Biomedical subjects

C D Davis

Publications and source records attributed to C D Davis.

At least 37 records · Page 2Linked to original sources

PubMed1998

Proliferation, development and DNA adduct levels in the mammary gland of rats given 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and a high fat diet.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine derived from cooked meat that is a mammary gland carcinogen in rats. A carcinogenic dose-regimen of PhIP (75 mg/kg, p.o., 10 doses, once per day) was administered to 43-day old female Sprague-Dawley rats, and the rats were then placed on a defined high fat (23.5% corn oil) or low fat (5% corn oil) diet for up to 6 weeks. At various times after carcinogen and diet, and prior to carcinogenesis, we examined the percentage of proliferating cells in terminal end bud (TEB) epithelial structures of the rat mammary gland by proliferating cell nuclear antigen staining, mammary gland architecture by whole mounting, and PhIP-DNA adduct levels in mammary epithelial cells by the 32P-post-labeling assay. Immediately after dosing, the percentage of proliferating epithelial cells in TEBs was significantly higher in PhIP-treated rats than in control rats receiving vehicle only [7.5 +/- 0.9% (n = 99) versus 4.2 +/- 0.6% (n = 127), respectively]. The mammary glands of PhIP-treated rats showed a significantly lower density of alveolar buds (ABs) and a higher density of TEBs than control rats, which suggests that PhIP exposure partially inhibited the normal glandular differentiation of TEBs to ABs. After 6 weeks on the diet, proliferation in TEBs was statistically higher in rats given PhIP plus a high fat diet than in rats given vehicle plus a low fat diet. The mammary glands from rats on a high fat diet also showed a statistically higher density of TEBs when compared with rats on a low fat diet [2.08 +/- 0.34% versus 1.04 +/- 0.20%, respectively (n = 6)]. PhIP-DNA adduct levels were relatively high in mammary epithelial cells of treated rats. At 3 h after the last dose of PhIP, DNA adduct levels [relative adduct labeling (RAL) x 10(7), mean +/- SE] were 10.5 +/- 1.7 (n = 8) and 0.9 +/- 0.2 (n = 7) in epithelial cells isolated from mammary gland and in the liver, respectively. DNA adduct removal rates from the mammary gland were not different between rats on the high fat and low fat diets. Adducts were still detected after 6 weeks on either diet. Thus, events that occurred prior to neoplasia in the mammary glands of PhIP-treated rats include formation of PhIP-DNA adducts at relatively high levels, and enhanced proliferation in TEBs (putative sites of origin of mammary gland carcinomas) and partial inhibition of TEB differentiation. The high fat diet, a promoter of PhIP-induced mammary gland carcinogenesis, appeared to sustain the proliferative effect of PhIP in mammary gland TEBs at a time when PhIP-DNA adducts are still detectable. These early events may contribute to the targeting and carcinogenicity of PhIP to the mammary gland of rats.

Animals↗

PubMed1998

Beneficial effect of selenium supplementation during murine infection with Trypanosoma cruzi.

Selenium (Se) has been shown to function as an antioxidant that may enhance immunity during microbial infection. To investigate the effect of elevated levels of Se on the course of experimental Chagas' disease, 5 groups of C3HeB/FeJ mice were infected with 10(3) bloodform trypomastigotes of a Brazil strain of Trypanosoma cruzi while receiving supplements of 0 ppm, 2 ppm, 4 ppm, 8 ppm, or 16 ppm Se as sodium selenate in drinking water. After 64 days of infection, survival ranged from 0 to 60%, with groups receiving 4 ppm and 8 ppm Se exhibiting 60% survival and the group without Se exhibiting 0% survival. In addition, parasitemia levels of mice supplemented with Se were significantly lower (P<0.01) than in nonsupplemented mice. The results of the present study suggest that Se supplementation does have a beneficial effect during murine infection with T. cruzi, resulting in decreased parasitemias and increased longevity.

Animals↗

PubMed1997

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.

Amines↗

PubMed1997

Bioactivation and inactivation of aflatoxin B1 by human, mouse and rat liver preparations: effect on SCE in human mononuclear leucocytes.

The purpose of this study was to investigate the use of human and animal subcellular liver fractions in an in vitro evaluation of carcinogenic risk. The bioactivation and bioinactivation of the known genotoxic carcinogen aflatoxin B1 by human, mouse and rat liver preparations was investigated using the SCE assay in human lymphocytes as a genotoxic endpoint. There was a 10-fold variation in SCE response (1.1-11.6 SCE/Cell) in human mononuclear leucocytes (MNLs) after aflatoxin B1 was activated by human liver microsomes (n = 6). Activation correlated with the CYP1A2 phenotype of livers (r = 0.8; p < 0.05), but there was no correlation with either GST M1 genotype or epoxide hydrolase phenotype. Mouse liver microsomes activated aflatoxin B1 to a greater extent [(1 micro M) 12.8 +/- 2.51 SCE/Cell] than either rat [(10 micro M) 12.0 +/- 3.84 SCE/Cell or human (L25) [(10 micro M) 8.8 +/- 2.00 SCE/Cell liver microsomes. The addition of mouse liver cytosol and reduced glutathione (GSH) significantly (p < 0.001) reduced aflatoxin B1-dependent genotoxicity, whereas the addition of either human or rat cytosol (+GSH) was without effect. These data indicate that species variation in both bioactivation and bioinactivation can exist. Therefore there is a necessity for careful selection of activation systems from species whose biochemical profile reflects that of man.

Aflatoxin B1↗

PubMed1997

Hepatic tumor induction in c-myc mono-transgenic and TGF-alpha/c-myc double-transgenic mice.

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc cDNA and mouse metallothionein 1 promoter-human TGF-alpha 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 as well as to produce an experimental model to test how environmental chemicals might interact with these genes during the neoplastic process. Coexpression of c-myc and TGF-alpha as transgenes in the mouse liver resulted in a tremendous acceleration of neoplastic development in this organ as compared to expression of either of these transgenes alone. The two distinct cellular reactions that occurred in the liver of the double transgenic mice prior to the appearance of liver tumors were dysplastic and apoptotic changes in the existing hepatocytes followed by emergence of multiple focal lesions composed of both hyperplastic and dysplastic cell populations. These observations suggest that the interaction of c-myc and TGF-alpha, during development of hepatic neoplasia contributes to the selection and expansion of the preneoplastic cell populations which consequently increases the probability of malignant conversion. Treatment of the double transgenic mice with both genotoxic agents such as diethylnitrosamine and IQ as well as the tumor promoter phenobarbital greatly accelerated the neoplastic process. These results suggest that selective 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.

Animals↗

PubMed1996

Characterisation of the toxic metabolite(s) of naphthalene.

The toxicity of naphthalene and its metabolites has been investigated in vitro. Both naphthalene and its metabolite 1-naphthol were bioactivated by human hepatic microsomes to metabolite(s) which were toxic to mononuclear leucocytes (MNL). However 1-naphthol was more cytotoxic than naphthalene (49.8 +/- 13.9% vs. 19.0 +/- 10.0% cell death; P < 0.01), indicating that the toxicity of naphthalene is dependent on the bioactivation of 1-naphthol. CYP2E1-induced rat liver microsomes increased metabolism of naphthalene by 13% compared to control microsomes with a concomitant increase in both 1-naphthol and dihydrodiol formation. The cytotoxicity of naphthalene but not of 1-naphthol was increased by CYP2E1 induction, indicating that separate enzymes are involved in the bioactivation of 1-naphthol. The metabolites of 1-naphthol, 1,2-naphthoquinone (51.4 +/- 6.6% cell death) and 1,4-naphthoquinone (49.1 +/- 3.4% cell death) were directly toxic to MNL and depleted glutathione to 1.0% of the control levels. Both quinones were also genotoxic to human lymphocytes. In contrast, the primary metabolite of naphthalene, the 1,2-epoxide (0-100 microM) was neither cytotoxic nor genotoxic, and did not deplete glutathione. In conclusion, our data suggests that the cytotoxicity and genotoxicity of naphthalene is associated with the formation of quinones from 1-naphthol rather than naphthalene-1,2-epoxide.

Adult↗

PubMed1996

In vivo mutagenicity and DNA adduct levels of heterocyclic amines in Muta mice and c-myc/lacZ 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.

Animals↗

PubMed1996

N-acetyltransferase expression and metabolic activation of the food-derived heterocyclic amines in the human mammary gland.

The heterocyclic amines (HCAs) found in cooked meat are procarcinogens that are metabolically activated by N-hydroxylation followed by O-acetylation by the N-acetyltransferases NAT1 and NAT2. Despite the importance of metabolic activation in HCA carcinogenicity and the finding that several HCAs are rodent mammary gland carcinogens, nothing was known about O-acetylation activity in the human mammary gland. The current study examines the expression and catalytic activity of NAT toward the N-hydroxy-HCAs 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP) and 2-hydroxy-amino-3-methylimidazo[4,5-f]quinoline (N-hydroxy-IQ) in the human mammary gland. Mammary gland cytosol from 10 women and lysates from a primary culture of human mammary epithelial cells metabolically activated 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline by NAT-mediated 0-acetyltransferase, as measured by the acetyl CoA-enhanced binding of the N-hydroxylamines to calf thymus DNA in vitro. N-acetylation of p-aminosalicylic, an activity specific to NAT1, but not N-acetylation of sulfamethazine, an activity specific to NAT2, was detected in the mammary gland cytosols and human mammary epithelial cell lysates. Immunohistochemical analysis of human mammary gland sections showed positive staining for NAT1 protein in the epithelial cells lining the mammary gland ducts. Reverse transcription-PCR analysis showed that mRNA transcripts for both NAT1 and NAT2 were present in human mammary gland; however, no NAT2 catalytic activity was detectable. Our data demonstrate for the first time that the human mammary gland is catalytically active toward the metabolic activation of HCA food mutagens, and that this activity is most likely contributed by NAT1 expressed in the ductular epithelial cells of the mammary gland.

Acetylation↗

PubMed1996

Ahr locus phenotype in congenic mice influences hepatic and pulmonary DNA adduct levels of 2-amino-3-methylimidazo[4,5-f]quinoline in the absence of cytochrome P450 induction.

The potent food mutagen/carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) undergoes metabolic N-hydroxylation by cytochromes P450, including cytochrome P450 1A2, followed by generation of an unstable ester catalyzed by acetyltransferases; promutagenic DNA adducts result. Genetic polymorphisms in these enzymes have been implicated in human cancer risk related to arylamine exposure. We investigated the effects of Ahr locus and acetylator polymorphisms on 32P-postlabeled IQ/DNA adducts in lungs and livers of female C57BL/6 mice congenic for slow acetylation and/or Ahr-nonresponsiveness; some groups were pretreated with beta-naphthoflavone (beta NF), a cytochrome P450 1A inducer. Total adducts in lung were doubled by beta NF pretreatment in Ahr-responsive mice only and consisted of < or = 30% adduct 2 and < or = 60% adduct 3. In contrast, in Ahr-nonresponsive mice, adducts 2 and 3 were each < or= 7% of the total. Livers of noninduced Ahr-responsive mice formed 6-18-fold more adducts than those of nonresponsive mice. This striking difference was not due to altered levels of cyp1a-2, as indicated by specific enzyme assays and immunoblotting, and was not accompanied by a comparable increase in the ability of liver preparations to activate IQ to a mutagen in the Ames test. Pretreatment of responsive mice with beta NF to induce cyp1a-1 and cyp1a-2 led to a reduction in liver adduct levels. Acetylation phenotype also had a significant effect in Ahr-responsive mice, with 3-fold more adducts in slow than in rapid acetylators. These results indicate that in uninduced mice, the normal Ah receptor facilitates formation of IQ/DNA adducts in liver and alters the profile of adducts in lung, via an unknown mechanism, whereas the Ah receptor-dependent enzyme induction reduces adducts in liver, probably due to increased detoxification, but increases them in lung.

Acetylation↗

PubMed1995

Kinetic parameters of lymphocyte microsomal epoxide hydrolase in carbamazepine hypersensitive patients. Assessment by radiometric HPLC.

Idiosyncratic hypersensitivity reactions with carbamazepine have been postulated to be due to a deficiency of microsomal epoxide hydrolase (HYL1), although this is based on indirect evidence. Using 3H-cis stilbene oxide (0.5 Ci/mmol) as a substrate, we have developed a radiometric HPLC assay sensitive enough to measure the kinetic parameters of HYL1 in lymphocytes. The intra-assay coefficient of variation was 8%. Enzyme activity has been measured in lymphocytes from six carbamazepine hypersensitive patients, six patients on carbamazepine without any adverse effects, and twelve drug-naive healthy volunteers. No significant difference was observed in three kinetic parameters of the enzyme among these three groups. The values for Km, Vmax, and intrinsic clearance ranged from 6.1-89.9 microM, 3.0-23.2 pmoles diol formed/min/mg protein, and 0.147-0.493 microliter/min/mg protein. There was no difference in enzyme activity between patients currently on carbamazepine and healthy volunteers, indicating a lack of induction of lymphocyte HYL1 by carbamazepine. Co-incubation of lymphocytes with 1,1,1-trichloropropene oxide, an inhibitor of hepatic HYL1, resulted in an 82% inhibition of activity, similar to that observed with the hepatic enzyme. The healthy volunteers were genotyped as being either GSTM1 positive (n = 6) or GSTM1 negative (n = 6). This did not affect the kinetic parameters of lymphocyte microsomal epoxide hydrolase. Our results suggest that there is normal HYL1 activity in lymphocytes of hypersensitive patients using cis-stilbene oxide as a substrate.

Adolescent↗

PubMed1995

Protective effect of N-acetylcysteine against heterocyclic amine-induced cardiotoxicity in cultured myocytes and in rats.

Cooked meat contains many mutagenic/carcinogenic heterocyclic amines (HAs), including 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The reactive N-hydroxylamine metabolites N-hydroxy-IQ and N-hydroxy-PhIP are toxic to isolated rat cardiomyocytes. This study examined whether antioxidant agents protect against N-hydroxylamine-induced cardiotoxicity. In isolated rat cardiomyocytes, N-acetylcysteine, alpha-tocopherol and glutathione were protective against N-hydroxylamine-mediated lactate dehydrogenase release into the medium, suggesting that a free radical mechanism may be partly involved in HA-induced cardiotoxicity. Since N-acetylcysteine was by far the most protective of the agents investigated, the effects of N-acetylcysteine on HA-induced ultrastructural damage were further examined both in vitro and in vivo. Isolated cardiomyocytes treated with 1.2 mM N-acetylcysteine before and during exposure to N-hydroxy-IQ or N-hydroxy-PhIP showed a smaller percentage of ultrastructural abnormalities, such as myofilament loss, sarcoplasmic reticulum swelling and abnormal mitochondria. N-Acetylcysteine pretreatment also significantly reduced the percentage of cardiac cells with T-tubule dilation and myelin figures in adult rats dosed with IQ. The protective effect of N-acetylcysteine was not associated with a reduction in HA-DNA adducts, as assessed by 32P-postlabelling analysis of DNA from isolated cardiomyocytes treated with N-hydroxylamines. DNA adduct formation per se, therefore, may not be associated with the observed cardiotoxic effects of the HAs. Further studies are required to confirm the involvement of a free radical mechanism in HA cardiotoxicity.

Acetylcysteine↗

PubMed1995

Possible mechanisms for PhIP-DNA adduct formation in the mammary gland of female Sprague-Dawley rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine in fried beef, is mammary gland carcinogen in rats. Using the 32P-postlabeling method, PhIP-DNA adduct levels were measured in mammary epithelial cells isolated from female Sprague-Dawley rats given 10 daily doses of PhIP (75 mg/kg, p.o.) according to a protocol previously shown to induce mammary gland cancer. At 24 h, 48 h, 1 week and 5 weeks after the last dose of PhIP, PhIP-DNA adduct levels [relative adduct labeling (RAL) x 10(7), mean +/- SD] were 10.2 +/- 0.7, 7.9 +/- 2.7, 2.2 +/- 0.6 and 0.9 +/- 0.03 respectively. When isolated rat mammary epithelial cells (from untreated rats) were incubated in vitro with N-hydroxy-PhIP (45 microM, 1 h, 37 degrees C), PhIP-DNA adducts were detected in cell DNA (RAL = approximately 97 x 10(7); however, no adducts were detected in cells incubated with PhIP (200 microM, 15 h, 37 degrees C). Incubating cells with pentachlorophenol, an inhibitor of acetyltransferase, or incubating cells at 0-4 degrees C, reduced N-hydroxy-PhIP adduct levels by 45 and 75% respectively, indicating that formation of N-hydroxy-PhIP adducts was largely due to metabolic activation. Further studies showed that rat mammary gland microsomes had little capacity to N-hydroxylate PhIP, as assayed by the mutagenic activation of PhIP in the Ames Salmonella assay. In contrast, N-hydroxy-PhIP was metabolically activated by cytosol-catalyzed PhIP-DNA adduct formation to calf thymus DNA incubated in vitro with N-hydroxy-PhIP (2 microM) in the presence of acetyl CoA. Notably, mammary cytosolic O-acetyltransferase activation of N-hydroxy-IQ or N-hydroxy-MeIQx. All three N-hydroxylamines were activated via cytosolic proline aminoacyl-tRNA synthetase and phosphorylase, although the activities of these enzymes were approximately 100-fold lower than O-acetyltransferase. No mammary cytosolic sulfotransferase activation could be detected with any of the N-hydroxylamines. Our results are consistent with the notion that PhIP-DNA adduct formation and initiation of carcinogenesis in the rat mammary gland may be associated with N-hydroxylation of PhIP outside the mammary gland, transport of the N-hydroxylamine to the mammary gland and subsequent in situ O-acetyltransferase-catalyzed activation of N-hydroxy-PhIP.

Acetyltransferases↗

PubMed1995

Protection by chlorophyllin and indole-3-carbinol against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced DNA adducts and colonic aberrant crypts in the F344 rat.

The most abundant heterocyclic amine in fried ground beef, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), induces colon carcinomas in the male F344 rat. The potential chemopreventive effects of two compounds, namely, the 'interceptor molecule' chlorophyllin (CHL) and a modulator of carcinogen activation, indole-3-carbinol (I3C), were examined in a PhIP colon carcinogenesis model. During weeks 3 and 4 of a 16-week study, F344 rats were given PhIP by oral gavage (50 mg/kg body weight, alternating days). Inhibitors were given either before and during PhIP exposure, after PhIP treatment, or continuously for 16 weeks. Treatment of rats with 0.1% CHL in the drinking water inhibited the formation of aberrant crypt foci (ACF) with > or = 4 crypts/focus, from 1.4 +/- 0.9 in controls to 0.7 +/- 0.3 following post-initiation CHL treatment, and to 0.3 +/- 0.5 in rats given CHL continuously for 16 weeks (mean +/- SD; P < 0.05). Potent inhibition of PhIP-induced ACF occurred following initiation, post-initiation and continuous exposure to 0.1% I3C in the diet. Using the initiation protocol, I3C completely inhibited the induction of the ACF with > or = 4 crypts/focus. In a separate experiment, rats were given 0.1% CHL in the drinking water or 0.1% I3C in the diet for 4 weeks. At the end of week 3, animals received 50 mg PhIP/kg body weight by single oral gavage and PhIP-DNA adducts were quantified in the colon and several other tissues by 32P-postlabeling analysis. In addition, the urine and feces were collected to study the effects of inhibitor treatment on PhIP metabolism and excretion. No significant protection against PhIP-DNA adduct formation was detected in the colon after CHL dosing, nor was a consistent pattern of CHL inhibition observed in several other tissues. In contrast, I3C shifted the time-course of adducts in all tissue; compared with controls, adducts were increased by I3C at 6 h but decreased at 24 h and 7 days following PhIP treatment. Analysis of urine metabolites revealed that I3C and CHL decreased the excretion of unmetabolized PhIP and 4'-hydroxy- << PhIP but increased the phase II detoxification products PhIP-4'-O-glucuronide and PhIP-4'- sulfate. In the feces, the elimination of unmetabolized PhIP was increased from 54.5% in controls to approximately 67% in CHL-treated rats and decreased to 28% in rats given I3C (P < 0.05). These results support a protective role for CHL and I3C against PhIP-induced colon carcinogenesis through mechanisms which alter the uptake or metabolism of the carcinogen, and by suppression in the post-initiation phase.

Animals↗

PubMed1995

Analysis of EGFR, TGF-alpha, neu and c-myc in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mammary tumors using RT-PCR.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a mutagen found in cooked meat, has been shown to induce mammary gland tumors in rats. Our laboratory recently observed that a high fat diet enhances the incidence and severity of PhIP-induced mammary gland cancer in rats. In the current study, reverse transcription followed by polymerase chain reaction amplification was used to determine whether EGFR, TGF-alpha, neu and c-myc are differentially expressed in PhIP-induced mammary gland tumors classified histologically as benign or malignant and to evaluate whether dietary fat intake influences the expression of these genes. Of 23 total PhIP-induced mammary tumors examined, 43%, 57% and 74% had increased expression of EGFR, TGF-alpha and neu mRNA respectively. Increased expression of these genes appeared to be consistently present in tumors displaying papillomatosis. In contrast, to the other three genes, c-myc mRNA levels were infrequently elevated. The percentage of dietary fat did not appear to influence the expression of EGFR, TGF-alpha or neu in either tumors or mammary gland from control rats. However, the levels of c-myc mRNA were 1.8- and 2.9-fold higher in the control mammary gland and benign PhIP-induced tumors respectively in rats fed the high-fat diet than in rats fed the low-fat diet, suggesting a slight effect of dietary fat (P < 0.08) on c-myc expression. These results suggest that increased expression of EGFR, TGF-alpha and especially neu is associated with PhIP-induced mammary gland cancer in rats.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1995

Metabolism of the food-derived carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in nonhuman primates.

The metabolism and disposition of the food mutagen and rodent carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline was investigated in cynomolgus monkeys. Monkeys were administered a single dose of radiolabeled [14C]MeIQx (2.2 or 50 mumol/kg). Peak blood levels of radioactivity were observed within 1-3 h after dosing and declined rapidly thereafter. By 72 h after dosing, approximately 50% and 70% of the 2.2 mumol/kg, and 50 mumol/kg dose, respectively, was excreted in the urine. Approximately 15-20% of either dose was recovered in the feces. Eight metabolites and the parent compound were detected in urine by HPLC. The parent compound accounted for approximately 15-25% of the dose excreted in the urine. Seven MeIQx urinary metabolites were identified. Five metabolites were identical to MeIQx metabolites previously found in rats: MeIQx-N2-glucuronide, MeIQx-N2-sulfamate, MeIQx-5-sulfate, MeIQx-5-O-glucuronide, and 8-CH2OH-MeIQx-5-sulfate. Cynomolgus monkeys, however, metabolized MeIQx to a novel glucuronide conjugate of MeIQx not found in rats. Based upon mass spectroscopy and proton NMR analyses, the structure of this metabolite was consistent with an N1-glucuronide of MeIQx. This metabolite was the major urinary metabolite found in monkeys, accounting for 31-37% of the dose excreted in the urine over a 24 h period. One additional metabolite identified in urine and feces of MeIQx treated cynomolgus monkeys, that has not been found previously in any other animal model, was 7-oxo-MeIQx, a likely enteric bacterial metabolite of MeIQx. 7-Oxo-MeIQx accounted for 20-25% of the dose of MeIQx found in the urine and was the major fecal metabolite. The N2-glucuronide conjugate of the carcinogenic metabolite 2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline (NHOH-MeIQx) was not detected in urine or bile of monkeys, even after 10 daily doses of MeIQx (100 mumol/kg) were given. The results indicate that MeIQx is metabolically processed in monkeys via multiple pathways of detoxification. However, MeIQx is poorly metabolically activated via cytochrome P450 mediated N-oxidation. The in vivo metabolism of MeIQx in cynomolgus monkeys is different from that of the structurally related food-derived mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), which is readily metabolically activated by this species and in contrast to MeIQx, has been shown to be a powerful hepatic carcinogen.

Animals↗

PubMed1995

Possible relationship between tissue distribution of DNA adducts and genotoxicity of food-derived heterocyclic amines.

During the past decade and a half, a number of potent mutagens belonging to the class of heterocyclic aromatic amines (HCA) have been isolated and identified from cooked fish, beef, fowl and other meat, and from beef extracts. Several of these HCA mutagens have also been found to be carcinogenic in rodent bioassays, and one of these compounds, 2-amino-3-methylimidazo-[4,5-f]-quinoline (IQ), has recently been found to cause hepatocellular carcinoma in cynomolgus monkeys. The potential etiological role of these mutagens and carcinogens in human cancer prompted us to evaluate the genotoxicity of these compounds in both nonhuman primates and rodents, with particular emphasis on the formation and tissue distribution of DNA adducts. We selected three compounds for this study based on several factors including chemical structure, mutagenic activity in vitro, concentration in cooked food, and carcinogenic activity in the rodent test system. These were IQ, 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (8-MeIQx), and 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine (PhIP). To maximize the sensitivity of the DNA adduct measurements, we have employed the 32P-postlabeling method to analyze levels and tissue distribution of DNA adducts derived from IQ 8-MeIQx, and PhIP. We have measured DNA adduct formation for all three compounds in various tissues and white blood cells of monkeys following both acute and chronic administration. Both IQ and PhIP form high levels of adducts in a number of organs, particularly liver, kidney, and heart. In contrast, administration of 8-MeIQx to the cynomolgus monkeys results in the formation of only low adduct levels with many tissues showing no detectable levels of adducts. Studies in rodents have also shown that IQ and PhIP from DNA adducts in a number of organs in addition to those that are targets for carcinogenic effects of these agents. Although high DNA adduct levels of HCA are generally found in target organs for HCA induced carcinogenesis the widespread distribution of the adducts in organs not associated with the carcinogenic effects suggest that HCA exposure may be a possible etiological factor in cardiovascular diseases and other degenerative ailments.

Amines↗

PubMed1994

Metabolic activation of heterocyclic amine food mutagens in the mammary gland of lactating Fischer 344 rats.

We investigated the ability of the mammary gland to metabolically activate 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Although mammary gland microsomes had almost no capacity to metabolically activate the parent compounds, mammary gland cytosol was able to esterify the N-hydroxylamines. Acetyltransferase was the primary enzyme responsible for the phase II activation of the N-hydroxylamines. The level of acetyl CoA-stimulated binding when N-hydroxy PhIP served as the substrate was approximately 3- and 17-fold higher than when IQ and MeIQx served as substrates, respectively. N-Hydroxy-IQ and N-hydroxy PhIP can also be activated by tRNA synthetase and phosphatase, but not by sulfotransferase. However, the levels of proline- and ATP-enhanced DNA binding was approximately 30- and 60-fold lower than the acetyl CoA-enhanced DNA binding of IQ and PhIP, respectively. Differences observed in the phase II activation of the various heterocyclic amines in the mammary gland may explain why the mammary gland is a target organ for PhIP-induced carcinogenicity but not for IQ- or MeIQx-induced carcinogenicity in Fischer 344 rats.

Acetyl-CoA C-Acetyltransferase↗

PubMed1994

Metabolism of the food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by lactating Fischer 344 rats and their nursing pups.

BACKGROUND: An important class of dietary mutagens and carcinogens are the heterocyclic arylamine compounds that have been identified in a variety of cooked, protein-containing foods. Among these heterocyclic amines, 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP) is potentially the most important carcinogen for human cancer risk. We have recently observed that PhIP-derived radioactivity is excreted into the breast milk of lactating rats administered [3H]PhIP. PURPOSE: To better assess the significance of breast milk as a route of exposure of the newborn to dietary heterocyclic amines, we examined the metabolites of PhIP in breast milk and in urine of nursing pups. METHODS: Lactating Fischer 344 rats with 5-day-old pups were given a single oral dose of 10 mg/kg of [3H]PhIP. We collected milk from the dams and urine from the pups and then analyzed the samples for metabolites of PhIP, using high-pressure liquid chromatography (HPLC). PhIP-DNA adduct levels in the tissues of the pups were determined by 32P-postlabeling analysis. RESULTS: Three radioactive peaks were observed by HPLC separation of milk samples: an unidentified early eluting peak, 4'-hydroxy-PhIP, and PhIP. Four metabolites and the parent compound were found in urine of the pups nursed by dams given radiolabeled PhIP: PhIP-4'-O-glucuronide, PhIP-4'-sulfate, 4'-hydroxy-PhIP, and N2-hydroxy-PhIP-N3-glucuronide. 4'-Hydroxy-PhIP and its conjugates contributed approximately 60% of the radioactivity found in the urine. By 32P-postlabeling analysis, PhIP-DNA adducts were detected in spleen, lung, heart, kidney, liver, and stomach of pups at mean levels ranging from 0.06 to 0.55 adducts/10(7) nucleotides. CONCLUSIONS: The large percentage of 4-hydroxy-PhIP and its conjugates in the urine indicates that 5-day-old pups detoxify PhIP and further metabolize 4'-hydroxy-PhIP obtained from the breast milk. The presence of the glucuronide conjugate of N-hydroxy-PhIP in the urine of pups and the lack of detectable conjugate or N-hydroxylamine itself in breast milk suggest that PhIP from breast milk undergoes metabolic activation via N-hydroxylation in 5-day-old rat pups. This conclusion was further supported by the observation that hepatic S9 fractions from the pups activated PhIP to a mutagen in the Ames Salmonella mutagenicity assay and by the presence of PhIP-DNA adducts in the tissues of the pups. IMPLICATIONS: The findings reported here may have carcinogenic and toxicologic implications for the offspring of women who breast-feed and consume a diet rich in cooked meat.

Animals↗