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Single Molecule Analysis of Laser Localized Psoralen Adducts
Published on: April 20, 2017
Quantitative analysis of DNA interstrand cross-links and monoadducts formed in human cells induced by psoralens and
Congfang Lai1, Huachuan Cao, John E Hearst
1Department of Chemistry, University of California, Riverside, California 92521-0403, USA.
Abstract:
Upon exposure to UVA light, psoralens can induce DNA interstrand cross-links (ICLs), which can block DNA replication and transcription. Among the psoralen derivatives, 8-methoxypsoralen (8-MOP) is conventionally applied for psoriasis therapy, and amotosalen S59 is used to inactivate bacterial and viral pathogens in blood components. In addition to the ICL formation, psoralens also readily form various monoadducts (MAs) with thymidine residues in DNA when exposed to UVA light, and the biological implications for these monoadducts remain unclear. Here, we reported a method that encompassed digestion with a single enzyme (nuclease P1) and LC-MS/MS, for the simultaneous quantification of ICL and MAs induced in human cells exposed with 8-MOP or S59 and UVA light. Our results showed that the yield of ICL induced by S59, which increased from 3.9 to 12.8 lesions/10(3) nucleotides as the dose of UVA light increased from 0.5 to 10.0 J/cm(2), was approximately 100 fold more than that induced by 8-MOP. In addition, three and five products were identified as 8-MOP- and S59-MAs, respectively, and the yields of MAs were significantly lower than that for ICL. The yields of the three 8-MOP-MAs were 7.6-2.2, 1.9-9.9, and 7.2-51 per 10(6) nucleotides and those of the five S59-MAs were 215-19, 106-39, 25-21, 32-146, and 22-26 per 10(6) nucleotides as the dose of UVA light increased from 0.5 to 10.0 J/cm(2). Although the yields of MAs induced by 8-MOP and S59 were lower than those of the respective ICLs under the same exposure conditions, the formation of appreciable amounts of MAs might account for some of the mutations induced by psoralens.
Insights
Psoralens like 8-methoxypsoralen and amotosalen S59 form DNA interstrand cross-links (ICLs) and monoadducts (MAs) under UVA light. A new method quantifies these, revealing S59 forms more ICLs than 8-MOP, and MAs may contribute to mutations.
Area of Science:
- Molecular Biology
- Photochemistry
- Toxicology
Background:
- Psoralens, upon UVA light exposure, induce DNA interstrand cross-links (ICLs), impacting DNA replication and transcription.
- 8-methoxypsoralen (8-MOP) is used for psoriasis, while amotosalen S59 inactivates pathogens in blood products.
- The biological significance of psoralen-induced DNA monoadducts (MAs) remains largely undetermined.
Purpose of the Study:
- To develop and validate a method for simultaneous quantification of ICLs and MAs induced by 8-MOP and S59 in human cells.
- To compare the yields of ICLs and MAs induced by 8-MOP and S59 under varying UVA light doses.
- To investigate the potential role of MAs in psoralen-induced mutations.
Main Methods:
- Development of a method involving single-enzyme (nuclease P1) digestion followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
- Application of the method for simultaneous quantification of ICLs and MAs in human cells exposed to 8-MOP or S59 and UVA light.
- Systematic variation of UVA light dose (0.5 to 10.0 J/cm²) to assess its impact on adduct formation.
Main Results:
- Amotosalen S59 induced approximately 100-fold more ICLs than 8-MOP, with yields increasing significantly with UVA dose.
- Three 8-MOP-MAs and five S59-MAs were identified, with MA yields consistently lower than ICL yields under identical conditions.
- Appreciable yields of MAs were observed for both psoralen derivatives, even at lower UVA doses.
Conclusions:
- The developed LC-MS/MS method enables simultaneous quantification of psoralen-induced ICLs and MAs.
- Amotosalen S59 is a more potent inducer of ICLs compared to 8-MOP.
- The formation of significant amounts of MAs, despite lower yields than ICLs, may contribute to the mutagenic potential of psoralens.
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