Effects of Extracellular and Intracellular pH on Repair of Potentially Lethal Damage, Chromosome Aberrations and DNA Double-Strand Breaks in Irradiated Plateau-Phase A549 Cells

Abstract

Plateau-phase A549 cells exhibit a high capacity for repair of potentially lethal radiation damage (PLD). Previously it was found that PLD repair could be partially inhibited by increasing the extracellular pH (pHe) of the spent medium from its normal value of 6.7-6.8 to 7.6 during postirradiation holding. The present study shows that PLD repair is also inhibited by reducing the pHe of the spent medium to 6.0. The effects of altering pHe on rejoining of DNA double-strand breaks (DSBs) as measured by neutral filter elution and on mitotic delay and chromosome aberrations seen after releasing cells from the plateau phase were investigated. Neither increasing nor decreasing the pHe of the spent medium had an effect on radiation-induced mitotic delay. Rejoining of DSBs was significantly inhibited by holding at pHe 6.0 but not affected by holding at pHe 7.6. At 2 h after irradiation about 51% of unrejoined breaks remained at pHe 6.0, compared to about 15% at pHe 6.7 or 7.6. However, holding at pHe 7.6 appeared to cause a marginal change in the kinetics of rejoining of DSBs. Repair of lesions leading to dicentric and acentric chromosome aberrations did not occur when cells were held at pHe 6.0, since less than 10% of these aberrations disappeared from cells held for 24 h before subculture. In contrast, holding plateau-phase cells at pHe 7.6 vs 6.7 caused a small but significant reduction in the disappearance of dicentrics but had no effect on the rate or extent of the disappearance of acentrics. These data have led us to hypothesize that inhibition of PLD repair by holding at pHe 6.0 is related both to inhibition of pH-dependent DNA repair enzymes and to induction of changes in DNA which lead to misrepair when the cells are released from plateau phase. Inhibition of PLD repair by holding at pHe 7.6, on the other hand, is related primarily to changes in DNA structure which promote misrepair.

Na+, K+-ATPase inhibitor, ouabain accentuates irradiation damage in human tumour cell lines

“…Radiocurability may depend upon a combination of sublethal and potential lethal damage repair [28] and both mechanisms may have a similar molecular basis [29]. The higher recovery ratio for A549 cells reported here falls within the range of 40 to 70 found for potential lethal damage repair [9,30].…”

“…Recovery of sublethal damage as a function of time was obtained from: S(t) ‫ס‬ S 0 + (S max − S 0 )(1 − e −k.t ) [9], where S 0 is the surviving fraction at time t ‫ס‬ 0 and S max is the maximum surviving fraction reached in the split dose experiment and k is the repair rate. Halftimes for repair (T 1 /2 ) were calculated as follows [10]: T 1 /2 ‫ס‬ Ln2/k ‫ס‬ 0.693/k, where k is the repair rate as calculated above.…”

Characterization of the APLF FHA–XRCC1 phosphopeptide interaction and its structural and functional implications

“…Cells exposed to hypoxia and low pH have been reported to exhibit a diminished capacity for DNA repair compared with control cells grown under standard culture conditions ( 52 ). Significant inhibition of rejoining double-stand DNA breaks (DSBs) also has been observed when irradiated cells are maintained at pH 6.0 ( 53 ). Although other effects, including the pH-dependence of polymerase enzymes ( 54 , 55 ) contribute to this behavior, pH-dependent interactions between XRCC1/4 and FHA domain proteins, including PNKP, APTX and APLF, may be an important contributor under some circumstances.…”

Disturbances in H+ dynamics during environmental carcinogenesis

“…clastogenicity [118]. The maintenance of such aberrations would be facilitated by the fact that a low pH e could also alter the DNA repair process [119,120]. Interestingly, data obtained by Massonneau and coworkers [121] have recently indicated that even rather small variations of pH e (from 7.2 to 6.9) could lead to impairment of the DNA repair; as a result, the genotoxicity of double-stranded breaks would be enhanced, leading to genetic instability.…”