Glutathione and DNA repair -
Min Liu, Yunxue Zhao, Xiumei Zhang. Strahlenschutz von Normalgewebszellen Radioprotection of normal tissue cells. Patrick Maier, Frederik Wenz, Carsten Herskind.
Appraisal of mechanisms of radioprotection and therapeutic approaches of radiation countermeasures. Krishna N. Mishra, Belal A. Moftah, Ghazi A. An ultrasensitive fluorogenic probe for revealing the role of glutathione in chemotherapy resistance. Yuejing Jiang, Juan Cheng, Chengyu Yang, Yongzhou Hu, Jia Li, Yifeng Han, Yi Zang, Xin Li.
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M Dafre, Protein S-thiolation and regulation of microsomal glutathione transferase activity by the glutathione redox couple, Arch. Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article.
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Rights and permissions Reprints and permissions. About this article Cite this article Singhal, R. Copy to clipboard. The exposure to carbon ions resulted in a lower surviving fraction compared with X-rays in both cell lines.
The survival fraction at 2 Gy SF2 was 0. SQ20B cells were systematically more resistant than SCC61 cells, even in response to carbon ions. Such a radiosensitization was reversed in the presence of 5 µM NaC, a highly powerful antioxidant agent, as evidenced by the SF2 value 0.
We first evaluated the efficiency of DSB repair according to the kinetics of γH2AX foci. As shown in Fig. After carbon ion exposure Fig. In contrast to the X-ray irradiation response, the repair kinetics was similar between the two cell lines.
Finally, the number of residual γH2AX foci measured in sensitive cells 24 h after carbon ion exposure was equivalent to that observed after the biological isodose of X-rays 7. Cells were irradiated with 2 Gy of X-rays A, C or 1 Gy of carbon ions B, D.
One hundred cells were scored for each time and the measurements were made in triplicate and repeated three times. The increase in the number of initial DNA lesions led to residual DSB 9. Interestingly, after carbon ion exposure, the response of GSH-depleted SQ20B cells matched perfectly that of irradiated SCC61 cells in terms of the initial DNA damage, repair kinetics, and residual DSB.
These results indicate that the depletion of the endogenous GSH pool in resistant cells combined with X-ray or carbon ion exposure at a biologically equivalent dose led to the persistence of DNA lesions at a level similar to that in the sensitive SCC61 cells.
To confirm the role of redox changes on DNA damage, we incubated SQ20B cells with NaC. Under these experimental conditions, NaC did not induce γH2AX foci in control experiments Fig. NaC may thus reverse the effect of GSH depletion. The level of alkali-labile sites and SSB in DNA was investigated using the SCGE assay.
No signal was obtained from SQ20B cells during the time studied with either type of irradiation. By contrast, SCC61 cells were highly responsive and showed a high level of breaks at the shortest times after irradiation.
Rapid repair occurred after X-ray exposure, as shown by the decreasing number of breaks with time. Although carbon ion irradiation induced a similar initial percentage of tail DNA compared with X-ray irradiation, the repair kinetics in SCC61 cells was considerably slower and showed a sustained increase up to 2 h followed by a decrease for a longer time.
Interestingly, GSH-depleted SQ20B cells showed a similar pattern to that observed for SCC61 cells and the rate was similar after exposure to either type of radiation.
This suggests that high endogenous GSH levels protect DNA against radiation in SQ20B cells. In a second set of experiments, the percentage of tail DNA identified using SCGE alone was subtracted from that obtained after treatment with the Fpg enzyme to study the spatial distribution of oxidized bases.
The results shown in Fig. However, GSH-depleted SQ20B cells displayed more scattered oxidative damage at the shortest time after X-ray irradiation, whereas a less variable pattern of damage after exposure to carbon ions suggested the local production of free radicals.
Comet assays were performed in alkaline conditions without A or in the presence of Fpg enzyme B. To determine to what extent GSH depletion and the residual DSB could affect the cellular response to X-ray or carbon ion irradiation through cell cycle redistribution, the relative number of SCC61, SQ20B, and GSH-depleted SQ20B cells in the sub-G1 Fig.
By contrast, no significant level of apoptosis was measured in SQ20B cells after either type of irradiation. This increase was slightly delayed 96 h after carbon exposure but reached the same level at h.
A The percentage of cells in sub-G1 phase. Unrepaired or misrepaired DNA damage can lead to chromosome changes in surviving cancer cells. The yield of MN was estimated by calculating the Ymn value. More MN were produced in sensitive SCC61 compared with SQ20B cells after both types of irradiation.
The maximum yield in SCC61 cells did not differ significantly between the two types of irradiation 2. The maximum value was slightly delayed after carbon ion irradiation 96 h , a time corresponding to the triggering of apoptosis, as described above.
By contrast, the yield of MN induced in resistant SQ20B cells did not exceed 0. Although the radiosensitization of SQ20B cells through GSH depletion led to residual DSB identical to those observed in SCC61 cells after irradiation, it did not induce the same pattern of MN.
The Ymn values measured in GSH-depleted SQ20B were equal to those in undepleted SQ20B cells after X-irradiation excepted at h post-irradiation , but were lower after carbon ion exposure for the majority of the kinetic time points.
Finally, only carbon ion irradiation induced an obvious decrease in the number of radioinduced MN in GSH-depleted SQ20B cells. This might correspond to a specific signature of carbon ion irradiation.
Two types of rearrangements were considered: apparently dicentric chromosomes, which were visualized as nucleoplasmic bridges NPB , and the more complex rearrangements, which were visualized by simultaneous appearance of NPB and MN.
GSH depletion in SQ20B cells did not alter significantly any values regardless of the type of irradiation. The expression of dicentric chromosomes in surviving cancer cells seemed to be independent of the intrinsic radiosensitivity and type of radiation.
No differences were observed in complex rearrangements between SCC61 and SQ20B, as evidenced by the simultaneous observation of NPB and MN after X-ray exposure Fig. GSH depletion in SQ20B cells had no significant effect on this type of rearrangement.
GSH depletion in SQ20B cells led to a strong and significant decrease in CCs at all times. The induction of complex rearrangements was independent of cell radiosensitivity, but these rearrangements differed according to the radiation type and GSH depletion in surviving cancer cells.
Percentage of cells displaying chromosomal rearrangements including dicentric chromosome formation A and complex rearrangements B. The aim of our study was to highlight the relationship between the nature of DNA damage and the consecutive chromosomal aberrations in response to low- and high-LET irradiation after a transient depletion of endogenous glutathione in resistant HNSCC cancer cells.
To address this issue, X-ray and carbon ion irradiation were performed at a biologically equivalent dose to focus on events leading to an equivalent level of cell death evaluated with the relative biological effectiveness and clonogenic assays ; this was performed to enable a comparison of the nature of DNA damage and the consequences on the transmission of chromosomal changes according to the type of radiation, radioresistance status, and endogenous GSH content.
The resistant and sensitive HNSCC cell lines displayed different responses in terms of DNA lesions and repair capacity in relation to their GSH content. In response to X-ray exposure, the resistant SQ20B cell line, with a higher endogenous GSH content, showed a higher DNA repair capacity that enabled fast disappearance of DSB and SSB.
By contrast, the repair capacity of sensitive SCC61 cells was slower and led to the persistence of residual DSB. These results confirm the previously suggested notion [18] , [22] , [23] that GSH level correlates with DNA repair capacity.
By contrast, a biologically equivalent dose of carbon ions 1 Gy carbon ions compared with 2 Gy X-rays induced fewer initial breaks. The repair kinetics were slower than after X-ray exposure, as confirmed by Schmid et al. Lesions were more difficult to repair, and the endogenous GSH level had no influence on the repair capacity after high-LET radiation, as previously reported [23].
These observations support the concept that breaks produced by particle tracks are more clustered and complex than are those produced by X-rays [2] , [41]. Interestingly, regardless of the number and the complexity of the initial lesions, sensitive cells were characterized by the same level of residual DSB 24 h after the equivalent biological dose of X-rays and carbon ions.
This damage reflects defects in the repair processes and correlates with apoptotic induction [42]. The transient GSH depletion in SQ20B cells led to the persistence of equal numbers of residual DSB after low- or high-LET exposure performed at a biologically equivalent dose.
Interestingly, the number of unrepaired DSB and the percentage of apoptotic cells were similar to those measured in radiosensitive cells.
The sensitizing effect induced by GSH depletion confirms its role in the mechanisms of radioresistance [11] , [43] against both high- and low-LET radiation.
To focus on the DNA radioprotective role of GSH, shorter kinetics points should be compared. After X-ray irradiation, GSH depletion increased the number of sparse DSB and SSB, and oxidized DNA bases.
Experiments using NaC demonstrated the reverse effect of the GSH depletion on DSB and confirmed that redox changes induced by this depletion are responsible for the effect on DNA damage.
The appearance of these sparse lesions corroborates the predominance of an indirect effect that produces long-life free radicals that react with DNA to generate DSB [22] , [44].
By contrast, after carbon ion irradiation, DNA lesions such as DSB or SSB require a much longer time to repair. The reverse effect of NaC suggests the involvement of oxidative stress in the induction of DNA lesions. Additional experiments are needed to demonstrate the involvement of oxidative stress.
However, our results suggest that GSH modulation impacts more on the quality of DNA lesions than on the quantity. The distribution of oxidized bases was not scattered, but appeared to colocalize with SSB, or even DSB, as evidenced by the results obtained after the addition of Fpg enzyme during the comet assays.
The hypothesis of localized clusters of oxidized bases is strengthened by the work of Bergeron et al. This suggests that short-life free radicals are produced along the carbon ion track and that these free radicals increase the complexity of DNA lesions after GSH depletion combined with carbon ion irradiation.
These data confirm the importance of an indirect effect at a local scale in response to carbon ion irradiation; in contrast to previous reports [19] , [22] , our data demonstrate the protective role of GSH against oxidative clustered DNA damage formation after carbon ion radiation.
Most studies have investigated the radioprotective role of GSH by adding exogenous GSH or other scavengers, but without considering the endogenous level of GSH in their cellular model [18] , [19] , [22] , [46]. Considering the results presented in this paper, the inclusion of this parameter now seems to be fundamental.
In our cellular models, we agreed that a high endogenous GSH content correlates with a faster repair process, suggesting that GSH helps in DSB rejoining, as reported previously by others [18] , [19] , [22] , [23] who did not propose any speculation about a possible specific intrinsic mechanism.
Our data did not enable to further speculate about rejoining. However, GSH depletion modifies the kinetics of repair after X-ray radiation, suggesting that GSH had a direct effect on the repair systems in our cellular models and probably on DNA damage recognition.
The relationship between DNA repair capacity and endogenous GSH level probably proceeds from inducing cell adaptation to protecting against oxidative stress [12]. In SQ20B cells, transient GSH depletion favored radiosensitization through the induction of supernumerary and highly complex lesions after X-ray and carbon ion irradiation, respectively, leading to a defect in repair, as observed in sensitive SCC61 cells.
DNA damage and its management are crucial in determining cell fate. The more severe lesions should guide cells toward death, and misrepaired lesions should lead to chromosomal changes.
Cancer cells have a highly rearranged genome and may survive even after a part of the genome is lost or modified. By contrast, transmission of chromosomal changes to cancer cell progeny may lead to adaptation and acquisition of additional resistance mechanisms.
The direct consequence is that first mitosis occurs with delays. Using the cytome assay, we have assessed the consequences of misrepaired and residual DNA lesions from the first mitosis after irradiation for up to five days.
As suggested by Johannes et al. Four classes of CCs were considered. First, in contrast to a previous study [4] , our analysis showed that dicentric chromosome expression NPB formation was unchanged regardless of the intrinsic radiosensitivity of the cell line and the type of radiation applied.
We also demonstrated that dicentric chromosome expression did not vary according to the endogenous GSH level. Second, complex rearrangements simultaneous expression of NPB and MN were considered. Contrary to a previous suggestion [4] , such complex aberrations do not depend on the sensitivity of cells to radiation, but do depend on the type of radiation and the radioinduced DNA lesions produced.
We demonstrated in GSH-depleted SQ20B cells that an increase in the number of sparse DNA lesions after X-ray irradiation did not modify the expression of complex rearrangements. By contrast, after GSH depletion combined with carbon ion exposure in SQ20B cells, the increasing complexity of lesions decreased the number of complex rearrangements at the same time as triggering apoptosis, but it was not equivalent to what happened in SCC61 cells.
These findings suggest that only a modification of the complexity along with highly clustered lesions could prevent the transmission of complex rearrangements to surviving cancer cells. This may be one advantage of hadrontherapy, i. a treatment minimizing genomic instability and tumor escape.
In this set of experiments, high-LET radiation induced a higher frequency of MN with centromeres in SQ20B and SCC61 cell lines compared with X-ray radiation.
To our knowledge, such a molecular signature has, up to now, never been reported [2] , [19]. We note that cancer cells display different numbers of chromosomes that may influence the yield of MN, as higher chromosome numbers may be linked to higher MN production.
However, as a clear estimation of chromosome number was not possible at the time of experimentation, it is difficult to weight these values. Globally, SQ20B cells are known to display a higher chromosome number than SCC cells [49].
The sensitive SCC61 cell line displayed a higher level of MN compared with the resistant SQ20B cell line. The percentage of cells with MN reflected the radiosensitivity of the cell lines assayed. A decrease in the frequency of deletions was previously associated with high levels of GSH [18] , [19] , [23].
Our results in SQ20B cells, which displayed a high GSH content, confirm these previous findings. In all cases, the GSH-depleted SQ20B cells did not show the same profile of MN as the SCC61 cells, suggesting that the MN profile is dependent on the intrinsic radioresistance of the SQ20B cell line.
This finding may contradict the data from previous studies based on sister chromatid staining that reported an increase in deletion frequency after GSH depletion [23] or a decrease after GSH addition [18] in human lymphocytes. This discrepancy may reflect the status of the cells, which were studied before the completion of the first metaphase.
We hypothesize that GSH modulation is part of regulation of the balance between cell cycle arrest and damage repair on the one hand and initiation of cell death on the other.
GSH depletion may reactivate the correct function of DNA-damage checkpoints, favoring cell death before mitosis, and may minimize the transmission of MN in the progeny following carbon ion irradiation, but not after X-ray irradiation. The prevention of transmissible MN and rearrangements is essential for guaranteeing the absence of chromosomal changes and consequently for limiting the genomic instability in surviving cells.
Hexavalent chromium Cr VI causes various toxic and Glutathions effects. Glutathione and DNA repair main carcinogenic Forskolin and inflammation is observed in the pulmonary system through inhalation route. Reduction of Glutathione and DNA repair VI Glutathioen Cr V, IV, and Repaif reactive intermediates within the cells by intracellular reducing agents such as glutathione is an important event leading to oxidative stress and oxidative DNA damage. This study evaluated the effects of intraperitoneal administration of Cr VI and GSH on total oxidant status TOStotal antioxidant capacity TACoxidative stress index, and oxidative DNA damage by evaluating the level of 8-hydroxy-2́-deoxyguanosine 8-OHdG in Swiss-Albino mice. No difference in TAC was observed among the groups. Thank you for visiting Glutathione and DNA repair. You are using a browser repakr with limited support for CSS. To Gljtathione the best Glutathoone, we Metabolism boosting pills you use a more up to date Glutatbione or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Human preterm neonates suffer from respiratory distress syndrome RDS due to immature lungs and require assisted ventilation with high concentrations of oxygen. Prolonged exposure to oxygen results in Bronchopulmonary dysplasia BPDa chronic lung disease and the leading cause of death in premature neonates.
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