Low extracellular pH inhibits nucleotide excision repair

https://doi.org/10.1016/j.mrgentox.2021.503374Get rights and content

Highlights

  • Low extracellular pH inhibits nucleotide excision repair (NER) pathway, leaving pyrimidine dimers unrepaired.

  • NER protein TFIIH was recruited normally but the release was delayed as the pH was lower.

  • Both accumulation and release of XPG were delayed at low pH.

  • Gap filling was retarded at low pH.

  • The dysregulated gap filling and suppressed release of accumulated XPG might be related to each other.

Abstract

Nucleotide excision repair (NER) is the main pathway to repair bulky DNA damages including pyrimidine dimers, and the genetic dysregulation of NER associated proteins is well known to cause diseases such as cancer and neurological disorder. Other than the genetic defects, ‘external factors’ such as oxidative stress and environmental chemicals also affect NER. In this study, we examined the impact of extracellular pH on NER. We prepared the culture media, whose pH values are 8.4 (normal condition), 7.6, 6.6 and 6.2 under atmospheric CO2 conditions. Human keratinocytes, HaCaT, slightly died after 48 h incubation in DMEM at pH 8.4, 7.6 and 6.6, while in pH 6.2 condition, marked cell death was induced. UV-induced pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs), were effectively repaired at 60 min and 24 h, respectively, which were remarkably inhibited at pH 6.6 and 6.2. The associated repair molecule, TFIIH, was accumulated to the damaged sites 5 min after UVC irradiation in all pH conditions, but the release was delayed as the pH got lower. Furthermore, accumulation of XPG at 5 min was delayed at pH 6.2 and 6.6, and the release at 60 min was completely suppressed. At the low pH, the DNA synthesis at the gaps created by incision of oligonucleotides containing pyrimidine dimers was significantly delayed. In this study, we found that the low extracellular pH inhibited NER pathway. This might partially contribute to carcinogenesis in inflamed tissues, which exhibit acidic pH.

Introduction

Nucleotide excision repair (NER) is the main pathway used by mammals to remove bulky DNA lesions induced by ultraviolet (UV) irradiation, environmental mutagens, and certain chemotherapeutic agents [1]. The repair process starts with damage recognition and then incisions 5′ and 3′ to the lesion to allow for the removal of oligonucleotides containing lesion, creating an ssDNA gap. This gap will be filled by newly synthesized damage-free DNA by polymerases and finally ligated to seal the nick. Defect in this pathway can contribute to accumulation of mutation [2], leading to cancer initiation and progression. The genetic dysfunctions of many proteins associated with the NER pathway are well known to cause diseases such as xeroderma pigmentosum, cockayne syndrome, and trichothiodystrophy [3]. Other than those genetic defects, ‘external factors’ such as oxidative stress and environmental chemicals also affect NER. Oxidative damage to NER proteins such as RPA, TFIIH, and XPA limited the NER capacity and enhanced the mutation risk [4,5]. RPA was vulnerable to oxidation; oxidized forms of RPA were associated with impaired NER [4]. We also reported that formaldehyde and unsaturated aldehydes contained in cigarette smoke delayed the NER by degradation of TFIIH [6,7].

Variations in pH may be an external factor to have a possibility to impact the DNA damage repair efficiency. Culturing mammalian cells in low pH medium induced clastogenic effects such as sister-chromatid exchanges and chromosomal aberrations [8,9]. Low extracellular pH is typically observed in tumor tissues, where hypoxia frequently occurs as a consequence of tumor cell metabolism producing lactate from anaerobic glycolysis [10]. Those tumor microenvironments cause increase of mutagenicity, genetic instability, metastatic capacity and resistance to chemotherapy [11,12]. Exposure to both hypoxia and low pH suppressed NER and elevated UV-induced mutagenesis [13]. However, the detailed mechanism by which the tumor microenvironment induces those NER inhibition has not been established. In normal physiological conditions, drop of pH below 7.4 is rare, but ischemia and inflammation result in extracellular acidification in tissue microenvironment [14,15].

The stratum corneum (SC), the outmost epidermal layer, exhibits an acidic surface pH, which is required for competent permeability barrier homeostasis [16]. The pH of skin surface in young humans is around 5. About a week to months are needed to achieve the SC acidification after birth [17,18], which is impaired in moderately aged humans and mice [16]. The cause for SC acidification includes byproducts of microbial metabolism, lactic acid and lactate from sweat, free fatty acids, and generation of cis-urocanic acid from filaggrin, whereas recent studies show that the SC is acidified largely by endogenous causes including Na+/H+ antiporter, sodium-proton exchanger 1 (NHE1) and secretory phospholipase A2 enzymes [18,19]. NHE1 is present in all epidermal cell layer, especially in the apical surface of the layer, and disappears in the enucleated cells above the SC/ stratum granulosum interface [19]. Skin is usually exposed to sunlight containing UV, and two types of bulky modifications are mainly produced, namely cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). SC, which receives UV radiation the most, is in acidic condition, especially in young humans [16]. As described above, extracellular pH might affect DNA damage repair. However, the effect of extracellular pH associated with repair of pyrimidine dimers has not been addressed.

In this study, we examined the repair of 6-4PPs and CPDs in low pH conditions. Low extracellular pH delayed the repair of both pyrimidine dimers, which was caused by the impaired accumulation and release of NER proteins and the delay of gap filling after excision of nucleotides containing pyrimidine dimers.

Section snippets

Preparation of low pH medium

The culture medium used was Dulbecco’s modified Eagle’s medium (DMEM) (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10 % fetal bovine serum (FBS, HyClone Laboratories, Inc., South Logan, UT, USA) and 100 units/mL of penicillin/streptomycin. The pH was adjusted by adding 10 % sodium bicarbonate. The pH of baseline DMEM used for normal cell culturing was 8.4 in ambient air. The pH at 6.2, 6.6, and 7.6 was obtained by adding reduced amount of sodium bicarbonate. The pH of 8.4,

Influence of medium pH to UV-induced cell death

Cell survival after culture in DMEM with different pH was determined (Fig. 1A). The treatment at pH6.6, 7.6 and 8.4 for up to 48 h only slightly decreased the survival, whereas incubating cells at pH 6.2 reduced cell viability by about 20 % at 24 h and 80 % at 48 h. Fig. 1B shows the intracellular pH after culture for 1 h in DMEM at four different pH. The intracellular pH changed according to the extracellular pH, but the extent of intracellular pH change was narrower than that of the

Discussion

Previous studies showed that changes in extracellular pH induced corresponding intracellular pH change [21,22]. In human pulmonary artery endothelial cells, incubation in buffer with the pH adjusted from 6.6 to 8.0 for only 30 min resulted in the intracellular pH from 7.12 to 7.65 [21]. We observed similar change of intracellular pH by incubating cells for 1 h. On the other hand, fetal human skin incubated in the medium at different pH for 72 h did not show significant change in intracellular

Funding source

This work was supported by JSPS KAKENHI Grant Number JP15H02828.

Declaration of Competing Interest

The authors report no declarations of interest.

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