An automated alkaline elution system: DNA damage induced by 1,2-dibromo-3-chloropropane in vivo and in vitro.
An automated alkaline elution system for the detection of DNA damage has been developed. After manual application of samples, which is completed within 5 min, the subsequent supply of liquids, changes in flow rates, and temperature are controlled automatically. The system operates 16 filters and may easily be expanded. The sensitivity of the fluorometric DNA determinations with the Hoechst 33258 dye is increased by using an elution buffer (20 mM Na2EDTA, pH 12.50) with low background fluorescence. DNA is determined using an automated setup similar to the one recently presented by Sterzel et al. (1985, Anal. Biochem. 147, 462-467). The most significant modification is the use of a neutralization buffer which allows variations in the pH of eluted fractions. This change increases the sensitivity of the DNA measurements. The automated alkaline elution system was evaluated using the nematocide 1,2-dibromo-3-chloropropane (DBCP) in a study of its genotoxic effects in the testes and the kidneys. Significant DNA damage was induced in testicular cells by 2.5 microM DBCP (1 h) in vitro and 85 mumol/kg DBCP ip (3 h) in vivo. The damage appeared after short treatment times (10 min in vivo). Variations in the observed DBCP response in vivo were largely due to interanimal variations. The automated alkaline elution system proved to be a sensitive assay also for the detection of DNA damage in kidney nuclei prepared from rats exposed to DBCP. Provided that kidney nuclei from untreated rats, mice, or hamster were kept ice-cold until lysing, 85-100% of their DNA was retained after 16 h of elution, indicating highly intact DNA. Under the same conditions, guinea pig DNA was rapidly degraded unless the nuclei were prepared in a buffer with a higher concentration of Na2EDTA (20 mM).
Brunborg
G
G
Department of Toxicology, National Institute of Public Health, Oslo, Norway.
Holme
J A
JA
Søderlund
E J
EJ
Omichinski
J G
JG
Dybing
E
E
D000468:Alkalies; D000818:Animals; D001322:Autoanalysis; D002467:Cell Nucleus / Q000187:drug effects; D006224:Cricetinae; D004249:DNA Damage; D005453:Fluorescence; D006168:Guinea Pigs; D007668:Kidney / Q000648:ultrastructure; D008297:Male; D008722:Methods; D051379:Mice; D011407:Propane / Q000031:analogs & derivatives* / Q000494:pharmacology; D051381:Rats; D013737:Testis / Q000166:cytology
D000468:Alkalies; C007318:1,2-dibromo-3-chloropropane; D011407:Propane