Use of Electronic Nicotine Delivery Systems (ENDS) by pregnant women I: Risk of small-for-gestational-age birth.

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.

Morbidity and mortality from farm tractor-related injuries in Arkansas.

This study applied a text string search algorithm to ascertain suspect farm tractor or agricultural machinery-related injuries in data sources available for 2000-2014 in the state of Arkansas. The occurrences of tractor or other agricultural machinery-related injuries were compared with data available from the Centers for Disease Control and Prevention's National Center for Health Statistics (NCHS) and the Bureau of Labor Statistics' Census of Fatal Occupational Injuries (CFOI). For death certificates that assigned an external cause of death, the authors first collected all those that were coded as related to agricultural machinery, based on search strings for occupation and industry and a description of how the injury occurred. They then inspected each case individually and removed those that were likely unrelated to agricultural machinery. This approach significantly increased (by 7.8 times) the number of suspect agricultural machinery-related fatalities compared to the number reported to CFOI, but there was only a 17% (not statistically significant) increase compared to NCHS. All hospital records with any discharge diagnosis coded as related to agricultural machinery were selected. Descriptive analysis of the fatalities and hospital records showed a significantly increased risk among men above retirement age, peaks during the summer, and an increased risk in the Mississippi delta region. About one-third of the agricultural machinery-related fatalities were due to overturns. The use of the algorithm can improve ascertainment of fatal agricultural machinery-related injuries in Arkansas. The death records were found to be rich in data on the circumstances of the injuries, which can be used to screen for tractor-related fatalities and, if confirmed, translated into action to improve the safety of Arkansas farmers.

Electronic Nicotine Delivery Systems and Smoking Cessation in Arkansas, 2014.

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.

Use of electronic nicotine delivery systems and recent initiation of smoking among US youth.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: Use of ENDS by adolescents was associated with initiation of cigarette smoking in the last year. This association was stronger in younger adolescents.

Transcriptome profiling of peripheral blood cells identifies potential biomarkers for doxorubicin cardiotoxicity in a rat model.

AIMS: Doxorubicin (DOX), a widely used anticancer agent, can cause an unpredictable cardiac toxicity which remains a major limitation in cancer chemotherapy. There is a need for noninvasive, sensitive and specific biomarkers which will allow identifying patients at risk for DOX-induced cardiotoxicity to prevent permanent cardiac damage. The aim of this study was to investigate whether the expression of specific genes in the peripheral blood can be used as surrogate marker(s) for DOX-induced cardiotoxicity. METHODS/RESULTS: Rats were treated with a single dose of DOX similar to one single dose that is often administered in humans. The cardiac and peripheral blood mononuclear cells (PBMCs) genome-wide expression profiling were examined using Illumina microarrays. The results showed 4,409 differentially regulated genes (DRG) in the hearts and 4,120 DRG in PBMC. Of these 2411 genes were similarly DRG (SDRG) in both the heart and PBMC. Pathway analysis of the three datasets of DRG using Gene Ontology (GO) enrichment analysis and Ingenuity Pathways Analysis (IPA) showed that most of the genes in these datasets fell into pathways related to oxidative stress response and protein ubiquination. IPA search for potential eligible biomarkers for cardiovascular disease within the SDRG list revealed 188 molecules. CONCLUSIONS: We report the first in-depth comparison of DOX-induced global gene expression profiles of hearts and PBMCs. The high similarity between the gene expression profiles of the heart and PBMC induced by DOX indicates that the PBMC transcriptome may serve as a surrogate marker of DOX-induced cardiotoxicity. Future directions of this research will include analysis of PBMC expression profiles of cancer patients treated with DOX-based chemotherapy to identify the cardiotoxicity risk, predict DOX-treatment response and ultimately to allow individualized anti-cancer therapy.

Gene categories differentially expressed in C. elegans age-1 mutants of extraordinary longevity: new insights from novel data-mining procedures.

Two nonsense mutants of age-1, the Caenorhabditis elegans gene encoding phosphoinositide 3-kinase, live nearly 10-fold longer than wild-type controls and are exceptionally resistant to several stresses. Genome-wide expression analyses implicated downregulation of many more genes than were upregulated in second-generation age-1 homozygotes. Functional-annotation analysis, based on Gene Ontology terms, suggested that novel mechanisms may mediate the stronger phenotypes observed for these worms than with milder age-1 disruption. For the current study, the same microarray data were reanalyzed using novel meta-analytic procedures that we developed recently. First, gene p values were corrected for systematic biases based on the observed distribution for nonexpressed genes; these values were then combined to derive an aggregate p value for each functional-annotation term while adjusting for intergene covariance. This resulted in much better coverage of relevant gene categories, including many that were independently supported by other data. The number of nonredundant GO categories significantly distinguishing age-1 alleles of exceptional longevity increased from sevenfold to greater than ninefold, improving both sensitivity and specificity of selection for altered pathways and implicating previously unsuspected longevity mechanisms. Of 150 genes whose differential expression underlay significant GO terms in both comparisons, over half were up- or down-regulated in accord with longevity, whereas one third showed altered expression uniquely in the longest-lived age-1-null strains, consistent with the activation or suppression of pathways peculiar to strong age-1 mutants.

In vivo mutation analysis using the ΦX174 transgenic mouse and comparisons with other transgenes and endogenous genes.

The ΦX174 transgenic mouse was first developed as an in vivo Ames test, detecting base pair substitution (bps) at a single bp in a reversion assay. A forward mutational assay was also developed, which is a gain of function assay that also detects bps exclusively. Later work with both assays focused on establishing that a mutation was fixed in vivo using single-burst analysis: determining the number of mutant progeny virus from an electroporated cell by dividing the culture into aliquots before scoring mutants. We review results obtained from single-burst analysis, including testing the hypothesis that high mutant frequencies (MFs) of G:C to A:T mutation recovered by transgenic targets include significant numbers of unrepaired G:T mismatches. Comparison between the ΦX174 and lacI transgenes in mouse spleen indicates that the spontaneous bps mutation frequency per nucleotide (mf(n)) is not significantly lower for ΦX174 than for lacI; the response to ENU is also comparable. For the lacI transgene, the spontaneous bps mf(n) is highly age-dependent up to 12 weeks of age and the linear trend extrapolates at conception to a frequency close to the human bps mf(n) per generation of 1.7 × 10(-8). Unexpectedly, we found that the lacI somatic (spleen) bps mf(n) per cell division at early ages was estimated to be the same as for the human germ-line. The bps mf(n) in bone marrow for the gpt transgene is comparable to spleen for the lacI and ΦX174 transgenes. We conclude that the G:C to A:T transition is characteristic of spontaneous in vivo mutation and that the MFs measured in these transgenes at early ages reflect the expected accumulation of in vivo mutation typical of endogenous mammalian mutation rates. However, spontaneous and induced mf(n)s per nucleotide for the cII gene in spleen are 5-10 times higher than for these other transgenes.

ACB-PCR quantification of K-RAS codon 12 GAT and GTT mutant fraction in colon tumor and non-tumor tissue.

K-RAS mutation is being developed as a cancer biomarker and tumor K-RAS is being used to predict therapeutic response. Yet, levels of K-RAS mutation in normal and pathological tissue samples have not been determined rigorously, nor inter-individual variation in these levels characterized. Therefore, K-RAS codon 12 GAT and GTT mutant fractions were measured in colonic mucosa of individuals without colon cancer, tumor-distal mucosa, tumor-proximal mucosa, normal tumor-adjacent tissues, colonic adenomas, and carcinomas. The results indicate K-RAS codon 12 GAT mutation is present at measurable levels in normal appearing mucosa. All tumors carried K-RAS mutation, in most cases as a mutant subpopulation.

The mRNA expression and histological integrity in rat forebrain motor and sensory regions are minimally affected by acrylamide exposure through drinking water.

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.

Testing for treatment effects on gene ontology.

In studies that use DNA arrays to assess changes in gene expression, it is preferable to measure the significance of treatment effects on a group of genes from a pathway or functional category such as gene ontology terms (GO terms, http://www.geneontology.org) because this facilitates the interpretation of effects and may markedly increase significance. A modified meta-analysis method to combine p-values was developed to measure the significance of an overall treatment effect on such functionally-defined groups of genes, taking into account the correlation structure among genes. For hypothesis testing that allows gene expression to change in both directions, p-values are calculated under the null distribution generated by a Monte Carlo method. As a test of this procedure, we attempted to distinguish altered pathways in microarray studies performed with Mitochips, oligonucleotide microarrays specific to mitochondrial DNA-encoded transcripts. We found that our analytic method improves the specificity of selection for altered pathways, due to incorporation of the inter-gene correlation structure in each pathway. It is thus a practical method to measure treatment effects on GO groups. In many actual applications, microarray experiments measure treatment effects under complicated design structures and with small sample sizes. For such applications to real data of limited statistical power, and also in computer simulations, we demonstrate that our method gives reasonable test results.

Frequency and spectrum of ENU-induced mutation in the Phi X174 transgene in mouse splenic lymphocytes and their significance to spontaneous transgenic rodent mutation frequencies.

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.

Populations of p53 codon 270 CGT to TGT mutant cells in SKH-1 mouse skin tumors induced by simulated solar light.

The p53 codon 270 CGT to TGT mutation was investigated as a biomarker of sunlight-induced mutagenesis and carcinogenesis. The relationship between tumor development and abundance of this hotspot mutation was analyzed in mouse skin tumors induced by chronic exposure to simulated solar light (SSL). The 24 tumors analyzed had similar growth kinetics, with an average doubling time of approximately 16.4 d. Levels of the p53 codon 270 mutation were quantified in the 24 mouse skin tumors using allele-specific competitive blocker-polymerase chain reaction (ACB-PCR). All tumors contained measurable amounts of the mutation. The p53 codon 270 CGT to TGT mutant fraction (MF) ranged from 2.29 x 10(-3) to 9.42 x 10(-2), with 3.26 x 10(-2) as the median. These p53 MF measurements are lower than expected for an initiating mutation involved in the development of tumors of monoclonal origin. There was no evidence of a correlation between p53 codon 270 MF and either tumor area or an estimate of tumor cell number. Thus, the data do not support the idea that p53 mutation accumulates linearly during tumor development. To investigate how p53 mutation was distributed within tumors, 19 needle biopsies from seven different tumors were analyzed by ACB-PCR. This analysis demonstrated that p53 codon 270 mutation is heterogeneously distributed within tumors. The long-term goal of this research is to combine morphological and p53 MF measurements from tissues corresponding to the various stages of tumor development, in order to derive mathematical models relating the p53 codon 270 mutation to the development of SSL-induced skin tumors.

Simulated solar light-induced p53 mutagenesis in SKH-1 mouse skin: a dose-response assessment.

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.

Nucleoside reverse transcriptase inhibitors (NRTIs)-induced expression profile of mitochondria-related genes in the mouse liver.

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 100mg/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.

Designing toxicogenomics studies that use DNA array technology.

BACKGROUND: Bioassays are routinely used to evaluate the toxicity of test agents. Experimental designs for bioassays are largely encompassed by fixed effects linear models. In toxicogenomics studies where DNA arrays measure mRNA levels, the tissue samples are typically generated in a bioassay. These measurements introduce additional sources of variation, which must be properly managed to obtain valid tests of treatment effects. RESULTS: An analysis of covariance model is developed which combines a fixed-effects linear model for the bioassay with important variance components associated with DNA array measurements. These models can accommodate the dominant characteristics of measurements from DNA arrays, and they account for technical variation associated with normalization, spots, dyes, and batches as well as the biological variation associated with the bioassay. An example illustrates how the model is used to identify valid designs and to compare competing designs. CONCLUSIONS: Many toxicogenomics studies are bioassays which measure gene expression using DNA arrays. These studies can be designed and analyzed using standard methods with a few modifications to account for characteristics of array measurements, such as multiple endpoints and normalization. As much as possible, technical variation associated with probes, dyes, and batches are managed by blocking treatments within these sources of variation. An example shows how some practical constraints can be accommodated by this modelling and how it allows one to objectively compare competing designs.

Reproducibility of microarray data: a further analysis of microarray quality control (MAQC) data.

BACKGROUND: Many researchers are concerned with the comparability and reliability of microarray gene expression data. Recent completion of the MicroArray Quality Control (MAQC) project provides a unique opportunity to assess reproducibility across multiple sites and the comparability across multiple platforms. The MAQC analysis presented for the conclusion of inter- and intra-platform comparability/reproducibility of microarray gene expression measurements is inadequate. We evaluate the reproducibility/comparability of the MAQC data for 12901 common genes in four titration samples generated from five high-density one-color microarray platforms and the TaqMan technology. We discuss some of the problems with the use of correlation coefficient as metric to evaluate the inter- and intra-platform reproducibility and the percent of overlapping genes (POG) as a measure for evaluation of a gene selection procedure by MAQC. RESULTS: A total of 293 arrays were used in the intra- and inter-platform analysis. A hierarchical cluster analysis shows distinct differences in the measured intensities among the five platforms. A number of genes show a small fold-change in one platform and a large fold-change in another platform, even though the correlations between platforms are high. An analysis of variance shows thirty percent of gene expressions of the samples show inconsistent patterns across the five platforms. We illustrated that POG does not reflect the accuracy of a selected gene list. A non-overlapping gene can be truly differentially expressed with a stringent cut, and an overlapping gene can be non-differentially expressed with non-stringent cutoff. In addition, POG is an unusable selection criterion. POG can increase or decrease irregularly as cutoff changes; there is no criterion to determine a cutoff so that POG is optimized. CONCLUSION: Using various statistical methods we demonstrate that there are differences in the intensities measured by different platforms and different sites within platform. Within each platform, the patterns of expression are generally consistent, but there is site-by-site variability. Evaluation of data analysis methods for use in regulatory decision should take no treatment effect into consideration, when there is no treatment effect, "a fold-change cutoff with a non-stringent p-value cutoff" could result in 100% false positive error selection.

Significance analysis of groups of genes in expression profiling studies.

MOTIVATION: Gene class testing (GCT) is a statistical approach to determine whether some functionally predefined classes of genes express differently under two experimental conditions. GCT computes the P-value of each gene class based on the null distribution and the gene classes are ranked for importance in accordance with their P-values. Currently, two null hypotheses have been considered: the Q1 hypothesis tests the relative strength of association with the phenotypes among the gene classes, and the Q2 hypothesis assesses the statistical significance. These two hypotheses are related but not equivalent. METHOD: We investigate three one-sided and two two-sided test statistics under Q1 and Q2. The null distributions of gene classes under Q1 are generated by permuting gene labels and the null distributions under Q2 are generated by permuting samples. RESULTS: We applied the five statistics to a diabetes dataset with 143 gene classes and to a breast cancer dataset with 508 GO (Gene Ontology) terms. In each statistic, the null distributions of the gene classes under Q1 are different from those under Q2 in both datasets, and their rankings can be different too. We clarify the one-sided and two-sided hypotheses, and discuss some issues regarding the Q1 and Q2 hypotheses for gene class ranking in the GCT. Because Q1 does not deal with correlations among genes, we prefer test based on Q2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Development of mitochondria-specific mouse oligonucleotide microarray and validation of data by real-time PCR.

This study describes the development of a mitochondria-specific microarray, MitoChip, to measure transcripts of mitochondria-associated genes in various diseases and drug-induced toxicities in the mouse. The array consists of 542 oligonucleotides that represent genes from the mitochondrial and nuclear genomes associated with mitochondrial structure and functions. The expression of mitochondrial genes was measured in the liver of both p53 haplodeficient (+/-) and wild-type (+/+) C3B6F(1) female mice exposed to antiretroviral agents, Zidovudine (AZT) and Lamivudine (3TC). Among genes whose expression was significantly altered, a set was selected for real-time PCR analysis to verify their differential gene expression. The real-time PCR data confirmed the observations by microarray analysis suggesting that the MitoChip may be an important tool for examining mitochondrial involvement in diseases and drug-induced toxicities.

ACB-PCR measurement of K-ras codon 12 mutant fractions in livers of Big Blue rats treated with N-hydroxy-2-acetylaminofluorene.

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.