Understanding resolution limit of displacement Talbot lithography.

Displacement Talbot lithography (DTL) is a new technique for patterning large areas with sub-micron periodic features with low cost. It has applications in fields that cannot justify the cost of deep-UV photolithography, such as plasmonics, photonic crystals, and metamaterials and competes with techniques, such as nanoimprint and laser interference lithography. It is based on the interference of coherent light through a periodically patterned photomask. However, the factors affecting the technique's resolution limit are unknown. Through computer simulations, we show the mask parameter's impact on the features' size that can be achieved and describe the separate figures of merit that should be optimized for successful patterning. Both amplitude and phase masks are considered for hexagonal and square arrays of mask openings. For large pitches, amplitude masks are shown to give the best resolution; whereas, for small pitches, phase masks are superior because the required exposure time is shorter. We also show how small changes in the mask pitch can dramatically affect the resolution achievable. As a result, this study provides important information for choosing new masks for DTL for targeted applications.

Angle dependent optical properties of polymer films with a biomimetic anti-reflecting surface replicated from cylindrical and tapered nanoporous alumina.

Anodic porous alumina nanostructures have been fabricated with tapered and cylindrical pores with a spacing of 100 and 200 nm and depth of 180-500 nm. The porous nanostructures were replicated into polymer films to create a moth-eye anti-reflecting surface by a roll-to-roll UV replication process. The angle dependent optical transmission of the resulting polymer films exhibited up to a 2% increase in transmission at a normal angle and up to a 5% increase in transmission at a 70° angle of incidence to an equivalent film with a surface replicated from polished aluminum. No significant difference was observed between the optical performance of moth-eye surfaces formed from cylindrical and tapered nano-pores.

Selenium levels in cows fed pasture and concentrates or a total mixed ration and supplemented with selenized yeast to produce milk with supra-nutritional selenium concentrations.

Seventy multiparous Holstein-Friesian cows were fed different amounts of pasture and concentrates, or a total mixed ration (TMR), for 42 d in mid-lactation to test the hypothesis that the concentration of Se in milk would depend on the amount of Se consumed, when the Se is primarily organic in nature, regardless of the diet of the cows. Of the 70 cows, 60 grazed irrigated perennial pasture at daily allowances of either 20 or 40 kg of dry matter (DM)/cow. These cows received 1 of 3 amounts of concentrates, either 1, 3, or 6 kg of DM/cow per day of pellets, and at each level of concentrate feeding, the pellets were formulated to provide 1 of 2 quantities of Se from Se yeast, either about 16 or 32 mg of Se/d. The other 10 cows were included in 2 additional treatments where a TMR diet was supplemented with 1 kg of DM/d of pellets formulated to include 1 of the 2 quantities of supplemental Se. Total Se intakes ranged from 14.5 to 35.9 mg/d, and of this, the Se-enriched pellets provided 93, 91, and 72% of the Se for cows allocated 20 and 40 kg of pasture DM/d or the TMR, respectively. No effects of the amount of Se consumed on any milk production variable, or on somatic cell count, body weight, and body condition score, for either the pasture-fed or TMR-fed cows were found. Milk Se concentrations responded quickly to the commencement of Se supplementation, reaching 89% of steady state levels at d 5. When milk Se concentrations were at steady state (d 12 to 40), each 1mg of Se eaten increased the Se concentration of milk by 5.0 µg/kg (R(2)=0.97), and this response did not seem to be affected by the diet of the cows or their milk production. The concentration of Se in whole blood was more variable than that in milk, and took much longer to respond to change in Se status, but it was not affected by diet at any time either. For the on-farm production of Se-enriched milk, it is important to be able to predict milk Se concentration from Se input. In our study, type of diet did not affect this relationship.

Familial and racial determinants of tumour suppressor genes promoter hypermethylation in breast tissues from healthy women.

To determine the hypermethylation status of the promoter regions of tumour suppressor genes in breast tissues from healthy women and identify the determinants of these epigenetic changes. Questionnaires and breast tissues were collected from healthy women without a history of cancer and undergoing reduction mammoplasty (N= 141). Methylation for p16(INK4), BRCA1, ERalpha and RAR-beta promoter regions from breast tissues were determined by methylation specific PCR. Associations were examined with chi-square and Fisher's exact test as well as logistic regression. All statistical tests were two-sided. p16(INK4), BRCA1, ERalpha and RAR-beta hypermethylation were identified in 31%, 17%, 9% and 0% of the women, respectively. Women with BRCA1 hypermethylation had an eight-fold increase in the risk of ERalpha hypermethylation (P= 0.007). p16(INK4) hypermethylation was present in 28% of African-Americans, but 65% in European-Americans (P= 0.02). There was an increased likelihood of p16(INK4) or BRCA1 hypermethylation for women with family history of cancer (OR 2.3; 95%CI: 1.05-4.85 and OR 5.0; 95%CI: 1.55-15.81, respectively). ERalpha hypermethylation was associated with family history of breast cancer (OR 6.6; 95%CI: 1.58-27.71). After stratification by race, p16(INK4) in European-Americans and BRCA1 hypermethylation in African-Americans were associated with family history of cancer (OR 3.8; 95%CI: 1.21-12.03 and OR 6.5; 95%CI: 1.33-31.32, respectively). Gene promoter hypermethylation was commonly found in healthy breast tissues from women without cancer, indicating that these events are frequent and early lesions. Race and family history of cancer increase the likelihood of these early events.

Newly diagnosed patients with advanced non-small cell lung cancer: A clinical description of those with moderate to severe depressive symptoms.

OBJECTIVES: The aims of this observational study were to 1) accrue newly diagnosed patients with advanced-stage non-small cell lung cancer (NSCLC) awaiting the start of first-line treatment and identify those with moderate to severe depressive symptoms and, 2) provide a clinical description of the multiple, co-occurring psychological and behavioral difficulties and physical symptoms that potentially exacerbate and maintain depressive symptoms. MATERIALS AND METHODS: Patients with stage IV NSCLC (N = 186) were enrolled in an observational study (ClinicalTrials.gov Identifier: NCT03199651) and completed the American Society of Clinical Oncology-recommended screening measure for depression (Patient Health Questionnaire-9 [PHQ-9]). Individuals with none/mild (n = 119; 64 %), moderate (n = 52; 28 %), and severe (n = 15; 8 %) depressive symptoms were identified. Patients also completed measures of hopelessness, generalized anxiety disorder (GAD) symptoms, stress, illness perceptions, functional status, and symptoms. RESULTS: Patients with severe depressive symptoms reported concomitant feelings of hopelessness (elevating risk for suicidal behavior), anxiety symptoms suggestive of GAD, and traumatic, cancer-specific stress. They perceived lung cancer as consequential for their lives and not controllable with treatment. Pain and multiple severe symptoms were present along with substantial functional impairment. Patients with moderate depressive symptoms had generally lower levels of disturbance, though still substantial. The most salient differences were low GAD symptom severity and fewer functional impairments for those with moderate symptoms. CONCLUSIONS: Depressive symptoms of moderate to severe levels co-occur in a matrix of clinical levels of anxiety symptoms, traumatic stress, impaired functional status, and pain and other physical symptoms. All of the latter factors have been shown, individually and collectively, to contribute to the maintenance or exacerbation of depressive symptoms. As life-extending targeted and immunotherapy use expands, prompt identification of patients with moderate to severe depressive symptoms, referral for evaluation, and psychological/behavioral treatment are key to maximizing treatment outcomes and quality of life for individuals with advanced NSCLC.

Increasing selenium concentration in milk: effects of amount of selenium from yeast and cereal grain supplements.

Two experiments were conducted to establish responses in milk Se concentrations in grazing dairy cows to different amounts of dietary Se yeast, and to determine the effects of the Se concentration of the basal diet. The hypothesis tested was that the response in milk, blood, and tissue Se concentrations to supplemental Se would not be affected by whether the Se was from the basal diet or from Se yeast. In addition, by conducting a similar experiment in either early (spring; experiment 1) or late (autumn; experiment 2) lactation, we hypothesized that different Se input-output relationships would result. Both 6-wk experiments involved 60 multiparous Holstein-Friesian cows, all of which had calved in spring. They were allocated to 1 of 10 dietary Se treatments that included 2 types of crushed triticale grain (low Se, approximately 165 microg of Se/kg of DM; or high Se, approximately 580 microg/kg of DM) fed at 4 kg of DM/d, and 1 kg of DM/d of pellets formulated to carry 5 quantities of Se yeast (0, 4, 8, 12, or 16 mg of Se). Daily total Se intakes ranged from <2 to >18 mg/cow in both experiments. Milk Se concentrations plateaued after 15 and 7 d of supplementation in experiments 1 and 2, respectively, and then remained at plateau concentrations. Average milk Se concentrations for the plateau period increased as the amount of Se yeast increased, and low- and high-Se grain treatments were different at all quantities of Se yeast, although there was a tendency for this difference to diminish at the greatest concentrations of yeast. There were significant positive, linear relationships between Se intake and the concentrations of Se in milk, which were not affected by the source of Se, and the relationships were similar for both experiments. Therefore, the output of Se in milk in experiment 1 was greater than that in experiment 2 because the milk yield of the cows in early lactation was greater. The estimated proportions of Se partitioned to destinations other than milk and feces increased with the amount of Se in the diet and were greater in experiment 2 than in experiment 1, a result that was supported by Se concentrations in whole blood and plasma and in semitendinosus muscle tissue. If high-Se products are to be produced for human nutrition, it is important to be able to develop feeding systems that produce milk with consistent and predictable Se concentrations so that products can consistently meet specifications. The results indicate that this objective is achievable.

Human lung carcinogen-DNA adduct levels mediated by genetic polymorphisms in vivo.

BACKGROUND: Cancer risk from exposure to tobacco smoke varies widely from person to person, depending in part on the status of particular genes and acquired susceptibilities. Certain genes determine how cells activate and detoxify carcinogens. Activated carcinogen metabolites may bind to DNA and form DNA adducts (e.g., 7-methyl-2'-deoxyguanosine-3'-monophosphate [7-methyl-dGMP] and polycyclic aromatic hydrocarbons-dGMP [PAHs-dGMP]), many of which can induce genetic mutations. Thus, if individuals have an increased capacity to activate carcinogens, they might form more carcinogen-DNA adducts and subsequently have an increased risk of cancer. PURPOSE: Using DNA-adduct detection methods specific for 7-methyl-dGMP and PAH-dGMP, we sought to determine whether an inherited genetic susceptibility to cancer associated with certain carcinogen-metabolizing and detoxifying genes (e.g., cytochrome P450 and glutathione S-transferase) is related to DNA adduct formation in lung tissue. METHODS: Human lung tissues were collected randomly from 90 autopsy donors who were free of cancer. Levels of 7-methyl-dGMP, a metabolic product of N-nitrosamines, and PAH-dGMP adducts were determined in lung tissue specimens by use of micropreparative DNA purification steps combined with a 32P-postlabeling assay. Genetic polymorphisms (the presence of different genes and/or alleles) were determined for the cytochrome P450 genes, CYP2D6, CYP2E1, and CYP1A1, as well as for glutathione S-transferase M1 (GSTM1). Statistical differences among adduct levels for the study variables, including genotypes, were assessed by the two-sided Student's t test or the Mann-Whitney U test. RESULTS: Higher 7-methyl-dGMP adduct levels were associated with CYP2D6 genotypes (P = .01), consistent with the reports of the increased risk of lung cancer associated with this genotype. Higher adduct levels were also associated with CYP2E1 minor alleles (P = .05). In both cases, the association was attributed mostly to individuals with low serum cotinine levels (P = .004 and P = .05, respectively), suggesting that the effect of the genotypes is mostly in nonsmokers exposed to either passive tobacco smoke or to N-nitrosamine exposures other than tobacco smoke or to N-nitrosamine exposures other than tobacco smoke. Separately, the presence of PAH-dGMP adducts was associated with the GSTM1 null genotype (absence of the gene) (odds ratio = 8.6; 95% confidence interval = 1.03-100). CONCLUSIONS: This study finds that the levels of two different carcinogen-DNA adducts vary in lung tissue (an important target tissue) in association with three separate genetic polymorphisms (i.e., CYP2D6, CYP2E1, and GSTM1). CYP2D6 and CYP2E1 genotypes are associated with higher 7-methyl-dGMP levels, while the GSTM1 null genotype is associated with higher numbers of PAH-dGMP adducts. These findings suggest that genetic polymorphisms are predictive of carcinogen-DNA adduct levels and would thus be predictive of an individual's lifetime response to carcinogen exposure.

Mutagens from heated Chinese and U.S. cooking oils.

BACKGROUND: The lung cancer incidence in Chinese women is among the highest in the world, but tobacco smoking accounts for only a minority of the cancers. Epidemiologic investigations of lung cancer among Chinese women have implicated exposure to indoor air pollution from wok cooking, where the volatile emissions from unrefined cooking oils are mutagenic. PURPOSE: This study was conducted to identify and quantify the potentially mutagenic substances emitted from a variety of cooking oils heated to the temperatures typically used in wok cooking. METHODS: Several cooking oils and fatty acids were heated in a wok to boiling, at temperatures (for the cooking oils) that ranged from 240 degrees C to 280 degrees C (typical cooking temperatures in Shanghai, China). The oils tested were unrefined Chinese rapeseed, refined U.S. rapeseed (known as canola), Chinese soybean, and Chinese peanut in addition to linolenic, linoleic, and erucic fatty acids. Condensates of the emissions were collected and tested in the Salmonella mutation assay (using Salmonella typhimurium tester strains TA98 and TA104). Volatile decomposition products also were subjected to gas chromatography and mass spectroscopy. Aldehydes were detected using high-performance liquid chromatography and UV spectroscopy. RESULTS: 1,3-Butadiene, benzene, acrolein, formaldehyde, and other related compounds were qualitatively and quantitatively detected, with emissions tending to be highest for unrefined Chinese rapeseed oil and lowest for peanut oil. The emission of 1,3-butadiene and benzene was approximately 22-fold and 12-fold higher, respectively, from heated unrefined Chinese rapeseed oil than from heated peanut oil. Lowering the cooking temperatures or adding an antioxidant, such as butylated hydroxyanisole, before cooking decreased the amount of these volatile emissions. Among the individual fatty acids tested, heated linolenic acid produced the greatest quantities of 1,3-butadiene, benzene, and acrolein. Separately, the mutagenicity of individual volatile emission condensates was correlated with linolenic acid content (r = .83; P = .0004). Condensates from heated linolenic acid, but not linoleic or erucic acid, were highly mutagenic. CONCLUSIONS: These studies, combined with experimental and epidemiologic findings, suggest that high-temperature wok cooking with unrefined Chinese rapeseed oil may increase lung cancer risk. This study indicates methods that may reduce that risk. IMPLICATIONS: The common use of wok cooking in China might be an important but controllable risk factor in the etiology of lung cancer. In the United States, where cooking oils are usually refined for purity, additional studies should be conducted to further quantify the potential risks of such methods of cooking.

Alcohol dehydrogenase 3 genotype and risk of oral cavity and pharyngeal cancers.

BACKGROUND: The consumption of alcoholic beverages is a strong risk factor for cancers of the oral cavity and pharynx (oral cancers). Alcohol dehydrogenase type 3 (ADH3) metabolizes ethanol to acetaldehyde, a carcinogen. We evaluated whether individuals homozygous for the fast-metabolizing ADH3(1) allele (ADH3[1-1]) have a greater risk of developing oral cancer in the presence of alcoholic beverage consumption than those with the slow-metabolizing ADH3(2) allele (ADH3[1-2] and ADH3[2-2]). METHODS: As part of a population-based study of oral cancer conducted in Puerto Rico, the ADH3 genotypes of 137 patients with histologically confirmed oral cancer and of 146 control subjects (i.e., individuals with no history of oral cancer) were determined by molecular genetic analysis of oral epithelial cell samples. Risks were estimated by use of multiple logistic regression analyses. RESULTS: Compared with nondrinkers with the ADH3(1-1) genotype, consumers of at least 57 alcoholic drinks per week with the ADH3(1-1), ADH3(1-2), and ADH3(2-2) genotypes had 40.1-fold (95% confidence interval [CI] = 5.4-296.0), 7.0-fold (95% CI = 1.4-35.0), and 4.4-fold (95% CI = 0.6-33.0) increased risks of oral cancer, respectively; the risk associated with the ADH3(1-1) genotype, compared with the ADH3(1-2) and ADH3(2-2) genotypes combined, was 5.3 (95% CI = 1.0-28.8) among such drinkers. Considering all levels of alcohol consumption, the risk of oral cancer per additional alcoholic drink per week increased 3.6% (95% CI = 1.9%-5.4%) for subjects with the ADH3(1-1) genotype and 2.0% (95% CI = 0.9%-3.0%) for subjects with the ADH3(1-2) or ADH3(2-2) genotype (two-sided P = .04). CONCLUSIONS: The ADH3(1-1) genotype appears to substantially increase the risk of ethanol-related oral cancer, thus providing further evidence for the carcinogenicity of acetaldehyde.

Combined high-performance liquid chromatography/32P-postlabeling assay of N7-methyldeoxyguanosine.

A highly sensitive and specific assay for the detection of N7-methyl-2'-deoxyguanosine (N7methyldG) has been developed by combining high-performance liquid chromatography, 32P-postlabeling, and nucleotide chromatography. Separation of normal nucleotides and adducts by high-performance liquid chromatography and then combining a portion of 2'-deoxyguanosine to the N7methyldG allows for quantitation using an internal standard. The directly determined molar ratio is not subject to errors in digestion, variable ATP-specific activity, or assumptions in relative adduct-labeling efficiency. The detection limit was one N7methyldG adduct in 10(7) unmodified 2'-deoxyguanosine bases. N7methyldG adducts have been detected in 5 human lung samples in which O6-methyl-2'-deoxyguanosine adducts had been previously determined. The mean ratio of N7methyldG to O6-methyl-2'-deoxyguanosine was determined to be approximately 10. The current assay complements the high-performance liquid chromatography/32P-postlabeling assay for O6-methyl-2'-deoxyguanosine and increases the detection sensitivity of DNA methylated by exogenous alkylating agents.

Polycyclic aromatic hydrocarbon-DNA adducts in human lung and cancer susceptibility genes.

Molecular dosimetry for polycyclic aromatic hydrocarbon-DNA adducts, genetic predisposition to cancer, and their interrelationships are under study in numerous laboratories. This report describes a modified 32P-postlabeling assay for the detection of polycyclic aromatic hydrocarbon-DNA adducts that uses immunoaffinity chromatography to enhance chemical specificity and quantitative reliability. The assay incorporates internal standards to determine direct molar ratios of adducts to unmodified nucleotides and to assess T4 polynucleotide kinase labeling efficiency. High performance liquid chromatography is used to assure adequacy of DNA enzymatic digestion. The assay was validated using radiolabeled benzo(a)pyrene-diol-epoxide modified DNA (r = 0.76, P < 0.05) thereby assessing all variables from enzymatic digestion to detection. Thirty-eight human lung samples were examined and adducts were detected in seven. A subset of samples also was examined for benzo(a)pyrene-diol-epoxide-DNA adducts by immunoaffinity chromatography, high performance liquid chromatography, and synchronous fluorescence spectroscopy. A high correlation between the two assays was found (P = 0.006). The lung samples were then analyzed by the polymerase chain reaction for the presence of mutations in the cytochrome P-450 (CYP) 1A1 and glutathione S-transferase mu (GST mu) genes. A positive association was identified for adduct levels and GST mu null genotypes (P = 0.038). No correlation was found between polycyclic aromatic hydrocarbon-adduct levels and CYP1A1 exon 7 mutations. Age, race, and serum cotinine were not related to adduct levels. Multivariate analysis indicated that only the GST mu genotype was associated with polycyclic aromatic hydrocarbon-DNA adduct levels. This work demonstrates that the 32P-postlabeling assay can be modified for chemically specific adduct detection and that it can be used in the assessment of potentially important genetic factors for cancer risk. The absence of a functional GST mu gene in humans is likely one such factor.

Mutability of p53 hotspot codons to benzo(a)pyrene diol epoxide (BPDE) and the frequency of p53 mutations in nontumorous human lung.

p53 mutations are common in lung cancer. In smoking-associated lung cancer,the occurrence of G:C to T:A transversions at hotspot codons, e.g., 157, 248, 249,and 273, has been linked to the presence of carcinogenic chemicalsin tobacco smoke including polycyclic aromatic hydrocarbons suchas benzo(a)pyrene (BP). In the present study, we have used a highly sensitive mutation assay to determine the p53 mutation load in nontumorous human lung and to study the mutability of p53 codons 157, 248, 249, and 250 to benzo(a)pyrene-diol-epoxide (BPDE), an active metabolite of BP in human bronchial epithelial BEAS-2B cells. We determined the p53 mutational load at codons 157, 248, 249, and 250 in nontumorous peripheral lung tissue either from lung cancer cases among smokers or noncancer controls among smokers and nonsmokers. A 5-25-fold higher frequency of GTC(val) to TTC(phe) transversions at codon 157 was found in nontumorous samples (57%) from cancer cases (n = 14) when compared with noncancer controls (n = 8; P < 0.01). Fifty percent (7/14) of the nontumorous samples from lung cancer cases showed a high frequency of codon 249 AGG(arg) to AGT(ser) mutations (P < 0.02). Four of these seven samples with AGT(ser) mutations also showed a high frequency of codon 249 AGG(arg) to ATG(met) mutations, whereas only one sample showed a codon 250 CCC to ACC transversion. Tumor tissue from these lung cancer cases (38%) contained p53 mutations but were different from the above mutations found in the nontumorous pair. Noncancer control samples from smokers or nonsmokers did not contain any detectable mutations at codons 248, 249, or 250. BEAS-2B bronchial epithelial cells exposed to doses of 0.125, 0.5, and 1.0 microM BPDE, showed G:C to T:A transversions at codon 157 at a frequency of 3.5 x 10(-7), 4.4 x 10(-7), and 8.9 x 10(-7), respectively. No mutations at codon 157 were found in the DMSO-treated controls. These doses of BPDE induced higher frequencies, ranging from 4-12-fold, of G:C to T:A transversions at codon 248, G:C to T:A transversions and G:C to A:T transitions at codon 249, and C:G to T:A transitions at codon 250 when compared with the DMSO-treated controls. These data are consistent with the hypothesis that chemical carcinogens such as BP in cigarette smoke cause G:C to T:A transversions at p53 codons 157, 248, and 249 and that nontumorous lung tissues from smokers with lung cancer carry a high p53 mutational load at these codons.

Smoking increases carcinogenic polycyclic aromatic hydrocarbons in human lung tissue.

Tobacco smoke is a major source of human exposure to polycyclic aromatic hydrocarbons (PAHs). The concentration of PAHs in lung tissue would reflect an individual's dose, and its variation could perhaps reflect cancer risk. Eleven PAHs were measured in 70 lung tissue samples from cancer-free autopsy donors by gas chromatography-mass spectrometry. There were 37 smokers and 33 nonsmokers as estimated by serum cotinine concentration. The sum of PAH concentrations was higher in smokers (P = 0.01), and there was a dose-response relationship for greater smoking (P < 0.01). Smoking increased the concentration of five PAHs including benzo(a)pyrene, which increased approximately 2-fold. The risk for increasing carcinogenic PAHs (odds ratio, 8.20; 95% confidence interval, 2.39-28.09) was 3-fold compared with noncarcinogenic PAHs (odds ratio, 2.61; 95% confidence interval, 0.75-9.12). A higher concentration of PAHs was detected in the lung tissue of males, although the estimated smoking was similar in males and females. Race was not associated with PAH concentrations overall, but PAH concentrations appeared to be higher in African-American males than in any other group. Age was weakly correlated with an increase in fluoranthene and pyrene. The measurement of PAHs in human lung tissue can be used to estimate the actual dose to the target organ.

Cytochrome P450IIE1 genetic polymorphisms, racial variation, and lung cancer risk.

Cytochrome P450IIE1 is responsible for the activation of carcinogenic N-nitrosamines, benzene, urethane, and other low-molecular-weight compounds. Restriction fragment length polymorphisms (PstI and RsaI restriction enzymes) have been identified in the cytochrome P450IIE1 transcription regulatory region that may affect expression. This study describes the PstI and RsaI polymorphisms in different racial populations and in a case-control study of lung cancer. The allelic frequencies were markedly different in Japanese, African-Americans, and Caucasians: the PstI rare allele was present at a frequency of 2% in Caucasians, 5% in African-Americans, and 24% in Japanese (P < 0.05). For the RsaI rare allele, frequencies were 2% in Caucasians, 2% in African-Americans, and 27% in Japanese (P < 0.05). The assay was also applied to 128 individuals enrolled in a case-control study of lung cancer. Although limited in statistical power, the data indicate no evidence for an association in the aggregate of cytochrome P450IIE1 PstI [for which the odds ratio was 0.7 (95% confidence interval (C.I.) = 0.2-2.8)] or RsaI [for which the odds ratio was 0.9 (95% C.I. = 0.2-5.4)] restriction fragment length polymorphisms with lung cancer in this U.S. population. When analyzed by race, the lung cancer odds ratio for the PstI mutant allele in African-Americans was 0.19 (95% C.I. = 0.03-1.38), and in Caucasians it was 4.13 (95% C.I. = 0.34-48.8). For the RsaI mutant allele, the odds ratios were 0.20 (95% C.I. = 0.02-2.43) and 4.28 (95% C.I. = 0.35-50.6), respectively. The ethnic differences of these restriction fragment length polymorphisms might be related to genetic susceptibilities for lung cancer among Caucasians and for gastric or esophageal cancer among Japanese.

Elevated frequency and functional activity of a specific germ-line p53 intron mutation in familial breast cancer.

Previous studies have determined that the frequency of germ-line p53 mutations in familial breast cancer patients is 1% or less, but these reports have not investigated the importance of polymorphic intron base changes in the p53 gene. Therefore, we investigated the frequency of both exon and intron germ-line p53 base changes in 42 breast cancer patients with a strong family history of breast cancer. The mean age of presentation of these patients was 44.0 years (range, 29-69), and 12 of 42 (29%) were of known Ashkenazi ancestry. Purified DNA obtained from the 42 index cases was screened for germ-line p53 mutations in exons 2-11 and surrounding introns using a combination of intron based primers for PCR-single strand conformation polymorphism analysis, direct sequencing, and microarray sequencing using the Affymetrix p53 gene chip methodology. Morphological analysis of apoptosis and cell survival determination were performed on EBV-immortalized lymphoblastoid cell lines from two patients with the p53 intron 6 mutation. A germ-line mutation in the p53 gene at nucleotide 13964 with a G to C base change (13964GC) was identified in 3 of 42 (7.1%) hereditary breast cancer patients. Two patients were heterozygous for this mutation, and one patient had a homozygous mutation. In comparison, 0 of 171 (0%) of sporadic breast cancer patients had the p53 13964GC mutation (P = 0.0003). We found that 0 of 42 (0%) of these hereditary breast cancer patients had other germ-line p53 mutation in exons 2-11. However, pedigree analysis demonstrated that all three patients had strong family histories of multiple types of cancers consistent with Li-Fraumeni syndrome but with late age of onset. Comprehensive BRCA1 and BRCA2 nucleotide analysis from patients with the p53 13964GC mutation revealed no concomitant deleterious BRCA1 or BRCA2 mutations, although they were found in the other hereditary breast cancer patients. Functional analysis of two immortalized lymphoblastoid cell lines derived from patients with the p53 13964GC mutation demonstrated prolonged in vitro survival in response to cisplatinum treatment and showed decreased chemotherapy-induced apoptosis. Immunohistochemical analysis of breast tumors from these patients revealed high levels of mutant p53 protein, suggesting a functional mutation in the p53 gene. In summary, we have identified a single p53 intron mutation in familial breast cancer patients that is present at elevated frequency and has functional activity.

Manganese superoxide dismutase (MnSOD) genetic polymorphisms, dietary antioxidants, and risk of breast cancer.

Oxidative stress, resulting from the imbalance between prooxidant and antioxidant states, damages DNA, proteins, cell membranes, and mitochondria and seems to play a role in human breast carcinogenesis. Dietary sources of antioxidants (chemical) and endogenous antioxidants (enzymatic), including the polymorphic manganese superoxide dismutase (MnSOD), can act to reduce the load of oxidative stress. We hypothesized that the valine-to-alanine substitution that seems to alter transport of the enzyme into the mitochondrion, changing its efficacy in fighting oxidative stress, was associated with breast cancer risk and that a diet rich in sources of antioxidants could ameliorate the effects on risk. Data were collected in a case-control study of diet and breast cancer in western New York from 1986 to 1991. Caucasian women with incident, primary, histologically confirmed breast cancer were frequency-matched on age and county of residence to community controls. Blood specimens were collected and processed from a subset of participants in the study (266 cases and 295 controls). Using a RFLP that distinguishes a valine (V) to alanine (A) change in the -9 position in the signal sequence of the protein for MnSOD, we characterized MnSOD genotypes in relation to breast cancer risk. We also evaluated the effect of the polymorphism on risk among low and high consumers of fruits and vegetables. Premenopausal women who were homozygous for the A allele had a 4-fold increase in breast cancer risk in comparison to those with 1 or 2 V alleles (odds ratio, 4.3; 95% confidence interval, 1.7-10.8). Risk was most pronounced among women below the median consumption of fruits and vegetables and of dietary ascorbic acid and alpha-tocopherol, with little increased risk for those with diets rich in these foods. Relationships were weaker among postmenopausal women, although the MnSOD AA genotype was associated with an almost 2-fold increase in risk (odds ratio, 1.8; confidence interval, 0.9-3.6). No appreciable modification of risk by diet was detected for these older women. These data support the hypothesis that MnSOD and oxidative stress play a significant role in breast cancer risk, particularly in premenopausal women. The finding that risk was greatest among women who consumed lower amounts of dietary antioxidants and was minimal among high consumers indicates that a diet rich in sources of antioxidants may minimize the deleterious effects of the MnSOD polymorphism, thereby supporting public health recommendations for the consumption of diets rich in fruits and vegetables as a preventive measure against cancer.

Increased p53 mutation load in noncancerous colon tissue from ulcerative colitis: a cancer-prone chronic inflammatory disease.

Ulcerative colitis (UC) is a chronic inflammatory disease that produces reactive oxygen and nitrogen species and increases the risk of colorectal cancer (CRC). The p53 tumor suppressor gene is frequently mutated in UC-associated dysplastic lesions and CRC. We are exploring the hypothesis that p53 mutations in the nontumorous colonic tissue in noncancerous UC cases indicate genetic damage from exposure to exogenous and endogenous carcinogens and may identify individuals at increased cancer risk. We are reporting, for the first time, the frequency of specific p53 mutated alleles in nontumorous colon tissue from donors either with or without UC by using a highly sensitive genotypic mutation assay. Higher p53 mutation frequencies of both G:C to A:T transitions at the CpG site of codon 248 and C:G to T:A transitions at codon 247 were observed in colon from UC cases when compared with normal adult controls (P = 0.001 and P = 0.001, respectively). In the UC cases, higher p53 codon 247 and 248 mutation frequencies were observed in the inflamed lesional regions when compared with the nonlesional regions of their colon (P < 0.001 and P = 0.001). The colonic nitric oxide synthase-2 activity was higher in UC cases than in non-UC adult controls (P = 0.02). Our data are consistent with the hypothesis that a higher frequency of p53 mutant cells can be generated under oxidative stress in people with UC. The increased frequency of specific p53 mutated alleles in noncancerous UC colon tissue may confer susceptibility to the development of CRC in an inflammatory microenvironment.

Frequent nitric oxide synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon cancer progression.

An increased expression of nitric oxide synthase (NOS) has been observed in human colon carcinoma cell lines as well as in human gynecological, breast, and central nervous system tumors. This observation suggests a pathobiological role of tumor-associated NO production. Hence, we investigated NOS expression in human colon cancer in respect to tumor staging, NOS-expressing cell type(s), nitrotyrosine formation, inflammation, and vascular endothelial growth factor expression. Ca2+-dependent NOS activity was found in normal colon and in tumors but was significantly decreased in adenomas (P < 0.001) and carcinomas (Dukes' stages A-D: P < 0.002). Ca2+-independent NOS activity, indicating inducible NOS (NOS2), is markedly expressed in approximately 60% of human colon adenomas (P < 0.001 versus normal tissues) and in 20-25% of colon carcinomas (P < 0.01 versus normal tissues). Only low levels were found in the surrounding normal tissue. NOS2 activity decreased with increasing tumor stage (Dukes' A-D) and was lowest in colon metastases to liver and lung. NOS2 was detected in tissue mononuclear cells (TMCs), endothelium, and tumor epithelium. There was a statistically significant correlation between NOS2 enzymatic activity and the level of NOS2 protein detected by immunohistochemistry (P < 0.01). Western blot analysis of tumor extracts with Ca2+-independent NOS activity showed up to three distinct NOS2 protein bands at Mr 125,000-Mr 138,000. The same protein bands were heavily tyrosine-phosphorylated in some tumor tissues. TMCs, but not the tumor epithelium, were immunopositive using a polyclonal anti-nitrotyrosine antibody. However, only a subset of the NOS2-expressing TMCs stained positively for 3-nitrotyrosine, which is a marker for peroxynitrite formation. Furthermore, vascular endothelial growth factor expression was detected in adenomas expressing NOS2. These data are consistent with the hypothesis that excessive NO production by NOS2 may contribute to the pathogenesis of colon cancer progression at the transition of colon adenoma to carcinoma in situ.

Genetic polymorphisms in catechol-O-methyltransferase, menopausal status, and breast cancer risk.

Polymorphic catechol-O-methyltransferase (COMT) catalyzes the O-methylation of estrogen catechols. In a case-control study, we evaluated the association of the low-activity allele (COMT(Met)) with breast cancer risk. Compared to women with COMT(Val/Val), COMT(Met/Met) was associated with an increased risk among premenopausal women [odds ratio (OR), 2.1; confidence interval (CI), 1.4-4.3] but was inversely associated with postmenopausal risk (OR, 0.4; CI, 0.2-0.7). The association of risk with at least one low-activity COMT(Met) allele was strongest among the heaviest premenopausal women (OR, 5.7; CI, 1.1-30.1) and among the leanest postmenopausal women (OR, 0.3; CI, 0.1-0.7), suggesting that COMT, mediated by body mass index, may be playing differential roles in human breast carcinogenesis, dependent upon menopausal status.