Hydroquinone Induces NLRP3-Independent IL-18 Release from ARPE-19 Cells

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Article Hydroquinone Induces NLRP3-Independent IL-18 Release from ARPE-19 Cells

Niina Bhattarai 1,* , Eveliina Korhonen 1,2 , Yashavanthi Mysore 1 , Kai Kaarniranta 3,4 and Anu Kauppinen 1,*

1 School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, 70210 Kuopio, Finland; eveliina.korhonen@uef.fi (E.K.); yashavanthi.mysore@uef.fi (Y.M.) 2 Department of Clinical Chemistry, University of Helsinki and Helsinki University Hospital, 00290 Helsinki, Finland 3 Department of Ophthalmology, Institute of Clinical Medicine, University of Eastern Finland, 70210 Kuopio, Finland; kai.kaarniranta@uef.fi 4 Department of Ophthalmology, Kuopio University Hospital, 70210 Kuopio, Finland * Correspondence: niina.bhattarai@uef.fi (N.B.); anu.kauppinen@uef.fi (A.K.); Tel.: +358-44-983-0424 (N.B.); +358-40-355-3216 (A.K.)

Abstract: Age-related macular degeneration (AMD) is a retinal disease leading to impaired vision. Cigarette smoke increases the risk for developing AMD by causing increased reactive oxygen species (ROS) production and damage in the retinal pigment epithelium (RPE). We have previously shown that the cigarette tar component hydroquinone causes oxidative stress in human RPE cells. In the present study, we investigated the propensity of hydroquinone to induce the secretion of interleukin (IL)-1β and IL-18. The activation of these cytokines is usually regulated by the Nucleotide-binding domain, Leucine-rich repeat, and Pyrin domain 3 (NLRP3) inflammasome. ARPE-19 cells were Citation: Bhattarai, N.; Korhonen, E.; exposed to hydroquinone, and cell viability was monitored using the lactate dehydrogenase (LDH) Mysore, Y.; Kaarniranta, K.; and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt (MTT) assays. Enzyme-linked Kauppinen, A. Hydroquinone immunosorbent assays (ELISAs) were used to measure the levels of proinflammatory cytokines IL-1β Induces NLRP3-Independent IL-18 and IL-18 as well as NLRP3, caspase-1, and poly (ADP-ribose) polymerase (PARP). Hydroquinone did Release from ARPE-19 Cells. Cells not change IL-1β release but significantly increased the secretion of IL-18. Cytoplasmic NLRP3 levels 2021, 10, 1405. https://doi.org/ increased after the hydroquinone treatment of IL-1α-primed RPE cells, but IL-18 was equally released 10.3390/cells10061405 from primed and nonprimed cells. Hydroquinone reduced the intracellular levels of PARP, which were restored by treatment with the ROS scavenger N-acetyl-cysteine (NAC). NAC concurrently Academic Editor: Juan Pablo de Rivero Vaccari reduced the NLRP3 levels but had no effect on IL-18 release. In contrast, the NADPH oxidase inhibitor ammonium pyrrolidinedithiocarbamate (APDC) reduced the release of IL-18 but had no effect on the Received: 24 April 2021 NLRP3 levels. Collectively, hydroquinone caused DNA damage seen as reduced intracellular PARP Accepted: 4 June 2021 levels and induced NLRP3-independent IL-18 secretion in human RPE cells. Published: 6 June 2021 Keywords: hydroquinone; oxidative stress; IL-1β; IL-18; NLRP3; RPE cell; PARP; DNA damage; Publisher’s Note: MDPI stays neutral NAC; APDC with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Cigarette smoke is a risk factor of age-related macular degeneration (AMD) that induces apoptosis, DNA damage, endoplasmic reticulum (ER) stress, mitochondrial frag- Copyright: © 2021 by the authors. mentation, reactive oxygen species (ROS) production and vascular endothelial growth Licensee MDPI, Basel, Switzerland. factor (VEGF) expression in retinal pigment epithelial (RPE) cells [1–5]. RPE cells main- This article is an open access article tain retinal homeostasis and support the functionality of photoreceptor cells, i.e., these distributed under the terms and cells are essential for good vision [6–8]. AMD can develop either into a dry (85–90%) conditions of the Creative Commons or wet (10–15%) disease form [6,7]. The late forms of AMD manifest with a geographic Attribution (CC BY) license (https:// atrophy in dry AMD or with an exudative form in wet AMD [7,9,10]. RPE degeneration creativecommons.org/licenses/by/ and inflammation are involved in both disease forms [7,11,12]. RPE cell degeneration 4.0/).

Cells 2021, 10, 1405. https://doi.org/10.3390/cells10061405 https://www.mdpi.com/journal/cells Cells 2021, 10, 1405 2 of 14

is a typical finding, especially in atrophic AMD, whereas choroidal neovascularization, hemorrhages, and swelling of the retina are hallmarks of wet AMD [7,9,10]. Oxidative stress and nucleotide-binding domain, leucine-rich repeat, and pyrin domain 3 (NLRP3) inflammasome activation are fundamental in the pathogenesis of AMD [5,7,10,12–14]. Hydroquinone, a component included in cigarette smoke, induces oxidative stress but prevents nuclear factor kappa B (NF-κB) activity and the release of interleukin (IL)-6 and IL-8 cytokines from RPE cells [5,15–17]. Increased ROS production is a known activator of NLRP3 inflammasome, leading to the cleavage and release of IL-1β with or without concur- rent maturation of IL-18 in human RPE cells [14,18]. The NLRP3 receptor is a pattern recog- nition receptor (PRR) that senses a wide variety of danger signals (pathogen or damage- associated molecular patterns i.e., PAMPs or DAMPs, respectively) [12,19,20]. Along with ROS production, its activation mechanisms include lysosomal destabilization and adeno- sine triphosphate (ATP) release-associated purinergic receptor X7/P2X purinoceptor 7 (P2X7) activation, resulting in K+-efflux and Ca2+-influx [13,14,18,21–23]. NLRP3 inflammasome activation requires consecutive priming and activation signals [13,19,24]. The priming signal, typically mediated through a Toll-like receptor (TLR) or a cytokine receptor, leads to the expression of the NLRP3 protein as well as the proforms of IL-1β and caspase- 1 [13,25–27]. Epithelial cells tend to express pro-IL-18 continuously without priming [25,27]. An activation signal induces the assembly of NLRP3 inflammasome complex where pro- caspase-1 becomes attached to oligomerised NLRP3 via the apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). The formation of this complex results in caspase-1 activation and subsequent cleavage of IL-1β and IL-18 into their mature and secreted forms [12,19,20,28]. IL-1β is involved in inflammatory diseases but it also possesses angiogenic properties [29–31]. IL-18 is a pleiotropic cytokine with distinct effects in different AMD models [32,33]. In mice, it has been observed to be protective against tuberculosis but harmful in combatting Ehrlichia infection [34,35]. In this study, we investigated the propensity of hydroquinone to activate NLRP3 inflammasome signaling in human RPE cells since we have previously shown that hydroquinone can induce ROS production in ARPE-19 cells, and oxidative stress is one of the best-known mechanisms of NLRP3 inflammasome activation [14,15,18]. Our results show that in addition to DNA damage, hydroquinone induced both NLRP3 inflammasome and IL-1α priming-independent secretion of IL-18 in human ARPE-19 cells.

2. Materials and Methods 2.1. Cell Culture and Treatments Experiments were performed using ARPE-19 cells (ATCC) that were cultured in DMEM/F12 (1:1) medium (Life Technologies, Paisley, UK) with added 10% fetal bovine serum (GE Healthcare Life Sciences, South Logan, UT, USA), penicillin 100 U/mL, strepto- mycin 100 µg/mL (Pen Strep, Life Technologies, Grand Island, NY, USA), and L-glutamine 2 mM (Life Technologies, Paisley, UK). The experiments were conducted in the same medium without serum supplement, and L-glutamine was added separately in each independent experiment just prior to the IL-1α-priming. Hydroquinone was dissolved in the medium before each experiment. In the experiments, cells were seeded onto 12-well plates (Costar, Corning Inc., Ken- nebunk, ME, USA) at the density of 200,000 cells per well and incubated for three days at ◦ +37 C under 5% CO2. The cells were ﬁrst washed once using serum-free medium and then primed using recombinant human IL-1α (4 ng/mL, R&D Systems, Minneapolis, MN, USA) ◦ for 24 h at +37 C and 5% CO2. Thereafter, the cells were washed once again with medium and exposed to hydroquinone (HQ, 10 µM to 500 µM; Sigma-Aldrich, Saint Louis, MO, ◦ USA) for 18 h at +37 C and 5% CO2. After the treatments, medium samples were collected and centrifuged at 381× g and +4 ◦C for 10 min (Biofuge Fresco Heraeus Instruments, New- port Pagnell, UK). Cells were either used directly for a viability assay or collected and lysed for other analyses. Optimal hydroquinone concentrations were probed by exposing cells to 10–500 µM hydroquinone, and cell viability and the secretion of mature IL-1β and IL-18 Cells 2021, 10, 1405 3 of 14

were determined. Two hydroquinone concentrations, 10 µM and 200 µM, were selected for the subsequent NLRP3 assays using only 200 µM for ATP and caspase-1 measurements. Caspase-1 inhibitor (casp.-1 inhib.; 20 µM in DMSO 0.22% v/v, Ac-YVAD-cmk, Sigma, St. Louis, MO, USA) was added 1 h before hydroquinone exposure when the role of caspase-1 in IL-18 release was investigated. Hydroquinone (200 µM) was also tested on unprimed cells. In experiments of NLRP3 silencing, ARPE-19 cells were seeded onto 12-well plates ◦ at the density of 150,000 cells per well and incubated for 24 h at +37 C and 5% CO2. Cells were washed once with DMEM/F12 medium without serum and antibiotics, and 500 µL of the same medium was added into each well. Cells were transfected by adding lipofectamine (lipofectamine RNAiMAX/lipofectamine 2000, Invitrogen by Thermo Life Technologies Corp., Carlsbad, CA, USA) with NLRP3 siRNA (siNLRP3 10 pmol; NLRP3 Silencer select predesigned siRNA, id:s41556, Ambion, Austin, TX, USA) diluted in Opti- MEM medium (Thermo Fisher Scientiﬁc, Grand Island, NY, USA) for 24 h at +37 ◦C and 5% CO2. Negative siRNA (Silencer select Negative control #1 siRNA, Ambion, Austin, TX, USA) and transfection reagent (Tr. reag.; Opti-MEM with lipofectamine) without siRNAs served as controls. After transfection, cells were washed, primed with IL-1α, and exposed to 200 µM hydroquinone, as described above. The effects of siNLRP3 transfection on the release of IL-18 and lactate dehydrogenase (LDH) were determined. When determining the effects of antioxidants, cells were primed with IL-1α for 24 h at ◦ +37 C and 5% CO2, washed, and exposed to the NADPH oxidase inhibitor ammonium pyrrolidinedithiocarbamate (APDC, 2 µM; Sigma-Aldrich, St. Louis, MO, USA) for 5 min, or to the ROS scavenger N-acetyl-cysteine (NAC, 5 mM; Sigma-Aldrich, St. Louis, MO, USA) for 1 h as in our previous investigation of hydroquinone-mediated ROS production [15]. Thereafter, 200 µM hydroquinone was added for an additional 18 h. Untreated and IL-1α- primed cells served as controls.

2.2. Cell Viability Assays Before sample collection, the cells were visually observed and photographed using an Axio Vert A1 Zeiss microscope with AxioCam MRm camera and Zen 2011 program (Carl Zeiss Microscopy GmbH, Jena, Germany). Thereafter, half (500 µL) of the cell culture medium was collected and centrifuged, as described above. LDH release was measured from medium samples immediately after the collection using the CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Absorbance values were measured at a wavelength of 490 nm with a spectrophotometer BioTek, ELx808 and the Gen-5 2.04 program (Instruments Inc., Winooski, VT, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt (MTT; 0.5 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added to cells remained on 12-well culture plates ◦ in the rest of medium (500 µL) and incubated for 3 h in the dark at +37 C and 5% CO2, as described previously [15]. Absorbance values were measured at the wavelength of 560 nm using the spectrophotometer BioTek, ELx808.

2.3. Enzyme-Linked Immunosorbent Assay The enzyme-linked immunosorbent assay (ELISA) technique was used to determine the levels of active IL-1β and IL-18, intra- and extracellular NLRP3, as well as cleaved caspase-1 (p20) and poly (ADP-ribose) polymerase (PARP) levels. IL-1β, IL-18, NLRP3, and caspase-1 were measured using the IL-1β BD OptEIATM Human ELISA Kit (BD Bio- sciences, San Diego, CA, USA), the Human IL-18 ELISA kit (Invitrogen by Thermo Fisher Scientiﬁc, Vienna, Austria), the Human NACHT, LRR and PYD domains-containing protein 3 (NLRP3/c1orf7/CIAS1/NALP3/PYPAF1) ELISA kit (Cusabio Biotech co., LTD, Wuhan, Hubei Province, China), and the Quantikine ELISA Human caspase-1/ICE immunoassay (R&D systems, McKinley, MN, USA), respectively. Absorbance values were determined at the wavelength of 450 nm with the correction wavelength of 655 nm in a spectrophotometer (Bio-Rad Model 550 with the Microplate Manager 5.2 program, Bio-Rad Laboratories Inc., Cells 2021, 10, 1405 4 of 14

Hercules, CA, USA). DNA damage was determined by measuring the PARP levels with the PARP/Apoptosis Colorimetric Assay Kit (R&D Systems, Minneapolis, MN, USA) and detecting absorbance values at the wavelength of 450 nm using a microplate reader Bio-Rad Model 550. For the detection of intracellular NLRP3, cells were lysed with 40 µL of 1× lysis buffer (Cell Lysis Buffer 10×, Cell Signaling Technology, Leiden, Netherlands) in a tube containing cells from one well of a 12-well plate and incubated on ice for 5 min. Thereafter, the lysates were sonicated (3 × 10 s) and centrifuged at 16,090× g and +4 ◦C for 10 min. Supernatants were moved into clean microtubes and their protein concentrations were measured utilizing a protocol based on the Bradford method [36]. Briefly, the bovine serum albumin fraction V (BSA 1 mg/mL; Roche, Mannheim, Germany) was used to create a standard curve (0.5–3.5 µg/µL). Thereafter, samples were pipetted (0.5–3.5 µL) into wells of a 96-well plate, Bradford solution (200 µL) was added to all wells, and absorbance values were measured at the wavelength of 595 nm in a spectrophotometer Bio-Rad Model 550. NLRP3 levels were determined from remaining cell lysate using the ELISA technique with the results normalized to the protein level of the respective sample. When measuring the levels of cleaved caspase-1, cells were lysed using the RIPA buffer (Thermo Scientific, Rockford, IL, USA), incubated for 5 min on ice, and centrifuged at 16,090× g and +4 ◦C for 10 min. The Pierce bicinchoninic acid (BCA) protein Assay Kit (Thermo Scientific, Rockford, IL, USA) was used to measure protein concentrations, and 15 or 25 µg of protein from each sample was used for the ELISA analysis. When measuring the PARP levels, cells were lysed according to the manufacturer’s protocol, and the Pierce BCA protein assay was used to determine protein levels. Thereafter, 5 µg of protein from sample was used for the ELISA assay.

2.4. Extracellular ATP The levels of ATP in culture medium were measured using the CellTiter-Glo Lumi- nescent Cell Viability Assay (Promega, Madison, WI, USA) following the manufacturer’s instructions. Luminescence values were measured using the BioTek Cytation3 imaging reader with the Gen-5 3.03 program (Instruments Inc., Winooski, VT, USA).

2.5. Statistical Analyses Results were analyzed using the GraphPad Prism program 7.04 (Graphpad Software, San Diego, CA, USA). Normality was determined using the Shapiro-Wilk and D’Agostino & Pearson normality tests (data not presented). Statistical differences between all groups were determined with the Kruskal-Wallis test, and post hoc comparisons using the Mann- Whitney U-test. Results are shown as mean ± standard error of mean (SEM) and differences were considered as statistically signiﬁcant with p-values below 0.05.

3. Results 3.1. Hydroquinone Induces IL-18 Secretion in ARPE-19 Cells Hydroquinone induces ROS production and cytotoxicity concentration-dependently in human ARPE-19 cells [15–17]. In order to investigate whether hydroquinone could induce the secretion of proinflammatory cytokines, IL-1β and IL-18, we determined their extracellular levels as well as their correlation with cell viability after an exposure of IL-1α- primed ARPE-19 cells to different hydroquinone concentrations ranging from 10 µM to 500 µM. Low hydroquinone concentrations (10–50 µM) were well tolerated by RPE cells since LDH release and MTT values remained unchanged when compared to IL-1α-primed ARPE-19 cells without hydroquinone (Figure1). LDH release was significantly increased in cells exposed to at least 100 µM hydroquinone, while metabolic activity was significantly reduced when the hydroquinone concentration was 150 µM or higher (Figure1). Cells 2021, 10, x FOR PEER REVIEW 5 of 16

Cells 2021, 10, x FOR PEER REVIEW 5 of 16

since LDH release and MTT values remained unchanged when compared to IL-1α-primed ARPE-19 cells without hydroquinone (Figure 1). LDH release was significantly increased in cells exposedsince to LDHat least release 100 and μM MTT hydroquinone, values remained while unchanged metabo whenlic activity compared was to signifi-IL-1α-primed ARPE-19 cells without hydroquinone (Figure 1). LDH release was significantly increased cantly reduced when the hydroquinone concentration was 150 μM or higher (Figure 1). in cells exposed to at least 100 μM hydroquinone, while metabolic activity was signifi- Cells 2021, 10, 1405 5 of 14 cantly reduced when the hydroquinone concentration was 150 μM or higher (Figure 1).

Figure 1. The effectFigure of hydroquinone 1. The effect of (HQ hydroquinone 10–500 µM; (HQ 18 10–500 h) on theμM; cell18 h) viability on the cell of viability IL-1α-primed of IL-1α ARPE-19-primed ARPE-19 cells measured cells measured Figureusing the1. The LDH effect (A)using andof hydroquinone MTTthe LDH (B) ( assays.A) and (HQ MTT Results 10–500 (B) assays. have μM; been 18Results h) normalizedon have the beencell viability tonormalized the mean of IL-1to of th theαe -primedmean untreated of the ARPE-19 untreated control cells group.control measured IL-1group.α- IL-1α- usingtreated the cells LDH served (A)treated and as a MTT control cells ( Bserved) forassays. hydroquinone as a Resultscontrol for have hydr exposure beenoquinone normalized in primed exposure cells.to in th primede Data mean werecells. of the Data combined untreated were combined from control three from group. independent three IL-1 independentα- treatedexperiments cells served with fourexperiments as a parallel control with samplesfor fourhydr parallel peroquinone group. samples exposure Results per gr areinoup. primed shown Results ascells. are mean shown Data± wereSEM.as mean combined **** ± SEM.p < 0.0001, **** from P < ns—notthree 0.0001, independent ns—not significant, significant, experiments with fourMann-Whitney parallel samples U-test. [Kruskal-Wallisper group. Results test, ( Aare) *** shown P < 0.001, as mean (B) *** ± P SEM. < 0.001]. **** P < 0.0001, ns—not significant, Mann-Whitney U-test. Mann-Whitney U-test. [Kruskal-Wallis test, (A) *** P < 0.001, (B) *** P < 0.001]. Next, we measuredNext, we the measured production the production of proinflammatory of proinflammatory cytokines cytokines IL-1β andIL-1β IL-18 and IL-18 fromNext, IL-1α we-primed frommeasured IL-1 ARPE-19α-primed the production cells ARPE-19 in response cellsof proinflammatory in toresponse hydroquinone to hydroquinone cytokines exposure exposureIL-1 (Figureβ and (Figure2 ).IL-18 Hy- 2). Hy- droquinone had no additional effect on the IL-1β release, which remained at the same fromdroquinone IL-1α-primed had no additionalARPE-19 cells effect in on response the IL-1 toβ release,hydroquinone which remained exposure at (Figure the same 2). levelHy- level as in IL-1α-treated cells (Figure 2A). Hydroquinone induced significant secretion of droquinoneas in IL-1α-treated had no cells additional (Figure 2effectA). Hydroquinone on the IL-1β release, induced which significant remained secretion at the of IL-18same at the 200 µMIL-18 and 300at theµ M200 concentrations, μM and 300 μM whereasconcentrations, lower whereas (≤150 µlowerM) or (≤higher 150 μM) (500 or higherµM) (500 level as in IL-1μαM)-treated concentrations cells (Figure had no 2A). effect Hydroquinone on the IL-18 levels induced (Figure significant 2B). secretion of IL-18concentrations at the 200 hadμM noand effect 300 μ onM theconcentrations, IL-18 levels (Figurewhereas2B). lower (≤ 150 μM) or higher (500 μM) concentrations had no effect on the IL-18 levels (Figure 2B).

Figure 2. The effect of hydroquinone (HQ 10–500 µM; 18 h) on the release of IL-1β (A) and IL-18 (B) from IL-1α-primed ARPE-19 cells. Data were combined from three independent experiments with four parallel samples per group in each experiment. Results are shown as mean ± SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001, ns—not signiﬁcant, Mann-Whitney U-test. [Kruskal-Wallis test, (A) ns p = 0.903, (B) *** p < 0.001].

3.2. Hydroquinone Increases the Intracellular Level of NLRP3 in IL-1α-Primed ARPE-19 Cells We showed previously that the oxidative stress induced either by impaired protein clearance or 4-hydroxynonenal (HNE) exposure promoted NLRP3 inﬂammasome signaling with caspase-1 activation in RPE cells favoring the secretion of IL-1β or IL-18, respectively [14,18]. In the present study, we investigated whether hydroquinone promoted NLRP3 inﬂammasome activation in human ARPE-19 cells. We have also showed that NLRP3 is partially degraded, but also secreted, out of the cells after the activation of Cells 2021, 10, x FOR PEER REVIEW 6 of 16

Figure 2. The effect of hydroquinone (HQ 10–500 μM; 18 h) on the release of IL-1β (A) and IL-18 (B) from IL-1α-primed ARPE-19 cells. Data were combined from three independent experiments with four parallel samples per group in each experiment. Results are shown as mean ± SEM. * P < 0.05, *** P < 0.001, **** P < 0.0001, ns—not significant, Mann-Whitney U-test. [Kruskal-Wallis test, (A) ns P = 0.903, (B) *** P < 0.001].

3.2. Hydroquinone Increases the Intracellular Level of NLRP3 in IL-1α-primed ARPE-19 Cells We showed previously that the oxidative stress induced either by impaired protein clearance or 4-hydroxynonenal (HNE) exposure promoted NLRP3 inflammasome signal- Cells 2021, 10, 1405 ing with caspase-1 activation in RPE cells favoring the secretion of IL-1β or IL-18,6 of respec- 14 tively [14,18]. In the present study, we investigated whether hydroquinone promoted NLRP3 inflammasome activation in human ARPE-19 cells. We have also showed that NLRP3 is partially degraded, but also secreted, out of the cells after the activation of the the inflammasomeinflammasome [37,38]. [37,38]. In the presentIn the present study, study, IL-1 αIL-1aloneα alone resulted resulted in in 2.8 2.8 and and 43.9-fold intra- intra- and extracellularand extracellular NLRP3 NLRP3 levels, levels, respectively, respectively when, when compared compared to to untreated untreated controlcontrol cells cells (Figure3(Figure). Hydroquinone 3). Hydroquinone at the at 10the 10µM μM concentration concentration had no no additional additional effect effect on the on intra- the intra- or extracellularor extracellular levels levels of of NLRP3NLRP3 when compared compared to toIL-1 IL-1α-treatedα-treated cellscells without without hydroqui- hydroquinonenone (Figure (Figure3). 3). In In contrast, contrast hydroquinone, hydroquinone at at the the 200 200 μMµ Mconcentration concentration significantly signifi- ele- cantly elevatedvated the the levels levels of of intracellular intracellular NLRP3,NLRP3, while while the the extracellular extracellular levels levels of NLRP3 of NLRP3 remained remained significantlysignificantly lower lower (Figure (Figure3 3).). InIn conclusion, we we did did not not detect detect any anyevidence evidence of the of release the release ofof NLRP3 NLRP3 at at the the 200 200µ MμM hydroquinone hydroquinone concentrationconcentration where the production of of IL-18 peaked (Figure 2). IL-18 peaked (Figure2).

Figure 3. The effect of hydroquinone (HQ 10 µM, 200 µM; 18 h) on the levels of NLRP3 in cell lysate Figure 3. The effect of hydroquinone (HQ 10 μM, 200 μM; 18 h) on the levels of NLRP3 in cell lysate (A) and medium (B) α samples in (IL-1A)α and-primed medium ARPE-19 (B) samples cell cultures. in IL-1 Results-primed (A) were ARPE-19 normalized cell cultures.to the total Results protein ( Alevel.) were Absorbance normalized values to in (A) werethe 0.263–0.507 total protein in the level.control, Absorbance 0.548–1.516 valuesin the IL-1 in (αA, )0.416–0.862 were 0.263–0.507 in the IL-1 inαthe + HQ control, 10 μM, 0.548–1.516 and 4.492–9.375 in the in the IL-1α +IL-1 HQα 200, 0.416–0.862 μM group. in NLRP3 the IL-1 concentrationsα + HQ 10 µ M,in andmedium 4.492–9.375 samples in(B the) were IL-1 0.590–2.054α + HQ 200 ng/mLµM group. in the NLRP3control, 11.81–161.453concentrations ng/mL in the inIL-1 mediumα, 10.345–28.244 samples ng/mL (B) were in the 0.590–2.054 IL-1α + HQ ng/mL 10 μM, in and the 1.268–6.303 control, 11.81–161.453 ng/mL in the ng/mLIL-1α + HQ 200 μM group. Data were combined from three independent experiments with two (A) or three (B) parallel samples in the IL-1α, 10.345–28.244 ng/mL in the IL-1α + HQ 10 µM, and 1.268–6.303 ng/mL in the IL-1α + per group in each experiment. Results are shown as mean ± SEM. *** P < 0.001, **** P < 0.0001, ns—not significant, Mann- Whitney U-test.HQ 200[Kruskal-WallisµM group. test, Data (A were) *** P combined < 0.001, (B from) *** P three< 0.001]. independent experiments with two (A) or three (B) parallel samples per group in each experiment. Results are shown as mean ± SEM. *** p < 0.001, **** p < 0.0001,3.3. ns—not Hydroquinone signiﬁcant, Exposure Mann-Whitney Does Not U-test.Induce Caspase-1 Activation in ARPE-19 Cells Next, we investigated whether caspase-1 would be activated and contributes to the 3.3. Hydroquinonesecretion Exposure of IL-18 Does upon Not hydr Induceoquinone Caspase-1 exposure Activation in IL-1 inα-primed ARPE-19 RPE Cells cells. Similarly to Next, wethe investigated situation with whether NLRP3, caspase-1 we previously would observed be activated that cleaved and contributescaspase-1 is partially to the re- secretion of IL-18leased upon from hydroquinone the cell upon inflammasome exposure in IL-1 activationα-primed [37,39]. RPE IL-1 cells.α did Similarly not increase to the the in- situation withtracellular NLRP3, welevels previously of cleaved observed caspase-1 that(p20) cleaved when compared caspase-1 to isthe partially untreated released control cells from the cell upon(Figure inﬂammasome 4A). Hydroquinone activation reduced [37 the,39 caspase-1]. IL-1α did (p20) not levels increase in cell the lysate intracellular samples of IL- levels of cleaved caspase-1 (p20) when compared to the untreated control cells (Figure4A). Hydroquinone reduced the caspase-1 (p20) levels in cell lysate samples of IL-1α-primed ARPE-19 cells when compared to the primed cells without hydroquinone (Figure4A).

Cleaved caspase-1 was not detected in medium samples of controls or hydroquinone- treated cells. Absorbance values were 0.182–0.197 in the control, 0.193–0.204 in the IL-1α, and 0.182–0.193 in the 200 µM hydroquinone group of IL-1α-primed cells. Caspase-1 inhibitor had no effect on the IL-18 release (Figure4B). These data suggest that hydroquinone did not activate caspase-1 in human ARPE-19 cells.

3.4. Hydroquinone Does Not Need Priming or NLRP3 Inﬂammasome Activation to Induce IL-18 Release We have showed previously that UVB irradiation-induced IL-1β secretion was NLRP3- dependent, but this was not the case for IL-18 release [39]. Next, we examined whether hydroquinone alone would be capable of inducing the release of IL-18 without IL-1α- priming. Hydroquinone signiﬁcantly increased IL-18 secretion but the levels of intracellular NLRP3 remained at the levels of untreated control cells (Figure5). In order to Cells 2021, 10, 1405 7 of 14 Cells 2021, 10, x FOR PEER REVIEW 7 of 16

1αconfirm-primed ARPE-19 the NLRP3-independent cells when compared IL-18 to the release, primed cells cells without were treatedhydroquinone with siNLRP3(Fig- before the urehydroquinone 4A). Cleaved caspase-1 exposure was of not IL-1 detectedα-primed in medium cells. Specificsamples of NLRP3 controls siRNA or hydroqui- did not reduce IL-18 none-treatedrelease when cells. comparedAbsorbance tovalues hydroquinone were 0.182–0.197 with in transfection the control, 0.193–0.204 reagent withoutin the siNLRP3 in IL-1IL-1α, andα-primed 0.182–0.193 cells in (Figurethe 200 μ6M). hydroquinone These findings group support of IL-1α the-primed data cells. shown Caspase- above that hydro- 1 quinoneinhibitor had was no able effect to on induce the IL-18 IL-18 release secretion (Figure from4B). These ARPE-19 data suggest cells withoutthat hydro- the activation of quinone did not activate caspase-1 in human ARPE-19 cells. NLRP3 inflammasome.

Figure 4. The effect of 200 µM hydroquinone (HQ; 18 h) on the cleaved caspase-1 (p20) levels detected Figure 4. The effect of 200 μM hydroquinone (HQ; 18 h) on the cleaved caspase-1 (p20) levels de- tectedfrom from cell cell lysate lysate (A ()A samples) samples andand the effect effect of of caspase-1 caspase-1 inhibitor inhibitor (casp.-1 (casp.-1 inhib.; inhib.; 20 μM 20in µM in DMSO 0.22% DMSOv/v) on0.22% the v/v hydroquinone-induced) on the hydroquinone-induced IL-18 IL-18 release release (B (B)) in in IL-1αα-primed-primed ARPE-19 ARPE-19 cells.cells. Absorbance Absorbancevalues in values (A) were in (A 0.225–0.288) were 0.225–0.288 in the in control,the control, 0.259–0.379 0.259–0.379 in in the the IL-1αα, ,and and 0.149–0.161 0.149–0.161 in the IL-1α + in the IL-1α + HQ 200 μM group. Data were combined from two (A) or one (B) independent exper- HQ 200 µM group. Data were combined from two (A) or one (B) independent experiments with two Cells 2021, 10, x FORiments PEER with REVIEW two ( A) or four (B) parallel samples per group. Results are shown as mean ± SEM. * P 8 of 16 < 0.05,(A) orns—not four significant, (B) parallel Mann-Whitney samples per U-test. group. [Kruskal-Wallis Results are test, shown (A) ** as P = mean 0.002, ±(B)SEM. ** P = * p < 0.05, ns—not 0.009].signiﬁcant, Mann-Whitney U-test.

3.4. Hydroquinone does not Need Priming or NLRP3 Inflammasome Activation to Induce IL-18 Release We have showed previously that UVB irradiation-induced IL-1β secretion was NLRP3-dependent, but this was not the case for IL-18 release [39]. Next, we examined whether hydroquinone alone would be capable of inducing the release of IL-18 without IL-1α-priming. Hydroquinone significantly increased IL-18 secretion but the levels of intracellular NLRP3 remained at the levels of untreated control cells (Figure 5). In order to confirm the NLRP3-independent IL-18 release, cells were treated with siNLRP3 before the hydroquinone exposure of IL-1α-primed cells. Specific NLRP3 siRNA did not reduce IL- 18 release when compared to hydroquinone with transfection reagent without siNLRP3 in IL-1α-primed cells (Figure 6). These findings support the data shown above that hydroquinone was able to induce IL-18 secretion from ARPE-19 cells without the activation of NLRP3 inflammasome.

Figure 5. The effect of 200 µM hydroquinone (HQ; 18 h) on the IL-18 secretion (A) and on the intracel- Figure 5. The effect of 200 μM hydroquinone (HQ; 18 h) on the IL-18 secretion (A) and on the intracellular NLRP3 level (B) in unprimedlular ARPE-19 NLRP3 levelcells. An (B) untreated in unprimed control ARPE-19 group wa cells.s used An as untreated the control control for hydroquinone group was treatment. used as theIL-18 control concentrationsfor in hydroquinone medium samples treatment. (A) were 4.552–10.556 IL-18 concentrations pg/mL in the in contro mediuml and samples 32.132–123.617 (A) were pg/mL4.552–10.556 in the HQ 200 pg/mL μM group. Absorbancein the control values and in 32.132–123.617(B) were 0.443–0.563 pg/mL in the control in the HQand 0.345–0.579 200 µM group. in the HQ Absorbance 200 μM group. values Data in were (B) were combined from two independent experiments containing four (A) or two (B) parallel samples per group. Results are shown as mean0.443–0.563 ± SEM. *** in P the< 0.001, control ns—not and significant, 0.345–0.579 Mann-Whitney in the HQ U-test. 200 µ M group. Data were combined from two independent experiments containing four (A) or two (B) parallel samples per group. Results are shown as mean ± SEM. *** p < 0.001, ns—not significant, Mann-Whitney U-test. 3.5. APDC Reduces the Release of IL-18 But Not NLRP3, While NAC Has the Opposite Effect upon Hydroquinone Exposure of IL-1α-Primed ARPE-19 Cells We showed previously that both NAC and APDC are able to reduce hydroquinone- induced ROS production in RPE cells [15]. Since it is known that IL-18 release from UVB-treated ARPE-19 cells is ROS-dependent [39], we tested whether antioxidants APDC or NAC would exert any effect on RPE cells exposed to hydroquinone. APDC had no effect on the LDH release, but NAC reduced it when compared to primed RPE cells after hydroquinone exposure without the supplementation with antioxidants (Figure7 ).

Figure 6. The effect of siNLRP3 on the hydroquinone-induced (HQ 200 μM; 18 h) release of IL-18 (A) and LDH (B) in IL-1α-primed ARPE-19 cells. Transfection reagent without siRNAs (Tr. reag.) was used as a control for the siNLRP3 treatment and negative siRNA (Neg. siRNA) was used to indicate if unspecific reduce of IL-18 release was detected. IL-18 concentrations in (A) were 1.342– 8.686 pg/mL in the IL-1α, 4.267–11.166 pg/mL in the Tr. reag. + IL-1α + HQ 200 μM, 10.187–12.171 pg/mL in the neg. siRNA + IL-1α + HQ 200 μM, and 6.233–12.171 pg/mL in the siNLRP3 + IL-1α + HQ 200 μM group. Data were combined from two independent experiments with four parallel samples per group in each experiment. Results are shown as mean ± SEM. * P < 0.05, *** P < 0.001, ns—not significant, Mann-Whitney U-test. [Kruskal-Wallis test, (A) *** P < 0.001, (B) *** P < 0.001].

3.5. APDC Reduces the Release of IL-18 but not NLRP3, while NAC has the Opposite Effect upon Hydroquinone Exposure of IL-1α-primed ARPE-19 Cells We showed previously that both NAC and APDC are able to reduce hydroquinone- induced ROS production in RPE cells [15]. Since it is known that IL-18 release from UVB- treated ARPE-19 cells is ROS-dependent [39], we tested whether antioxidants APDC or NAC would exert any effect on RPE cells exposed to hydroquinone. APDC had no effect

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Microscope observations were in line with the LDH data since APDC-treated cells were damaged, but the NAC-treated cells appeared healthy and viable (Figure8). APDC reduced the secretion of IL-18 but did not affect the intracellular NLRP3 levels (Figure7). In Figure 5. The effect of 200 μMcontrast, hydroquinone NAC (HQ; had 18 noh) on effect the IL-18 on thesecretion levels (A) ofand IL-18 on the in intracellular IL-1α-primed NLRP3 ARPE-19level cells exposed (B) in unprimed ARPE-19 cells.to An hydroquinone, untreated control butgroup the was levels used as of the NLRP3 control for as hy adroquinone part of thetreatment. total IL-18 protein concentration concentrations in medium samples (A) were 4.552–10.556 pg/mL in the control and 32.132–123.617 pg/mL in the HQ 200 μM group. Absorbance valueswere in (B) signiﬁcantlywere 0.443–0.563 lower in the control when and compared 0.345–0.579 toin the the HQ respective 200 μM group. cells Data without were NAC (Figure7). combined from two independentThese experiments results suggest containing that four the (A) releaseor two (B of) parallel IL-18 andsamples the per expression group. Results of NLRP3 are protein are not shown as mean ± SEM. *** P

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Figure 6. The effect of siNLRP3 on the hydroquinone-induced (HQ 200 µM; 18 h) release of IL-18 (A) Figure 6. The effect of siNLRP3on the LDH on the release, hydroquinone-induced but NAC reduced (HQ it 200 when μM; compared 18 h) release to of primed IL-18 RPE cells after hydro- (Aand) and LDH LDH (B)) in in IL-1 IL-1quinoneα-primedα-primed exposure ARPE-19 ARPE-19 cells.without Transfection cells. the Transfection supplementation reagent without reagent with siRNAs without antioxidants (Tr. siRNAsreag.) (Figure (Tr. reag.)7). Microscope was wasused used as as a a control control forforobservations the siNLRP3 siNLRP3 weretreatment treatment in line and with and negative negativethe LDH siRNA siRNAdata (Neg. since siRNA) (Neg. APDC-treated siRNA)was used was to cells used were to indicatedamaged, if but indicateunspeciﬁc if unspecific reduce reduce ofthe IL-18 NAC-treated of IL-18 release release was cells was detected. appeareddetected. IL-18 IL-18 healthy concentrations and viable in (Figure in(A) ( Awere) were 8). 1.342– APDC 1.342–8.686 reduced pg/mL the secre- 8.686 pg/mL in the IL-1αtion, 4.267–11.166 of IL-18 but pg/mL did innot the affect Tr. reag. the +intracellular IL-1α + HQ 200 NLRP3 μM, 10.187–12.171 levels (Figure 7). In contrast, NAC in the IL-1α, 4.267–11.166 pg/mL in the Tr. reag. + IL-1α + HQ 200 µM, 10.187–12.171 pg/mL in the pg/mL in the neg. siRNAhad + IL-1 noα effect + HQ on200 the μM, levels and 6.233–12.171 of IL-18 in pg/mLIL-1α -primedin the siNLRP3 ARPE-19 + IL-1 cellsα + exposed to hydroqui- α µ α µ HQneg. 200 siRNAμM group. + IL-1Datanone, were+ HQ butcombined 200 the levelsM, from and of two NLRP3 6.233–12.171 independent as a part experiments pg/mL of the total in thewith protein siNLRP3 four concentrationparallel + IL-1 + were HQ significantly 200 M samples per group in each experiment. Results are shown as mean ± SEM. * P < 0.05, *** P < 0.001, group. Data were combinedlower when from compared two independent to the respective experiments cells without with four NAC parallel (Figure samples 7). These per results group sug- ns—not significant, Mann-Whitney U-test. [Kruskal-Wallis test, (A) *** P < 0.001, (B) *** P < 0.001]. in each experiment.gest Results that the are release shown of as IL-18 mean and± theSEM. expression * p < 0.05, of ***NLRP3p < 0.001, protein ns—not are not signiﬁcant, dependent on each other in the cells’ response to hydroquinone exposure. 3.5.Mann-Whitney APDC Reduces U-test.the Release [Kruskal-Wallis of IL-18 but not test, NLRP3, (A) ***whilep < NAC 0.001, has ( Bthe) *** Oppositep < 0.001]. Effect upon Hydroquinone Exposure of IL-1α-primed ARPE-19 Cells We showed previously that both NAC and APDC are able to reduce hydroquinone- induced ROS production in RPE cells [15]. Since it is known that IL-18 release from UVB- treated ARPE-19 cells is ROS-dependent [39], we tested whether antioxidants APDC or NAC would exert any effect on RPE cells exposed to hydroquinone. APDC had no effect

Figure 7. The effect of antioxidants (2 µM APDC, 5 mM NAC) in conjunction with 200 µM hydroquinone Figure 7. The effect of antioxidants (2 μM APDC, 5 mM NAC) in conjunction with 200 μM hydroquinone (HQ; 18 h) on the release(HQ; of 18 LDH h) on (A) the and release IL-18 (B of), and LDH on ( Athe) andintracellular IL-18 (B NLRP3), and (C) on levels the intracellular in IL-1α-primed NLRP3 ARPE-19 (C) levelscells. Data in IL-1 wereα - combinedprimed from ARPE-19 four (A,B cells.) or 2 ( DataC) independent were combined experiments from fourcontaining (A,B) four or 2 ( (AC, )B independent) or two (C) parallel experiments samples containingper group. IL-18 fourconcentrations (A, B) or in two (B) ( Cwere) parallel 0.745–10.597 samples pg/mL per in group. the IL-1 IL-18α, 6.515–83.423 concentrations pg/mL in in (B the) were IL-1α 0.745–10.597 + HQ 200 μM, pg/mL 2.58– 26.253 pg/mL in the IL-1α + APDC + HQ 200 μM, and 7.721–26.506 pg/mL in the IL-1α + NAC + HQ 200 μM group. α α µ α Absorbancein the values IL-1 , in 6.515–83.423 (C) were 0.62–0.72 pg/mL in inthe the control, IL-1 2.171–2.824+ HQ 200 in theM, 2.58–26.253IL-1α + HQ 200 pg/mL μM, 1.816–2.564 in the IL-1 in the+ APDC IL-1α + + APDCHQ + HQ 200 200µ M,μM, and and 7.721–26.5061.266–2.25 in the pg/mL IL-1α + in NAC the IL-1+ HQα 200+ NAC μM group. + HQ Results 200 µ Mare group.presented Absorbance as mean ± SEM. values * P in< 0.05, ***(C )P were< 0.001, 0.62–0.72 **** P < 0.0001, in the control,ns = not significant, 2.171–2.824 Mann-Whitney in the IL-1α +U-test. HQ 200[Kruskal-WallisµM, 1.816–2.564 test, (A in) the*** P IL-1 < 0.001,α + APDC(B) *** P < 0.001, (C) *** P < 0.001]. + HQ 200 µM, and 1.266–2.25 in the IL-1α + NAC + HQ 200 µM group. Results are presented as mean ± SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001, ns = not significant, Mann-Whitney U-test.

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FigureFigure 8.8. The effect of of 200 200 μµMM hydroquinone hydroquinone (HQ; (HQ; 18 18h) h)on onAR ARPE-19PE-19 cells cells with with and andwithout without IL-1α IL-1- α- priming and antioxidants (2 μM APDC, 5 mM NAC). Cells were visualized using the Axio Vert priming and antioxidants (2 µM APDC, 5 mM NAC). Cells were visualized using the Axio Vert A1 A1 Zeiss microscope with AxioCam MRm camera and Zen 2011 program (Carl Zeiss Microscopy ZeissGmbH, microscope Jena, Germany). with AxioCam MRm camera and Zen 2011 program (Carl Zeiss Microscopy GmbH, Jena, Germany). 3.6. Hydroquinone Increases the Levels of Extracellular ATP 3.6. Hydroquinone Increases the Levels of Extracellular ATP Extracellular ATP is reported to be one potential activator of the NLRP3 inflam- Extracellular ATP is reported to be one potential activator of the NLRP3 inﬂammasome masome with the effect mediated through the P2X7 receptor, but it can also serve as a withsecondary the effect response mediated to NLRP3 through activation the P2X7 as well receptor, as a stimulant but it can for alsothe antioxidant serve as a secondarysystem responsein times of to oxidative NLRP3 activationstress [18,23,40]. as well In order as a stimulant to study the for potential the antioxidant role of ATP, system we meas- in times ofured oxidative the extracellular stress [18 ATP,23,40 levels]. In after order the to IL-1 studyα-primed the potential cells were role exposed of ATP, to wehydroqui- measured thenone. extracellular Hydroquinone ATP increased levels after the the extracellular IL-1α-primed ATP levels cells were concurrently exposed while to hydroquinone. elevating Hydroquinonethe intracellular increasedlevel of NLRP3 the extracellularand the amount ATP of IL-18 levels from concurrently IL-1α-primed while ARPE-19 elevating cells the intracellular(Figure 9). IL-1 levelα treatment of NLRP3 reduced and the the amount extracellular of IL-18 ATP from levels IL-1 in ARPE-19α-primed cell ARPE-19 cultures, cells (Figurewhereas9). hydroquinone IL-1 α treatment at 200 reduced μM concentration the extracellular significantly ATP levels increased in ARPE-19 the extracellular cell cultures, whereasATP levels hydroquinone in IL-1α-primed at 200 cellsµ M(Figure concentration 9). signiﬁcantly increased the extracellular ATP levels in IL-1α-primed cells (Figure9).

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Figure 9. The effect of hydroquinone (HQ 200 μM; 18 h) exposure on the levels of extracellular ATP in IL-1α-primed ARPE-19 cell cultures. Results were normalized to the untreated control group.Figure Data 9. The were effect combined of hydroquinone from three (HQindepend 200 µentM; experiments 18 h) exposure with on four the levels parallel of extracellularsamples per ATP groupFigurein IL-1 in 9. αeach The-primed effectexperiment. ARPE-19 of hydr Resultsoquinone cell cultures. are (HQ shown Results 200 as μ M;mean were 18 normalizedh)± SEM. exposure **** toPon < the the0.0001, untreated levels Mann-Whitney of controlextracellular group. U- Data test.ATPwere [Kruskal-Wallis in combinedIL-1α-primed from test, ARPE-19 three *** P independent < cell 0.001]. cultures. experiments Results were with normalized four parallel to the samples untreated per control group in each group. Data were combined from three independent experiments with four parallel samples per experiment. Results are shown as mean ± SEM. **** p < 0.0001, Mann-Whitney U-test. 3.7.group Hydroquinone-Induced in each experiment. Results Reduction are shown in PARP as mean Levels ± SEM.Reflect **** DNA P < 0.0001,Damage Mann-Whitney in ARPE-19 CellsU- test.3.7. [Kruskal-WallisDNA Hydroquinone-Induced damage inducestest, *** P NLRP3 < Reduction 0.001]. inflammasome in PARP Levels activation Reflect DNA in human Damage keratinocytes in ARPE-19 [41]. Cells In orderDNA to investigate damage induces hydroquinone-induced NLRP3 inflammasome DNA activationdamage and in humanto estimate keratinocytes the correla- [41]. 3.7. Hydroquinone-Induced Reduction in PARP Levels Reflect DNA Damage in ARPE-19 Cells tionIn orderbetween to investigate IL-18 release hydroquinone-induced and DNA damage, DNAwe determined damage and intracellular to estimate PARP the correlation levels afterbetween DNAhydroquinone damage IL-18 release induces exposure. and NLRP3 DNA Hydroquinone damage,inflammasome we significantly determined activation reduced intracellularin human the levels keratinocytes PARP of the levels DNA- [41]. after repairingInhydroquinone order to PARP investigate protein exposure. hydroquinone-induced in ARPE-19 Hydroquinone cells irrespective significantly DNA damageof the reduced presence and to the estimateof levelsIL-1α (Figurethe of thecorrela- DNA-10). APDCtionrepairing between boosted PARP IL-18 the proteineffect release of in hydroquinoneand ARPE-19 DNA cellsdamage, with irrespective thewe reduction determined of the being presence intracellular statistically of IL-1 PARPα significant(Figure levels 10 ). afterafterAPDC 30hydroquinone min boosted exposure. the exposure. effect In contrast, of hydroquinone Hydroquinone NAC treatm with significantlyent the increased reduction reduced the being PARP the statistically levels levels of indicating the significant DNA- thatrepairingafter it was 30 PARPmin able exposure. to protein alleviate in In ARPE-19DNA contrast, damage. cells NAC irrespective treatment of increased the presence the PARP of IL-1 levelsα (Figure indicating 10). APDCthat itboosted was able the to effect alleviate of hydroquinone DNA damage. with the reduction being statistically significant after 30 min exposure. In contrast, NAC treatment increased the PARP levels indicating that it was able to alleviate DNA damage.

Figure 10. The effect of 200 µM hydroquinone (HQ) on the intracellular PARP level with and without FigureIL-1α 10.-priming The effect and of antioxidants 200 μM hydroquinone (2 µM APDC, (HQ) 5 mM on the NAC) intrac inellular ARPE-19 PARP cells. level IL-1 withα and and untreated without IL-1α-priming and antioxidants (2 μM APDC, 5 mM NAC) in ARPE-19 cells. IL-1α and un- control group were used as the control for hydroquinone treatment. Data were collected from two treated control group were used as the control for hydroquinone treatment. Data were collected independent experiments containing three parallel samples per group. Results are shown as mean ± fromFigure two 10. independent The effect of experiments 200 μM hydroquinone containing (HQ)three onparallel the intrac samplesellular per PARP group. level Results with areand with- p shownoutSEM. IL-1 as **α -primingmean< 0.01, ± SEM. and Mann-Whitney antioxidants** P < 0.01, Mann-Whitney U-test. (2 μM APDC, U-test.5 mM NAC)[Kruskal-Wallis in ARPE-19 test, cells. *** IL-1P < α0.001]. and un- treated4. Discussion control group were used as the control for hydroquinone treatment. Data were collected from two independent experiments containing three parallel samples per group. Results are shown asAMD mean is ± a SEM. consequence ** P < 0.01, of Mann-Whitney RPE cell degeneration U-test. [Kruskal-Wallis that causes photoreceptortest, *** P < 0.001]. cell death and impaired vision [7]. Oxidative stress, a major factor in the pathogenesis of both dry and wet AMD, is a potent activator of the NLRP3 inﬂammasome in RPE cells [7,12–14,18].

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Hydroquinone is a genotoxic and cytotoxic compound that induces ROS production in RPE cells [15–17,42–44]. However, hydroquinone is sensitive to any kind of variation in experimental conditions, such as changes in diluent or medium supplements, which can influence the concentration needed to evoke cytotoxicity [16,17,43,45]. In the present study, signs of cellular membrane rupturing and reduced metabolic activity were evident in IL-1α- primed human RPE cells when the hydroquinone concentrations were greater than 100 µM and 150 µM, respectively. In our previous experiments with ARPE-19 cells without priming, the corresponding hydroquinone concentrations were 125 µM and 200 µM[15]. Thus, our data suggest that if ARPE-19 cells are exposed to the proinflammatory IL-1α cytokine, this makes them more sensitive to the adverse effects of hydroquinone. This finding is in line with the observation that treatment with an IL-1 receptor antagonist reduced hydroquinone-induced apoptosis in keratinocytes by preventing IL-1α activity [46]. NLRP3 inflammasome activation with subsequent caspase-1 activation is known to re- sult in the cleavage of pro-IL-1β and pro-IL-18 into their mature and secreted forms [12,19,20,28]. However, NLRP3 inflammasome activation does not always lead to the secretion of both cytokines [33,39,47]. The accumulation of Alu RNA has been shown to induce IL-18 but not IL-1β release from RPE cells [33]. The outcome in ARPE-19 cells, especially of IL-18 production, appears to be highly context-dependent. HNE induced the secretion of both cytokines but impaired protein clearance favored IL-1β secretion with undetectable IL-18 levels [14,47]. Upon exposure to UVB, the same cell cultures produced both cytokines but only IL-1β was NLRP3 inflammasome-dependent [39]. The present study revealed one more difference i.e., hydroquinone induced IL-18 secretion but had no effect on the production of IL-1β in IL-1α-primed ARPE-19 cells. The propensity of hydroquinone to promote IL-18 production has also been observed in a mouse model of contact hypersensitivity [48], which supports the concept that the regulation of IL-18 release strongly depends on the activation signal and may not be as strictly inflammasome- dependent as is the case for IL-1β. In our previous study, where UVB induced an NLRP3 inflammasome-independent release of IL-18, we observed that it was ROS that were responsible for the release of the cytokine [39]. We wished to examine another DNA-damaging factor and, therefore, we studied hydroquinone. We observed that APDC, but not NAC, reduced IL-18 secretion after the cells were exposed to hydroquinone. We previously showed that both the NADPH oxidase inhibitor APDC and the ROS scavenger NAC can reduce ROS production induced by hydroquinone in RPE cells [15]. The present data suggest that NADPH oxidase could contribute to the IL-18 production evoked by hydroquinone exposure. However, since NAC treatment exerted no response in RPE cells, APDC probably prevented IL-18 release by some other mechanism and not by inhibiting NADPH oxidase-mediated ROS production. Extracellular ATP can protect cells from DNA damage caused by chemical exposure; it mediates this effect by activating P2X7 receptors, leading to NADPH oxidase-mediated activation of caspase-4 and 5 [42,44,49,50]. In human intestinal endothelial cells, caspase-4 has been shown to increase mature form of IL-18 in response to an exposure to enteric pathogens [51]. Our results are line with the concept that the ATP-induced NADPH oxidase mediated caspase-4 or 5 activation could contribute to the production of IL-18, since along with increased IL-18 production, hydroquinone resulted in significant ATP release from RPE cells. In addition, the ability of ATP release to alleviate DNA damage would be compatible with this concept since hydroquinone reduced the PARP levels in RPE cells. PARP participates in the DNA damage repair response and induces poly (ADP-ribose) (PAR) production-related DNA repair [52]. Hydroquinone has been shown to induce DNA damage in human hepatoma HepG2 and TK6 lymphoblastoid cells [42,53,54]. In the latter cells, the extent of the DNA damage correlated with immediately reduced PARP levels as well as with increased PAR production [42]. In this study, we investigated the potential of hydroquinone exposure to induce NLRP3 inflammasome activation in ARPE-19 cells. Hydroquinone induced DNA damage and promoted IL-18 secretion, which was independent from the NLRP3 inflammasome. Since IL- Cells 2021, 10, 1405 12 of 14

18 has been reported to reduce DNA damage in UVB-irradiated mouse keratinocytes [55,56], the present results do not exclude the probability that the release of both ATP and IL-18 in response to hydroquinone exposure would be attempts by the human RPE cells to alleviate hydroquinone-induced DNA damage. That proposal will need to be examined in further studies.

5. Conclusions Hydroquinone induced DNA damage and promoted an NLRP3-independent IL-18 release from RPE cells. NADPH oxidase contributed to IL-18 release but the detailed mechanism still needs clariﬁcation.

Author Contributions: Conceptualization, A.K.; methodology, A.K., N.B. and E.K.; validation, A.K. and N.B.; formal analysis, N.B.; investigation, N.B.; resources, A.K.; data curation, A.K. and N.B.; writing— original draft preparation, A.K. and N.B.; writing—review and editing, A.K., N.B., E.K., Y.M. and K.K.; visualization, A.K. and N.B.; supervision, A.K.; project administration, A.K.; funding acquisition, A.K., K.K. and N.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Academy of Finland (297267, 307341, 328443, 296840 and 333302), the Emil Aaltonen Foundation, the Päivikki and Sakari Sohlberg Foundation, the Kuopio University Hospital (5503743), the Finnish Eye Foundation, The Sigrid Juselius Foundation, Sokeain Ystävät ry, and Silmä- ja Kudospankkisäätiö. The APC was funded by the Academy of Finland (328443). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding authors. Acknowledgments: We warmly acknowledge laboratory technician Anne Seppänen for her valuable support in the laboratory work and Res. Dir. Emeritus Antero Salminen for his valuable collaboration, discussions, and critical review of the manuscript, and Ewen MacDonald for the language revision. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analysis or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References 1. Velilla, S.; García-Medina, J.J.; García-Layana, A.; Dolz-Marco, R.; Pons-Vázquez, S.; Pinazo-Durán, M.D.; Gómez-Ulla, F.; Arévalo, J.F.; Díaz-Llopis, M.; Gallego-Pinazo, R. Smoking and Age-Related Macular Degeneration: Review and Update. J. Ophthalmol. 2013, 2013, 1–11. [CrossRef][PubMed] 2. Khan, J.C.; Thurlby, D.A.; Shahid, H.; Clayton, D.G.; Yates, J.R.W.; Bradley, M.; Moore, A.T.; Bird, A.C. Smoking and Age Related Macular Degeneration: The Number of Pack Years of Cigarette Smoking is a Major Determinant of Risk for both Geographic Atrophy and Choroidal Neovascularisation. Br. J. Ophthalmol. 2006, 90, 75–80. [CrossRef] 3. Fujihara, M.; Nagai, N.; Sussan, T.; Biswal, S.; Handa, J. Chronic Cigarette Smoke Causes Oxidative Damage and Apoptosis to Retinal Pigmented Epithelial Cells in Mice. PLoS ONE 2008, 3, e3119. [CrossRef][PubMed] 4. Huang, C.; Wang, J.J.; Ma, J.H.; Jin, C.; Yu, Q.; Zhang, S.X. Activation of the UPR Protects Against Cigarette Smoke-Induced RPE Apoptosis through Up-Regulation of Nrf2. J. Biol. Chem. 2015, 290, 5367–5380. [CrossRef][PubMed] 5. Bertram, K.M.; Baglole, C.J.; Phipps, R.P.; Libby, R.T. Molecular Regulation of Cigarette Smoke Induced-Oxidative Stress in Human Retinal Pigment Epithelial Cells: Implications for Age-Related Macular Degeneration. Am. J. Physiol. Cell Physiol. 2009, 297, 1200–1210. [CrossRef][PubMed] 6. Bhutto, I.; Gerard, L. Understanding Age-Related Macular Degeneration (AMD): Relationships between the Photorecep- tor/Retinal Pigment Epithelium/Bruch’smembrane/Choriocapillaris Complex. Mol. Aspects Med. 2012, 33, 295–317. [CrossRef] [PubMed] 7. Kaarniranta, K.; Sinha, D.; Blasiak, J.; Kauppinen, A.; Veréb, Z.; Salminen, A.; Boulton, M.E.; Petrovski, G. Autophagy and Heterophagy Dysregulation Leads to Retinal Pigment Epithelium Dysfunction and Development of Age-Related Macular Degeneration. Autophagy 2013, 9, 973–984. [CrossRef] 8. Qin, S.; Rodrigues, G.A. Progress and Perspectives on the Role of RPE Cell Inﬂammatory Responses in the Development of Age-Related Macular Degeneration. J. Inﬂamm. Res. 2008, 1, 49–65. [CrossRef] Cells 2021, 10, 1405 13 of 14

9. Zajac-Pytru, H.M.; Pilecka, A.; Turno-Krecicka, A.; Adamiec-Mroczek, J.; Misiuk-Hojlo, M. The Dry Form of Age-Related Macular Degeneration (AMD): The Current Concepts of Pathogenesis and Prospects for Treatment. Adv. Clin. Exp. Med. 2015, 24, 1099–1104. [CrossRef] 10. Gao, J.; Liu, R.T.; Cao, S.; Cui, J.Z.; Wang, A.; To, E.; Matsubara, J.A. NLRP3 Inflammasome: Activation and Regulation in Age-Related Macular Degeneration. Mediat. Inflamm. 2015, 2015, 1–11. [CrossRef][PubMed] 11. Kauppinen, A.; Kaarniranta, K.; Salminen, A. Potential Role of Myeloid-Derived Suppressor Cells (MDSCs) in Age-Related Macular Degeneration (AMD). Front. Immunol. 2020, 11, 384. [CrossRef] 12. Kauppinen, A.; Paterno, J.; Blasiak, J.; Salminen, A.; Kaarniranta, K. Inflammation and its Role in Age-Related Macular Degeneration. Cell. Mol. Life Sci. 2016, 73, 1765–1786. [CrossRef] 13. Tseng, W.A.; Thein, T.; Kinnunen, K.; Lashkari, K.; Gregory, M.S.; D’Amore, P.A.; Ksander, B.R. NLRP3 Inflammasome Activation in Retinal Pigment Epithelial Cells by Lysosomal Destabilization: Implications for Age-Related Macular Degeneration. Investig. Ophthalmol. Vis. Sci. 2013, 54, 110–120. [CrossRef] 14. Kauppinen, A.; Niskanen, H.; Suuronen, T.; Kinnunen, K.; Salminen, A.; Kaarniranta, K. Oxidative Stress Activates NLRP3 Inflammasomes in ARPE-19 Cells-Implications for Age-Related Degeneration (AMD). Immunol. Lett. 2012, 147, 29–33. [CrossRef] 15. Bhattarai, N.; Korhonen, E.; Toppila, M.; Koskela, A.; Kaarniranta, K.; Mysore, Y.; Kauppinen, A. Resvega Alleviates Hydroquinone-Induced Oxidative Stress in ARPE-19 Cells. Int. J. Mol. Sci. 2020, 21, 2066. [CrossRef][PubMed] 16. Ramírez, C.; Cáceres-del-Carpio, J.; Chu, J.; Chu, J.; Moustafa, M.T.; Chwa, M.; Limb, G.A.; Kuppermann, B.D.; Kenney, M.C. Brimonidine can Prevent in Vitro Hydroquinone Damage on Retinal Pigment Epithelium Cells and Retinal Müller Cells. J. Ocul. Pharmacol. Ther. 2016, 32, 102–108. [CrossRef][PubMed] 17. Moustafa, M.T.; Ramirez, C.; Schneider, K.; Atilano, S.R.; Limb, G.A.; Kuppermann, B.D.; Kenney, M.C. Protective Effects of Memantine on Hydroquinone-Treated Human Retinal Pigment Epithelium Cells and Human Retinal Müller Cells. J. Ocul. Pharmacol. Ther. 2017, 33, 610–619. [CrossRef] 18. Piippo, N.; Korhonen, E.; Hytti, M.; Kinnunen, K.; Kaarniranta, K.; Kauppinen, A. Oxidative Stress is the Principal Contributor to Inflammasome Activation in Retinal Pigment Epithelium Cells with Defunct Proteasomes and Autophagy. Cell. Physiol. Biochem. 2018, 49, 359–367. [CrossRef][PubMed] 19. McKee, C.M.; Coll, R.C. NLRP3 Inflammasome Priming: A Riddle Wrapped in a Mystery Inside an Enigma. J. Leukoc. Biol. 2020, 108, 937–952. [CrossRef] 20. Jha, S.; Ting, J.P.-Y. Inflammasome-Associated Nucleotide-Binding Domain, Leucine-Rich Repeat Proteins and Inflammatory Diseases. J. Immunol. 2009, 183, 7623–7629. [CrossRef] 21. Triantafilou, K.; Morga, P.; Hughes, T.; Triantafilou, M. The Complement Membrane Attack Complex Triggers Intracellular Ca2+ Fluxes Leading to NLRP3 Inflammasome Activation. J. Cell Sci. 2013, 126, 2903–2913. [CrossRef] 22. Di Virgilio, F.; Dal Ben, D.; Sarti, A.C.; Giuliani, A.L.; Falzoni, S. The P2 × 7 Receptor in Infection and Inflammation. Immunity 2017, 47, 15–31. [CrossRef][PubMed] 23. Amores-Iniesta, J.; Barberà-Cremades, M.; Martínez, C.M.; Pons, J.A.; Revilla-Nuin, B.; Martínez-Alarcón, L.; Di Virgilio, F.; Parrilla, P.; Baroja-Mazo, A.; Pelegrín, P. Extracellular ATP Activates the NLRP3 Inflammasome and is an Early Danger Signal of Skin Allograft Rejection. Cell Rep. 2017, 21, 3414–3426. [CrossRef] 24. Sutterwala, F.S.; Haasken, S.; Cassel, S.L. Mechanism of NLRP3 Inflammasome Activation. Ann. N. Y. Acad. Sci. 2014, 1319, 82–95. [CrossRef][PubMed] 25. Dinarello, C. Interleukin 1 and Interleukin 18 as Mediators of Inflammation and the Aging Process. Am. J. Clin. Nutr. 2006, 83, 447S–455S. [CrossRef] 26. Dinarello, C.A.; Novick, D.; Kim, S.; Kaplanski, G. Interleukin-18 and IL-18 Binding Protein. Front. Immunol. 2013, 4, 289. [CrossRef] 27. Zhu, Q.; Kannegant, T. Distinct Regulatory Mechanisms Control Proinflammatory Cytokines IL-18 and IL-1β. J. Immunol. 2017, 198, 4210–4215. [CrossRef] 28. Yang, Y.; Wang, H.; Kouadir, M.; Song, H.; Shi, F. Recent Advances in the Mechanisms of NLRP3 Inflammasome Activation and its Inhibitors. Cell Death Dis. 2019, 10, 128–139. [CrossRef] 29. Wooff, Y.; Man, S.M.; Aggio-Bruce, R.; Natoli, R.; Fernando, N. IL-1 Family Members Mediate Cell Death, Inflammation and Angiogenesis in Retinal Degenerative Diseases. Front. Immunol. 2019, 10, 1618. [CrossRef][PubMed] 30. Dinarello, C.A. Immunological and Inflammatory Functions of the Interleukin-1 Family. Annu. Rev. Immunol. 2009, 27, 519–550. [CrossRef][PubMed] 31. Voronov, E.; Shouval, D.S.; Krelin, Y.; Cagnano, E.; Benharroch, D.; Iwakura, Y.; Dinarello, C.A.; Apte, R.N. IL-1 is Required for Tumor Invasiveness and Angiogenesis. Proc. Natl. Acad. Sci. USA 2003, 100, 2645–2650. [CrossRef] 32. Doyle, S.L.; Campbell, M.; Ozaki, E.; Salomon, R.G.; Mori, A.; Kenna, P.F.; Farrar, G.J.; Kiang, A.; Humphries, M.M.; Lavelle, E.C.; et al. NLRP3 has a Protective Role in Age-Related Macular Degeneration through the Induction of IL-18 by Drusen Components. Nat. Med. 2012, 18, 791–798. [CrossRef] 33. Tarallo, V.; Hirano, Y.; Gelfand, B.; Dridi, S.; Kerur, N.; Kim, Y.; Cho, W.; Kaneko, H.; Fowler, B.; Bogdanovich, S.; et al. DICER1 Loss and Alu RNA Induce Age-Related Macular Degeneration Via the NLRP3 Inflammasome and MyD88. Cell 2012, 149, 847–859. [CrossRef][PubMed] Cells 2021, 10, 1405 14 of 14

34. Ghose, P.; Ali, A.Q.; Fang, R.; Forbes, D.; Ballard, B.; Ismail, N. The Interaction between IL-18 and IL-18R Limits the Magnitude of Protective Immunity and Enhances Pathogenic Responses Following Infection with Intracellular Bacteria. J. Immunol. 2011, 187, 1333–1346. [CrossRef] 35. Schneider, B.E.; Korbel, D.; Hagens, K.; Koch, M.; Raupach, B.; Enders, J.; Kaufmann, S.H.E.; Mittrücker, H.; Schaible, U.E. A Role for IL-18 in Protective Immunity Against Mycobacterium Tuberculosis. Eur. J. Immunol. 2010, 40, 396–405. [CrossRef] 36. Bradford, M.M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248–254. [CrossRef] 37. Bhattarai, N.; Piippo, N.; Ranta-Aho, S.; Mysore, Y.; Kaarniranta, K.; Kauppinen, A. Effects of Resvega on Inflammasome Activation in Conjunction with Dysfunctional Intracellular Clearance in Retinal Pigment Epithelial (RPE) Cells. Antioxidants 2021, 10, 67. [CrossRef][PubMed] 38. Piippo, N.; Korhonen, E.; Hytti, M.; Skottman, H.; Kinnunen, K.; Josifovska, N.; Petrovski, G.; Kaarniranta, K.; Kauppinen, A. Hsp90 Inhibition as a Means to Inhibit Activation of the NLRP3 Inflammasome. Sci. Rep. 2018, 8, 6720–6729. [CrossRef][PubMed] 39. Korhonen, E.; Piippo, N.; Hytti, M.; Hyttinen, J.M.T.; Kaarniranta, K.; Kauppinen, A. Only IL-1β Release is Inflammasome- Dependent upon Ultraviolet B Irradiation although IL-18 is also Secreted. FASEB J. 2020, 34, 6437–6448. [CrossRef] 40. Satoh, E.; Tsukimoto, M.; Kojima, S. Involvement of P2Y Receptors in the Protective Effect of ATP Towards the Cell Damage in HaCaT Cells Exposed to H2O2. J. Toxicol. Sci. 2011, 36, 741–750. [CrossRef] 41. Hasegawa, T.; Nakashima, M.; Suzuki, Y. Nuclear DNA Damage-Triggered NLRP3 Inflammasome Activation Promotes UVB- Induced Inflammatory Responses in Human Keratinocytes. Biochem. Biophys. Res. Commun. 2016, 477, 329–335. [CrossRef] 42. Ling, X.X.; Liu, J.X.; Yun, L.; Du, Y.J.; Chen, S.Q.; Chen, J.L.; Tang, H.W.; Liu, L.H. Poly(ADP-Ribosyl)Ation of Apoptosis Antagonizing Transcription Factor Involved in Hydroquinone-Induced DNA Damage Response. Biomed. Environ. Sci. 2016, 29, 80–84. [CrossRef] 43. Cho, J.Y. Suppressive Effect of Hydroquinone, a Benzene Metabolite, on in Vitro Inflammatory Responses Mediated by Macrophages, Monocytes, and Lymphocytes. Mediat. Inflamm. 2008, 2008, 1–11. [CrossRef] 44. Bolton, J.L.; Trush, M.A.; Penning, T.M.; Dryhurst, G.; Monks, T.J. Role of Quinones in Toxicology. Chem. Res. Toxicol. 2000, 13, 135–160. [CrossRef][PubMed] 45. Lee, J.; Yang, E.; Kim, I. Hydroquinone-Induced Apoptosis of Human Lymphocytes through Caspase 9/3 Pathway. Mol. Biol. Rep. 2012, 39, 6737–6743. [CrossRef][PubMed] 46. Lee, H.; Cheong, K.A.; Kim, J.; Kim, N.; Noh, M.; Lee, A. IL-1 Receptor Antagonist Reduced Chemical-Induced Keratinocyte Apoptosis through Antagonism to IL-1α/IL-1β. Biomol. Ther. 2018, 26, 417–423. [CrossRef] 47. Piippo, N.; Korkmaz, A.; Hytti, M.; Kinnunen, K.; Salminen, A.; Atalay, M.; Kaarniranta, K.; Kauppinen, A. Decline in Cellular Clearance Systems Induces Inflammasome Signaling in Human ARPE-19 Cells. Biochim. Biophys. Acta 2014, 1843, 3038–3046. [CrossRef][PubMed] 48. Bando, K.; Tanaka, Y.; Kuroishi, T.; Sasaki, K.; Takano-Yamamoto, T.; Sugawara, S.; Endo, Y. Mouse Model of Hydroquinone Hypersensitivity Via Innate and Acquired Immunity and its Promotion by Combined Reagents. J. Investig. Dermatol. 2017, 137, 1082–1093. [CrossRef] 49. Aho, J.; Helenius, M.; Vattulainen-Collanus, S.; Alastalo, T.; Koskenvuo, J. Extracellular ATP Protects Endothelial Cells Against DNA Damage. Purinergic Signal. 2016, 12, 575–581. [CrossRef][PubMed] 50. Roberts, J.S.; Yilmaz, Ö. Dangerous Liaisons: Caspase-11 and Reactive Oxygen Species Crosstalk in Pathogen Elimination. Int. J. Mol. Sci. 2015, 16, 23337–23354. [CrossRef] 51. Knodler, L.; Crowley, S.; Sham, H.; Yang, H.; Wrande, M.; Ma, C.; Ernst, R.; Steele-Mortimer, O.; Celli, J.; Vallance, B. Non- Canonical Inflammasome Activation of Caspase-4/Caspase-11 Mediates Epithelial Defenses Against Enteric Bacterial Pathogens. Cell Host Microbe 2014, 16, 249–256. [CrossRef] 52. Sousa, F.G.; Matuo, R.; Soares, D.G.; Escargueil, A.E.; Henriques, J.A.P.; Larsen, A.K.; Saffi, J. PARPs and the DNA Damage Response. Carcinogenesis 2012, 33, 1433–1440. [CrossRef] 53. Liu, J.; Yuan, Q.; Ling, X.; Tan, Q.; Liang, H.; Chen, J.; Lin, L.; Xiao, Y.; Chen, W.; Liu, L.; et al. PARP-1 may be Involved in Hydroquinone-Induced Apoptosis by Poly ADP-Ribosylation of ZO-2. Mol. Med. Rep. 2017, 16, 8076–8084. [CrossRef] 54. Luo, L.; Jiang, L.; Geng, C.; Cao, J.; Zhong, L. Hydroquinone-Induced Genotoxicity and Oxidative DNA Damage in HepG2 Cells. Chem. Biol. Interact. 2008, 173, 1–8. [CrossRef][PubMed] 55. Schwarz, T.; Schwarz, A. DNA Repair and Cytokine Responses. J. Investig. Dermatol. Symp. Proc. 2009, 14, 63–66. [CrossRef] [PubMed] 56. Schwarz, A.; Maeda, A.; Ständer, S.; van Steeg, H.; Schwarz, T. IL-18 Reduces Ultraviolet Radiation-Induced DNA Damage and Thereby Affects Photoimmunosuppression. J. Immunol. 2006, 176, 2896–2901. [CrossRef][PubMed]