A comparative study of the use of primary Chinese hamster liver cultures and genetically engineered immortal V79 Chinese hamster cell lines expressing rat liver CYP1A1, 1A2 and 2B1 cDNAs in micronucleus assays.
Liver microsome preparations (S9 mix) have been extensively used for in vitro genotoxicity studies to provide the capacity for the activation of indirect genotoxins. However, the use of S9 preparations with mammalian cell cultures has raised considerable toxicity problems which limit their use to exposure times which are only a small fraction of the cell cycle. In addition, false negative results may be obtained if reactive metabolites are unable to penetrate the cell membrane or have short half-lives. The generation and detection of a promutagen within a single cell would therefore be advantageous. To this end, we have studied the bioactivation of a panel of promutagens (benzo[a]pyrene, cyclophosphamide, 2-aminoanthracene and sterigmatocystin) in low passage Chinese hamster fibroblasts of hepatic origin (LiC2 cells) and in a series of V79 Chinese hamster cell lines genetically engineered to express rat liver cytochrome P450 cDNAs. These include strains XEM2 (expresses CYP1A1), SD1 (CYP2B1) and strains XEMd-MZ and XEMd-NH which express CYP1A2. The end point selected for study was the induction of micronuclei. The protocol incorporated a cytochalasin B-induced cytokinesis block and the enumeration of micronuclei in the resulting binucleate cells which have undergone one nuclear division following the induction of chromosome damage. Micronuclei containing whole chromosomes and chromosome fragments were distinguished by the use of CREST antibody specific for kinetochore protein as a measure for the presence of centromeres. Micronuclei were induced by the test agents in low passage liver fibroblasts and in immortal V79 cultures only in the presence of Aroclor-induced S9 preparations. The data obtained from micronucleus assays of the genetically engineered V79 cell lines demonstrated the utility of each strain for the optimal detection and quantification of the activity of the individual test compounds. Kinetochore antibody demonstrated differences in the kinetics of induction of micronuclei containing chromosome fragments and whole chromosomes with chemicals such as benzo[a]pyrene. As part of this cytogenetic study, we also conducted karyotypic analyses and spindle fidelity assays of the V79 cell lines to investigate the presence of chromosomal instabilities which may arise as a consequence of the genetic engineering procedure. Such studies represent an important quality control step in the validation of the suitability of each cell line prior to their use in genotoxicity studies.
Ellard
S
S
Molecular Biology Research Group, School of Biological Sciences, University College Swansea, UK.
Parry
J M
JM
D000818:Animals; D001711:Biotransformation; D002461:Cell Line, Transformed; D006224:Cricetinae; D003412:Cricetulus; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary; D008099:Liver / Q000201:enzymology*; D008297:Male; D015162:Micronucleus Tests; D009153:Mutagens / Q000493:pharmacokinetics / Q000633:toxicity; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000378:metabolism; D051381:Rats
D018076:DNA, Complementary; D009153:Mutagens; D003577:Cytochrome P-450 Enzyme System; D019388:Cytochrome P-450 CYP1A2; D010089:Oxidoreductases, N-Demethylating