222-Nanometer Far-UVC Exposure Results in DNA Damage and Transcriptional Changes to Mammalian Cells

Abstract

Ultraviolet (UV) germicidal tools have recently gained attention as a disinfection strategy against the COVID-19 pandemic, but the safety profile arising from their exposure has been controversial and impeded larger-scale implementation. We compare the emerging 222-nanometer far UVC and 277-nanometer UVC LED disinfection modules with the traditional UVC mercury lamp emitting at 254 nm to understand their effects on human retinal cell line ARPE-19 and HEK-A keratinocytes. Cells illuminated with 222-nanometer far UVC survived, while those treated with 254-nanometer and 277-nanometer wavelengths underwent apoptosis via the JNK/ATF2 pathway. However, cells exposed to 222-nanometer far UVC presented the highest degree of DNA damage as evidenced by yH2AX staining. Globally, these cells displayed transcriptional changes in cell-cycle and senescence pathways. Thus, the introduction of 222-nanometer far UVC lamps for disinfection purposes should be carefully considered and designed with the inherent dangers involved.

Keywords: DNA damage; UVC LED; far UVC; senescence; signaling pathways.

Figure 1

254-nanometer and 277-nanometer UVC sources result in cell death driven by apoptosis, while 222-nanometer far UVC results in decreased cell viability compared to control. (A) SRB assay for ARPE-19 cells exposed to either no UVC or 60 min of respective UVC wavelengths and then incubated for the indicated number of days. Values are reported as mean +/− S.D. from n = 3 experiments. (B) SRB assay for ARPE-19 cells exposed to either 0, 20 or 60 min of 222-nanometer far UVC and then incubated for the indicated number of days. Values are reported as mean +/− S.D. from n = 3 experiments. (C) Dynamic monitoring of cell numbers through xCelligence platform. Values are reported as mean +/− SD from n = 8 replicates. (D) Trypan blue staining assay performed on ARPE-19 cells exposed to either no UVC or 60 min of respective UVC wavelengths and then incubated for 1 day. Values are reported as mean +/− SD from n = 4 experiments. (E) Representative Western blot analysis of pro-caspase 3 versus cleaved caspase 3 after either no UVC or 60 min of respective UVC wavelengths. GAPDH immunoblotting was used as an internal control. Where applicable, Student’s t-test is performed and significance is represented as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 2

Differential activation of MAPK and ER-stress signaling pathways results in the differential activation of apoptotic pathways. (A) Schematic indicating the classical MAPK signaling pathways studied due to classical 254-nanometer UVC irradiation. (B) Representative Western blots of phosphor-ERK (Thr202, Tyr204), total ERK1/2, phosphor-JNK/SAPK (Thr183, Tyr185), total JNK1/2, phosphor-p38 (Thr180, Tyr182), p38, phosphor-ATF2 (Thr69), and GAPDH from ARPE-19 cells exposed to 0, 20, and 60 min of respective UVC wavelengths. (C) Schematic indicating the proteins involved in endoplasmic-reticulum-induced stress pathway. (D) Representative Western blots of phosphor-PERK (Thr980), total ERK1/2, phosphor-EIF2a (Ser49), EIF2a, and GAPDH from ARPE-19 cells exposed to 0, 20, and 60 min of respective UVC wavelengths.

Figure 3

DNA-associated damage or associated repair mechanisms were observed in 222-nanometer-lit cells. (A) γH2AX staining in ARPE19 cells upon 60 min of respective UVC irradiation. Scale bars = 50 µm. (B) Quantification of the extent of γH2AX activation in the ARPE19 cells upon 0, 20, and 60 min of UVC illumination. Values are reported as mean +/− SD from n = 3 experiments. (C) Quantification of the extent of γH2AX activation in the ARPE19 cells exposed to 60 min of 222-nanometer far-UVC illumination and then recovered 0, 2, and 7 days after the far-UVC treatment. Values are reported as mean +/− SD from n = 5 experiments. (D) Thymine dimer staining in ARPE19 cells upon 60 min of respective UVC irradiation. Scale bars = 20 µm. (E) Quantification of the extent of thymine dimer formation in the ARPE19 cells after 60 min of UVC illumination. Values are reported as mean +/− SD from n = 3–4 experiments. Where applicable, Student’s t-test is performed and significance is represented as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 4

Perturbation of transcription and cellular signaling events was observed in 222-nanometer-lit cells. RT-PCR and Western blot analysis of ARPE-19 cells after 222-nanometer far-UVC illumination. (A) RT-PCR analysis of selected genes 1 day after 60 min of 222-nanometer far-UVC illumination. (B) Representative Western blots of Rb1, p53, phosphor-ERK (Thr202, Tyr204), SNAI1, and GAPDH from ARPE-19 cells 1, 3, and 6 days after being exposed to 60 min of 222-nanometer far UVC illumination. (C) Quantification of the band intensity for Rb1, p53, phosphor-ERK (Thr202, Tyr204) and SNAI1 relative to internal-control GAPDH in the ARPE19 cells 1, 3, and 6 days after 60 min of UVC illumination. Values are reported as mean +/− SD from n = 3 experiments. Where applicable, Student’s t-test is performed and significance is represented as ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 5

Analysis of global RNA transcripts one week after 222-nanometer exposure (A) Highlighted in red are genes that are either upregulated with log2FoldChange at more than 2 and p < 0.05 (n = 140) or downregulated with log2FoldChange at less than 2 and p < 0.05 (n = 73). (B) Top ingenuity canonical pathways involved due to 222-nanometer illumination with ingenuity pathway analysis performed on 2361 statistically significant differentially expressed genes at p < 0.05. A positive activation zScore reflects upregulation of pathway in 222-nanometer illuminated ARPE-19 cells, while a negative activation zScore reflects the opposite. (C) Top diseases and disorders and molecular and cellular function categories derived from ingenuity pathway analysis out of 2361 statistically significant differentially expressed genes. (D) Ingenuity pathway analysis graphical summary providing a quick overview of the major biological themes and how they relate to each other. (E) Gene-set enrichment analysis of all 20358 genes detected in the RNA sequencing found upregulation of DNA damage and telomere-stress induced senescence pathway and a reduction in cell-cycle genes in 222-nanometer-illuminated ARPE-19 cells.