Preferential induction of the AhR gene battery in HepaRG cells after a single or repeated exposure to heterocyclic aromatic amines

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) are two of the most common heterocyclic aromatic amines (HAA) produced during cooking of meat, fish and poultry. Both HAA produce different tumor profiles in rodents and are suspected to be carcinogenic in humans. In order to better understand the molecular basis of HAA toxicity, we have analyzed gene expression profiles in the metabolically competent human HepaRG cells using pangenomic oligonucleotide microarrays, after either a single (24-h) or a repeated (28-day) exposure to 10 μM PhIP or MeIQx. The most responsive genes to both HAA were downstream targets of the arylhydrocarbon receptor (AhR): CYP1A1 and CYP1A2 after both time points and CYP1B1 and ALDH3A1 after 28 days. Accordingly, CYP1A1/1A2 induction in HAA-treated HepaRG cells was prevented by chemical inhibition or small interference RNA-mediated down-regulation of the AhR. Consistently, HAA induced activity of the CYP1A1 promoter, which contains a consensus AhR-related xenobiotic-responsive element (XRE). In addition, several other genes exhibited both time-dependent and compound-specific expression changes with, however, a smaller magnitude than previously reported for the prototypical AhR target genes. These changes concerned genes mainly related to cell growth and proliferation, apoptosis, and cancer. In conclusion, these results identify the AhR gene battery as the preferential target of PhIP and MeIQx in HepaRG cells and further support the hypothesis that intake of HAA in diet might increase human cancer risk.     

Protective effects of xanthohumol against the genotoxicity of heterocyclic aromatic amines MeIQx and PhIP in bacteria and in human hepatoma (HepG2) cells

Previous studies showed that xanthohumol (XN), a hop derived prenylflavonoid, very efficiently protects against genotoxicity and potential carcinogenicity of the food borne carcinogenic heterocyclic aromatic amine (HAA) 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). In this study, we showed that XN was not mutagenic in Salmonella typhimurium TA98 and did not induce genomic instability in human hepatoma HepG2 cells. In the bacteria XN suppressed the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8 dimethylimidazo[4,5-f]quinoxaline (MeIQx) induced mutations in a dose dependent manner and in HepG2 cells it completely prevented PhIP and MeIQx induced DNA strand breaks at nanomolar concentrations. With the QRT-PCR gene expression analysis of the main enzymes involved in the biotransformation of HAAs in HepG2 cells we found that XN upregulates the expression of phase I (CYP1A1 and CYP1A2) and phase II (UGT1A1) enzymes. Further gene expression analysis in cells exposed to MeIQx and PhIP in combination with XN revealed that XN mediated up-regulation of UGT1A1 expression may be important mechanism of XN mediated protection against HAAs induced genotoxicity. Our findings confirm the evidence that XN displays strong chemopreventive effects against genotoxicity of HAAs, and provides additional mechanistic information to assess its potential chemopreventive efficiency in humans.     

Antioxidant and antigenotoxic effects of rosemary (Rosmarinus officinalis L.) extracts in Salmonella typhimurium TA98 and HepG2 cells

In the present study the chemopreventive effects of water soluble AquaROX^® 15 and oil soluble VivOX^® 40 rosemary extracts against 4-nitroquinoline-N-oxide (NQNO) and 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline (IQ) induced mutagenicity in the reverse mutation assays with Salmonella typhimurium TA98 and against t-butyl hydroperoxide (t-BOOH), benzo(a)pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced DNA damage in HepG2 cells were studied, applying the comet assay. The results showed comparable protective effect of AquaROX and VivOX against oxidative DNA damage, whereas protection against indirect active genotoxic carcinogens was more efficient by VivOX.     

Influence of frying fat on the formation of heterocyclic amines in fried beefburgers and pan residues

The influence of six frying fats (butter, margarine, margarine fat phase, liquid margarine, rapeseed oil and sunflower seed oil) on the formation of mutagenic/carcinogenic heterocyclic amines (HAs) during the frying of beefburgers was investigated. Frying was performed at 165 and 200°C (i.e. under conditions that represented normal household cooking practices). The fried beefburgers and their corresponding pan residues were purified using solid-phase extraction and analysed for HAs using HPLC with photodiode array UV and fluorescence detection. The HAs 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MelQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 9H-pyrido[3,4-b]indole (norharman) and 1-methyl-9H-pyrido[3,4-b]indole (harman) were recovered. The amount increased with the temperature, and the content of HAs in the pan residue was much higher than in the corresponding beefburger. The amounts of MeIQx ranged from 0.2 to 1.6 ng/g in the beefburgers and from 0.8 to 4.3 ng/g in the pan residues. DiMeIQx ranged from undetectable to 0.4 ng/g in the beefburgers and from 0.4 to 1.3 ng/g in the residues. PhIP ranged from 0.08 to 1.5 ng/g in the meat and from 0.4 to 13.3 ng/g in the residues. The total amount of HAs in meat and pan residue combined was significantly lower after frying in sunflower seed oil or margarine than after frying with the other fats. The observed differences in MeIQx and DiMeIQx formation could be explained in terms of oxidation status (peroxide and anisidine value) and antioxidant content (vitamin A, vitamin E and tocopherols/tocotrienols) using partial least squares analysis.     

Deconjugation of N-glucuronide conjugated metabolites with hydrazine hydrate--biomarkers for exposure to the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

The metabolism of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been investigated in rabbit liver S9. Two phase I metabolites, N(2)-OH-PhIP and 4'-OH-PhIP were identified based on UV and mass spectra and co-elution with reference standards. Fortification of the incubation with UDGPA resulted in a complete glucuronidation of PhIP and N(2)-OH-PhIP, while 4'-OH-PhIP was only partly glucuronidated. Also, the PhIP metabolite 5-OH-PhIP was completely glucuronidated by rabbit liver S9, while 5-OH-PhIP was a poor substrate for CYP mediated hydroxylation. The glucuronic acid conjugates of PhIP metabolites were unsusceptible to treatment with beta-glucuronidase indicating that these are N-glucuronides. Treatment of the conjugates with hydrazine hydrate, however, resulted in complete hydrolysis of the glucuronic acid conjugates as well as in reduction to the parent amine of metabolites hydroxylated in the exocyclic amino group. Urine was collected from a male volunteer following consumption of fried chicken. Treatment of the urine with beta-glucuronidase/sulfatase resulted in release of 4'-OH-PhIP, while treatment with hydrazine hydrate in addition resulted in release of substantial amounts of PhIP and 5-OH-PhIP. The data show that hydrazine hydrate can hydrolyse N-glucuronides of metabolites of PhIP, glucuronides that are unsusceptible to enzymatic hydrolysis. In addition the data indicate that humans metabolise a large fraction of ingested PhIP to genotoxic metabolites. The chemical hydrolysis of glucuronide conjugates of PhIP metabolites with hydrazine hydrate observed in this study may also be a useful approach in the development of biomarkers for exposure and effect of other xenobiotics.     

Oral administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) yields PhIP-DNA adducts but not tumors in male Syrian hamsters congenic at the N-acetyltransferase 2 (NAT2) locus.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine carcinogen present in well-done meat. PhIP must undergo host-mediated bioactivation to exert its mutagenic and carcinogenic effects. Following N-hydroxylation, N-acetyltransferases catalyze the O-acetylation (activation) of N-hydroxy-PhIP to an electrophile causing DNA damage. A well-defined genetic polymorphism in N-acetyltransferase 2 (NAT2) activity exists in humans and the Syrian hamster. Since some human epidemiological studies suggest an association between acetylator genotype and cancer susceptibility in individuals who consume well done meats, this study was designed to investigate the specific role of acetylator genotype in PhIP-induced tumors using a Syrian hamster model congenic at the NAT2 locus. Following oral administration of PhIP to male rapid and slow acetylator Syrian hamsters, DNA adducts were identified in each tissue examined with levels in the relative order: pancreas > heart and urinary bladder > prostate, small intestine and transverse colon > ascending colon, liver, cecum, descending colon, and rectum. However, no tumors were observed in male rapid and slow acetylator congenic hamsters administered 11 oral doses of PhIP (75 mg/kg) and maintained on a high fat diet for one year.     

Biomonitoring of urinary metabolites of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) following human consumption of cooked chicken

Human risk assessment of exposure to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) through the diet may be improved by conducting biomonitoring studies comparing metabolism in humans and rodents. Eleven volunteers ingested a meal of cooked chicken containing 4 -OH-PhIP and PhIP in amounts of 0.6 and 0.8mug/kg, respectively and urine was collected for the next 16h. The large number of PhIP metabolites was by treatment of the urine samples with hydrazine hydrate and hydrolytic enzymes reduced to three substances, 4'-OH-PhIP, PhIP and 5-OH-PhIP of which the first is a biomarker for detoxification and the last a biomarker for activation. The eleven volunteers eliminated large amounts of 4'-OH-PhIP in the urine. The majority of which could be accounted for by the presence of 4'-OH-PhIP in the fried chicken, showing that PhIP only to a small extent (11%) was metabolised to 4'-OH-PhIP. A larger fraction of the PhIP exposure, 38%, was recovered as PhIP and the largest fraction (51%) was recovered as 5-OH-PhIP suggesting that PhIP in humans to a large extent is metabolised to reactive substances. In rats, less than 1% of the dose of PhIP was eliminated as 5-OH-PhIP, suggesting that human cancer risk from exposure to PhIP is considerable higher than risk estimations based on extrapolation from rodent bioassays.     

Enhancement of hepatocellular genotoxicity of several mutagens from amino acid pyrolysates and broiled foods following ethanol pretreatment

The effect of subchronic ethanol ingestion on the genotoxicity and metabolism of the mutagens 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,5-b]indole (Trp-P-2), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4- dimethylimidazo[4,5-f]quinoline (MeIQ) was evaluated in primary cultures of rat hepatocytes. Male Sprague-Dawley rats were pair-fed, for 8 days, liquid diets containing either ethanol (8%, v/v) or an isocaloric sucrose solution. Ethanol pretreatment significantly (P less than 0.05, Student's t test) enhanced the level of DNA repair stimulated by Glu-P-1, Glu-P-2, IQ and MeIQ. Statistically significant increases in DNA-repair activity ranged from 1.9-fold for IQ to 3.4-fold for Glu-P-2. Following a 16-hr exposure, the concentration of parent mutagen in the culture medium decreased by 75-98%. Neither the rate of mutagen metabolism in hepatocyte cultures nor the extent of mutagenic activation in microsome preparations was appreciably affected by ethanol pretreatment. The results suggest that ethanol pretreatment enhances the genotoxicity of Glu-P-1, Glu-P-2, IQ and MeIQ by inducing non-microsomal activation processes.     

A mechanistic basis for the role of cycle arrest in the genetic toxicology of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of meat, induces tumors of the prostate, colon, and mammary gland when fed to rats. PhIP is readily absorbed and efficiently metabolized to a genotoxic derivative by CYP1 enzymes. Although metabolism and mutational potential of PhIP have previously been well characterized, the intervening cellular and genomic responses to the chemical are not fully understood. We have examined the cellular response to PhIP exposure in human mammary epithelial MCF10A cells, which retain characteristics of normal breast epithelial cells. Because these cells fail to activate PhIP, they were cocultured with a human lymphoblastoid cell line MCL-5, which constitutively expresses CYP1A1, and have been transfected to express human CYPs1A2, 2A6, 3A4, and 2E1. The MCL-5 cells were irradiated (2,000 rads) prior to coculture, rendering them unable to replicate yet still retaining metabolic competency. MCF10A cells were treated (in the presence of MCL-5 cells) with PhIP (1-100 microM) and harvested at various time-points. Compared to DMSO control, treatment (24 or 48 h) with PhIP resulted in a significant dose-dependent fall in cell number. Cells treated for 48 h then cultured in the absence of PhIP (and MCL-5 cells) for a further 6 days showed a much greater dose-dependent reduction in cell number. Flow cytometric analysis indicated that PhIP treatment (48 h) resulted in a dose-dependent accumulation of cells in the G1 population. Western blotting revealed elevated expression of p53 and the cyclin dependent kinase inhibitor p21WAF1/CIP1 after PhIP treatment. Levels of MDM2, a negative regulator of p53, and the hypophosphorylated form of RB were also elevated, consistent with the triggering of G1 cell cycle checkpoint. These cell cycle effects are critical, as they enable cells to effect genome repair, accept mutation, or eliminate excessively damaged cells.     

Heterocyclic aromatic amines in domestically prepared chicken and fish from Singapore Chinese households.

Chicken and fish samples prepared by 42 Singapore Chinese in their homes were obtained. Researchers were present to collect data on raw sample weight, cooking time, maximum cooking surface temperature, and cooked sample weight. Each participant prepared one pan-fried fish sample and two pan-fried chicken samples, one marinated, one not marinated. The cooked samples were analyzed for five heterocyclic aromatic amine (HAA) mutagens, including MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline); 4,8-DiMeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline); 7,8-DiMeIQx (2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline); PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), and IFP (2-amino-(1,6-dimethylfuro[3,2-e]imidazo[4,5-b])pyridine). A paired Student's t-test showed that marinated chicken had lower concentrations of PhIP (p<0.05), but higher concentrations of MeIQx (p<0.05) and 4,8-DiMeIQx (p<0.001) than non-marinated chicken, and also that weight loss due to cooking was less in marinated chicken than in non-marinated chicken (p<0.001). Interestingly, the maximum cooking surface temperature was higher for fish than for either marinated or non-marinated chicken (p<0.001), yet fish was lower in 4,8-DiMeIQx per gram than marinated or non-marinated chicken (p<0.001), lower in PhIP than non-marinated chicken (p<0.05), and lost less weight due to cooking than either marinated or non-marinated chicken (p<0.001). Fish was also lower in MeIQx and 7,8-DiMeIQx than marinated chicken (p<0.05). This study provides new information on HAA content in the Singapore Chinese diet.     

Degradation of heterocyclic aromatic amines in oil under storage and frying conditions and reduction of their mutagenic potential.

Heterocyclic aromatic amines (HAA) were systematically studied concerning their partition behavior in water/oil-systems and their thermostability in different animal derived fats and vegetable oils. Partitioning of IQx-compounds and PhIP in water/oil systems was found to depend on the polarity defined by the molecular structure and on the pH-value of the aqueous phase. In particular, beta-carbolines norharman and harman showed a significant strong lipophilic character at alkaline pH. After heating in frying fats at 130 degrees C, contents of IQx compounds and PhIP were reduced by more than 40% and after heating at 180 degrees C less than 10% of the HAA initial concentration was recovered. By contrast, norharman and harman were much more stable under equivalent conditions. The present study leads for the first time to the conclusion that degradation of HAA in frying fats strongly correlates to the type of frying fat and is promoted by lipid oxidation products. Firstly, addition of hydroperoxides to model oils lead to a decrease of HAA during storage at 40 degrees C. Secondly, stability of HAA correlated with the content of unsaturated fatty acids in the oil, which is indicative for the oxidative stability of the medium. Degradation of HAA by heat treatment was associated with a reduction of their mutagenic potential towards strain TA98 of Salmonella typhimurium.     

Intestinal bacteria metabolize the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine following consumption of a single cooked chicken meal in humans.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic amine formed in meats during cooking. Although the formation of PhIP metabolites by mammalian enzymes has been extensively reported, the involvement of the intestinal bacteria remains unclear. This study examined the urinary and fecal excretion of a newly identified microbial PhIP metabolite 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetrahydropyrido[3',2':4,5]imidazo[1,2-a]pyrimidin-5-ium chloride (PhIP-M1) in humans. The subjects were fed 150 g of cooked chicken containing 0.88-4.7 microg PhIP, and urine and feces collections were obtained during 72 h after the meal. PhIP-M1 and its trideuterated derivate were synthesized and a LC/MS/MS method was developed for their quantification. The mutagenic activity of PhIP-M1, as analyzed using the Salmonella strains TA98, TA100 and TA102, yielded no significant response. Of the ingested PhIP dose, volunteers excreted 12-21% as PhIP and 1.2-15% as PhIP-M1 in urine, and 26-42% as PhIP and 0.9-11% as PhIP-M1 in feces. The rate of PhIP-M1 excretion varied among the subjects. Yet, an increase in urinary excretion was observed for successive time increments, whereas for PhIP the majority was excreted in the first 24h. These findings suggest that besides differences in digestion, metabolism and diet, the microbial composition of the gastrointestinal tract also strongly influences individual disposition and carcinogenic risk from PhIP.     

The cooked meat-derived mammary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) elicits estrogenic-like microRNA responses in breast cancer cells

The cooking of meat results in the generation of heterocyclic amines (HCA), the most abundant of which is 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Data from epidemiological, mechanistic, and animal studies indicate that PhIP could be causally linked to breast cancer incidence. Besides the established DNA damaging and mutagenic activities of PhIP, the chemical is reported to have oestrogenic activity that could contribute to its tissue specific carcinogenicity. In this study we investigated the effect of treatment with PhIP and 17-β-estradiol (E2) on global microRNA (miRNA) expression of the oestrogen responsive MCF-7 human breast adenocarcinoma cell line.     

Effects of (+)catechin and (−)epicatechin on heterocyclic amines‐induced oxidative DNA damage

The aim of the present study was to evaluate the protective effect of (+)‐catechin and (?)‐epicatechin against 2‐amino‐3,8‐ dimethylimidazo[4,5‐f]quinoxaline (8‐MeIQx), 2‐amino‐3,4,8‐trimethylimidazo[4,5‐f]‐quinoxaline (4,8‐diMeIQx) and 2‐amino‐1‐methyl‐6‐phenyl‐imidazo[4,5‐b]pyridine (PhIP)‐induced DNA damage in human hepatoma cells (HepG2). DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single‐cell gel electrophoresis or comet assay. Increasing concentrations of 8‐MeIQx, 4,8‐diMeIQx and PhIP induced a significant increase in DNA strand breaks and oxidized purines and pyrimidines in a dose‐dependent manner. Among those, PhIP (300 µm ) exerted the highest genotoxicity. (+)‐Catechin exerted protection against oxidized purines induced by 8‐MeIQx, 4,8‐diMeIQx and PhIP. Oxidized pyrimidines and DNA strand breaks induced by PhIP were also prevented by (+)‐catechin. Otherwise, (?)‐epicatechin protected against the oxidized pyrimidines induced by PhIP and the oxidized purines induced by 8‐MeIQx and 4,8‐diMeIQx. One feasible mechanism by which (+)‐catechin and (?)‐epicatechin exert their protective effect towards heterocyclic amines‐induced oxidative DNA damage may be by modulation of phase I and II enzyme activities. The ethoxyresorufin O‐deethylation (CYP1A1) activity was moderately inhibited by (+)‐catechin, while little effect was observed by (?)‐epicatechin. However, (+)‐catechin showed the greatest increase in UDP‐glucuronyltransferase activity. In conclusion, our results clearly indicate that (+)‐catechin was more efficient than (?)‐epicatechin in preventing DNA damage (strand breaks and oxidized purines/pyrimidines) induced by PhIP than that induced by 8‐MeIQx and 4,8‐diMeIQx. Copyright © 2010 John Wiley & Sons, Ltd.     

Carcinogenic risk of heterocyclic amines in combination – Assessment with a liver initiation model

Carcinogenic potential of heterocyclic amines (HCAs) was investigated using an in vivo 5-week initiation assay with quantitative evaluation of glutathione S-transferase placental form (GST-P) positive foci in rat liver. Numbers of GST-P positive foci were significantly increased with individual administration of six different HCAs, indicating utility of the assay. It was therefore applied to investigate risk with multiple HCAs in combination. Unexpectedly, concomitant treatment with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) did not result in any additive carcinogenicity. In the rats taking MeIQx prior to PhIP the value was almost equal to the sum total of individual data, indicating additive initiation activities. In contrast, simultaneous or prior administration of PhIP rather exerted inhibitory effects on the carcinogenic potential of MeIQx. Moreover, microarray and quantitative RT-PCR assessment revealed that PhIP induced cytochrome P450 1A1, responsible for both activation and detoxification of HCAs, more strongly than MeIQx. It is noteworthy that complex exposure to multiple HCAs is not necessarily associated with increased risk of carcinogenesis because they are simultaneously and continuously ingested under normal circumstances.     

Effect of chrysin,a flavonoid compound,on the mutagenic activity of 2-amino-1-methyl-6-phenylimidazo[4,5-<Emphasis Type="Italic">b</Emphasis>]pyridine (PhIP) and benzo(a)pyrene (B(a)P) in bacterial and human hepatoma (HepG2) cells

The aim of the present study was to investigate the antimutagenic effects of chrysin (CR), a flavonoid compound contained in many fruits, vegetables and honey. Earlier investigations with bacterial indicators showed that CR is one of the most potent antimutagens among the flavonoids. In the present study, we tested the compound in the Salmonella strains TA98 and TA100 in combination with benzo(a)pyrene (B(a)P) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and found pronounced protective activity over a concentration range between 10 and 100 g/ml. The compound itself was devoid of mutagenic activity at all concentrations tested. In the micronucleus (MN) assay with human-derived HepG2 cells, a different pattern of activity was seen. CR itself caused significant induction of MN at dose levels 15 g/ml; in combination experiments with B(a)P and PhIP, U-shaped dose-response curves were obtained and protection was found only in a narrow dose range (5 – 10 g/ml). Our findings indicate that the molecular mechanisms that account for the antimutagenic effects of CR in bacterial cells are different from those responsible for the effects in HepG2 cells. Earlier reports indicate that the antimutagenic effects of CR towards B(a)P and heterocyclic amines in bacterial indicators is due to inhibition of the activity of CYP1A. In contrast to this, we found a significant induction of CYP1A1 activity in HepG2 cells by CR. It can also be excluded that induction of GST, which is involved in the detoxification of polycyclic aromatic hydrocarbons accounts for the protective effects of CR against B(a)P since this enzyme was not significantly induced in the HepG2 cells. In the case of PhIP, induction of UDGPT and/or inhibition of sulfotransferase seen in human derived HepG2 cells after exposure to CR might play a role in the antimutagenic effects. In conclusion, our findings show that data from antimutagenicity studies with bacterial indicators cannot be extrapolated to HepG2 cells, and that CR causes genotoxic effects at higher dose levels in the latter cells. The implications of these observations for human chemoprevention strategies are discussed.     

DNA adduct formation of 4-aminobiphenyl and heterocyclic aromatic amines in human hepatocytes

DNA adduct formation of the aromatic amine, 4-aminobiphenyl (4-ABP), a known human carcinogen present in tobacco smoke, and the heterocyclic aromatic amines (HAAs), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), potential human carcinogens, which are also present in tobacco smoke or formed during the high-temperature cooking of meats, was investigated in freshly cultured human hepatocytes. The carcinogens (10 μM) were incubated with hepatocytes derived from eight different donors for time periods up to 24 h. The DNA adducts were quantified by liquid chromatography-electrospray ionization mass spectrometry with a linear quadrupole ion trap mass spectrometer. The principal DNA adducts formed for all of the carcinogens were N-(deoxyguanosin-8-yl) (dG-C8) adducts. The levels of adducts ranged from 3.4 to 140 adducts per 10(7) DNA bases. The highest level of adduct formation occurred with AαC, followed by 4-ABP, then by PhIP, MeIQx, and IQ. Human hepatocytes formed dG-C8-HAA-adducts at levels that were up to 100-fold greater than the amounts of adducts produced in rat hepatocytes. In contrast to HAA adducts, the levels of dG-C8-4-ABP adduct formation were similar in human and rat hepatocytes. These DNA binding data demonstrate that the rat, an animal model that is used for carcinogenesis bioassays, significantly underestimates the potential hepatic genotoxicity of HAAs in humans. The high level of DNA adducts formed by AαC, a carcinogen produced in tobacco smoke at levels that are up to 100-fold higher than the amounts of 4-ABP, is noteworthy. The possible causal role of AαC in tobacco-associated cancers warrants investigation.     

Effects of soy sauce and sugar on the formation of heterocyclic amines in marinated foods.

The effects of soy sauce and sugar on the formation of heterocyclic amines (HAs) in marinated pork, eggs, and bean cakes were studied. Food samples were immersed in water in the presence of various levels of soy sauce and sugar, and the mixtures were subjected to simmering at 98+/-2 degrees C for 1 h in a closed saucepan. The various HAs in marinated food samples were analyzed by HPLC with photodiode-array detection. Results showed that seven HAs: 2-amino-3-methylimidazo[4,5-f]quinoline (IQ); 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx); 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ); 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx); 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1); 2-amino-1-methyl-6-phenylimidazo[4,5-f]pyridine (PhIP); and 2-amino-9H-pyrido[2,3,-b]indole (AalphaC) were detected in marinated pork, while five HAs: IQ, MeIQx; 4,8-DiMeIQx; PhIP; and AalphaC in bean cakes, as well as four HAs, MeIQx, 4,8-DiMeIQx, Trp-P-1 and PhIP in eggs. In most samples PhIP was formed in largest amount, followed by MeIQx, 4,8-DiMeIQx, IQ, AalphaC, Trp-P-1 and MeIQ. The amounts of HAs produced in marinated food samples followed an increased order for each increasing level of soy sauce or sugar. Marinated juice was found to contain a higher content of HAs than marinated foods.     

4-hydroxynonenal induces mitochondrial oxidative stress, apoptosis and expression of glutathione S-transferase A4-4 and cytochrome P450 2E1 in PC12 cells

An excessive and sustained increase in reactive oxygen species (ROS) production and oxidative stress have been implicated in the pathogenesis of many diseases. In the present study, we have demonstrated that 4-hydroxynonenal (4-HNE), a product of lipid peroxidation, alters glutathione (GSH) pools and induces oxidative stress in PC12 cells in culture. This increase was accompanied by alterations in subcellular ROS and glutathione (GSH) metabolisms. The GSH homeostasis was affected as both mitochondrial and extramitochondrial GSH levels, GSH peroxidase and glutathione reductase activities were inhibited and glutathione S-transferase (GST) activity was increased after 4-HNE treatment. A concentration- and time-dependent increase in cytochrome P450 2E1 (CYP 2E1) activity in the mitochondria and postmitochondrial supernatant was also observed. 4-HNE-induced oxidative stress also caused an increase in the expression of GSTA4-4, CYP2E1 and Hsp70 proteins in the mitochondria. Increased oxidative stress in PC12 cells initiated apoptosis as indicated by the release of mitochondrial cytochrome c, activation of poly-(ADP-ribose) polymerase (PARP), DNA fragmentation and decreased expression of antiapoptotic Bcl-2 proteins. Mitochondrial respiratory and redox functions also appeared to be affected markedly by 4-HNE treatment. These results suggest that HNE-induced oxidative stress and apoptosis might be associated with altered mitochondrial functions and a compromised GSH metabolism and ROS clearance.     

Formation and inhibition of heterocyclic aromatic amines in fried ground beef patties.

The effect of vitamin E and oleoresin rosemary on heterocyclic aromatic amine (HAA) formation in fried ground beef patties was studied. Patties were fried at three temperatures (175 degrees C, 200 degrees C, 225 degrees C) for 6 and 10 min/side to determine the conditions for optimum HAA formation. HAAs were isolated by solid phase extraction and quantitated by HPLC. Greatest concentrations were generated when patties were fried at 225 degrees C for 10 min/side, 31.4 ng/g 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 5.8 ng/g 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). Vitamin E, when used at two concentrations (1% and 10% based on fat content) and added directly to the ground beef patties, reduced PhIP concentrations in the cooked patties by 69% and 72%, respectively. Smaller but more variable reductions were achieved for MeIQx. Comparable inhibition of HAA formation was achieved by the direct addition of vitamin E (1% based on fat content) to the surface of the patties before frying. Concentrations of five HAAs studied were all significantly reduced (P<0.006), with average reductions ranging from 45% to 75%. Oleoresin rosemary, when used at two concentrations (1% and 10% based on fat content), reduced PhIP formation by 44%.