Showing papers in "Carcinogenesis in 2002"

Sensing and repairing DNA double-strand breaks.

Abstract: The DNA double-strand break (DSB) is the principle cytotoxic lesion for ionizing radiation and radio-mimetic chemicals but can also be caused by mechanical stress on chromosomes or when a replicative DNA polymerase encounters a DNA single-strand break or other type of DNA lesion. DSBs also occur as intermediates in various biological events, such as V(D)J recombination in developing lymphoid cells. Inaccurate repair or lack of repair of a DSB can lead to mutations or to larger-scale genomic instability through the generation of dicentric or acentric chromosomal fragments. Such genome changes may have tumourigenic potential. In other instances, DSBs can be sufficient to induce apoptosis. Because of the threats posed by DSBs, eukaryotic cells have evolved complex and highly conserved systems to rapidly and efficiently detect these lesions, signal their presence and bring about their repair. Here, I provide an overview of these systems, with particular emphasis on the two major pathways of DSB repair: non-homologous end-joining and homologous recombination. Inherited or acquired defects in these pathways may lead to cancer or to other human diseases, and may affect the sensitivity of patients or tumour cells to radiotherapy and certain chemotherapies. An increased knowledge of DSB repair and of other DNA DSB responses may therefore provide opportunities for developing more effective treatments for cancer.

Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: its suppression by ectopic expression of Bcl-2 and Bcl-xl

Abstract: Pharmacologically safe compounds that can inhibit the proliferation of tumor cells have potential as anticancer agents. Curcumin, a diferuloylmethane, is a major active component of the food flavor turmeric (Curcuma longa) that has been shown to inhibit the proliferation of a wide variety of tumor cells. The apoptotic intermediates through which curcumin exhibits its cytotoxic effects against tumor cells are not known, and the participation of antiapoptotic proteins Bcl-2 or Bcl-xl in the curcumin-induced apoptosis pathway is not established. In the present report we investigated the effect of curcumin on the activation of the apoptotic pathway in human acute myelogenous leukemia HL-60 cells and in established stable cell lines expressing Bcl-2 and Bcl-xl. Curcumin inhibited the growth of HL-60 cells (neo) in a dose- and time-dependent manner, whereas Bcl-2 and Bcl-xl-transfected cells were relatively resistant. Curcumin activated caspase-8 and caspase-3 in HL-60 neo cells but not in Bcl-2 and Bcl-xl-transfected cells. Similarly, time-dependent poly(ADP)ribose polymerase (PARP) cleavage by curcumin was observed in neo cells but not in Bcl-2 and Bcl-xl-transfected cells. Curcumin treatment also induced BID cleavage and mitochondrial cytochrome c release in neo cells but not in Bcl-2 and Bcl-xl-transfected cells. In neo HL-60 cells, curcumin also downregulated the expression of cyclooxygenase-2. Because DN-FLICE blocked curcumin-induced apoptosis, caspase-8 must play a critical role. Overall, our results indicate that curcumin induces apoptosis through mitochondrial pathway involving caspase-8, BID cleavage, cytochrome c release, and caspase-3 activation. Our results also suggest that Bcl-2 and Bcl-xl are critical negative regulators of curcumin-induced apoptosis.

Adolescent and adult soy intake and risk of breast cancer in Asian-Americans

Abstract: The association between soyfood intake and breast cancer risk is controversial. Most of the epidemiologic studies published on this topic in the 1990s were not designed to specifically address this question. We conducted a population-based, case-control study of breast cancer among Chinese, Japanese and Filipino women in Los Angeles County to further investigate the role of soy. Our primary objective was to quantify breast cancer risks associated with intake of soy during adolescence and adult life among Asian-American women. During 1995-1998, we successfully interviewed 501 breast cancer patients and 594 control subjects. Intake of soy among Asian-Americans is still relatively high; the median intake was 12 mg isoflavones/day, approximately one-third of that reported in a recent study in Shanghai, China. The risk of breast cancer was significantly inversely associated with soy intake during adolescence and adult life. After adjusting for age, specific Asian ethnicity, education, migration history and menstrual and reproductive factors, women who reported soy intake at least once per week during adolescence showed a statistically significantly reduced risk of breast cancer. There was also a significant trend of decreasing risk with increasing soy intake during adult life. When we considered soy intake during both adolescence and adult life, subjects who were high-soy consumers during both time periods showed the lowest risk (OR=0.53, 95% CI=0.36-0.78) compared with those who were low consumers during both time periods. Risk of breast cancer was intermediate among subjects who were high-soy consumers during adolescence and low-soy consumers during adult life (OR=0.77, 95% CI=0.51-1.10). Based on a relatively small number of subjects, the risk did not appear to differ between those who were low consumers during adolescence and high consumers during adult life. Results remained similar after adjustment for other potential confounders including other dietary and non-dietary risk factors for breast cancer. These results show that high soy intake in childhood in Asian-Americans is associated with reduced breast cancer risk. Risk may be further reduced by intake as an adult.

Enterococcus faecalis produces extracellular superoxide and hydrogen peroxide that damages colonic epithelial cell DNA.

Abstract: Enterococcus faecalis is a commensal microorganism of the human intestinal tract that produces substantial extracellular superoxide (O(-)(2)), and derivative reactive oxygen species such as H(2)O(2) and hydroxyl radical, through autoxidation of membrane-associated demethylmenaquinone. Because these oxidants may be important as a cause of chromosomal instability (CIN) associated with sporadic adenomatous polyps and colorectal cancer, the ability of E.faecalis to damage eukaryotic cell DNA was examined using the alkaline lysis single cell gel electrophoresis (comet) assay. Both Chinese hamster ovary and HT-29 intestinal epithelial cells showed increased DNA damage after co-incubation with wild-type E. faecalis strain OG1RF, but not a transposon-inactivated mutant with attenuated extracellular O(-)(2) production. E. faecalis-mediated DNA damage was prevented by catalase, but not manganese superoxide dismutase, indicating H(2)O(2) arising from O(-)(2) was the genotoxin. In a rat model of intestinal colonization, OG1RF resulted in significantly higher stool concentrations of H(2)O(2) and 5,5-dimethyl-1-pyrroline N-oxide adducts of hydroxyl and thiyl radicals, as identified by electron spin resonance-spin trapping, compared with rats colonized with a mutant strain having attenuated O(-)(2) production. Using the comet assay, luminal cells from the colon of rats colonized with O(-)(2)-producing E. faecalis showed significantly increased DNA damage compared with control rats colonized with the mutant. These findings suggest a potentially profound role for extracellular free radical production by E. faecalis in promoting CIN associated with sporadic adenomatous polyps and colorectal cancer.

Human urinary carcinogen metabolites: biomarkers for investigating tobacco and cancer.

Abstract: Measurement of human urinary carcinogen metabolites is a practical approach for obtaining important information about tobacco and cancer. This review presents currently available methods and evaluates their utility. Carcinogens and their metabolites and related compounds that have been quantified in the urine of smokers or non-smokers exposed to environmental tobacco smoke (ETS) include trans,trans-muconic acid (tt-MA) and S-phenylmercapturic acid (metabolites of benzene), 1- and 2-naphthol, hydroxyphenanthrenes and phenanthrene dihydrodiols, 1-hydroxypyrene (1-HOP), metabolites of benzo[a]pyrene, aromatic amines and heterocyclic aromatic amines, N-nitrosoproline, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (NNAL and NNAL-Gluc), 8-oxodeoxyguanosine, thioethers, mercapturic acids, and alkyladenines. Nitrosamines and their metabolites have also been quantified in the urine of smokeless tobacco users. The utility of these assays to provide information about carcinogen dose, delineation of exposed vs. non-exposed individuals, and carcinogen metabolism in humans is discussed. NNAL and NNAL-Gluc are exceptionally useful biomarkers because they are derived from a carcinogen- 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- that is specific to tobacco products. The NNAL assay has high sensitivity and specificity, which are particularly important for studies on ETS exposure. Other useful assays that have been widely applied involve quantitation of 1-HOP and tt-MA. Urinary carcinogen metabolite biomarkers will be critical components of future studies on tobacco and human cancer, particularly with respect to new tobacco products and strategies for harm reduction, the role of metabolic polymorphisms in cancer, and further evaluation of human carcinogen exposure from ETS.

Low mitochondrial respiratory chain content correlates with tumor aggressiveness in renal cell carcinoma

Abstract: A mechanism decreasing oxidative metabolism during normal cell division and growth is expected to direct substrates toward biosyntheses rather than toward complete oxidation to CO(2). Hence, any event decreasing oxidative phosphorylations (OXPHOS) could provide a proliferating advantage to a transformed or tumor cell in an oxidative tissue. To test this hypothesis, we studied mitochondrial enzymes, DNA and OXPHOS protein content in three types of renal tumors from 25 patients. Renal cell carcinomas (RCCs) of clear cell type (CCRCCs) originate from the proximal tubule and are most aggressive. Chromophilic RCCs, from similar proximal origin, are less aggressive. The benign renal oncocytomas originate from collecting duct cells. Mitochondrial enzyme and DNA contents in all tumor types or grades differed significantly from normal tissue. Mitochondrial impairment increased from the less aggressive to the most aggressive RCCs, and correlated with a considerably decreased content of OXPHOS complexes (complexes II, III, and IV of the respiratory chain, and ATPase/ATP synthase) rather than to the mitochondrial content (citrate synthase and mitochondrial (mt)DNA). In benign oncocytoma, some mitochondrial parameters (mtDNA, citrate synthase, and complex IV) were increased 4- to 7-fold, and some were slightly increased by a factor of 2 (complex V) or close to normal (complexes II and III). A low content of complex V protein was found in all CCRCC and chromophilic tumors studied. However F(1)-ATPase activity was not consistently decreased and its impairment was associated with increased aggressiveness in CCRCCs. Immunodetection of free F(1)-sector of complex V demonstrated a disturbed assembly/stability of complex V in several CCRCC and chromophilic tumors. All results are in agreement with the hypothesis that a decreased OXPHOS capacity favors faster growth or increased invasiveness.

AKT proto-oncogene overexpression is an early event during sporadic colon carcinogenesis.

Abstract: The inhibition of apoptosis is a critical event in the development of colorectal malignancies, although the mechanism(s) remain incompletely understood. The anti-apoptotic proto-oncogene, AKT, has been implicated in the molecular pathogenesis of a variety of human malignancies; however, no data exist on the role of AKT in colon carcinogenesis. We therefore evaluated the presence of AKT in human and experimental colon neoplasms by immunohistochemistry. Normal colonic mucosa and hyperplastic polyps exhibited no significant AKT expression, in marked contrast to the dramatic AKT immunoreactivity seen in colorectal cancers (57% positive) and in both human colorectal cancer cell lines examined. Importantly, AKT was also detected in 57% of the adenomas examined, implicating overexpression of this proto-oncogene as an early event during colon carcinogenesis. Moreover, in the rodent-carcinogen model, azoxymethane (AOM)-treatment induced AKT expression in premalignant rat colonocytes. Tumors that evolve via different genetic pathways displayed a lower incidence of AKT overexpression. Indeed, only 22% of mismatch repair defective tumors and 42% of AOM-induced rodent tumors upregulated AKT. Staining with an antibody specific for AKT 2 duplicated findings with the AKT 1&2 antibody, suggesting that AKT 2 was the predominant isoform involved in colon carcinogenesis. Furthermore, utilizing an antibody that specifically recognizes the serine-473 phosphorylated form of AKT, we observed that activated AKT was detectable in the neoplastic but not normal epithelium. In summary, our immunohistochemical analysis indicates AKT overexpression occurs frequently during human colon carcinogenesis, but is less common in colon cancers with microsatellite instability. The early inhibition of apoptosis during sporadic colon carcinogenesis may be related, at least partly, to the overexpression of AKT.

Antitumorigenic activity of the prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis on azoxymethane-induced colon carcinogenesis in rats.

Abstract: Prebiotics such as fructans, and probiotics such as Lactobacilli or Bifidobacteria, or a combination of prebiotics and probiotics (synbiotics) are thought to be protective against colon cancer. Therefore, we studied whether the prebiotic inulin enriched with oligofructose (Raftilose-Synergy1, briefly, Synergy1, 10% of the diet), probiotics [Bifidobacterium lactis (Bb12) and Lactobacillus rhamnosus (LGG), each at 5x10(8) c.f.u./g diet] or synbiotics (a combination of the two) protect rats against azoxymethane (AOM)-induced colon cancer. Male F344 rats were divided into: Controls; PRE, which were fed a diet containing Synergy1; PRO, fed a diet containing LGG and Bb12; PREPRO, fed a diet containing Synergy1, LGG and BB12. Ten days after beginning the diets, rats were treated with AOM (15 mg/kg s.c. two times); dietary treatments were continued for the entire experiment. Thirty-one weeks after AOM, rats treated with Synergy1 (PRE and PREPRO groups) had a significantly lower (P < 0.001) number of tumours (adenomas and cancers) than rats without Synergy1 (colorectal tumours/rat were 1.9 +/- 1.7, 1.1 +/- 1.1, 2.2 +/- 1.4 and 0.9 +/- 1.2 in Controls, PRE, PRO and PREPRO groups, respectively, means +/- SD). A slight, not significant effect of probiotics in reducing malignant tumours was also observed (P = 0.079). Caecal short-chain fatty acids (SCFA) were higher (P < 0.001) in the groups treated with Synergy1. Apoptosis was increased in the normal mucosa of the PRO group, while no variation was observed in the tumours. Colonic proliferation was lower in the PRE group as compared with Controls. Glutathione S-transferase placental enzyme pi type expression, and to a lesser extent, inducible NO synthase were depressed in the tumours from rats in the PRE and PREPRO groups. Cycloxygenase-2 expression was increased in the tumours of control rats but not in those from PRE, PRO or PREPRO rats. In conclusion, prebiotic administration in the diet decreases AOM-induced carcinogenesis in rats.

Carnosol, an antioxidant in rosemary, suppresses inducible nitric oxide synthase through down-regulating nuclear factor-κB in mouse macrophages

Abstract: Carnosol is a naturally occurring phytopolyphenol found in rosemary. Carnosol functions as antioxidant and anticarcinogen. In the present study, we compared the antioxidant activity of carnosol and other compounds extracted from rosemary. Carnosol showed potent antioxidative activity in α,α-diphenyl-β-picrylhydrazyl (DPPH) free radicals scavenge and DNA protection from Fenton reaction. High concentrations of nitric oxide (NO) are produced by inducible NO synthase (iNOS) in inflammation and multiple stages of carcinogenesis. Treatment of mouse macrophage RAW 264.7 cell line with carnosol markly reduced lipopolysaccharide (LPS)-stimulated NO production in a concentration-related manner with an IC50 of 9.4 µM; but other tested compounds had slight effects. Western blot, reverse transcription‐polymerase chain reaction, and northern blot analyses demonstrated that carnosol decreased LPS-induced iNOS mRNA and protein expression. Carnosol treatment showed reduction of nuclear factor-κB (NF-κB) subunits translocation and NF-κB DNA binding activity in activated macrophages. Carnosol also showed inhibition of iNOS and NF-κB promoter activity in transient transfection assay. These activities were referred to down-regulation of inhibitor κ B( IκB) kinase (IKK) activity by carnosol (5 µM), thus inhibited LPSinduced phosphorylation as well as degradation of IκBα. Carnosol also inhibited LPS-induced p38 and p44/42 mitogen-activated protein kinase (MAPK) activation at a higher concentration (20 µM). These results suggest that carnosol suppresses the NO production and iNOS gene expression by inhibiting NF-κB activation, and provide possible mechanisms for its anti-inflammatory and chemopreventive action.

Zerumbone, a Southeast Asian ginger sesquiterpene, markedly suppresses free radical generation, proinflammatory protein production, and cancer cell proliferation accompanied by apoptosis: the α,β-unsaturated carbonyl group is a prerequisite

Abstract: Zerumbone (ZER), a sesquiterpene from the edible plant Zingiber zerumbet Smith, has recently been found to suppress tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus activation in a potent manner. In the present study, we evaluated the anti-inflammatory and chemopreventive potentials of ZER in a variety of cell culture experiments. ZER effectively suppressed TPA-induced superoxide anion generation from both NADPH oxidase in dimethylsulfoxide-differentiated HL-60 human acute promyelocytic leukemia cells and xanthine oxidase in AS52 Chinese hamster ovary cells. The combined lipopolysaccharide- and interferon-gamma-stimulated protein expressions of inducible nitric oxide synthase and cyclooxygenase (COX)-2, together with the release of tumor necrosis factor-alpha, in RAW 264.7 mouse macrophages were also markedly diminished. These suppressive events were accompanied with a combined decrease in the medium concentrations of nitrite and prostaglandin E(2), while the expression level of COX-1 was unchanged. ZER inhibited the proliferation of human colonic adenocarcinoma cell lines (LS174T, LS180, COLO205, and COLO320DM) in a dose-dependent manner, while the growth of normal human dermal (2F0-C25) and colon (CCD-18 Co) fibroblasts was less affected. It also induced apoptosis in COLO205 cells, as detected by dysfunction of the mitochondria transmembrane, Annexin V-detected translocation of phosphatidylserine, and chromatin condensation. Intriguingly, alpha-humulene, a structural analog lacking only the carbonyl group in ZER, was virtually inactive in all experiments conducted, indicating that the alpha,beta-unsaturated carbonyl group in ZER may play some pivotal roles in interactions with unidentified target molecule(s). Taken together, our results indicate that ZER is a food phytochemical that has distinct potentials for use in anti-inflammation, chemoprevention, and chemotherapy strategies.

Cellular background level of 8-oxo-7,8-dihydro-2′-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up

Abstract: The measurement of oxidative damage to cellular DNA is a challenging analytical problem requiring highly sensitive and specific methods. In addition, artefactual DNA oxidation during its extraction and subsequent work-up may give rise to overestimated levels of oxidized DNA bases. In the present study, we have used (18)O-labelled 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) as an internal standard to evaluate the extent of artefactual DNA oxidation during the critical steps preceding the measurement. The labelled oxidized purine nucleoside was specifically generated in cellular DNA using the recently available generator of (18)O-labelled singlet oxygen. Artefactual DNA oxidation that could take place during the work-up increases the level of 8-oxodGuo but not of the (18)O-oxidized nucleoside. Therefore, the ratio between the two compounds, as measured by high performance liquid chromatography coupled to tandem mass spectrometry, allows an unambiguous comparison of different methodologies. The comparison of different DNA extraction protocols led to the conclusion that artefactual DNA oxidation during the extraction step could be minimized if: (i) nuclei are isolated after cell lysis; (ii) desferrioxamine, a transition metal chelator is added to the different extraction buffers; and (iii) sodium iodide (or alternatively guanidine thiocyanate) is used for DNA precipitation. It was also demonstrated that sodium iodide does not decompose the targeted oxidized purine nucleoside. In addition, three different DNA digestion protocols were evaluated and they were found to give rise to similar results. Using the best-studied protocol, the steady-state cellular background level of 8-oxodGuo, in a lymphocyte cell line, was determined to be approximately 0.5 lesions/10(6) DNA nucleosides.

Smoking-related DNA and protein adducts in human tissues.

Abstract: Tobacco smoking causes not only lung cancer but also cancer of the oral and nasal cavities, oesophagus, larynx, pharynx, pancreas, liver, kidney, stomach, urinary tract and cervix. Tobacco smoke contains many carcinogens that exert their biological effects through interaction of reactive intermediates with DNA to form DNA adducts. The same electrophilic species also react with cellular proteins. The effects of smoking are evident by the detection of elevated levels of carcinogen-DNA adducts in many human tissues and of carcinogen-protein adducts in blood. Components of tobacco smoke also induce oxidative DNA damage. Systemic exposure to tobacco-derived carcinogens is demonstrated by the observation of elevated levels of DNA adducts in tissues not directly exposed to tobacco smoke. For many of these tissues there is epidemiological evidence, varying from comprehensive to preliminary, that smoking is a causative factor in cancer of that site. The effects of passive smoking, which also causes lung cancer in non-smokers, is also evident in elevated levels of protein adducts in exposed non-smokers so exposed, relative to non-exposed non-smokers. This paper reviews the literature on smoking-related DNA and protein adducts in human tissues and shows how such studies have provided mechanistic insight into the epidemiological associations between smoking and cancer.

A pharmacogenetic study to investigate the role of dietary carcinogens in the etiology of colorectal cancer.

Abstract: Susceptibility to colorectal cancer, one of the most common forms of cancer in the Western world, has been associated with several environmental and dietary risk factors. Dietary exposure to food derived heterocyclic amine carcinogens and polycyclic aromatic hydrocarbons have been proposed as specific risk factors. Many polymorphic Phase I and Phase II drug metabolizing enzymes are responsible for the metabolism and disposition of these compounds and it is therefore possible that inheritance of specific allelic variants of these enzymes may influence colorectal cancer susceptibility. In a multicenter case-control study, 490 colorectal cancer patients and 593 controls (433 matched case-control pairs) were genotyped for common polymorphisms in the cytochrome P450 (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C9, CYP2C19 and CYP2D6), glutathione S-transferase (GSTM1, GSTP1 and GSTT1), sulfotransferase (SULT1A1 and SULT1A2), N-acetyl transferase 2 (NAT2), NAD(P)H:quinone oxidoreductase (NQO1), methylenetetrahydrofolate reductase (MTHFR), and microsomal epoxide hydrolase (EPHX1) genes. Matched case-control analysis identified alleles associated with higher colorectal cancer risk as carriage of CYP1A1*2C (OR = 2.15, 95% CI 1.36-3.39) and homozygosity for GSTM1*2/*2 (OR = 1.53, 95% CI 1.16-2.02). In contrast, inheritance of the CYP2A6*2 (OR = 0.51, 95% CI 0.28-1.06), CYP2C19*2 (OR = 0.72, 95% CI 0.52-0.98) and the EPHX1(His113) alleles were associated with reduced cancer risk. We found no association with colorectal cancer risk with NAT2 genotype or any of the other polymorphic genes associated with the metabolism and disposition of heterocyclic amine carcinogens. This data suggests that heterocyclic amines do not play an important role in the aetiology of colorectal cancer but that exposure to other carcinogens such as polycyclic aromatic hydrocarbons may be important determinants of cancer risk.

Modulation of repair of ultraviolet damage in the host-cell reactivation assay by polymorphic XPC and XPD/ERCC2 genotypes.

Abstract: DNA repair capacity (DRC) plays an important role in genetic susceptibility to cancer. Polymorphisms of a number of DNA repair genes involved in several distinct pathways have been identified. However, their effects on repair function have not been well characterized. We demonstrated previously that DRC for removal of benzo[a]pyrene diol epoxide-induced DNA damage measured by a host-cell reactivation assay was modulated by two XPD/ERCC2 polymorphisms in lung cancer. In this report, we investigated the association between the repair phenotype of ultraviolet (UV)-induced damage and genotypes of three DNA repair genes, XPC and XPD [involved in nucleotide excision repair (NER)] and XRCC1 [involved in base excision repair (BER)]. We measured DRC for removal of UV photoproducts by the host-cell reactivation assay in cryopreserved lymphocytes from 102 healthy non-Hispanic white subjects. We also typed these subjects for five polymorphisms in these three DNA repair genes (at intron 9 of XPC; exons 6, 10 and 23 of XPD and exon 10 of XRCC1). Compared with wild-type homozygotes, subjects homozygous for polymorphisms of the two NER genes consistently had suboptimal DRC. The DRC was consistently lower in subjects homozygous for XPC, XPD or both than in subjects with other genotypes, although the difference was not statistically significant for XPD variants. In contrast, the polymorphic allele of the BER gene, XRCC1, had no consistent effect on DRC. We concluded that these NER polymorphisms may modulate DRC and may be useful biomarkers for identifying individuals at risk of developing cancer.

Cigarette smoke condensate activates nuclear transcription factor-κB through phosphorylation and degradation of IκBα: correlation with induction of cyclooxygenase-2

Abstract: Cigarette smoke (CS) contains several carcinogens known to initiate and promote tumorigenesis and metastasis. Because various genes that mediate carcinogenesis and tumorigenesis are regulated by nuclear factor-kappaB (NF-kappaB), we postulated that the effects of CS must be mediated through activation of this transcription factor. Therefore, in the present report we investigated whether cigarette smoke condensate (CSC) activates NF-kappaB, and whether the pathway employed for activation is similar to that of TNF, one of the potent activators of NF-kappaB. Our results show that the treatment of human histiocytic lymphoma U-937 cells with CSC activated NF-kappaB in a dose- and time-dependent manner. The kinetics of NF-kappaB activation by CSC was comparable with that of TNF. CSC-induced NF-kappaB activation was not cell type-specific, as it also activated NF-kappaB in T cells (Jurkat), lung cells (H1299), and head and neck squamous cell lines (1483 and 14B). Activation of NF-kappaB by CSC correlated with time-dependent degradation of IkappaB(alpha), an inhibitor of NF-kappaB. Further studies revealed that CSC induced phosphorylation of the serine residue at position 32 in IkappaB(alpha). In vitro immunocomplex kinase assays showed that CSC activated IkappaB(alpha) kinase (IKK). The suppression of CSC-activated NF-kappaB-dependent reporter gene expression by dominant negative form of IkappaB(alpha), TRAF2, NIK and IKK suggests a similarity to the TNF-induced pathway for NF-kappaB. CSC also induced the expression of cyclooxygenase-2, an NF-kappaB regulated gene product. Overall, our results indicate that through phosphorylation and degradation of IkappaB(alpha), CSC can activate NF-kappaB in a wide a variety of cells, and this may play a role in CS-induced carcinogenesis.

Meta- and pooled analyses of the effects of glutathione S-transferase M1 polymorphisms and smoking on lung cancer risk

Abstract: Susceptibility to lung cancer may in part be attributable to inter-individual variability in metabolic activation or detoxification of tobacco carcinogens. The glutathione S-transferase M1 (GSTM1) genetic polymorphism has been extensively studied in this context; two recent meta-analyses of case-control studies suggested an association between GSTM1 deletion and lung cancer. At least 15 studies have been published after these overviews. We undertook a new meta-analysis to summarize the results of 43 published case-control studies including >18 000 individuals. A slight excess of risk of lung cancer for individuals with the GSTM1 null genotype was found (odds ratio (OR) = 1.17, 95% confidence interval (CI) 1.07-1.27). No evidence of publication bias was found (P = 0.4), however, it is not easy to estimate the extent of such bias and we cannot rule out some degree of publication bias in our results. A pooled analysis of the original data of about 9500 subjects involved in 21 case-control studies from the International Collaborative Study on Genetic Susceptibility to Environmental Carcinogens (GSEC) data set was performed to assess the role of GSTM1 genotype as a modifier of the effect of smoking on lung cancer risk with adequate power. Analyses revealed no evidence of increased risk of lung cancer among carriers of the GSTM1 null genotype (age-, gender- and center-adjusted OR = 1.08, 95% CI 0.98-1.18) and no evidence of interaction between GSTM1 genotype and either smoking status or cumulative tobacco consumption.

The major lipid peroxidation product, trans- 4-hydroxy-2-nonenal, preferentially forms DNA adducts at codon 249 of human p53 gene, a unique mutational hotspot in hepatocellular carcinoma

Abstract: Trans-4-hydroxy-2-nonenal (4-HNE), a major electrophilic by-product of lipid peroxidation, is able to interact with DNA to form exocyclic guanine adducts. 4-HNE is a mutagen and a significant amount of 4-HNE-guanine adduct has been detected in normal cells. Recently, it has been reported that exposure of the wild-type p53 human lymphoblastoid cell line to 4-HNE causes a high frequency of G to T transversion mutations at the third base of codon 249 (-AGG*-) in the p53 gene, a mutational hotspot in human cancers, particularly hepatocellular carcinoma. These findings raise a possibility that 4-HNE could be an important etiological agent for human cancers that have a mutation at codon 249 of the p53 gene. However, to date, the sequence specificity of 4-HNE-DNA binding remains unclear due to the lack of methodology. To address this question, we have developed a method, using UvrABC nuclease, a nucleotide excision repair enzyme complex isolated from Escherichia coli, to map the distribution of 4-HNE-DNA adducts in human p53 gene at the nucleotide sequence level. We found that 4-HNE-DNA adducts are preferentially formed at the third base of codon 249 in the p53 gene. The preferential binding of 4-HNE was also observed at codon 174, which has the same sequence and the same nearest neighbor sequences (-GAGG*C-) as codon 249. These results suggest that 4-HNE may be an important etiological agent for human cancers that have a mutation at codon 249 of the p53 gene.

Overexpression of an ectopic H19 gene enhances the tumorigenic properties of breast cancer cells

Abstract: The maternally expressed H19 gene is transcribed as an untranslated RNA that serves as a riboregulator. We have previously reported that this transcript accumulates in epithelial cells in approximately 10% of breast cancers. To gain further insight on how the overexpression of the H19 gene affects the phenotype of human breast epithelial cells, we investigated the oncogenic potential of RNA that was abundantly expressed from MDA-MB-231 breast cancer cells stably transfected with the genomic sequence of the human H19 gene. The amount of H19 RNA did not affect cell proliferation capacity, timing of cell cycle phases or anchorage-dependent ability of H19-transfected clones in vitro. But in anchorage-independent growth assays the H19-recombined cells formed more and larger colonies in soft-agar versus control cells. To explore this phenotypic change, we analysed tumour development after subcutaneous injection of H19-recombined cells into scid mice. Results showed that H19 overexpression promotes tumour progression. These data support the hypothesis that an overload of H19 transcript is associated with cells exhibiting higher tumorigenic phenotypes and therefore we conclude that the H19 gene has oncogenic properties in breast epithelial cells.

The XPD variant alleles are associated with increased aromatic DNA adduct level and lung cancer risk.

Abstract: The DNA repair protein xeroderma pigmentosum complementation group D (XPD) is involved in the nucleotide excision repair of DNA lesions induced by many tobacco and environmental carcinogens. In order to study the functional impact of the common polymorphisms in XPD exon 10 (G > A, Asp312Asn) and exon 23 (A > C, Lys751Gln), we have genotyped 185 Swedish lung cancer cases (97 smokers and 88 never-smokers) and 162 matched population controls (83 smokers and 79 never-smokers). Presence of one or two variant alleles was associated with increased risk for lung cancer among never-smokers only, in particular younger (<70 years) never-smokers [odds ratio (OR) = 2.6, 95% confidence interval (CI) = 1.1-6.5 for exon 10; OR = 3.2, 95% CI = 1.3-8.0 for exon 23, adjusted for age, gender and environmental tobacco smoke]. Aromatic DNA adduct level (AL) in peripheral lymphocytes was found to be similar between cases and controls, but significantly increased by current or recent smoking. Overall, there was a significant trend for increasing AL with increasing number of variant alleles in exon 10 (P = 0.02) or in exon 23 (P = 0.001). In addition, subjects with the combined exon 10 AA and exon 23 CC genotype showed a significantly higher AL compared with all those with any of the other genotypes (P = 0.02). We conclude that the XPD variant alleles may be associated with reduced repair of aromatic DNA adducts in general and increased lung cancer risk among never-smokers.

p53–Mdm2—the affair that never ends

Abstract: The p53-Mdm2 paradigm represents the best-studied relationship between a tumor suppressor gene which functions as a transcription factor and an oncogene, which functions primarily as an E3 protein ligase. The intimate relationship between these two partners has expanded to include almost every cellular biological system - from development, to growth control and programmed cell death. The affair between Mdm2 and p53 is closely controlled by a complex array of post-translational modifications, which in turn dictates the stability and activity of p53 and Mdm2. Functional diversity depends on the association with a large subset of partner proteins, which dictates the type of activity and corresponding selectivity. Here we summarize the current understanding of post-translational modifications and their effect on conformation-based functional relationship between Mdm2 and p53, as it pertains to their diverse cellular biological functions.

Dietary isothiocyanates, glutathione S-transferase polymorphisms and colorectal cancer risk in the Singapore Chinese Health Study.

Abstract: Dietary intake of cruciferous vegetables (Brassica spp.) has been inversely related to colorectal cancer risk, and this has been attributed to their high content of glucosinolate degradation products such as isothiocyanates (ITCs). These compounds act as anticarcinogens by inducing phase II conjugating enzymes, in particular glutathione S-transferases (GSTs). These enzymes also metabolize ITCs, such that the protective effect of cruciferous vegetables may predicate on GST genotype. The Singapore Chinese Health Study is a prospective investigation among 63 257 middle-aged men and women, who were enrolled between April 1993 and December 1998. In this nested case-control analysis, we compared 213 incident cases of colorectal cancer with 1194 controls. Information on dietary ITC intake from cruciferous vegetables, collected at recruitment via a semi-quantitative food frequency questionnaire, was combined with GSTM1, T1 and P1 genotype from peripheral blood lymphocytes or buccal mucosa. When categorized into high (greater than median) and low (less than/equal to median) intake, dietary ITC was slightly lower in cases than controls but the difference was not significant [odds ratio (OR) 0.81, 95% confidence interval (CI) 0.59-1.12]. There were no overall associations between GSTM1, T1 or P1 genotypes and colorectal cancer risk. However, among individuals with both GSTM1 and T1 null genotypes, we observed a 57% reduction in risk among high versus low consumers of ITC (OR 0.43, 95% CI 0.20-0.96), in particular for colon cancer (OR 0.31, 0.12-0.84). Our results are compatible with the hypothesis that ITCs from cruciferous vegetables modify risk of colorectal cancer in individuals with low GST activity. Further, this gene-diet interaction may be important in studies evaluating the effect of risk-enhancing compounds in the colorectum.

Growth inhibition, cell-cycle arrest and apoptosis in human T-cell leukemia by the isothiocyanate sulforaphane

Abstract: Glucosinolates (GL) can inhibit, retard or reverse experimental multistage carcinogenesis. When brassica plant tissue is broken, GLs are hydrolyzed by the endogenous enzyme myrosinase (Myr), releasing many products including isothiocyanates (ITC). Synthetic ITCs like sulforaphane exert chemopreventive effects against chemically induced tumors in animals, modulating enzymes required for carcinogens' activation/detoxification and/or the induction of cell-cycle arrest and apoptosis in tumor cell lines. To investigate the chemopreventive potential of ITCs while reproducing the circumstances of dietary contact with sulforaphane, we studied proliferation, apoptosis induction and p53, bcl-2 and bax protein expression in Jurkat T-leukemia cells by sulforaphane, the ITC generated in situ in a quantitative manner by Myr starting from glucoraphanin (GRA). Jurkat cells were treated with different doses of GRA-Myr mixture. Effects on cell growth or survival were evaluated by counting trypan blue-excluding cells. Cell-cycle progression, apoptosis and expression of p53, bax and bcl-2 proteins were analyzed by flow cytometry. Results were analyzed by two-sided Fisher's exact test. Sulforaphane, but not GRA, caused G(2)/M-phase arrest (P = 0.028) and increase of apoptotic cell fraction (P < 0.0001) in a time- and dose-dependent manner. Necrosis was observed after prolonged exposure to elevated sulforaphane doses. Moreover, it markedly increased p53 and bax protein expression, and slightly affected bcl-2 expression. These findings indicate that sulforaphane but not the native GL GRA can exert both protective and toxic effects inhibiting leukemic cell growth. Sulforaphane therefore deserves study as a potential chemopreventive/chemotherapeutic antileukemic agent.

Comparative analysis of baseline 8-oxo-7,8-dihydroguanine in mammalian cell DNA, by different methods in different laboratories: an approach to consensus.

Abstract: The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up to resolve the problems associated with the measurement of background levels of oxidative DNA damage (in particular 8-oxo-7,8-dihydroguanine, or 8-oxoGua) in human cells. A tendency for DNA oxidation to occur during sample preparation prior to chromatography has been recognized as the source of a very substantial artefact. To assess the success of attempts to eliminate the artefact, ESCODD has distributed to its members standard samples of pig liver and HeLa cells for analysis. Estimates of 8-oxoGua in pig liver, using chromatographic techniques, ranged from 2.23 to 441 per 10*6 guanines, with a median of 10.47 per 10*6 guanines. Chromatographic analysis of HeLa cell DNA gave a range of 1.84 to 214 per 10*6 guanines with a median of 5.23 per 10*6 guanines. HeLa cell DNA was also analysed by an enzymic approach, in which whole cell DNA was treated with formamidopyrimidine DNA glycosylase, which nicks DNA at sites of 8-oxoGua, and the breaks measured with the comet assay, alkaline unwinding or alkaline elution. Values with these methods ranged from 0.06 to 4.988-oxoGua per 10*6 guanines, with a median of 0.79 per 10*6 guanines. Although there are clearly still serious discrepancies between methods and laboratories, the lowest estimates by chromatography (arguably those in which the artefact was best controlled) are only 2.5 times higher than the median value obtained with the enzymic approach.

Arylhydrocarbon receptor-dependent induction of liver and lung cytochromes P450 1A1, 1A2, and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in genetically engineered C57BL/6J mice.

Abstract: Arylhydrocarbon receptor knock-out, AhR(-/-), mice have recently been shown to be rather resistant to benzo[a]pyrene (B[a]P)-induced tumor formation, probably reflecting the inability of these mice to express significant levels of cytochrome P450 (P450 or CYP) 1A1 that activates B[a]P to reactive metabolites (Y. Shimizu, Y. Nakatsuru, M. Ichinose, Y. Takahashi, H. Kume, J. Mimura, Y. Fujii-Kuriyama and T. Ishikawa (2000) PROC: Natl Acad. Sci. USA, 97, 779-782). However, it is not precisely determined whether CYP1B1, another enzyme that is also active in activating B[a]P, plays a role in the B[a]P carcinogenesis in mice. To understand the basis of roles of CYP1A1 and CYP1B1 in the activation of chemical carcinogens, we compared levels of induction of liver and lung CYP1A1, 1A2, and 1B1 by various polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls in AhR(+/+) and AhR(-/-) mice. Liver and lung CYP1A1 and 1B1 mRNAs were highly induced in AhR(+/+) mice by a single intraperitoneal injection of each of the carcinogenic PAHs, such as B[a]P, 7,12-dimethylbenz[a]anthracene, dibenz[a,l]pyrene, 3-methylcholanthrene, 1,2,5,6-dibenzanthracene, benzo[b]fluoranthene, and benzo[a]anthracene and by a co-planar PCB congener 3,4,3',4'-tetrachlorobiphenyl. We also found that 6-aminochrysene, chrysene, benzo[e]pyrene, and 1-nitropyrene weakly induced the mRNA expression of CYP1A1 and 1B1, whereas anthracene, pyrene, and fluoranthene that have been reported to be non-carcinogenic in rodents, were very low or inactive in inducing these P450s. The extents of induction of liver CYP1A2 by these chemicals were less than those of CYP1A1 and 1B1 in AhR(+/-/+/-) mice. In AhR(-/-) mice, there was no induction of these P450s by PAHs and polychlorinated biphenyls. Liver microsomal activities of 7-ethoxyresorufin and 7-ethoxycoumarin O-deethylations and of mutagenic activation of (+/-)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P to DNA-damaging products were found to correlate with levels of CYP1A1 and 1B1 mRNAs in the liver. Our results suggest that carcinogenicity potencies of PAHs may relate to the potencies of these compounds to induce CYP1A1 and 1B1 through AhR-dependent manner and that these induced P450s participate in the activation of B[a]P and related carcinogens causing initiation of cancers in mice.

Gene-environment interaction and aetiology of cancer: what does it mean and how can we measure it?

Abstract: One form of defence against cancer development involves a series of genes whose role is to metabolize and excrete potentially toxic compounds and to repair subtle mistakes in DNA. Much laboratory and epidemiological research over the past decade has concentrated on the identification of these genes and an assessment of their role in cancer aetiology. Of particular interest has been whether the risk of cancer associated with a particular environmental exposure differs with respect to functionally different polymorphisms of these genes, i.e. gene-environment interaction. A large number of studies have been conducted for numerous genes and also for all common cancer sites, although results have been very inconsistent and therefore inconclusive. This is partially due to the inadequate sample size of most studies to detect modest effects and the over-reporting of positive associations identified in subgroups of the dataset. There is also much confusion about the meaning of "gene-environment interaction", what type of studies should be conducted to study it and also how it should be measured. Furthermore, the very purpose of these studies is not clear; are they attempting to identify high-risk individuals, or are they simply trying to further understand the cancer process? This review will explore these questions and provide some recommendations to help ensure that the next phase of gene-environment interaction studies, which are likely to be much larger and based on many more genes, also provide some clearer answers.

Genetic polymorphisms of alcohol and aldehyde dehydrogenases and glutathione S-transferase M1 and drinking, smoking, and diet in Japanese men with esophageal squamous cell carcinoma

Abstract: The genetic polymorphisms of aldehyde dehydrogenase-2 (ALDH2), alcohol dehydrogenase-2 (ADH2), ADH3, and glutathione S-transferase M1 (GSTM1) influence the metabolism of alcohol and other carcinogens. The ALDH2*1/2*2 genotype, which encodes inactive ALDH2, and ADH2*1/2*1, which encodes the low-activity form of ADH2, enhance the risk for esophageal cancer in East Asian alcoholics. This case-control study of whether the enzyme-related vulnerability for esophageal cancer can be extended to a general population involved 234 Japanese men with esophageal squamous cell carcinoma and 634 cancer-free Japanese men who received annual health checkups. The GSTM1 genotype was not associated with the risk for this cancer. Light drinkers (1-8.9 units/week) with ALDH2*1/2*2 had an esophageal cancer risk 5.82 times that of light drinkers with ALDH2*1/2*1 (reference category), and their risk was similar to that of moderate drinkers (9-17.9 units/week) with ALDH2*1/2*1 (odds ratio = 5.58). The risk for moderate drinkers with ALDH2*1/2*2 (OR = 55.84) exceeded that for heavy drinkers (18+ units/week) with ALDH2*1/2*1 (OR = 10.38). Similar increased risks were observed for those with ADH2*1/2*1. A multiple logistic model including ALDH2, ADH2, and ADH3 genotypes showed that the ADH3 genotype does not significantly affect the risk for esophageal cancer. For individuals with both ALDH2*1/2*2 and ADH2*1/2*1, the risk of esophageal cancer was enhanced in a multiplicative fashion (OR = 30.12), whereas for those with either ALDH2*1/2*2 or ADH2*1/2*1 alone the ORs were 7.36 and 4.11. In comparison with the estimated population-attributable risks for preference for strong alcoholic beverages (30.7%), smoking (53.6%) and for lower intake of green and yellow vegetables (25.7%) and fruit (37.6%), an extraordinarily high proportion of the excessive risk for esophageal cancer in the Japanese males can be attributed to drinking (90.9%), particularly drinking by persons with inactive heterozygous ALDH2 (68.5%). Education regarding these risky conditions in connection with ALDH2 and ADH2 is vitally important in a new strategic approach aimed at preventing esophageal cancer in East Asians.

Generation and characterization of p53 null transformed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma

Abstract: Oval cells are bipotential liver stem cells able to differentiate into hepatocytes and bile duct epithelia. In normal adult liver oval cells are quiescent, existing in low numbers around the periportal region, and proliferate following severe, prolonged liver trauma. There is evidence implicating oval cells in the development of hepatocellular carcinoma, and hence the availability of an immortalized oval cell line would be invaluable for the study of liver cell lineage differentiation and carcinogenesis. A novel approach in the generation of cell lines is the use of the p53 knockout mouse. Absence of p53 allows a cell to cycle past the normal Hayflick limit, rendering it immortalized, although subsequent genetic alterations are thought necessary for transformation, p53 knockout mice were fed a choline-deficient, ethionine-supplemented diet, previously shown to increase oval cell numbers in wild-type mice. The oval cells were isolated by centrifugal elutriation and maintained in culture. Colonies of hepatic cells were isolated and characterized with respect to phenotype, growth characteristics and tumorigenicity. Analysis of gene expression by Northern blotting and immunocytochemistry suggests they are oval-like cells by virtue of albumin and transferrin expression, as well as the oval cell markers alpha fetoprotein, M 2 -pyruvate kinase and A6. Injection into athymic nude mice shows the cell lines are capable of forming tumors which phenotypically resemble hepatocellular carcinoma. Thus, the use of p53 null hepatic cells successfully generated immortalized and tumorigenic hepatic stem cell lines. The results presented support the idea that deleting p53 allows immortalization and contributes to the transformation of the oval-like cell lines. Further, the tumorigenic status of the cell lines is direct evidence for the participation of oval cells in the formation of hepatocellular carcinoma.

Carcinogenicity of dimethylarsinic acid in male F344 rats and genetic alterations in induced urinary bladder tumors

Abstract: Arsenic is a well-documented human carcinogen, and contamination with this heavy metal is of global concern, presenting a major issue in environmental health. However, the mechanism by which arsenic induces cancer is unknown, in large part due to the lack of an appropriate animal model. In the present set of experiments, we focused on dimethylarsinic acid (DMA), a major metabolite of arsenic in most mammals including humans. We provide, for the first time, the full data, including detailed pathology, of the carcinogenicity of DMA in male F344 rats in a 2-year bioassay, along with the first assessment of the genetic alteration patterns in the induced rat urinary bladder tumors. Additionally, to test the hypothesis that reactive oxygen species (ROS) may play a role in DMA carcinogenesis, 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in urinary bladder was examined. In experiment 1, a total of 144 male F344 rats at 10 weeks of age were randomly divided into four groups that received DMA at concentrations of 0, 12.5, 50 and 200 p.p.m. in the drinking water, respectively, for 104 weeks. From weeks 97-104, urinary bladder tumors were observed in 8 of 31 and 12 of 31 rats in groups treated with 50 and 200 p.p.m. DMA, respectively, and the preneoplastic lesion, papillary or nodular hyperplasias (PN hyperplasia), was noted in 12 and 14 rats, respectively. DMA treatment did not cause tumors in other organs and no urinary bladder tumors or preneoplastic lesions were evident in the 0 and 12.5 p.p.m.-treated groups. Urinary levels of arsenicals increased significantly in a dose-responsive manner except for arsenobetaine (AsBe). DMA and trimethylarsine oxide (TMAO) were the major compounds detected in the urine, with small amounts of monomethylarsonic acid (MMA) and tetramethylarsonium (TeMa) also detected. Significantly increased 5-bromo-2'-deoxyuridine (BrdU) labeling indices were observed in the morphologically normal epithelium of the groups treated with 50 and 200 p.p.m. DMA. Mutation analysis showed that DMA-induced rat urinary bladder tumors had a low rate of H-ras mutations (2 of 20, 10%). No alterations of the p53, K-ras or beta-catenin genes were detected. Only one TCC (6%) demonstrated nuclear accumulation of p53 protein by immunohistochemistry. In 16 of 18 (89%) of the TTCs and 3 of 4 (75%) of the papillomas, decreased p27(kip1) expression could be demonstrated. Cyclin D1 overexpression was observed in 26 of 47 (55%) PN hyperplasias, 3 of 4 (75%) papillomas, and 10 of 18 (56%) TCCs. As a molecular marker of oxidative stress, increased COX-2 expression was noted in 17 of 18 (94%) TCCs, 4 of 4 (100%) papillomas, and 39 of 47 (83%) PN hyperplasias. In experiment 2, 8-OHdG formation in urinary bladder was significantly increased after treatment with 200 p.p.m. DMA in the drinking water for 2 weeks compared with the controls. The studies demonstrated DMA to be a carcinogen for the rat urinary bladder and suggested that DMA exposure may be relevant to the carcinogenic risk of inorganic arsenic in humans. Diverse genetic alterations observed in DMA-induced urinary bladder tumors imply that multiple genes are involved in stages of DMA-induced tumor development. Furthermore, generation of ROS is likely to play an important role in the early stages of DMA carcinogenesis.

Inhibition of 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamsters by tea and curcumin.

Abstract: Tea is one of the most popular beverages consumed in the world. Curcumin, the major yellow pigment in turmeric, is used widely as a spice and food-coloring agent. In this study, we studied the effects of tea and curcumin on 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamsters. DMBA solution (0.5% in mineral oil, 0.1 ml) was applied topically to the left cheek pouch of male Syrian golden hamsters 3 times/week for 6 weeks. Two days after the last treatment of DMBA, the animals received green tea (6 mg tea solids/ml) as drinking fluid, or 10 mmol curcumin applied topically 3 times/week, or the combination of green tea and curcumin treatment, or no treatment for 18 weeks. The combination of tea and curcumin significantly decreased the oral visible tumor incidence from 92.3% (24/26) to 69.2% (18/26) and the squamous cell carcinoma (SCC) incidence from 76.9% (20/26) to 42.3% (11/26). The combination of tea and curcumin also decreased the number of visible tumors and the tumor volume by 52.4 and 69.8%, as well as the numbers of SCC, dysplasic lesions and papillomas by 62.0, 37.5 and 48.7%, respectively. Green tea or curcumin treatment decreased the number of visible tumors by 35.1 or 39.6%, the tumor volume by 41.6 or 61.3% and the number of SCC by 53.3 or 51.3%, respectively. Green tea also decreased the number of dysplasic lesions. Curcumin also significantly decreased the SCC incidence. Tea and curcumin, singly or in combination, decreased the proliferation index in hyperplasia, dysplasia and papillomas. Only the combination treatment decreased the proliferation index in SCC. Tea alone and in combination with curcumin significantly increased the apoptotic index in dysplasia and SCC. Curcumin, alone and in combination with tea, significantly inhibited the angiogenesis in papilloma and SCC. The results suggested that green tea and curcumin had inhibitory effects against oral carcinogenesis at the post-initiation stage and such inhibition may be related to the suppression of cell proliferation, induction of apoptosis and inhibition of angiogenesis.

Catechol ortho-quinones: the electrophilic compounds that form depurinating DNA adducts and could initiate cancer and other diseases.

Abstract: Catechol estrogens and catecholamines are metabolized to quinones, and the metabolite catechol (1,2-dihydroxybenzene) of the leukemogenic benzene can also be oxidized to its quinone. We report here that quinones obtained by enzymatic oxidation of catechol and dopamine with horseradish peroxidase, tyrosinase or phenobarbital-induced rat liver microsomes react with DNA by 1,4-Michael addition to form predominantly depurinating adducts at the N-7 of guanine and the N-3 of adenine. These adducts are analogous to the ones formed with DNA by enzymatically oxidized 4-catechol estrogens (Cavalieri,E.L., et al. (1997) PROC: Natl Acad. Sci., 94, 10937). The adducts were identified by comparison with standard adducts synthesized by reaction of catechol quinone or dopamine quinone with deoxyguanosine or adenine. We hypothesize that mutations induced by apurinic sites, generated by the depurinating adducts, may initiate cancer by benzene and estrogens, and some neurodegenerative diseases (e.g. Parkinson's disease) by dopamine. These data suggest that there is a unifying molecular mechanism, namely, formation of specific depurinating DNA adducts at the N-7 of guanine and N-3 of adenine, that could initiate many cancers and neurodegenerative diseases.