A comparison of the methods applied to detect apoptosis in genotoxically-damaged lymphocytes cultured in the presence of four antimutagens.
The sensitivity of the available methods of apoptosis detection in lymphocyte cultures was tested. Cells were preincubated with genotoxic agents: hydrogen peroxide (0.2 mM; 20 min.) and benzo[a]pyrene (40 microM; 90min.), and then cultured for 36h in the presence of a lectin (PHA-M; 1% v/v) and one of the following potentially antimutagenic agents: alkylresorcinols, anthocyanins, todralazine and fluphenazine. It was established that staining with a mixture of fluorochromes (ethidium bromide and acridine orange) provided the highest amount of detected apoptotic cells, and the best repeatability of the results in subsequent experiments. Calculation of the Spearman's rank correlation coefficients proved that there was a high correlation between the results obtained by the ethidium bromide/acridine orange method and those obtained by identifying genomic DNA fragmentation by means of FIGE-electrophoresis. Therefore, these two methods were chosen for further studies of the tested antimutagens' impact on apoptosis in genotoxically-damaged lymphocytes.
Gasiorowski
K
K
Wrocław Medical University, Department of Basic Medical Sciences, Poland. kaz@basmed.am.wroc.pl
Brokos
B
B
Kulma
A
A
Ogorzałek
A
A
Skórkowska
K
K
D000165:Acridine Orange; D000328:Adult; D016587:Antimutagenic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D003584:Cytological Techniques / Q000379:methods*; D004247:DNA / Q000032:analysis / Q000737:chemistry; D004249:DNA Damage / Q000187:drug effects*; D003720:Densitometry; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004996:Ethidium; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods; D008875:Middle Aged; D009153:Mutagens / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
D016587:Antimutagenic Agents; D009153:Mutagens; D004247:DNA; D004996:Ethidium; D000165:Acridine Orange