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Purpose Metabolic syndrome has a high prevalence in the United States. P-wave duration is a valuable indicator for atrial electrical conduction. Abnormalities in atrial electrical conduction can predispose to atrial fibrillation. Our objective was to estimate the effect of metabolic syndrome on P-wave duration in a nationally representative sample. Methods We included 6499 adults who participated in the National Health and Nutrition Examination Survey III (1988–1994). We estimated the effect of metabolic syndrome and its components on P-wave duration after adjusting for confounders such as demographic and lifestyle variables. We stratified the analyses by gender. Results Irrespective of gender, participants with metabolic syndrome had a longer P-wave duration than that of those without it. In addition, we observed a positive linear dose–response relation between metabolic syndrome components and P-wave duration. Conclusions Patients with metabolic syndrome had a longer P-wave duration. They might have been at a higher risk for atrial fibrillation and mortality; they need to be periodically checked by their health care providers. However, the results of this study should be confirmed in prospective studies.

Studies reported a positive relationship between visit-to-visit blood pressure variability (VVBPV) and cardiovascular morbidity and mortality independently of the mean arterial blood pressure across clinical visits. The literature is scarce on the genes and biological mechanisms that regulate long-term VVBPV. We sought to identify biological pathways that regulate visit-to-visit blood pressure variability. We used phenotypic and genotype data from the Women’s Health Initiatives and Cardiovascular Health Studies. We defined VVBPV of systolic and diastolic blood pressure phenotypes as the standard deviation about the participant’s regression line with systolic and diastolic blood pressure regressed separately across visits. We imputed missing genotypes and then conducted a genome-wide association analysis to identify genomic variants related to the VVBPV and detect biological pathways. For systolic VVBPV, we identified a neurological pathway, the GABAergic pathway (P values = 1.1E − 2), and a vascular pathway, the RAP1 signaling pathway (P values = 5.8E − 2). For diastolic VVBPV, the hippo signaling (P values = 4.1E − 2), CDO myogenesis (P values = 7.0E − 2), and O-glycosylation of TSR domain-containing protein pathways (P values = 9.0E − 2) were the significant pathways. Future studies are warranted to validate these results. Further understanding of the roles of the genes regulating the identified pathways will help researchers to improve future pharmacological interventions to treat VVBPV in clinical practice.

The QRS-T angle is a key ECG predictor for ventricular arrhythmia. Lead has a strong relation with cardiovascular diseases; however, no study has been conducted to investigate the association between lead exposure and QRS-T angle. Thus, we explored this association in a population-based representative sample: NHANES III. We used the standard 12-lead ECGs to calculate Spatial QRS-T angles. Blood lead concentration was measured using graphite furnace atomic absorption spectrophotometry method. We conducted multivariate weighted logistic regression to adjust for impaired fasting glucose, hypertension, poverty index, age, race, and smoking status. We found that when the log of blood lead increased by one unit, the odds of an abnormal QRS-T angle increased by 34% among men and 4% among women. We suggest the use of QRS-T angle deviation among those who are exposed to lead to detect individuals at risk for adverse cardiovascular outcomes such as arrhythmias.

Objective Cadmium's effect on QRS|T angle has not been studied. An abnormal QRS|T angle deviation may increase the risk for ventricular dysrhythmias. Methods We calculated the orientation of spatial QRS|T angle using QRS and T amplitudes of leads V2, V5, V6, and AVF from the National Health and Nutritional Examination Survey III. Cadmium concentration was measured in urine. We fit weighted unadjusted and adjusted logistic regressions to calculate odds ratios and their corresponding 95% confidence intervals. Results A unit increase in the logarithm of urinary cadmium increased the odds of QRS|T angle deviation by 30% [1.30 (1.01 to 1.61)]. Conclusions Cadmium exposure was associated with an abnormal QRS|T angle in women but not in men. Women exposed to cadmium should be periodically evaluated to detect QRS|T angle deviation, which can predispose them to ventricular dysrhythmias.

Arkansas (AR) consistently has higher ischemic heart disease (IHD) death rates than the US, which is believed to be due to a higher prevalence in AR of major, modifiable risk factors. We examined the difference in IHD death rates between AR and the US as consequences of differences in the prevalence of nine risk factors between pre-Medicare age group (45-64) and Medicare age group (65-84). We modeled IHD deaths attributable to differential prevalence between AR and the US using mortality data and prevalence data from AR and US for years 2000-2010, and relative risk measures obtained from the INTERHEART and Atherosclerosis Risk in Communities studies. From 2000-2010, our study showed that if we were to reduce the prevalence of significant risk factors to US levels, we would reduce AR IHD deaths by 26.6% in the pre-Medicare age group and 15.9% in the Medicare age group. Most of the increased mortality was explained by higher prevalence of smoking and hypertension in AR. Other socioeconomic factors that contributed to an increased risk of poor health outcomes were education, income, and the lack of health insurance; with AR having worse outcomes than the US for the pre-Medicare age group. The importance of risk factors depended on race, sex, and age. The excess mortality in AR relative to the US for the two age groups can largely be explained by prevalence differences in smoking, hypertension, cholesterol, education and income.

A power calculation for a study with a quantitative outcome requires information on the outcome distribution under the alternative hypothesis. Researchers face challenges when they concisely specify alternative distributions in genetic studies because power depends on genotype frequencies and the average effect of each genotype. In GWAS, investigators evaluate hundreds of thousands of associations; therefore it is unrealistic to specify gene frequencies and gene effects for each test and some simplification is needed. Software packages are available to calculate power, but many of them have limited flexibility and / or may have a steep learning curve. In this review, we describe to researchers and graduate students the essentials of a power calculation for testing for an association between a quantitative trait and genotypes. In addition, we provide them with the codes of the different available software packages—free and commercial—to calculate this power. The calculations can be carried out using virtually any computer language that computes the cumulative distribution function of a non-central F-distribution.

Background: The process by which salt affects the gastric precancerous process has not been adequately studied in humans. Methods: We investigated the effects of salt on gastric inflammation, epithelial damage, the density of Helicobacter pylori infection, and gastric epithelial cell proliferation, all of which may be mediators between salt and gastric precancerous/cancerous lesions. These potential mediators were measured using gastric biopsies as: (a) the density of polymorphonuclear and mononuclear cells (gastric inflammation), (b) mucus depletion (gastric epithelial damage), and (c) the severity of H. pylori infection. Salt intake was measured with spot urine samples (using urinary sodium/creatinine ratios), self-reported frequency of adding salt to food, and as total added salt. Results: The average sodium/creatinine ratio (at baseline and post-treatment at five months) was associated with increased epithelial damage over the 12-year follow-up period among those with a greater severity of chronic inflammation and among those with continued H. pylori infection after treatment at five months. This association was stronger when both severe gastric inflammation and H. pylori infection were present at five months (ß: 1.112, 95% CI: 0.377, 1.848). Conclusion: In humans, salt was associated with an increase in epithelial damage in stomachs with more severe previous H. pylori-induced chronic inflammation.

Gastric cancer is the third leading cause of cancer mortality worldwide. Studies investigating the effect of salt on gastric cancer have mainly used self-reported measures, which are not as accurate as sodium/creatinine ratios because individuals may not know the amount of salt in their food. Using data from a prospective cohort study, we investigated the effect of salt intake on progression to gastric precancerous lesions. Salt intake was estimated by urinary sodium/creatinine ratios, self-reported frequencies of adding salt to food, and total added table salt. We repeated the analyses among groups with and without Helicobacter pylori infection. We did not observe a positive association between salt intake, measured by urinary sodium/creatinine ratio, and overall progression in the gastric precancerous process (adjusted risk ratio (RR): 0.94, 95% confidence interval (CI) 0.76&ndash, 1.15). We did observe an association between salt intake and increased risk for progression to dysplasia or gastric cancer overall (adjusted risk ratio (RR): 1.32, 95% confidence interval (CI): 0.96&ndash, 1.81), especially among those who continued to have H. pylori infection at the five-month follow-up (adjusted RR: 1.53, 95% CI: 1.12&ndash, 2.09), and among those who had persistent H. pylori infection over 12 years (adjusted RR: 1.49, 95% CI: 1.09&ndash, 2.05). Salt intake may increase the risk of gastric dysplasia or gastric cancer in individuals with H. pylori infection.

Background. Gastric cancer is the fourth most common cancer and the third most common cause of cancer deaths worldwide. Morbidity and mortality from gastric cancer may be decreased by identification of those that are at high risk for progression in the gastric precancerous process so that they can be monitored over time for early detection and implementation of preventive strategies. Method. Using machine learning, we developed prediction models for gastric precancerous progression in a population from a developing country with a high rate of gastric cancer who underwent gastroscopies for dyspeptic symptoms. In the data imputed for completeness, we divided the data into a training and a validation test set. Using the training set, we used the random forest method to rank potential predictors based on their predictive importance. Using predictors identified by the random forest method, we conducted best subset linear regressions with the leave-one-out cross-validation approach to select predictors for overall progression and progression to dysplasia or cancer. We validated the models in the test set using leave-one-out cross-validation. Results. We observed for all models that complete intestinal metaplasia and incomplete intestinal metaplasia were the strongest predictors for further progression in the precancerous process. We also observed that a diagnosis of no gastritis, superficial gastritis, or antral diffuse gastritis at baseline was a predictor of no progression in the gastric precancerous process. The sensitivities and specificities were 86% and 79% for the general model and 100% and 82% for the location-specific model, respectively. Conclusion. We developed prediction models to identify gastroscopy patients that are more likely to progress in the gastric precancerous process, among whom routine follow-up gastroscopies can be targeted to prevent gastric cancer. Future external validation is needed.

Background Spatial QRS|T angle is a predictor of cardiovascular events. Those with metabolic syndrome have an increased risk of cardiovascular morbidity and mortality. This study investigated the association between metabolic syndrome and spatial QRS|T angle. Methods We obtained data from the National Health and Nutritional Examination Survey III on 6,249 adults. We calculated spatial QRS|T angle from the standard 12-lead electrocardiogram and classified it as abnormal, borderline, or normal. We identified metabolic syndrome if at least three of the following were present: abdominal obesity, elevated blood pressure, elevated triglycerides, decreased high-density lipoprotein (HDL), and impaired fasting glucose. We used weighted logistic regression to estimate the effect of metabolic syndrome and its components on QRS|T angle while stratifying by gender and adjusting for age, race, smoking status, heart rate, PR, QT, and QRS interval, and QRS amplitude. Results Among men and women, metabolic syndrome, the number of components present, elevated blood pressure, and impaired fasting glucose were positively associated with QRS|T angle. Among women, decreased HDL and abdominal obesity were also positively associated with QRS|T angle. Conclusions This study suggests that persons with metabolic syndrome may be at increased risk for ventricular arrhythmias. The use of spatial QRS|T angle to assess this cardiovascular risk is warranted.

High-normal blood pressure and visit-to-visit blood pressure variability are common in clinical settings. They are associated with cardiovascular outcomes. No population based studies have assessed the association between these two phenomena. Our objective was to test the relationship of high-normal blood pressure with visit-to-visit blood pressure variability.A cross-sectional study.We used data from the cross-sectional Third National Health and Nutrition Examination Survey to test the relationship between high-normal blood pressure and visit-to-visit blood pressure variability; we conducted multivariable regression analyses to evaluate the relationship between these two variables.The analysis included 6,071 participants. The participants' mean age was 37.16 years. The means of visit-to-visit systolic and diastolic blood pressure variability were 5.84 mmHg and 5.26 mmHg. High-normal blood pressure was significantly associated with systolic and diastolic blood pressure variability (p values0.05).High-normal blood pressure is associated with visit-to-visit blood pressure variability. Additional research is required to replicate the reported results in prospective studies and evaluate approaches to reduce blood pressure variability observed in clinical settings among patients with high-normal blood pressure to reduce the subsequent complications of blood pressure variability.

We assessed whether the prevalence of recent (within a year) initiation of cigarette smoking was associated with reports of ever using electronic delivery systems (ENDS) in the National Youth Tobacco Survey (NYTS) and whether the association varied by age. Weighted cross-sectional analysis of use of ENDS, cigarette smoking, age at interview and age at initiation of smoking collected systematically through the 2011–2013 NYTS cycles. In multivariate analyses those who ever used ENDS were twice as likely as nonusers of ENDS to have tried cigarette smoking in the last year (multivariate PR: 2.3; 95 % CI 1.9, 2.7). This average hid significant variations by age: a 4.1-fold increase (95 %; 2.6, 6.4) among those 11–13 years of age, compared to a smaller increase among those 16–18 years: 1.4-fold (95 % CI 1.1, 1.8). Use of ENDS by adolescents was associated with initiation of cigarette smoking in the last year. This association was stronger in younger adolescents.

Introduction The 2016 US Surgeon General's Report suggests that the use of electronic nicotine delivery systems (ENDS) is a fetal risk factor. However, no previous study has estimated their effect on adverse pregnancy outcomes. We assessed the prevalence of current ENDS use in pregnant women and explored the effect on birth weight and smallness-for-gestational-age (SGA), correcting for misclassification from nondisclosure of smoking status. Methods We conducted a cohort study with 248 pregnant women using questionnaire data and biomarkers (salivary cotinine, exhaled carbon monoxide, and hair nicotine). We evaluated the association between birth weight and the risk of SGA by applying multivariate linear and log-binomial regression to reproductive outcome data for 232 participants. Participants who did not disclose their smoking status were excluded from the referent group. Sensitivity analysis corrected for misclassification of smoking/ENDS use status. Results The prevalence of current ENDS use among pregnant women was 6.8% (95% CI: 4.4-10.2%); most of these (75%) were concurrent smokers. Using self-reports, the estimated risk ratio of SGA for ENDS users was nearly two times the risk in the unexposed (RR=1.9, 95% CI: 0.6-5.5), and over three times that for ENDS-only users versus the unexposed (RR=3.1, 95% CI: 0.8-11.7). Excluding from the referent group smokers who did not disclose their smoking status, the risk of SGA for ENDS-only use was 5 times the risk in the unexposed (RR=5.1, 95% CI: 1.1- 22.2), and almost four times for all types of ENDS users (RR=3.8, 95% CI: 1.3-11.2). SGA risk ratios for ENDS users, corrected for misclassification due to self-report, were 6.5-8.5 times that of the unexposed. Conclusions Our data suggest that ENDS use is associated with an increased risk of SGA.

Certain viruses naturally infect and cause cancer in chickens and turkeys. Humans are widely exposed. The viruses cause cancer in primates, and transform human cells in vitro, but it is not known if they cause cancer in humans, mainly because of the lack of epidemiologic evidence. We conducted cohort mortality studies of workers in poultry slaughtering/processing plants across the United States, because they have the highest human exposures. An excess of lung cancer and other deaths was recorded in the poultry workers. Here, we report on a case-cohort study of the lung cancer deaths nested within these cohorts, that was conducted to provide epidemiologic evidence linking these viruses with human cancer occurrence, while adjusting for possible confounders, including workplace chemical carcinogens. We obtained interviews for 339 lung cancer deaths and 457 controls, selected from our combined cohorts of 30,411 poultry plant workers and 16,405 non-poultry workers, belonging to United Food & Commercial Workers unions. Data was analyzed by both logistic regression and Cox regression, adjusting for smoking and other confounders. Lung cancer risk was independently associated with tasks or work areas indicative of exposure to both poultry oncogenic viruses and to workplace chemical carcinogens. The study provides an incremental piece of evidence (epidemiologic), indirectly linking the oncogenic viruses of poultry with the occurrence of cancer in humans, and thus may have public health implications, but the limitations highlighted must be considered. Confirmatory studies, particularly molecular studies providing definitive proof of poultry oncogenic retrovirus integration in human DNA are needed, before the findings observed in this study can be put into proper perspective.

Microarray and RNA-sequencing technologies measure thousands of genes per biological sample. Because of the large number of genes, empirical distributions over genes for statistics computed over samples resolve properties of the data that can be exploited to define the expressed genes, diagnose problems with hypothesis tests, and even remedy some of these problems. The empirical distribution of the average expressions for genes was first used to partition the interrogated genes into two subsets, ‘non-expressed’ genes and ‘expressed’ genes. The p-values from tests for treatment effects were computed for both subsets and their empirical distributions were examined next. A plot of empirical distributions of p-values (p-value plot) indicated that the ‘non-expressed’ genes do not follow the anticipated null distribution, which implies that the p-values for expressed genes may also misrepresent their true significance. In simulations we were able to produce a similar departure from the null distribution with dye effects, suggesting that comparable confounding may account for the observed discrepancies. By using the empirical distribution of non-expressed genes as the null distribution, p-values for the expressed genes were adjusted with the goal of mitigating biases introduced by systematic distortions such as a dye effect. A plot of the empirical distribution for a statistic computed per endpoint provides a succinct visualization of the distributional properties, which can be compared to expected properties. Such comparisons are effective at identifying problems with analyses, and indicating adjustments that can be applied to generate more reliable lists of affected genes based on false-discovery criteria.

Animal studies showed that the use of metformin after myocardial infarction (MI) resulted in a protective effect on cardiac myocytes. In this study, we examined the effect of metformin in patients with diabetes mellitus (DM) on left ventricular ejection fraction (LVEF) and post-MI mortality. We reviewed charts of patients with MI admitted to the UAMS medical center. Baseline characteristics and 12-month follow up data were collected. Patients were classified into three groups: Control group- no DM (n = 464), Metformin group- DM + MI (n = 88) and No-Metformin group- DM + MI (n = 168). First, we compared Metformin and No-Metformin groups to the Control group. Second, we performed propensity-score matching in patients with DM, and compared Metformin to No-Metformin groups. All-cause 30-day and 12-month mortality was significantly higher in the No-Metformin group compared to controls (13.5 vs 9.3 % p = 0.03 at 30 days, 23.7 vs 15.9 % p = 0.03 at 12 months). However, all-cause 30-day and 12-month mortality were similar in the Controls and Metformin group (9.3 vs 6.8 % p = 0.93 at 30 days, 15.9 vs 11.4 % p = 0.97 at 12 months). Mean LVEF on presentation (45 % in the three groups) and at follow up (47.84, 46.38 and 43.62 % in Control, Metformin, and No-Metformin groups, respectively) were not statistically different. There were no significant differences in regard to re-hospitalization, re-intervention, new stroke, CHF development, new MI, or identifiable arrhythmias. Metformin was an independent predictor of lower 30-day and 12-month all-cause mortality in patients with DM (HR 0.25, p = 0.02 and HR 0.32, p = 0.01, respectively). In the matched analysis, 30-day all-cause mortality was significantly higher in the No-Metformin compared to the Metformin group (21.1 vs 8.8 %, p = 0.05). However the difference in 12-month all-cause mortality did not reach statistical significance (24.6 vs 15.8 %, p = 0.15). This proof-of-concept study shows that use of metformin in patients with DM is associated with lower 30-day all-cause mortality and tendency for a lower 12-month all-cause mortality following MI without discernible improvement in LVEF.

Objectives: As of October 2015, evidence needed to make a recommendation about the use of electronic nicotine delivery systems (ENDS) for smoking cessation was limited. We used the 2014 Arkansas Behavioral Risk Factor Surveillance System with additional state-specific questions to determine the prevalence of ENDS use, the impact of ENDS use on smoking cessation, and beliefs about ENDS use in Arkansas. Our objectives were to determine if (1) ENDS use was associated with lower odds of quitting smoking, (2) ENDS users believed that ENDS use was not harmful to their health, and (3) ENDS users believed that switching to ENDS reduced their tobacco-related health risks. Methods: We conducted a cross-sectional study of 4465 respondents to the Arkansas Behavioral Risk Factor Surveillance System and used weighted analyses to account for the complex survey design. We used a subset of records formed by (1) formers smokers who quitted smoking in the last 5 years and (2) current smokers to assess the odds of quitting. Results: In 2014, 6.1% (95% confidence interval [CI], 5.0%-7.4%) of Arkansas adults were currently using ENDS. Of the 1083 participants who were current smokers or had quit smoking within the past 5 years, 515 (54.1%) had used ENDS. Of the 515 ENDS users, 404 (80.3%) had continued smoking. ENDS use was significantly associated with reduced odds of quitting smoking (weighted odds ratio = 0.53; 95% CI, 0.34-0.83). Although 2437 of 3808 participants (62.5%) believed that it was harmful for nonsmokers to start using ENDS and 1793 of 3658 participants (47.0%) believed that switching to ENDS did not reduce tobacco-related health risks, only 80 of 165 (41.3%) and 50 of 168 (33.9%) ENDS users shared these same respective beliefs. Conclusions: Most smokers who indicated smoking in the past 5 years and who tried ENDS did not stop smoking. ENDS use was inversely associated with smoking cessation. Tobacco cessation programs should tell cigarette smokers that ENDS use may not help them quit smoking.

Chronic infections have been shown to enhance atherogenicity. However, the association between chronic hepatitis C (HCV) and coronary heart disease (CHD) remains controversial. We examined the risk for CHD events in patients with HCV with an emphasis on the risk of CHD events with active infection. We conducted a retrospective cohort study using the enterprise data warehouse at the University of Arkansas for Medical Sciences. HCV positive and negative patients were identified based on serology, and incident CHD events were studied. Patient characteristics at entry were compared either by the analysis of variance or F test (continuous variables) or by a chi-square test (categorical variables). The joint effect of risk factors for incident CHD was evaluated using logistic regression. A total of 8,251 HCV antibody positive, 1,434 HCV RNA positive, and 14,799 HCV negative patients were identified. Patients with HCV antibody and RNA positivity had a higher incidence of hypertension, diabetes mellitus, obesity, and chronic lung disease, but lower serum cholesterol levels compared with patients who were HCV negative (p0.001). HCV seropositive patients had a higher incidence of CHD events compared with controls (4.9% vs 3.2%, p0.001). In the HCV cohort, patients with detectable HCV RNA had a significantly higher incidence of CHD events compared with patients who were only HCV antibody positive with no detectable RNA (5.9% vs 4.7%, p = 0.04). In multivariate logistic regression analyses, both HCV antibody positivity (odds ratio 1.32, 95% confidence interval 1.09 to 1.60, p0.001) and HCV RNA positivity (odds ratio 1.59, 95% confidence interval 1.13 to 2.26, p0.001) were independent risk factors for incident CHD events. In conclusion, there is an increased incidence of CHD events in patients with HCV seropositivity and the incidence is much higher in patients with detectable HCV RNA compared with patients with remote infection who are only antibody positive. Lipid profile does not appear to be a good cardiovascular risk stratification tool in patients with HVC.

Epidemiological studies have shown that blood pressure is not a constant variable. Evidence has accumulated showing that the blood pressure variability is associated with organ damage. A substantial increase in the prevalence of the metabolic syndrome has been documented globally. We examined the association of visit-to-visit blood pressure variability with the metabolic syndrome and its components, using data collected in the Third National Health and Nutrition Examination Survey. A multivariable generalized linear model was performed. The metabolic syndrome and its components, particularly hypertension, increased waist circumference and hyperglycemia, were significantly associated with systolic blood pressure variability across study visits (P0.05). After adjusting for the effect of age, gender, race and antihypertensive medication, the multivariable analyses did not show significant relationships between the metabolic syndrome and diastolic blood pressure variability (P-values0.05). Additional research is required to verify the observed results in prospective studies and evaluate approaches to reduce blood pressure variability observed in clinical settings among persons with the metabolic syndrome to reduce its subsequent complications.

Background— The excess stroke mortality in the southeastern states of the United States (stroke-belt states) is well known; however, the factors associated with this pattern have not been fully elucidated. We measured the contribution of several demographic factors by analyzing stroke mortality data (2005–2009) at the census block group (BG) level in the state of Arkansas. Methods and Results— Census BGs were used as proxies for neighborhoods. BGs were stratified using 5 census measures: poverty (percent of population below federal poverty level), population density (population per square mile), education (percent of population aged >25 years who did not graduate from high school), population mobility (percent of population who resided at the same address 1 year ago), and the percent of non-Hispanic blacks (percent of population that is black). Generalized additive models were used to estimate the variation in stroke mortality among BGs and to assess the impact of different demographic variables. From 2005 to 2009, there were 8930 stroke deaths in Arkansas. There was considerable variation in the relative risk even between adjacent BGs within a single county. The geographically weighted regression analyses indicated that 4.5% to 9% of deviance in stroke mortality among BGs could be explained by poverty, education, population density, and population mobility. Race/ethnicity (non-Hispanic blacks) explains Conclusions— Our study shows that primordial risk factors such as poverty and education drive disparities in stroke mortality among neighborhoods in Arkansas.

Background Driving time to a percutaneous coronary intervention ( PCI )–capable hospital is important in timely treatment of acute myocardial infarction ( AMI ). Our objective was to determine whether driving time from one's residence to a PCI ‐capable hospital contributes to AMI deaths. We conducted a cross‐sectional study of age‐ and sex‐adjusted mortality in census block groups to evaluate this question. Methods and Results We studied all (14 027) AMI deaths that occurred during 2008–2012 in Arkansas to assess the relationship between driving time from the population center of a block group (neighborhood) to the nearest PCI ‐capable hospital. We estimated standardized mortality ratios in block groups that were adjusted for education (population over 25 years of age who did not graduate from high school), poverty (population living below federal poverty level), population density (population per square mile), mobility (population residing at the same address as 1 year ago), black (population that is black), rurality (rural households), geodesic distance, and driving time. The median geodesic distance and driving time were 12.8 miles (interquartile range 3.6–30.1) and 28.3 minutes (interquartile range 9.6–58.7), respectively. Risks in neighborhoods with long driving times (90th percentile) were 26% greater than risks in neighborhoods with short driving times (10th percentile), even after adjusting for education, poverty, population density, rurality, and black race ( P Conclusions AMI mortality increases with increasing driving time to the nearest PCI ‐capable hospital. Improving the healthcare system by reducing time to arrive at a PCI ‐capable hospital could reduce AMI deaths.

Large numbers of mRNA transcripts, proteins, metabolites, and single nucleotide polymorphisms can be measured in a single tissue sample using new molecular biological techniques. Accordingly, the interpretation of ensuing hypothesis tests should manage the number of comparisons. For example, cDNA microarray experiments generate large multiplicity problems in which thousands of hypotheses are tested simultaneously. In this context, the false discovery rate (FDR) and false non-discovery rate (FNR) are used to account for multiple comparisons. In this study, we propose non-parametric estimates of FDR and FNR that are conceptually and computationally straightforward. Additionally, to illustrate their properties and use in a procedure for an optimum subset of significant tests, an example from a functional genomics study is presented.

A study was undertaken to determine whether alterations in the gene expression or overt histological signs of neurotoxicity in selected regions of the forebrain might occur from acrylamide exposure via drinking water. Gene expression at the mRNA level was evaluated by cDNA array and/or RT-PCR analysis in the striatum, substantia nigra and parietal cortex of rat after a 2-week acrylamide exposure. The highest dose tested (maximally tolerated) of approximately 44 mg/kg/day resulted in a significant decreased body weight, sluggishness, and locomotor activity reduction. These physiological effects were not accompanied by prominent changes in gene expression in the forebrain. All the expression changes seen in the 1200 genes that were evaluated in the three brain regions were < or =1.5-fold, and most not significant. Very few, if any, statistically significant changes were seen in mRNA levels of the more than 50 genes directly related to the cholinergic, noradrenergic, GABAergic or glutamatergic neurotransmitter systems in the striatum, substantia nigra or parietal cortex. All the expression changes observed in genes related to dopaminergic function were less than 1.5-fold and not statistically significant and the 5HT1b receptor was the only serotonin-related gene affected. Therefore, gene expression changes were few and modest in basal ganglia and sensory cortex at a time when the behavioral manifestations of acrylamide toxicity had become prominent. No histological evidence of axonal, dendritic or neuronal cell body damage was found in the forebrain due to the acrylamide exposure. As well, microglial activation was not present. These findings are consistent with the absence of expression changes in genes related to changes in neuroinflammation or neurotoxicity. Over all, these data suggest that oral ingestion of acrylamide in drinking water or food, even at maximally tolerable levels, induced neither marked changes in gene expression nor neurotoxicity in the motor and somatosensory areas of the central nervous system.

Sunlight and ultraviolet-induced mutation of the p53 gene is a frequent, possibly obligate step in skin cancer development, making quantitative measurement of p53 mutation an ideal biomarker for sunlight-induced skin carcinogenesis. To understand how the appearance of p53 mutation relates to skin tumor development, SKH-1 hairless mice were exposed 5 d per week to one of four different doses of simulated solar light (SSL; 0, 6.85, 13.70, 20.55 mJ x CIE/cm(2)) previously characterized for their tumorigenic potential. Allele-specific competitive blocker-PCR (ACB-PCR) was used to measure levels of p53 codon 270 CGT to TGT mutation within DNA isolated from dorsal skin of exposed mice. For each dose, p53 mutant fraction (MF) was measured after 4, 16, and 28 wk of exposure. Significant dose- and time-dependent increases in p53 MF were identified. All p53 MF measurements were integrated by relating the observed p53 MF to the cumulative dose of SSL. The increase in the logarithm of p53 MF was described by the linear function: log(10) MF = alpha + 0.0016 x d, where alpha is the spontaneous log(10) MF after a particular time point and d is the dose of SSL in mJ x CIE/cm(2). The p53 MF induced in nontumor bearing skin by 28 wk of exposure at the high dose of SSL was significantly lower than that found in skin tumors induced by approximately 32 wk of exposure to the same dose of SSL. p53 MF showed a strong negative correlation with tumor latency, suggesting this quantitative biomarker has the potential to predict tumorigenicity.

A perceived disadvantage of transgenic rodent mutation assays is that spontaneous mutant frequencies are high compared to those of endogenous genes and may consequently reduce sensitivity to induced mutation. We have previously argued that unrepaired G:T mismatches from spontaneous deamination of 5-methylcytosine at CpG sites could be converted to apparent in vivo mutations in the bacterial recovery systems because of rapid, random, mismatch repair in Escherichia coli. In this study, we have measured mutation frequencies in spleen of male mice induced by N-ethyl-N-nitrosourea (ENU) using the PhiX174 transgene, which is not subject to mismatch repair in E.coli, using single-burst analysis, a unique method to identify in vivo mutation. In order to compare our results to those using the lacI and cII transgenes, we converted all mutant frequencies to base pair substitution (bps) mutation frequencies per nucleotide based on mutant spectra from this study and published literature. We found this frequency in control spleen to be similar for lacI (3.8 +/- 0.7 x 10(-8)) and PhiX174 (3.1 +/- 1.2 x 10(-8)) at 6 weeks of age. We found a strong age dependence for spontaneous lacI mutation that extrapolated to a value at conception (1.8 +/- 0.9 x 10(-8)) that was not significantly different from the human germ line bps mutation frequency per nucleotide of 1.7 +/- 0.2 x 10(-8). These two transgenes provided similar mutational responses to 40 mg/kg ENU, 7- to 9-fold. In contrast, the cII target gene in the same tissue produces both spontaneous and induced mutation frequencies approximately 10 times higher, for unknown reasons. We conclude that the spontaneous mutant frequencies measured by the lacI and PhiX174 transgenes in this moderately dividing tissue accurately measure in vivo mutation frequencies at early ages. For these two transgenes, seemingly high mutant frequencies may reflect the expected accumulation of somatic mutation with age.

Mitochondrial dysfunction has been implicated in the adverse effects of nucleoside reverse transcriptase inhibitors (NRTIs) used to treat HIV-1 infections. To gain insight into the mechanism by which NRTIs alter mitochondrial function, the expression level of 542 genes associated with mitochondrial structure and functions was determined in the livers of p53 haplodeficient (+/−) C3B6F1 female mouse pups using mouse mitochondria-specific oligonucleotide microarray. The pups were transplacentally exposed to zidovudine (AZT) at 240 mg/kg bw/day or a combination of AZT and lamivudine (3TC) at 160 and 100 mg/kg bw/day, respectively, from gestation day 12 through 18, followed by continuous treatment by oral administration from postnatal day 1–28. In addition, AZT/3TC effect was investigated in wild-type (+/+) C3B6F1 female mice. The genotype did not significantly affect the gene expression profile induced by AZT/3TC treatment. However, the transcriptional level of several genes associated with oxidative phosphorylation, mitochondrial tRNAs, fatty acid oxidation, steroid biosynthesis, and a few transport proteins were significantly altered in pups treated with AZT and AZT/3TC compared to their vehicle counterparts. Interestingly, AZT/3TC altered the expression level of 153 genes with false discovery rate of less than 0.05, in contrast to only 20 genes by AZT alone. These results suggest that NRTI-related effect on expression level of genes associated with mitochondrial functions was much greater in response to AZT/3TC combination treatment than AZT alone.

Objectives: To determine if driving time to the percutaneous coronary intervention (PCI) capable hospital is an important factor in deaths of patients with acute myocardial infarction (AMI). Methods: We studied the relationship between driving time to the nearest PCI-capable hospital and AMI mortality (2008-2012) in census block groups (BG), N=2147, in the state of Arkansas. Population center of a BG was used as a proxy for an individual’s place of residence. The driving time to the nearest PCI-capable hospital was calculated from the population center of a BG. The risk of AMI death was then calculated using five census measures: poverty (% of population below federal poverty level); population density (population per square mile); education (% of population over 25 years of age that did not graduate from high school); mobility (% of population who resided at the same address 1 year ago) and the percent of non-Hispanic blacks (% of population that is Black); and the calculated driving time to the nearest PCI-capable hospital. Arc GIS was used to calculate the driving time to the nearest PCI-capable hospital. Generalized additive models were used to estimate the variation in AMI mortality among BGs. Findings: During 2008-2012, there were 14,027 AMI deaths in Arkansas. There was considerable variation in the relative risk between BGs and AMI mortality based on the distance to the PCI hospital. The median distance to the nearest PCI-capable hospital was 12.7 miles (Interquartile range 3.5 - 30.1); and the median driving time to the nearest PCI-capable hospital was 28.3 minutes (Interquartile range 9.6-58.7). However, driving time (Range- 58.7 to 149.3 minutes) among residents in BGs (n=536) that are in the fourth quartile was much higher than the optimal timeframe to receive PCI for AMI. The regression analyses indicated that the driving time to the nearest PCI-capable hospital is a significant predictor of AMI mortality (p < 0.0001) in all BGs, after adjusting for education, poverty, race, and population density. Conclusions: Our study shows that the driving time to the nearest PCI-capable hospital is an independent predictor of AMI mortality. Improving health care system by reducing time to arrive at a PCI-capable hospital could reduce AMI deaths in underserved communities.

K-ras codon 12 GGT-->GAT and GGT-->GTT mutations are the most frequently observed K-ras point mutations in human and rodent tumors and therefore are implicated in carcinogenesis for many tissues. Measurement of these mutations in rat models and human tissue could facilitate a more logical extrapolation of rodent tumorigenesis data to human disease. We have developed allele-specific competitive blocker PCR (ACB-PCR) assays for rat K-ras codon 12 GGT-->GTT and GGT-->GAT mutations that parallel the already published assays for human K-ras codon 12 mutations. Liver K-ras codon 12 mutant allele fractions were measured in vehicle-treated and N-hydroxy-2-acetylaminofluorene (N-OH-AAF)-treated Big Blue rats. The average K-ras codon 12 GGT-->GTT mutant fraction (MF) for four control rats was 50 x 10(-6) (95% CI: 27 x 10(-6), 95 x 10(-6)) and for four treated rats was 165 x 10(-6) (95% CI: 87 x 10(-6), 312 x 10(-6)), indicating a 3.3-fold increase with treatment (95% CI: 1.3-8.1). The average MF of K-ras codon 12 GGT-->GAT for control rats was 1320 x 10(-6) (95% CI: 498 x 10(-6), 3500 x 10(-6)) and for treated rats was 8450 x 10(-6) (95% CI: 3180 x 10(-6), 22 400 x 10(-6)), indicating a 6.4-fold increase with treatment (95% CI: 1.6-25.4). These transgenic rats were part of a study that included analysis of liver lacI mutations. Although data from lacI determinations show that this compound induces mostly G-->T mutations, using the ACB-PCR method both K-ras codon 12 GGT-->GTT and GGT-->GAT MFs were significantly increased in treated rats versus control rats. This data raises the possibility that N-OH-AAF may not only induce mutations by a genotoxic mechanism, but also by amplification of both de novo and pre-existing K-ras mutation.

Probabilistic risk assessment is gaining acceptance as the most appropriate way to characterize and communicate uncertainties in estimates of human health risk and/or reference levels of exposure such as benchmark doses. Although probabilistic techniques are well established in the exposure-assessment component of the National Research Council's risk-assessment paradigm, they are less well developed in the dose-response-assessment component. This paper proposes the use of hierarchical statistical models as tools for implementing probabilistic dose-response assessments, in that such models provide a natural connection between the pharmacokinetic (PK) and pharmacodynamic (PD) components of dose-response models. The results show that incorporating internal dose information into dose-response assessments via the coupling of PK and PD models in a hierarchical structure can reduce the uncertainty in the dose-response assessment of risk. However, information on the mean of the internal dose distribution is sufficient; having information on the variance of internal dose does not affect the uncertainty in the resulting estimates of excess risks or benchmark doses. In addition, the complexity of a PK model of internal dose does not affect how the variability in risk is measured via the ultimate endpoint.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Testing (IWGT), comprised of experts from Japan, Europe, and the United States, met on August 29, 2003, in Aberdeen, Scotland, United Kingdom. This meeting of the MLA Workgroup was devoted to reaching a consensus on the appropriate approach to data evaluation and on acceptance criteria for both the positive and negative/vehicle controls. The Workgroup reached consensus on the acceptance criteria for both the agar and microwell versions of the MLA. Recommendations include acceptable ranges for mutant frequency, cloning efficiency, and suspension growth of the negative/vehicle controls and on criteria to define an acceptable positive control response. The recommendation for the determination of a positive/negative test chemical response includes both the requirement that the response exceeds a defined value [the global evaluation factor (GEF)] and that there also be a positive dose-response (evaluated by an appropriate statistical method).

DNA mismatch repair (MMR) deficiencies result in increased frequencies of spontaneous mutation and tumor formation. In the present study, we tested the hypothesis that a chemically-induced mutational response would be greater in a mouse with an MMR-deficiency than in the MMR-proficient mouse models commonly used to assay for chemical carcinogenicity. To accomplish this, the induction of H-ras codon 61 CAA-->AAA mutation was examined in Pms2 knockout mice (Pms2-/-, C57BL/6 background) and sibling wild-type mice (Pms2+/+). Groups of five or six neonatal male mice were treated with 0.3 micromol 4-aminobiphenyl (4-ABP) or the vehicle control, dimethylsulfoxide. Eight months after treatment, liver DNAs were isolated and analysed for levels of H-ras codon 61 CAA-->AAA mutation using allele-specific competitive blocker-PCR. In Pms2-proficient and Pms2-deficient mice, 4-ABP treatment caused an increase in mutant fraction (MF) from 1.65x10(-5) to 2.91x10(-5) and from 3.40x10(-5) to 4.70x10(-5), respectively. Pooling data from 4-ABP-treated and control mice, the approximately 2-fold increase in MF observed in Pms2-deficient as compared with Pms2-proficient mice was statistically significant (P=0.0207) and consistent with what has been reported previously in terms of induction of G:C-->T:A mutation in a Pms2-deficient background. Pooling data from both genotypes, the increase in H-ras MF in 4-ABP-treated mice, as compared with control mice, did not reach the 95% confidence level of statistical significance (P=0.0606). The 4-ABP treatment caused a 1.76-fold and 1.38-fold increase in average H-ras MF in Pms2-proficient and Pms2-deficient mice, respectively. Furthermore, the levels of induced mutation in Pms2-proficient and Pms2-deficient mice were nearly identical (1.26x10(-5) and 1.30x10(-5), respectively). We conclude that Pms2-deficiency does not result in an amplification of the H-ras codon 61 CAA-->AAA mutational response induced by 4-ABP.

The utility of liver H-ras codon 61 CAA to AAA mutant fraction as a biomarker of liver tumor development was investigated using neonatal male mice treated with 4-aminobiphenyl (4-ABP). Treatment with 0.1, 0.3, or 1.0 mumol 4-ABP produced dose-dependent increases in liver DNA adducts in B6C3F(1) and C57BL/6N mice. Eight months after treatment with 0.3 mumol 4-ABP or the DMSO vehicle, H-ras codon 61 CAA to AAA mutant fraction was measured in liver DNA samples (n = 12) by allele-specific competitive blocker-polymerase chain reaction (ACB-PCR). A significant increase in average mutant fraction was found in DNA of 4-ABP-treated mice, with an increase from 1.3 x 10(-5) (control) to 44.9 x 10(-5) (treated) in B6C3F(1) mice and from 1.4 x 10(-5) to 7.0 x 10(-5) in C57BL/6N mice. Compared with C57BL/6N mutant fractions, B6C3F(1) mutant fractions were more variable and included some particularly high mutant fractions, consistent with the more rapid development of liver foci expected in B6C3F(1) mouse liver. Twelve months after treatment, liver tumors developed in 79.2% of 4-ABP-treated and 22.2% of control B6C3F(1) mice; thus measurement of H-ras mutant fraction correlated with subsequent tumor development. This study demonstrates that ACB-PCR can directly measure background levels of somatic oncogene mutation and detect a carcinogen-induced increase in such mutation.

A statistical method for parametric density estimation based upon a mixture-of-genotypes model is developed for the thermostable phenol sulfotransferase (SULT1A1) activity which has a putative role in modifying risk for colon and prostate cancer/polyps. The EM algorithm for the general mixture model is modified to accommodate the genetic constraints and is used to estimate genotype frequencies from the distribution of the SULT1A1 phenotype. A parametric bootstrap likelihood ratio test is considered as a testing method for the number of mixing components. The size and power of the test is then investigated and compared with the conventional chi-squared test. The relative risk associated with genotypes defined by this model is also investigated through the generalized linear model. This analysis revealed that a genotype with the highest mean value of SULT1A1 activity has greater impact on cancer risk than others. This result suggests that the phenotype with a higher SULT1A1 activity might be important in studying the association between the cancer risk and SULT1A1 activity. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

cDNA arrays allow quantitative measurement of expression levels for thousands of genes simultaneously. The measurements are affected by many sources of variation, and substantial improvements in the precision of estimatedeffects accompany adjustments for these effects. Two generic nuisance variations, one associated with the magnitude of expression and the other associated with array location, are common in data from filter arrays. Procedures, like normalization using lowess regression, are effective at reducing variation associated with magnitude, and they have been widely adopted. However, variation associated with location has received less attention. Here, a simple, but effective method based on localized median is expounded for dealing with these nuisance effects, and its properties are discussed. The proposed methodology handles location-dependent variation ("splotches") and magnitude-dependent variation (background and/or saturation) effectively. The procedure is related to lowess when implemented to adjust magnitude-dependent variation, and it performs similarly. The proposed methodology is illustrated with data from the National Center for Toxicological Research (NCTR), where treatment differences in levels of mRNA from rat hepatocytes were assessed using 3 3 P-labeled samples hybridized to cDNA spotted arrays. Normalizing intensities by the median-of-subsets removes systematic variation associated with the location of a gene on the array and/or the level of its expression. This procedure is easy to implement using iteratively reweighted least-squares algorithms. Although less sophisticated than lowess, this procedure works nearly as well for normalizing intensities based upon their magnitude. Unlike lowess, it can adjust for location-dependent effects.

Single-burst analysis was applied to a forward assay for gene A mutation in splenic lymphocytes of phiX174 transgenic mice for the purpose of optimizing analytical parameters for identifying in vivo mutations. The effect of varying the cutoff value for an in vivo burst on induced mutant frequency, fold increase, and the significance of the difference between control and N-ethyl-N-nitrosourea (ENU)-treated mice was calculated by two different methods. The plating density was reduced to an average of less than 10 background mutant plaques per aliquot in order to separate in vitro bursts. The spectrum of mutations contributing60 plaques per aliquot from control animals was not significantly different from the control spectra from E. coli or transgenic phiX174 cells in culture. The mutant spectra from ENU-treated animals was highly different between mutant bursts of80 plaques per aliquot compared to mutations contributing60 plaques per aliquot (P0.000001), the former fitting the spectrum expected for ENU-induced mutations. The latter spectrum was also different from control animals and E. coli (P0.000001), suggesting the difference was caused by ex vivo mutation. With the mutations found in this study, the total number of reported target sites for gene A is now 33. The results support the interpretation that, in contrast to results for the lacI transgene, 100% of mutants isolated in gene A from control animals and cells were fixed in E. coli. We attribute the difference between the two genes to hot-spot sites for mutation in gene A and to a testable hypothesis that the mosaic plaque assay for the lacI transgene underestimates the frequency of ex vivo mutants.

Cultured primary hepatocytes are one of the most suitable in vitro models for hepatic toxicological studies. Unfortunately, there is a temporal loss of liver-specific function in culture that limits their utility for some applications. Plating hepatocytes on a substratum has been shown to stabilize the differentiated phenotype for short-term culture. In order to identify the substratum that best supports in vivo basal hepatocyte gene expression profiles in vitro, the gene expression profiles of primary rat hepatocytes plated on collagen I in hepatocyte maintenance medium (HMM) or hepatocyte culture medium (HCM), or on matrigel in HMM medium for 2 h, 16 h, or 72 h were compared to the expression profiles of freshly isolated rat hepatocytes using the Atlas rat stress array. After 16 h in culture, there were differences in gene expression between cultured primary hepatocytes and freshly isolated hepatocytes, but no apparent substratum effects. At 72 h, the expression of 9 genes was altered in hepatocytes plated on either substratum compared to expression in freshly isolated hepatocytes. However, there were an additional 13 genes with increased expression in hepatocytes plated on collagen I that were expressed at low or non-detectable levels in freshly isolated hepatocytes or primary hepatocytes plated on matrigel. In summary, after 72 h, primary hepatocytes plated on matrigel had basal gene expression patterns more similar to patterns in freshly isolated hepatocytes than did hepatocytes cultured on collagen. In addition, culture on matrigel suppressed the expression of atypical genes in culture. These preliminary studies suggest that culture on matrigel may be preferable for longer-term in vitro toxicological studies.

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.

Transgenic systems for measuring mammalian mutagenesis often use recoverable viral vectors. We hypothesize that mutations in these transgenic systems can arise from three different origins of DNA damage and replication errors and that these three origins of mutations (in vivo, ex vivo, and in vitro) can be differentiated in the PhiX174 am3, cs70 single burst assay (SBA) on the basis of burst size (BS). In vivo mutations are fixed in the animal, ex vivo mutations are fixed in bacterial cells during recovery of the phage, and in vitro revertants arise during the first replications of nonmutant phages under selective conditions. PX-2 cells, derived from a homozygous embryo of a PhiX174 transgenic mouse, were treated with vehicle or N-ethyl-N-nitrosourea (ENU). An algorithm was developed to estimate the BS that resulted in the highest induced revertant frequency; the estimate was 56. In vivo revertants were defined as having BS >55, ex vivo revertants as having a BS of 13-56, and in vitro revertants as having a BS of