This study investigated the effect of glucose and fructose, and advanced glycation end-products (AGEs) on genome damage in WIL2-NS cells, measured using the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. The effect of AGEs was investigated using the bovine serum albumin (AGE-BSA) model system induced either with glucose (Glu–BSA) or with fructose (Fru–BSA). Liquid chromatography-mass spectrometry (LC-MS/MS) analysis showed higher Nε-carboxymethyllysine (CML; 26.76 ± 1.09 nmol/mg BSA) levels in the Glu–BSA model. Nε-Carboxyethyllysine (CEL; 7.87 ± 0.19 nmol/mg BSA) and methylglyoxal-derived hydroimidazolone-1 (MG-H1; 69.77 ± 3.74 nmol/mg BSA) levels were higher in the Fru–BSA model. Genotoxic effects were measured using CBMN-Cyt assay biomarkers [binucleated(BN) cells with micronuclei (MNi), BN with nucleoplasmic bridges (NPBs) and BN with nuclear buds (NBuds)] following 9 days of treatment with either glucose, fructose, Glu–BSA or Fru–BSA. Fructose treatment exerted a significant genotoxic dose–response effect including increases of BN with MNi (R2 = 0.7704; P = 0.0031), BN with NPBs (R2 = 0.9311; P < 0.0001) and BN with NBuds (R2 = 0.7118; P = 0.0091) on cells, whereas the DNA damaging effects of glucose were less evident. High concentrations of AGEs (400–600 µg/ml) induced DNA damage; however, there was no effect on cytotoxicity indices (necrosis and apoptosis). In conclusion, this study demonstrates a potential link between physiologically high concentrations of reducing sugars or AGEs with increased chromosomal damage which is an important emerging aspect of the pathology that may be induced by diabetes. Ultimately, loss of genome integrity could accelerate the rate of ageing and increase the risk of age-related diseases over the long term. These findings indicate the need for further research on the effects of glycation on chromosomal instability and to establish whether this effect is replicated in humans in vivo.

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Abstract This study investigated the effect of dietary sugars and advanced glycation end-products (AGE) on telomere dynamics in WIL2-NS cells. Dietary sugars [glucose (Glu) and fructose (Fru); 0.1 M each] were incubated with bovine serum albumin (BSA) (10 mg/ml) at 60 ± 1°C for 6 weeks to generate AGE-BSA. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis showed total AGE levels as 87.74 ± 4.46 nmol/mg and 84.94 ± 4.28 nmol/mg respectively in Glu-BSA and Fru-BSA model. Cell treatment studies using WIL2-NS cells were based on either glucose, fructose (each 2.5–40 mM) or AGE-BSA (200–600 µg/ml) in a dose-dependent manner for 9 days. Telomere length (TL) was measured using qPCR. Nitric oxide (NO) production and tumour necrosis factor-α (TNF-α) levels were measured in WIL2-NS culture medium. An increasing trend for TNF-α and NO production was observed with higher concentration of glucose (R2 = 0.358; P = 0.019; R2 = 0.307; P = 0.027) and fructose (R2 = 0.669; P = 0.001; R2 = 0.339; P = 0.006). A decreasing trend for TL (R2 = 0.828; P = 0.000), and an increasing trend for NO production (R2 = 0.352; P = 0.031) were observed with increasing Glu-BSA concentrations. Fru-BSA treatment did not show significant trend on TL (R2 = 0.135; P = 0.352) with increasing concentration, however, a significant reduction was observed at 600 µg/ml (P < 0.01) when compared to BSA treatment. No trends for TNF-α levels and a decreasing trend on NO production (R2 = 0.5201; P = 0.019) was observed with increasing Fru-BSA treatment. In conclusion, this study demonstrates a potential relationship between dietary sugars, AGEs and telomere attrition. AGEs may also exert telomere shortening through the production of pro-inflammatory metabolites, which ultimately increase the risk of diabetes complications and age-related disease throughout lifespan.

The cytokinesis-block micronucleus cytome (CBMNcyt) assay is a comprehensive method to measure DNA damage, cytostasis and cytotoxicity caused by nutritional, radiation and chemical factors. A slide imaging technique has been identified as a new method to assist with the visual scoring of cells for the CBMNcyt assay. A NanoZoomer S60 Digital Pathology slide scanner was used to view WIL2-NS cells treated with hydrogen peroxide (H2O2) and measure CBMNcyt assay biomarkers using a high-definition desktop computer screen. The H2O2-treated WIL2-NS cells were also scored visually using a standard light microscope, and the two visual scoring methods were compared. Good agreement was found between the scoring methods for all DNA damage indices (micronuclei, nucleoplasmic bridges and nuclear buds) and nuclear division index with correlation R values ranging from 0.438 to 0.789, P < 0.05. Apoptotic and necrotic cell frequency was lower for the NanoZoomer scoring method, but necrotic frequency correlated well with the direct visual microscope method (R = 0.703, P < 0.0001). Considerable advantages of the NanoZoomer scoring method compared to direct visual microscopy includes reduced scoring time, improved ergonomics and a reduction in scorer fatigue. This study indicates that a digital slide scanning and viewing technique may assist with visual scoring for the CBMNcyt assay and provides similar results to conventional direct visual scoring.

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