The use of in vitro transcriptional data to identify thresholds of effects in a human lens epithelial cell-line exposed to ionizing radiation

Abstract

Abstract Purpose: The International Commission on Radiological Protection (ICRP) recently recommended reducing the occupational equivalent dose limit for the lens of the eye. Based primarily on a review of epidemiological data, the absorbed dose threshold is now considered to be 0.5\u2009Gy independent of dose-rate and severity of opacification, reduced from the previous threshold of 2\u2009Gy. However, direct mechanistic evidence to support an understanding of the underlying molecular mechanisms of damage is still lacking. To this end, we explored the effects of a broad dose-range of ionizing radiation exposure on gene expression changes in a human lens epithelial (HLE) cell-line in order to better understand the shape of the dose–response relationship and identify transcriptional thresholds of effects. Methods: HLE cells were exposed to doses of 0, 0.01, 0.05, 0.25, 0.5, 2, and 5\u2009Gy of X-ray radiation at two dose rates (1.62 cGy/min and 38.2 cGy/min). Cell culture lysates were collected 20\u2009h post-exposure and analyzed using whole-genome RNA-sequencing. Pathways and dose-thresholds of biological effects were identified using benchmark dose (BMD) modeling. Results: Transcriptional responses were minimal at doses less than 2\u2009Gy. At higher doses, there were a significant number of differentially expressed genes (DEGs) (p≤.05, fold change≥|1.5|) at both dose rates, with 1308 DEGs for the low dose rate (LDR) and 840 DEGs for the high dose rate (HDR) exposure. Dose–response modeling showed that a number of genes exhibited non-linear bi-phasic responses, which was verified by digital droplet PCR. BMD analysis showed the majority of the pathways responded at BMD median values in the dose range of 1.5–2.5\u2009Gy, with the lowest BMD median value being 0.6\u2009Gy for the HDR exposure. The minimum pathway BMD median value for LDR exposure, however, was 2.5\u2009Gy. Although the LDR and HDR exposures shared pathways involved in extracellular matrix reorganization and collagen production with BMD median value of 2.9\u2009Gy, HDR exposures were more effective in activating pathways associated with DNA damage response, apoptosis, and cell cycling relative to LDR exposure. Conclusions: Overall, the results suggest that radiation induces complex non-linear transcriptional dose–response relationships that are dose-rate dependent. Pathways shared between the two dose rates may be important contributors to radiation-induced cataractogenesis. BMD analysis suggests that the majority of pathways are activated above 0.6\u2009Gy, which supports current ICRP identified dose thresholds for deterministic effects to the lens of the eye of 0.5\u2009Gy.