NADPH oxidase DUOX1 promotes long-term persistence of oxidative stress after an exposure to irradiation

Significance

Increasing evidence supports the role of chronic oxidative stress in late radiation-induced effects, including malignancy and genetic instability. To date, elevated levels of reactive oxygen species (ROS) have been considered a cause of persistent instability, but until now the mechanism(s) underlying the perpetuation of ROS generation in irradiated cells and their progeny was undetermined. Cells can produce ROS through activation and/or induction of NADPH oxidases. The present investigation identifies the DUOX1-based NADPH oxidase as a ROS-generating system induced after irradiation, causing delayed DNA breakage. Overexpression of DUOX1 in radio-induced thyroid tumors suggests that DUOX1 may contribute to a chronic oxidative stress promoting genomic instability and tumorigenesis.

Abstract

Ionizing radiation (IR) causes not only acute tissue damage, but also late effects in several cell generations after the initial exposure. The thyroid gland is one of the most sensitive organs to the carcinogenic effects of IR, and we have recently highlighted that an oxidative stress is responsible for the chromosomal rearrangements found in radio-induced papillary thyroid carcinoma. Using both a human thyroid cell line and primary thyrocytes, we investigated the mechanism by which IR induces the generation of reactive oxygen species (ROS) several days after irradiation. We focused on NADPH oxidases, which are specialized ROS-generating enzymes known as NOX/DUOX. Our results show that IR induces delayed NADPH oxidase DUOX1-dependent H₂O₂ production in a dose-dependent manner, which is sustained for several days. We report that p38 MAPK, activated after IR, increased DUOX1 via IL-13 expression, leading to persistent DNA damage and growth arrest. Pretreatment of cells with catalase, a scavenger of H₂O₂, or DUOX1 down-regulation by siRNA abrogated IR-induced DNA damage. Analysis of human thyroid tissues showed that DUOX1 is elevated not only in human radio-induced thyroid tumors, but also in sporadic thyroid tumors. Taken together, our data reveal a key role of DUOX1-dependent H₂O₂ production in long-term persistent radio-induced DNA damage. Our data also show that DUOX1-dependent H₂O₂ production, which induces DNA double-strand breaks, can cause genomic instability and promote the generation of neoplastic cells through its mutagenic effect.

Ionizing radiation (IR) can cause various delayed effects in cells, including genomic instability that leads to the accumulation of gene mutations and chromosomal rearrangements, which are thought to play a pivotal role in radiation-induced carcinogenesis. The persistence of such effects in progeny cells has profound implications for long-term health risks, including emergence of a second malignancy after radiotherapy (1). The thyroid gland is one of the most sensitive organs to the carcinogenetic effects of IR. The risk of thyroid tumors is maximal for exposure at a younger age and increases linearly with radiation dose (2). More than 90% of these cancers are papillary, presenting a RET/PTC chromosomal rearrangement in 70% of cases. Thus, the thyroid can serve as a paradigm for analyzing the long-term delayed effects of IR.

The mechanism by which radiation exposure is memorized and leads to delayed DNA breakage remains to be determined. Hypoxia and antioxidant therapy reduce the X-ray–induced delayed effects, suggesting that radio-induced oxidative stress plays a significant role in determining the susceptibility of irradiated cells to genetic instability (3–5). We recently showed that H₂O₂ is able to cause RET/PTC1 rearrangement in thyroid cells, indicating that oxidative stress could be responsible for the RET/PTC rearrangement frequently found in radiation-induced thyroid tumors (6).

Cells can produce ROS through activation and/or induction of NADPH oxidases, which constitute a family of enzymes known as NOX/DUOX (7). Unlike other oxidoreductases, NADPH oxidases are “professional” ROS producers, whereas the other enzymes produce ROS only as by-products along with their specific catalytic pathways. ROS produced by NOXs participate in the regulation of many cell functions and have been implicated in various pathological conditions, including the late side effects induced by IR and chemotherapy (8–10). Thyroid cells express three of these NADPH oxidases, including two H₂O₂-generating systems located at the apical plasma membrane of the thyroid cells: DUOX2, which is implicated in thyroid hormone biosynthesis, and DUOX1, whose role in the thyroid is still unknown (11, 12). Furthermore, recently NOX4 was found to be expressed inside these cells (13).

Because ROS may contribute to the late effects observed after radiation exposure, we hypothesized that IR induces a delayed oxidative stress in thyroid cells via the activation and/or induction of NADPH oxidase. In the present study, we demonstrate that DUOX1 expression, induced via the IL-13 pathway in response to IR, is the primary source of sustained ROS production that causes persistent DNA damage. We show that p38 MAPK activation is required for the increased radio-induced DUOX1 expression. Finally, our analysis of human thyroid tissues shows that DUOX1 is overexpressed in both radio-induced and sporadic tumors, suggesting that radiation exposure by inducing DUOX1-based oxidative stress might favor a neoplastic process that can occur naturally. Our findings assign the NADPH oxidase DUOX1 a previously unidentified role in radio-induced genetic instability.

Results

Radiation Exposure Induces Chronic DUOX1-Dependent H₂O₂ Production in Human Thyroid Cells.

The concentration of extracellular H₂O₂ produced by thyroid cells (HThy-ori) after γ-ray irradiation at 10 Gy increased from day 3 up to day 4, and then remained stable until day 7 (Fig. 1A). DUOX1 protein level also increased from day 1 to day 10 after irradiation (Fig. 1B). Strikingly, irradiation (10 Gy) of HThy-ori cells preferentially resulted in the up-regulation of DUOX1 mRNA level (1- to 14-fold) compared not only with levels of NOX4 and DUOX2 mRNA, two NADPH oxidases expressed in the normal thyroid gland (Fig. 1C), but also with the other NOXs (Table S1). The increase in DUOX1 mRNA level was dose-dependent (Fig. S1B).

Fig. 1.

Radiation increases oxidative stress and DUOX1 expression in the HThy-ori3.1 cell line. (A) Radiation exposure-induced extracellular H₂O₂ production at the indicated days. (B) Time induction of DUOX1 protein from whole-cell lysates of irradiated cells analyzed by Western blot analysis. Vinculin served as a loading control. (C) Comparative expression of DUOX1, DUOX2, and NOX4 genes in irradiated HThy-ori cells, analyzed by real-time qRT-PCR. (D) (Upper) Effect of siDUOX1 vs. siControl on extracellular H₂O₂ production activity measured at day 4 post-IR. (Lower) DUOX1 protein expression of the corresponding experiment from particulate fractions. Values are mean ± SE. **P < 0.01.

DUOX1 needs the maturation factor DUOXA1 to exit the endoplasmic reticulum and be active on cell surface. The DUOX1/DUOXA1 genes are aligned head-to-head in a compressed genomic locus on chromosome 15, suggesting that expression of DUOX1 oxidase and its maturation factor are coordinated by a common bidirectional promoter (14). Several alternative splicing variants of DUOXA1 mRNA have been identified, and the lack of coding exon 6 has been shown to generate inactive forms of DUOXA1 (15).

We designed an oligonucleotide primer set in the DUOXA1 mRNA region containing exon 6. Real time quantitative RT-PCR (qRT-PCR) analysis performed at 4 d after a 10-Gy exposure of HThy-ori cells showed that a spliced variant of DUOXA1 mRNA encoding an active form was selectively increased in this condition. This mRNA variant was up-regulated in a dose-dependent manner (Fig. S1C). Knocking down DUOX1 with specific siRNA reduced the level of H₂O₂ produced by irradiated cells (Fig. 1D), indicating that irradiation induces chronic H₂O₂ production via DUOX1 up-regulation. Irradiation induced an increase in cytosolic [Ca²⁺] in thyroid cells at day 4, consistent with activation of the calcium-dependent H₂O₂-generating activity of DUOX1 (16) (Fig. S1D).

Radiation Induces IL-13 in Human Thyroid Cells.

To identify the molecular mechanism underlying chronic DUOX1 expression after irradiation, we analyzed the gene expression profile of immune-related genes in HThy-ori cells at different intervals after γ-ray exposure at 10 Gy. Among the cytokine genes analyzed, IL-1β, IL-13, IL-6, IL-8, and TNF-α were found to be up-regulated after irradiation in a time-dependent manner (Fig. S1 E and F). The induction of IL-13 mRNA was correlated with an increased level of IL-13 protein in HThy-ori cells assayed by Western blot analysis at days 4 and 7 after irradiation (Fig. 2A). A neutralizing IL-13 monoclonal antibody (clone 32116) that blocks the binding of IL-13 to its receptor abrogated radiation-induced H₂O₂ production (Fig. 2B). In addition, IL-13 down-regulation by RNA interference resulted in a significant reduction of DUOX1 mRNA level (Fig. S1G). Our data indicate that IL-13 regulates the radiation-induced increase in DUOX1 expression.

Fig. 2.

IL-13 and p38 MAPK regulate radiation-induced DUOX1 expression. (A) Immunoblot detection of IL-13 protein in nonirradiated and irradiated HThy-ori3.1 cells at days 4 and 7. (B) HThy-ori3.1 cells were incubated with increasing concentrations of IL-13–neutralizing antibody from day 2 after γ-irradiation (10 Gy), and the H₂O₂-generating activity was measured at day 4. (C) Increase in p38 MAPK phosphorylation after irradiation. Cells were irradiated, and whole-cell lysates were collected at the indicated days. Downstream target of p38 MAPK (Hsp27) was also analyzed by Western blot analysis. (D) p38 MAPK knockdown with interference RNA decrease the level of DUOX1 mRNA expression in irradiated HThy-ori3.1 cells at day 4, as analyzed by real-time qRT-PCR. (E) Persistent DNA damage foci. HThy-ori3.1 cells were untreated or irradiated (10 Gy), then fixed and stained for 53BP1 (green), γH2AX (red), and DNA (DAPI; blue) at day 4. (F) Time course analysis of γH2AX in HThy-ori3.1 cells untreated or irradiated (10 Gy). Whole-cell lysates were collected at the indicated days thereafter. Vinculin served as a loading control. Values are mean ± SE. *P < 0.05; **P < 0.01; ***P < 0.001.

p38 MAPK Regulates DUOX1 Expression.

To define the upstream mechanisms that regulate IL-13–induced DUOX1 expression in irradiated cells, we tested the effect of pharmacologic inhibitors of NF-KB and canonical mitogen-associated protein kinase (MAPK) pathways. Of these inhibitors, only SB203580 (SB), which specifically inhibits p38 MAPK, attenuated the induction of DUOX1 mRNA expression at day 4 after irradiation (Fig. S1H).

Because these data supported a role for p38 MAPK in radiation-induced DUOX1 expression, we analyzed the time course levels of total and phosphorylated p38 MAPK and its downstream target HSP27 after radiation exposure (10 Gy) (17). Western blot analysis showed that p38 MAPK and HSP27 phosphorylation rose substantially at 24 h and remained elevated until day 10 (Fig. 2C). Importantly, the kinetics of p38 MAPK activation closely paralleled changes in DUOX1 expression. Knocking down p38 MAPK with specific siRNA affected radiation-induced DUOX1 mRNA expression (Fig. 2D). Treatment of thyroid cells with the p38 MAPK inhibitor SB decreased radio-induced H₂O₂ production (Fig. S2A), and p38 MAPK down-regulation by RNA interference also affected the increased expression of IL-13 protein at day 4 (Fig. S2B). Taken together, these data indicate that p38 MAPK activation plays a key role in radiation-induced up-regulation of DUOX1 via IL-13.

DUOX1 Is Involved in Radio-Induced DNA Damage.

A high radiation dose generates persistent DNA damage foci, leading to prolonged DNA damage response activation for several days (18). One of the first proteins to respond to DNA double-strand breaks (DSBs) is Ataxia Telangiectasia Mutated (ATM), a member of the phosphoinosityl-3 kinase-like kinase (PIKK) family. ATM substrates include H2AX, a nucleosomal histone variant, and p53-binding protein 1 (53BP1). At day 4 postirradiation, phosphorylated form of H2AX (γH2AX) and 53BP1 localized to DSBs, forming characteristic foci (Fig. 2E). Immunoblot analysis of the time-dependent stimulation of γH2AX in nonirradiated and irradiated cells showed that it increased starting at day 2 after irradiation (Fig. 2F).

ATM also phosphorylates the DDR kinase checkpoint kinase 2 (Chk2), which promotes growth arrest. Kinetic analysis of Chk2 phosphorylation after 10-Gy irradiation showed an increase at 1 d after irradiation that was sustained for 10 d (Fig. S2C). We performed a cell cycle profile analysis of HThy-ori cells after irradiation. As shown in Fig. 3A, irradiated cells exhibited an increased percentage of cells in G2/M phase, indicating a G2/M arrest. Many cells showed aberrantly enhanced DNA content (>4n), likely reflecting partial re-replication of the genome uncoupled from cell division. This was accompanied by up-regulation of p21, a cell-cycle inhibitor (Fig. S2D).

Fig. 3.

DUOX1 is involved in delayed radio-induced DNA damage. (A) Cell cycle phase distribution in untreated and irradiated (10 Gy) cells analyzed at day 4. The percentage of cells was determined by flow cytometry. Values are mean ± SE from three independent measurements. (B) Confocal analysis of H₂O₂-dependent fluorescence in HThy-ori3.1 cells expressing pHyper-nuc at 4 d after irradiation. (C) Redox Western blot of nuc-rxYFP. (D) Immunoblot for γH2AX in untreated and irradiated (10 Gy) cells. The cells were transfected with different siRNAs against DUOX1, IL-13, p38 MAPK, or ATM 2 d before being collected at day 4 for analysis. Catalase (250 U/mL) was added in the cell medium 2 d before cells were collected for Western blot analysis. γH2AX was quantified by densitometry. Values are mean ± SE. *P < 0.05; **P < 0.01; ***P < 0.001.

A genetically encoded highly specific fluorescent probe has been developed for detecting H₂O₂ inside living cells (19). This biosensor, known as Hyper, has been shown to have submicromolar affinity to H₂O₂ and to be insensitive to other oxidants. Using the mammalian expression vector encoding nuclear-targeted Hyper, we observed changes in the fluorescence of Hyper in nuclei at day 4 postirradiation (Fig. 3B). In addition, we targeted a yellow fluorescent protein-based redox sensor (rxYFP) to the nucleus of thyroid cells and monitored the nuclear redox changes in response to H₂O₂ by analyzing both reduced and oxidized forms of rxYFP after 30 min. In the presence of 12.5 µM H₂O₂, which is the dose of H₂O₂ produced by DUOX1 (Fig. 1D), nucleus-rxYFP became oxidized, confirming changes in the nuclear redox environment in response to extracellular H₂O₂ (Fig. 3C).

Exogenous H₂O₂ induces DNA damage in thyroid cells (6, 20). To establish a link between H₂O₂-generating NADPH oxidase DUOX1 activation and DNA damage observed several days after irradiation, we performed RNA interference experiments (Fig. 3D and Fig. S2E). ATM depletion prevented phosphorylation of its substrate H2AX. Knockdown of DUOX1, DUOXA1, IL-13, or p38 MAPK significantly reduced the level of γH2AX and the number of 53BP1 nuclear foci (Fig. S2F). Inhibition of both p38 and IL-13 affected IL-13 expression more significantly, providing better protection against DNA damage (Fig. S3A). In contrast, depletion of p22^phox, the NOX functional partner (except for NOX5), had no effect on the expression of γH2AX and phospho-p38 MAPK (Fig. S3B). Finally, treatment of cells with catalase, a scavenger of H₂O₂, protected DNA from DSBs induced at postirradiation (Fig. 3D). Taken together, these data indicate involvement of DUOX1-dependent H₂O₂ generation in delayed radio-induced DNA damage. Overexpression of both DUOX1 and DUOXA1 in thyroid cells at a level producing extracellular concentration of H₂O₂ comparable to that measured at postirradiation also increased expression of γH2AX (Fig. S3C).

Extracellular H₂O₂ Reproduces the Effect of Irradiation on DUOX1.

Preincubation of HThy-ori cells for 4 h before irradiation or treatment of cells with catalase at days 2 and 3 postirradiation significantly decreased the level of γH2AX analyzed at day 4 postirradiation (Fig. 4A). This was related to a significant decrease in DUOX1 mRNA level (Fig. 4B). Conversely, treatment of cells with H₂O₂ induced a dose-dependent increase of DUOX1 expression at day 4 after treatment, which was associated with increases in both H2AX and p38 MAPK phosphorylation (Fig. 4C).

Fig. 4.

H₂O₂ mediates the radiation effect. (A) Effect of pretreatment or treatment with 250 U/mL catalase on γH2AX expression analyzed at day 4 post-IR. (B) Decreased level of DUOX1 mRNA expression at day 4 after treatment of irradiated cells with catalase, as analyzed by real-time qRT-PCR. (C) Dose-dependent increases in DUOX1, γH2AX, and phospho-p38 MAPK expression treated with H₂O₂ and analyzed after 4 d.

DUOX1 Mediates Radiation-Induced H₂O₂ Production in Primary Human Thyrocytes.

After radiation exposure, primary human thyrocytes increased H₂O₂ production at day 4. This effect was independent of serum (Fig. S3D). DUOX1 mRNA was found to be selectively up-regulated after irradiation, and this was correlated with an increase in DUOX1 protein level (Fig. 5A, Fig. S3E, and Table S2). DUOX1 inactivation led to a significant reduction in radiation-induced H₂O₂ production (Fig. 5B). p38 MAPK and IL-13 were activated and up-regulated, respectively, several days after 10-Gy irradiation, and their depletion also affected the radio-induced expression of DUOX1 (Fig. 5 C and D).

Fig. 5.

DUOX1 is involved in radio-induced H₂O₂ production in human primary thyrocytes. (A) Time course analysis of DUOX1 protein expression in irradiated human thyrocytes. Nonirradiated cells served as a negative control. Vinculin served as a loading control. (B) Effect of siDUOX1 vs. siControl on the extracellular H₂O₂ production activity of untreated and irradiated thyrocytes. DUOX1 protein expression of the corresponding experiment is shown. (C) Immunoblot for IL-13 and for p38 MAPK phosphorylation in untreated and irradiated (10 Gy) thyrocytes. Whole-cell lysates were collected at the indicated days after irradiation. (D) Immunoblot for DUOX1 in untreated and irradiated (10 Gy) cells. The cells were transfected with different siRNAs against DUOX1, IL-13, p38 MAPK, or SB (p38 MAPK inhibitor). Catalase (250 U/mL) was added in the cell medium 2 d before cells were collected for Western blot analysis. Protein expression was quantified by densitometry. The mean ± SE value from three independent measurements is reported. (E) Induction of p21 at day 4 (10 Gy) in human thyrocytes analyzed by Western blot analysis. β-actin served as a loading control. Values are mean ± SE. *P < 0.05; **P < 0.01; ***P < 0.001.

p38 MAPK down-regulation by RNA interference was able to counteract the selective up-regulation of DUOXA1 (+ exon 6) mRNA expression by irradiation (Fig. S4A). H₂O₂ reproduced the effect of irradiation on DUOX1 expression in human thyrocytes (Fig. S4B), and its degradation by the catalase prevented up-regulation of DUOX1 (Fig. 5D). Interestingly, DUOX1 inactivation itself affected the level of p38 MAPK phosphorylation analyzed at day 7 postirradiation, indicating that DUOX1 contributes to long-term maintenance of the radiation-induced effect (Fig. S4C). The DNA damage response was activated in human thyrocytes at day 4. γH2AX and 53BP1 formed characteristic foci in the nucleus of irradiated cells, suggesting the presence of DNA DSBs (Fig. S4D). This was associated with cell growth arrest, as evidenced by activation of Chk2 via an increase in its phosphorylated form, increase in G2/M phase arrest, and up-regulation of p21 (Fig. 5E and Fig. S4 E and F). The DNA damage response was also activated when the thyrocytes were treated with H₂O₂ (Fig. S4G).

Immunofluorescence analysis showed that DUOX1 was expressed both at the plasma membrane and in the perinuclear compartment (Fig. 6A). A significant reduction in radio-induced DNA damage detected by γH2AX was observed after DUOX1 inactivation (Fig. 6B). This was confirmed by immunofluorescence analysis showing that DUOX1-inactivated cells displayed 50% reduction of DNA-damage foci (Fig. 6 C and D). Taken together, these data indicate that DUOX1 is involved in radio-induced DSBs in primary thyroid cells.

Fig. 6.

DUOX1 is involved in persistent radio-induced DNA damage in human thyrocytes. (A) Localization of membrane protein (lamin A/C; red) and DUOX1 (green) analyzed by immunofluorescence in untreated and irradiated (10 Gy) thyrocytes. The merged red and green channels show colocalization in yellow. Thyrocytes were transfected with siRNA against DUOX1 at days 2 and 4 post-IR and analyzed at day 7. (B) Affect of inhibition of DUOX1 on irradiation-induced γH2AX expression. DUOX1 and γH2AX were visualized by Western blot analysis in thyrocytes at day 7. (C and D) Quantification of the number of merged γH2AX/53BP1 foci in nuclei of untreated and irradiated thyrocytes at days 4 and 7. Thyrocytes were transfected with siRNA against DUOX1 as described above. Cells were fixed and stained for 53BP1 foci (green) and γH2AX foci (red) at days 4 and 7 post-IR.

DUOX1 Is Increased in Thyroid Cancers.

Human exposure to IR is a strong risk factor for the development of thyroid tumors. To investigate whether our findings in human thyrocytes are relevant to radio-induced human thyroid tumors, we analyzed the expression of DUOX1 and IL-13 in 20 thyroid tumor tissues from patients with a history of radiation exposure during childhood (Table S3). DUOX1 and IL-13 mRNA levels were measured in normal (n = 6) and tumor tissues by real-time qRT-PCR, and the DUOX1 gene expression level was significantly higher in radio-induced thyroid tumors than in normal thyroid tissues (Fig. 7). In sporadic thyroid tumors (Table S4), the increase in DUOX1 level was of borderline significance. Expression of both DUOX1 and IL-13 was detected by immunohistochemistry in normal thyrocytes, but clear overexpression of both proteins was observed in sporadic and radio-induced thyroid tumors (Fig. S5).

Fig. 7.

Comparative expression of DUOX1 (A) and IL-13 (B) genes in human thyroid tissues analyzed by real-time qRT-PCR. Data are expressed as mRNA relative expression levels, determined as x-fold of calibrator corresponding to a pool of thyroid tissue samples. RI, radio-induced thyroid cancers (n = 20; Table S3); SP, sporadic thyroid cancers (n = 9; Table S4); normal tissues, n = 6.

Discussion

A growing body of evidence appears to support the concept that chronic oxidative stress might drive the progression of radiation-induced late effects (4). A radiation-induced increase in ROS generation and/or an oxidative stress has been observed in vivo (21). Our results reveal that both DUOX1 and its maturation factor DUOXA1 are up-regulated several days after irradiation in human thyrocytes, supporting the role of DUOX1-based NADPH oxidase in a chronic oxidative stress. DUOX1 has been originally identified in the thyroid gland (12). Like its counterpart DUOX2, it was first characterized as active only at the apical cell surface of thyrocytes, where it produces H₂O₂ in the extracellular colloid space. Because exposure of thyroid cells to exogenous H₂O₂ can induce DNA breaks (20) and produce RET/PTC1 rearrangement (6), it was conceivable that extracellular H₂O₂ produced by DUOX could be implicated in DNA damage. Although H₂O₂ is relatively stable and has a high membrane-diffusible capacity, a paracrine effect of H₂O₂ most likely explains H₂O₂ accumulation in the nucleus (6) (Fig. 3B), which was recently shown to be associated with nuclear redox changes (22) (Fig. 3C).

The generation of DNA damage leads to the accumulation of characteristic foci of DNA damage response (DDR) factors. At low radiation doses, these foci disappear within hours, indicating the presence of repairable DNA lesions. In contrast, at higher radiation doses, a few clearly detectable DDR foci persist for many days, especially DNA DSBs (18). Importantly, siRNA-mediated abrogation of both DUOX1 expression and DUOXA1 expression resulted in a significant decrease in postirradiation DNA damage in thyroid cells (Figs. 3D and 6 B–D, and Fig. S2 E and F), identifying for the first time, to our knowledge, a key role of DUOX1-dependent H₂O₂ production in persistent radio-induced DNA damage and, consequently, in DDR signaling.

Although DNA damage can arise through the direct interaction of oxidants with genomic DNA, it also can be generated by oxidation of DNA precursors in the nucleotide pool (23) or by an imbalance in the dNTP pools caused by inhibition of enzymes involved in nucleotide synthesis by ROS (24). Chronic depletion or imbalance in the nucleotide pool inflicted by radio-induced H₂O₂ production may lead to replication stress with generation of DNA strand breaks and genomic instability (25). Thus, further studies are needed to determine how DUOX1 induces DNA damage.

Persistent changes precede the establishment of senescence-associated phenotypes, including growth arrest (26–28) and senescence-associated secretory phenotype (SASP), with potent autocrine and paracrine activities (18, 29). In the present study, persistent DNA damage foci in thyroid cells were associated with increased expression of cytokines, including IL-13. Importantly, this cytokine, which has been implicated in the development of late effects of radiation (30), mediates radio-induced DUOX1 expression in thyroid cells. Until recently, the Th2 cytokines IL-4 and IL-13 were considered to selectively up-regulate DUOX1 (31, 32); however, recent data show that human thyrocytes exposed to IL-4 or IL-13 present a selective up-regulation of DUOX2 and DUOXA2 genes (33), indicating that, depending on cell context, these cytokines regulate both DUOX genes.

In human fibroblasts, p38 MAPK induces the senescence growth arrest and cytokine secretion in response to X-ray exposure (34). In this case, p38 MAPK phosphorylation increased only slightly over the 24 h after X-ray irradiation (10 Gy) and reached a peak after several days. We also observed a delayed p38 MAPK response to irradiation in human thyroid cells, which controlled both IL-13 and DUOX1 expression (Fig. 5D and Fig. S2B). A feedback loop involving ROS in permanent growth arrest has been described previously (35). Knockdown of DUOX1 expression affected p38 activation, IL-13 expression, and DNA damage, confirming the involvement of DUOX1-derived H₂O₂ in the maintenance of DDR in irradiated thyroid cells (Figs. S2B and S4C). Catalase not only protected cells from the toxic effects of H₂O₂, but also suppressed radio-induced DUOX1 expression (Figs. 4 A and B and 5D). Conversely, H₂O₂ induced a delayed increase in DUOX1 expression (Fig. 4C), indicating that H₂O₂ produced during irradiation through water radiolysis may mediate part of the radiation effect.

Partially transformed and tumorigenic cells systematically and spontaneously emerge from senescent cultures (36). Senescence-associated ROS may be a cause of both senescence, through their deleterious effects, and of the emergence of pretumoral cells, through their mutagenicity. Thus, DNA-damaged thyroid cells resulting from radio-induced DUOX1-dependent H₂O₂ generation could emerge from senescence and propagate chromosome abnormalities and mutations that lead to tumorigenesis. This may explain why radio-induced thyroid cancers overexpress DUOX1.

In conclusion, our findings demonstrate that chronic H₂O₂ production in human thyroid cells in response to irradiation exposure is mediated by DUOX1 (Fig. S6). The p38 MAPK pathway is involved in IL-13–induced DUOX1 expression. Thus, DUOX1 as a major source of radio-induced H₂O₂ may cause substantial DNA damage in progeny of irradiated cells and their neighboring bystanders and, consequently, participate in the initiation and development of thyroid tumors. Therefore, DUOX1 might constitute a potential target for specific inhibitors to mitigate the side effects of radiotherapy.

Materials and Methods

Cell Culture and Treatments with Inhibitors and Cytokines.

HThy-ori cells and primary human thyroid cells were cultured as described previously (13); details are provided in SI Materials and Methods. At 2 d after irradiation, thyroid cells were treated for 24 h with different doses of inhibitors. Inhibitors of the MAPK pathway (U0126, SB, InSolution JNK inhibitor II) and NFκB pathway (IKK Inhibitor II) were purchased from Calbiochem.

In Vitro Irradiation.

Exponentially growing HThy-ori cells were plated at 24 h before irradiation at 2.5 × 10⁵/well in six-well plates in 10% (vol/vol) serum-containing medium. Immediately before irradiation, cell culture medium was replaced by fresh culture medium, and cells were exposed to a single dose of γ-irradiation from a generator operating at 200 KV and 15 mA at a dose rate of 3 Gy/min. At 24 h after irradiation, cells were cultured in 3% (vol/vol) serum-containing medium at different times before being harvested. Human primary thyrocytes were first plated in a 75-cm² flask. When cells reached 70% of confluence, culture medium was replaced by 0.2% FCS culture medium without TSH and insulin. One week later, thyrocytes were plated at 2.5 × 10⁵/well in six-well plates in the same medium. After 24 h, cells were irradiated as described above and harvested at different times.

ROS Detection.

Extracellular H₂O₂ generation was quantified by the Amplex Red/HRP assay (Molecular Probes, Invitrogen), which detects the accumulation of a fluorescent oxidized product. The H₂O₂ released was quantified (nmol H₂O₂/h/10⁵ cells) using standard calibration curves. For nuclear ROS detection, pHyPer-transfected cells were plated onto glass-bottom dishes, and the fluorescence (excitation/emission: 488/530 nm) was examined at day 4 post-IR under a Zeiss LSM 510 confocal microscope.

Western Blot Analysis.

Particulate cell fractions were solubilized in 20 mM Tris⋅HCl pH 7.8 containing 135 mM NaCl, 1 mM MgCl₂, 2.7 mM KCl, 1% Triton X100, 10% (vol/vol) glycerol, and a mixture of protease and phosphatase inhibitors (Calbiochem). Immunodetection was performed as described previously (13) using as primary antibody either anti-DUOX (1/1,000) (12) or a rabbit polyclonal anti-DUOX1 antiserum (1/1,000) raised against residues 988–1011 (Eurogentec) that does not detect DUOX2 (Fig. S1A). Immune complexes were detected with an AP-coupled anti-rabbit IgG antibody (1/4,500; Promega) or HRP-coupled anti-rabbit antibody (1/15,000; Southern Biotech).

For total cell lysates, cells were solubilized in 100 mM Tris⋅HCl pH 7.0 containing 2.5% (wt/vol) SDS, 1 mM EDTA, 1 mM EGTA, 4 M urea, and a mixture of phosphatase and protease inhibitors (Calbiochem). Primary antibodies were p-p38 MAPK Thr18 0/tyr182 XP rabbit, p38αMAP kinase, p-Chk2 (Thr-68), ChK2, p-HSP27 (ser 82), HSP27, p-ATM (ser1981), and ATM (all from Cell Signaling Technology); γH2AX Ser-139 mouse and IL-13 (both from Millipore); H2AX and Vinculin (both from Abcam); p21 (sc-397; Santa Cruz Biotechnology); and actin (Sigma-Aldrich).

Transfection of siRNAs.

Cells were transfected at 60% confluence at 2 d after radiation exposure with siRNA against DUOX1 (stealth RNAi duplex HSS182411), scrambled siRNA control (Invitrogen), or siRNA against IL-13, p38 MAPK, and ATM (smart pool, from Dharmacon) using INTERFERIN transfection reagent (Polyplus-Transfection) according to the manufacturer’s protocol. Cells harvested on day 7 after irradiation were transfected twice, at day 2 and day 4.

Tissue Samples and Immunohistochemistry.

Thirty-five frozen tissue specimens were obtained from the Institut Gustave Roussy (Tables S3 and S4). Histopathological diagnosis was performed according to World Health Organization guidelines. The series comprises 20 radio-induced thyroid tumors, 9 sporadic tumors, and 6 normal tissues. Sporadic tumors were matched by histology and TNM classification. Immunohistochemistry is described in SI Materials and Methods.

Statistical Analysis.

Statistical analyses were performed using GraphPad Instat software for ANOVA and the Student t test, with the level of significance set at P < 0.05.