4-Ipomeanol and 2-aminoanthracene cytotoxicity in C3H/10T12 cells expressing rabbit cytochrome P450 4B1

doi:10.1016/0006-2952(95)02029-2

Biochemical Pharmacology

Volume 50, Issue 10, 9 November 1995, Pages 1567-1575

https://doi.org/10.1016/0006-2952(95)02029-2 Get rights and content

Abstract

In the present study, retroviral vectors were used to stably transfer and express the cDNA encoding rabbit CYP4B1 in mouse $C3H/10T 12$ cells. The replication defective retroviral vector was packaged in the ecotropic packaging cell line, GP + E-86, with infectious titer of ~1 × 10⁶ cfu/mL. Infection, followed by selection with G418, showed an infection efficiency of approximately 70% for the recipient $C3H/10T 12$ cells. Analysis of ten G418 resistant clones showed that the number of vector inserts ranged from 4 to 13 copies per cell genome. Each clone was positive for microsomal CYP4B1 protein as determined by immunoblotting. Cytochrome P450 4B1 activity was assessed by the cytotoxicity of 4-ipomeanol, a known substrate for P450 4B1 and a model compound for chemical-induced injury to the lung. The initial clonigenic assays showed that 100% toxicity occurred in all the clones after a 96-hr exposure to 250 μM 4-ipomeanol. Parental $C3H/10T 12$ cells were resistant to 4-ipomeanol at concentrations as high as 1 mM. Two clones, designated No. 2 and No. 19, differing in levels of P450 4B1 protein, were characterized further for 4-ipomeanol and other chemical toxicities. A concentration-response study indicated 50% cytotoxicity at 4-ipomeanol concentrations of 1.5 μg/mL for clone No. 2 and 2.5 μg/mL for clone No. 19. A panel of agents representing the aromatic amines, some of which are known or suspected P450 4B1 substrates, were tested for cytotoxicity in clone No. 2. These agents included 2-aminoanthracene, 2-aminonaphthalene, 2-aminofluorene, 2-acetylaminofluorene and 4-aminobiphenyl. Only 2-aminoanthracene gave a clear cytotoxic response reducing the survival fraction of clone No. 2 to 50% at 0.2 μg/mL while affecting parental cells minimally. In vitro expression of CYP4B1 provides a new experimental system for further elucidating the cytotoxic and mutagenic effects of P450 4B1 substrates.

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Research paper4-Ipomeanol and 2-aminoanthracene cytotoxicity in C3H/10T12 cells expressing rabbit cytochrome P450 4B1

Abstract

Toxicol Appl Pharmacol

Pharmacol Ther

Pharmacol Ther

Methods Enzymol

Mutat Res

Mutat Res

J Biol Chem

Anal Biochem

Arch Biochem Biophys

Pharmacol Ther

Cancer Lett

Searches for ultimate chemical carcinogens and their reactions with cellular macromolecules

Cancer

Calcium-mediated mechanisms in chemically induced cell death

Annu Rev Pharmacol Toxicol

Interindividual variation among humans in carcinogen metabolism, DNA adduct formation and DNA repair

Carcinogenesis

Recent studies on the structure and function of multisubstrate flavin-containing monooxgenases

Annu Rev Pharmacol Toxicol

Prostaglandin H synthase and xenobiotic oxidation

Annu Rev Pharmacol Toxicol

The P450 superfamily: Update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature

DNA Cell Biol

Respiratory tract uptake of inhalants and metabolism of xenobiotics

Annu Rev Pharmacol Toxicol

Induction and tissue-specific expression of rabbit cytochrome P450IIE1 and IIE2 genes

Mol Pharmacol

Evidence for the Clara cell as a site of cytochrome P450-dependent mixed-function oxidase activity in lung

Nature

Immunohistochemical demonstration of cytochrome P-450 monooxygenase in Clara cells throughout the tracheobronchial airways of the rabbit

Exp Lung Res

Clara cells in the lung

Cytochrome P-450: Localization in rabbit lung

Science

Distribution of cytochrome P450 1A1 and NADPH-cytochrome P450 reductase in lungs of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin: Ultrastructural immunolocalization and in situ hybridization

Mol Pharmacol

Tissue, species and substrate concentration differences in the position selective hydroxylation of N-nitrosodibutylamine

Drug Metab Dispos

Research paper
4-Ipomeanol and 2-aminoanthracene cytotoxicity in $C3H/10T 12$ cells expressing rabbit cytochrome P450 4B1