Carcinogenicity and mutagenicity of heterocyclic amines in transgenic mouse models.

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc cDNA and mouse metallothionein1 promoter-human TGFalpha cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing both the interaction of nuclear oncogenes and growth factors in tumorigenesis. In addition, these mice provide an experimental model to test how environmental chemicals might interact with the c-myc and TGFalpha transgenes during the neoplastic process. Treatment of the double transgenic mice with both genotoxic agents such as diethylnitrosamine and 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) as well as the tumor promoter phenobarbital greatly accelerated the neoplastic process. To investigate the role of mutagenesis in the carcinogenic process, 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) induced mutagenesis and hepatocarcinogenicity was examined in C57BL/lacZ (Muta Mice) and double transgenic c-myc/lacZ mice that carry the lacZ mutation reporter gene. The MelQx hepatocarcinogenicity was associated with an increase in in vivo mutagenicity as scored by mutations in the lacZ reporter gene. These results suggest that transgenic mouse models may provide important tools for testing both the carcinogenic potential of environmental chemicals and the interaction/cooperation of these compounds with specific genes during the neoplastic process.     

Carcinogenicity of 4-hydroxybutyl-butylnitrosamine in Syrian hamsters.

4-Hydroxybutyl-butylnitrosamine (HBBN), a urinary metabolite of dibutylnitrosamine (DBN) was shown to induce respiratory and bile duct tumors as well as carcinomas of the urinary bladder in Syrian golden hamsters. This contrasts with results previously obtained in rats and demonstrates that HBBN is not a specific bladder carcinogen. Bronchogenic tumors, seen in DBN-treated hamsters, were not observed after injection of HBBN.     

Carcinogenicity of subcutaneously injected N-nitrosoheptamethyleneimine in European hamsters.

Male and female European hamsters (45 of each sex) recieved sc injections once weekly for life of N-nitrosoheptamethyleneimine (NHMI) at one-fifth the median lethal dose (LD50) (females: 44 mg/kg body wt; males: 66 mg/kg body wt), one-tenth the LD50 (females: 22 mg/kg body wt; males: 33 mg/kg body wt), or one-twentieth the LD50 (females: 11 mg/kg body wt; males: 16.5 mg/kg body wt). Survival times for both males and females were dependent on the dose of NHMI. Pulmonary neoplasms were induced in almost all the treated animals. They were histologically diagnosed as adenocarcinomas, squamous cell carcinomas, and mixed cell carcinomas. In addition, nasal cavity tumors developed in all hamsters of all treatment groups; these were papillomas, squamous cell carcinomas, and a few adenocarcinomas. Only 1 tumor of the larynx and 1 tumor of the trachea were observed. Several papillomas and a few carcinomas were also detected in the forestomach. The results were discussed with reference to previous findings in rats and Syrian golden hamsters.     

Sulfotransferase catalyzing sulfation of heterocyclic amines.

Cytosolic sulfation of arylamines to form sulfamates is found to be mediated by sulfotransferases of three gene families (SULT1 to 3). Among them, a SULT3 form (ST3A1) showed a high selectivity for N-sulfation of N-substituted aryl and alicyclic compounds. SULT1 (phenol) and SULT2 (hydroxysteroid) sulfotransferases showed N-sulfating activities of carcinogenic heterocyclic amines. For N-hydroxyarylamine O-sulfation, SULT1 forms showed high activity. In rats, ST1C1 mediated the metabolic activation of N-hydroxyarylamines. However, the related form (ST1C2) in humans showed the negligible activity. Instead, ST1A3 showed high metabolic activating abilities among human sulfotransferases.     

Carcinogenicity of methylated nitrosopiperazines in rats and hamsters

A comparison has been made of the carcinogenic activities ofdinitroso-2,6-dimethylpiperazine (Me₂DNP) and nitroso-3-4,5-trimethylpiperazine(Me₃NP) in F344 rats and Syrian golden hamsters. The compoundswere administered in drinking water to the rats and by gavageto the hamsters, at similar dose rates. As measured by the increasedrate of mortality from tumors induced, Me₂DNP was a more potentcarcinogen in rats than Me₃NP; Me₂DNP induced mainly esophagealtumors, while Me₃NP induced tumors of the nasal cavity, butno esophageal tumors. In contrast, in Syrian hamsters, by thesame criterion, Me₃NP was at least as potent as Me₂DNP. Me₃NPwas more potent in hamsters than Me₂DNP as measured by the numberand multiplicity of tumors induced. The most numerous tumorsinduced by both compounds in hamsters were papillomas of theforestomas, but in addition Me₃NP induced a high incidence oflung tumors. A small number of hamsters treated with eithercompound had liver angiosarcomas. Two hamsters given Me₃NP hadpapillomas of the esophagus, a highly unusual tumor in Syrianhamsters.     

Dietary intake of heterocyclic amines and benzo(a)pyrene: associations with pancreatic cancer.

OBJECTIVE: Heterocyclic amines (HCA) and polycyclic aromatic hydrocarbons, formed in temperature- and time-dependent manners during the cooking of meat, are mutagens and carcinogens. We sought to assess the association between dietary intake of HCA and benzo(a)pyrene [B(a)P] and exocrine pancreatic cancer in a population-based case-control study. METHODS: Subjects (193 cases and 674 controls) provided information on their usual meat intake and preparation method, e.g., stewed, fried, or grilled/barbecued, etc. Meat doneness preferences were measured using photographs that showed internal doneness and external brownness. We used a meat-derived HCA, B(a)P, and mutagen database with a questionnaire to estimate intake of PhIP, DiMeIQx, MeIQx, B(a)P, and mutagenic activity (revertants/g of daily meat intake). Data were analyzed with unconditional logistic regression. RESULTS: In analyses adjusted for age, sex, smoking, education, race, and diabetes, the odds ratio and 95% confidence interval for the highest compared with the lowest quintile were as follows: PhIP, 1.8 (1.0-3.1); DiMeIQx, 2.0 (1.2-3.5); MeIQx, 1.5 (0.9-2.7); B(a)P, 2.2 (1.2-4.0); and mutagenic activity, 2.4 (1.3-4.3). CONCLUSIONS: HCAs and B(a)P from well-done barbecued and pan-fried meats may be associated with increased risk for pancreatic cancer.     

Carcinogenicity of diallylnitrosamine following intragastric administration to Syrian hamsters

Single and multiple intragastric doses of diallylnitrosamine [(DAN) CAS: 16338-97-9] administered to Syrian golden hamsters induced tumors, primarily of the respiratory tract, in which the nasal cavity epithelium was the preferred site. When compared to the effect of DAN after subcutaneous administration at equal doses, the incidence of respiratory tract tumors was lower but that of hepatic tumors was higher, suggesting partial metabolism of DAN in the liver. Comparative metabolic and mutagenesis studies in BD IX rats (which reportedly are refractory to the carcinogenic effects of DAN), in Wistar rats, and in Syrian hamsters showed that a greater proportion of orally administered DAN was exhaled by both rat strains (12-19%) than by hamsters (2-4%). The activity of the microsomal fraction of the hamster liver for metabolizing DAN to allyl alcohol was about 10 times higher than that in rats, whereas no significant species differences were found with the cytosolic fraction. Pretreatment of animals with phenobarbital (PB) or pregnenolone-16 alpha-carbonitrile (PCN) did not influence either microsomal or cytosolic enzyme activities in hamsters, whereas about a tenfold increase in enzyme activities was seen after pretreatment with PB in both rat strains and following PCN in Wistar rats. Moreover, in bacterial mutagenesis assays, hamster liver microsomes were twice as active as those in BD IX rats. The results are discussed in relation to the carcinogenicity of DAN in rats and hamsters.     

Carcinogenicity tests of acetoxymethylphenylnitrosamine and benzenediazonium tetrafluoroborate in Syrian hamsters

The metabolic activation of the esophageal carcinogen methylphenylnitrosamine (MPhN) via α-hydroxylation to hydroxymethylphenylnitrosamine (HO-MPhN) should afford benzenediazonium ion (BDI) as the ultimate electrophilic metabolite. To determine if this proposed activation pathway is accurate, BDI, as its tetrafluoroborate (BF₄) salt, was tested by chronic subcutaneous injection and gavage in Syrian golden hamsters. Acetoxymethylphenylnitrosamine (AMPhN), which is rapidly hydrolyzed to HO-MPhN in vivo, was similarly tested by s.c. injection. AMPhN was weakly carcinogenic, while BDI-BF₄ did not induce a significant tumor incidence by subcutaneous administration. When orally administered, BDI was inactive. Both AMPhN and BDI-BF₄ were mutagenic only in Salmonella typhimurium strain TA1537 without enzymic activation. The parent nitrosamine, MPhN was also mutagenic in TA1537, but only with enzymic activation. The mechanistic and environmental significance of these results are discussed.     

Assessment of the human exposure to heterocyclic amines

Heterocyclic amines are possible human carcinogens and fried meat is an important source of exposure in the Western diet. To study the effect of heterocyclic amines in humans, accurate assessment of individual food consumption is essential. Parameters influencing the intake include the amount and type of meat ingested, frequency of consumption, cooking method, cooking temperature and the duration of cooking. The aim of the present study was to develop a practical method for assessing individual intakes of specific heterocyclic amines in a large sample of people. This has been done by combining information on food consumption and laboratory findings of heterocyclic amines in food products. Diet was assessed using a semi-quantitative food frequency questionnaire including photos of fried meat and, in all, 22 dishes were cooked and chemically analyzed. The method was employed in an elderly population in Stockholm to estimate the daily mean intake of the five heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8- dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8- trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP). The total daily intake ranged from none to 1816 ng, with a mean intake of 160 ng, which is well below estimates reported previously. Highest amounts ingested were of PhIP (mean 72, range 0-865 ng/day) and MeIQx (mean 72, range 0-1388 ng/day), followed by DiMeIQx (mean 16, range 0-171 ng/day), while MeIQ and IQ were ingested only in very small amounts (mean <1 ng/day).      

Genotoxicity of heterocyclic amines in the Salmonella/hepatocyte system

The Salmonella/hepatocyte system was employed to determine the mutagenicity in bacteria as well as the DNA damage induced in mouse hepatocytes following exposure to heterocyclic amines. With hepatocytes from C57BL/6N mice, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) showed a clear mutagenic effect in the Salmonella, while weak mutagenic effects were observed with 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 2-amino-6-methyldipyrido[1,2-a:3',2'-b]imidazole (Glu-P-1), and 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2). All the compounds induced low levels of DNA damage in the hepatocytes. In vivo pretreatment of mice with the potent monooxygenase inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 50 micrograms/kg) clearly increased both the mutagenicity in the bacteria and the DNA damage induced in the hepatocytes in vitro. Glu-P-2 showed the lowest mutagenic effect but induced more DNA damage at low concentrations than the other compounds when TCDD-pretreated hepatocytes were used. These data indicate that the genotoxic potency of Glu-P-2 in the intact hepatocyte differs from that observed in the bacteria. Treatment of hepatocytes with a-naphthoflavone, a selective inhibitor of polycyclic hydrocarbon-inducible cytochrome P-450 form(s), prior to exposure to the heterocyclic amines completely inhibited the mutagenic effect in the bacteria. In vivo administration of all the heterocyclic amines 4 hr prior to isolating the hepatocytes resulted in DNA damage, and this effect was augmented by TCDD pretreatment of mice. Our data suggest that agents modulating the activity and composition of the cytochrome P-450 system may greatly influence both toxicity and carcinogenicity of these heterocyclic amines.     

Prostaglandin hydroperoxidase-dependent activation of heterocyclic aromatic amines

Heterocyclic aromatic amines, derived from the pyrolysis of amino acids and proteins, are potent mutagens in the Ames Salmonella assay with rodent liver activation. Additionally, heterocyclic aromatic amines are multipotent carcinogens. We report evidence that these compounds are substrates for the hydroperoxidase activity of prostaglandin H synthase, as measured by alterations in UV/visible spectra, and are bioactivated to macromolecule-reactive species by this enzyme. Indirect electron paramagnetic resonance studies indicate that this activation may occur via a one-electron mechanism. 2-Amino-3-methylimidazo[4,5f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) are direct-acting mutagens in TA98. The mutagenicity of IQ and MeIQ, but not Trp-P-2, were enhanced by activation with ram seminal vesicle microsomes (a rich source of prostaglandin H synthase). Subsequent experiments utilized the newly constructed tester strain TA1538/1,8-DNP6 (pYG 121), which has enhanced arylamine N-acetyltransferase activity. In this strain IQ, MeIQ and 2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole (Glu-P-1) were mutagenic with ram seminal vesicle microsome activation. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was a weak direct-acting mutagen, and was not activated by the ram seminal vesicles (RSV) system. The responses of IQ and MeIQ were markedly enhanced in TA1538/1.8-DNP6 (pYG 121), relative to TA98. These data are consistent with the involvement of prostaglandin H synthase-catalyzed activation in heterocyclic aromatic amine-induced extrahepatic neoplasia.     

Simple methods for quantifying mutagenic heterocyclic aromatic amines in food products

Two solid-phase extraction methods were developed for the determinationof mutagenic heterocyclic aromatic amines in heated meat products.The copper phthalocyanine (CPC) tandem extraction was performedon coupled cartridges of diatomaceous earth and CPC-derivatizedSephasorb HP, followed by further clean-up on Sephasorb HP.Parts per billion levels of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline(MeIQx) and its homologs as well as 2-amino-3-methylimidazo[4,5-f]quinoline(IQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoline (MeIQ), 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine(PhIP), amino--carboline (AC), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole(Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2),harman (H) and norharman (NH) can then be simultaneously quantifiedby HPLC with UV detection. The propylsulfonyl silica gel (PRS)tandem extraction is a one-step clean-up method on coupled cartridgesof diatomaceous earth and PRS, suitable for the determinationof MeIQx, IQ and their homologs, as well as the glutamic acidpyrolysates 2-amlno-6-methyldipyrido[1,2-a:3',2']imidazole (Glu-P-1)and 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2). 4,7,8-TriMeIQxor 7,8-DiMeIQx were used as internal standards. Four grams ofsample or less are required for analysis. The recovery of theamineswas between 46 and 83% and the detection limit was in the lowp.p.b. range with coefficients of variation ranging between5 and 18%. The major mutagenic contaminant found in meat extractswas MeIQx (from <1 to 44 p.p.b.), followed by 4,8-DiMeIQx(1.3–5 p.p.b.) whereas the major contaminant in friedmeat was PhIP (23–48 p.p.b.), followed by MeIQx (5.1–8.3p.p.b.), AC (3.2–8.9 p.p.b.) and 4,8-DiMeIQx (1.3–2p.p.b.).The co-mutagens NH and H were found in fried meat atlevels of 8.7–19 p.p.b. and 3–4.8 p.p.b. respectively.     

Carcinogenicity test of quercetin and rutin in golden hamsters by oral administration

Quercetin and its glycoside, rutin were tested for carcinogenicityin non-inbred golden hamsters of both sexes. In Experiment I,10% quercetin, 10% rutin, or control diet was given to animalsfor 735 days. In this experiment, tumors appeared mainly inthe forestomach, but the incidence was not statistically differentamong the three groups. Quercetin and rutin were not carcinogenicunder these conditions. In Experiment II, Group 1 was given4% quercetin diet for 709 days. Group 2 was given 1% quercetindiet for 351 days and then the basal diet for 350 days. Group3 was given 1% quercetin diet and then 1% croton oil diet andGroup 4 was given the basal diet followed by 1% croton oil diet,for the same periods as Group 2. Group 5 was given the basaldiet for 701 days. In Experiment II, papillomas of the forestomachappeared in Groups 1, 2, and 5, and papillomatosis in Group3 and 4. There were no statistical differences among experimentalgroups and respective controls. Thus, quercetin was not carcinogenicwhen given at the concentrations of 4% and 1%; even with theadministration of 1% croton oil after 1% quercetin, there wasno increase in tumor incidence.     

A case of pancreatic serous cystadenoma obstructing the distal pancreatic duct.

We present a case of resected serous cystadenoma of the pancreas inducing marked dilatation of the main distal pancreatic duct. A 68-year-old woman, previously diagnosed with chronic pancreatitis, presented with upper abdominal pain. Abdominal US revealed a highly echoic mass in the pancreas. A CT scan disclosed a low density mass in the pancreas and dilatation of the main peripheral pancreatic duct. The mass demonstrated homogeneous and high signal intensity on T2-weighted magnetic resonance imaging (MRI). Selective abdominal arteriography showed the mass strained by the celiac artery. The tumor markers were CEA (2.4 ng/ml) and CA19-9 (6.1 U/ml). After the diagnosis of serous cystadenoma of the pancreas, the patient underwent distal pancreatectomy and splenectomy. The tumor (2.5 cm in diameter) consisted of grayish-white nodules and occupied the body of the pancreas. The tail of the pancreas was atrophic. Histopathological examination of the specimen showed a multilocular lesion containing numerous cysts with the inner surfaces evenly lined by one layer of cuboid or flat epithelial cells which stained positive for periodic acid-Schiff (PAS), evidencing serous cystadenoma. The patient is doing quite well one and a half years after the operation.     

Involvement of free radicals in the formation of heterocyclic amines and prevention by antioxidants.

It was found that free radical intermediates, pyrazine cation radial and carbon-centered radicals, generated in the Maillard reaction of sugars/amino acids, were involved in the production of mutagenic and carcinogenic imidazoquinoquizaline heterocyclic amines. Prevention of the generation of these radical intermediates by phenolic antioxidants effectively inhibited the generation of the heterocyclic amines.