Abstract
Xeroderma Pigmentosum (XP) is a DNA repair disease characterized by nucleotide excision repair (NER) malfunction, leading to photosensitivity and increased incidence of skin malignancies. The role of XP-A in NER pathways has been well studied while discrepancies associated with ROS levels and the role of radical species between normal and deficient XPA cell lines have been observed. Using liquid chromatography tandem mass spectrometry we have determined the four 5’,8-cyclopurines (cPu) lesions (i.e., 5′R-cdG, 5′S-cdG, 5′R-cdA and 5′S-cdA), 8-oxo-dA and 8-oxo-dG in wt (EUE-pBD650) and XPA-deficient (EUE-siXPA) human embryonic epithelial cell lines, under different oxygen tension (hyperoxic 21%, physioxic 5% and hypoxic 1%). The levels of Fe and Cu were also measured. The main findings of our study were: (i) the total amount of cPu (1.82–2.52 lesions/106 nucleotides) is the same order of magnitude as 8-oxo-Pu (3.10–4.11 lesions/106 nucleotides) in both cell types, (ii) the four cPu levels are similar in hyperoxic and physioxic conditions for both wt and deficient cell lines, whereas 8-oxo-Pu increases in all cases, (iii) both wt and deficient cell lines accumulated high levels of cPu under hypoxic compared to physioxic conditions, whereas the 8-oxo-Pu levels show an opposite trend, (iv) the diastereoisomeric ratios 5′R/5′S are independent of oxygen concentration being 0.29 for cdG and 2.69 for cdA for EUE-pBD650 (wt) and 0.32 for cdG and 2.94 for cdA for EUE-siXPA (deficient), (v) in deficient cell lines Fe levels were significantly higher. The data show for the first time the connection of oxygen concentration in cells with different DNA repair ability and the levels of different DNA lesions highlighting the significance of cPu. Membrane lipidomic data at 21% O2 indicated differences in the fatty acid contents between wild type and deficient cells, envisaging functional effects on membranes associated with the different repair capabilities, to be further investigated.
Highlights
Xeroderma Pigmentosum (XP-complementation group A–G) is a rare autosomal recessive disorder, which presents itself with an increased incidence of sun-induced skin cancer and of internal cancer [1].Patients of the complementation group XP-A are prone to neurodegeneration
LC-MS/MS analysis revealed that both EUE-pBD650 and EUE-siXPA cells accumulated high levels of cPu under hypoxic conditions (1%) compared with physioxic (5%) and hyperoxic conditions (21%) (Figure 3)
Elevated levels of 50 R-cyclo-20 -deoxyguanosine (cdG) (p = 0.033) and 50 R-cyclo-20 -deoxyadenosine (cdA) (p = 0.032) were observed in EUE-pBD650 cells under hypoxia while 50 R-cdA, 50 S-cdG and 50 S-cdA were found significantly increased in EUE-siXPA cells under hypoxic conditions (p = 0.037, p = 0.002, p = 0.020, respectively)
Summary
Xeroderma Pigmentosum (XP-complementation group A–G) is a rare autosomal recessive disorder, which presents itself with an increased incidence of sun-induced skin cancer and of internal cancer [1]. Patients of the complementation group XP-A are prone to neurodegeneration. XP-A is one of the 30 proteins involved in the nucleotide excision repair (NER), a repair pathway responsible for the removal of those lesions that distort the DNA helix. NER operates by two distinct pathways: global genome repair (GGR). That removes lesions from the genome overall and transcription-coupled repair (TCR) that repairs transcriptionally active domains [3]. XP-A is involved in the transcription-domain associated repair (DAR), a DNA repair system that repairs bulky lesions in both transcribed and non-transcribed strands of active genes, whereas
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