When chlorotetracycline (CTC) was introduced into the DNA-ethidium bromide (EB) complex, the CTC fluorescence was interpolated into the EB fluorescence of DNA. The fluorescence parameters of EB and CTC could be measured biparametrically in relation to DNA structures. The EB fluorescence enhanced by intercalation into DNA was decreased by addition of CTC in vitro. Similar decrease of fluorescence occurred in cell nuclei isolated from rat liver cells and their chromatin.
On CsCl density gradient centrifugation, the peak position of the CTC-treated DNA was at a lower density than that of untreated DNA. On the sucrose density gradient centrifugation, CTC-treated chromatin was also at a lower density than untreated chromatin. When rats were treated with CTC in vivo, the fluorescence response of their liver DNA appeared to be similar to that in vitro.
Thus, biparametric fluoro-assay could be used for following structural change of DNA caused by CTC in vitro or in vivo.