Nuclear DNA and Mitochondrial Damage of the Cooked Meat Carcinogen 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Human Neuroblastoma Cells.

Animal fat and iron-rich diets are risk factors for Parkinson's disease (PD). The heterocyclic aromatic amines (HAAs) harman and norharman are neurotoxicants formed in many foods and beverages, including cooked meats, suggesting a role for red meat in PD. The structurally related carcinogenic HAAs 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-9H-pyrido[2,3-b]indole (AαC) also form in cooked meats. We investigated the cytotoxicity, DNA-damaging potential, and mitochondrial damage of HAAs and their genotoxic HONH-HAA metabolites in galactose-dependent SH-SY5Y cells, a human neuroblastoma cell line relevant for PD-related neurotoxicity. All HAAs and HONH-HAAs induced weak toxicity except HONH-PhIP, which was 1000-fold more potent than the other chemicals. HONH-PhIP DNA adduct formation occurred at 300-fold higher levels than adducts formed with HONH-MeIQx and HONH-AαC, assuming similar cellular uptake rates. PhIP-DNA adduct levels occurred at concentrations as low as 1 nM and were threefold or higher and more persistent in mitochondrial DNA than nuclear DNA. N-Acetyltransferases (NATs), sulfotransferases, and kinases catalyzed PhIP-DNA binding and converted HONH-PhIP to highly reactive ester intermediates. DNA binding assays with cytosolic, mitochondrial, and nuclear fractions of SH-SY5Y fortified with cofactors revealed that cytosolic AcCoA-dependent enzymes, including NAT1, mainly carried out HONH-PhIP bioactivation to form N-acetoxy-PhIP, which binds to DNA. Furthermore, HONH-PHIP and N-acetoxy-PhIP inhibited mitochondrial complex-I, -II, and -III activities in isolated SH-SY5Y mitochondria. Mitochondrial respiratory chain complex dysfunction and DNA damage are major mechanisms in PD pathogenesis. Our data support the possible role of PhIP in PD etiology.

Anthracyclines React with Apurinic/Apyrimidinic Sites in DNA.

The combination of doxorubicin (Adriamycin) and cyclophosphamide, referred to as AC chemotherapy, is commonly used for the clinical treatment of breast and other cancers. Both agents target DNA with cyclophosphamide causing alkylation damage and doxorubicin stabilizing the topoisomerase II-DNA complex. We hypothesize a new mechanism of action whereby both agents work in concert. DNA alkylating agents, such as nitrogen mustards, increase the number of apurinic/apyrimidinic (AP) sites through deglycosylation of labile alkylated bases. Herein, we demonstrate that anthracyclines with aldehyde-reactive primary and secondary amines form covalent Schiff base adducts with AP sites in a 12-mer DNA duplex, calf thymus DNA, and MDA-MB-231 human breast cancer cells treated with nor-nitrogen mustard and the anthracycline mitoxantrone. The anthracycline-AP site conjugates are characterized and quantified by mass spectrometry after NaB(CN)H3 or NaBH4 reduction of the Schiff base. If stable, the anthracycline-AP site conjugates represent bulky adducts that may block DNA replication and contribute to the cytotoxic mechanism of therapies involving combinations of anthracyclines and DNA alkylating agents.

DNA Damage and Oxidative Stress of Tobacco Smoke Condensate in Human Bladder Epithelial Cells.

Smoking is a major risk factor for bladder cancer (BC), with up to 50% of BC cases being attributed to smoking. There are 70 known carcinogens in tobacco smoke; however, the principal chemicals responsible for BC remain uncertain. The aromatic amines 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA) are implicated in BC pathogenesis of smokers on the basis of the elevated BC risk in factory workers exposed to these chemicals. However, 4-ABP and 2-NA only occur at several nanograms per cigarette and may be insufficient to induce BC. In contrast, other genotoxicants, including acrolein, occur at 1000-fold or higher levels in tobacco smoke. There is limited data on the toxicological effects of tobacco smoke in human bladder cells. We have assessed the cytotoxicity, oxidative stress, and DNA damage of tobacco smoke condensate (TSC) in human RT4 bladder cells. TSC was fractionated by liquid-liquid extraction into an acid-neutral fraction (NF), containing polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, phenols, and aldehydes, and a basic fraction (BF) containing aromatic amines, heterocyclic aromatic amines, and N-nitroso compounds. The TSC and NF induced a time- and concentration-dependent cytotoxicity associated with oxidative stress, lipid peroxide formation, glutathione (GSH) depletion, and apurinic/apyrimidinic (AP) site formation, while the BF showed weak effects. LC/MS-based metabolomic approaches showed that TSC and NF altered GSH biosynthesis pathways and induced more than 40 GSH conjugates. GSH conjugates of several hydroquinones were among the most abundant conjugates. RT4 cell treatment with synthetic hydroquinones and cresol mixtures at levels present in tobacco smoke accounted for most of the TSC-induced cytotoxicity and the AP sites formed. GSH conjugates of acrolein, methyl vinyl ketone, and crotonaldehyde levels also increased owing to TSC-induced oxidative stress. Thus, TSC is a potent toxicant and DNA-damaging agent, inducing deleterious effects in human bladder cells at concentrations of <1% of a cigarette in cell culture media.

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

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), which are present in urine of smokers and omnivores. 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 bio-activation in RT4 cells, whereas CYP1A1, another enzyme implicated in AA-I toxicity, had a lesser role in bio-activation 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.