2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)‐DNA adducts in benign prostate and subsequent risk for prostate cancer

Despite convincing evidence that 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)—a heterocyclic amine generated by cooking meats at high temperatures—is carcinogenic in animal models, it remains unclear whether PhIP exposure leads to increased cancer risk in humans. PhIP‐DNA adduct levels were measured in specimens from 534 prostate cancer case‐control pairs nested within a historical cohort of men with histopathologically benign prostate specimens. We estimated the overall and race‐stratified risk of subsequent prostate cancer associated with higher adduct levels. PhIP‐DNA adduct levels in benign prostate were significantly higher in Whites than African Americans (0.274 optical density units (OD) ±0.059 vs. 0.256 OD ±0.054; p<0.0001). Prostate cancer risk for men in the highest quartile of PhIP‐DNA adduct levels was modestly increased [odds ratio (OR) = 1.25; 95% confidence interval (CI) = 0.76–2.07]. In subset analyses, the highest risk estimates were observed in White patients diagnosed more than 4 years after cohort entry (OR = 2.74; 95% CI = 1.01–7.42) or under age 65 (OR = 2.80; 95% CI = 0.87–8.97). In Whites, cancer risk associated with high‐grade prostatic intraepithelial neoplasia combined with elevated PhIP‐DNA adduct levels (OR = 3.89; 95% CI = 1.56–9.73) was greater than risk associated with either factor alone. Overall, elevated levels of PhIP‐DNA adducts do not significantly increase prostate cancer risk. However, our data show that White men have higher PhIP‐DNA adduct levels in benign prostate tissue than African American men, and suggest that in certain subgroups of White men high PhIP‐DNA adduct levels may predispose to an increased risk for prostate cancer.     

NADPH oxidase overexpression in human colon cancers and rat colon tumors induced by 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)

NADPH oxidase/dual‐oxidase (Nox/Duox) family members have been implicated in nuclear factor kappa‐B (NFκB)‐mediated inflammation and inflammation‐associated pathologies. We sought to examine, for the first time, the role of Nox/Duox and NFκB in rats treated with the cooked meat heterocyclic amine carcinogen 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP). In the PhIP‐induced colon tumors obtained after 1 year, Nox1, Nox4, NFκB‐p50 and NFκB‐p65 were all highly overexpressed compared with their levels in adjacent normal‐looking colonic mucosa. Nox1 and Nox4 mRNA and protein levels also were markedly elevated in a panel of primary human colon cancers, compared with their matched controls. In HT29 human colon cancer cells, Nox1 knockdown induced G1 cell cycle arrest, whereas in Caco‐2 cells there was a strong apoptotic response, with increased levels of cleaved caspase‐3, ‐6, ‐7 and poly(ADP‐ribose)polymerase. Nox1 knockdown blocked lipopolysaccharide‐induced phosphorylation of IκB kinase, inhibited the nuclear translocation of NFκB (p50 and p65) proteins, and attenuated NFκB DNA binding activity. There was a corresponding reduction in the expression of downstream NFκB targets, such as MYC, CCND1 and IL1β. The results provide the first evidence for a role of Nox1, Nox4 and NFκB in PhIP‐induced colon carcinogenesis, including during the early stages before tumor onset. Collectively, the findings from this investigation and others suggest that further work is warranted on the role of Nox/Duox family members and NFκB in colon cancer development.     

Distribution of the DNA adducts of 2-amino-3–methylimidazo[4,5–f]quinoline and 2–amino-1–methyl-6–phenylimidazo[4,5–b]pyridine in the supF gene as determined by polymerase arrest assay

The distribution of the adducts of the cooked meat-derived heterocyclic amines 2–amino-3–methylimidazo 4,5–flquinoline (a) and 2–amino-1–methyl-6–phenylimidazo[4,5–b]pyridine (a) was examined in the supF gene of PSP 189 by a polymerase-arrest assay using thermal-cycle sequencing. The reactive N-acetoxy metabolites of both compounds showed an overwhelming preference for reacting with guanine residues in the supF gene of the shuttle vector pSP189. The distribution of the IQ and PhIP guanine adducts was not random; instead, patterns of adduct hot-spots and cold-spots were observed. There was a striking similarity between both compounds in their preferred sites of adduct formation. The finding that IQ and PhIP adducted to guanine concurred with previous results showing that the target sites for IQ and PhIP mutations in supF were also at guanine. However, the adduct hot-spot sites were not predictive of the known sites of mutation hot-spots. In addition, despite the similarity in adduct hot-spots for IQ and PhIP, their reported mutation spectra in the supF gene were different. Factors in addition to adduct location therefore appear to play a role in the mutation spectra induced by the heterocyclic amines in the supF gene. © 1995 Wiley- Liss, Inc.     

Atorvastatin inhibits pancreatic carcinogenesis and increases survival in LSL‐KrasG12D‐LSL‐Trp53R172H‐Pdx1‐Cre mice

There are several studies supporting the role of HMG‐CoA reductase inhibitors such as atorvastatin against carcinogenesis, in which inhibiting the generation of prenyl intermediates involved in protein prenylation plays the crucial role. Mutation of Kras gene is the most common genetic alteration in pancreatic cancer and the Ras protein requires prenylation for its membrane localization and activity. In the present study, the effectiveness of atorvastatin against pancreatic carcinogenesis and its effect on protein prenylation were determined using the LSL‐Kras^G12D‐LSL‐Trp53^R172H‐Pdx1‐Cre mouse model (called Pan^kras/p53 mice). Five‐week‐old Pan^kras/p53 mice were fed either an AIN93M diet or a diet supplemented with 100 ppm atorvastatin. Kaplan–Meier survival analysis with Log‐Rank test revealed a significant increase in survival in mice fed 100 ppm atorvastatin (171.9 ± 6.2 d) compared to the control mice (144.9 ± 8.4 d, P < 0.05). Histologic and immunohistochemical analysis showed that atorvastatin treatment resulted in a significant reduction in tumor volume and Ki‐67‐labeled cell proliferation. Mechanistic studies on primary pancreatic tumors and the cultured murine pancreatic carcinoma cells revealed that atorvastatin inhibited prenylation in several key proteins, including Kras protein and its activities, and similar effect was observed in pancreatic carcinoma cells treated with farnesyltransferase inhibitor R115777. Microarray assay on the global gene expression profile demonstrated that a total of 132 genes were significantly modulated by atorvastatin; and Waf1p21, cyp51A1, and soluble epoxide hydrolase were crucial atorvastatin‐targeted genes which involve in inflammation and carcinogenesis. This study indicates that atorvastatin has the potential to serve as a chemopreventive agent against pancreatic carcinogenesis. © 2012 Wiley Periodicals, Inc.     

Mouse lung CYP1A1 catalyzes the metabolic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) carcinogenesis is initiated by N(2)-hydroxylation, mediated by several cytochromes P450, including CYP1A1. However, the role of CYP1A1 in PhIP metabolic activation in vivo is unclear. In this study, Cyp1a1-null and wild-type (WT) mice were used to investigate the potential role of CYP1A1 in PhIP metabolic activation in vivo. PhIP N(2)-hydroxylation was actively catalyzed by lung homogenates of WT mice, at a rate of 14.9 +/- 5.0 pmol/min/g tissue, but <1 pmol/min/g tissue in stomach and small intestine, and almost undetectable in mammary gland and colon. PhIP N(2)-hydroxylation catalyzed by lung homogenates of Cyp1a1-null mice was approximately 10-fold lower than that of WT mice. In contrast, PhIP N(2)-hydroxylation activity in lung homogenates of Cyp1a2-null versus WT mice was not decreased. Pretreatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin increased lung Cyp1a1 mRNA and lung homogenate PhIP N(2)-hydroxylase activity approximately 50-fold in WT mice, where the activity was substantially inhibited (70%) by monoclonal antibodies against CYP1A1. In vivo, 30 min after oral treatment with PhIP, PhIP levels in lung were similar to those in liver. After a single dose of 0.1 mg/kg [(14)C]PhIP, lung PhIP-DNA adduct levels in Cyp1a1-null mice, but not in Cyp1a2-null mice, were significantly lower (P = 0.0028) than in WT mice. These results reveal that mouse lung has basal and inducible PhIP N(2)-hydroxylase activity predominantly catalyzed by CYP1A1. Because of the high inducibility of human CYP1A1, especially in cigarette smokers, the role of lung CYP1A1 in PhIP carcinogenesis should be considered. (237 words).     

Potential role of cytochrome P450‐1B1 in the metabolic activation of 4‐aminobiphenyl in humans

Metabolites of the human carcinogen 4‐aminobiphenyl (4‐ABP) form hemoglobin (Hb) adducts, which represent a useful biomarker for exposure. However, not every individual responds to a similar degree to 4‐ABP exposure, and variations in 4‐ABP‐Hb adduct formation might be explained by genetic polymorphisms in genes coding for enzymes involved in 4‐ABP metabolism. 4‐ABP‐Hb adducts were measured in blood samples from 57 smoking and 10 non‐smoking volunteers. An association was found between cigarette smoking and 4‐ABP‐Hb adduct levels in smokers (R² = 0.5, P < 0.001). Subsequently, subjects were genotyped for 12 polymorphisms in seven genes involved in biotransformation reactions. From this selection of polymorphisms, a significant impact was found for the CYP1B1 Leu⁴³²Val polymorphism (P = 0.021), which has been reported to lead to a decrease in enzyme activity. Indeed higher levels of 4‐ABP‐Hb adducts were observed in homo‐ and heterozygous carriers of the CYP1B1 ⁴³²Leu as compared to the double CYP1B1 ⁴³²Val genotype. A significant interaction between these CYP1B1 genotypes and the level of exposure was found (P = 0.003). Noteworthy, a saturation effect was observed for 4‐ABP‐Hb adduct formation at high smoking doses limited to carriers of the CYP1B1 ⁴³²Leu allele. No effect of polymorphisms in other genes were found. This is the first study in humans suggesting a crucial role of the CYP1B1 enzyme in 4‐ABP metabolism, indicating a protective effect of the CYP1B1 Leu⁴³²Val polymorphism against the formation of 4‐ABP‐Hb adduct levels, depending on the smoking dose. © 2009 Wiley‐Liss, Inc.     

Higher susceptibility to aflatoxin B1‐related hepatocellular carcinoma in glycine N‐methyltransferase knockout mice

In both humans and rodents, males are known to be more susceptible than females to hepatocarcinogenesis. We have previously reported that glycine N‐methyltransferase (GNMT) interacts with aflatoxin B₁ (AFB₁) and reduces both AFB₁‐DNA adduct formation and hepatocellular carcinoma (HCC) in mice. We also reported that 50% of the males and 100% of the females in a small group of Gnmt null (Gnmt?/?) mice developed HCC, with first dysplastic hepatocellular nodules detected at mean ages of 17 and 16.5 months, respectively. In our study, we tested our hypothesis that male and female Gnmt?/? mice are susceptible to AFB₁ carcinogenesis, and that the absence of Gnmt expression may accelerate AFB₁‐induced liver tumorigenesis. We inoculated Gnmt?/? and wild‐type mice intraperitoneally with AFB₁ at 7 days and 9 weeks of age and periodically examined them using ultrasound. Dysplastic hepatocellular nodules were detected in six of eight males and five of five females at 12.7 and 12 months of ages, respectively. Dysplastic hepatocellular nodules from 5/8 (62.5%) male and 4/5 (80%) female Gnmt?/? mice were diagnosed as having HCC, ～6 months earlier than AFB₁‐treated wild‐type mice. Results from microarray and real‐time PCR analyses indicate that five detoxification pathway‐related genes were downregulated in AFB₁‐treated Gnmt?/? mice: Cyp1a2, Cyp3a44, Cyp2d22, Gsta4 and Abca8a. In summary, we observed overall higher susceptibility to AFB₁‐related HCC in Gnmt?/? mice, further evidence that GNMT overexpression is an important contributing factor to liver cancer resistance.     

Oxidative DNA damage and reporter gene mutation in the livers of gpt delta rats given non‐genotoxic hepatocarcinogens with cytochrome P450‐inducible potency

Previous reports have proposed that reactive oxygen species resulting from induction of cytochrome P450 (CYP) isozymes might be involved in the modes of action of hepatocarcinogens with CYP‐inducible potency. In the present study, we investigated 8‐hydroxydeoxyguanosine (8‐OHdG) levels, in vivo mutagenicity and glutathione S‐transferase placental form (GST‐P)‐positive foci in the livers of gpt delta rats treated with piperonyl butoxide (PBO) or phenobarbital (PhB) for 4 and 13 weeks. Significant elevations in Cyp 1A1 and Cyp 1A2 mRNA levels after PBO treatment, and in Cyp 2B1 mRNA levels after PBO or PhB treatment, appeared together with remarkable hepatomegaly through the experimental period. Time‐dependent and statistically significant increases in 8‐OHdG levels were observed in the PBO treatment group along with significant increases in proliferating cell nuclear antigen (PCNA)‐positive hepatocytes at 4 weeks, while no increase in 8‐OHdG levels was found in PhB‐treated rats. No changes in mutant frequencies of gpt and red/gam (Spi^‐) genes in liver DNA from PBO‐ or PhB‐treated rats were observed at 4 or 13 weeks. A 13‐week exposure to either PBO or PhB did not affect the number and area of GST‐P‐positive hepatocytes. CYP 1A1 and 1A2 induction may be responsible for elevated levels of 8‐OHdG in PBO‐treated rats. However, neither GC:TA transversions nor deletion mutations, typically regarded as 8‐OHdG‐related mutations, were observed in any of the treated rats. We conclude that reactive oxygen species, possibly produced through CYP catalytic pathways, likely induced genomic DNA damage but did not give rise to permanent gene mutation. (Cancer Sci 2010; 101: 2525–2530)     

Augmentation of 3‐methylcholanthrene‐induced bioactivation in the human hepatoma cell line HepG2 by the calcium channel blocker nicardipine

(Cancer Sci 2010; 101: 652–657) The abilities of the dihydropyridine calcium channel blocker nicardipine (Nic) to induce cytochrome P450 1 family enzymes (CYP1s) and to enhance the 3‐methylcholanthrene (MC)‐mediated induction of CYP1s and formation of MC‐DNA adduct were examined in the human hepatoma cell line HepG2. The results from real time RT‐PCR analysis demonstrated that Nic could induce CYP1 mRNAs and enhance the MC‐mediated induction of the CYP1 mRNAs. The luciferase‐reporter gene assay using the HepG2‐A10 cell line, which has been previously established for the screening of aryl hydrocarbon receptor (AhR) activators, also indicated the augmentation of MC‐mediated activation of AhR (induction of luciferase) by Nic, although Nic showed limited capacity for the activation of AhR. Furthermore, the results from the Western blot analysis of CYP1s, the enzyme activity assay, and the assay for MC‐DNA adduct formation indicated that Nic could enhance the MC‐mediated induction of CYP1s, especially CYP1A1. Furthermore, the intracellular accumulation level of [³H]MC after treatment of HepG2 cells with [³H]MC significantly increased in the presence of Nic. The present findings demonstrate that Nic can enhance the MC‐mediated induction of CYP1s, especially CYP1A1, and the formation of MC‐DNA adduct in HepG2 cells. Furthermore, the augmentation of the MC‐mediated bioactivation by Nic is demonstrated to occur mainly through an increase in intracellular accumulation of MC.     

Oral benzo[a]pyrene in Cyp1a1/1b1(–/–) double‐knockout mice: Microarray analysis during squamous cell carcinoma formation in preputial gland duct

Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH) found in combustion processes. Cytochrome P450 1A1 and 1B1 enzymes (CYP1A1, CYP1B1) and other enzymes can activate PAHs to reactive oxygenated intermediates involved in mutagenesis and tumor initiation; also, CYP1 enzymes can detoxify PAHs. Cyp1(+/+) wild‐type (WT) and Cyp1b1(–/–) knockout mice receiving oral BaP (12.5 mg/kg/day) remain healthy for >12 months. In contrast, we found that global knockout of the Cyp1a1 gene (1a1KO) results in proximal small intestine (PSI) adenocarcinoma within 8–12 weeks on this BaP regimen; striking compensatory increases in PSI CYP1B1 likely participate in initiation of adenocarcinoma in 1a1KO mice. Cyp1a1/1b1(–/–) double‐knockout (DKO) mice on this BaP regimen show no PSI adenocarcinoma, but instead preputial gland duct (PGD) squamous cell carcinoma (SCC) occurs by 12 weeks. Herein, we compare microarray expression of PGD genes in WT, 1a1KO and DKO mice at 0, 4, 8, 12 and 16 weeks of oral BaP; about four dozen genes up‐ or down‐regulated during most critical time‐points were further verified by qRT‐PCR. In DKO mice, CYP3A59 was unequivocally identified as the BaP‐inducible and BaP‐metabolizing best candidate responsible for initiation of BaP‐induced SCC. Striking increases or decreases were found in 26 cancer‐related genes plus eight Serpin genes in DKO, but not in 1a1KO or WT, mice on this BaP regimen; of the 26, 8 were RAS‐related oncogenes. The mechanism by which cancer‐related genes are responsible for SCC tumor progression in the PGD remains to be elucidated.     

Oral benzo[a]pyrene‐induced cancer: Two distinct types in different target organs depend on the mouse Cyp1 genotype

Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH) found in combustion processes. Cytochrome P450 1A1 and 1B1 enzymes (CYP1A1 and CYP1B1) can both detoxify PAHs and activate them to cancer‐causing reactive intermediates. Following high dosage of oral BaP (125 mg/kg/day), ablation of the mouse Cyp1a1 gene causes immunosuppression and death within ～28 days, whereas Cyp1(+/+) wild‐type mice remain healthy for >12 months on this regimen. In this study, male Cyp1(+/+) wild‐type, Cyp1a1(?/?) and Cyp1b1(?/?) single‐knockout and Cyp1a1/1b1(?/?) double‐knockout mice received a lower dose (12.5 mg/kg/day) of oral BaP. Tissues from 16 different organs—including proximal small intestine (PSI), liver and preputial gland duct (PGD)—were evaluated; microarray cDNA expression and >30 mRNA levels were measured. Cyp1a1(?/?) mice revealed markedly increased CYP1B1 mRNA levels in the PSI, and between 8 and 12 weeks developed unique PSI adenomas and adenocarcinomas. Cyp1a1/1b1(?/?) mice showed no PSI tumors but instead developed squamous cell carcinoma of the PGD. Cyp1(+/+) and Cyp1b1(?/?) mice remained healthy with no remarkable abnormalities in any tissue examined. PSI adenocarcinomas exhibited striking upregulation of the Xist gene, suggesting epigenetic silencing of specific genes on the Y‐chromosome; the Rab30 oncogene was upregulated; the Nr0b2 tumor suppressor gene was downregulated; paradoxical overexpression of numerous immunoglobulin kappa‐ and heavy‐chain variable genes was found—although the adenocarcinoma showed no immunohistochemical evidence of being lymphatic in origin. This oral BaP mouse paradigm represents an example of “gene‐environment interactions” in which the same exposure of carcinogen results in altered target organ and tumor type, as a function of just 1 or 2 globally absent genes.     

Different properties of mammary carcinogenesis induced by two chemical carcinogens,DMBA and PhIP,in heterozygous BALB/c Trp53 knockout mice

Chemical carcinogens, such as 7,12-dimethylbenz[a]anthracene (DMBA) and 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), are known to induce mammary carcinomas in mice and rats. In the present study, the phenotypic and genotypic characteristics of carcinogen-induced mammary carcinogenesis in heterozygous BALB/c tumor protein p53 (Trp53) knockout mice were examined with reference to published data surrounding human breast cancer. A significantly accelerated induction of mammary carcinomas was observed following a single dose of DMBA (50 mg/kg of body weight at 7 weeks of age), and a modest acceleration was induced by PhIP (50 mg/kg of body weight) administered by gavage 6 times/2 weeks from 7 weeks of age. DMBA-induced mammary carcinomas were histopathologically characterized by distinct biphasic structures with luminal and myoepithelial cells, as well as a frequent estrogen receptor expression, and PhIP-induced carcinomas with solid/microacinar structures consisted of pleomorphic cells. Of note, DMBA-induced mammary carcinomas were characterized by a HRas proto-oncogene (Hras) mutation at codon 61, and gene/protein expression indicating MAPK stimulation. PhIP-induced lesions were suspected to be caused by different molecular mechanisms, including Wnt/β-catenin signaling and/or gene mutation-independent PI3K/AKT signaling activation. In conclusion, the present mouse mammary carcinogenesis models, induced by a combination of genetic and exogenous factors, may be utilized (such as the DMBA-induced model with Trp53 gene function deficiency as a model of adenomyoepithelioma, characterized by distinct biphasic cell constituents and Hras mutations), but PhIP-induced models are required to further analyze the genetic/epigenetic mechanisms promoting mammary carcinomas.     

P‐glycoprotein and cytochrome P450 3A act together in restricting the oral bioavailability of paclitaxel

Paclitaxel is avidly transported by P‐glycoprotein (P‐gp/MDR1/ABCB1). This results in low oral bioavailability, which can be boosted by coadministration of P‐gp inhibitors. Unlike paclitaxel, docetaxel is extensively metabolized by CYP3A4 and its oral bioavailability can be enhanced in mice and humans by coadministration of the potent CYP3A inhibitor ritonavir. Unexpectedly, ritonavir also enhances the oral bioavailability of paclitaxel in humans. We aimed to resolve the mechanism underlying this enhancement. Using mice lacking Cyp3a and/or P‐gp, we investigated the combined and separate restricting roles of Cyp3a and P‐gp in the oral bioavailability of paclitaxel, and the boosting effect of ritonavir. CYP3A4‐humanized mice were used for translation to the human situation. P‐gp had a dominant effect (11.6‐fold, p < 0.001) over Cyp3a (<1.5‐fold, n.s.) in limiting plasma concentrations of oral paclitaxel. However, in the absence of P‐gp, Cyp3a decreased paclitaxel plasma concentrations twofold (p < 0.001). Coadministered ritonavir inhibited Cyp3a‐mediated metabolism, but not P‐gp‐mediated transport of paclitaxel. Owing to the dominant effect of P‐gp, ritonavir enhanced only paclitaxel plasma concentrations in P‐gp‐deficient mice. Mouse liver microsomes metabolized paclitaxel far less efficiently than human or CYP3A4‐transgenic liver microsomes, revealing much lower efficiency of paclitaxel metabolism by mouse than by human CYP3As. Accordingly, ritonavir could enhance the oral bioavailability of paclitaxel in CYP3A4‐humanized mice, despite the fact that these mice are P‐gp‐proficient. Our results show that CYP3A4 inhibition most likely underlies the boosting effect of ritonavir on oral paclitaxel bioavailability in humans. Furthermore, CYP3A4‐humanized mice allow improved understanding of CYP3A4‐mediated paclitaxel metabolism in humans.     

Genotype-specific Trp53 mutational analysis in ultraviolet B radiation-induced skin cancers in Xpc and Xpc Trp53 mutant mice

We have examined the mutational spectrum in the Trp53 gene from UVB radiation-induced skin cancers in Trp53+/+ and Trp53+/- mutant mice of all three possible Xpc genotypes. Mutations were detected in exons 2-10 of the Trp53 coding region in approximately 90% of >80 different skin cancers examined. In contrast to Trp53+/+ mice in which most mutations in the Trp53 gene were located in exons 5-8, the majority of the mutations in Trp53+/- mice were at other exons. We observed a high predilection for C-->T transition mutations at a unique CpG site in codon 122 (exon 4) of the Trp53 gene in Xpc-/- Trp53+/- mice. This site is not part of a pyrimidine dinucleotide. Mutations at this codon, as well as in codons 124 and 210, were observed exclusively in Xpc-/- or Xpc+/- mice. Mutations at the corresponding codons (127 and 213) in the human p53 gene have been reported in skin tumors from human patients with xeroderma pigmentosum. Hence, mutations at codons 122 (125), 124 (127), and 210 (213) may constitute signatures for defective or deficient nucleotide excision repair in mice (humans). In Xpc-/- mice, the majority of mutations were located at C residues in CpG sites, in which the C is presumably methylated. A similar bias can be deduced from studies in human XP individuals.     

Carcinogenesis of the food mutagen PhIP in mice is independent of CYP1A2

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant of the heterocyclic amines found in cooked meat. Based on in vitro studies with rats and humans, CYP1A2 is believed to be the primary enzyme responsible for N(2)-hydroxylation, the initial step in the metabolic activation of PhIP. To determine whether CYP1A2 is the primary P450 responsible for metabolic activation of PhIP in mice that leads to tumor formation, neonatal Cyp1a2-null and wild-type mice were treated with approximately 11 (low dose) and approximately 22 (high dose) mg/kg PhIP at days 8 and 15, corresponding cumulatively to 600 and 1200 nmol PhIP, and analyzed at 19-21 months of age. Three major induced tumors were found; lymphomas and tumors in lung and liver. The incidence of lymphoma was higher in Cyp1a2-null females than wild-type females treated with low dose (600 nmol) PhIP whereas no significant differences were observed in other treatment groups of mice. Overall differences in incidences of lung adenoma/adenocarcinoma were in general not consistent among sexes, genotypes and PhIP doses used, although reduced incidences of lung tumors were found in Cyp1a2-null males with low dose (600 nmol) and null females with high dose (1200 nmol) PhIP. Higher incidences of hepatocellular adenoma were observed in Cyp1a2-null female and male mice as compared with wild-type mice. In vitro studies using Cyp1a2-null and wild-type mouse liver microsomes revealed that CYP1A2 is the major enzyme required for PhIP N2-hydroxylation in mouse, the initial metabolic activation of PhIP that is thought to lead to tumor formation. These in vivo and in vitro results suggest that although the metabolic activation of PhIP is carried out primarily by CYP1A2, an unknown pathway unrelated to CYP1A2 appears to be responsible for PhIP carcinogenesis in mouse when examined in the neonatal bioassay. In fact, CYP1A2 may even be protective against all transformation, especially in females.     

Curcumin‐induced apoptosis in ovarian carcinoma cells is p53‐independent and involves p38 mitogen‐activated protein kinase activation and downregulation of Bcl‐2 and survivin expression and Akt signaling

New cytotoxic agents are urgently needed for the treatment of advanced ovarian cancer because of the poor long‐term response of this disease to conventional chemotherapy. Curcumin, obtained from the rhizome of Curcuma longa, has potent anticancer activity; however, the mechanism of curcumin‐induced cytotoxicity in ovarian cancer cells remains a mystery. In this study we show that curcumin exhibited time‐ and dose‐dependent cytotoxicity against monolayer cultures of ovarian carcinoma cell lines with differing p53 status (wild‐type p53: HEY, OVCA429; mutant p53: OCC1; null p53: SKOV3). In addition, p53 knockdown or p53 inhibition did not diminish curcumin killing of HEY cells, confirming p53‐independent cytotoxicity. Curcumin also killed OVCA429, and SKOV3 cells grown as multicellular spheroids. Nuclear condensation and fragmentation, as well as DNA fragmentation and poly (ADP‐ribose) polymerase‐1 cleavage in curcumin‐treated HEY cells, indicated cell death by apoptosis. Procaspase‐3, procaspase‐8, and procaspase‐9 cleavage, in addition to cytochrome c release and Bid cleavage into truncated Bid, revealed that curcumin activated both the extrinsic and intrinsic pathways of apoptosis. Bax expression was unchanged but Bcl‐2, survivin, phosphorylated Akt (on serine 473), and total Akt were downregulated in curcumin‐treated HEY cells. Curcumin also activated p38 mitogen‐activated protein kinase (MAPK) without altering extracellular signal‐regulated kinase 1/2 activity. We conclude that p53‐independent curcumin‐induced apoptosis in ovarian carcinoma cells involves p38 MAPK activation, ablation of prosurvival Akt signaling, and reduced expression of the antiapoptotic proteins Bcl‐2 and survivin. These data provide a mechanistic rationale for the potential use of curcumin in the treatment of ovarian cancer. © 2009 Wiley‐Liss, Inc.