Metabolites of the Carcinogen 2-Amino-α-carboline Formed in Male Sprague−Dawley Rats in Vivo and in Rat Hepatocyte and Human HepG2 Cell Incubates (original) (raw)
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Environmental and chemical carcinogenesis
Seminars in Cancer Biology, 2004
People are continuously exposed exogenously to varying amounts of chemicals that have been shown to have carcinogenic or mutagenic properties in experimental systems. Exposure can occur exogenously when these agents are present in food, air or water, and also endogenously when they are products of metabolism or pathophysiologic states such as inflammation. It has been estimated that exposure to environmental chemical carcinogens may contribute significantly to the causation of a sizable fraction, perhaps a majority, of human cancers, when exposures are related to "life-style" factors such as diet, tobacco use, etc. This chapter summarizes several aspects of environmental chemical carcinogenesis that have been extensively studied and illustrates the power of mechanistic investigation combined with molecular epidemiologic approaches in establishing causative linkages between environmental exposures and increased cancer risks.
Chemical Research in Toxicology, 2016
2-Amino-9H-pyrido[2,3-b]indole (AαC) is the most abundant carcinogenic heterocyclic aromatic amine (HAA) formed in mainstream tobacco smoke. AαC is a liver carcinogen in rodents, but its carcinogenic potential in humans is not known. To obtain a better understanding of the genotoxicity of AαC in humans, we have investigated its metabolism and its ability to form DNA adducts in human hepatocytes. Primary human hepatocytes were treated with AαC at doses ranging from 0.1 to 50 µM and the metabolites were characterized by UPLC/ ion trap multistage mass spectrometry (UPLC/MS n). Six major metabolites were identified: a ring-oxidized doubly conjugated metabolite, N 2-acetyl-2-amino-9H-pyrido[2,3b]indole-6-yl-oxo-(β-D-glucuronic acid) (N 2-acetyl-AαC-6-O-Gluc); two ring-oxidized glucuronide (Gluc) conjugates: 2-amino-9H-pyrido[2,3-b]indol-3-yl-oxo-(β-D-glucuronic acid) (AαC-3-O-Gluc) and 2-amino-9H-pyrido[2,3-b]indol-6-yl-oxo-(β-D-glucuronic acid) (AαC-6-O-Gluc); two sulfate conjugates, 2-amino-9H-pyrido[2,3-b]indol-3-yl sulfate (AαC-3-O-SO 3 H) and 2-amino-9H-pyrido[2,3-b]indol-6-yl sulfate (AαC-6-O-SO 3 H); and the Gluc conjugate, N 2-(β-D-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N 2-Gluc). In addition, four minor metabolites were identified: N 2-acetyl-9H-pyrido[2,3-b]indol-3-yl sulfate (N 2-acetyl-AαC-3-O-SO 3 H); N 2-acetyl-9H-pyrido[2,3-b]indol-6-yl sulfate (N 2-acetyl-AαC-6-O-SO 3 H), N 2-acetyl-2-amino-9H-pyrido[2,3-b]indol-3-yl-oxo-(β-D-glucuronic acid) (N 2-acetyl-AαC-3-O-Gluc), and O-(β-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3b]indole (AαC-HN 2-O-Gluc). The latter metabolite, AαC-HN 2-O-Gluc is a reactive intermediate which binds to DNA to form the covalent adduct N-(2′-deoxyguanosin-8-yl)-2amino-9H-pyrido[2,3-b]indole (dG-C8-AαC). Pre-incubation of hepatocytes with furafylline, a selective mechanism-based inhibitor of P450 1A2, resulted in a strong decrease in the formation of AαC-HN 2-O-Gluc and a concomitant decrease in DNA adduct formation. Our findings describe the major pathways of metabolism of AαC in primary human hepatocytes Page 3 of 41 ACS Paragon Plus Environment Chemical Research in Toxicology and reveal the importance of N-acetylation and glucuronidation in metabolism of AαC. P450 1A2 is a major isoform involved in the bioactivation of AαC to form the reactive AαC-HN 2-O-Gluc conjugate and AαC-DNA adducts.
Toxicological Sciences, 2013
Tobacco smoking is a risk factor for cancers of the liver and gastrointestinal (GI) tract, but the causal agents responsible for these cancers are uncertain. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is an abundant heterocyclic aromatic amine present in tobacco smoke. AαC is a liver carcinogen and both a transgene mutagen and inducer of aberrant crypt foci in the colon of mice. We hypothesize that AαC may contribute to DNA damage and tumorigenesis in these organs of smokers. The potential of AαC to induce DNA adduct formation in liver, organs of the GI tract, lung, and urinary bladder, which are target organs of cancer in smokers, was examined using the C57BL/6 mouse as an animal model. AαC (400 or 800 ppm) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) (300 ppm), a liver and colon carcinogen in C57BL/6 mice, were given in the diet for up to 12 weeks. Liquid chromatography/mass spectrometry was employed to measure DNA adducts. The major DNA adducts of both carcinogens were identified as deoxyguanosine-C8 adducts. The levels of formation of AαC-and MeIQ-DNA adducts were similar in liver and extrahepatic tissues when adjusted for dose. The highest levels of adducts occurred in liver, followed by urinary bladder, and then in cecum and colon; lower DNA adduct levels were formed in the lung and pancreas following 12 weeks of feeding. The high levels of AαC adduct formed in liver, GI tract, and bladder of C57BL/6 mice reinforce the notion that AαC may contribute to DNA damage and cancer of these organs in smokers.
Mutagenesis, 2010
To elucidate the mechanism underlying suppression of N-nitrosobis(2-oxopropyl)amine (BOP)-induced hamster pancreatic carcinogenesis by cigarette smoke (CS), hepatic levels of microsomal cytochrome P450 (CYP) enzymes, mutagenic activation of environmental carcinogens and three types of uridine diphosphate-glucuronyltransferase (UDPGT) and sulphotransferase (ST) activities were assayed in male Syrian golden hamsters and F344 rats exposed to CS. Immunoblot analyses of microsomal CYP proteins revealed induction of constitutive CYP1A2 (2.6fold increase) and 2A8 (4.0-fold increase) and induction of CYP1A1 and constitutive CYP1A2 (3.9-fold increase) in rats following exposure to CS for 4 weeks using a Hamburg type II smoking machine. CS exposure enhanced mutagenicities of four heterocyclic amines in the presence of liver S9 in both species, whereas the mutagenicities of aflatoxin B 1 (AFB 1), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAaC) and 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK) were significantly increased by CS in hamsters but not in rats. However, no CS-induced alterations in the mutagenic activities of other carcinogens, including BOP and other pancreatic carcinogens, were observed in either species. Application of several CYP inhibitors revealed that the mutagenic activities of MeAaC, AFB 1 and NNK in the hamster liver S9 were partly associated with CYP2A8, whereas those of the three pancreatic carcinogens were selectively associated with CYP2B. CS enhanced UDPGT activities towards 4-nitrophenol (4-NP) (1.9-to 2.0-fold) but did not affect those of bilirubin, testosterone UDPGTs and three STs in both species. Together with the previous findings that BOP does not induce tumourigenesis in rats and that the glucuronidation of b-oxypropylnitrosamines is higher in rats than in hamsters, suppression of BOP-induced pancreatic carcinogenesis by CS might be attributed to increased detoxification by 4-NP UDPGT and not decreased CYP2B activation. This is the first demonstration of the induction of CYP2A protein by CS; CYP2A protein polymorphisms have been associated with oral and pulmonary carcinogenesis in smokers.
DNA Adduct Formation of 4-Aminobiphenyl and Heterocyclic Aromatic Amines in Human Hepatocytes
Chemical Research in Toxicology, 2011
DNA adduct formation of the aromatic amine, 4-aminobiphenyl (4-ABP), a known human carcinogen present in tobacco smoke, and the heterocyclic aromatic amines (HAAs), 2-amino-9Hpyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), potential human carcinogens, which are also present in tobacco smoke or formed during the hightemperature cooking of meats, was investigated in freshly cultured human hepatocytes. The carcinogens (10 μM) were incubated with hepatocytes derived from eight different donors for time periods up to 24 h. The DNA adducts were quantified by liquid chromatography-electrospray ionization mass spectrometry with a linear quadrupole ion trap mass spectrometer. The principal DNA adducts formed for all of the carcinogens were N-(deoxyguanosin-8-yl) (dG-C8) adducts. The levels of adducts ranged from 3.4 to 140 adducts per 10 7 DNA bases. The highest level of adduct formation occurred with AαC, followed by 4-ABP, then by PhIP, MeIQx, and IQ. Human hepatocytes formed dG-C8-HAA-adducts at levels that were up to 100-fold greater than the amounts of adducts produced in rat hepatocytes. In contrast to HAA adducts, the levels of dG-C8-4-ABP adduct formation were similar in human and rat hepatocytes. These DNA binding data demonstrate that the rat, an animal model that is used for carcinogenesis bioassays, significantly underestimates the potential hepatic genotoxicity of HAAs in humans. The high level of DNA adducts formed by AαC, a carcinogen produced in tobacco smoke at levels that are up to 100-fold higher than the amounts of 4-ABP, is noteworthy. The possible causal role of AαC in tobaccoassociated cancers warrants investigation.
Urinary metabolites as indicators of human exposure to chemical carcinogens
Zdravstvena zastita, 2021
Population exposure to environmental chemical carcinogens is a growing public health problem. Carcinogenic chemicals may be classified into two groups: genotoxic and non-genotoxic. A genotoxic chemical has a potential to induce the development of cancer, either in direct interaction with DNA or with cell structures, which are responsible for the maintenance of genome integrity. A non-genotoxic chemical has a potential to induce cancer indirectly by entering the processes of cancer etiopathogenesis. Previous research studies indicate that inorganic arsenic compounds may be associated with various malign diseases (lung cancer, urinary bladder cancer, skin, kidney, liver and prostate cancer). Inorganic arsenic is mainly present in meat, dairy products and grains, while organic arsenic (arsenobetaine) is present in seafood, fruit and vegetables. Benzene metabolites are associated with different types of leukemias and lymphomas, benzidine with bladder cancer, nickel with lung cancer, chr...
Chemical & pharmaceutical bulletin, 2016
The major toxicants in cigarette smoke, α,β-unsaturated aldehydes, such as acrolein (ACR) and crotonaldehyde (CA), and α,β-unsaturated ketone, methyl vinyl ketone (MVK), are known to form Michael-type adducts with glutathione (GSH) and consequently cause intracellular GSH depletion, which is involved in cigarette smoke-induced cytotoxicity. We have previously clarified that exposure to cigarette smoke extract (CSE) of a mouse melanoma cell culture medium causes rapid reduction of intracellular GSH levels, and that the GSH-MVK adduct can be detected by LC/MS analysis while the GSH-CA adduct is hardly detected. In the present study, to clarify why the GSH-CA adduct is difficult to detect in the cell medium, we conducted detailed investigation of the structures of the reaction products of ACR, CA, MVK and CSE in the GSH solution or the cell culture medium. The mass spectra indicated that in the presence of the cells, the GSH-CA and GSH-ACR adducts were almost not detected while their c...
Chemical Research in Toxicology, 1990
The metabolism of 2-amino-3,8-dimethylimidazo[4,5-flquinoxaline, a potent bacterial mutagen and rodent carcinogen formed in low quantities in cooked meat and fish, was studied in freshly isolated rat hepatocytes. Ten metabolites were characterized by various spectroscopic methods. Sulfamate formation was the major route of metabolism in hepatocytes of untreated rats whereas ring-hydroxylated sulfuric and glucuronic acid conjugates were major metabolites in animals pretreated with the enzyme inducers Aroclor-1254,P-naphthoflavone, or isosafrole. The formation of a mutagenic metabolite through N-oxidation, 2-(hydroxyamino)-3,8-dimethylimidazo[4,5flquinoxaline (HNOH-MeIQx), was a n important route of metabolism in hepatocytes of pretreated animals. Its metastable derivative, the N-hydroxy-N-glucuronide, also was detected. T h e nitro derivative of MeIQx, a direct-acting bacterial mutagen, was readily detoxified by glutathione transferase, forming a conjugate where the thiol group of glutathione displaced the nitro moiety. Low but detectable levels of N-acetyltransferase activity were observed for MeIQx and sulfamethazine in hepatocytes. HNOH-MeIQx and 4-(hydroxyamino)biphenyl (HNOH-ABP), a recognized human carcinogen, displayed acetyl coenzyme A dependent DNA binding in hepatic cytosol assays. Sulfamethazine decreased the DNA binding of HNOH-MeIQx in hepatocytes, suggesting a competition for acetyltransferase. However, the binding of HNOH-MeIQx to DNA in hepatocytes was independent of sulfotransferase since inhibitors of this enzyme, 2,6-dichloro-4-nitrophenol (DCNP) and pentachlorophenol (PCP), did not diminish DNA binding. In contrast, binding of HNOH-ABP to DNA was not decreased by sulfamethazine, but binding was diminished by both sulfotransferase inhibitors. From these inhibition experiments it appears that a major route of binding of HNOH-MeIQx to DNA in hepatocytes is mediated through 0-acetyltransferase while a significant portion of HNOH-ABP bound to DNA is catalyzed by sulfotransferase.