Tepair antioxidants a. More metrics information. and Ames,B. Slides intended for oxidized DNA base measurements were washed with the enzyme buffer 0.

Glutathione and DNA repair -

Such transmissible aberrations may induce genomic instability in surviving cells, leading to tumor escape. The aim of the present study was first to determine the quality and the quantity of DNA lesions in relation to endogenous GSH level and radiation quality, and second to determine the consecutive CCs in surviving cancer cells after X-ray and carbon ion irradiation in order to evaluate the risk of radioinduced instability and then tumor escape.

A transient GSH-depletion strategy was applied before irradiation, based on the use of dimethylfumarate DMF , a glutathione-depleting agent, and buthionine sulfoximine BSO , a glutathione biosynthesis inhibitor.

This strategy has been previously tested [14] in terms of in vitro toxicity and the activated pathways leading SQ20B cells to death after irradiation were clearly demonstrated. It allowed SQ20B cells to display the same sensitivity as SCC61 cells, a radiosensitive HNSCC cell line that displays a low endogenous GSH content [14] , [34].

In this paper, the data obtained from radioresistant SQ20B cells and GSH-depleted SQ20B cells were therefore compared with sensitive SCC61 cells. Taken together, our results lead to a new understanding of the quality and the number of DNA lesions in relation to GSH level and radiation quality and thus clarify some divergent results reported in the literature.

In this regard, the cytome assay gave a clear overview of chromosomal aberrations transmitted in the surviving cancer cells. It enabled the assertion that an increase of the DNA lesion complexity obtained by GSH-depletion adjuvant therapy combined with hadrontherapy may minimize genomic instability in resistant cancer cells and thus reduce the phenomenon of tumor escape after radiotherapy.

Cells were cultured for no more than 12 passages. Dimethylfumarate DMF, µM , a GSH-depleting agent, and l -buthionine sulfoximine BSO, µM , an inhibitor of GSH biosynthesis, were added to the SQ20B culture medium 4 h before irradiation to deplete GSH, as described previously [14].

Primary γH2AX and centromeric protein A CENPA mouse antibodies were obtained from Upstate and Abcam, respectively, and the secondary antibody AlexaFluor goat anti-mouse IgG was obtained from Invitrogen. Antifade mounting medium was purchased from Dako. Low-melting point agarose and SYBR Green solution were from Sigma, and the formamidopyrimidine glycosylase Fpg was obtained from Trevigen.

Clonogenic cell survival was monitored after X-ray and carbon ion exposure at doses ranging from 1 to 5 Gy. The cells were seeded before irradiation and reseeded immediately after exposure into flasks of 25 cm 2 at different concentrations. Cell survival was assessed by the standard colony formation assay as described in [35].

Total glutathione was quantified by HPLC analysis. Briefly, proteins were precipitated from the cellular homogenate with sulfosalicylic acid and centrifuged at The supernatant was then derivatized with o -phthalaldehyde.

Chromatographic separation was achieved on a 5 µm Spherisorb Ccolumn, with a mobile phase composed of methanol—0. Fluorescence of the glutathione- o -phthalaldehyde derivatives was detected at an emission wavelength of nm and an excitation at nm [36]. The detection of γH2AX foci or CENPA was assayed by immunohistochemistry.

The CENPA detection was performed with the cytome assay described below. Digital images were obtained using a fluorescent microscope Axio Imager Z1 Zeiss microscope, × magnification. A minimum of nuclei were scored at each time to calculate the average number of γH2AX foci using ImageJ software.

After defrosting, the samples were centrifuged rpm, 4°C , and the pellet was suspended in cold PBS and mixed with 0. Gels were spread onto microscope slides and the SCGE technique was used as described by Tice et al. Slides intended for oxidized DNA base measurements were washed with the enzyme buffer 0.

The slides were rinsed with mM Tris base pH 7. A total of comets were observed visually and scored on each slide using free CASP software. The tail intensity, defined as the percentage of DNA migrating from the head of the comet into the tail, was measured for each scored nucleus.

Propidium iodide PI staining was used to analyze the cell cycle distribution, as previously described [34]. The multiendpoint cytokinesis-blocked micronucleus assay was used to assess chromosome aberrations [32]. No binucleated cells are numbered in the 5 h following addition of cytochalasin B.

Cytochalasin B was added 4 h before irradiation in order to cumulate the most representative cell population at a binucleated stage 24 h later. These markers are described here in ascending order of complexity. Cells with MN.

These cells are characterized by the presence of both a main nucleus and one or more smaller nuclei called MN. The frequency of MN in the cell population is the yield of MN Ymn , which is calculated as: where MN1 is the number of cells with one micronucleus, MN2 the number of cells with two MN, MNn the number of cells with n MN, and BN is the total number of binucleated cells.

Cells with NPB. NPB are continuous DNA-containing structures linking the nuclei in a binucleated cell. NPB originate from dicentric chromosomes in which the centromeres are pulled to opposite poles during anaphase and are therefore representative of misrepaired DNA, chromosome rearrangement or telomere end fusion.

Cells with simultaneous expression of NPB and MN. All scoring criteria were used according to the morphological parameters described by Fenech [33]. The data from at least three independent experiments are presented as the mean and standard deviation. The data were analyzed using Student's t test.

In the first set of experiments Table 1A , we have quantified the total GSH content in SQ20B cells treated under different conditions. When used in combination, DMF and BSO treatment resulted in total GSH depletion after 4 h of incubation with a slow restoration of the GSH level at 24 h.

No significant variations of oxidized glutathione were detected under our experimental conditions data not shown. This protocol was therefore considered as optimal to efficiently and transiently deplete SQ20B GSH stores.

The results for clonogenic cell survival in the radiosensitive SCC61 and the radioresistant SQ20B cell lines after X-ray or carbon ion irradiation are shown in Fig.

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. The urinary 8-OHdG levels were measured by using a commercial ELISA kit. No significant difference was found between the patient and control groups for GSH level, GPx activity, and basal and H2O2-induced DNA damage.

Post-repair DNA damage was found to be higher in the patient group than those in the control group. Urinary 8-OHdG level was lower in the patient group compared to the control group. In the control group, GSH level and post-repair DNA damage were higher in the vaccinated individuals.

In conclusion, oxidative stress formed due to the immune response against SARS-COV-2 may impair DNA repair mechanisms. Defective DNA repair may be an underlying pathological mechanism of post-COVID conditions.

Cell Sci. M Jacob, Aspects of the biological redox chemistry of cysteine: From simple redox responses to sophisticated signalling pathways, Biol. M Dafre, Protein S-thiolation and regulation of microsomal glutathione transferase activity by the glutathione redox couple, Arch.

M Holmgren, Antioxidant function of thioredoxin and glutaredoxin systems, Antioxid. Redox Signal. Cancer Suppl. Cell Biochem. M Lillig, Short interfering RNA-mediated silencing of glutaredoxin 2 increases the sensitivity of HeLa cells towards doxorubicin and phenylarsine oxide, Proc.

Crossref citations: Protection of radiation induced DNA damage by a newly developed molybdenum complex Md. Crossref citations: 1. Influence of reduced glutathione on end-joining of DNA double-strand breaks: Cytogenetical and molecular approach Nitin Ghoshal, Sheetal Sharma, Atanu Banerjee, Sillarine Kurkalang, Sathees C.

Crossref citations: 6. Crossref citations: 9.

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