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OBJECTIVE To develop a generalized “discovery-confirmation” strategy for DNA adducts based on liquid chromatography-triple quadrupole mass spectrometry (LC-QQQ-MS) and liquid chromatography-high resolution mass spectrometry (LC-HRMS), study the direct and metabolic reactivity of the potentially genotoxic substance lucidin (Luc) with three 2'-deoxynucleosides in Rubia cordifolia L., and screen and confirm possible Luc-specific DNA adducts. METHODS Three 2'-deoxynucleosides with the same fragmentation pathways with DNA adducts in mass spectrometry were used to develop and optimize a non-targeted screening method for unknown DNA adducts in QQQ-MS neutral loss and pseudo-neutral loss scanning modes, as well as a confirmatory method for adduct targeting in HRMS data-dependent scanning mode. Luc was incubated with 2'-deoxynucleoside under phase I metabolically activated and inactivated conditions, respectively, and the resulting specific DNA adducts were screened and then structurally verified by characteristic MS spectra of fragmentations. RESULTS The optimized pseudo-neutral loss scanning is sensitive to the detection of deoxyribonucleoside deglycosylation at the pg·mL-1 level. Six Luc-DNA adducts, containing two 2'-deoxycytidine (dC) adducts, two 2'-deoxyadenosine (dA) adducts, and two 2'-deoxyguanosine (dG) adducts, were identified and confirmed in vitro incubation model of Luc and 2'-deoxynucleoside, and their structures were characterized. Luc-DNA adduct production increased with increasing Luc exposure and exposure time, and there were significant dose-response and time-response relationships, and the binding did not require metabolic activation to occur. CONCLUSION The established discovery-confirmation strategy for DNA adducts is highly sensitive and accurate. It can provide structural information of the adducts at the molecular level. This is suitable for the assessment of DNA damage caused by the presence of Luc and provides strong data support for the study and re-evaluation of its toxicity mechanism. It also offers an important methodological reference for the rapid screening of potential genotoxic components in Chinese herbal medicines.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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