Covalent binding of the carcinogen trichloroethylene to hepatic microsomal proteins and to exogenous DNA in vitro

Studies were carried out on the in vitro covalent binding of the carcinogen trichloroethylene (TCE) to liver microsomal preparations and to exogenous DNA. The binding of TCE to liver microsomal proteins of male C57BL/6 X C3H/He F1 (hereafter called B6C3F1) hybrid mice, a species and strain susceptible to TCE-induced liver tumorigenesis, was 46% higher than that of [14C]TCE to microsomal proteins from male Osborne-Mendel rats, a species and strain resistant to TCE-induced hepatocellular carcinoma. The in vitro binding of [14C]TCE to liver microsomal proteins was 37% higher for male B6C3F1 mice; female B6C3F1 mice that have been reported to show a lower incidence of TCE-induced hepatocellular carcinoma than do males. Microsomal proteins from the lung, stomach, and kidney of B6C3F1 hybrid mice also metabolized TCE, as indicated by the covalent binding of [14C]TCE to microsomal proteins from these organs. For rats the binding of TCE to liver microsomal proteins of Sprague-Dawley animals was higher than that of Osborne-Mendel and Fischer 344 rats. Incubation of [14C]TCE with salmon sperm DNA in the presence of microsomal preparations from B6C3F1 hybrid mice resulted in covalent binding of [14C]TCE to DNA. This binding was much higher in the presence of microsomal proteins from male rather than female mice. The binding to DNA and protein was enhanced by in vivo phenobarbital administration. The effects of 1,2-epoxy-3,3,3-trichloropropane on the covalent binding of [14C]TCE to protein and DNA were also examined.     

The effect of dose and enzyme inducers on the metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rats.

Male Fischer 344 rats were given a single dose of 0.03-30 mg/kg of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ([14C]PhIP), the radioactivity in urine and feces was determined over 48 h, and the major metabolites were identified and quantified. Dose had little effect on the profile of metabolites in the urine but did influence the profile in the feces. PhIP was more efficiently metabolized at higher doses. In addition, rats were pretreated with Aroclor 1254 (PCB), 3-methylcholanthrene (MC), phenobarbital (PB), PhIP and corn oil prior to a single dose of [14C]PhIP, and compared with a control group receiving [14C]PhIP only. The major metabolites in the urine and feces were quantitated for each group, as well as PhIP binding to serum proteins, hemoglobin and selected tissues. Pretreatment with MC and PCB resulted in an increase in the amount of 4'-hydroxylation of PhIP and a decrease in the amount of N-hydroxylated metabolites in the urine. Pretreatment with PB resulted in an increase in the amount of N-hydroxylated metabolites, but a decrease in 4'-hydroxylation. Pretreatment with either MC or PCB resulted in an increase in PhIP binding to the liver and kidney, while reducing the binding in other tissues. Animals pretreated with PhIP showed few significant differences from the untreated group, while pretreatment with PB in general resulted in a decrease of PhIP binding in tissues.     

Fate and distribution of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in mice at a human dietary equivalent dose.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine rodent carcinogen that is found at the ppb level in cooked meat. Most laboratory studies are at 10(4)-10(7)-fold greater concentrations than actual ingested human doses. We report the first study of the bioavailability and fate of this heterocyclic amine at a human dietary equivalent dose using the high sensitivity offered by accelerator mass spectrometry. [2-14C]PhIP was administered to C57BL/6 male mice (41 ng/kg) by gavage. Tissues and excreta were collected over the subsequent 96 h. One hundred % of the administered dose was excreted in urine (90%) and feces (10%) over the length of the study. Absorption of the radiocarbon-tagged PhIP from the gastrointestinal tract was rapid, with radiocarbon levels peaking in the whole blood and urine within 1 h of exposure. Fecal 14C levels peaked at 12 h. Tissue levels peaked by 3 h with the highest concentrations of radiolabel in the intestine, stomach, and liver, followed by the kidney, pancreas, lung, and spleen. Low levels of 14C from PhIP (0.01-0.04% of the administered dose) could be detected in the tissues 48-96 h after exposure, possibly due to covalent binding to protein or DNA. The calculated half-life of PhIP at this dose was 1.14 h. This study is the first example of how accelerator mass spectrometry can be used to gather biological information about carcinogenic compounds at environmental levels of exposure.     

Disposition and metabolism of aniline in Fischer 344 rats and C57BL/6 X C3H F1 mice

We examined the metabolism and disposition of aniline, which induces spleen hemangiosarcomas in rats but no tumors in mice, in normal and predosed Fischer 344 rats, and C57BL/6 X C3H F1 mice administered low (50 and 100 mg/kg, respectively) or high (250 and 500 mg/kg, respectively) doses. Of 11 tissues examined, the highest levels of binding of [14C]aniline to DNA were in the kidney, large intestine, and spleen of high-dose rats that had received prior dosing; these tissues had covalent binding indices of 14.2, 4.3, and 3.7 mumol/mol nucleotides/dose, respectively. Protein and RNA were the major macromolecular targets for binding of radioactivity from [14C]aniline. Relative to controls, most tissues from predosed mice (low dose and high dose) showed less binding to protein and RNA; but for most tissues from predosed rats administered 50-mg/kg doses of [14C]aniline, there was more extensive binding. Also relative to controls, binding of radioactivity in the spleen of predosed rats given [14C]aniline (50 mg/kg) was 148% greater for protein and 302% greater for RNA. For rats administered 250 mg of [14C]aniline per kg, however, there were no outstanding differences in binding to RNA and protein between normal and predosed animals. The profiles of urinary metabolites produced by rats and mice were not appreciably different in animals predosed with aniline. For rats, however, the profiles were different for the low and high doses, suggesting that the main metabolic pathway was saturated at the higher dose. p-Acetamidophenyl sulfate represented over 70% of the total radioactivity recovered from the urine of rats dosed with 50 mg of aniline per kg but only 30% in the urine of those dosed with 250 mg/kg. The urine of the high-dose rats contained greater percentages of p-aminophenyl sulfate, p-acetamidophenyl glucuronide, and unconjugated metabolites. In mouse urine, p-acetamidophenyl glucuronide, representing 29 to 32% of the total radioactivity, was the major metabolite. Nevertheless, mice produced more ortho derivatives than did rats, for in acid-treated urine, the ratio of p- to o-aminophenol was 8.1 for rats and 1.6 for mice. Predosing of rats and mice did not change the kinetic values for liver aniline p-hydroxylase or N-hydroxylase but increased the amount of mouse liver cytochrome P-450 from 0.231 to 0.491 nmol/mg protein. For p-hydroxylase of rat liver, the apparent Km value was higher, and the apparent Vmax value lower than in mouse liver. Kinetic values for rat and mouse N-hydroxylase were similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in mice.

The metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP), a heterocyclic amine carcinogen detected in cooked meats, was investigated in mice. In 3-methylcholanthrene-induced mice administered 0.1, 1.0 and 10 mg/kg [14C]PhIP (i.p.), urinary and fecal excretion over 24 h accounted for 16% and 42-56% of the dose respectively. Urinary excretion of unchanged parent compound accounted for only 0.5-0.8% of the administered dose. At all doses, the major urinary metabolite was identified as 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate and this metabolite comprised approximately 5% of the dose. Uninduced mice excreted greater than 13% of a 10 mg/kg dose as the sulfate conjugate. Urinary excretion of both 2-amino-1-methyl-6-(4'-hydroxy)-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP) and a glucuronide conjugate of 2-hydroxyamino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (N-hydroxy-PhIP) was also higher (4-fold) in uninduced versus induced mice. The decreased urinary excretion of P450-derived metabolites via induction contrasted with increased metabolite formation by hepatic microsomal preparations. 4'-Hydroxy-PhIP and N-hydroxy-PhIP were produced in amounts nearly 7- and 3-fold higher respectively by induced versus uninduced microsomal incubations at 50 microM [3H]PhIP. At concentrations less than 10 microM, PhIP was almost exclusively converted by the induced preparations to an unidentified metabolite that was not retained by the C18 column. This metabolite, which also was formed in incubations with either 4'-hydroxy-PhIP or N-hydroxy-PhIP, was produced by microsomes from uninduced animals at a much slower rate. Covalent binding to microsomal protein in incubations with [3H]PhIP was concentration-dependent and 2- to 4-fold higher in induced than uninduced preparations. Covalent binding in liver and kidney of induced mice administered [14C]PhIP was dose dependent. At 10 mg/kg PhIP, adducts were produced at 1.7-fold higher levels in livers of induced versus uninduced mice, but renal binding was higher in uninduced animals. These studies indicate the importance of cytochrome P450 and other xenobiotic enzymes in the metabolism, disposition and activation of PhIP.     

Autoradiographic distribution of 14C-labeled 3H-imidazo[4,5-f]quinoline-2-amines in mice

The highly mutagenic heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), are formed during heating of protein-rich foods. In order to gain information about the distribution and fate of IQ and MeIQ in vivo, a whole-body autoradiographic study of i.v.-injected 14C-labeled IQ and MeIQ has been performed in male NMRI, pregnant NMRI, and female C3H mice. IQ and MeIQ showed similar distribution patterns. At short survival times, the autoradiograms were characterized by an accumulation of radioactivity in metabolic and excretory organs (liver, kidney, bile, urine, gastric and intestinal contents, salivary glands, nasal mucosa, and Harder's gland), as well as in lymphomyeloid tissues (bone marrow, thymus, spleen and lymph nodes) and in endocrine and reproductive tissues (adrenal medulla, pancreatic islets, thyroid, hypophysis, testis, epididymis, seminal vesicles, ampulla, and prostate). The liver and kidney cortex were identified as sites of retention of nonextractable radioactivity. IQ and MeIQ showed a strong affinity for melanin. IQ and MeIQ passed the placenta, but no radioactivity was retained in fetal tissues. The results pinpoint the liver as a site of IQ- and MeIQ-mediated toxicity. Future studies of IQ and MeIQ may be guided by and clarify the role of other tissue localizations in the toxicity of IQ and MeIQ.     

The identification of [2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine metabolites in humans

[2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ([14C]PhIP), a putative human carcinogenic heterocyclic amine found in well-done cooked meat, was administered orally to three colon cancer patients undergoing a partial colonectomy. Forty-eight to seventy-two hours prior to surgery, subjects received a 70-84 microg dose of 14C. Urine and blood were analyzed by HPLC for PhIP and PhIP metabolites. Metabolites were identified based on HPLC co-elution with authentic PhIP metabolite standards, mass spectral analysis and susceptibility to enzymatic cleavage. In two subjects, approximately 90% of the administered [14C]PhIP dose was eliminated in the urine, whereas in the other, only 50% of the dose was found in the urine. One subject excreted three times more radioactivity in the first 4 h than did the others. Twelve radioactive peaks associated with PhIP were detected in the urine samples. The relative amount of each metabolite varied by subject, and the amounts of each metabolite within subjects changed over time. In all three subjects the most abundant urinary metabolite was identified as 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine-N2-glucuron ide (N-hydroxy-PhIP-N2-glucuronide), accounting for 47-60% of the recovered counts in 24 h. PhIP accounted for <1% of the excreted radiolabel in all three patients. Other metabolites detected in the urine at significant amounts were 4-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate, N-hydroxy-PhIP-N3-glucuronide and PhIP-N2-glucuronide. In the plasma, N-hydroxy-PhIP-N2-glucuronide accounted for 60, 18 and 20% of the recovered plasma radioactivity at 1 h post PhIP dose in subjects 1, 2 and 3 respectively. Plasma PhIP was 56-17% of the recovered dose at 1 h post exposure. The relatively high concentration of N-hydroxy-PhIP-N2-glucuronide and the fact that it is an indicator of bioactivation make this metabolite a potential biomarker for PhIP exposure and activation. Determining the relative differences in PhIP metabolites among individuals will indicate metabolic differences that may predict individual susceptibility to carcinogenic risk from this suspected dietary carcinogen.     

Fate and distribution of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in rats

Fischer 344 rats were given a single dose of 0.60 mg/animal of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by gavage; and radioactivity contained in feces, urine, blood, serum proteins, hemoglobin, and tissues was determined at 12, 24, 48 and 96 h after dosing. One major and four minor radioactivity-containing fractions were found in the urine and one major and two minor radioactivity-containing fractions were found in the feces. The feces was the major route of excretion, representing 78% of dose during the first 24 h, and unchanged PhIP in the feces accounted for 51% of the dose. Unmetabolized PhIP was also shown to be the major radioactive fraction in bile and feces from animals given a single dose by i.p. injection. Blood contained a small fraction of the dose and the major, persistently-bound form of PhIP in the blood was to hemoglobin. At 12 h after administration of the dose the colon and cecum contained the highest concentration of radioactivity, while at later times the kidney and liver showed the highest concentration. Of the tissue-contained radioactivity 80-90% was ethanol insoluble at time points later than 24 h, suggesting that it was covalently bound to macromolecules.     

Distribution of the carcinogenic tryptophan pyrolysis product Trp-P-1 in control, 9-hydroxyellipticine and {beta}-naphthoflavone pretreated mice

Autoradiograms obtained 1–4 h after i.v. injection ofthe ¹⁴C-labelled carcinogenic tryptophan pyrolysis product Trp-P-1to albino and pigmented mice showed a pronounced uptake of radioactivityin the lymphatic system (thymus, lymphnodes, bone marrow andspleen), in the endocrine system (hypophysis, thyroid, adrenalmedulla) and in the liver, kidney medulla and brain. High radioactivitywas present in the excretory pathways, predominantly in thebile/intestinal contents. At longer post-injection times (24h to 6 days) most of the labelled substance had left the tissues,except for the liver which still retained a high concentrationof radioactivity. Trp-P-1 is known to be activated by cytochromeP-448. The uptake of radioactivity in the liver could be reducedby pretreatment with the cylochrome P448 inhibitor 9-hydroxy-ellipticinesuggesting that the observed accumulation of radioactivity inthe liver was partly due to metabolites of Trp-P-1. After pretreatmentwith the cytochrome P-448 inducer ß-naphthoflavone,the administration of Trp-P-1 resulted in a highly selectiveaccumulation of radioactivity in the lung parenchyma, exceedingall other tissues in the body. ß-Naphthoflavone pretreatmentalso increased the uptake of radioactivity in the kidney cortexand small intestinal mucosa. As indicated by a high labellingof the pigmented tissues of the maternal and fetal eye, thecarcinogen and/or its metabolites were accumulated in melanin.     

Anti-tumor activity of a 3-oxo derivative of oleanolic acid

We have studied the impact of genetic background on susceptibility to spontaneous or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced intestinal tumorigenesis. The increase in small intestinal tumor number after PhIP exposure was 3.8- and 3.7-fold above the spontaneous levels in multiple intestinal neoplasia (Min)/+ F1 mice with AKR/J and A/J backgrounds, respectively, compared with only 3-fold in C57BL/6J mice. In the colon, PhIP increased the number of tumors slightly more in C57BL/6J mice (3.3-fold) than in A/J mice (3.0-fold). AKR/J mice had no colonic tumors. Most of the tumors were located in the distal two-thirds of the small intestine in all three strains.     

Strain-dependent differences in the metabolism of 3-methylcholanthrene by maternal, placental, and fetal tissues of C57BL/6J and DBA/2J mice

C57BL/6J (B6) and DBA/2J (D2) mice have different susceptibilities to developmental toxicity and transplacental carcinogenesis induced by in utero exposure to polycyclic aromatic hydrocarbons, which has been associated with polycyclic aromatic hydrocarbon metabolism and inducibility at the Ah locus. The distribution of total 3-methylcholanthrene (3-MC)-associated radioactivity in maternal, placental, and fetal tissues of beta-naphthoflavone-pretreated pregnant B6 and D2 mice was determined up to 12 h after p.o. exposure to [6-14C]-3-MC (63 mg/kg, 20 mu Ci) on gestational day 17. 3-MC-associated radioactivity in maternal plasma was not significantly different in the two strains. However, D2 tissue homogenates had consistently higher levels of 3-MC-associated radioactivity, which included both bound and free parent compound and metabolites. Increased metabolism of 3-MC by B6 maternal liver was suggested by the induced levels of aryl hydrocarbon hydroxylase activity in that tissue and by the observation that levels of total radioactivity decreased more rapidly in B6 tissues than in D2 tissues. The D2 fetal lung, the target tissue for 3-MC-induced transplacental carcinogenesis, appeared to accumulate 3-MC-associated radioactivity for a longer period of time than either the D2 fetal liver or the B6 fetal tissues. This study suggests that the genetic differences in fetal susceptibility to the developmental toxicity and transplacental carcinogenesis of 3-MC may be related to the presystemic elimination of the compound from both maternal and fetal tissues.     

Autoradiographic distribution of [14C]-labelled pimonidazole in rhabdomyosarcoma-bearing rats and pigmented mice

Summary The hypoxic cell radiosensitizer [2-¹⁴C] pimonidazole (2-nitro--(piperidinomethyl)-l-imidazole ethanol) was injected i.p. into pigmented mice and rats bearing transplanted rhabdomyosarcoma. The injected dose level was 200 mg/kg, and the delivered activity was 96 Ci/kg. Whole-body autoradiography was carried out on all animals. We noted an extensive whole-body distribution of radioactivity. At short intervals, the autoradiograms were characterized by an accumulation of radioactivity in the metabolic and exretory organs (liver, kidney, urinary tract, and intestinal content) as well as in lymphomyeloid tissues (thyroid gland, suprarenal gland, and hypophysis) and salivary glands. In pigmented mice, the uveal and biliary tracts were the highest labelled. The liver and particularly the renal medulla were identified as sites of retention of radioactivity. In the tumor the radioactivity was detected only in peripheral regions, with higher uptake in viable zones than in necrotic islets.Abbreviations used MISO misonidazole - [¹⁴C] ou [¹⁴C] Ro, [¹⁴C] Ro 03-8799 plus Ro 03-8799-derived [¹⁴C]-labelled metabolites     

Curcumin modifies Apc(min) apoptosis resistance and inhibits 2-amino 1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced tumour formation in Apc(min) mice

Curcumin, the active ingredient of the rhizome of Curcuma longa, promotes apoptosis and may have chemopreventive properties. This study investigates the effects of curcumin on apoptosis and tumorigenesis in male Apc(min) mice treated with the human dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Intestinal epithelial apoptotic index in response to PhIP treatment was approximately twice as great in the wild-type C57BL/6 APC(+/+) strain than in Apc(min) mice (3.7% Apc(+/+) versus 1.9% Apc(min); P < 0.001). PhIP promoted tumour formation in Apc(min) proximal small intestine (4.6 tumours per mouse, PhIP treated versus 2.1 tumours per mouse, control untreated; P < 0.05). Curcumin enhanced PhIP-induced apoptosis (4.0% curcumin + PhIP versus 2.1% PhIP alone; P < 0.01) and inhibited PhIP-induced tumorigenesis in the proximal small intestine of Apc(min) mice (2.2 tumours per mouse, curcumin + PhIP versus 4.6 tumours per mouse PhIP alone; P < 0.05). This study shows that the Apc(min) genotype is associated with resistance to PhIP-induced apoptosis in intestinal epithelium. Curcumin attenuates Apc(min) resistance to PhIP-induced apoptosis and inhibits PhIP-induced tumorigenesis in proximal Apc(min) mouse small intestine.     

Induction of intestinal tumors and lymphomas in C57BL/6N mice by a food-borne carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic amine contained in cooked meat and fish. Although PhIP has been demonstrated to induce various types of tumors in rats, lymphomas predominated in mice using the CDF1 strain. To investigate the carcinogenic activity of PhIP on other organs in mice with a different genetic background, PhIP was administered to C57BL / 6N mice. After a 40-week administration of 300 ppm of PhIP in a high-fat diet followed by continuous feeding with a high fat diet, C57BL / 6N mice developed adenomas and adenocarcinomas in the small intestine, the incidences being 52% in males and 68% in females at weeks 95 and 70, respectively. Lymphomas of B-cell origin also developed in both sexes as frequently as in the CDF1 strain, incidences being 48% in males and 32% in females. Although the incidence in PhIP-treated female mice did not differ from that in the control mice, lymphomas developed significantly earlier in the PhIP-treated mice. The present study demonstrated that the intestinal tract is another potential target of PhIP-induced carcinogenesis in mice, and that the carcinogenic activity of PhIP could be affected by the genetic background of the animals.     

Dose-response study on covalent binding to forestomach protein from male F344 rats following oral administration of [14C]3-BHA.

A dose-response study on covalent binding to forestomach protein was performed using male F344 rats following oral administration of [14C]3-tert-butyl-4-hydroxyanisole (3-BHA). The order of tissue distribution of radioactivity 6 h after oral administration of 1% [14C]3-BHA was forestomach greater than glandular stomach greater than liver greater than kidney greater than plasma. The covalent binding levels to forestomach protein were very low until 0.1% 3-BHA, but rapidly increased at concentrations of 1% and 2% 3-BHA. The dose-response relations of 3-BHA levels to the covalent binding to protein coincided well with the incidence of forestomach papilloma reported previously. The binding levels of forestomach and glandular stomach were compared. In case of i.v. administration, both binding levels were almost the same, however, in case of 0.1% p.o. administration, the forestomach level was approximately 8-fold higher than the glandular stomach level. The binding level of forestomach protein by p.o. administration was approximately 54-fold higher than that by i.v. administration. Although the amount of tert-butylhydroquinone (BHQ) was very low compared with the amount of covalent binding, the BHQ levels in forestomach were dependent upon the dose levels of 3-BHA. Our study indicates that the dose-response study on covalent binding to target tissue protein is an efficient method for the quantitative estimation of the active metabolites coming from the chemicals which form the quinone metabolites.     

The fate of [14C]streptozotocin in nicotinamide-pretreated mice: observations on pancreatic islet radioactivity and urinary N1-methyl-14C]nicotinamide-excretion.

A high labelling of the pancreatic islets was found 3 and 24 h after a diabetogenic dose of [14C]streptozotocin to mice in which the acids islet injury had been prevented by nicotinamide-pretreatment. In non-pretreated [14C]streptozotocin-injected mice, a much lower radioactivity was observed in the pancreatic islets; at 3 h and at 24 h, there was no detectable radioactivity in the islets. No evidence was found to indicate that nicotinamide-pretreatment had any marked effect on the uptake or retention of radioactivity in other tissues. N1-[methyl-14C]nicotinamide was not found in the urine of non-pretreated [14C]streptozotocin-injected mice. When the animals were pretreated with nicotinamide, N1-[methyl-14C]nicotinamide was detected in the urine, but this represented only a small fraction of the injected radioactivity and of the excreted N1-methylnicotinamide. This result does not support the hypothesis that the disturbance of the NAD-metabolism, which streptozotocin causes, is due to a methylation of nicotinamide.     

Induction of Intestinal Tumors and Lymphomas in C57BL/6N Mice by a Food-borne Carcinogen, 2-Amino-l-methyl-6-phenylimidazo[4,5-b]pyridine

2-Amino-l-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP) is the most abundant heterocyclic amine contained in cooked meat and fish. Although PhIP has been demonstrated to induce various types of tumors in rats, lymphomas predominated in mice using the CDF1 strain. To investigate the carcinogenic activity of PhIP on other organs in mice with a different genetic background, PhIP was administered to C57BL/6N mice. After a 40-week administration of 300 ppm of PhIP in a high-fat diet followed by continuous feeding with a high fat diet, C57BL/6N mice developed adenomas and adenocarcinomas in the small intestine, the incidences being 52% in males and 68% in females at weeks 95 and 70, respectively. Lymphomas of B-cell origin also developed in both sexes as frequently as in the CDF1 strain, incidences being 48% in males and 32% in females. Although the incidence in PhIP-treated female mice did not differ from that in the control mice, lymphomas developed significantly earlier in the PhIP-treated mice. The present study demonstrated that the intestinal tract is another potential target of PhIP-induced carcinogenesis in mice, and that the carcinogenic activity of PhIP could be affected by the genetic background of the animals.     

Mouse strain differences in inflammatory responses of colonic mucosa induced by dextran sulfate sodium cause differential susceptibility to PhIP-induced large bowel carcinogenesis

In mice, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces a high incidence of malignant lymphoma and leukemia, but exhibits little, if any, carcinogenic activity in the large intestine after long-term exposure. However, recent studies have revealed that colonic adenocarcinomas can be efficiently and rapidly induced by combined treatment with PhIP and dextran sulfate sodium (DSS), a potent inducer of colitis. In the present study, the authors investigated the effects of inflammation on PhIP-induced carcinogenesis using two mouse strains, C57BL/6J and MSM/Ms, showing distinct temporal profiles of inflammatory responses to DSS. A long-term carcinogenesis experiment conducted with a single i.g. administration of PhIP (200 mg/kg body weight), followed by DSS treatment in drinking water for 4-6 days, revealed an increase in tumor incidence in C57BL/6J mice in accordance with the DSS intake. In contrast, neoplastic lesions were rarely observed in the MSM/Ms strain. From the short-term exposure to DSS for 4 days, C57BL/6J mice demonstrated severe chronic colitis, accompanied by hyperplastic cryptal epithelium and extensive cellular infiltration. Splenomegaly and swelling of mesenteric lymph nodes were also evident for over 1 month as chronic symptoms of systemic immunological disturbance. However, no inflammatory lesions were detected in MSM/Ms mice. The present results provide strong evidence that prolonged chronic inflammatory responses induced by DSS are directly responsible for the observed enhancement of PhIP-induced large bowel carcinogenicity.     

Metabolic activation of the food mutagen Trp-P-1 in endothelial cells of heart and kidney in cytochrome P450-induced mice

Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole) is a carcinogenmetabolized by hepatic cytochrome P4501A (P4501A). This studyshowed that there was a highly selective solvent-resistant bindingof radioactive substance in endothelial cells of heart and kidney1 day following injection of [³H]-Try-P-1 (0.1 or 1.5 mg/kg)in NMRI mice treated with the P450-inducing agent ß-naphthoflavone(BNF). In the heart, the binding was highest in capillariesand coronary vessels. In the kidney, the binding was highestin afferent and efferent arterioles and glomerular and peritubularcapillaries. A corresponding localization of radioactivity didnot occur in corn oil-treated mice injected with [³H]-Trp-P-1.On incubation of heart and kidney slices with [³H]-Trp-P-1,there was a binding of radioactivity in endothelial cells ofBNF-treated mice, but not in corn oil-treated mice. The P4501Ainhibitor effipticine abolished the BNF-induced endothelialbinding of [³H]-Trp-P-1 in vivo and in vitro, whereas the effectsof     

Excretion of DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, PhIP- dG, PhIP-DNA and DiMeIQx-DNA from the rat

The heterocyclic aromatic amines, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) are formed during frying of meat. PhIP and 4,8-DiMeIQx have, after metabolic activation, been shown to form adducts with DNA at the C8 of guanine both in vitro and in vivo. In order to investigate possible urinary biomarkers for estimation of the genotoxic dose of PhIP and 4,8-DiMeIQx, [3H]PhIP-dG, [3H]PhIP-DNA and [14C]4,8-DiMeIQx- DNA were injected i.p. to rats and the excretion of radioactivity in urine and faeces were measured. For all three [3H]PhIP-dG, [3H]PhIP-DNA and [14C]4,8-DiMeIQx-DNA 15-20% of the dose were excreted in the urine and 80-85% of the dose were excreted in the faeces. Urinary excretion showed maximum to 24 h (90%) with a rapid decline, 10% to 48 h and 0% to 72 h. Faecal excretion also showed maximum to 24 h (60%) with a slower decline, 30% to 48 h and 10% to 72 h. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [3H]PhIP-dG, showed that approximately 90% of the radioactivity co-eluted with PhIP- dG, indicating that PhIP-dG is excreted unmetabolized. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [3H]PhIP-DNA, showed that approximately 85% of the radioactivity co- eluted with PhIP-dG, indicating that PhIP-DNA adducts is mainly excreted as nucleoside adducts. Approximately 5% of the radioactivity excreted in the urine co-eluted with PhIP-G, indicating loss of deoxyribose. HPLC analysis of samples of urine and extracts from faeces, from rats dosed with [14C]4,8-DiMeIQx-DNA, showed that approximately 90% of the radioactivity co-eluted with 4,8-DiMeIQx-dG, indicating that 4,8-DiMeIQx-DNA adducts is mainly excreted as nucleoside adducts. Man is able to eliminate compounds of a higher mol. wt in the urine than the rat, the percentage of PhIP-dG and 4,8-DiMeIQx eliminated in the urine of man would therefore be expected to be higher than in the rat. Measurement of urinary nucleoside adducts of PhIP and 4,8-DiMeIQx could therefore provide a basis for the development of a biomonitoring strategy for the genotoxic dose of these food derived HAA.