Multi-DNA Adduct and Abasic Site Quantitation In Vivo by Nano-Liquid Chromatography/High-Resolution Orbitrap Tandem Mass Spectrometry: Methodology for Biomonitoring Colorectal DNA Damage

Abstract

Epidemiological and mechanistic studies suggest that processed and red meat consumption and tobacco smoking are associated with colorectal cancer (CRC) risk. Several classes of carcinogens, including N-nitroso compounds (NOCs) in processed meats and heterocyclic aromatic amines (HAAs) and polycyclic aromatic hydrocarbons (PAHs) in grilled meats and tobacco smoke, undergo metabolism to reactive intermediates that may form mutation-inducing DNA adducts in the colorectum. Heme iron in red meat may contribute to oxidative DNA damage and endogenous NOC formation. However, the chemicals involved in colorectal DNA damage and the paradigms of CRC etiology remain unproven. There is a critical need to establish physicochemical methods for identifying and quantitating DNA damage induced by genotoxicants in the human colorectum. We established robust nano-liquid chromatography/high-resolution accurate mass Orbitrap tandem mass spectrometry (LC/HRAMS²) methods to measure DNA adducts of nine meat and tobacco-associated carcinogens and lipid peroxidation products in the liver, colon, and rectum of carcinogen-treated rats employing fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues. Some NOCs form O⁶-carboxymethyl-2'-deoxyguanosine, O⁶-methyl-2'-deoxyguanosine, and unstable quaternary N-linked purine/pyrimidine adducts, which generate apurinic/apyrimidinic (AP) sites. AP sites were quantitated following derivatization with O-(pyridin-3-yl-methyl)hydroxylamine. DNA adduct quantitation was conducted with stable isotope-labeled internal standards, and method performance was validated for accuracy and reproducibility. Limits of quantitation ranged from 0.1 to 1.1 adducts per 10⁸ bases using 3 μg of DNA. Adduct formation in animals ranged from ∼1 in 10⁸ to ∼1 in 10⁵ bases, occurring at comparable levels in fresh-frozen and FFPE specimens for most adducts. AP sites increased by 25- to 75-fold in the colorectum and liver, respectively. Endogenous lipid peroxide-derived 3-(2-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M₁dG) and 6-oxo-M₁dG adduct levels were not increased by carcinogen dosing but increased in FFPE tissues. Human biomonitoring studies can implement LC/HRAMS² assays for DNA adducts and AP sites outlined in this work to advance our understanding of CRC etiology.

Figure 1.

DNA adducts formed with cooked meat and environmental carcinogens (dR = 2′-deoxyribose).

Figure 2.

DNA adduct levels in MC-dosed rats (PhIP, AαC, MeIQx, 4-ABP, AOM, and AS at 5 mg/kg body weight, and B[a]P at 2.5 mg/kg) in fresh-frozen and FFPE liver, colon, and rectum measured by LC/HRAMS² (*p < 0.05, **p < 0.01, ***p < 0.005; unpaired t-test).

Figure 3.

LC/HRAMS2 EIC (trapping fraction) of DNA adducts in (A) negative control, (B) fresh-frozen MC-treated rat colon, and (C) FFPE MC-treated rat colon. The internal standard level was 5 adducts in 108 bases. RT: Retention time; A: Peak area

Figure 4.

LC/HRAMS² EIC (direct injection fraction) of alkylated DNA adducts in (A) negative control, (B) fresh-frozen MC-treated rat colon, and (C) FFPE MC-treated rat colon. Internal standard level 5 adducts in 10⁸ bases. *Loss of deoxyribose (116.0473 Da) in-source. RT: Retention time; A: Peak area.

Figure 5.

DNA adduct levels in AOM/AS-dosed rats at two lower dosing levels (AS (1 mg/kg and 5 mg/kg) and AOM (0.01 mg/kg and 0.05 mg/kg) in fresh-frozen and FFPE liver, colon and rectum measured by LC/HRAMS² (*p < 0.05, **p < 0.01, ***p < 0.005; unpaired t-test).

Figure 6.

(A) PMOA-derivatized AP site levels in liver, colon, and rectum of rats treated with AOM/AS and (B) LC/HRAMS² EIC of PMOA-dR and [¹³C₅]-PMOA-dR in negative control (NC), MC-treated (5 mg/kg) rat colon, and low level alkylating agent (0.05 mg/kg AOM; 5 mg/kg AS) treated rat colon. Internal standard level 3.3 adducts in 10⁶ bases (*p < 0.05, **p < 0.01, ***p < 0.005; one-way ANOVA with Dunnett’s test). RT: Retention time; A: Peak area.

Scheme 1.

Formation of O⁶MedG, O⁶CMdG, and AP sites from NOCs following P450 bioactivation.