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. Author manuscript; available in PMC: 2022 Jun 1.
Published in final edited form as: Arch Toxicol. 2021 May 3;95(6):2189–2199. doi: 10.1007/s00204-021-03059-3

Cytotoxicity and genotoxicity of the carcinogen Aristolochic acid I (AA-I) in human bladder RT4 cells

Medjda Bellamri 1, Kyle Brandt 1, Christina V Brown 1, Ming-Tsang Wu 2, Robert J Turesky 1
PMCID: PMC8284306  NIHMSID: NIHMS1712669  PMID: 33938965

Abstract

Aristolochic acid (AA-I) induces upper urothelial tract cancer (UUTC) and bladder cancer (BC) in humans. AA-I forms the 7-(2′-deoxyadenosin-N6-yl)aristolactam I (dA-AL-I) adduct, which induces multiple A:T-to-T:A transversion mutations in TP53 of AA-I exposed UTUC patients. This mutation is rarely reported in TP53 of other transitional cell carcinomas and thus recognized as an AA-I mutational signature. A:T-to-T:A transversion mutations were recently detected in bladder tumors of patients in Asia with known AA-I-exposure, implying that AA-I contributes to BC. Mechanistic studies on AA-I genotoxicity have not been reported in human bladder. In this study, we examined AA-I DNA adduct formation and mechanisms of toxicity in the human RT4 bladder cell line. The biological potencies of AA-I were compared to 4-aminobiphenyl, a recognized human bladder carcinogen, and several structurally related carcinogenic heterocyclic aromatic amines (HAA). AA-I (0.05 –10 μM) induced a concentration-and time-dependent cytotoxicity. AA-I (100 nM) DNA adduct formation occurred at over a thousand higher levels than the principal DNA adducts formed with 4-ABP or HAAs (1 μM). dA-AL-I adduct formation was detected down to a 1 nM concentration. Studies with selective chemical inhibitors provided evidence that NQO1 is the major enzyme involved in AA-I bioactivation in RT4 cells, whereas CYP1A1, another enzyme implicated in AA-I toxicity, had a lesser role in bioactivation or detoxification of AA-I. AA-I DNA damage also induced genotoxic stress leading to p53-dependent apoptosis. These biochemical data support the human mutation data and a role for AA-I in BC.

Keywords: Bladder cancer, Aristolochic acid I, Genotoxicity, p53, DNA adducts

Introduction

Aristolochic acid (AA) is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC) (IARC 2012). Elevated exposure to AA causes rapidly progressing interstitial renal fibrosis that evolves into an end-stage renal failure, a pathology named aristolochic acid nephropathy (AAN) (Debelle et al. 2008). AA exposure is also a high-risk factor for developing upper urothelial tract cancer (UUTC) and bladder cancer (BC) (Debelle et al. 2008). AAN is a global disease with cases reported in Belgium, the United Kingdom, France, Croatia, Serbia and Romania, China, and Taiwan (Grollman 2013). In Belgium, AAN occurred in women taking a slimming health supplement inadvertently contaminated with Aristolochia fangchi, a plant species that contains AA (Vanherweghem et al. 1993). In the Balkans, AAN is prevalent in the endemic farming villages along the tributaries of the Danube river where consumption of bread products containing wheat cross-contaminated with Aristolochia clematitis is a likely cause of the disease (Jelakovic et al. 2019). In Asia, AAN and elevated UUTC incidences are attributed to the frequent usage of traditional herbal medicines containing AA (Debelle et al. 2008). Aristolochia species contain a mixture of 8-methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-I) and 6-nitrophenathrene-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-II) as major toxic components (Stiborova et al. 2017). The carcinogenicity of AA-I is marked by its ability to form persistent DNA adducts in rodents and humans (Stiborova et al. 2017; Yun et al. 2014). The dA-AL-I adduct leads to an A:T→T:A transversion at the genome-wide level (Hoang et al. 2013; Moriya et al. 2011) and in the tumor suppressor gene TP53 (Grollman 2013). The mutational spectrum of the TP53 gene in AAN-associated patients with UUTC is dominated by A:T-to-T:A mutations located predominantly on the non-transcribed strand at codons 131, codon 179, and the splice acceptor site for intron 6 as hot spots (Grollman 2013). The A:T-to-T:A transversion is rarely observed in the TP53 mutations of other transitional cell carcinomas reported in the IARC database (http://p53.iarc.fr/) and recognized as an AA-I mutational signature (Grollman 2013; Hollstein et al. 2013). The mutational signature of AA-I was also recently detected in liver and bladder tumors of patients in Taiwan and other Asian countries, indicating AA exposure is widespread and contributes to these malignancies (Ng et al. 2017; Poon et al. 2015)

AA-I and AA-II undergo bioactivation by enzymatic reduction of the nitro moieties of the phenanthrene rings by NAD(P)H:quinone oxidoreductase 1 (NQO1), cytochrome P450 (CYP)1, xanthine oxidase, or prostaglandin H synthase to produce the N-hydroxyaristolactams (HON-AL), the postulated reactive nitrenium intermediates, which covalently bind to DNA and form aristolactam (AL)-DNA adducts (Stiborova et al. 2017). AA-I and AA-II form 7-(2′-deoxyadenosin-N6-yl)aristolactam I (dA-AL-I), 7-(2′-deoxyguanosin-N2-yl) aristolactam I (dG-AL-I), 7-(2′-deoxyadenosin-N6-yl)aristolactam II (dA-AL-II), and 7-(2′-deoxyguanosin-N2-yl)aristolactam II (dG-AL-II) as the main AL-DNA adducts in rodents (Arlt et al. 2002b; Dong et al. 2006; Shibutani et al. 2007). dA-AL-I is the predominant adduct detected in human renal tissues and is associated with the distinctive A:T-to-T:A mutations (Arlt et al. 2002a; Bieler et al. 1997; Grollman et al. 2007; Yun et al. 2014).

The genotoxic effects of AA-I in renal tissues have been studied extensively in rodent and human (Arlt et al. 2007); however, to our knowledge, there are no published studies investigating the biological effects of AA-I in human bladder. In this study, we investigated the capacity of AA-I to form DNA adducts and its mechanisms of toxicity mediated through p53 employing RT4 cells, a well-characterized human bladder cell line (O’Toole et al. 1983). We compared AA-I DNA damage and cytotoxic potency to those of 4-aminobiphenyl (4-ABP), a human bladder carcinogen (Group 1) (IARC 2004), and several structurally related carcinogenic heterocyclic aromatic amines (HAA) formed in cooked meats and tobacco smoke, including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-9H-pyrido[2,3-b]indole (AαC) (Sugimura et al. 2004). Our findings show that AA-I is efficiently bioactivated in RT4 cells leading to DNA adduct formation at levels that are ~1000-fold higher than adduct levels formed by 4-ABP, HAAs or their genotoxic N-hydroxylated metabolites. AA-I bioactivation is mainly carried out by NQO1 while CYP1A1 plays a minor role in RT4 cells. Moreover, the high levels of AA-I DNA adduct formed in RT4 cells resulted in the activation of TP53 leading to the induction of apoptosis.

Materials and methods

Chemicals.

Aristolochic acid I (AA-I), dimethyl sulfoxide (DMSO), ethoxyresorufin, methoxyresorufin, α-naphthoflavone (α-NF), nicotinamide adenine dinucleotide phosphate (NADPH), bovine serum albumin (BSA), ethylenediaminetetraacetic acid (EDTA), β-mercaptoethanol (BME), Dicumarol (DIC), Pifithrin-α (PFT-α), 2,6-Dichlorophenolindophenol (DCPIP), Tween 20, RIPA buffer, RNase A (bovine pancreas), RNase T1 (Aspergillus oryzae), proteinase K (Tritirachium album), sodium dodecyl sulfate (SDS), DNase I (type IV, bovine pancreas), alkaline phosphatase (Escherichia coli), and nuclease P1 (Penicillium citrinum) were purchased from Sigma Aldrich (St. Louis, MO, USA). Halt protease and phosphatase inhibitor, goat anti-mouse secondary antibody, and goat antirabbit secondary antibody were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Primary monoclonal antibodies anti-NQO, GAPDH, P53, and phosphor-P53 were purchased from cell signaling (Danvers, MA, USA). PhIP, MeIQx, and AαC were purchased from Toronto Research Chemicals (Toronto, Canada). N-hydroxyamino-4-aminobiphenyl (HONH-4-ABP) and HONH-HAAs were synthesized as described (Pathak et al. 2016). ES936 was purchased from Santa Cruz Biotechnology (TS, USA). Z-VAD-FMK was purchased from Selleckchem (Houston, TX, USA). Phosphodiesterase I (Crotalus adamanteus venom) was purchased from Worthington Biochemical Corp. (Newark, NJ). 7-(2′-dA-AL-I and [15N5]-dA-AL-I N-(2′-deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8–4-ABP), and [13C10]-dG-C8–4-ABP, N-(2′-deoxyguanosin-8-yl)-AαC (dG-C8-AαC), [13C 10]-dG-C8-AαC, N-(2′-deoxyguanosin-8-yl)-MeIQx(dG-C8-MeIQx),[2H3C]-dG-C8-MeIQx,N-(2′-deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP), and [13C10]-dG-C8-PhIP were synthesized as described (Bessette et al. 2009).

Cell culture.

RT4 cell (ATCC, Manassas, VA) were cultured in McCoy’s 5A medium (ATCC, Manassas, VA) containing 10% fetal calf serum (Sigma Aldrich, St. Louis, MO, USA), penicillin (100 IU/ml) (Gibco, Life Technologies, Carlsbad, CA, USA), and streptomycin (100 μg/ml) (Gibco, Life Technologies, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere of 5% CO2.

Cell Viability Assays.

Cell viability was performed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St Louis, MO, USA). Cells (50 × 104) were seeded in MW96 plates and cultured in complete media. After 48 h, cells were washed with pre-warmed phosphate-buffered saline (PBS) and treated with AA-I (50 nM – 10 μM), or AA-I (5 μM) with various concentrations of chemical inhibitors (α-FN, DIC, PFT-α, or Z-VAD-FMK), or 0.1% DMSO as a solvent control for up to 24 h. After treatment, cells were washed with pre-warmed PBS and incubated with 100 μL of media containing MTT (0.5 mg/ml) at 37°C. After 1 h, the media was removed, and the formazan crystals were dissolved in 100 μL DMSO. The absorbance was measured at 570 nm using a SpectraMax ID3 plate reader (Molecular Devices, San Jose, CA).

Cell treatment for DNA adducts.

Cells (3.0 × 106) were seeded in 20 cm2 plates and cultured for 48 h. At 80–90% confluence, the cells were washed with pre-warmed PBS, and fresh media containing AA-I (1 nM - 100 nM), 4-ABP (1 μM), HAAs (1 μM), HONH-4ABP (1 μM), HONH-HAA (1 μM), AA-I (100 nM) with chemical inhibitor (α-FN, DIC, ES936, PFT-α) or DMSO (0.1% v/v). After treatment periods, cells were washed on ice with ice-chilled PBS. Cells were then scraped with 1 ml ice-chilled PBS and pelleted at 250 g for 10 min at 4 °C. After centrifugation, the PBS was removed, and the cell pellets were stored at −80 °C until DNA extraction.

DNA isolation and enzymatic digestion.

Cell pellets (~ 3 x 106 cells) were homogenized in TE buffer (300 μL, 50 mM Tris-HCl containing 10 mM EDTA, and 10 mM BME, pH 8.0) and DNA was extracted and DNA concentration was measured as described (Bellamri et al. 2018). Ten μg of DNA were spiked with [15N5]-dA-AL-I, [13C10]-dG-C8–4-ABP, [13C10]-dG-C8-AαC, [2H 3C]-dG-C8-MeIQx, or [13C10]-dG-C8-PhIP at a level of 5 adducts per 107 nucleotides and digested with a cocktail of nucleases in 5 mM Bis-Tris-HCl buffer (pH 7.1) (Bellamri et al. 2018).

Ultraperformance liquid chromatography-electrospray ionization multistage scan mass spectrometry (UPLC-ESI/MS3) of DNA adducts.

The DNA adducts were measured by UPLC-ESI/MS3 employing a Dionex Ultimate 3000 LC (Thermo Fisher, San Jose, CA) equipped with a Thermo Acclaim PepMap trap cartridge RP C18 (0.3 x 5 mm, 5 μm particle size, 100 Å), a Michrom Magic C18 AQ column (0.3 ×150 mm, 3 μm particle size), and a Michrom Captive Spray source (Auburn, CA) interfaced with Velos Pro ion trap mass spectrometer (Thermo Scientific, San Jose, CA). The chromatography conditions, MS parameters, and the MS3 transitions used to measure DNA adducts were reported (Bellamri et al. 2019; Yun et al. 2012).

CYP and NQO1 activities.

Ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activity associated with CYP1A1/2 and CYP1A2, respectively (Burke and Mayer 1983) were measured in RT4 cells as described (Bellamri et al. 2019). NQO1 activity was measured in the RT4 cell lysate employing DCPIP as a substrate (Reshetnikova et al. 2016). The reaction mixture consisted of RT4 cell lysate (1 mg/ml) in 25 mM Tris-HCl (pH 7.5) containing 0.01% Tween 20, 0.7 mg/ml BSA, and 80 μM DCPIP in a final volume of 200 μl. The reaction was initiated by the addition of 200 μM NADH. The DCPIP reduction was monitored at 37 °C at 600 nm over 10 min in 30 s intervals. Reaction rates were linear over time and proportional to protein concentration.

Total Protein Extraction and Western Blot Analysis.

Cells were washed with ice-chilled PBS and harvested in RIPA buffer containing 1% Halt protease and phosphatase inhibitor. After 15 minutes of incubation on ice, cell lysates were centrifuged for 20 min at 14,000 g, and the supernatants were harvested. Protein content was measured using Bradford assay (BioRad, Hercules, CA, USA). Twenty μg of protein was premixed with Bolt LDS sample buffer and Bolt sample reducing agent (ThermoFisher Scientific) and heated to 95 °C for 5 min. Proteins were then separated on Bolt 4 to 12% Bis-Tris gels employing Bolt MOPS running buffer and transferred to nitrocellulose membranes employing Bolt transfer buffer (ThermoFisher Scientific). Membranes were blocked for 2 h at room temperature with 5% fat-free milk powder in Tris-buffered saline with 1% (v/v) Tween 20 (TBST buffer) and then incubated overnight at 4 °C with the primary antibody raised against NQO1, p53, phosphor-p53, or GAPDH (1:1000 dilution, in TBST buffer with 5% milk powder). After 3 washes with TBS for 10 min, the membranes were incubated with the secondary antibody diluted 1:2000 in TBST buffer with 5% fat-free milk powder for 1 h at room temperature. Detection was done using Pierce ECL Western Blotting Substrate (ThermoFisher Scientific) and an LI-COR Odyssey Fc imaging system (LI-COR Biotechnology, Lincoln, NE, USA).

Apoptosis assay.

Cells (50 × 104) were seeded in MW96 plate and cultured in complete media. After 48 h, cells were washed with pre-warmed PBS and treated with AA-I (2 μM, 5 μM, 10 μM), or AA-I (5 μM) with various concentrations of PFT-α or Z-VAD-FMK, or 0.1% DMSO. After 16h of treatment, caspase-3/7 activity was monitored over 4 h employing cellevent™ caspase-3/7 green detection reagent according to the manufacturer’s instructions (ThermoFisher Scientific). The fluorescence was measured at absorption/emission maxima of 530 nm and 570 nm respectively using a SpectraMax ID3 plate reader (Molecular Devices, San Jose, CA).

Statistics.

Statistics were performed with Prism 5.03 (GraphPad Software, La Jolla, CA). The statistical significance was determined by the Student’s t-test to determine the effect of treatment within a group. The data are reported as the mean ± SD (* P < 0.05; **P < 0.01, ***P < 0.005 versus control). All studies were performed with at least three independent experiments in triplicate

Results

AA-I cytotoxicity in RT4 cells.

Increasing concentrations of AA-I (0.05 –10 μM) for 24 h resulted in concentration-and time-dependent cytotoxicity in RT4 cells (Fig. 1A and 1B). In contrast, no cytotoxicity was observed in RT4 cells exposed to 4-ABP, AαC, PhIP, or MeIQx at similar concentrations (data not shown).

Fig. 1: AA-I cytotoxicity and DNA adduct formation of AA-I, 4-ABP, AαC, MeIQx, PhIP in RT4 cells.

Fig. 1:

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(A) Concentration- (B) and time-dependent cytotoxicity of AA-I in RT4 cells treated with AA-I (50 nM – 10 μM) for up to 24h. (C) AA-I, 4-ABP, AαC, MeIQx, PhIP and (D) HONH-4-ABP, HONH-AαC, HONH-MeIQx, and HONH-PhIP DNA adduct formation in RT4 cells after 24 h of carcinogen treatment. (E) Concentration- and (F) time-dependent formation of dA-AL-I in RT4 cells treated with AA-I (1 nM – 100 nM) for up to 24 h

DNA adduct formation in RT4 cells.

The DNA adducts formed by AA-I (100 nM), 4-ABP, AαC, MeIQx, and PhIP, or their synthetic HONH-metabolites (1 μM) were measured after 24 h of cell treatment. AA-I undergoes efficient bioactivation in RT4 cells leading to very high levels of DNA adducts compared to 4-ABP, and the HAAs (Fig. 1C). The CYP2A family involved in 4-ABP activation, by N-oxidation, is expressed in RT4 cells, but the CYP1A2 involved in HAA bioactivation is not (Bellamri et al. 2019). Thus, we compared AA-I DNA adduct formation to those levels formed by the direct-acting N-hydroxylated metabolites of 4-ABP, AαC, MeIQx, and PhIP (Fig. 1D). The levels of dA-AL-I adducts formed were ~1000-fold or greater than adduct levels formed with HONH-4-ABP or HONH-HAAs. UPLC-ESI/MS3 chromatograms of the dG-C8 adducts of 4-ABP, AαC, MeIQx, and PhIP adduct and their MS3 scan-stage mass spectra are shown in Supporting Information (Supplemental Fig. 1)

Kinetics and dose effects of AA-I DNA adduct formation in RT4 cells.

The dA-AL-I adduct was formed in RT4 cells within 2 h of treatment with AA-I (100 nM) and increased over 48 h (Fig. 1E). dA-AL-I also formed in a concentration-dependent manner down to 1 nM treatment with AA-I (Fig. 1F).

Role of CYP1 in AA-I bioactivation in RT4 cells.

NQO1 and CYP1 enzymes catalyze the first AA-I bioactivation step through formation of the HONH-AL-I (Scheme 1) (Stiborova et al. 2017). We assessed the role of these enzymes in AA-I bioactivation in RT4 cells. We previously detected EROD activity attributed to CYP1A1/2 (2.46 ± 0.22 pmol/min/mg protein), but not MROD activity (<0.2 pmol/min/mg protein) attributed to CYP1A2 in RT4 cells (Bellamri et al. 2019). AA-I (100 nM) significantly induced EROD activity by 2.5-fold in RT4 cells after 24 h of treatment (Fig 2A). Moreover, treatment with α-NF, a specific CYP1 inhibitor (Tassaneeyakul et al. 1993) resulted in a concentration-dependent inhibition of both basal and AA-I-induced EROD activity in cells (Fig. 2A). α-NF treatment resulted in a modest yet significant concentration-dependent 25% reduction in dA-AL-I levels (Fig. 2B), associated with a modest decrease in AA-I induced cytotoxicity (Fig. 2C). These data demonstrate that CYP1A1 contributes to dA-AL-I adduct formation, but CYP1A1 is not the major bioactivation pathway of AA-I in RT4 cells.

Scheme 1:

Scheme 1:

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Activation and detoxication pathways of AA-I and the role of p53-dependent apoptosis in RT4 cells.

Fig. 2: Role of NQO1 and CYP1 in the bioactivation of AA-I in RT4 cells.

Fig. 2:

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(A) Effects of α-NF, and AA-I on CYP1 activity in RT4 cells after 24 h of treatment. (B) Effect of α-NF on AA-I DNA adduct formation in RT4 cells after 24 h of treatment. (C) Effect of α-NF on AA-I induced cytotoxicity in RT4 cells after 24 h of treatment.(D) Effects of DIC and AA-I on NQO1 activity in RT4 cells lysates after 24h of treatment. (E) Effect of DIC on AA-I DNA adduct formation in RT4 cells after 24 h of treatment. (F) Effects of DIC on AA-induced cytotoxicity in RT4 cells after 24 h of treatment. (G) Effect of DIC and AA-I on NQO1 expression. Western blot analysis of NQO1 in RT4 cells treated with DIC in the absence or the presence AA-I. GAPDH expression served as a loading control. (H) Effects of ES963 and AA-I on NQO1 activity in RT4 cells lysates after 24 h of treatment. (I) Effect of ES963 on AA-I DNA adduct formation in RT4 cells after 24 h of treatment.

Role of NQO1 in AA-I bioactivation in RT4 cells.

DCPIP is a specific substrate to measure NQO1 activity (Reshetnikova et al. 2016). The NQO1 basal activity in RT4 cell lysate was 97.4 ± 2.43 pmol/min/mg protein and increasing concentrations of DIC, a competitive inhibitor of NQO1 (Cheng et al. 2021), resulted in a concentration-dependent inhibition of NQO1 activity (Fig. 2D). DIC (50 μM) decreased NQO1 activity decreased by 65%; higher concentrations of DIC were not employed due to cytotoxicity. α-NF did not alter NQO1 activity. Cell treatment with AA-I (100 nM) did not modulate NQO1 activity; however, co-treatment of cells with DIC (25 or 50 μM) and AA-I (100 nM) lead to a significant concentration-dependent induction of NQO1 activity (Fig. 2D) and a modest increase in NQO1 protein levels (Fig. 2G). DIC and AA-I co-treatment induced NQO1 activity and resulted in a significant concentration-dependent increase in dA-AL-I adduct formation (Fig. 2E) and increased cytotoxicity of AA-I (Fig. 2F). These data signify that NQO1 is a major enzyme in AA-I bioactivation in RT4 cells.

We employed ES936, a mechanism-based specific NQO1 inhibitor to further characterize the role of NQO1 in AA-I bioactivation in RT4 cells (Dehn et al. 2003). ES963 treatment resulted in a potent concentration-dependent decrease in NQO1 activity in RT4 cell lysate. In contrast to the synergistic effects of AA-I with DIC, AA-I did not alter ES963 efficacy to inhibit NQO1 activity (Fig. 2H). The treatment of RT4 cells with ES963 led to concentration-dependent decrease in dA-AL-I adduct levels (Fig. 2I), confirming NQO1 is a major enzyme in AA-I bioactivation in RT4 cells.

Role of p53 in AA-I DNA adducts formation and cytotoxicity.

Previous studies have shown the impact of p53 on the bioactivation of several carcinogens including AA-I, through the modulation of protein expression and enzymatic activity of xenobiotic metabolizing enzymes such as CYP1 and sulfotransferases 1A1 and 1A3 (Krais et al. 2016; Wohak et al. 2018; Wohak et al. 2019). We examined the impact of p53 on AA-I DNA adduct formation and repair in RT4 cells which harbors a wild-type p53 (Cooper et al. 1994). AA-I treatment resulted in the activation of p53 marked by an increased protein level of both total p53 and phosphorylated p53 in RT4 cells (Fig. 3A). PFT-α, a known p53 inhibitor (Walton et al. 2005), was employed to investigate the impact of p53 on AA-I cytotoxicity and DNA adduct formation and repair in RT4 cells. PFT-α resulted in a potent to complete protection against AA-I-induced cytotoxicity in RT4 cells (Fig. 3B) and a concentration-dependent increase in dA-AL-I adduct formation (Fig. 3C). Thus, high levels of dA-AL-I adducts leads to the activation of p53 and cell death.

Fig. 3. AA-I induces a P53 dependent apoptosis in RT4 cells.

Fig. 3.

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(A) Effect of PFT-α and AA-I on p53 and P-p53 expression in RT4 cells. Western blot analysis of p53 and P-p53 in RT4 cells treated with PFT-α in the absence or the presence AA-I. GAPDH expression served as a loading control (B) Effects of PFT-α on AA-I induced cytotoxicity in RT4 cells after 16, 20, and 24 h of treatment. (C) Effects of PFT-α on AA-I bioactivation in RT4 cells after 24 h of treatment. (D) Induction of Caspase 3/7 activity by AA-I in RT4 cells. Caspase 3/7 activity was measured after 16 h of treatment with AA-I for over 240 min. (E) Effects of PFT-α and ZVAD-FMK on AA-I induced caspase 3/7 activity in RT4 cells. Caspase 3/7 activity was measured after 24 h of treatment with AA-I in the presence or the absence of PFT-α and ZVAD-FMK. (F) Effects of ZVAD-FMK on AA-I-induced cytotoxicity in RT4 cells after 16, 20, and 24 h of treatment with AA-I in the presence or the absence of PFT-α.

AA-I induced a p53 dependent apoptosis.

p53 is activated in response to many stress stimuli, including DNA damage. Upon activation, p53 regulates the activities of up to 500 genes that regulate cellular events such as cell arrest, DNA damage repair, and apoptosis (Aubrey et al. 2018). We investigated the potential of AA-I to induce apoptosis in RT4 cells. Apoptosis induction depends on the proteolytic activity of caspase3/7. AA-I treatment resulted in a concentration- and time-dependent increase in caspase3/7 activity in RT4 cells (Fig. 3D). The presence of PFT-α or ZVAD-FMK, a well-known apoptosis inhibitor (Van Noorden 2001), resulted in a concentration-dependent decrease in AA-I-induced caspase3/7 activity (Fig. 3E) and correlated with a potent protection against AA-I-induced cytotoxicity in RT4 cells (Fig. 3F).

Discussion

To our knowledge, this study is the first to reveal the strong cytotoxicity and potent genotoxicity of AA-I in RT4 cells, a well characterized human epithelial bladder cell line (O’Toole et al. 1983). AA-I is efficiently bioactivated compared to the human bladder carcinogen 4-ABP, and structurally related HAA carcinogens AαC, PhIP, and MeIQx or their direct-acting genotoxic N-hydroxylated metabolites in RT4 cells. The mutation-prone dA-AL-I adduct occurred at levels that were up to ~1000 fold higher than the principal dG-C8 adducts formed by 4-ABP, AαC, PhIP, and MeIQx. 3-Nitrobenzanthrone (10 μM) another structurally related carcinogen to AA-I is bioactivated by overlapping metabolic enzymes to form DNA adducts. However, the apparent DNA adduct levels are 100-folder lower than we report here for AA-I (100 nM) in RT4 cells (Reshetnikova et al. 2016)

Prior to our study, AA-I DNA adduct formation was reported in the bladder of C57BL/6 and CH3/He wild-type mice and humanized CYP1A1 and CYP1A2 (hCYP1A1-Cyp1A2) transgenic mice (Sborchia et al. 2019; Shibutani et al. 2007; Stiborova et al. 2012). AA-I requires enzymatic reduction of the nitrophenanthrene ring moiety to covalently bind to DNA (Stiborova et al. 2017). In humans, CYP1A1 and CYP1A2 are major microsomal enzymes involved in AA-I metabolism (Stiborova et al. 2017). Both enzymes catalyze the reduction AA-I to form the genotoxic HON-ALI intermediate but also the O-demethylation of AA-I, leading to its detoxication (Scheme 1). (Stiborova et al. 2015; Stiborova et al. 2012).

We previously reported CYP1A1 but not CYP1A2 activity in RT4 cells (Bellamri et al. 2019). In this study, we observed a significant induction of EROD activity in RT4 cells following treatment with AA-I. CYP1A activity was also induced in the liver and kidney of AA-I- treated mice (Sborchia et al. 2019; Stiborova et al. 2012), but not in the liver of hCYP1A1-Cyp1A2 transgenic mice, or liver, kidney, and lung of Wistar rats (Dracinska et al. 2016; Stiborova et al. 2012). However, the complete inhibition of CYP1A1 activity by α-NF in RT4 cells only led to a 25% decrease in AA-I DNA adduct levels in RT4 cells, demonstrating CYP1A1 is a minor contributor to both AA-I bioactivation and detoxification in this cell line. Renal and lung microsomes of hCYP1 mice showed considerably lower AA-I bioactivation than hepatic hCYP1 mice microsomes (Stiborova et al. 2012). CYP1A2 is exclusively expressed in the liver, while CYP1A1 is mainly expressed in extrahepatic tissues, including the kidney and lung. Thus, these data are consistent with our findings on the minor contribution of CYP1A1 in AA-I bioactivation. The poor detoxification of AA-I by CYP1A1 in RT4 cells is also suggested by the kinetics of dA-AL-I adduct formation, which occurred at a constant rate over 48 h.

NQO1 is the major cytosolic reductase involved in AA-I bioactivation in humans (Stiborova et al. 2017). NQO1 protein and activity were detected in normal and tumoral human bladder biopsies, and several human bladder cell lines (Choudry et al. 2001), and detected in this study using RT4 cells. The employment of DIC as an NQO1 inhibitor resulted in a concentration-dependent decrease of NQO1 activity. However, when used in the presence of AA-I, DIC resulted in an induction of NQO1 activity leading to a significant increase in dA-AL-I levels and AA-I-mediated cytotoxicity in RT4 cells. The induction of NQO1 activity by DIC and AA-I mixture leading to higher AA-I DNA adduct levels was previously observed in Wistar rat liver and kidney (Stiborova et al. 2014). In contrast, Chen et al. report that DIC and AA-I co-exposure in C57BL/6 mice led to lower NQO1 activity providing protection against AA-I-induced nephropathy (Chen et al. 2011). Although DIC treatment did not serve its purpose as an NQO1 inhibitor in RT4 cells treated with AA-I, the induction of NQO1 activity by co-treatment with AA-I reinforces the proposed role of NQO1 as a major enzyme involved in AA-I bioactivation in RT4 cells. These conclusions were further supported by the data obtained with ES963, a mechanism-based specific NQO1 inhibitor (Dehn et al. 2003), which decreased NQO1 activity by 75% leading to ~50% lower levels of dA-AL-I adducts.

HON-AL-I is further bioactivated by SULT1A1, SULT1A3, NAT1, and NAT2 in bacterial cells to form the penultimate intermediates that covalently bind to DNA (Okuno et al. 2019). In contrast, these enzymes do not participate in HONH-AL-I bioactivation in human kidney HK-2 cells, human liver and kidney cytosols, and other phase II enzyme(s) likely contribute to HON-AL-I bioactivation (Scheme I) (Okuno et al. 2019; Stiborova et al. 2011). SULTs and NATs are expressed in human bladder, and studies are warranted to determine if these enzymes are involved in HON-AA-I bioactivation in the bladder (Kirlin et al. 1989; Pacifici et al. 1988).

The tumor suppressor gene, TP53 acts as a gatekeeper of the genome through the regulation of several tumor-suppressive events including cell cycle arrest, DNA repair, and apoptosis to prevent the proliferation of mutated cells (Vousden and Lane 2007). TP53 gene is targeted by AA-I genotoxicity and mutations in human kidney, liver, and bladder (Grollman 2013; Ng et al. 2017; Poon et al. 2015). Our data show that AA-I induces apoptosis through p53 activation in RT4 cells (Scheme 1). Although the exact molecular mechanism connecting AA-I DNA adducts to apoptosis remains unknown, AA-I DNA adducts induce a significant genotoxic stress in RT4 cells, leading to p53 activation by post-translational modifications. Similar observations were reported in vitro in the normal rat kidney cell line (NRK52E) and human renal epithelial cell line (HK-2) but also in vivo with C57BL/6 wild-type and TP53 KO mice (Romanov et al. 2015; Zhou et al. 2010). In contrast, Sborchia et al. reported a significant higher level of renal injury in Tp53 (-/-) C57BL/6 mice compared to Tp53 (+/+) (Sborchia et al. 2019). The discrepancy TP53 genotype effects on the biological outcome of AA-I exposure in mice may be explained by the higher doses of AA-I employed for study by Zhou, leading to more severe DNA damage. In human renal epithelial HK-2 cells, high concentrations of AA-I activate p53 leading to apoptosis while lower concentrations of AA-I activate p53 leading to cell cycle via the activation of ATM, Chk2, p53 and p21 DNA damage checkpoint pathway (Romanov et al. 2015).

NQO1 is polymorphic (Nakajima and Aoyama 2000). In one study, individuals with AAN who harbor the NQO1*2 (C609T) genotype, which is associated with lower NQO1 activity, were unexpectedly at elevated susceptibility to develop UUTC; however, other cohorts showed no elevated risk with this genotype (Stiborova et al. 2016). The impact of NQO1 polymorphisms on TP53 mutations and AA-I associated bladder carcinogenesis requires further study.

Supplementary Material

1712669_Supp_Info

Acknowledgment.

The Turesky laboratory gratefully acknowledges the support of the Masonic Chair in Cancer Causation.

Funding. This research was supported by R01ES030559 (RJT) from the National Institute of Environmental Health Sciences and by R01CA220367 (RJT) from the National Cancer Institute, National Institutes of Health.

Footnotes

Conflict of interest. The authors have no conflict of interest to declare.

Publisher's Disclaimer: This Author Accepted Manuscript is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication but has not been copyedited or corrected. The official version of record that is published in the journal is kept up to date and so may therefore differ from this version.

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