The use of genetically engineered V79 Chinese hamster cultures expressing rat liver CYP1A1, 1A2 and 2B1 cDNAs in micronucleus assays.
We have undertaken a comparative study of the bioactivation of a panel of promutagens by V79 Chinese hamster cells genetically engineered to metabolic competence. In vitro micronucleus assays of the test agents in V79 cultures in the presence of an Aroclor induced rat S9 yielded positive results. In the genetically engineered cell lines, benzo[a]pyrene was metabolized specifically by the 3-methylcholanthrene inducible rat liver CYP1A1 (cell line XEM2) whereas cyclophosphamide increased the micronucleus frequency only in cultures expressing the phenobarbital inducible CYP2B1 (SD1). Following exposure to the mycotoxin sterigmatocystin, elevated frequencies of micronucleated cells were recorded in XEM2, SD1 and XEMd-MZ (expresses the isosafrole inducible CYP1A2) cells. The aromatic amine 2-amino-anthracene elicited a weak response in the cell line XEMd-MZ which expressed CYP1A2. This response was enhanced when this cDNA was expressed in a V79 variant cell strain which also possessed endogenous acetyltransferase activity. Upon exposure to tobacco particulate matter, a greater induction of micronuclei was observed in the XEM2 cell line compared to V79 cultures, implicating polycyclic aromatic hydrocarbons in addition to direct-acting compounds as causal agents in the genotoxicity of tobacco particulate matter. The cytokinesis blocked in vitro micronucleus assay provides a faster, simpler alternative to metaphase analysis, and kinetochore labelling techniques enable the discernment of both structural and numerical chromosome changes. The inclusion of metabolically competent test strains in the in vitro micronucleus assay therefore creates a powerful system for detecting genotoxins and may be extended to elucidate both mechanisms of bioactivation and modes of genotoxic insult.
Ellard
S
S
Molecular Biology Research Group, School of Biological Sciences, University College of Swansea, UK.
Mohammed
Y
Y
Dogra
S
S
Wölfel
C
C
Doehmer
J
J
Parry
J M
JM
D000818:Animals; D000873:Anthracenes / Q000378:metabolism / Q000633:toxicity; D001564:Benzo(a)pyrene / Q000378:metabolism / Q000633:toxicity; D001711:Biotransformation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003520:Cyclophosphamide / Q000378:metabolism / Q000633:toxicity; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005069:Evaluation Studies as Topic; D005455:Fluorescent Antibody Technique; D005818:Genetic Engineering; D008099:Liver / Q000201:enzymology; D015162:Micronucleus Tests / Q000379:methods*; D009153:Mutagens / Q000378:metabolism / Q000633:toxicity*; D010947:Plants, Toxic; D051381:Rats; D013241:Sterigmatocystin / Q000378:metabolism / Q000633:toxicity; D014026:Nicotiana
D000873:Anthracenes; D009153:Mutagens; D013241:Sterigmatocystin; D001564:Benzo(a)pyrene; C006593:2-anthramine; D003520:Cyclophosphamide; D003577:Cytochrome P-450 Enzyme System