Exogenous 8-hydroxydeoxyguanosine attenuates doxorubicin-induced cardiotoxicity by decreasing pyroptosis in H9c2 cardiomyocytes

Doxorubicin (DOX), which is widely used in cancer treatment, can induce cardiomyopathy. One of the main mechanisms whereby DOX induces cardiotoxicity involves pyroptosis through the NLR family pyrin domain containing 3 (NLRP3) inflammasome and gasdermin D (GSDMD). Increased NAPDH oxidase (NOX) and oxidative stress trigger pyroptosis. Exogenous 8-hydroxydeoxyguanosine (8-OHdG) decreases reactive oxygen species (ROS) production by inactivating NOX. Here, we examined whether 8-OHdG treatment can attenuate DOX-induced pyroptosis in H9c2 cardiomyocytes. Exposure to DOX increased the peroxidative glutathione redox status and NOX1/2/4, toll-like receptor (TLR)2/4, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression, while an additional 8-OHdG treatment attenuated these effects. Furthermore, DOX induced higher expression of NLRP3 inflammasome components, including NLRP3, apoptosis-associated speck-like protein containing a c-terminal caspase recruitment domain (ASC), and pro-caspase-1. Moreover, it increased caspase-1 activity, a marker of pyroptosis, and interleukin (IL)-1β expression. All these effects were attenuated by 8-OHdG treatment. In addition, the expression of the cardiotoxicity markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was increased by DOX, whereas the increase of ANP and BNP induced by DOX treatment was reversed by 8-OHdG. In conclusion, exogenous 8-OHdG attenuated DOX-induced pyroptosis by decreasing the expression of NOX1/2/3, TLR2/4, and NF-κB. Thus, 8-OHdG may attenuate DOX-induced cardiotoxicity through the inhibition of pyroptosis. Supplementary Information The online version contains supplementary material available at 10.1186/s12860-022-00454-1.


Introduction
Doxorubicin (DOX) is widely used as a treatment for numerous cancers, such as breast cancer, soft tissue sarcomas, lymphomas, and leukemia. Despite its effectiveness, DOX can induce serious complications such as cumulative cardiotoxicity [1,2]. It has been known that DOX-induced cardiotoxicity is mainly caused by cumulative effect, thus it has been recommended that accumulation doses should not exceed 500 mg/m 2 [3]. However, resent studies showed that Dox-induced cardiotoxicity could develop even by single administration of DOX [4,5]. DOX-induced cardiotoxicity leads to irreversible myocardial injury or congestive heart failure [6]. The pathophysiology of DOX-induced cardiotoxicity has not been fully revealed; however, oxidative stress, apoptosis, inflammation, and impaired regulation of autophagy may be involved in its development [7][8][9][10]. Moreover, NLR family pyrin domain containing 3 (NLRP3) inflammasome formation, which leads to gasdermin D (GSDMD)dependent pyroptosis, has recently been identified as one of the main mechanisms whereby DOX induces cardiotoxicity [11].
Tumor necrosis factor (TNF)-α expression is increased in DOX-induced cardiotoxicity [26,27], and this is also known to lead to pyroptosis [28]. Toll-like receptors (TLRs) are highly expressed in cardiomyocytes and related to DOX-induced cardiotoxicity [28]. In DOXinduced cardiotoxicity, the activation of TLR-4 and TLR-2 leads to the upregulation of nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB), which results in increased expression of various pro-inflammatory cytokines, including TNF-α and IL-6 [29,30]. TLR/ NF-κB signaling increases NLRP3 binding to ASC [31]. Furthermore, increased oxidative stress is involved in DOX-induced cardiotoxicity [32]. Oxidative stress is the result of both an increased production of reactive oxygen species (ROS) and a decreased production of endogenous antioxidants such as catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH) in the cellular system [32,33]. NADPH oxidase (NOX)1 and NOX4 are also involved in the activation of the NLRP3 inflammasome through the increase in ROS production during DOX-induced cardiotoxicity [34]. NOX2 is also involved in the activation of the NLRP3 inflammasome in brain injuries [35]. In addition, TLR4 promotes NOX4-mediated ROS production [36], and TLR2 stimulates NOX1 and NOX2 to generate ROS [28,37]. Increased expression of TLR2 also lead to pyroptosis via TLR2/Myd88/ NF-κB pathway [38].
Here, we examined whether exogenous 8-OHdG can attenuate DOX-induced pyroptosis in H9c2 cardiomyocytes. We hypothesized that treatment with exogenous 8-OHdG would lead to the downregulation of NOX, and NF-κB expression, and decreased TNF-α production by decreasing Rac1, eventually leading to the attenuation of DOX-induced pyroptosis.

Glutathione assay
Cells were plated in 100 mm dishes and treated with DOX and 8-OHdG for 24 h. A glutathione assay kit (Abcam, Cambridge, UK) was used as per the manufacturer's instructions. Briefly, cell suspension from lysed 10 6 cells using lysis buffer was incubated in medium containing thiol or GSSG probe at room temperature for 10-60 m. Then, we measured concentrations of GSH and total glutathione (GSH + GSSG) at Ex/Em = 490/520 nm using a fluorescence microplate reader (Thermo Scientific, Waltham, MA, USA). The GSH/GSSG ratio was calculated from concentrations of GSH and GSSG.

Statistical analysis
All experiments were repeated more than three times, and the average values are presented unless otherwise stated. Data are presented as mean ± standard deviation. The statistical significance of the results was determined with Prism ® software (GraphPad, San Diego, CA, USA) using one-way ANOVA and post-hoc Dunnett's test.
The results showed that cell viability gradually decreased as the concentration of DOX increased (Fig. 1A). The IC 50 values of DOX over the study period were 20.6 μM (24 h), 0.4778 μM (48 h), and 0.02895 μM (72 h). After 24 h culture, the viability of 8-OHdGtreated H2c9 cells did not significantly differ from that of the control when the 8-OHdG concentration ranged from 0 to 250 μg/mL; however, cell viability was significantly decreased when the concentration was ≥ 500 μg/ mL (Fig. 1B). After 48 and 72 h culture, the viability of 8-OHdG-treated H2c9 cells did not significantly differ from that of the control when the 8-OHdG concentration ranged from 0 to 50 μg/mL (Fig. 1B). The IC 50 values of 8-OHdG were 699.4 μg/mL (24 h), 595.5 μg/mL (48 h), and 544.4 μg/mL (72 h). To evaluate the effect on pyroptosis, we used 1 μM DOX as in previous reports [41]. In addition, we examined cell proliferation and cytotoxicity in 1 μM DOX-treated cells by treating them with various concentrations (50, 100, 250, and 500 μg/mL) of 8-OHdG. 50, 100, and 250 μg/mL of 8-OHdG augmented the proliferation of DOX-treated H9c2 cells and conversely alleviated the cytotoxicity of these cells (Fig. 1C  and D). Therefore, we treated these H9c2 cells with 1 μM of DOX and 100 and 250 μg/mL of 8-OHdG for following investigation.

Exogenous 8-OHdG decreases the expression of cardiotoxicity-related markers in DOX-treated H9c2 cells
Cytotoxicity of DOX-treated H9c2 cells was reduced by 8-OHdG treatment (Fig. 1). Thus, we tested whether exogenous 8-OHdG affects the expression of cardiac hypertrophy markers, ANP and BNP in DOX-treated H9c2 cells. DOX increased ANP and BNP mRNA levels by 258-479% and 337-428%, respectively, compared to  attenuate DOX-induced cardiotoxicity in H9c2 cells, and that GATA6 may be involved.

Exogenous 8-OHdG decreases NOX1/2/4 expression and NF-κB phosphorylation, and increases the GSH/GSSG ratio in DOX-treated H9c2 cells
Given that 8-OHdG is involved in oxidative stress via Rac1, we examined the regulatory effects of DOX and 8-OHdG on expression of NOX1/2/4 and the Rac1 activity in H9c2 cells. We performed the Rac1 activation assay using immunoprecipitation and found that 250 μg/mL 8-OHdG decreased the rat Rac1 activity in DOX-exposed H9c2 cells (Fig. 3A). Additionally, when cells were treated with both DOX and 8-OHdG, the protein levels of NOX1, 2, and 4 were decreased compared to DOXtreated cells (Fig. 3B). However, 8-OHdG suppressed the expression of NOX2 and NOX4 more than that of NOX1 in DOX-treated cells. In addition, exogenous 8-OHdG repressed the phosphorylation of p65 compared to the levels in DOX-treated H9c2 cells (Fig. 3C). These findings suggest that 8-OHdG may mitigate DOX-induced NOX1/2/4 upregulation through the inactivation of p65 and Rac1.
Oxidative stress is the result of both the increased production of ROS and decreased content of endogenous antioxidants such as CAT, SOD, and GSH in the cellular system [32,33]. Thus, we examined whether 8-OHdG treatment affects the GSH/GSSG ratio in DOX-treated H9c2 cells. Exogenous 8-OHdG recovered the ratio of GSH/GSSG by 13.8-389% in DOX-treated H92C cells (Fig. 3D). Collectively, the results demonstrated that 8-OHdG treatment decreases NOX1/2/4 expression and increases the GSH/GSSG ratio in DOX-treated H9c2 cells, suggesting that 8-OHdG may alleviate DOXinduced oxidative stress in cardiomyocytes.
To evaluate whether 8-OHdG decreased NOX1/2/4 and TLR2/4 similar to Rac1 siRNA, H9c2 were transfected with siRNAs for negative control and two kinds of rat Rac1, and then treated with DOX and/or 8-OHdG for 24 h. The inhibitory effects of 8-OHdG on the regulation of NOX1/2/4, and TLR2/4 in DOX-exposed H9c2 cells were similar to that of two kinds of Rac1 siRNA (Suppl. Fig. 1).
Induced NLRP3 expression (55-70%) by DOX was reduced by 54.8-91.2% in additional treatment of 8-OHdG (Fig. 4A). In addition, DOX increased the protein levels of ASC and caspase-1 compared to those in the control group, whereas 8-OHdG treatment decreased ASC and active caspase-1 levels compared to those in DOX-treated cells (Fig. 4B). Collectively, these findings suggest that 8-OHdG may be effective in suppressing DOX-induced inflammasome activation in cardiomyocytes.

Discussion
Cardiotoxicity is the most serious complication after chemotherapy, which leads to a reduction in left ventricular ejection fraction (LVEF) of more than 10% to 50% [42]. DOX-induced cardiotoxicity is dose-dependent and occurs in 3 to 18% of patients [43]. Furthermore, DOX treatment leads to congestive heart failure, which is the most severe form of cardiotoxicity, in around 5% of patients [44].
The prognosis of DOX-induced congestive heart failure is very poor [45]. To prevent DOX-induced cardiotoxicity, regular monitoring of LVEF is recommended, and cessation of chemotherapy is also recommended when LVEF drops below 40% [46]. Currently, prophylactic or curative drugs for treating DOX-induced cardiotoxicity are sparse [47,48]. Several standard heart failure medications including renin-angiotensin system blockers or beta blockers have been used; however, they are not very effective in the treatment or prevention of DOX-induced cardiotoxicity [47,48]. Therefore, other targeted cardioprotective therapies or prophylactic treatments for DOX-induced cardiotoxicity should be developed [49].
The NLRP3 inflammasome has been shown to be involved in DOX-induced cardiotoxicity. DOX treatment increases NLRP3 inflammasome and IL-1β secretion in the myocardium of mice [50]. Increased pyroptosis is also involved in DOX-induced cardiotoxicity in both in vitro and in vivo models [51]. Moreover, the inhibition of the NLRP3 inflammatory response leads to decreased DOX-induced cardiotoxicity [52]. Thus, decreasing NLRP3 inflammasome or pyroptosis could be promising therapeutic methods to prevent or treat DOX-induced cardiotoxicity [53]. Increased ROS or NOX activity is known to increase NLRP3 inflammasome [34,35]. Exogenous 8-OHdG leads to decreasing production of ROS and NOX activity via Rac1 downregulation [39]. Thus, we evaluated whether exogenous 8-OHdG decreases NLRP3 inflammasome-related inflammation and pyroptosis in DOXtreated cardiomyocytes by Rac1 inhibition. H9c2 cells are subclonal line which derived from embryonic rat heart tissue and have similarities with differentiated adult cardiac cells [54,55], but H9c2 cells do not have ability of contraction [55]. However, H9c2 is most extensively used during preclinical studies of anticancer drug development such as evaluation of cardiotoxicity and safety [56]. Particularly, DOX-treated H9c2 model could mimic relevant mechanisms of DOX induced cardiac injuries such as oxidative stress, apoptosis, sarcoplasmic reticulum stress, and cell death [33,57,58]. Thus, we thought that H9c2 would be proper cell line to evaluated DOXinduced pyroptosis.
In our study, 8-OHdG decreased Rac1 activity in DOXtreated H9c2 cells ( Fig. 2A). Previous study showed that DOX induced Rac1 medicated NOX activation [59]. NOX1/2/4 expression was significantly increased by DOX in H9c2 cells, and was significantly decreased by additional treatment with 8-OHdG. Furthermore, we evaluated oxidative stress by measuring the GSH/GSSG ratio and found that, while DOX treatment increased oxidative stress, 8-OHdG decreased it.
In our study, DOX treatment increased the expression of NLRP3 inflammasome components (NLRP3, ASC, and pro-caspase-1) and caspase-1, whereas 8-OHdG treatment decreased it. DOX treatment led to increased expression of both pro-IL-1β and IL-1β in H9c2 cells. GSDMD-NT levels were increased by DOX treatment and decreased by 8-OHdG treatment. Moreover, the amount of pyroptotic cells as evaluated by a caspase-1 activity assay was increased by DOX treatment and decreased by 8-OHdG treatment.
Both ANP and BNP are known to be useful predictors and prognostic markers of heart failure [60][61][62]. ANP is secreted from atria when the atrium is dilated [63][64][65]. BNP is synthesized in the ventricle depending on end diastolic pressure and volume, and is therefore a more sensitive marker for heart failure than ANP [63][64][65]. Moreover, BNP production starts to increase from week 6 to 12 of DOX-induced cardiotoxicity [65]. Several studies have shown that BNP can be used as a marker of DOX-induced cardiotoxicity [61,62]. Both BNP and LVEF are effective predictors of hospitalization with heart failure after chemotherapy with DOX [66]. However, BNP predicts overall death after DOX-induced cardiotoxicity more accurately than LVEF [66]. On the other hand, GATA4 is a member of the GATA family of proteins, and is essential for the adaptive response of cardiomyocytes [67][68][69]. DOX treatment leads to GATA4 depletion, and decreased GATA4 levels are associated with DOX-induced cardiotoxicity. A 50% reduction in GATA4 levels results in a hyper-response to DOX in mice, and results in higher myocyte loss than in wildtype mice [70]. GATA6 levels are also decreased by DOX. Furthermore, the expression of GATA4/6 is decreased in DOX-treated H9c2 cells [71].
In our study, the expression of ANP and BNP was increased by DOX treatment and decreased by 8-OHdG treatment. However, the expression of GATA4/6 was decreased by DOX treatment, and 8-OHdG treatment only increased GATA6 expression. Thus, it seems that 8-OHdG decreases the expression of cardiotoxicity makers in DOX-treated H9c2 cells which was accompanied with decreased expression of TLR2/4, NF-κB, and NOX 1/2/4 and pyroptosis. DOX-treated H9c2 cells also exhibited increased expression of IL-6 which induced cell injuries as well as IL-1β or TNF-α [72]. Since exogenous 8-OHdG also leads to decrease IL-1β, TNF-α, and IL-6 [39], it is possible that 8-OHdG could the decrease expression of cardiotoxicity makers by directly reducing IL-6, IL-1β, and TNF-α as well as decreasing pyroptosis. Immune checkpoint inhibitors (ICIs) which show effect of anti-PD-1 (nivolumab and pembrolizumab), anti-PD-L1 (atezolizumab, avelumab, and durvalumab), and anti-CTLA-4 antibodies (ipilimumab and tremelimumab) [73][74][75] also result in side effect of myocarditis [76]. Those ICIs lead to increased NLRP3 and MyD88 in the microcytes [77]. Thus, there is a possibility that 8-OHdG could decrease ICIs induced myocarditis by inhibiting NRLP3. We will evaluate whether 8-OHdG could decrease ICIs induced myocarditis as a future study. Ferroptosis is a non-apoptotic cell death which induced by excessive lipid peroxidation via iron-dependent activation of lipoxygenase [78,79]. Moreover, ferroptosis is one of pathophysiology of DOX-induced cardiotoxicity [79]. DOX induces increased iron mediated ferroptosis by upregulation of NOX4 signaling [80]. Thus, we will evaluate whether 8-OHdG could decrease DOX-induced cardiotoxicity by decreasing ferroptosis as a future study.

Conclusions
Collectively, exogenous 8-OHdG decreased the expression of NOX1/2/4, TLR2/4, and NF-κB by decreasing Rac1 activity in DOX-treated cells, which eventually led to reduced NLRP3 inflammasome, IL-1β, and GSDMD-NT levels. Furthermore, the decrease in the levels of pyroptosis-related factors led to the decreased expression of myocarditis-related markers such as ANP and BNP (Fig. 5D). Thus, exogenous 8-OHdG might potentially be used to attenuate DOX-induced cardiotoxicity through the inhibition of pyroptosis.