Effects Of PhIP Research Articles

Potential of Fiber and Probiotics to Fight Against the Effects of PhIP + DSS-Induced Carcinogenic Process of the Large Intestine.

We determined the in vivo counteracting effect of fiber and probiotic supplementation on colonic mucosal damage and alterations in gut microbiota caused by 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) and sodium dextran sulfate (DSS). Male Fischer-344 rats were randomly divided into 4 groups: control (standard diet), PhIP + DSS group (standard diet + PhIP + DSS), fiber (fiber diet + PhIP + DSS), and probiotic (probiotic diet + PhIP + DSS). The intake of PhIP + DSS for 3 weeks induced colonic mucosal erosion, crypt loss, and inflammation, and the distal colon was more severely damaged. Fiber alleviated colonic mucosal damage by reducing crypt loss and inflammation, while the probiotic increased colon length. The intake of PhIP + DSS increased the fecal relative abundance of Clostridia UCG014 along the intervention, in contrast to the lower abundances of these taxa found after PhIP + DSS administration in the rats supplemented with probiotics or fiber. Fiber supplementation mitigated the histological damage caused by PhIP + DSS shifting the gut microbiota toward a reduction of pro-inflammatory taxa.

The food-borne carcinogenic 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) disrupts circadian rhythms and ameliorated by pterostilbene (PSB) in Caenorhabditis elegans.

The food-borne 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a potential human carcinogen abundant in cooked meat. While circadian rhythms are crucial biological oscillations, the negative impact of PhIP on circadian systems and the potential of mitigation remain underexplored. We investigated the effects of PhIP on circadian rhythms and the mitigating effects of the phytochemical antioxidant pterostilbene (PSB) in Caenorhabditis elegans. We show that exposure to 10μM PhIP disrupts the 24-h circadian rhythms of C. elegans, an effect mitigated by co-exposure to 100μM PSB. In addition, PhIP-induced circadian disruption can be linked to defective oxidative stress resistance, which is associated with the DAF-16/FOXO pathway and is modulated by PSB. Molecular docking suggested that PhIP and PSB bind similarly to DAF-16. Moreover, 10μM PhIP abolished the rhythmic expression of the core clock gene prdx-2, which is restored by 100μM PSB. Findings from this study provide novel insight of how food-borne contaminant like PhIP may contribute to the disruption of circadian rhythms and suggest potential for PSB to mitigate these effects in higher organisms.

EGFR/MAPK signaling pathway acts as a potential therapeutic target for sulforaphane-rescued heart tube malformation induced by various concentrations of PhIP exposure

Background2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyrimidine (PhIP) is a known carcinogen generated mainly from cooking meat and environmental pollutants. It is worth exploring the potential of natural small-molecule drugs to protect against adverse effects on embryonic development. PurposeIn this study, we investigated the potential toxicological effects of PhIP on embryonic heart tube formation and the effect of Sulforaphane (SFN) administration on the anti-toxicological effects of PhIP on embryonic cardiogenesis. Study design and methodsFirst, the chicken embryo model was used to investigate the different phenotypes of embryonic heart tubes induced by various concentrations of PhIP exposure. We also proved that SFN rescues PhIP-induced embryonic heart tube malformation. Second, immunofluorescence, western blot, Polymerase Chain Reaction (PCR) and flow cytometry experiments were employed to explore the mechanisms by which SFN protects cardiac cells from oxidative damage in the presence of PhIP. We used RNA-seq analysis, molecular docking, in situ hybridization, cellular thermal shift assay and solution nuclear magnetic resonance spectroscopy to explore whether SFN protects cardiogenesis through the EGFR/MAPK signaling pathway. ResultsThe study showed that PhIP might dose-dependently interfere with the C-looping heart tube (mild) or the fusion of a pair of bilateral endocardial tubes (severe) in chick embryos, while SFN administration prevented cardiac cells from oxidative damage in the presence of high-level PhIP. Furthermore, we found that excessive reactive oxygen species (ROS) production and subsequent apoptosis were not the principal mechanisms by which low-level PhIP induced malformation of heart tubes. This is due to PhIP-disturbed Mitogen-activated protein kinase (MAPK) signaling pathway could be corrected by SFN administration. ConclusionsThis study provided novel insight that PhIP exposure could increase the risk of abnormalities in early cardiogenesis and that SFN could partially rescue various concentrations of PhIP-induced abnormal heart tube formation by targeting EGFR and mediating EGFR/MAPK signaling pathways.

Sub-chronic exposure to PhIP induces oxidative damage and DNA damage, and disrupts the amino acid metabolism in the colons of Wistar rats

Heterocyclic amines (HCAs) are a group of mutagenic compounds produced during thermal processing of protein-rich foods. One of the most abundant HCAs, 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) has potential carcinogenic and mutagenic effects on human organs, especially the colon. This study aimed to explore the toxic effects of PhIP on amino acid metabolism in the colon of Wistar rats using RNA-seq and LC-MS/MS. Exposure to PhIP for 4 weeks induced oxidative damage and DNA damage in the colons, and disrupted the expression of related genes involved in tryptophan metabolism, beta(β)-alanine metabolism, valine, leucine, and isoleucine degradation, and glutathione metabolic pathways. Moreover, the levels of fecal metabolites related to amino acid metabolism were affected by PhIP. Cumulatively, these results indicate that PhIP can induce colonic oxidative injury and disorders related to amino acid metabolism, thereby providing a new theoretical basis for the study of PhIP toxicity.

Abstract 2346: Curcumin suppresses PhIP induced inflammatory protein expression in breast epithelial cells

Abstract PhIP (2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine), a prominent mutagen/carcinogen is a heterocyclic amine found in meat cooked at high temperature and in tobacco. PhIP is known to induce liver, colon, skin, prostate, and breast cancer as evident from several in vivo and in vitro studies. Our previous studies have demonstrated that curcumin significantly inhibits PhIP-induced DNA adduct formation and DNA double-strand breaks with the concomitant decrease in reactive oxygen species (ROS) production via multiple molecular targets. We hypothesize that PhIP induced ROS production induces inflammatory proteins and co-treatment with curcumin would suppress the induced inflammation. Therefore, human breast epithelial cells (MCF 10A) were used to test the expression of inflammatory proteins. MCF 10A cells were treated with PhIP alone and in presence of curcumin. Untreated cells and treated only with curcumin served as control. Human Inflammation array (Ray Biotech) was used to analyze the expression of forty inflammatory proteins. Results show that the expression of twenty inflammatory proteins is induced in MCF 10 A cells when treated with PhIP as compared to the controls. Presence of curcumin reduces the induced expression of these twenty inflammatory proteins. However, three inflammatory proteins (IL-8, IL 12p40 and ICAM-1) showed a significantly higher level of expression with PhIP and curcumin co-treated cells as compared to cells treated with PhIP alone. It is shown that higher expression of IL-8, IL 12p-40 and ICAM-1 is involved in chemotactic factor involve in attracting the neutrophils, basophils; activation of T and NK cells which might be helping in coordinating the inflammation process. Hence the present study suggests that curcumin is a promising natural compound to revert the effect of PhIP induced inflammation. Citation Format: Ashok Jain. Curcumin suppresses PhIP induced inflammatory protein expression in breast epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2346.

Abstract LB-270: Screening of novel phytochemical/s that can protect breast epithelial cells from PhIP induced cytotoxicity

Abstract Amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) is a known food carcinogen abundantly found in cooked meat. Several studies have shown that PhIP can induce tumors in breast, prostate and colon cells and rodent models. Metabolism of PhIP results in the formation of free radicals (ROS) and PhIP metabolites are known to produce DNA adduct and DNA strand breaks. The metabolism and mutational effects of PhIP are well defined. Phytochemicals are known to inhibit cytotoxic and genotoxic effects. Therefore, we hypothesized that the right combination of antioxidants and or phytochemical (naturally present in fruits, vegetables and spices) along with grilled meat should be capable of suppressing the PhIP induced cytotoxicity and development of breast cancer. Therefore, a model system using human breast epithelial cells (MCF 10A) was developed to mimic the cellular changes and test various antioxidants in presence or absence of PhIP. We have tested four vitamins (C, K3, D3, and E), Gingerol (6 and 10), N-acetyl cysteine, glutathione and curcumin at varying concentrations. The protective effect of these compounds was evaluated using cell viability assay, DCF assay to quantify ROS production, Comet assay to quantify the DNA damage and DNA adduct formation by immunofluorescence method. Results indicate that presence of these compounds improves cell viability as compared to PhIP treated group. However, curcumin showed significant differences and PhIP induced cell cytotoxicity was consistently reverted to normal in presence of Curcumin. Gene expression analysis using RT PCR technique indicates that curcumin interact via multi molecular targets. Hence, the present study suggests that Curcumin is a promising natural compound to regress the effect of PhIP induced cell cytotoxicity in breast cells. Citation Format: Ashok K. Jain. Screening of novel phytochemical/s that can protect breast epithelial cells from PhIP induced cytotoxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-270. doi:10.1158/1538-7445.AM2015-LB-270

Development of an online-SPE-LC-MS method for the investigation of the intestinal absorption of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PHIP) and its bacterial metabolite PHIP-M1 in a Caco-2 Transwell system

Heterocyclic aromatic amines such as PHIP are formed during the heat processing of food. PHIP undergoes bacterial metabolism leading to 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) as main metabolite. We developed an LC-MS method with automated sample preparation by online-solid-phase-extraction for the simultaneous quantification of PHIP and its mammalian and bacterial metabolites N-hydroxy-PHIP, 4-OH-PHIP and PHIP-M1 in biological samples. The method was used to investigate the transport of PHIP-M1 through a Caco-2 cell monolayer. The experiments show that PHIP-M1 rapidly crosses the cell monolayer and that PHIP-M1 is a substrate for P-glycoprotein and the multiple drug resistance 2 transporter. The intestinal absorption of PHIP-M1 is comparable with that of PHIP and a moderate to high bioavailability has to be expected. Thus, not only the human metabolites of PHIP but also the bacterial metabolite PHIP-M1 formed in the gut could contribute to the toxic effects of PhIP.

Dietary Restriction and Toremifene on PhIP induced Carcinogenesis in rats

Toremifene inhibits estrogen receptor (ER) alpha which is found in prostate stroma and epithelium, recurring prostate cancer, and metastasis. Exposure to 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), the cooked meat heterocyclic amine, results in binding to ER alpha and cancer. Our hypothesis is the estrogenic effects of PhIP are responsible for carcinogenicity and blocking ERα via toremifene would prevent the effects of PhIP. We provided PhIP (70mg/kg) with or without toremifene (~8.6 mg/kg) in AIN93G diets to male Fischer rats for 20 weeks and then AIN93G diet for 32 weeks before collecting prostates, seminal vesicles, testes, skin, and intestines. Toremifene + PhIP rats consumed 37% less calories than PhIP rats thus a 37% dietary restricted (DR) + PhIP group was used as a control. Decreases in prostate and seminal vesicle weights, histological changes, and lack of mature sperm in the testes’ tubules were seen with toremifene. Ventral prostates showed decreased androgen receptor levels with toremifene, while testes showed no positivity. CK5 increased with toremifene, whereas PhIP had the opposite effect. PhIP rats had the greatest incidences of skin, intestinal, and prostate carcinomas. While some reductions were seen with DR the greatest effects were in rats given toremifene. We conclude that in the rat toremifene has marked anti‐androgenic and anticancer effects.

Effects of 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) on histopathology, oxidative stress, and expression of c-fos, c-jun and p16 in rat stomachs

2-Amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP) is one of the most abundant heterocyclic amines (HCAs) generated from overcooking meat at high temperatures. To understand the possible mechanism of PhIP-associated stomach cancer, the effects of PhIP on morphology, oxidative stress, gene expression of c-fos, c-jun and p16 in rat stomachs were investigated. The results showed that (1) 15mg/kg body weight PhIP induced obvious histopathological changes in gastric mucosa; (2) PhIP (10 and/or 15mg/kg) significantly decreased superoxide dismutase (SOD) and glutathioneperoxidase (GPx) activities, while increased catalase (CAT) activity compared with the control. With the elevated doses of PhIP, malondialdehyde (MDA) contents, protein carbonyl (PCO) contents and DNA-protein crosslinks (DPC) coefficients were significantly raised in a dose-dependent manner; (3) PhIP at the doses of 10mg/kg and/or 15mg/kg significantly inhibited p16 mRNA and protein expression, whereas enhanced c-fos and c-jun expression relative to control. The data indicated that PhIP could cause stomach injury, oxidative stress in rat stomachs as well as the activation of c-fos and c-jun and inactivation of p16, which may play a role in the pathogenesis of PhIP-associated stomach cancer.

Computational Characterization of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5b]pyridine at Androgen Receptor: Mechanistic support for its role in Prostate Cancer

The cooked meat‐derived carcinogenic heterocyclic amine 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5b]pyridine (PhIP) has been shown to produce tissue specific tumors in animal models. The mechanism for this specificity has not been fully elucidated and has been attributed to organ differences. In order to evaluate the modifying effects of PhIP on carcinogenesis we analyzed its ability to activate or inhibit an androgenic response. The present study seeks to evaluate the interactions of PhIP and identify molecular events associated with PhIP‐mediated disruption of the androgen receptor (AR) function. We demonstrate that PhIP binds with the ligand‐binding domain by computational analysis. Using molecular docking simulations, of ArgusLab and Molegro, we have identified a possible binding mode of PhIP to the AR based on the predicted binding free energy, compared to that of DHT at the AR. This molecular docking simulation gives insight into the molecular mechanism by which PhIP is able to initiate transduction of the activation signal. Additional studies using receptor‐binding experiments will be used to confirm modeling data. Our Docking results indicate tht PhIP may play an important role in the modification of AR gene expression and initiation of prostate cancer. Research supported by NCRR/RCMI G12 RR03020, NIGMS/MBRS/SCORE GM08111, and HRSA SD34HP0 4018.

Intestinal carcinogenesis of two food processing contaminants, 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine and 5‐hydroxymethylfurfural, in transgenic FVB min mice expressing human sulfotransferases

Humans express sulfotransferases (SULTs) of the SULT1A subfamily in many tissues, whilst the single SULT1A gene present in rodents is mainly expressed in liver. The food processing contaminants, 5-hydroxymethylfurfural (HMF) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), are bioactivated by human SULT1A1 and SULT1A2. FVB multiple intestinal neoplasia (Min) mice, which spontaneously develop tumors and flat aberrant crypt foci (ACF) in intestine, were crossed with transgenic FVB mice expressing human SULT1A1 and 1A2 (hSULT) in several tissues, giving rise to wild-type and Min mice with and without hSULT. One-week-old Min mice with or without hSULT were given HMF (375 or 750 mg/kg bw) or saline by gavage three times a week for 11 wk. In another experiment, the F1 generation received subcutaneous injections of 50 mg/kg bw PhIP or saline 1 wk before birth, and 1, 2, and 3 wk after birth. HMF did not affect the formation of tumors, but may have induced some flat ACF (incidence 15-20%) in Min mice with and without hSULT. No control mouse developed any flat ACF. With the limitation that these putative effects were weak, they were unaffected by hSULT expression. The carcinogenic effect of PhIP increased in the presence of hSULT, with a significant increase in both incidence (31-80%) and number of colonic tumors (0.4-1.3 per animal). Thus, intestinal expression of human SULT1A1 and 1A2 might increase the susceptibility to compounds bioactivated via this pathway implying that humans might be more susceptible than conventional rodent models.

Differential toxicity of heterocyclic aromatic amines and their mixture in metabolically competent HepaRG cells

Human exposure to heterocyclic aromatic amines (HAA) usually occurs through mixtures rather than individual compounds. However, the toxic effects and related mechanisms of co-exposure to HAA in humans remain unknown. We compared the effects of two of the most common HAA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), individually or in combination, in the metabolically competent human hepatoma HepaRG cells. Various endpoints were measured including cytotoxicity, apoptosis, oxidative stress and DNA damage by the comet assay. Moreover, the effects of PhIP and/or MeIQx on mRNA expression and activities of enzymes involved in their activation and detoxification pathways were evaluated. After a 24 h treatment, PhIP and MeIQx, individually and in combination, exerted differential effects on apoptosis, oxidative stress, DNA damage and cytochrome P450 (CYP) activities. Only PhIP induced DNA damage. It was also a stronger inducer of CYP1A1 and CYP1B1 expression and activity than MeIQx. In contrast, only MeIQx exposure resulted in a significant induction of CYP1A2 activity. The combination of PhIP with MeIQx induced an oxidative stress and showed synergistic effects on apoptosis. However, PhIP-induced genotoxicity was abolished by a co-exposure with MeIQx. Such an inhibitory effect could be explained by a significant decrease in CYP1A2 activity which is responsible for PhIP genotoxicity. Our findings highlight the need to investigate interactions between HAA when assessing risks for human health and provide new insights in the mechanisms of interaction between PhIP and MeIQx.

Responses of PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) in MCF-7 cells are culture condition dependent

To compare the effects of the food toxin 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) and estradiol in hormone-responsive MCF-7 cells, the cells were exposed to different concentrations of either PhIP or estradiol. The effect of various culture conditions (e.g. phenol red, FBS, vehicle (DMSO/EtOH) and seeding density) on responses was studied. Cells were continuously grown with steroid-containing or -deprived medium, or switched from steroid-containing to -deprived medium for the experiments to minimize the effect of background estrogenicity. Effects of PhIP and estradiol on cell viability and proliferation were determined by ATP analysis and Ki-67 immunocytochemistry. Expression of estrogen receptor alpha, cell stress markers (p53 and ERK) and estrogen responsive proteins (c-myc and ERK) were immunoblotted. All concentrations of estradiol induced cell proliferation, viability and changes in protein expression, typical for estrogenic responses. PhIP, however, increased viability only at low concentrations and depending on culture conditions. No changes in protein expressions by PhIP were noted, not even when switching cells from steroid-containing to -deprived medium which down-regulated the expression of proteins at basal level. Vehicle affected significantly viability, especially after exposure to PhIP, but not protein expression while medium changes affected both. In conclusion, the effects of PhIP and estradiol in MCF-7 cells are dependent on culture conditions. The detected PhIP-induced changes are weaker compared to those induced by estradiol.

A food-born heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), suppresses tumor necrosis factor-α expression in lipoteichoic acid-stimulated RAW 264.7 cells

2-Amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), a heterocyclic amine with strong carcinogenic and mutagenic potential, is created abundantly in the overcooking of meat and fish. Carcinogenic toxicants are often implicated in immunosuppression, where cancer cells are not easily eliminated by the host immune system. Here, we investigated the effect of PhIP on tumor necrosis factor-alpha (TNF-α) expression by murine macrophage-like cells (RAW 264.7) stimulated with lipoteichoic acid (LTA), a major virulence factor of Gram-positive bacteria. Upon exposure to LTA purified from Staphylococcus aureus, TNF-α expression was substantially induced, whereas pretreatment with PhIP significantly inhibited LTA-induced TNF-α expression. LTA is known to activate Toll-like receptor 2 (TLR2) and NF-κB, resulting in TNF-α expression. Interestingly, PhIP did not interfere with LTA-binding to TLR2, its stimulation of TLR2, or the DNA-binding activity of NF-κB. However, treatment with actinomycin D facilitated the PhIP-induced attenuation of TNF-α mRNA expression, implying that PhIP might decrease TNF-α mRNA stability rather than its biosynthesis. Furthermore, Western blot analysis demonstrated that PhIP reduced the phosphorylation of ERK1/2 and JNK but not p38 kinase in LTA-stimulated cells. The addition of a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, rescued PhIP-inhibited TNF-α expression in LTA-stimulated cells. These results suggest that PhIP downregulates TNF-α expression in LTA-stimulated macrophages by decreasing TNF-α mRNA stability and signaling pathways related to PKC, ERK1/2, and JNK activation.

The Cooked Meat Carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]Pyridine Activates the Extracellular Signal–Regulated Kinase Mitogen-Activated Protein Kinase Pathway

During the cooking of meat, mutagenic and carcinogenic heterocyclic amines are formed, the most abundant of which, 2-amino-1-methyl-6-phenylimidazo[4-5-b]pyridine (PhIP), induces tumors of the prostate, colon, and mammary gland in rats. Humans consuming cooked meat are exposed to PhIP on a daily basis, yet few studies have assessed the effects of PhIP at dietary relevant concentrations. In addition to its genotoxic properties, recent studies have shown that PhIP can activate estrogen receptor-mediated signaling pathways at doses that are similar to those that may be present in the body following consumption of a cooked meat meal. In the present study, we examined whether such doses of PhIP can affect estrogen receptor-independent signal transduction via the mitogen-activated protein kinase (MAPK) extracellular signal-related kinase (ERK) pathway to influence proliferation and migration in the human mammary epithelial cell line MCF10A and the prostate cancer cell line PC-3. At doses shown to have a proliferative effect on MCF10A cells (10(-11)-10(-7) mol/L), PhIP induced a rapid, transient increase in phosphorylation of both MAPK/ERK kinase 1/2 and ERKs. Inhibition of this pathway significantly reduced the PhIP-induced proliferation of MCF10A cells and the migration of PC-3 cells. The data presented here show that levels of PhIP that approximate to human dietary exposure stimulate cellular signaling pathways and result in increased growth and migration, processes linked to the promotion and progression of neoplastic disease. These findings provide strong evidence that PhIP acts as a tumor initiator and promoter and that dietary exposure to this compound could contribute to carcinogenesis in humans.

Mechanisms of action of the carcinogenic heterocyclic amine PhIP

Formed during the cooking of meat, the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4-5- b]pyridine (PhIP) is mutagenic and carcinogenic. Although the metabolism and mutational effects of PhIP are well defined, the early cellular and genomic events by which it can induce neoplastic transformation are not yet fully characterised. These early cellular responses to genotoxic doses of PhIP were examined in a human mammary epithelial cell, MCF10A. Using Western blotting, PhIP was shown to induce expression of the DNA damage response proteins p53 and p21 WAF1/CIP1, and to inhibit cell growth while activating G1 cell cycle checkpoint, a consequence of PhIP-induced DNA damage. Using low doses of PhIP (previously shown to activate oestrogenic signalling), PhIP increased proliferation in the oestrogen receptor (ER)-negative MCF10A cell line and to activate the mitogen-activated protein kinase (MAPK) pathway. Inhibition of this pathway significantly reduced the PhIP-induced cell growth of MCF10A cells. The work presented here suggests that, further to its genotoxic properties, at levels close to human exposure PhIP stimulates cellular signalling pathways that are linked to the promotion and progression of neoplastic disease. It is possible that a combination of these DNA damaging and growth promoting properties provide a mechanism for the tumourigenicity of PhIP, and may be key determinants for the tissue specificity of PhIP-induced carcinogenesis.

Comparison of gene expression in cell culture to that in the intact animal: relevance to drugs and environmental toxicants. Focus on “Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes”

to study the effects of drugs and other environmental toxicants on the liver, many believe that the use of hepatocyte-derived cultures offers advantages over studies in the liver of intact animals. Cell cultures can be manipulated more easily, and they are far less expensive to maintain than a

Molecular Dynamics of a Food Carcinogen−DNA Adduct in a Replicative DNA Polymerase Suggest Hindered Nucleotide Incorporation and Extension

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant of the carcinogenic heterocyclic aromatic amines in the human diet, and the major mutagenic effect of dietary PhIP is G-->T transversions. The major PhIP-derived DNA adduct is to C8 of guanine. We have investigated this adduct in a PhIP-induced mutational hotspot 5'-GGGA-3' of the Apc tumor suppressor gene, frequently mutated in mammalian colon tumors. We have carried out a molecular dynamics study to elucidate on a structural level nucleotide incorporation and extension opposite this major adduct during replication. The PhIP adduct was modeled into the ternary complex closed conformation of DNA polymerase RB69, at incorporation and extension positions, with normal cytosine or mismatched partner adenine. RB69 polymerase is a member of the B family as are most replicative eukaryotic DNA polymerases such as DNA polymerase alpha. These systems were subjected to molecular dynamics simulations with AMBER. Our results show that the adduct can reside on the major groove side of the modified DNA template opposite an incoming dCTP or dATP. In the case of the normal partner, disturbance to the active site is observed at the incorporation step, but there is less perturbance in the extension simulation. In the case of the mismatched partner, a less disturbed active site is observed during the incorporation step, but extension appears to be more difficult. Disturbances include adverse impacts on Watson-Crick hydrogen bonding in the nascent base pair, on the distance between the alpha-phosphate of the incoming dNTP and the primer terminus 3'-OH, and on critical protein interactions with the dNTP. However, in all of these cases, a near reaction ready distance (within 3.5 angstroms) between the 3'-terminal oxygen of the primer and the Palpha of the incoming nucleotide triphosphate is sampled occasionally (0.4-23.5% of the time). Thus, error-free bypass or the induction of a G-->T transversion mutation could occur at times and contribute to an extent to the mutagenic effect of PhIP. Polymerase stalling would be the more common outcome and in vivo could lead to switch to an error-prone bypass polymerase.

The effect of UDP-glucuronosyltransferase 1A1 expression on the mutagenicity and metabolism of the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine in CHO cells

UDP-glucuronosyltransferase proteins (UGT) catalyze the glucuronidation of both endogenous and xenobiotic compounds. In previous studies, UGT1A1 has been implicated in the detoxification of certain food-borne carcinogenic-heterocyclic amines. To determine the importance of UDP-glucuronosyltransferase 1A1 (UGT1A1) in the biotransformation of the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), genetically modified CHO cells that are nucleotide excision repair-deficient, and express cytochrome P4501A2 (UV5P3 cell line) were transfected with a cDNA plasmid of human UGT1A1 to establish the UDP-glucuronosyltransferase 1A1 expressing 5P3hUGT1A1 cell line. Expression of the UGT1A1 gene was verified by screening neo gene expressing clonal isolates (G-418 resistant) for their sensitivity to cell killing from PhIP exposure. Five of 11 clones were chosen for further analysis due to their resistance to cell killing. Western blot analysis was used to confirm the presence of the UGT1A1 and CYP1A2 proteins. All five clones displayed a 52-kDa protein band, which corresponded to a UGT1A1 control protein. Only four of the clones had a protein band that corresponded to the CYP1A2 control protein. Correct fragment size of the cDNAs in the remaining four clones was confirmed by RT-PCR and quantification of the mRNA product was accomplished by real-time RT-PCR. Expression of UGT1A1 in the transfected cells was 10 4–10 5-fold higher relative to the UV5P3 parental cells. One clone (#14) had a 10-fold higher increase in expression at 1.47 × 10 5 over the other three clones. This clone was also the most active in converting N-hydroxy-PhIP to N-hydroxy-PhIP glucuronide conjugates in microsomal metabolism assays. Based on the D 50 values, the cytotoxic effect of PhIP was decreased ∼350-fold in the 5P3hUGT1A1 cells compared to the UV5P3 control cells. In addition, no significant increase in mutation frequency was observed in the transfected cells. These results clearly indicate that UGT1A1 plays a critical role in PhIP biotransformation, providing protection against PhIP-mediated cytotoxicity and mutagenicity.

Role of p53 and mismatch repair in PhIP-induced perturbations of the cell cycle

Heterocyclic amines, found ubiquitously in our diet, are carcinogenic and mutagenic. Among this class of compounds, 2-amino-1-methyl-6-phenylimidazo [4,5- b]pyridine (PhIP) is the most abundant. To further understand the carcinogenesis of this compound, we studied the effects of PhIP on the progression of human lymphoblastoid cells through the cell-cycle. Cells differing in p53 or mismatch repair status were used to evaluate the role of those proteins. Following PhIP-treatment, a dose and time-dependent accumulation of p53 was found in cells containing functional p53. The augmentation of the p53 protein, accompanied by increases in p21-WAF1, confirms that the p53 is activated. The increase in p53 was independent of the mismatch repair status of the cells. Perturbations in the cell-cycle were also observed. Twenty-four hours after PhIP treatment, the activation of the G2-M checkpoint was evident. Functional p53 and mismatch repair were not required for the PhIP-induced G2-M arrest. The G2-M arrests were reversible and are interpreted as necessary for the repair of the PhIP-DNA lesions. Under treatment conditions where less than 5% of the cells survived, the G2-M arrests were absent.