Cloning and expression of rat CYP2E1 in Saccharomyces cerevisiae: detection of genotoxicity of N-alkylformamides.
A cDNA coding for rat cytochrome P450 2E1 was cloned into the multicopy vector pYeDP60 and expressed in haploid RSY6 and diploid RS112 yeast strains of Saccharomyces cerevisiae under control of the GAL10-CYC1 promoter. Spectral and catalytic properties of the expressed 2E1 were examined in whole cells or microsomes of both strains. The level of CYP2E1 obtained in RS112 (200 pmol/mg microsomal protein) was the highest among CYP2E1 produced in the various expression systems. The monooxygenase activity in the microsomes of both strains, measured as aniline hydroxylase, was found comparable to that of control rat hepatic microsomes. In a reconstituted system in the presence of exogenous rat P450 reductase, their activity increased about 10-fold. When exposed to the carcinogen NDMA, a known 2E1 substrate, the recombination frequency determined in the 2E1-expressing RS112 cells was enhanced, in a dose-dependent manner, up to 20-fold. The exposure of the same cells to the hepatotoxic solvents, N-methyl- and N-ethylformamide, resulted in an induction of recombination frequency, which was not observed in the void plasmid containing RS112 cells in the presence of S9 hepatic fractions from pyrazole-induced rats, as a specific exogenous metabolic activation system. These results demonstrate that the 2E1-expressing cells metabolize the two N-alkylformamides to genotoxic intermediates and, therefore, they provide an useful tool to study the bioactivation mechanism of potential P450 2E1 substrates.
Del Carratore
M R
MR
Institute of Mutagenesis and Differentiation, National Council of Research, Pisa, Italy. r.delcarratore@imd.pi.cnr.it
Mezzatesta
C
C
Hidestrand
M
M
Neve
P
P
Amato
G
G
Gervasi
P G
PG
D000818:Animals; D001711:Biotransformation; D003001:Cloning, Molecular; D019392:Cytochrome P-450 CYP2E1 / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D005559:Formamides / Q000493:pharmacokinetics / Q000633:toxicity*; D005822:Genetic Vectors; D008297:Male; D009603:Nitroso Compounds / Q000633:toxicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D018675:Toxicity Tests / Q000379:methods*
D005559:Formamides; D009603:Nitroso Compounds; D011994:Recombinant Proteins; C008651:4-nitrosodimethylaniline; C035404:N-ethylformamide; D019392:Cytochrome P-450 CYP2E1; C002950:methylformamide