An in vitro method for detecting genetic toxicity based on inhibition of RNA synthesis by DNA lesions

Abstract

Introduction: A wide variety of DNA lesions such as ultraviolet light-induced photoproducts and chemically induced bulky adducts and crosslinks (intrastrand and interstrand) interfere with replication and lead to mutations and cell death. In the human body, these damages may cause cancer, inborn diseases, and aging. So far, mutation-related actions of DNA polymerases during replication have been intensively studied. However, DNA lesions also block RNA synthesis, making the detection of their effects on transcription equally important for chemical safety assessment. Previously, we established an in vivo method for detecting DNA damage induced by ultraviolet light and/or chemicals via inhibition of RNA polymerase by visualizing transcription. Results: Here, we present an in vitro method for detecting the effects of chemically induced DNA lesions using in vitro transcription with T7 RNA polymerase and real-time reverse transcription polymerase chain reaction (PCR) based on inhibition of in vitro RNA synthesis. Conventional PCR and real-time reverse transcription PCR without in vitro transcription can detect DNA lesions such as complicated cisplatin DNA adducts but not UV-induced lesions. We found that only this combination of in vitro transcription and real-time reverse transcription PCR can detect both cisplatin- And UV-induced DNA lesions that interfere with transcription. Conclusions: We anticipate that this method will be useful for estimating the potential transcriptional toxicity of chemicals in terminally differentiated cells engaged in active transcription and translation but not in replication.