Evaluation of the Mechanisms Involved in the Development of Bladder Toxicity following Exposure to Occupational Bladder Cancer Causative Chemicals Using DNA Adductome Analysis

Abstract

Occupational exposure to aromatic amines (AAs) is an important risk factor for urinary bladder cancer. This study aimed to evaluate the toxicity of AAs and analyze the carcinogenic mechanisms in rat bladder by comprehensive analysis of DNA adducts (DNA adductome). DNA was extracted from the bladder epithelia of rats treated with AAs, including acetoacet-o-toluidine (AAOT) and o-toluidine (OTD), and adductome analysis was performed. Principal component analysis-discriminant analysis revealed that OTD and AAOT observed in urinary bladder hyperplasia could be clearly separated from the controls and other AAs. After confirming the intensity of each adduct, four adducts were screened as having characteristics of the OTD/AAOT treatment. Comparing with the in-house DNA adduct database, three of four candidates were identified as oxidative DNA adducts, including 8-OH-dG, based on mass fragmentation together with high-resolution accurate mass (HRAM) spectrometry data. Therefore, findings suggested that oxidative stress may be involved in the toxicity of rat bladder epithelium exposed to AAs. Consequently, the administration of apocynin, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, in six-week-old rats fed with 0.6% OTD in their diet resulted in simple hyperplastic lesions in the bladder that were suppressed by apocynin. The labeling indices of Ki67, γ-H2AX, and 8-OHdG were significantly decreased in an apocynin concentration-dependent manner. These findings indicate that oxidative stress may have contributed to the development of urinary cancer induced by OTD.

Keywords: DNA adductome; bladder; o-toluidine; oxidative stress.

Figure 1

Histology of the rat urinary bladder epithelium treated with aromatic amines (AAs). Typical histopathological changes by AAs in the urinary bladder epithelium of the (A) untreated control, (B) aniline-treated rats, (C) p-toluidine-treated rats, (D) acetoaceto-o-toluidine-treated rats, and (E) o-toluidine-treated rats. Scale bar = 25 µm.

Figure 2

PCA−DA scores and loading plots. (A) 2D PCA–DA scores of DNA adducts obtained from the adductome analysis. Blue, control; red, OTD; yellow, AAOT; orange, PT; and green, ANL. The clusters are indicated by ellipses: red for OTD, yellow for AAOT, and blue for others. (B) Variable loading plots. Each red spot represents DNA adducts observed in the DNA adductome analysis. The intensity of each DNA adduct enclosed in a square region was confirmed. Arrowheads indicate the candidate characteristic DNA adducts for OTD/AAOT exposure.

Figure 3

Quantitative analysis of three DNA adducts screened as characteristic DNA adducts for OTD/AAOT treatment. The intensity of DNA adducts (A) adduct 32, (B) adduct 242, (C) adduct 489, and (D) adduct 557 screened as characteristic DNA adducts for OTD/AAOT treatment. * p < 0.05, ** p < 0.01, Student’s t-test.

Figure 4

Product ion scan and MS/MS fragmentation data of characteristic DNA adducts. Using HRAM data, a candidate formula of the adduct was automatically calculated using the PeakView^® software (SCIEX), as described above. (A) MS/MS fragmentation data of adduct 32 with a retention time of 2.04 min. The m/z [M + H]⁺ value of the precursor ion was nearly identical to that of 8-OH-dA (m/z [M + H]⁺ 268.1046), and a fragment ion with m/z 136.0606 [M + H]⁺ showing a similar m/z value to adenine with a value of m/z 136.0630 [A + H]⁺ was observed. (B) MS/MS fragmentation data of adduct 242 with a retention time of 2.19 min. The m/z [M + H]⁺ value of the precursor ion was nearly identical to that of 8-OH-dG (m/z [M + H]⁺ 284.0994), and a fragment ion with m/z 152.0566 [M + H]⁺ showing a similar m/z value to guanine with a value of m/z 152.0580 [G + H]⁺ was observed. (C) MS/MS fragmentation data of adduct 489 with a retention time of 1.76 min. The m/z [M + H]⁺ value of the precursor ion was nearly identical to that of 5-OH-dC (m/z [M + H]⁺ 244.0933), and a fragment ion with m/z 112.0504 [M + H]⁺ showing a similar m/z value to cytosine with a value of m/z 112.0518 [C + H]⁺ was observed. (D) MS/MS fragmentation data of adduct 557 with a retention time of 2.67 min. A peak corresponding to the loss of dR moiety (−116.0467) from the precursor ion peak (m/z 290.0860) and daughter ion peak (m/z 391.1090) was observed. An ion peak with m/z [M + H]⁺ 127.0493 was nearly identical to that of thymine moiety (m/z [M + H]⁺ 127.0515).

Figure 4

Product ion scan and MS/MS fragmentation data of characteristic DNA adducts. Using HRAM data, a candidate formula of the adduct was automatically calculated using the PeakView^® software (SCIEX), as described above. (A) MS/MS fragmentation data of adduct 32 with a retention time of 2.04 min. The m/z [M + H]⁺ value of the precursor ion was nearly identical to that of 8-OH-dA (m/z [M + H]⁺ 268.1046), and a fragment ion with m/z 136.0606 [M + H]⁺ showing a similar m/z value to adenine with a value of m/z 136.0630 [A + H]⁺ was observed. (B) MS/MS fragmentation data of adduct 242 with a retention time of 2.19 min. The m/z [M + H]⁺ value of the precursor ion was nearly identical to that of 8-OH-dG (m/z [M + H]⁺ 284.0994), and a fragment ion with m/z 152.0566 [M + H]⁺ showing a similar m/z value to guanine with a value of m/z 152.0580 [G + H]⁺ was observed. (C) MS/MS fragmentation data of adduct 489 with a retention time of 1.76 min. The m/z [M + H]⁺ value of the precursor ion was nearly identical to that of 5-OH-dC (m/z [M + H]⁺ 244.0933), and a fragment ion with m/z 112.0504 [M + H]⁺ showing a similar m/z value to cytosine with a value of m/z 112.0518 [C + H]⁺ was observed. (D) MS/MS fragmentation data of adduct 557 with a retention time of 2.67 min. A peak corresponding to the loss of dR moiety (−116.0467) from the precursor ion peak (m/z 290.0860) and daughter ion peak (m/z 391.1090) was observed. An ion peak with m/z [M + H]⁺ 127.0493 was nearly identical to that of thymine moiety (m/z [M + H]⁺ 127.0515).

Figure 5

Histology of the rat urinary bladder epithelium treated with OTD and/or apocynin. Typical and specific histopathological changes by OTD and/or apocynin in the urinary bladder epithelia of rats from groups (A) treated with OTD, (B) treated with OTD + low dose apocynin, (C) treated with OTD + high dose apocynin, (D) Untreated control, and (E) treated with high dose apocynin. Moderate and mild simple hyperplasia were detected in groups (A) and (B), respectively. One rat in group (C) showed no hyperplasia. Scale bar = 50 µm.

Figure 6

Schematic illustration of the possible genotoxic mechanisms of OTD.