Iron-chlorophyllin-mediated conversion of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)) into its nitroso derivative

Early work from our laboratory has shown that the mutagenicity of heterocyclic amines in Salmonella can be inhibited by hemin and chlorophyllins. We have speculated that the inhibition is a result of complex formation between heterocyclic amines and the pigments, and the speculation has been given a line of experimental evidence. We have now found that ferric-chlorophyllin (Fe-chlorophyllin) can modify the mutagenicity of 3-hydroxyamino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2(NHOH)), a metabolically activated form of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The mutagenicity of Trp-P-2(NHOH) in Salmonella typhimurium TA 98 (without S9) was strongly inhibited by an addition of an equimolar Fe-chlorophyllin in the pre-incubation mixture. Fe-chlorophyllin also inhibited the mutagenicity of 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d] imidazole (Glu-P-1(NHOH)). A rapid change in the UV spectrum of a mixture of Trp-P-2(NHOH) and Fe-chlorophyllin was observed. Analysis by high performance liquid chromatography showed that Trp-P-2(NHOH) was converted into 3-nitroso-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NO)), the mutagenic potency of which is a quarter of that of Trp-P-2(NHOH). Furthermore, the mutagenicity of Trp-P-2(NO), in turn, was inhibited by Fe-chlorophyllin. We conclude that the suppression of the mutagenicity of Trp-P-2(NHOH) is ascribable to the oxidative function of Fe-chlorophyllin, coupled with its ability to form complex formation with the planar surface of the heterocyclic amine molecules.     

Determination of glycyrrhizic acid and 18-beta-glycyrrhetinic acid in biological fluids by micellar electrokinetic chromatography

Human urine is known to contain substances that strongly inhibit bacterial mutagenicity of aromatic and heterocyclic amines in vitro. The biological relevance of these anti-mutagens was examined by comparing levels of tobacco-related DNA adducts in exfoliated urothelial cells from smokers with the anti-mutagenic activity in corresponding 24-h urine samples. An inverse relationship was found between the inhibition of PhIP-mutagenicity by urine extracts in vitro and two DNA adduct measurements: the level of the putatively identified ABP-dG adduct and the total level of all tobacco-smoke-related carcinogen adducts including those probably derived from PhIP. These substances appear to be dietary phenolics and/or their metabolites because (i) the anti-mutagenic activity of urine extracts (n=18) was linearly related to their content in phenolics; (ii) the concentration ranges of these substances in urine extracts were similar to those of various plant phenols (e.g., quercetin, isorhamnetin) for which an inhibitory effect on the liver S9-mediated mutagenicity of PhIP was obtained; (iii) treatment of urines with beta-glucuronidase and arylsulfatase enhanced both anti-mutagenicity and the levels of phenolics in urinary extracts; (iv) urinary extracts inhibited non-competitively the liver S9-mediated mutagenicity of PhIP as did quercetin, used as a model phenolics. Onion, lettuce, apples and red wine are important sources of dietary flavonoids which are probably responsible for the anti-mutagenicity associated with foods and beverages. After HPLC fractionation of urinary extracts, the distribution profile of anti-mutagenic activity corresponded roughly to that of onion and wine extract combined. Overall, our study strongly suggests that smokers ingesting dietary phenolics, probably flavonoids, are partially protected against the harmful effects by tobacco carcinogens within their bladder mucosal cells.     

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by human cytochrome P4501B1

Cytochrome P4501B1 (CYP1B1) is the most recently identified member of the dioxin-inducible CYP1 family. CYP1B1 is constitutively expressed in most human tissues, including colon and breast, and can activate numerous chemically diverse carcinogens. We evaluated the metabolism of the dietary heterocyclic amine carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by microsomes from yeast expressing the human CYP1B1 protein. PhIP metabolites were analysed by HPLC with fluorescence and absorbance detection. We found that human CYP1B1 metabolizes PhIP to three products: N2-OH-PhIP, a mutagenic activation product; 4'-OH-PhIP, a detoxification product; and 2-OH-PhIP, the mutagenic potential of which is unknown. Metabolite identity was confirmed by co-elution with authentic standards and synchronous fluorescence spectroscopy. The identity of the 2-OH-PhIP standard was additionally confirmed by mass spectrometry. Kinetic studies of the formation of N2-OH-PhIP, 4'-OH-PhIP and 2-OH-PhIP by CYP1B1 indicated apparent Km values of 5.7 +/- 1.3, 2.2 +/- 0.5 and 1.3 +/- 0.2 microM, respectively. Apparent turnover rates were 0.40 +/- 0.03, 0.93 +/- 0.02 and 0.04 +/- 0.00 nmol product/min nmol P450, respectively. At saturating levels of substrate, CYP1B1-mediated formation of the non-mutagenic metabolite 4'-OH-PhIP was favored two-fold over that of the mutagenic metabolite, N2-OH-PhIP and >10-fold over that of 2-OH-PhIP. The formation of N2-OH-PhIP, a potent mutagen implicated in the etiology of human colon and breast cancer, indicates that CYP1B1 may play an important role in PhIP-mediated carcinogenesis.     

Antimutagenicity of flavones and flavonols to heterocyclic amines by specific and strong inhibition of the cytochrome P450 1A family

We found the mechanism in flavonoids that can strongly suppress the mutagenicity of one of the food-derived and carcinogenic heterocyclic amines, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The antimutagenicity was evaluated by IC50 value, the amount required for 50% inhibition of the mutagenicity of 0.1 nmol Trp-P-2, with Salmonella typhimurium TA98 strain in the presence of S9 mix. The flavones and flavonols were two orders stronger as antimutagens that such antimutagenic phytochemicals as chlorophylls and catechins. We had previously found flavonoids to be a desmutagen to neutralize Trp-P-2 before or during attack of DNA, because they had no effect on either the ultimate mutagenic form of Trp-P-2 (N-hydroxy-Trp-P-2) or the mutated cells. The desmutagenicity of the flavonoids did not depend on the hydroxy number or position that should be associated with antioxidative potency, and was also unaffected by the solubility of Trp-P-2 in the assay solution. The inhibitory effect of the flavonoids on the metabolic activation of Trp-P-2 to N-hydroxy-Trp-P-2 was almost in parallel with the antimutagenic IC50 value, when determined with a Saccharomyces cerevisiae AH22 cell simultaneously expressing both rat cytochrome P450 1A1 and yeast reductase. The Ki values of flavones and flavonols for the enzyme were less than 1 microM, while the K(m) value of Trp-P-2 was 25 microM. The antimutagenicity of the flavones and flavonols was thus concluded to be due to inhibition of the activation process of Trp-P-2 by P450 1A1 to the ultimate carcinogenic form. They were also able to act as antimutagens toward other indirect mutagens that were activated by P450 1A1.     

Differential effect of acetyltransferase expression on the genotoxicity of heterocyclic amines in CHO cells

We earlier developed the Chinese hamster ovary UV5P3 cell line that expresses cytochrome P4501A2 and lacks nucleotide excision repair for studying metabolism and mutagenicity of heterocyclic amines. The Chinese hamster ovary UV5P3 cells are approximately 50-fold more sensitive to the cooked food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) than 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), another genotoxic compound found in cooked food, with respect to cytotoxicity and mutation induction at the adenine phosphoribosyltransferase (aprt) locus. To test the hypothesis that the important missing activity in our CHO system for IQ genotoxicity was acetyltransferase, we transfected the UV5P3 cells with cDNA plasmids of either the human NAT2 N-acetyltransferase gene or a bacterial O-acetyltransferase gene. Functionally transformed clones were determined by the differential cytotoxicity assay using IQ, and confirmed by measuring the enzyme activity with isoniazid as substrate. Two clones designated 5P3NAT2 and 5P3YG (expressing human and bacterial transferases, respectively) were characterized. Both cell lines were sensitive to killing by IQ at concentrations as low as 4 ng/ml. Based on the D37 value, the dose that reduced the survival to 37% relative to untreated controls, the acetyltransferase expressing lines showed approximately 1000-fold increase in sensitivity to the killing effect of IQ over the parental UV5P3 cell line. The same dramatic change in sensitivity was also seen in mutation response at the aprt locus and with chromosomal aberrations and sister chromatid exchanges. In contrast, these cell lines showed cytotoxicity to PhIP similar to that of the parental line UV5P3. These results suggest that PhIP does not require acetyltransferase for metabolic activation leading to genotoxicity in these cells. These new cell lines constitute a sensitive cell system for assessing genotoxicity of compounds requiring metabolic activation by both P450IA2 and acetyltransferase, as well as for studying the molecular processes by which DNA damage can lead to mutation and cancer.     

Comparative mutagenicity of N-nitrosamines in a semi-solid and in a liquid incubation system in the presence of rat or human tissue fractions

The rat liver microsome-mediated mutagenicities of a series of N-nitrosodialkylamines and heterocyclic N-nitrosamines were determined in a liquid incubation system using Salmonella typhimurium TA1530. The influence on mutation frequency of the concentration of co-factors for mixed-function oxidase and composition and molarity of the buffer was investigated, using N-nitrosomorpholine as substrate. The mutagenicity of the N-nitroso compounds in the liquid incubation system under optimal reaction conditions at equimolar concentration was compared quantitatively with that obtained in a soft-agar incorporation assay. N-Nitrosodi-n-pentylamine and N-nitrosodi-n-butylamine showed no enzyme-mediated mutagenicity in the liquid incubation system, and metabolically activated N-nitroso-dimethylamine and N-nitroso-diethylamine showed negligible mutagenic activity in the soft-agar assays. In contrast with these results with the N-nitrosodialkylamines, the mutagenic effects of heterocyclic N-nitrosamines were similar in the liquid incubation system and in soft-agar incorporation assays. The heterocyclic N-nitrosamines showed rat-liver microsome-mediated mutagenicity in the following descending order: N-nitrosomorpholine greater than N-nitrosopyrrolidine greater than N-nitrosopiperidine greater than N-nitroso-N'-methylpiperazine. Seven human liver specimens converted all heterocyclic N-nitrosamines into mutagens; this activity was similar to that of rat liver, except that for N-nitroso-N'-methylpiperazine, fractions from three human liver biopsies were three to 30 times more active than those from untreated rats. The specific reversion of S. typhimurium TA1530 to histidine prototrophy provides experimental evidence that all the N-nitrosamines studied were converted by liver microsomal enzymes into monofunctional alkylating agents.     

Inhibitory activity of cigarette-smoke condensate on the mutagenicity of heterocyclic amines

Cigarette-smoke condensate (CSC) is a complex mixture containing over 3800 identified chemicals including nicotine, water, mutagens, antimutagens, cytotoxins and inert chemicals. Although CSC is mutagenic in the Ames test, its effect on the activity of other mutagens has not been characterized. Using the Ames Salmonella bacterial mutagenesis assay, we found CSC exerts a significant inhibitory effect on mutagens requiring bioactivation. Those studied included heterocyclic amines (Glu-P-1, Glu-P-2, IQ, MeIQ, Trp-P-1 and Trp-P-2), benzo[a]pyrene (B[a]P) and aflatoxin B1. However, CSC had no effect on the activity of direct-acting mutagens (2-nitrofluorene, sodium azide, 4-nitro-1,2-phenylenediamine, 4-nitroquinoline N-oxide and methyl methanesulfonate). With indirect-acting mutagens, the reduced number of revertants observed in the presence of CSC was not attributable to cytotoxicity. CSC exhibited a potent inhibitory effect on the cytochrome P-450 dependent monooxygenases, ethoxyresorufin O-deethylase and B[a]P hydroxylase. This suggests inhibition of the cytochrome P-450 isozymes as one possible mechanism for the antimutagenicity of CSC. Fractionation studies of CSC revealed that the neutral, weakly acidic (phenolic) and basic fractions are all effective as antimutagens against Glu-P-1, a representative heterocyclic amine. This indicates that several classes of chemicals contribute to the inhibitory effect of CSC on the mutagenicity of the heterocyclic amines.     

Effect of glutathione depletion and inhibition of glucuronidation and sulfation on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) metabolism, PhIP-DNA adduct formation and unscheduled DNA synthesis in primary rat hepatocytes

The potent rat colon carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), unlike other food-borne heterocyclic amines, does not induce tumors in rat liver. This correlates with an extremely low level of PhIP-DNA adducts formed in this tissue, and together these observations suggest that PhIP is efficiently detoxified in the liver. In order to identify possible detoxification mechanisms, we assessed the effect of inhibition of glucuronidation, glutathione (GSH) conjugation and sulfation on PhIP metabolism and PhIP-induced DNA damage in rat hepatocytes. Hepatocytes isolated from rats pretreated with Aroclor 1254 metabolized PhIP to the same products found in vivo. N-Hydroxy-PhIP N3-glucuronide and N-hydroxy-PhIP N2-glucuronide were major and minor metabolites respectively. 32P-Postlabeling analysis of DNA from the PhIP-treated hepatocytes indicated the presence of two major adducts, one of which was identified as N-(deoxyguanosin-8-yl)-PhIP, and one minor adduct. There was no unscheduled DNA synthesis (UDS) in these cells. However, pretreatment of the hepatocytes with 1-bromoheptane and buthionine sulfoximine, which depletes GSH and prevents its resynthesis, resulted in a 15-fold increase in the formation of PhIP-DNA adducts, as well as in a high level of UDS. GSH depletion had no effect on the formation of detectable PhIP metabolites. Hepatocyte pretreatment with D-galactosamine, which inhibits glucuronidation, increased the formation of DNA adducts two-fold and UDS was increased similarly. D-Galactosamine decreased the formation of the two N-glucuronides of N-hydroxy-PhIP by 50-60%, but had no effect on other metabolites. Pentachlorophenol, which strongly inhibits sulfotransferases, decreased adduct formation slightly, but had essentially no effect on UDS or on the formation of PhIP metabolites. These results indicate that metabolic conjugation pathways involving GSH and glucuronidation may play an important role in protecting rat liver against PhIP carcinogenesis.     

The metabolism and DNA binding of the cooked-food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in precision-cut rat liver slices

Precision-cut liver slices prepared from Aroclor 1254 pretreated male rats were used to investigate the metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The acetyltransferase and sulfotransferase inhibitors, pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP), and the cytochrome P450 inhibitor, alpha-naphthoflavone (ANF), were used to modulate PhIP metabolism and DNA and protein adduct formation. PCP and DCNP had similar effects on the formation of some PhIP metabolites. PCP and DCNP decreased the formation of 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate (4'-PhIP-sulfate) and 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP)-glucuronide to 10% and 55% of controls, respectively. 2-Amino-1-methyl-4'-hydroxy-6-phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP) was increased by 50% relative to control levels due to PCP and DCNP treatment. PCP and DCNP had different effects on the formation of other PhIP metabolites. Metabolite formation as percent of control for the uncharacterized metabolite, 'Peak A', was 50% and 100% in incubations with PCP and DCNP, respectively. Formation of 4'-hydroxy-PhIP-glucuronide was decreased to 10% of controls with PCP and increased to 147% of controls with DCNP. PCP and DCNP had no effect on the formation of an unidentified metabolite, 'Peak B'. ANF decreased metabolite formation by 60-95%. None of the enzyme inhibitors had a statistically significant effect on PhIP-DNA binding. Covalent binding of PhIP to protein was slightly decreased in incubations containing DCNP or PCP. The lack of significant changes in covalent binding to either DNA or protein suggests that additional pathways may be important in PhIP bioactivation in rat liver slices. With ANF, there was a significant decrease (35%) in protein binding. These observations on the effects of PCP, DCNP and ANF on PhIP metabolism as well as on covalent binding of PhIP to tissue macromolecules are in close agreement with what was reported earlier in hepatocytes. This indicates that tissue slices from various target tissues for tumorigenesis will be a useful in vitro tool for future studies on heterocyclic amine metabolism. This study provides another important example of the utility of precision-cut tissue slices to investigate xenobiotic metabolism and toxicity.     

Human liver arylacetamide deacetylase. Molecular cloning of a novel esterase involved in the metabolic activation of arylamine carcinogens with high sequence similarity to hormone-sensitive lipase

Microsomal arylacetamide deacetylase (DAC) competes against the activity of cytosolic arylamine N-acetyltransferase, which catalyzes one of the initial biotransformation pathways for arylamine and heterocyclic amine carcinogens in many species and tissues. Activity determination and immunoblot analysis of DAC in human target tissues for arylamine carcinogens revealed that in extrahepatic tissues, additional enzymes are responsible for any deacetylation activity, whereas a single enzyme predominantly catalyzes this hydrolytic reaction in liver. We isolated and characterized a full-length cDNA from a human liver lambda gt11 library. This clone encodes an open reading frame of 400 amino acids with a deduced molecular mass of 45.7 kDa and contains two putative glycosylation sites. The 3'-untranslated region contains two putative polyadenylation signals. The cDNA was confirmed to be that for DAC in tryptic peptides from the purified human liver protein. Highest sequence similarity of DAC was found in a series of prokaryotic esterases encompassing the putative active site. Two extended regions of significant sequence homology with hormone-sensitive lipase and with lipase 2 from Moraxella TA144 were identified, whereas similarity to carboxyl esterases was restricted to the region encompassing the putative active site, indicating that DAC should be classified as esterase. This cDNA provides an important tool to study deacetylation and its effects on the metabolic activation of arylamine and heterocyclic amine carcinogens.     

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.     

Interindividual variation in the metabolic activation of heterocyclic amines and their N-hydroxy derivatives in primary cultures of human mammary epithelial cells

The heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are pyrolysis products formed when meat is cooked and are rodent mammary carcinogens. They are thought to be metabolically activated by N-hydroxylation, catalysed by cytochrome P450 (CYP), followed by O-acetylation catalysed by N-acetyltransferases. Primary cultures of human mammary epithelial cells (HMECs) prepared from up to 26 individuals for each compound, were treated with IQ, MeIQ, or PhIP (500 microM) or with N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) or N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline (N-OH-IQ) (20 microM) and the levels of adduct formation in their DNA analysed by 32P-post-labelling. In order to investigate whether pharmacogenetic polymorphisms influence DNA adduct formation, the NAT2 genotype of each individual was determined by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that distinguishes between the wild-type and four variant alleles. Presence of two variant alleles designates a slow NAT2 acetylator, whereas individuals with one or two wild-type alleles are designated fast NAT2 acetylators. Interindividual variations in total DNA adduct levels ranged for IQ from 0.64-63.1 DNA adducts per 10(8) nucleotides (mean 7.80), for MeIQ from 1.99-17.8 (mean 6.63), for PhIP from 0.13-4.0 (mean 0.96), for N-OH-PhIP from 6.32-497 (mean 176) and for N-OH-IQ from 0.92-30.6 (mean 9.24). The higher adduct levels observed in cells treated with the N-OH metabolites suggests that N-hydroxylation is the rate-limiting step in HMECs and this may be due to low CYP levels. In contrast, the Phase II reaction catalysed by N-acetyltransferases is probably the major step in the metabolic activation of heterocyclic amines that occurs in the breast. Higher mean levels of heterocyclic amine-DNA adduct formation were detected in the cells of NAT2 fast acetylators compared with slow acetylators, with mean adduct levels per 10(8) nucleotides following IQ treatment, of 12.74 and 3.57 respectively, following PhIP treatment, of 1.20 and 0.74, respectively, following MeIQ treatment, of 7.90 and 5.08, respectively and following N-OH-PhIP-treatment, of 243.1 and 130.0, respectively. However, due to the large variations in adduct levels, these differences in mean values were not statistically significant with the limited number of individuals studied. This appears to be the first pilot study to demonstrate interindividual variations in the metabolic activation of heterocyclic amines and their metabolic intermediates in primary cultures of HMECs in vitro.     

Missed opportunities for prevention: smoking cessation counseling and the competing demands of practice

Activating mutations in and expression of the Ha-ras gene were examined in benign and malignant female Sprague-Dawley rat mammary gland tumors induced by the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and promoted by a diet high in polyunsaturated fat. Ha-ras mutations were detected in codons 12 and 13 by selective polymerase chain reaction amplification of mutated sequences and nucleotide sequencing. The percentage of Ha-ras mutations in carcinomas from PhIP-treated rats was significantly higher in rats on a low-fat diet than in rats on a high-fat diet (82% (nine of 11) vs 26% (seven of 27), respectively, P < 0.01). In addition, whereas 56% of the carcinomas with Ha-ras mutations from rats on a low-fat diet carried double Ha-ras mutations, none of the carcinomas from rats on a high-fat diet had double mutations. Ha-ras mutations were also detected in benign tumors (largely adenomas) induced by PhIP in rats on different diets; two of eight and three of four benign tumors examined from rats on low-fat and high-fat diets, respectively, had Ha-ras mutations, suggesting that activating Ha-ras mutations alone are not sufficient for PhIP-induced tumors to become malignant. No differences were observed in the level of Ha-ras mRNA expression in the different groups. In our animal model, a high-fat diet increased the incidence and percentage of malignant PhIP-induced mammary gland tumors yet decreased the percentage of carcinomas showing Ha-ras mutations. Thus, the complement of genetic alterations associated with PhIP-induced mammary gland carcinogenesis is probably altered by the level of dietary fat.     

Uptake of the food-derived heterocyclic amine carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its N-hydroxy metabolite into rat pancreatic acini and hepatocytes in vitro

Sinc DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed at relatively high levels in the rat pancreas but not liver, we examined the uptake of PhIP and its N-hydroxy metabolite (N-OH-PhIP) into pancreatic acini and hepatocytes to determine if differential tissue uptake was a factor modulating the formation of PhIP-DNA adducts. In addition, since the precursors of PhIP formation are two amino acids and since various amino acid transporters have been identified in the pancreas, the possible involvement of these transporters in the uptake of PhIP and N-OH-PhIP was investigated. The uptake both heterocyclic compounds into both tissue preparations was rapid, with maximal uptake occurring with 1-2 min. However, PhIP uptake into pancreatic acini was significantly (2-way ANOVA, P < 0.05) greater than uptake of N-OH-PhIP into pancreatic acini and the uptake of both PhIP and N-OH-PhIP into hepatocytes. Although uptake was rapid, efflux of both compounds from both tissue preparations was also rapid. However, the efflux rate constant (1.86 +/- 0.6/min, mean +/- SEM) for PhIP) was significantly lower (Student's t-test, P < 0.05) than that for N-OH-PhIP (4.14 +/- 0.04/min) from pancreatic acini. This, combined with the increased uptake of PhIP into pancreatic acini , suggests that there is substantial but reversible binding of PhIP in the pancreas. The uptake of both PhIP and N-OH-PhIP into pancreatic acini and hepatocytes was not affected by the presence of various amino acids in the incubation buffer, indicating that amino acid transporters are not involved in uptake of these compounds. Furthermore, uptake of both compounds did not appear to be dependent on metabolic energy supply. The above data, together with the high octanol:buffer partition coefficients (logP = 1.322 and 1.301 for PhiP and N-OH-PhIP respectively) suggest that both uptake and efflux of PhIP and N-OH-PhIP are consistent with a process of passive diffusion. The tissue binding characteristics for PhIP in the pancreas may create conditions whereby pancreatic cytochrome P450 1A1 can catalyse the formation of N-OH-PhIP. While N-OH-PhIP is not the ultimate reactive DNA binding species, it has been shown to directly bind to and form DNA adducts. Therefore, it is possible that the apparent selective accumulation of PhIP may contribute to the high level of PhIP-DNA adducts formed in the rat pancreas.     

Transcellular transport of benzoic acid across Caco-2 cells by a pH-dependent and carrier-mediated transport mechanism

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are two members of a family of carcinogenic heterocyclic amines (HAs) found in cooked meats that form DNA adducts after activation to N-acetoxy derivatives. The ability of IQ- and PhIP-DNA adducts to inhibit gene expression was investigated using a human growth hormone (hGH) reporter gene in a pUC12-based mammalian expression vector under the control of either the herpes simplex virus-1 thymidine kinase promoter or the human immunodeficiency virus-1 long terminal repeat. The plasmids were treated in vitro with 0, 5, 10, or 40 microM N-hydroxy-IQ or N-hydroxy-PhIP in the presence of a 10-fold molar excess of acetic anhydride to generate the N-acetoxy derivatives in situ. The adduct levels in the plasmids were quantitated by the 32P-postlabeling method. The adducted (and control) plasmids were each transfected into repair-deficient or -proficient Chinese hamster ovary cells, and expression of hGH was measured by immunoassay of growth hormone secreted into the cell medium. The results showed that IQ- and PhIP-DNA adducts inhibited gene expression in both plasmids and that the degree of inhibition of hGH production was proportional to the levels of IQ- and PhIP-DNA adducts. The degree of inhibition, however, was independent of the promoter, despite the differences in the strengths of the two promoters to drive hGH production. Repair capacity influenced the extent of inhibition of gene expression by HA adducts since, in general, fewer adducts were needed to inhibit reporter gene expression in repair-deficient cells than in repair-proficient cells. In both cell lines, DNA adducts of PhIP appeared to be more potent in inhibiting hGH expression than adducts of IQ. Whether alteration of gene expression by HA adducts plays a role in the carcinogenicity of these compounds deserves further study.     

Prevention of heterocyclic amine formation by tea and tea polyphenols

Heterocyclic amines (HCAs), formed during the cooking of meats and fish, are thought to be the genotoxic carcinogens associated with important types of human cancer in meat-eating populations, such as cancer of the breast, colon or pancreas. We studied the effect of black or green tea, and of the tea polyphenols theaflavine gallate (TFG, black tea) and epigallocatechin gallate (EGCG, green tea) on the formation of typical HCAs, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), using the model in vitro systems of J?gerstad. Green tea and black tea and solutions of TFG and EGCG lower the formation of PhIP by 62-85% during 1 h heating at 160 degrees C of 1 mmol creatinine, 1 mmol phenylalanine and 0.5 mmol glucose in 3.3 ml diethylene glycol-water (10:1), where the inhibitors replaced 0.3 ml of the water. The production of MeIQx and related HCAs, in the same system but with 1 mmol glycine instead of phenylalanine was likewise reduced, determined by amounts of mutagens formed. In the latter systems, the teas were not, or less effective, but the polyphenols were inhibitory. Thus, the tea products represent another approach to lower the formation of HCAs.     

Influence of carcinogen binding by lactic acid-producing bacteria on tissue distribution and in vivo mutagenicity of dietary carcinogens

The aims of this investigation were to determine whether viable cultures of lactic acid-producing organisms (LAB) can bind dietary carcinogens and to assess the consequences of binding for the absorption from the gut, distribution in the body and in vivo genotoxicity of ingested carcinogens. The carcinogens used in this study were ones known to be present in the human diet, namely benzo[a]pyrene (B(a)P, aflatoxin B1 (AFB1) and the cooked food carcinogens 2-amino-3-methyl-3H-imidazo[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), 5-phenyl-2-amino-1-methylimidazo [4,5-f]pyridine (PhIP) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). They represent a range of structural types so that the specificity of any binding effects could be addressed. Of the carcinogens tested, B(a)P and Trp-P-2 were bound most effectively by the two LAB strains Bifidobacterium longum and Lactobacillus acidophilus. AFB1 was poorly bound, while MeIQx, MeIQ, PhIP and IQ were bound to an intermediate degree. The extent of the binding of the heterocyclic amine carcinogens was dependent on the pH conditions during incubation and this effect was more apparent with B. longum than with L. acidophilus. Using the host-mediated assay (HMA), an in vivo bacterial mutation assay, it was demonstrated that the administration of bacterial cell suspensions of B. longum and L. acidophilus did not lead to a reduction in induced mutagenicity by MeIQ, MeIQx or Trp-P-2, detectable in the liver of treated mice compared with controls. The lack of a protective effect could not be attributed to a short period of contact between bacterial cells and mutagens, since similar results were obtained after preincubating bacteria and mutagens together at pH 5 for 50-60 min, to maximize the binding, before gavaging the mice. Lack of activity of B(a)P in the HMA prevented the determination of the effect of LAB on genotoxicity of the polycyclic aromatic hydrocarbon. However, it is clear from the radiolabel distribution study that the amount of the carcinogen entering the blood was not significantly reduced by B. longum administration. In addition, the amount of radiolabelled B(a)P that reached the target organs (liver, lungs and heart) was also not affected by the LAB administration. A similar lack of inhibitory effect of B. longum on blood concentration and accumulation in the liver of Trp-P-2 was apparent. The results of the present study suggest that although LAB may bind carcinogens in vitro, this does not lead to major changes in absorption and distribution of carcinogens in the body, or in their genotoxic activity in the liver.     

Differential effect of triiodothyronine and thyroxine on liposomes containing cholesterol: physiological speculations

Chemopreventive effects of the antioxidants 1-O-hexyl-2,3,5- trimethylhydroquinone (HTHQ), 3-O-ethylascorbic acid (EAsA), 3-O-dodecylcarbomethylascorbic acid (DAsA), green tea catechins (GTC) and ellagic acid on 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis were examined in female F344 rats. Groups of 20-21 6-week-old rats were maintained on a powdered diet containing 0.02% PhIP alone, PhIP together with 0.5% HTHQ, 1% EAsA, 1% DAsA, 1% GTC or 0.1% ellagic acid, these antioxidants alone or basal diet alone without supplement for 52 weeks. The survival rates of PhIP plus antioxidant groups at the end of the experiment were higher than that of the PhIP alone group. Sequential observation of palpable mammary tumors demonstrated only one tumor by week 52 in the PhIP plus HTHQ group, whereas 40% of the rats receiving PhIP alone had tumors by this time point. The final incidence of mammary adenocarcinomas was significantly decreased in the PhIP plus HTHQ group (4.8%, P < 0.01) as compared to the PhIP alone value (40%). Although statistically not significant, incidences of adenocarcinomas in the other antioxidant-treated groups (23.8-28.6%) were also lower than in the PhIP alone group. Furthermore, the incidence of large intestinal tumors in the PhIP plus HTHQ group (0%) showed a tendency to decrease relative to the PhIP alone group (16.7%). These results indicate that antioxidants, particularly HTHQ, exert a potent chemopreventive action against PhIP-induced carcinogenesis.     

Characterization of the gntT gene encoding a high-affinity gluconate permease in Escherichia coli

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.     

A human sound transducer/reproducer: temporal capabilities of a profoundly echolalic child

The effect of pO2 on the monoamine oxidase activity of mitochondria from rabbit brain and liver was investigated using the substrates tyramine, dopamine, tryptamine and serotonin. The effect of the second substrate (oxygen) was dependent upon the concentration of the first substrate (the amine). At amine concentrations below 50 micronM, the reaction rate as measured by a radiometric assay, was not affected by variations in the pO2. It was found that both phenazine methosulfate (PMS) and chlorpromazine (CPZ) are reversible inhibitors of monoamine oxidase, the former was a potent inhibitor (Ki=3X10(-6) M) and the latter relatively weak (Ki=5X10(-4) M). Inhibition by both compounds was non-competitive with respect to the amine substrate. Imipramine was a weak inhibitor of purified MAO from beef kidney and of the MAO activity of mitochrondria from brain and liver. Using tyramine or dopamine as the substrate (0.5-1.0 mM), inhibition ranging from 6-30% was observed at 5X10(-4) M imipramine. With tryptamine or serotonin (0.5-1.0 mM) as the substrate in the presence of 5X10(-4) M imipramine the drug seemed to have no net effect on MAO activity since the average value in the presence of imipramine for a number of experiences was the same as the average for control experiments. For p-iodo-phenyl-3-p-nitrophenyl tetrazolium chloride, a Ki of 43X10(-6) M was found using dopamine as the substrate and oxygen as the gas phase.