Detection of Adriamycin-DNA adducts by accelerator mass spectrometry at clinically relevant Adriamycin concentrations
Kate E Coldwell1, Suzanne M Cutts, Ted J Ognibene
1Department of Biochemistry, La Trobe University, Bundoora, Victoria 3086, Australia.
Abstract:
Limited sensitivity of existing assays has prevented investigation of whether Adriamycin-DNA adducts are involved in the anti-tumour potential of Adriamycin. Previous detection has achieved a sensitivity of a few Adriamycin-DNA adducts/10(4) bp DNA, but has required the use of supra-clinical drug concentrations. This work sought to measure Adriamycin-DNA adducts at sub-micromolar doses using accelerator mass spectrometry (AMS), a technique with origins in geochemistry for radiocarbon dating. We have used conditions previously validated (by less sensitive decay counting) to extract [(14)C]Adriamycin-DNA adducts from cells and adapted the methodology to AMS detection. Here we show the first direct evidence of Adriamycin-DNA adducts at clinically-relevant Adriamycin concentrations. [(14)C]Adriamycin treatment (25 nM) resulted in 4.4 +/- 1.0 adducts/10(7) bp ( approximately 1300 adducts/cell) in MCF-7 breast cancer cells, representing the best sensitivity and precision reported to date for the covalent binding of Adriamycin to DNA. The exceedingly sensitive nature of AMS has enabled over three orders of magnitude increased sensitivity of Adriamycin-DNA adduct detection and revealed adduct formation within an hour of drug treatment. This method has been shown to be highly reproducible for the measurement of Adriamycin-DNA adducts in tumour cells in culture and can now be applied to the detection of these adducts in human tissues.
Insights
Researchers used accelerator mass spectrometry (AMS) to detect Adriamycin-DNA adducts at low drug doses. This breakthrough enables studying Adriamycin
Area of Science:
- Pharmacology
- Molecular Biology
- Analytical Chemistry
Background:
- Adriamycin's anti-tumour potential is linked to Adriamycin-DNA adducts.
- Existing assays lack sensitivity to detect adducts at clinical drug concentrations.
Purpose of the Study:
- To measure Adriamycin-DNA adducts at sub-micromolar doses using accelerator mass spectrometry (AMS).
- To establish a highly sensitive method for detecting Adriamycin-DNA adducts.
Main Methods:
- Utilized [(14)C]Adriamycin and accelerator mass spectrometry (AMS) for adduct detection.
- Adapted existing cell extraction methods for AMS sensitivity.
- Measured adducts in MCF-7 breast cancer cells at 25 nM Adriamycin concentration.
Main Results:
- First direct evidence of Adriamycin-DNA adducts at clinically relevant concentrations.
- Detected 4.4 +/- 1.0 adducts/10(7) bp, a >3-fold increase in sensitivity.
- Adduct formation observed within one hour of drug treatment.
Conclusions:
- AMS provides unprecedented sensitivity for Adriamycin-DNA adduct detection.
- The method is reproducible and applicable to human tissues.
- Enables investigation of Adriamycin's anti-tumour mechanisms at therapeutic doses.
