Albumin mRNA levels remained stable, indicating specific P450 loss, not general dedifferentiation.
- Revertant cells regained P450IIB1 expression and AFB1 sensitivity.
Conclusions:
- Cytochrome P450IIB1 is a major enzyme responsible for activating aflatoxin B1 in rat hepatoma cells.
- The cell-resistance strategy is effective for identifying P450 enzymes involved in procarcinogen activation.
- This approach can be broadly applied to other CYP-activated cytotoxic compounds.