An RNA synthesis inhibition assay for detecting toxic substances using click chemistry

Abstract

-Biological risk assessment studies of chemical substances that induce DNA lesions have been primarily based on the action of DNA polymerases during replication. However, DNA lesions interfere not only with replication, but also with transcription. There is no simple method for the detection of the DNA lesion-induced inhibition of transcription. Here, we report an assay for estimating the toxicity of chemical substances by visualizing transcription in mammalian cells using nucleotide analog 5-ethynyluridine (EU) and its click chemistry reaction. Ultraviolet light and representative chemical substances (camptothecin, 4-nitroquinoline-1-oxide, mitomycin C, and cisplatin, but not etoposide) of DNAdamaging agents show toxicity, as indicated by RNA synthesis inhibition in response to DNA damage in HeLa cells. Using titanium dioxide, we observed RNA synthesis inhibition in response to the rutile form, but not the anatase form, indicating that rutile titanium dioxide is a toxic substance. Because this method is based on the transcriptional response to DNA lesions, we can use terminally differentiated neuronlike PC12 cells, the differentiation of which can be induced by nerve growth factors, for evaluating chemical substances. Ultraviolet light and some chemicals (camptothecin, 4-nitroquinoline-1-oxide, mitomycin C, and cisplatin, but not etoposide) inhibited RNA synthesis in non-differentiated PC12 cells. Conversely, camptothecin and cisplatin did not inhibit RNA synthesis in differentiated PC12 cells, but 4-nitroquinoline-1-oxide, mitomycin C, and etoposide did. And using titanium dioxide, we did not observed any RNA synthesis inhibition. These data suggest that this method might be used to estimate the potential risk of chemical substances in differentiated mammalian cells, which are the most common cell type found in the human body.

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

“…Previously, we demonstrated that our in vivo method for visualizing transcription in mammalian cells can detect UV- and/or chemically (e.g., camptothecin, etoposide, 4NQO, cisplatin) induced damage in genomic DNA by inhibiting RNA polymerase during transcription elongation [ 20 , 22 , 23 ]. Here, we modified this in vivo method to establish a new in vitro method (Fig.…”

“…In our previous work, we established a method for detecting the effects of chemically induced DNA damage on transcription using the nucleotide analog 5-bromouridine and a anti-5-bromouridine antibody and/or the nucleotide analog 5-ethynyluridine [ 20 ] and a click chemistry reaction [ 21 , 22 ] without radioisotopes [ 23 ]. Our method is based on a model for transcription-coupled nucleotide excision repair (NER) triggered by blocked transcription at DNA lesions [ 24 , 25 ].…”

Current techniques for visualizing RNA in cells

“…RNA labeling has seen extensive use in imaging of infection by RNA viruses (e.g., 58 ). Another interesting application of RNA imaging has been to monitor transcription and this has been used, for example, to determine the toxicity of certain substances that inhibit transcription 59 .…”