Herman A.J. Schut

Formation of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in male Fischer-344 rats

2-Amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP) is known to induce colon tumors in male Fischer-344 rats. Using 32P-postlabeling assays, we have examined PhIP-DNA adduct formation in various organs and white blood cells (WBCs) of the male Fischer-344 rat 24 h after a single oral dose of 0, 0.5, 5 or 50 mg PhIP/kg. Three PhIP-DNA adducts were detected in WBCs and in all organs, except in the liver and stomach which had only two adducts. The extent of adduct formation was dose-related, but at 0.5 mg/kg no adducts could be detected in any of the organs. At 50 mg/kg, adduct levels, expressed as relative adduct labeling values (RAL x 10(7), or adducts per 10(7) nucleotides assuming complete labeling) were highest in the large intestine (5.66), followed by WBCs (5.04), stomach (1.44), small intestine (1.32), kidney (1.16), liver (0.67) and lungs (0.52). It is concluded that orally administered PhIP forms high levels of specific DNA adducts in the large intestine, the target organ in PhIP carcinogenesis in the male Fischer-344 rat, and that the high level of adducts in WBCs indicates that significant amounts of the ultimate carcinogenic form of PhIP are present in the circulation.

Metabolism of food-derived heterocyclic amines in nonhuman primates.

During the cooking of meats, several highly mutagenic heterocyclic amines (HCAs) are produced. Three HCAs, IQ, MeIQx, and PhIP have been under study for carcinogenicity in cynomolgus monkeys, and to date, IQ has been shown to be a potent hepatocarcinogen. Concomitantly, the metabolic processing of these HCAs has been examined. Metabolism studies show that the potent hepatocarcinogenicity of IQ is associated with the in vivo metabolic activation of IQ via N-hydroxylation and the formation of DNA adducts. In monkeys undergoing carcinogen bioassay with IQ, N-hydroxylation was confirmed by the presence of the N-hydroxy-N-glucuronide conjugate of IQ in urine. The N-hydroxylation of IQ appears to be carried out largely by hepatic CYP3A4 and/or CYP2C9/10, and not by CYP1A2, an isoform not expressed in liver of this species. Notably MeIQx is poorly activated in cynomolgus monkeys and lacks the potency of IQ to induce hepatocellular carcinoma after a 5-year dosing period. The poor activation of MeIQx appears to be due to the lack of constitutive expression of CYP1A2 and an inability of other cytochromes P450, such as CYP3A4 and CYP2C9/10, to N-hydroxylate the quinoxalines. MeIQx is detoxified in monkeys largely by conjugation with glucuronide at the N-1 position. Although the carcinogenicity of PhIP is not yet known, the metabolic data suggest that PhIP will be carcinogenic in this species. PhIP is metabolically activated in vivo in monkeys by N-hydroxylation, as discerned by the presence of the N-hydroxy-N-glucuronide conjugate in urine, bile, and plasma. PhIP also produces DNA adducts that are widely distributed in tissues. The results from these studies support the importance of N-hydroxylation in the carcinogenicity of HCAs in nonhuman primates and by analogy, the importance of this metabolic activation step in the possible carcinogenicity of dietary HCAs in humans.

Indole-3-carbinol as a chemopreventive agent in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) carcinogenesis: inhibition of PhIP–DNA adduct formation, acceleration of PhIP metabolism, and induction of cytochrome P450 in female F344 rats

The chemopreventive properties of dietary indole-3-carbinol (I3C) were evaluated by assessing its effect on DNA adduct formation and metabolism of the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and the induction of cytochromes P450 1A1 and -1A2 in female F344 rats. In experiment 1, animals on I3C diets (0, 0.02% or 0.1%, w/w) were treated by gavage with 1mg/kg/day of PhIP for 23 days. On days 2, 9, 16 and 23, their 24-hr urine was collected and unmetabolized PhIP was measured by GC/MS. On day 24, the animals were sacrificed, and DNA from pancreas, spleen, white blood cells (WBCs), lung, colon, kidney, mammary epithelial cells, caecum, heart, small intestine, liver and stomach was isolated for determination of PhIP-DNA adduct levels by (32)P-postlabelling assays. Except in the mammary gland, I3C diets significantly inhibited PhIP-DNA adduct formation in WBCs and in all organs, ranging from 34.7 to 67.7% with the 0.02% I3C diet to 68.4 to 95.3% with the 0.1% I3C diet. I3C diets also significantly decreased the concentration of urinary unmetabolized PhIP to 29.5-38.4% (0.02% I3C) and 12.8-17.8% (0.1% I3C) of values obtained with the I3C-free diet. In experiment 2, animals were either treated by intubation of I3C at 100 or 200mg/kg for 2 consecutive days or given an I3C-containing diet (0.02% or 0.1%, w/w) for 2 weeks. The expression and activity of cytochromes P450 1A1 and -1A2 were studied by Northern blots, Western blots, and in vitro enzyme determinations. Both the expression and activity of these cytochromes were induced by all of the I3C treatments. It is concluded that, in the female F344 rat, dietary I3C inhibits PhIP-DNA adduct formation and accelerates PhIP metabolism, probably through induction of cytochromes P450 1A1 and -1A2. The chemopreventive properties of I3C in PhIP-induced carcinogenesis are probably mediated through enhancement of PhIP detoxification pathways.

Tea as a Potential Chemopreventive Agent in PhIP Carcinogenesis: Effects of Green Tea and Black Tea on PhIP-DNA Adduct Formation in Female F-344 Rats

The heterocyclic amine 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of proteinaceous animal foods (meat, chicken, and fish). PhIP is a carcinogen in the Fischer 344 (F-344) rat; it induces mammary tumors in female rats and lymphomas and colon and prostate tumors in male rats. In F-344 rats, PhIP forms DNA adducts in various organs, including the target organs. Inhibition of PhIP-DNA adduct formation is likely to lead to inhibition of PhIP tumorigenicity. We have examined the chemopreventive properties of green tea and black tea in PhIP carcinogenesis by evaluating their effects on PhIP-DNA adduct formation in the female F-344 rat. Young adult animals were maintained on powdered AIN-76A diet while receiving regular drinking water or 2% (wt/vol) infusions of green tea or black tea for a total of six weeks. During Weeks 3, 4, and 5, all animals received PhIP by gavage (1 mg/kg/day). Three rats per group were euthanized on Days 1 and 8 after termination of PhIP exposure. DNA was isolated from a number of organs and analyzed for PhIP-DNA adducts by 32P-postlabeling assays. Compared with animals on regular drinking water, PhIP-DNA adduct formation was inhibited in small intestine, colon, liver, and mammary epithelial cells (MECs) of animals receiving green tea or black tea as the sole source of drinking fluid. Green tea inhibited adduct formation in colon, liver, and MECs (33.3-80.0%) on both days, but only on Day 8 (54.4%) in small intestine. Black tea inhibited adduct formation on both days in liver (71.4-80.0%), on Day 1 in colon (40.0%), and on Day 8 in small intestine (81.8%); it had no effect on MEC adducts. Neither green tea nor black tea had an effect on adduct levels in pancreas, lungs, white blood cells, heart, kidneys, spleen, cecum, or stomach. Similarly, these teas did not affect the rate of adduct removal (percent change from Day 1 to Day 8) in any organ. It is concluded that green tea and black tea are potential chemopreventive agents in PhIP-induced tumorigenesis in the F-344 rat.

DNA adducts of heterocyclic amines: formation, removal and inhibition by dietary components

The dietary mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogenic in rodents. In F344 rats PhIP induces mammary tumors in females and colon tumors in males, while IQ induces tumors principally in the liver, Zymbal gland and intestines. In CDF1 mice, IQ induces liver, lung and forestomach tumors. We have evaluated the dynamics of formation, removal and inhibition of PhIP- and IQ-DNA adducts in these rodents. After bolus doses (50 mg/kg, by gavage) of IQ or PhIP, both IQ- and PhIP-DNA adducts were removed rapidly from both target and nontarget organs, while after 3-4 weeks of feeding IQ or PhIP (0.01-0.04%) adduct removal was much slower. Gavaging of male F344 rats with PhIP (0.1-1000 micrograms/kg/day) for 23 days resulted in accumulation of PhIP-DNA adducts in various organs, but adducts were detectable only at 100 or 1000 micrograms/kg/day. Urinary excretion of unchanged PhIP was a constant proportion (1.6-2.1%) of the daily dose over the entire dose range and was independent of duration of exposure. When weanling female F344 rats were exposed to dietary PhIP (0.01-0.04%) for 1-4 weeks, the presence of either conjugated linoleic acid (CLA; 0.1-1.0%) or indole-3-carbinol (13C; 0.1%) in the diet inhibited PhIP-DNA adduct formation (58-99%) in various organs, including the mammary gland and the colon. Similarly, the inclusion of 0.075% 4-ipomeanol (IPO) in the diet of male CDF1 mice exposed for 3 weeks to dietary IQ (0.01%) resulted in inhibition of IQ-DNA adduct formation (30-59%) in the target organs (liver, lungs, stomach) but not in a number of other organs. It is concluded that (1) the rate of PhIP- and IQ-DNA adduct removal depends on the dose and frequency of administration, (2) urinary PhIP may be a good biomarker of recent PhIP exposure and (3) CLA, I3C and IPO are potential chemopreventive agents against PhIP- or IQ-induced tumors in rodents.

DNA adduct formation of the carcinogen 2-amino-3-methylimidazo [4,5-f]-quinoline in target tissues of the F-344 rat

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), a potent bacterial mutagen present in broiled sardines, cooked beef and beef extract, is a carcinogen in the F-344 rat, affecting mainly the liver and small and large intestines. Using 32P-postlabeling assays, the formation of IQ-DNA adducts was examined in male F-344 rats. Twenty-four hours after i.p. doses (5-50 mg IQ/kg) the liver, small and large intestine each showed the presence of 5 adducts, the liver and large intestine having an average of 18.1 and 2.4 times as many adducts, respectively, as the small intestine. None of these adducts could be detected in vehicle-treated animals. It is concluded that IQ forms specific DNA adducts in target tissues of the F-344 rat.

In vivo mutagenicity and DNA adduct levels of heterocyclic amines in Muta™Mice and c-myclacZ double transgenic mice

The cooked meat derived heterocyclic amines (HCAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-9H-pyrido[2,3-b]indole (A alpha C) are established mutagens in the Salmonella assay and hepatocarcinogens in mice. The current study uses transgenic mice to examine hepatic HCA-DNA adduct formation and mutagenesis in vivo and the impact of hepatic overexpression of the c-myc oncogene on HCA-induced mutagenesis. C57B1/lacZ and c-myc/lacZ mice strains, produced by crossbreeding Muta Mice (carrying the lacZ mutation target gene) with either C57B1 control or c-myc transgenic mice, respectively, were treated with 10 daily doses of IQ, MelQx or A alpha C (20 micrograms/g, p.o.). Four weeks after dosing, the frequency of mutations in the lacZ gene in liver of either C57B1/lacZ or c-myc/lacZ mice was significantly higher in mice treated with any one of the three HCAs than in mice given vehicle only. In addition, all three HCAs formed hepatic DNA adducts, as measured by the 32P-postlabeling analysis 24 h after dosing. In both strains of mice, hepatic DNA adduct levels were 2-3-fold higher with A alpha C than with either IQ or MeIQx, although the mutant frequencies in the lacZ gene were 30-40% lower in mice dosed with A alpha C. These results suggest that A alpha C-DNA adducts may be less mutagenic in vivo than either IQ- or MeIQx-DNA adducts. The lacZ mutant frequencies observed with all three HCAs appeared to be influenced by c-myc transgene expression: after HCA treatment, transgenic mice carrying the c-myc gene showed a 30-40% higher lacZ mutant frequency than mice not carrying this transgene. Notably, lacZ mutant frequencies were not different among C57B1/lacZ and c-myc/lacZ mice that received vehicle control. DNA adduct studies showed that the levels of IQ- and MeIQx-DNA adducts were 2-3-fold higher in c-myc/lacZ mice than in C57B1/lacZ mice; however, A alpha C DNA adducts were not statistically different between the two strains. In addition, phase 1 metabolic activation of these HCAs, as assessed by hepatic microsomal mutagenic activation, was also similar in both strains of mice. These results support the notion that overexpression of the c-myc oncogene cooperates with the HCAs to enhance in vivo mutagenicity. Further studies are needed to assess the mechanisms of this cooperative effect.

Effect of CYP1A2 deficiency on heterocyclic amine DNA adduct levels in mice

The contribution of CYP1A2 to the formation of DNA adducts of the cooked meat-derived heterocyclic amines (HCAs) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined in CYP1A2-null (knock-out, KO) and wild-type (WT) mice. IQ (25 mg and 75 mg/kg) and PhIP (150 mg/kg) were administered by gavage to mice and DNA adduct levels in liver, kidney, mammary gland and colon were examined by the 32P-postlabeling assay. Three hours after either dose of IQ, adducts levels in liver and kidney of KO mice were 20-30% of the levels in WT mice, a difference that was statistically significant (Students t-test, P < 0.05). In the colon, adduct levels in KO mice were significantly lower than in the WT mice only at the lowest dose of IQ (1.6+/-0.6 vs 4.6+/-0.7, respectively, relative adduct labeling (RAL) x 10(8), mean+/-S.E.M., n = 3-5 mice). In the mammary gland, however, there was no difference in IQ-DNA adduct levels in KO and WT mice at either dose of IQ. Three hours after dosing with PhIP, PhIP-DNA adduct levels were statistically significantly lower in KO mice than in WT mice in all tissues examined. PhIP-DNA adducts in liver and kidney of WT mice were 9.9+/-1.1 and 22.5+/-6.9, respectively, whereas no PhIP-DNA adducts were detected in either organ of KO mice (limit of detection, 1.4-2.8 x 10(9)). PhIP-DNA adduct levels in mammary gland and colon of WT mice were 47.1+/-9.5 and 58.0+/-21.7, respectively, but accordingly only 3.8+/-0.7 and 5.4+/-0.9 in KO mice. The findings indicate that CYP1A2, responsible for IQ and PhIP N-hydroxylation, the first step in the metabolic action, significantly effects DNA adduct formation in vivo. However, the data raise the possibility that other cytochromes P450 as well as other pathways of activation potentially contribute to DNA adduct formation in specific organs, depending on the HCA substrate.

Inhibition of 2-amino-3-methylimidazo[4,5-ƒ]quinoline-DNA adduct formation in CDF1 mice by heat-altered derivatives of linoleic acid

Grilled ground beef contains a number of carcinogens, including aminoimidazoazaarenes, such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), as well as anticarcinogenic substances, such as heat-generated derivatives of linoleic acid (CLA). In the present study, CLA was administered by gavage every other day to young adult CDF1 mice for a period of 45 days (50 microliters/48 hr for days 1-24 and 100 microliters/48 hr for days 25-45), using trioctanoin as a control. On day 46 all animals received a single oral dose (50 mg/kg) of IQ and tissues were collected 24 hr later. Tissue DNA was purified and analysed for IQ-DNA adducts by 32P-postlabelling assays. Compared with controls, CLA treatment caused a 43.1 and 31.8% inhibition of adduct formation in the livers of male and female mice, respectively. In the lung and large intestine CLA had a 74.2 and 39.4% inhibitory effect, respectively, in the female only, whereas there was no effect in the stomach or small intestine of either sex. In the kidneys of females, CLA treatment inhibited IQ-DNA adduct formation almost completely (95.2%), whereas in the kidneys of males CLA had no effect. It is concluded that CLA inhibits IQ-DNA adduct formation in certain IQ target organs (liver and lung) and non-target organs (large intestine, kidney), but is inactive in other target organs (stomach) and non-target organs (small intestine) of the CDF1 mouse.

A comparison of the lung adenoma response in strain A/J mice after intraperitoneal and oral administration of carcinogens

This study was undertaken to compare the ability of a series of compounds from different chemical classes to induce lung tumors in strain A/J mice after either ip or po administration. 3-Methylcholanthrene, benzo(a)pyrene, urethan, diethylnitrosamine, ethylnitrosourea, and dimethylhydrazine induced a significant (p less than 0.05; t test) increase in the lung tumor response when given both ip and po. 2,4-Dinitrotoluene, 2,6-dinitrotoluene, and a 2:1 mixture of 2,4-dinitrotoluene and 2,6-dinitrotoluene were inactive by both routes of administration and at all dose levels. The lung tumor response to all doses of 3-methylcholanthrene and benzo(a)pyrene, the highest dose of diethylnitrosamine, and the middle doses of both ethylnitrosourea and dimethylhydrazine varied as a function of the route of administration. This finding was most evident for the polycyclic hydrocarbons, e.g., the average number of lung tumors per mouse in animals that received the middle dose of 3-methylcholanthrene or the highest dose of benzo(a)pyrene by the ip route exceeded that by the po route by factors of 12 and 13, respectively. Tissue distribution and elimination studies were conducted in an effort to determine the basis for the observed difference in lung tumor response to 3-methylcholanthrene after ip or po administration. The data indicated that 3-methylcholanthrene persists for longer periods in the animals when given ip, thus potentially providing an extended carcinogenic stimulus. Extrapulmonary lesions observed at a higher than normal frequency at necropsy included peritoneal sarcomas (in 3-methylcholanthrene-treated mice), and both squamous cell carcinomas of the forestomach and abnormal lesions of the liver (in diethylnitrosamine-treated mice).