• The parity-related protection against breast cancer is compromised by cigarette smoke during rat pregnancy: observations on tumorigenesis and immunological defenses of the neonate.

      Steinetz, Bernard G.; Gordon, Terry; Lasano, Salamia; Horton, Lori; Ng, Sheung Pui; Zelikoff, Judith T.; Nadas, Arthur; Bosland, Maarten C. (2006-06)
      Early pregnancy is a powerful negative risk factor for breast cancer (BCa) in women. Pregnancy also protects rats against induction of BCa by carcinogens such as N-methyl-N-nitrosourea (MNU), making the parous rat a useful model for studying this phenomenon. Smoking during early pregnancy may lead to an increased risk of BCa in later life, possibly attributable to carcinogens in cigarette smoke (CS), or to reversal of the parity-related protection against BCa. To investigate these possibilities, 50-day-old timed first-pregnancy rats were exposed to standardized mainstream CS (particle concentration = 50 mg/m3) or to filtered air (FA) 4 h/day, Day 2-20 of gestation. Age-matched virgin rats were similarly exposed to CS or FA. At age 100 days, the CS or FA-exposed, parous and virgin rats were injected s.c. with MNU (50 mg/kg body wt), or with MNU vehicle. Mammary tumors (MTs) first appeared in virgin rats 9 weeks post-MNU injection. While no MTs were detected in FA-exposed parous rats until 18 weeks post-MNU, MTs appeared in the CS-exposed parous rats as early as 10 wks (P < 0.02). As no MTs developed in CS-exposed rats not injected with MNU, CS did not act as a direct mammary carcinogen. Serum prolactin concentration on Day 19 of pregnancy in CS-exposed dams was reduced by 50% compared with FA-exposed dams (P < 0.005). CS exposure during a pregnancy may thus 'deprotect' rats, enhancing their vulnerability to MNU-induced BCa. Prenatal CS exposure had no detectable effect on the immune responses of the pups examined at 3, 8 or 19 weeks of age. However, prolactin concentration in stomach contents (milk) of 3-day-old pups suckled by CS-exposed dams was decreased when compared with that of FA-exposed dams (P < 0.032). As milk-borne prolactin modulates development of the central nervous and immune systems of neonatal rats, CS exposure of the dams could adversely affect later maturation of these systems by reducing milk prolactin.
    • Pendimethalin exposure and cancer incidence among pesticide applicators.

      Hou, Lifang; Lee, Won Jin; Rusiecki, Jennifer; Hoppin, Jane A.; Blair, Aaron; Bonner, Matthew R.; Lubin, Jay H.; Samanic, Claudine; Sandler, Dale P.; Dosemeci, Mustafa; et al. (2006-05)
      BACKGROUND: Pendimethalin, a widely used herbicide, has been classified as a group C possible human carcinogen by the U.S. Environmental Protection Agency. We evaluated the incidence of cancer in relation to reported pendimethelin use among pesticide applicators in the Agricultural Health Study, a prospective cohort of licensed pesticide applicators in Iowa and North Carolina. METHODS: Information on pesticide use came from two questionnaires (enrollment and take-home). The present analysis includes 9089 pendimethalin-exposed and 15,285 nonpendimethalin-exposed pesticide applicators with complete information on pendimethalin use and covariates from a take-home questionnaire. We conducted Poisson regression analyses to evaluate the association of pendimethalin exposure with cancer incidence (mean follow-up = 7.5 years) using two exposure metrics: tertiles of lifetime days of exposure and tertiles of intensity-weighted lifetime days of exposure. RESULTS: Overall cancer incidence did not increase with increasing lifetime pendimethalin use, and there was no clear evidence of an association between pendimethalin use and risks for specific cancers. The risk for rectal cancer rose with increasing lifetime pendimethalin exposure when using nonexposed as the reference (rate ratio = 4.3; 95% confidence interval = 1.5-12.7 for the highest exposed subjects; P for trend = 0.007), but the association was attenuated when using the low exposed as the referent group (P for trend = 0.08). Similar patterns for rectal cancer were observed when using intensity-weighted exposure-days. The number of rectal cancer cases among the pendimethalin-exposed was small (n = 19). There was some evidence for an elevated risk for lung cancer, but the excess occurred only in the highest exposure category for lifetime pendimethalin exposure. The trends for lung cancer risk were inconsistent for different exposure metrics. CONCLUSIONS: We did not find a clear association of lifetime pendimethalin exposure either with overall cancer incidence or with specific cancer sites.
    • Phenylethyl isothiocyanate and its N-acetylcysteine conjugate suppress the metastasis of SK-Hep1 human hepatoma cells.

      Hwang, Eun-Sun; Lee, Hyong Joo (2006-12)
      Phenylethyl isothiocyanate (PEITC), a hydrolysis compound of gluconasturtiin, is metabolized to N-acetylcysteine (NAC)-PEITC in the body after the consumption of cruciferous vegetables. We observed an inhibitory effect of PEITC and its metabolite NAC-PEITC on cancer cell proliferation, adhesion, invasion, migration and metastasis in SK-Hep1 human hepatoma cells. PEITC and NAC-PEITC suppressed SK-Hep1 cell proliferation in a dose-dependent manner, and exposure to 10 microM PEITC or NAC-PEITC reduced cell proliferation by 25% and 30%, respectively. NAC-PEITC inhibited cancer cell adhesion, invasion and migration to a similar or to an even larger degree than PEITC. The expression of matrix metalloproteinase (MMP) 2, MMP-9 and membrane type 1 matrix metalloproteinase (MT1-MMP) is a known risk factor for metastatic disease. Gelatin zymography analysis revealed a significant downregulation of MMP-2/MMP-9 protein expression in SK-Hep1 cells treated with 0.1-5 microM PEITC or NAC-PEITC. PEITC and NAC-PEITC treatment caused dose-dependent decreases in MMP-2/MMP-9 and MT1-MMP mRNA levels, as determined by reverse transcription polymerase chain reaction. PEITC and NAC-PEITC also increased the mRNA levels of tissue inhibitors of matrix metalloproteinase (TIMPs) 1 and 2. Our data suggest that this inhibition is mediated by downregulation of MMP and upregulation of TIMPs.
    • Photoprotective effect of isoflavone genistein on ultraviolet B-induced pyrimidine dimer formation and PCNA expression in human reconstituted skin and its implications in dermatology and prevention of cutaneous carcinogenesis.

      Moore, Julian O.; Wang, Yongyin; Stebbins, William G.; Gao, Dayuan; Zhou, Xueyan; Phelps, Robert; Lebwohl, Mark; Wei, Huachen (2006-08)
      Genistein, the most abundant isoflavone of the soy derived phytoestrogen compounds, is a potent antioxidant and inhibitor of tyrosine kinase. We previously reported the antiphotocarcinogenic effects of genistein in SKH-1 murine skin, including its capacity for scavenging reactive oxygen species, inhibiting photodynamic DNA damage and downregulating UVB(ultra violet B)-induced signal transduction cascades in carcinogenesis. In this study we elucidate genistein's photoprotective efficacy within the context of full thickness human reconstituted skin relative to acute challenges with ultraviolet-B irradiation. Skin samples were pre-treated with three concentrations of genistein (10, 20 and 50 microM) 1 h prior to UVB radiation at 20 and 60 mJ/cm2. Proliferating cell nuclear antigen (PCNA) and pyrimidine dimer (PD) expression profiles were localized using immunohistochemical analysis on paraffin embedded samples 6 and 12 h post UVB exposure. Genistein dose dependently preserved cutaneous proliferation and repair mechanics at 20 and 60 mJ/cm2, as evidenced by the preservation of proliferating cell populations with increasing genistein concentrations and noticeable paucity in PCNA immunoreactivity in the absence of genistein. Genistein inhibited UV-induced DNA damage, evaluated with PD immunohistochemical expression profiles, demonstrated an inverse relationship with increasing topical genistein concentrations. Irradiation at 20 and 60 mJ/cm2 substantially induced PD formation in the absence of genistein, and a dose dependent inhibition of UVB-induced PD formation was observed relative to increasing genistein concentrations. Collectively all genistein pre-treated samples demonstrated appreciable histologic architectural preservation when compared with untreated specimens. These findings represent a critical link between our animal and cell culture studies with those of human skin and represent the first characterization of the dynamic alterations of UV-induced DNA damage and proliferating cell populations relative to pretreatment with genistein in human reconstituted skin. The implications of our findings serve as compelling validation to our conclusions that genistein may serve as a potent chemopreventive agent against photocarcinogenesis.
    • Phytoestrogen exposure, polymorphisms in COMT, CYP19, ESR1, and SHBG genes, and their associations with prostate cancer risk.

      Low, Yen-Ling; Taylor, James I.; Grace, Philip B.; Mulligan, Angela A.; Welch, Ailsa A.; Scollen, Serena; Dunning, Alison M.; Luben, Robert N.; Khaw, Kay-Tee; Day, Nick E.; et al. (2006)
      Prospective phytoestrogen exposure was assessed using both biomarkers and estimates of intake in 89 British men recruited into the Norfolk arm of the European Prospective Investigation into Cancer and Nutrition study, men who subsequently developed prostate cancer. Results were compared with those from 178 healthy men matched by age and date of recruitment. Levels of seven phytoestrogens (daidzein, genistein, glycitein, O-desmethylangolensin, equol, enterodiol, and enterolactone) were measured in spot urine and serum samples. Five single-nucleotide polymorphisms in COMT, CYP19, ESR1, and SHBG genes were genotyped. Urinary levels of all phytoestrogens correlated strongly with serum levels. Correlation coefficients ranged from 0.63 (glycitein) to 0.88 (daidzein) (P < 0.001). Urinary and serum levels correlated significantly with isoflavone intake assessed from food diaries (R = 0.15-0.20; P < 0.05) but not with that from a food-frequency questionnaire. Odds ratios for phytoestrogen exposure, as assessed using the four methods, were not significantly associated with prostate cancer risk (P = 0.15-0.94). Men with the CC genotype for the ESRI PvuII polymorphism had significantly higher risk for prostate cancer compared with men with the TT genotype [adjusted odds ratio = 4.65 (1.60-13.49); P = 0.005]. Our results utilizing a combined prospective exposure provide no evidence that phytoestrogens alter prostate cancer risk in British men, whereas the C allele for the PvuII polymorphism may be associated with increased risk.
    • Point mutations in the juxtamembrane domain of FLT3 define a new class of activating mutations in AML.

      Reindl, Carola; Bagrintseva, Ksenia; Vempati, Sridhar; Schnittger, Susanne; Ellwart, Joachim W.; Wenig, Katja; Hopfner, Karl-Peter; Hiddemann, Wolfgang; Spiekermann, Karsten (2006-05-01)
      In acute myeloid leukemia (AML), two clusters of activating mutations are known in the FMS-like tyrosine kinase-3 (FLT3) gene: FLT3-internal tandem duplications (FLT3-ITDs) in the juxtamembrane (JM) domain in 20% to 25% of patients, and FLT3 point mutations in the tyrosine-kinase domain (FLT3-TKD) in 7% to 10% of patients, respectively. Here, we have characterized a new class of activating point mutations (PMs) that cluster in a 16-amino acid stretch of the juxtamembrane domain of FLT3 (FLT3-JM-PMs). Expression of 4 FLT3-JM-PMs in interleukin-3 (IL-3)-dependent Ba/F3 cells led to factor-independent growth, hyperresponsiveness to FLT3 ligand, and resistance to apoptotic cell death. FLT3-JM-PM receptors were autophosphorylated and showed a higher constitutive dimerization rate compared with the FLT3-wild-type (WT) receptor. As a molecular mechanism, we could show activation of STAT5 and up-regulation of Bcl-x(L) by all FLT3-JM-PMs. The FLT3 inhibitor PKC412 abrogated the factor-independent growth of FLT3-JM-PM-expressing cells. Compared with FLT3-ITD and FLT3-TKD mutants, the FLT3-JM-PMs showed a weaker transforming potential related to lower autophosphorylation of the receptor and its downstream target STAT5. Mapping of the FLT3-JM-PMs on the crystal structure of FLT3 showed that these mutations reduce the stability of the autoinhibitory JM domain, and provides a structural basis for the transforming capacity of this new class of gain-of-function mutations of FLT3.
    • A polymorphism of the methionine synthase reductase gene increases chromosomal damage in peripheral lymphocytes in smokers.

      Ishikawa, Hitoshi; Ishikawa, Takashi; Miyatsu, Yu; Kurihara, Kazuo; Fukao, Akira; Yokoyama, Kazuhito (2006-07-25)
      The cytogenetic effects of cigarette smoke has been evaluated as one of many potential confounders in a large number of biomonitoring studies of occupationally or environmentally exposed populations and control subjects. Despite the well-known presence of carcinogens in the cigarette smoke, the results in the scientific literature linking smoking habits to micronuclei (MN) frequency, one of the cytogenetic markers, are rather controversial. Here, we investigated the relationships among MN frequency, smoking habits and five folate metabolic enzyme gene polymorphisms (MTHFR C677T and A1298C, MTR A2756G, MTRR A66G and TYMS 3'UTR) in 132 healthy Japanese men who were non-habitual drinkers. In never- and former-smokers, no statistically significant differences in the mean MN frequencies were observed according to the five folate metabolic enzyme gene polymorphisms. In current-smokers, however, subjects with the AA genotype for MTRR had a significantly higher mean MN frequency than the AG genotypes for MTRR (p<0.05). Furthermore, among subjects with the AA genotype for MTRR, current-smokers were found to have a significantly higher mean MN frequency than never- and former-smokers (p<0.05). To further characterize this association, we stratified the smoking status into five groups: non-smokers (never-smokers and former-smokers), 1-10 cigarettes/day, 11-20 cigarettes/day, 21-30 cigarettes/day and >or=31 cigarettes/day. There was an overall trend for the mean MN frequency in subjects with the MTRR AA genotype to increase as the number of cigarettes smoked per day increased (p<0.01, Jonckheere-Terpstra test). The results of our preliminary study suggest that the MTRR AA genotype acts to increase the MN frequency resulting from cigarette smoking. Therefore, studies on human genotoxicity based on cytogenetic markers of MN should take into account both the MTRR polymorphism and the potential confounding effect of smoking, although these preliminary findings need to be validated in larger populations because of the relatively small sample size.
    • Polymorphisms and haplotypes of the NBS1 gene are associated with risk of sporadic breast cancer in non-Hispanic white women <or=55 years.

      Lu, Jiachun; Wei, Qingyi; Bondy, Melissa L.; Li, Donghui; Brewster, Abenaa; Shete, Sanjay; Yu, Tse-Kuan; Sahin, Aysegul; Meric-Bernstam, Funda; Hunt, Kelly K.; et al. (2006-11)
      DNA double-strand breaks (DSBs) may lead to genomic instability and cancer if unrepaired. Nijmegen breakage syndrome 1 (NBS1) protein is one of the key proteins that participates in recognition and repair of DSBs in humans. We hypothesized that polymorphisms of NBS1 are associated with breast cancer risk. We selected three NBS1 haplotype-tagging polymorphisms (i.e. 924T>C, 8360G>C and 30537G>C) to represent all common (>or=5%) haplotypes reported in the National Institute of Environmental Health Sciences database and to reconstruct haplotypes. In a hospital-based case-control study of 421 non-Hispanic white patients with sporadic breast cancer (C polymorphism. Furthermore, the derived haplotypes were associated with risk in a dose-response manner as the number of variant (risk) alleles (i.e. 8360C, 924C or 30537C) increased (adjusted OR = 1.07, 95% CI = 0.78-1.46 for 1-2 variant alleles; adjusted OR = 2.47, 95% CI = 1.48-4.14 for 3-6 variant alleles; P(trend) = 0.006). These findings suggest that NBS1 polymorphisms and haplotypes may contribute to the etiology of sporadic breast cancer in young non-Hispanic white women. Large studies are warranted to confirm these findings.
    • Polymorphisms in genes involved in DNA double-strand break repair pathway and susceptibility to benzene-induced hematotoxicity.

      Shen, Min; Lan, Qing; Zhang, Luoping; Chanock, Stephen; Li, Guilan; Vermeulen, Roel; Rappaport, Stephen M.; Guo, Weihong; Hayes, Richard B.; Linet, Martha; et al. (2006-10)
      Benzene is a recognized hematotoxicant and carcinogen that produces genotoxic damage. DNA double-strand breaks (DSB) are one of the most severe DNA lesions caused directly and indirectly by benzene metabolites. DSB may lead to chromosome aberrations, apoptosis and hematopoietic progenitor cell suppression. We hypothesized that genetic polymorphisms in genes involved in DNA DSB repair may modify benzene-induced hematotoxicity. We analyzed one or more single nucleotide polymorphisms (SNPs) in each of seven candidate genes (WRN, TP53, NBS1, BRCA1, BRCA2, XRCC3 and XRCC4) in a study of 250 workers exposed to benzene and 140 controls in China. Four SNPs in WRN (Ex4 -16 G > A, Ex6 +9 C > T, Ex20 -88 G > T and Ex26 -12 T > G), one SNP in TP53 (Ex4 +119 C > G) and one SNP in BRCA2 (Ex11 +1487 A > G) were associated with a statistically significant decrease in total white blood cell (WBC) counts among exposed workers. The SNPs in WRN and TP53 remained significant after accounting for multiple comparisons. One or more SNPs in WRN had broad effects on WBC subtypes, with significantly decreased granulocyte, total lymphocyte, CD4(+)-T cell, CD8(+)-T cell and monocyte counts. Haplotypes of WRN were associated with decreased WBC counts among benzene-exposed subjects. Likewise, subjects with TP53 Ex4 +119 C > G variant had reduced granulocyte, CD4(+)-T cell and B cell counts. The effect of BRCA2 Ex11 +1487 A > G polymorphism was limited to granulocytes. These results suggest that genetic polymorphisms in WRN, TP53 and BRCA2 that maintain genomic stability impact benzene-induced hematotoxicity.
    • A pooled analysis of 12 cohort studies of dietary fat, cholesterol and egg intake and ovarian cancer.

      Genkinger, Jeanine M.; Hunter, David J.; Spiegelman, Donna; Anderson, Kristin E.; Beeson, W. Lawrence; Buring, Julie E.; Colditz, Graham A.; Fraser, Gary E.; Freudenheim, Jo L.; Goldbohm, R. Alexandra; et al. (2006-04)
      Fat and cholesterol are theorized to promote ovarian carcinogenesis by increasing circulating estrogen levels. Although case-control studies have reported positive associations between total and saturated fat intake and ovarian cancer risk, two cohort studies have observed null associations. Dietary cholesterol and eggs have been positively associated with ovarian cancer risk. A pooled analysis was conducted on 12 cohort studies. Among 523,217 women, 2,132 incident epithelial ovarian cancer cases were identified. Study-specific relative risks (RR) and 95% confidence intervals (CI) were calculated by Cox proportional hazards models, and then pooled using a random effects model. Total fat intake was not associated with ovarian cancer risk (pooled multivariate RR = 1.08, 95% CI 0.86-1.34 comparing > or =45 to 30-<35% of calories). No association was observed for monounsaturated, polyunsaturated, trans-unsaturated, animal and vegetable fat, cholesterol and egg intakes with ovarian cancer risk. A weakly positive, but non-linear association, was observed for saturated fat intake (pooled multivariate RR = 1.29, 95% CI: 1.01-1.66 comparing highest versus lowest decile). Results for histologic subtypes were similar. Overall, fat, cholesterol and egg intakes were not associated with ovarian cancer risk. The positive association for saturated fat intake at very high intakes merits further investigation.
    • Preventive effects of chrysin on the development of azoxymethane-induced colonic aberrant crypt foci in rats.

      Miyamoto, Shingo; Kohno, Hiroyuki; Suzuki, Rikako; Sugie, Shigeyuki; Murakami, Akira; Ohigashi, Hajime; Tanaka, Takuji (2006-05)
      The modifying effects of dietary feeding with chrysin (5,7-dihydroxyflavone) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. We also assessed the effect of chrysin on mitosis and apoptosis in 'normal appearing' crypts. To induce ACF, rats were given two weekly subcutaneous injections of AOM (20 mg/kg body weight). They also received an experimental diet containing chrysin (0.001 or 0.01%) for 4 weeks, starting 1 week before the first dose of AOM. AOM exposure produced a substantial number of ACF (73+/-13/rat) at the end of the study (week 4). Dietary administration of chrysin caused significant reduction in the frequency of ACF: 0.001% chrysin, 37+/-17/rat (49% reduction, P<0.001); and 0.01% chrysin, 40+/-10/rat (45% reduction, P<0.001). In addition, chrysin administration significantly reduced the mitotic index and significantly increased the apoptotic index in 'normal appearing' crypts. These findings might suggest a possible chemopreventive activity of chrysin in the early step of colon tumorigenesis through modulation of cryptal cell proliferation activity and apoptosis.
    • Prospective study of mesothelioma mortality in Turkish villages with exposure to fibrous zeolite.

      Baris, Y. Izzettin; Grandjean, Philippe (2006-03-15)
      Mesothelioma incidence is high in certain villages on the Anatolian plateau in Turkey, where environmental exposure includes erionite, a form of zeolite fibers, from the local volcanic tuff. Previous studies of this exposure were cross-sectional or with a follow-up period of only a few years. A prospective study of residents of two exposed and one nearby control village was initiated in 1979 and continued through December 31, 2003. A total of 891 men and women, aged 20 years or older, were included, 230 of them residing in the village without known exposure to erionite. Mortality data were obtained from hospital records and death certificates. During the 23-year follow-up, 372 deaths occurred; 119 of these were from mesothelioma, which was the cause of 44.5% of all deaths in the exposed villages. Seventeen patients had peritoneal mesothelioma; the rest had pleural mesothelioma. Only two cases of mesothelioma, one of each type, occurred in the control village-both in women born elsewhere. When standardized to the world population, the pleural mesothelioma incidence was approximately 700 and 200 cases per 100,000 people annually in the two exposed villages and about 10 cases per 100,000 people in the control village. When we used Danish data for comparison, the standardized pleural mesothelioma mortality rate was 485 (95% confidence interval = 395 to 590). Our results emphasize the severity of the mesothelioma endemic in erionite-exposed areas of Turkey and call for intensified prevention of mesothelioma by limiting environmental exposures to these fibers.
    • Prostate cancer risk from occupational exposure to polycyclic aromatic hydrocarbons interacting with the GSTP1 Ile105Val polymorphism.

      Rybicki, Benjamin A.; Neslund-Dudas, Christine; Nock, Nora L.; Schultz, Lonni R.; Eklund, Ludmila; Rosbolt, James; Bock, Cathryn H.; Monaghan, Kristin G. (2006)
      BACKGROUND: Variation in the glutathione S-transferase (GSTP1) gene and occupational polycyclic aromatic hydrocarbons (PAH) exposure are putative prostate cancer risk factors. An Ile/Val polymorphism in codon 105 of GSTP1 affects its enzymatic activity toward PAH detoxification, a possible mechanism in prostate carcinogenesis. METHODS: To determine whether the GSTP1 Ile105Val polymorphism modifies prostate cancer risk associated with occupational PAH exposure, we studied 637 prostate cancer cases and 244 controls of White and African-American race from the Henry Ford Health System in Detroit, Michigan. Occupational exposure to PAH from wood, petroleum, coal or other sources through respiratory and cutaneous routes was retrospectively assessed by expert review of job histories. The association of occupational PAH exposure and GSTP1 Ile105Val polymorphism with prostate cancer was tested in multiple logistic regression models adjusting for potential confounders. Cases were over sampled compared with controls to evaluate gene-environment interaction with the statistically efficient case-only analytic approach. RESULTS: Neither carriage of the GSTP1 Val(105) variant allele nor occupational PAH exposure was significantly associated with prostate cancer. However, case-only analyses revealed that carriage of the GSTP1 Val(105) variant allele was associated with increasing levels of occupational respiratory PAH exposures from any source and from petroleum (trend test p=0.01 for both). The GSTP1 Val(105) allele was observed most frequently in cases in the highest quartile of occupational respiratory PAH exposures from petroleum (OR=1.74; 95% CI=1.11-2.72) or from any source (OR=1.85; 95% CI=1.19-2.89). The gene-environment risk estimate in the highest PAH petroleum exposure quartile was greatest in men under age 60 (OR=4.52; 95% CI=1.96-10.41) or with a positive family history of prostate cancer (OR=3.02; 95% CI=1.15-7.92). CONCLUSIONS: Our results suggest men who carry the GSTP1 Val(105) variant and are exposed at high levels to occupational PAH have increased risk for prostate cancer. This increased risk is more pronounced in men under age 60 or with a family history of prostate cancer.
    • Protection against esophageal cancer in rodents with lyophilized berries: potential mechanisms.

      Stoner, Gary D.; Chen, Tong; Kresty, Laura A.; Aziz, Robeena M.; Reinemann, Tiffany; Nines, Ronald (2006)
      For several years, our laboratory has been evaluating the ability of lyophilized (freeze-dried) black raspberries (Rubus occidentalis, BRBs), blackberries (R. fructicosus, BBs), and strawberries (Fragaria ananasia, STRWs) to inhibit carcinogen-induced cancer in the rodent esophagus. To assure "standardized" berry preparations for study, each berry type is of the same cultivar, picked at about the same degree of ripeness, washed and frozen within 2-4 h of the time of picking, and freeze-dried under conditions that preserve the components in the berries. Some of the known chemopreventive agents in berries include vitamins A, C, and E and folic acid; calcium and selenium; beta-carotene, alpha-carotene, and lutein; polyphenols such as ellagic acid, ferulic acid, p-coumaric acid, quercetin, and several anthocyanins; and phytosterols such as beta-sitosterol, stigmasterol, and kaempferol. In initial bioassays, freeze-dried STRW, BRB, and BB powders were mixed into AIN-76A synthetic diet at concentrations of 5% and 10% and fed to Fischer 344 rats before, during, and after treatment with the esophageal carcinogen N-nitrosomethylbenzylamine (NMBA). At 25 wk of the bioassay, all three berry types were found to inhibit the number of esophageal tumors (papillomas) in NMBA-treated animals by 24-56% relative to NMBA controls. This inhibition correlated with reductions in the formation of the NMBA-induced O6-methylguanine adduct in esophageal DNA, suggesting that the berries influenced the metabolism of NMBA leading to reduced DNA damage. Studies are ongoing to determine the mechanisms by which berries influence NMBA metabolism and DNA adduct formation. BRBs and STRWs were also tested in a postinitiation scheme and were found to inhibit NMBA-induced esophageal tumorigenesis by 31-64% when administered in the diet following treatment of the animals with NMBA. Berries, therefore, inhibit tumor promotion and progression events as well as tumor initiation. In vivo mechanistic studies with BRBs indicate that they reduce the growth rate of premalignant esophageal cells, in part, through down-regulation of cyclooxygenase-2 leading to reduced prostaglandin production and of inducible nitric oxide synthase leading to reduced nitrate/nitrite levels in the esophagus. Based upon the preclinical data on rodents, we have initiated prevention trials in humans to determine if berries might exhibit chemopreventive effects in the esophagus.
    • Quinone reductase induction as a biomarker for cancer chemoprevention.

      Cuendet, Muriel; Oteham, Carol P.; Moon, Richard C.; Pezzuto, John M. (2006-03)
      Chemoprevention involves the use of natural or synthetic substances to reduce the risk of developing cancer. Strategies for protecting cells from initiation events include decreasing metabolic enzymes responsible for generating reactive species (phase I enzymes) while increasing phase II enzymes that can deactivate radicals and electrophiles known to intercede in normal cellular processes. Reduction of electrophilic quinones by quinone reductase is an important detoxification pathway. Following evaluation of approximately 3000 plant and marine organism extracts, the number characterized as "active" was established in the range of 12% of the total, and over 60 active compounds have been isolated as quinone reductase inducers. One of them, isoliquiritigenin (1), isolated from tonka bean, was shown to be a monofunctional inducer by having similar quinone reductase inducing ability in wild-type Hepa 1c1c7 cells and two mutant cell lines. To further investigate the mechanism of induction, HepG2 human hepatoma cells stably transfected with ARE-luciferase plasmid were used. Isoliquiritigenin (1) significantly induced the luciferase activity in a dose-dependent manner. On the basis of these results, a full-term cancer chemoprevention study was conducted with 7,12-dimethylbenz[a]anthracene (DMBA)-treated female Sprague-Dawley rats. Dietary administration of 1 increased tumor latency. Based on these promising preliminary results, additional mechanistic studies are underway, as well as full-term carcinogenesis studies with chronic administration schedules.
    • Rapid analysis of XRCC1 polymorphisms using real-time polymerase chain reaction.

      Schneider, Joachim; Classen, Vera; Philipp, Monika; Helmig, Simone (2009-03-13)
      DNA repair plays a critical role in protecting the genome from carcinogens or ionizing radiation. Three coding polymorphisms at codons 194, 280, and 399 in X-ray cross-complementing group 1 (XRCC1) DNA repair gene have been identified that may affect DNA repair and alter cancer susceptibility. In order to study their role in molecular-epidemiology studies we developed a single-step procedure for genotyping these polymorphisms using real-time polymerase chain reaction (rt-PCR) and subsequent melting curve analysis. Genotypes of 622 unrelated Caucasians without prior history of cancer were determined by real-time PCR and compared to genotypes obtained by restriction fragment length polymorphism PCR. In the population studied, the allele frequency of the XRCC1 26304 site (C-->T) of codon 194 in exon 6 was 0.065, the allele frequency of the XRCC1 27466 site (G-->A) of codon 280 in exon 9 was 0.048 and of the XRCC1 28152 site (G-->A) of codon 399 in exon 10 was 0.35. There was no disagreement between the two methods. These findings confirm the real-time fluorescence PCR method as a rapid and reliable assay for the analysis of large numbers of samples.
    • The reaction of flavanols with nitrous acid protects against N-nitrosamine formation and leads to the formation of nitroso derivatives which inhibit cancer cell growth.

      Lee, Stephanie Y. H.; Munerol, Bibiana; Pollard, Susan; Youdim, Kuresh A.; Pannala, Ananth S.; Kuhnle, Gunter G. C.; Debnam, Edward S.; Rice-Evans, Catherine; Spencer, Jeremy P. E. (2006-01-15)
      Studies have suggested that diets rich in polyphenols such as flavonoids may lead to a reduced risk of gastrointestinal cancers. We demonstrate the ability of monomeric and dimeric flavanols to scavenge reactive nitrogen species derived from nitrous acid. Both epicatechin and dimer B2 (epicatechin dimer) inhibited nitrous acid-induced formation of 3-nitrotyrosine and the formation of the carcinogenic N-nitrosamine, N-nitrosodimethylamine. The reaction of monomeric and dimeric epicatechin with nitrous acid led to the formation of mono- and di-nitroso flavanols, whereas the reaction with hesperetin resulted primarily in the formation of nitrated products. Although, epicatechin was transferred across the jejunum of the small intestine yielding metabolites, its nitroso form was not absorbed. Dimer B2 but not epicatechin monomer inhibited the proliferation of, and triggered apoptosis in, Caco-2 cells. The latter was accompanied by caspase-3 activation and reductions in Akt phosphorylation, suggesting activation of apoptosis via inhibition of prosurvival signaling. Furthermore, the dinitroso derivative of dimer B2, and to a lesser extent the dinitroso-epicatechin, also induced significant toxic effects in Caco-2 cells. The inhibitory effects on cellular proliferation were paralleled by early inhibition of ERK 1/2 phosphorylation and later reductions in cyclin D1 levels, indicating modulation of cell cycle regulation in Caco-2 cells. These effects highlight multiple routes in which dietary derived flavanols may exert beneficial effects in the gastrointestinal tract.
    • Red palm oil suppresses the formation of azoxymethane (AOM) induced aberrant crypt foci (ACF) in Fisher 344 male rats.

      Boateng, J.; Verghese, M.; Chawan, C.B.; Shackelford, L.; Walker, L.T.; Khatiwada, J.; Williams, D.S. (2006-10)
      Red palm oil (RPO) contains significant levels of carotenoids and Vitamin E. In this experiment we compared the inhibitory effects of RPO (7% and 14% levels) and soybean oil (7% and 14%) on azoxymethane (AOM) induced aberrant crypt foci (ACF). Thirty-two male Fisher 344 rats were randomly assigned to four groups. Two groups received AIN-93 G control (C) diet containing 7% and 14% soybean oil (SBO), respectively. Groups 3 and 4 received a treatment diet consisting of 7% and 14% RPO, respectively. The rats received subcutaneous injections of AOM at 16 mg/kg body weight at 7 and 8 weeks of age. At 17 weeks of age rats were killed by CO(2) asphyxiation. Numbers of ACF (mean+/-SE) in the proximal and distal colon were: 39.9 +/- 0.9, 53.8 +/- 2.8, 26.0 +/- 3.0, 27.5 +/- 1.5 and 118.2 +/- 1.7, 125.6 +/- 3.2, 41 +/- 7, 52.3 +/- 1.8 in rats fed 7% SBO, 14% SBO, 7% RPO and 14% RPO, respectively. The results of this study showed that RPO reduced the incidence of AOM induced ACF and may therefore have a beneficial effect in reducing the incidence of colon cancer.
    • Relationship between genotoxicity biomarkers in somatic and germ cells: findings from a biomonitoring study.

      Migliore, L.; Colognato, R.; Naccarati, A.; Bergamaschi, E. (2006-03)
      A biomonitoring study to evaluate chromosome and DNA damage respectively in somatic and germ cells of a group of male workers exposed to styrene by using biomarkers of genotoxicity was carried out. Styrene-exposed workers from three different areas of Tuscany and healthy subjects, of comparable mean age, sex and lifestyle characteristics, as a control group not exposed to chemicals, have been enrolled. In addition to previous reports [L. Migliore, A. Naccarati, A. Zanello, R. Scarpato, L. Bramanti and M. Mariani (2002) Hum. Reprod., 17, 2912-2918; L. Migliore, A. Naccarati, F. Coppedè et al. (2006) Pharmacogenet. Genomics, 16, 87-99] we present now data on a cross-sectional investigation involving a homogeneous group of subjects for which data on both somatic and germ cells have been obtained from individuals (42 exposed and 25 controls). Somatic cell genotoxicity was assessed by analysing the frequency of micronucleated binucleated cells (MNBN) in blood lymphocytes. The micronucleus assay was coupled with centromeric fluorescence in situ hybridization (FISH) analysis. Primary DNA damage in germ cells was evaluated by alkaline single-cell gel electrophoresis (Comet assay) and the percentage of the tail DNA (%TD) was used as parameter of Comet evaluation. Moreover, to investigate the frequencies of aneuploidy and diploidy in sperm, we performed multicolour FISH, using DNA probes specific for the centromeric regions of sex chromosomes and chromosome 2, in decondensed sperm nuclei of samples with normal semen parameters in a subgroup of individuals. Mandelic and phenylglyoxylic acids (MAPGA) in end of shift samples were determined as biomarkers of internal dose. MAPGA excretion was consistent with an exposure to styrene above the threshold limit value-time weighted average concentration of 20 p.p.m. Styrene workers showed significantly higher frequency of MNBN as compared to controls (13.8 +/- 5.2 versus 6.2 +/- 5.1; P < 0.001), due to higher proportions of both micronuclei (MN) arising from chromosomal breakage (C-MN) and harbouring whole chromosomes (C+MN). DNA damage in sperm cells was also higher among styrene-exposed, the %TD being 11.02 +/- 2.99 versus 7.42 +/- 2.30 in controls (P < 0.001). The incidence of aneuploidy and diploidy for the tested chromosomes in sperm did not show a statistically significant difference between workers and controls. However, a positive correlation was found between genotoxic damage detected in somatic and in germ cells, even after removing the effect of age (r = 0.475; P < 0.001). Although cytogenetic biomarkers detected both in somatic and germ cells were interrelated, no relationships were apparent with exposure parameters. Styrene exposure may increase the likelihood of both chromosome and DNA damage in somatic and germ cells, thus supporting the hypothesis of an interference on reproductive capacity among exposed workers. This is the first time that a field study shows a correlation between two biomarkers of genotoxicity evaluated at the same time in somatic and germ cells.