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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. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none was reported.

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.

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.

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.

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.

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., (Published by Elsevier B.V.)

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.

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.

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.

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.

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.

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., ((c) 2008 Wiley-Liss, Inc.)

Acrylamide (AA) is an important industrial chemical that is neurotoxic in rodents and humans and carcinogenic in rodents. The observation of cancer in endocrine-responsive tissues in Fischer 344 rats has prompted hypotheses of hormonal dysregulation, as opposed to DNA damage, as the mechanism for tumor induction by AA. The current investigation examines possible evidence for disruption of the hypothalamic-pituitary-thyroid axis from 14 days of repeated exposure of male Fischer 344 rats to doses of AA that range from one that is carcinogenic after lifetime exposure (2.5 mg/kg/d), an intermediate dose (10 mg/kg/d), and a high dose (50 mg/kg/d) that is neurotoxic for this exposure time. The endpoints selected include: serum levels of thyroid and pituitary hormones; target tissue expression of genes involved in hormone synthesis, release, and receptors; neurotransmitters in the CNS that affect hormone homeostasis; and histopathological evaluation of target tissues. These studies showed virtually no evidence for systematic alteration of the hypothalamic-pituitary-thyroid axis and do not support hormone dysregulation as a plausible mechanism for AA-induced thyroid cancer in the Fischer 344 rat. Specifically, there were no significant changes in: 1) mRNA levels in hypothalamus or pituitary for TRH, TSH, thyroid hormone receptor alpha and beta, as well 10 other hormones or releasing factors; 2) mRNA levels in thyroid for thyroglobulin, thyroid peroxidase, sodium iodide symporter, or type I deiodinases; 3) serum TSH or T3 levels (T4 was decreased at high dose only); 4) dopaminergic tone in the hypothalamus and pituitary or importantly 5) increased cell proliferation (Mki67 mRNA and Ki-67 protein levels were not increased) in thyroid or pituitary. These negative findings are consistent with a genotoxic mechanism of AA carcinogenicity based on metabolism to glycidamide and DNA adduct formation. Clarification of this mechanistic dichotomy may be useful in human cancer risk assessments for AA.

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.

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.

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.

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.

Compulsive drug abuse has been conceptualized as a behavioral state where behavioral stimuli override normal decision making. Clinical studies of methamphetamine users have detailed decision making changes and imaging studies have found altered metabolism and activation in the parietal cortex. To examine the molecular effects of amphetamine (AMPH) on the parietal cortex, gene expression responses to amphetamine challenge (7.5 mg/kg) were examined in the parietal cortex of rats pretreated for nine days with either saline, non-neurotoxic amphetamine, or neurotoxic AMPH dosing regimens. The neurotoxic AMPH exposure [three doses of 7.5 mg/kg/day AMPH (6 h between doses), for nine days] produced histological signs of neurotoxicity in the parietal cortex while a non-neurotoxic dosing regimen (2.0 mg/kg/day x 3) did not. Neurotoxic AMPH pretreatment resulted in significantly diminished AMPH challenge-induced mRNA increases of activity-regulated cytoskeletal protein (ARC), nerve growth-factor inducible protein A (NGFI-A), and nerve growth-factor inducible protein B (NGFI-B) in the parietal cortex while neither saline pretreatment nor non-neurotoxic AMPH pretreatment did. This effect was specific to these genes as tissue plasminogen activator (t-PA), neuropeptide Y (NPY) and c-jun expression in response to AMPH challenge was unaltered or enhanced by amphetamine pretreatments. In the striatum, there were no differences between saline, neurotoxic AMPH, and non-neurotoxic AMPH pretreatments on ARC, NGFI-A or NGFI-B expression elicited by the AMPH challenge. These data indicate that the responsiveness of synaptic plasticity-related genes is sensitive to disruption specifically in the parietal cortex by threshold neurotoxic AMPH exposures.

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.

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.

Background: Gender differences in gene expression were estimated in liver samples from 9 males and 9 females. The study tested 31,110 genes for a gender difference using a design that adjusted for sources of variation associated with cDNA arrays, normalization, hybridizations and processing conditions., Results: The genes were split into 2,800 that were clearly expressed (expressed genes) and 28,310 that had expression levels in the background range (not expressed genes). The distribution of p-values from the 'not expressed' group was consistent with no gender differences. The distribution of p-values from the 'expressed' group suggested that 8% of these genes differed by gender, but the estimated fold-changes (expression in males/expression in females) were small. The largest observed fold-change was 1.55. The 95% confidence bounds on the estimated fold-changes were less than 1.4 fold for 79.3%, and few (1.1%) exceed 2-fold., Conclusion: Observed gender differences in gene expression were small. When selecting genes with gender differences based upon their p-values, false discovery rates exceed 80% for any set of genes, essentially making it impossible to identify any specific genes with a gender difference.

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., (Published 2005 Wiley-Liss, Inc.)

The roles that glutamate N-methyl-D-aspartate (NMDA) and dopamine D1-like and D2-like receptors play in the cortical neurotoxicity occurring in rats exposed to multiple doses of amphetamine (AMPH) for 2 days was evaluated. Neurodegeneration in rats that did not become hyperthermic during AMPH exposure was quantified by counting isolectin B4-labeled phagocytic microglia and Fluoro-Jade (F-J)-labeled neurons in the somatosensory parietal cortex, piriform cortex and posterolateral cortical amygdaloid nucleus (PLCo). The NMDA receptor antagonist, dizocilpine (0.63 mg/kg day) blocked AMPH-induced neurodegeneration in the somatosensory cortex. However, it did not affect degeneration in the piriform cortex and PLCo indicating that limbic degeneration was not NMDA-mediated. The dopamine antagonists, eticlopride (D2/3, 0.25 mg/kg day) and SCH-23390 (D1, 0.25 mg/kg day), blocked the stereotypic behavior and neurodegeneration in the somatosensory cortex. However, eticlopride had a lesser protective effect in the limbic regions. As well, the dopamine D2/D3 agonist quinpirole (1.5 mg/kg day) protected against cortical neurodegeneration when it was given during AMPH exposure and continued until sacrifice. The dopamine D1 agonist (SKF-38393, 12.5 mg/kg day) had no significant effect on neurodegeneration. These data indicate that there are significant differences in NMDA and dopamine D2 modulation of AMPH-induced neurodegeneration in the somatosensory cortex compared to the limbic cortices, and limbic cortical degeneration is not necessarily dependent on excessive stimulation of NMDA receptors as it is in the somatosensory cortex. Although excessive dopamine receptor stimulation during amphetamine exposure may trigger the neurodegenerative processes, continued D2 stimulation after AMPH exposure is neuroprotective in the cortex.

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 estimated effects 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 33P-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.

An objective of many functional genomics studies is to estimate treatment-induced changes in gene expression. cDNA arrays interrogate each tissue sample for the levels of mRNA for hundreds to tens of thousands of genes, and the use of this technology leads to a multitude of treatment contrasts. By-gene hypotheses tests evaluate the evidence supporting no effect, but selecting a significance level requires dealing with the multitude of comparisons. The p-values from these tests order the genes such that a p-value cutoff divides the genes into two sets. Ideally one set would contain the affected genes and the other would contain the unaffected genes. However, the set of genes selected as affected will have false positives, i.e., genes that are not affected by treatment. Likewise, the other set of genes, selected as unaffected, will contain false negatives, i.e., genes that are affected. A plot of the observed p-values (1 - p) versus their expectation under a uniform [0, 1] distribution allows one to estimate the number of true null hypotheses. With this estimate, the false positive rates and false negative rates associated with any p-value cutoff can be estimated. When computed for a range of cutoffs, these rates summarize the ability of the study to resolve effects. In our work, we are more interested in selecting most of the affected genes rather than protecting against a few false positives. An optimum cutoff, i.e., the best set given the data, depends upon the relative cost of falsely classifying a gene as affected versus the cost of falsely classifying a gene as unaffected. We select the cutoff by a decision-theoretic method analogous to methods developed for receiver operating characteristic curves. In addition, we estimate the false discovery rate and the false nondiscovery rate associated with any cutoff value. Two functional genomics studies that were designed to assess a treatment effect are used to illustrate how the methods allowed the investigators to determine a cutoff to suit their research goals.

Motivation: A microarray experiment is a multi-step process, and each step is a potential source of variation. There are two major sources of variation: biological variation and technical variation. This study presents a variance-components approach to investigating animal-to-animal, between-array, within-array and day-to-day variations for two data sets. The first data set involved estimation of technical variances for pooled control and pooled treated RNA samples. The variance components included between-array, and two nested within-array variances: between-section (the upper- and lower-sections of the array are replicates) and within-section (two adjacent spots of the same gene are printed within each section). The second experiment was conducted on four different weeks. Each week there were reference and test samples with a dye-flip replicate in two hybridization days. The variance components included week-to-week, animal-to-animal and between-array and within-array variances., Results: We applied the linear mixed-effects model to quantify different sources of variation. In the first data set, we found that the between-array variance is greater than the between-section variance, which, in turn, is greater than the within-section variance. In the second data set, for the reference samples, the week-to-week variance is larger than the between-array variance, which, in turn, is slightly larger than the within-array variance. For the test samples, the week-to-week variance has the largest variation. The animal-to-animal variance is slightly larger than the between-array and within-array variances. However, in a gene-by-gene analysis, the animal-to-animal variance is smaller than the between-array variance in four out of five housekeeping genes. In summary, the largest variation observed is the week-to-week effect. Another important source of variability is the animal-to-animal variation. Finally, we describe the use of variance-component estimates to determine optimal numbers of animals, arrays per animal and sections per array in planning microarray experiments.

Gene expression profiles in several brain regions of adult male rats were evaluated following a d-amphetamine (AMPH) exposure paradigm previously established to produce AMPH neurotoxicity. Escalating doses of AMPH (5-30 mg/kg) were given over the course of 16 h per day in an 18 degrees C environment for 2 days. This paradigm produces neurotoxicity but eliminates or minimizes the hyperthermia and seizure activity that might influence gene expression in a manner unrelated to the neurotoxic effects of AMPH. The expression of 1185 genes was monitored in the striatum, parietal cortex, piriform cortex and posteriolateral cortical amygdaloid nucleus (PLCo) using cDNA array technology, and potentially significant changes were verified by RT-PCR. Gene expression was determined at time points after AMPH when neurodegeneration was beginning to appear (16 h) or maximal (64 h). Expression was also determined 14 days after AMPH to find long-term changes in gene expression that might be biomarkers of a neurotoxic event. In the parietal cortex there was a two-fold increase in neuropeptide Y precursor protein mRNA whereas nerve growth factor-induced receptor protein I-A and I-B mRNA decreased 50% at 16 h after the end of AMPH exposure. Although these changes in expression were not observed in the PLCo, insulin-like growth factor binding protein 1 mRNA was increased two-fold in the PLCo at 16 and 64 h after AMPH. Changes in gene expression in the cortical regions were all between 1.2- and 1.5-fold 14 days after AMPH but some of these changes, such as annexin V increases, may be relevant to neurotoxicity. Gene expression was not affected by more than 1.5-fold at the time points in the striatum, although 65% dopamine depletions occurred, but the plasma membrane-associated dopamine transporter and dopamine D2 receptor were decreased about 40% in the substantia nigra at 64 h and 14 days post-AMPH. Thus, the 2-day AMPH treatment produced a few changes in gene expression in the two-fold range at time points 16 h or more after exposure but the majority of expression changes were less than 1.5-fold of control. Nonetheless, some of these lesser fold-changes appeared to be relevant to the neurotoxic process.

Daily, rhythmic variation in various biochemical, physiological, and behavioral events is a fundamental property of biological organization. Here, we report analysis of relative levels of gene expression in the liver of 16 Fischer 344 rats as a function of time of day. Expression levels were determined for 3906 genes using high-density oligonucleotide microarrays. Of the 3906 genes, 1171 (30%) were clearly expressed while 2735 (70%) were not expressed or the expression was too low to distinguish from background levels. The maximum estimated changes observed for most genes (1029, 88%) were less than 1.5-fold. Analysis of variance and the Kruskal-Wallis tests were used to identify 67 genes whose expression was significantly altered as a function of time of day. These significantly altered genes were classified according to their functions and fall into key cellular pathways including drug metabolism, ion transport, signal transduction, DNA binding and regulation of transcription, and immune response.

The use of transgenic targets for measuring mutant frequencies in mammalian tissue requires an estimate of the mutant frequency that results from recovery of the transgene in bacterial recovery systems. In this study, we have determined the spontaneous mutant frequency, estimated the mutation rate, and ascertained the mutation spectrum for gene A of phiX174 grown in E. coli strain CQ2 from 156 small independent cultures. The mutant frequency of 12 of the 156 cultures was 17 +/- 1.0 x 10(-6) and the estimated mutation rate per gene replication was 7.4 +/- 2.3 x 10(-6). The mutant frequency and spectrum from E. coli were not significantly different from that of solvent-treated embryonic mouse cells in culture, 19 +/- 0.5 x 10(-6) (Valentine CR et al. [2002]: Environ Mol Mutagen 39:55-68), indicating that those spontaneous mutants were primarily derived from E. coli. The E. coli spectrum was heavily weighted toward two major target sites (hot spots), 4225A-->G (56%) and 4218G-->A or C (20%). Four new target sites and one new mutational event were recovered by the gene A forward assay. A mutant spectrum from an expanded phage stock was also determined to assess the effects of propagating the virus. This mutant frequency was higher (6 x 10(-4)), contained more double mutants (15% compared to 0.6%), and had a significantly different spectrum from the spectrum for independent cultures (fewer A:T-->G:C and G:C-->C:G changes and more G:C-->A:T; P < 0.002). The E. coli mutation spectrum will be useful for determining the origin of gene A mutation in tissues of phiX174 transgenic mice., (Copyright 2004 Wiley-Liss, Inc.)

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 contributing < 60 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 of > 80 plaques per aliquot compared to mutations contributing < 60 plaques per aliquot (P < 0.000001), the former fitting the spectrum expected for ENU-induced mutations. The latter spectrum was also different from control animals and E. coli (P < 0.000001), 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., (Copyright 2004 Wiley-Liss, Inc.)

Modest changes in gene expression of three-fold or less might be expected after mild to moderate neurotoxic exposure to classes of compounds, such as the substituted amphetamines, or at time points that are weeks after more severe neurotoxic exposures. When many genes appear to change expression by less than two-fold, it is crucial to run several pairs of arrays and use statistical analysis to determine which genes are really changing. This limits the number of genes that have to undergo the time consuming task of performing RT-PCR to validate change in expression levels. We describe here methods for statistically determining which genes are being expressed above background levels. These methods are used to compare expression differences among the striatum, parietal cortex, posterior lateral amygdaloid nucleus, and substantia nigra brain regions, all of which differ significantly in their gene expression profiles. In these comparisons, it was possible to distinguish differences among hundreds of genes with manageable estimated false discovery rates. The effect of amphetamine treatment on gene expression in posterior lateral amygdaloid nucleus was also evaluated. The expression data indicate that many genes have changed, but in this case it is more difficult to separate affected genes from false positives. The optimum list has 50 genes, of which 32% are expected to be false positives.

Breast development, one of the first signs of puberty, is closely associated with age at menarche; and early menarche is in turn a well-established risk factor for female breast cancer. We examined the relationships between the onset of puberty and gene variants for certain enzymes that regulate hormone metabolism among 137 healthy nine-year-old girls from two pediatric clinics. High-activity CYP17 alleles, involved in estrogen formation, and high-activity CYP1A2 and CYP1B1 alleles, whose gene products metabolize estradiol, were not associated with pubertal stage. High activity CYP3A4, but not CYP3A5, which primarily metabolizes testosterone, showed a striking association with the onset of puberty (adjusted odds ratio, 3.21; 95% confidence interval, 1.62-6.89 for the genotype 0-1-2 rapid alleles). Of the homozygous CYP3A4*1B/1B girls, 90% had reached puberty; whereas, for the low-activity homozygous CYP3A4*1A/1A individuals, only 40% had done so. In heterozygotes, 56% had reached puberty. CYP1B1, CYP3A4, and CYP3A5 rapid variants were more common in African-American than in Hispanic or Caucasian girls.

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 <14. The frequencies of in vivo revertants at 0, 100, and 200 mg/kg ENU were 0.06, 0.36, and 4.10 x 10(-6) (dose response, P = 0.004); ex vivo revertants were 0.36, 0.46, and 0.41 x 10(-6) (P = 0.37), and in vitro revertants were 0.39, 0.46, and 0.41 x 10(-6) (P = 0.55), respectively. These results show that only in vivo revertants reflect mutagen treatment. They also provide a basis for identifying PhiX174 am3 revertants induced in vivo and may increase the sensitivity of the assay for in vivo mutation.

The sensitivity of in vivo transgenic mutation assays benefits from the sequencing of mutations, although the large number of possible mutations hinders high throughput sequencing. A forward mutational assay exists for Phi X174 that requires an altered, functional Phi X174 protein and therefore should have fewer targets (sense, base-pair substitutions) than forward assays that inactivate a protein. We investigated this assay to determine the number of targets and their suitability for detecting a known mutagen, N-ethyl-N-nitrosourea (ENU). We identified 25 target sites and 33 different mutations in Phi X174 gene A after sequencing over 350 spontaneous and ENU-induced mutants, mostly from mouse embryonic cell line PX-2 isolated from mice transgenic for Phi X174 am3, cs70 (line 54). All six types of base-pair substitution were represented among both the spontaneous and ENU-treated mutant spectra. The mutant spectra from cells treated with 200 and 400 microg/ml ENU were both highly different from the spontaneous spectrum (P < 0.000001) but not from each other. The dose trend was significant (P < 0.0001) for a linear regression of mutant frequencies (R(2) = 0.79), with a ninefold increase in mutant frequency at the 400 microg/ml dose. The spontaneous mutant frequency was 1.9 x 10(-5) and the spontaneous spectrum occurred at 11 target base pairs with 15 different mutations. Thirteen mutations at 12 targets were identified only from ENU-treated cells. Seven mutations had highly significant increases with ENU treatment (P < 0.0001) and 15 showed significant increases. The results suggest that the Phi X174 forward assay might be developed into a sensitive, inexpensive in vivo mutagenicity assay.

Genotypic selection methods detect rare sequence changes in populations of DNA molecules. These methods have been used to investigate the chemical induction of mutation and for the detection and diagnosis of cancer. The possible use of genotypic selection for improving current risk assessment practices is based on the premise that the frequency of somatic mutation is of critical importance in understanding and modeling carcinogenesis. If genotypic selection can measure the induction of specific mutations that disrupt normal cell/tissue homeostasis, then it could provide key mechanistic information for cancer risk assessment. For example, genotypic selection data might support a particular low-dose extrapolation method or characterize the relationship between rodent and human cancer risk. Strategies for evaluating the use of genotypic selection in cancer risk assessment include the concept of developing a battery of targets that detect a range of agent-specific effects. Ideal targets to examine by genotypic selection are the oncogene and tumor suppressor gene mutations frequently detected in human tumors because these are thought to represent tumor-initiating events. The most commonly occurring basepair (bp) substitutions within the ras and p53 genes are identified. Also, the battery of genotypic selection methods is defined in terms of the most important mutational specificities to include. In theory, the major basepair substitution mutations induced by 29 of 31 chemical carcinogens could be detected by analyzing three different mutations: G:C-->T:A, G:C-->A:T, and A:T-->T:A. Genotypic selection will have the greatest impact on risk assessment if measurement of spontaneous mutation is possible. Data from phenotypic selection assays suggest this corresponds to detection of mutant fractions of approximately 10(-7), and this would necessitate examining DNA samples containing >10(7) target molecules. Despite its apparent potential, considerable development and validation is needed before genotypic selection data can be applied to cancer risk assessment.

The erythrocyte concentrations of the body's chief physiologic methyl donor S-adenosylmethionine (SAM) and of its metabolite and inhibitor S-adenosylhomocysteine (SAH), the plasma concentrations of total homocysteine (tHcy), and the activity of N(5,10) methylenetetrahydrofolate reductase (MTHFR) in lymphocytes were determined in healthy subjects and patients with diabetes mellitus without complications and at various stages of diabetic nephropathy, categorized according to the degree of progression of the disease. These groups were as follows: 1, control; 2, diabetics with no complications; 3, patients with albuminuria; 4, patients with an elevated plasma creatinine; and 5, patients on dialysis. No parameter studied exhibited significant differences between the type 1 and the type 2 diabetics. In control subjects, the blood concentrations of SAM were proportional to the activity of MTHFR; in diabetics, it was not. Consistent with previous observations, progression of nephropathy was accompanied by increased concentrations of tHcy. Increased erythrocyte concentrations of SAH, decreased erythrocyte concentrations of SAM, SAM/SAH ratios, and lymphocyte MTHFR activity also accompanied disease progression. The blood concentrations of SAH paralleled those of tHcy, while the concentrations of SAM showed a bimodal relationship with those of tHcy. These results provide further evidence that alterations in the blood concentrations of SAM and related compounds are abnormal in patients with diabetes, particularly in those with nephropathy. The deficiency of SAM may lead to methyl deficiencies, which may contribute to the high morbidity and mortality in patients with diabetic nephropathy. We have also demonstrated a decrease in lymphocyte MTHFR activity in patients with advanced nephropathy, suggesting that hyperhomocysteinemia in these patients may be due to a generalized metabolic abnormality. Further studies are needed to determine the pathogenesis of these abnormalities and whether they are present in renal failure due to causes other than diabetes or whether they are specific to diabetic nephropathy., (Copyright 2001 by W.B. Saunders Company)

An increasing number of both clinical and experimental studies have shown an association between deficiencies of the dietary sources of physiological methyl groups and cancer formation. The critical metabolic intermediate in a determination of methylation status is S-adenosylmethionine (SAM), the body's chief physiological methyl donor. The present study examined the erythrocyte levels of SAM and of its demethylated metabolite S-adenosylhomocysteine (SAH) in 66 normal subjects (33 men and 33 women), whose blood had been drawn at days 0, 7 and 14 of an experimental period during which they were fed a fixed diet. The plasma levels of homocysteine (HCys) were also determined in the same individuals at the same time points. In addition, the subjects had completed a food frequency questionnaire (FFQ) describing their usual dietary habits before being placed on the dietary regimen. The blood levels of SAM, SAH, and HCys were compared with the dietary intakes of folate, vitamin B(6), fats, and calories, both prior to using the FFQ and during the experimental period. The results indicated that the intraindividual differences were very low, but the interindividual differences were large for the values of SAM, SAH, SAM:SAH ratios, and HCys. Interestingly, the blood levels of SAM and HCys were higher in men than in women and generally showed the expected correlations with folate intake i.e., positive for SAM and negative for HCys. The intakes of folate (276 microg/days) and B(6) (1.87 mg/days) during the 2-week experimental period were relatively low compared with the usual intakes of these vitamins (375 and 2.06 mg/day for folate and B(6), respectively) but correlated well with each other during both periods of the study. Surprisingly, both men and women showed a significant rise in erythrocyte SAM:SAH ratios as a function of age. In addition, the combined results from men and women, even adjusted for gender, showed significant correlations between HCys and both weight and body mass index. On the other hand, during the experimental period of the study, blood SAM levels were inversely correlated with the intakes of both fat and calories when the data for both men and women were combined and adjusted for gender. The blood determinations of SAM and related compounds showed a high degree of reproducibility over time and thus appear to provide a practical marker of methylation status for the assessment of cancer risk from dietary, environmental, and genetic factors.

Purpose: To investigate the associations between inflammation tests and radiation dose in A-bomb survivors., Subjects and Methods: Subjects were A-bomb survivors who underwent inflammation tests of leukocyte counts, neutrophil counts, erythrocyte sedimentation rate, corrected erythrocyte sedimentation rate, alpha-1 globulin, alpha-2 globulin and sialic acid between 1988 and 1992. Associations with radiation dose (DS86) were analyzed by regression analysis and heterogeneity among inflammatory diseases, anaemia at examination, or history of cancer was also tested., Results: The associations with radiation dose were statistically significant for leukocyte counts (71.0mm(-3) Gy(-1), p=0.015), erythrocyte sedimentation rate (1.58 mm h(-1) Gy(-1) , p = 0.0001), corrected erythrocyte sedimentation rate (1.14mm h(-1) Gy(-1), p=0.0001), alpha-1 globulin (0.0057 g dl(-1) Gy(-1), p=0.0001), alpha-2 globulin (0.0128 g dl(-1) Gy(-1), p=0.0001), and sialic acid (1.2711 mg dl(-1) Gy(-1), p=0.0001) but not for neutrophil counts (29.9 mm(-3) Gy(-1), p=0.17). Heterogeneity was not statistically significant. Among inflammatory diseases, associations were the strongest for chronic thyroiditis and chronic liver diseases., Conclusions: This study suggests statistically significant association between inflammation in A-bomb survivors and radiation dose of during 1988-1992. The association might contribute, as an epigenetic and/or bystander effect, to development of several radiation-induced disorders.

Data from the National Toxicology Program's carcinogenesis study of fumonisin B1 in B6C3F1 mice, conducted at the National Center for Toxicological Research, were used to fit the Moolgavkar-Venzon-Knudson (MVK) two-stage, clonal-expansion model of carcinogenesis. In addition to tumour data from the conventional 2-year bioassay, the study included data on tissue weights, cell proliferation, cell death, and sphingolipid metabolism in primary target organs. The model was used to predict 2-year liver tumour rates in female and male mice based on differences among dose groups in the effect of fumonisin B1 on the growth of normal tissue and on the proliferation of preneoplastic cells as a compensatory response to sphinganine-induced cell death. Fumonisin B1 was assumed to be non-genotoxic, i.e. the model did not include any effect of fumonisin B1 on either of the two mutation rates of the MVK model. The model was able to reproduce reasonably well the observed tumour rates in both female and male mice, predicting substantially increased rates above background only at the highest doses of fumonisin B1 in females.

NCTR measured sphinganine concentrations in the livers of mice and in the livers and kidneys of rats in conjunction with a tumour bioassay. In our model of the tumour incidence, target-tissue levels of sphinganine serve as a biomarker for a dose response of fumonisin B1 on cell death. Initially we questioned the utility of sphinganine levels in this role because they were highly variable when compared across time points. In spite of this concern, a conceptual framework and data are presented that support the use of sphinganine as a biomarker for a dose response of fumonisin B1 on cell death. This framework is reasonably consistent with observed sphinganine concentrations in the examined tissues, the literature on fumonisin's effects on sphingolipid synthesis, and our hypothesized mechanism through which fumonisin B1 increases age-specific tumour incidence.

Target genes in most transgenic systems have higher spontaneous mutation frequencies than do endogenous mammalian genes. Spontaneous mutations in transgenes predominantly arise from three sources: (1) mutations fixed in the animals, (2) mutations arising from replication errors caused by damage to the DNA that may have occurred in vivo or in vitro and then was fixed during amplification of the vector in vitro, and (3) mutations arising during replication of non-revertant phages in non-permissive bacteria. An assay based on single bursts was developed to directly distinguish between the in vivo and in vitro origins of revertants. The size of the aliquot is determined by mutant frequency and is adjusted so that ideally no more than 10 to 20% of the aliquots contain a bacterial cell transformed with a mutant phage. Mutations are detected as revertants of an amber mutation (am3) in phiX174 am3, cs70. The minimum burst size of non-revertant phiX am3, cs70 from splenic DNA on a permissive bacterial strain was larger than 30 plaque-forming units (pfu). Based on this observation, a burst size of 31 plaque-forming revertants was chosen as the minimum burst size of a fixed mutation. The single burst assay was tested on DNA from spleens of animals that were treated with 150 mg/kg 1-ethyl-1 nitrosurea. Only the fraction of aliquots with single bursts of revertants (> 30) increased in the treated animals compared to the controls. In contrast, there was no difference between treated and control animals for revertant frequencies calculated for burst sizes < or =30 pfu. Among the spontaneous mutations, only 30% were caused by mutations fixed in animals (i.e. burst size >30 pfu). Total average revertant frequency measured in DNA from treated animals was less than twofold more than the average spontaneous frequency (P = 0.048). When frequencies were based on burst sizes >30, there was a 4.6-fold increase among treated animals compared with controls (P = 0.026). The single burst-assay resulted in a more sensitive test for mutagenicity because it eliminated noise from in-vitro mutations.

In mutation assays using transgenic mice, with recoverable vectors such as PhiX174 am3, cs70, mutations originate from two sources: (1) in vivo mutations, that is, mutations that were fixed in the mouse, or (2) ex vivo mutations, that is, mutations that were fixed during recovery or plating. When a bacteriophage infects a bacterium, it multiplies and bursts the cell, releasing a number of phages referred to as the burst size. Our method for distinguishing between in vivo mutations and ex vivo mutations estimates the average number of bursts, the denominator of in vivo mutant frequencies, by dividing the total plaque-forming units (PFU) by the average number of phages in a burst. Herein, we outline a probability model relating observed plaque counts to the burst size and present the statistical method used to estimate the burst size. The average size of a single burst from nonrevertant phages was estimated in eight studies under the conditions of our mutation assay. The average burst size was stable across studies at 182.5 plaques per burst (standard error, 14.25). The probability that a burst is a specific size was approximated by a negative binomial distribution, which implies a Poisson-Pascal distribution for the observed plaque counts. The observed plaque counts were adequately fit by this approximation. Environ. Mol. Mutagen. 37:356-360, 2001 Published 2001 Wiley-Liss, Inc.

A thermostable phenol sulfotransferase, SULT1A1, has been implicated in numerous detoxification and bioactivation pathways; however, little is known regarding its endogenous function or its putative role in mediating risk for human environmental disease. A simple endpoint colorimetric assay is described that can be used for rapid phenotyping of SULT1A1 activity in human populations. The assay utilizes a microtiter-plate format and relatively small amounts of platelet cytosol-derived enzyme. The enzyme catalyzes the synthesis of 2-naphthylsulfate from 2-naphthol and 5'-phosphoadenosine 3'-phosphosulfate (PAPS), whereas addition of p-nitrophenyl sulfate to the assay contributes to an effective PAPS-regenerating system. In contrast to other sulfotransferase assay methods, 3'-phosphoadenosine 5'-phosphate (PAP) does not accumulate during the incubation to interfere with enzyme activity, but instead serves as a cofactor to cause the removal of sulfate from p-nitrophenyl sulfate to regenerate PAPS. This reaction concomitantly results in generation of p-nitrophenol that can be quantified colorimetrically at 405 nm (epsilon = 18,200 M(-1)) to give an indirect measure of sulfotransferase activity. Using platelet enzyme preparations from adult human subjects, sulfation rates of two prototypical thermostable phenol sulfotransferase substrates (2-naphthol and p-nitrophenol) and one thermolabile phenol sulfotransferase substrate (dopamine) were determined using standard radiochemical protocols. These data were then compared with results from the colorimetric assay using 2-naphthol as substrate. There was a good correlation between the phenotyping assay and radiochemical assays for both 2-naphthol sulfotransferase and p-nitrophenol sulfotransferase activity (r = 0.85 and 0.69, respectively). However, SULT1A1 activity was approximately 10 to 20 times higher with the colorimetric determination. As anticipated, there was no correlation between SULT1A1 activity and dopamine sulfotransferase activity (r = 0.07) in these human platelet preparations. This inexpensive and rapid method for phenotyping SULT1A1 activity may help investigators assess a role for this enzyme in disease susceptibility.

The Poisson distribution is a fundamental probability model for count data, and is a natural model for the observed plaque counts in mutation assays using animals with lambda or PhiX174 transgenes. The Poisson likelihood for observed counts is a function of the mutant fraction, and it is straightforward to derive the associated maximum likelihood estimate of the mutant fraction and its variance. The estimate is easy to calculate, and if not the same, very similar to ad hoc estimates in current use. The model indicates the proper way to combine data from a number of plates, possibly prepared with different sample dilutions. The estimator of the mutant fraction is biased as a consequence of dividing by a random variable, the plaque count used to calculate the total recovered plaque-forming units. Fortunately, the bias becomes negligible as this count becomes large. On the other hand, increasing this count can increase the variance by decreasing the amount of sample assayed for mutant phages. Concurrent heed to the bias and the variance provides some guidance as to the optimum allocation of a sample into portions assayed for mutant phages and total recovered phages. The distribution of the estimate of the mutant fraction is related to the binomial distribution. This relationship implies a binomial distribution for the mutant count conditional on an overall count (either the sum of mutant and counted total plaques or the sum of counted mutant and non-mutant plaques). A special but important case occurs when each plate can be evaluated for mutant plaques and non-mutant plaques. Then, the observed proportion of mutants estimates the mutant fraction. More generally, the relationship to a binomial distribution provides a procedure for calculating a confidence interval., (Copyright 1999 Elsevier Science B.V.)

Many studies have suggested a relationship between certain alleles of the human leukocyte antigen (HLA) and the prevalence of some diseases or the immunological responsiveness to certain antigens. Furthermore, our studies in the past have demonstrated decreased immune function among atomic-bomb survivors who were exposed to high doses of radiation. However, no studies have addressed the possibility of various degrees of radiation-induced immune suppression being dependent on HLA type. To investigate the possibility of differing frequency distributions of HLA type in the Hiroshima atomic bomb survivors, HLA-DQA1 alleles and HLA-DR antigens were typed for 291 survivors in a high-dose group (>1.5 Gy), 339 survivors in an intermediate-dose group (0.005-1.5 Gy), and 388 in a distally exposed control group (<0.005 Gy). These doses are whole-body exposures, mainly from gamma-rays but with a small neutron component. When examinees were grouped by distinct pairs of HLA-DQA1 allele or HLA-DR antigen, no sex- or dose-related differences were found. However, when subjects were grouped by the presence of a specific allele or antigen, males carrying DQA1*0103 in at least one of their two HLA-DQA1 loci exhibited frequency distributions that decreased as radiation dose increased. These results suggest, although weakly, a possible population bias among male survivors with respect to HLA polymorphism. However, this bias is unlikely to be great enough to have a substantial effect on the cancer risk estimates.

Cancer mortality for the period from October 1950 through May 1992 was analyzed in atomic bomb survivors exposed in utero. Risk estimates for this group were also compared to those for survivors who were less than 6 years old at the time of exposure. The cohorts studied include 807 in utero survivors and 5,545 persons exposed during childhood with all members of both groups having estimated doses of at least 0.01 Sv. The comparison group includes 10,453 persons with little (<0.01 Sv) or no exposure. Analyses were limited mainly to cancer deaths occurring between the ages of 17 and 46. Only 10 cancer deaths were observed among persons exposed in utero. However, there is a significant dose response with an estimate of excess relative risk per sievert (ERR/Sv) of 2.1 (90% confidence interval of 0.2 to 6.0). This estimate does not differ significantly from that for survivors exposed during the first 5 years of life. The cancer deaths among those exposed in utero involved leukemia (2), female-specific organs (3) and digestive organs (5). Nine deaths occurred in females, where the excess risk for all solid cancers has a 90% confidence interval on the ERR/Sv of 1.6 to 17. Significant risks were found for cancers of the digestive system [90% confidence interval (CI) on the ERR/Sv of 0.7 to 20] and for female-specific cancers (90% CI on the ERR/Sv of 0.7 to 42). These risks do not differ significantly from those seen in females exposed as children. There were no deaths from solid cancer in men exposed in utero. The ERR/Sv has an upper 95% confidence bound of 2.5 which does not differ from that for exposed children, where the upper 95% confidence bound is 1.5. The sexes differ even when female-specific cancers are excluded from the comparison. Although there were only two leukemia deaths among those exposed in utero, the leukemia death rate for this group is higher than that in the comparison group (P = 0.054) with an exposure effect that is about half the magnitude and not significantly different from that seen after childhood exposure (P = 0.103). However, there is no evidence of a dose response among those exposed in utero because no high-dose leukemia deaths were observed, a result that differs considerably from that for those exposed as children. There is a need for caution in the interpretation of these data. First, the number of cancer deaths is small; second, there is unexplained significant difference in the mortality from solid cancer between the sexes; and third, the excess of leukemia in those exposed in utero is not reflected in an increasing dose response.