Inhaled ozone (O3)-induces changes in serum metabolomic and liver transcriptomic profiles in rats.

Air pollution has been linked to increased incidence of diabetes. Recently, we showed that ozone (O3) induces glucose intolerance, and increases serum leptin and epinephrine in Brown Norway rats. In this study, we hypothesized that O3 exposure will cause systemic changes in metabolic homeostasis and that serum metabolomic and liver transcriptomic profiling will provide mechanistic insights. In the first experiment, male Wistar Kyoto (WKY) rats were exposed to filtered air (FA) or O3 at 0.25, 0.50, or 1.0ppm, 6h/day for two days to establish concentration-related effects on glucose tolerance and lung injury. In a second experiment, rats were exposed to FA or 1.0ppm O3, 6h/day for either one or two consecutive days, and systemic metabolic responses were determined immediately after or 18h post-exposure. O3 increased serum glucose and leptin on day 1. Glucose intolerance persisted through two days of exposure but reversed 18h-post second exposure. O3 increased circulating metabolites of glycolysis, long-chain free fatty acids, branched-chain amino acids and cholesterol, while 1,5-anhydroglucitol, bile acids and metabolites of TCA cycle were decreased, indicating impaired glycemic control, proteolysis and lipolysis. Liver gene expression increased for markers of glycolysis, TCA cycle and gluconeogenesis, and decreased for markers of steroid and fat biosynthesis. Genes involved in apoptosis and mitochondrial function were also impacted by O3. In conclusion, short-term O3 exposure induces global metabolic derangement involving glucose, lipid, and amino acid metabolism, typical of a stress-response. It remains to be examined if these alterations contribute to insulin resistance upon chronic exposure.

Signaling Pathways and MicroRNA Changes in Nano-TiO2 Treated Human Lung Epithelial (BEAS-2B) Cells.

The effect of titanium dioxide nanoparticles (nano-TiO2 Degussa p25) treatment of human lung epithelial cells (BEAS-2B) was examined by analyzing changes in messenger [mRNA] and microRNA [miRNA]. BEAS-2B cells were treated with 0, 3, 10, 30 or 100 µg/ml nano-TiO2 for 1 day (for mRNA analysis) or 3 days (for miRNA analysis). Differentially expressed mRNA and miRNA were analyzed using Affymetrix microarrays and Affymetrix miRNA microarrays, respectively. Although, the tested doses were not cytotoxic, there were alterations in both mRNA and miRNA expression. The expression of mRNA/miRNA changes were examined in MetaCore (GeneGo) and IPA (Ingenuity Pathway Analysis) to delineate associated canonical/signaling pathways. Canonical/signaling pathways altered by nano-TiO2 treatments included: cell cycle regulation, apoptosis, calcium signaling, translation, NRF2-mediated oxidative response, IGF1 signaling, RAS signaling, PI3K/AKT signaling, cytoskeleton remodeling, cell adhesion, BMP signaling, and inflammatory response. Many of the genes in these pathways are known to be regulated by the miRNAs whose expressions were altered by the nano-TiO2 treatment. The miRNA 17-92 cluster and let-7 miRNA family that are involved in lung cancer formation were altered by nano-TiO2 treatment. The miR-17-92 cluster, an oncogenic microRNA cluster, is induced while the tumor suppressor microRNA, let-7 family, is suppressed. The changes of let-7/KRAS signaling pathway was observed in all the doses treated. The observed changes in miRNA expression introduces an additional mechanistic dimension that supports the significance of the observed mRNA expression changes, and demonstrated that the nano-TiO2 in vitro treatment in human lung cells can cause diverse but coordinated pathway alterations associated with changes in in vivo response to tumorigenes.

Left ventricular gene expression profile of healthy and cardiovascular compromised rat models used in air pollution studies.

The link between pollutant exposure and cardiovascular disease (CVD) has prompted mechanistic research with animal models of CVD. We hypothesized that the cardiac gene expression patterns of healthy and genetically compromised, CVD-prone rat models, with or without metabolic impairment, will reveal underlying disease processes that facilitate understanding of the mechanisms of air pollution susceptibility differences. Left ventricular gene expression was examined using Affymetrix rat 230A-gene arrays in male, age-matched (12-14 weeks old) healthy Wistar Kyoto (WKY) and CV-compromised spontaneously hypertensive (SH), stroke-prone SH (SHSP), obese SH heart failure (SHHF) and obese insulin-resistant (JCR) rats. Principle component analysis separated strains in three clusters: (1) WKY, (2) JCR and )3) SH, SHSP and SHHF. Gene expression pattern in JCR differed from all other CVD strains. Both SHHF and JCR strains presented the most differentially expressed genes from WKY, but generally with opposing directional pattern suggesting that the CVD in these strains arise through different mechanisms. Hierarchical clustering of nuclear factor-kappaB target genes indicated varying degrees of, but similar directional changes, in SH, SHSP and SHHF relative to WKY rats, which may relate to the severity of their CVD. The JCR strain had less pronounced expressions of these genes suggesting milder cardiac disease. No unique expression pattern could be identified for genes implicated in stroke and heart failure in SHSP and SHHF rats, respectively. The data show that the CVD pathophysiological mechanisms differ in models with different genetic backgrounds, and therefore, the mechanisms by which air pollutants affect the cardiopulmonary system are likely to vary.

Pulmonary transcriptional response to ozone in healthy and cardiovascular compromised rat models.

The genetic cardiovascular disease (CVD) and associated metabolic impairments can influence the lung injury from inhaled pollutants. We hypothesized that comparative assessment of global pulmonary expression profile of healthy and CVD-prone rat models will provide mechanistic insights into susceptibility differences to ozone. The lung expression profiles of healthy Wistar Kyoto (WKY) and CVD-compromised spontaneously hypertensive (SH), stroke-prone SH (SHSP), obese SH heart failure (SHHF) and obese, atherosclerosis-prone JCR rats were analyzed using Affymetrix platform immediately after 4-h air or 1 ppm ozone exposure. At baseline, the JCR exhibited the largest difference in the number of genes among all strains when compared with WKY. Interestingly, the number of genes affected by ozone was inversely correlated with genes different at baseline relative to WKY. A cluster of NFkB target genes involved in cell-adhesion, antioxidant response, inflammation and apoptosis was induced in all strains, albeit at different levels (JCR < WKY < SHHF < SH < SHSP). The lung metabolic syndrome gene cluster indicated expressions in opposite directions for SHHF and JCR suggesting different mechanisms for common disease phenotype and perhaps obesity-independent contribution to exacerbated lung disease. The differences in expression of adrenergic receptors and ion-channel genes suggested distinct mechanisms by which ozone might induce protein leakage in CVD models, especially SHHF and JCR. Thus, the pulmonary response to ozone in CVD strains was likely linked to the defining gene expression profiles. Differential transcriptional patterns between healthy and CVD rat strains at baseline, and after ozone suggests that lung inflammation and injury might be influenced by multiple biological pathways affecting inflammation gene signatures.

Lung transcriptional profiling: insights into the mechanisms of ozone-induced pulmonary injury in Wistar Kyoto rats.

Acute ozone-induced pulmonary injury and inflammation are well characterized in rats; however, mechanistic understanding of the pathways involved is limited. We hypothesized that acute exposure of healthy rats to ozone will cause transcriptional alterations, and comprehensive analysis of these changes will allow us to better understand the mechanism of pulmonary injury and inflammation. Male Wistar Kyoto rats (10-12 week) were exposed to air, or ozone (0.25, 0.5 or 1.0 ppm) for 4 h and pulmonary injury and inflammation were assessed at 0-h or 20-h (n = 8/group). Lung gene expression profiling was assessed at 0-h (air and 1.0 ppm ozone, n = 3-4/group). At 20-h bronchoalveolar lavage, fluid protein and neutrophils increased at 1 ppm ozone. Numerous genes involved in acute inflammatory response were up-regulated along with changes in genes involved in cell adhesion and migration, steroid metabolism, apoptosis, cell cycle control and cell growth. A number of NRF2 target genes were also induced after ozone exposure. Based on expression changes, Rela, SP1 and TP3-mediated signaling were identified to be mediating downstream changes. Remarkable changes in the processes of endocytosis provide the insight that ozone-induced lung injury and inflammation are likely initiated by changes in cell membrane components and receptors likely from oxidatively modified lung lining lipids and proteins. In conclusion, ozone-induced injury and inflammation are preceded by changes in gene targets for cell adhesion/migration, apoptosis, cell cycle control and growth regulated by Rela, SP1 and TP53, likely mediated by the process of endocytosis and altered steroid receptor signaling.

Variability in ozone-induced pulmonary injury and inflammation in healthy and cardiovascular-compromised rat models.

The molecular bases for variability in air pollutant-induced pulmonary injury due to underlying cardiovascular (CVD) and/or metabolic diseases are unknown. We hypothesized that healthy and genetic CVD-prone rat models will exhibit exacerbated response to acute ozone exposure dependent on the type and severity of disease. Healthy male 12-14-week-old Wistar Kyoto (WKY), Wistar (WS) and Sprague Dawley (SD); and CVD-compromised spontaneously hypertensive (SH), Fawn-Hooded hypertensive (FHH), stroke-prone spontaneously hypertensive (SHSP), obese spontaneously hypertensive heart failure (SHHF) and obese JCR (JCR) rats were exposed to 0.0, 0.25, 0.5, or 1.0 ppm ozone for 4 h; pulmonary injury and inflammation were analyzed immediately following (0-h) or 20-h later. Baseline bronchoalveolar lavage fluid (BALF) protein was higher in CVD strains except for FHH when compared to healthy. Ozone-induced increases in protein and inflammation were concentration-dependent within each strain but the degree of response varied from strain to strain and with time. Among healthy rats, SD were least affected. Among CVD strains, lean rats were more susceptible to protein leakage from ozone than obese rats. Ozone caused least neutrophilic inflammation in SH and SHHF while SHSP and FHH were most affected. BALF neutrophils and protein were poorly correlated when considering the entire dataset (r = 0.55). The baseline and ozone-induced increases in cytokine mRNA varied markedly between strains and did not correlate with inflammation. These data illustrate that the degree of ozone-induced lung injury/inflammation response is likely influenced by both genetic and physiological factors that govern the nature of cardiovascular compromise in CVD models.

Strain differences in antioxidants in rat models of cardiovascular disease exposed to ozone.

We examined the hypothesis that antioxidant substances and enzymes in lung, heart and in bronchoalveolar lavage fluid (BALF) are altered in response to O3 in cardiovascular disease and/or metabolic syndrome (CVD)-prone rat models. CVD strains [spontaneously hypertensive (SH), SH stroke-prone (SHSP), SHHF/Mcc heart failure obese (SHHF), insulin-resistant JCR:LA-cp obese (JCR) and Fawn-Hooded hypertensive (FHH)] were compared with normal strains [Wistar, Sprague-Dawley (SD) and Wistar Kyoto (WKY)]. Total glutathione (GSH + GSSG or GSx), reduced ascorbate (AH2), uric acid (UA) and antioxidant enzymes were determined in lung, heart and BALF immediately (0 h) or 20-h post 4-h nose-only exposure to 0.0, 0.25, 0.5 and 1.0 ppm O3. Basal- and O3-induced antioxidant substances in tissues varied widely among strains. Wistar rats had a robust O3-induced increase in GSx and AH2 in the lung. Two CVD strains (JCR and SHHF) had high basal levels of AH2 and GSx in BALF as well as high basal lung UA. Across all strains, high BALF GSx was only observed when high BALF AH2 was present. CVD rats tended to respond less to O3 than normal. High-basal BALF AH2 levels were associated with decreased O3 toxicity. In summary, large differences were observed between both normal and CVD rat strains in low-molecular weight antioxidant concentrations in lung, BALF and heart tissue. Wistar (normal) and JCR and SHHF (CVD) rats appeared to stand out as peculiar in terms of basal- or O3-induced changes. Results elucidate interactions among antioxidants and air pollutants that could enhance understanding of cardiopulmonary disease.

Arsenic and the epigenome: interindividual differences in arsenic metabolism related to distinct patterns of DNA methylation.

Biotransformation of inorganic arsenic (iAs) is one of the factors that determines the character and magnitude of the diverse detrimental health effects associated with chronic iAs exposure, but it is unknown how iAs biotransformation may impact the epigenome. Here, we integrated analyses of genome-wide, gene-specific promoter DNA methylation levels of peripheral blood leukocytes with urinary arsenical concentrations of subjects from a region of Mexico with high levels of iAs in drinking water. These analyses revealed dramatic differences in DNA methylation profiles associated with concentrations of specific urinary metabolites of arsenic (As). The majority of individuals in this study had positive indicators of As-related disease, namely pre-diabetes mellitus or diabetes mellitus (DM). Methylation patterns of genes with known associations with DM were associated with urinary concentrations of specific iAs metabolites. Future studies will determine whether these DNA methylation profiles provide mechanistic insight into the development of iAs-associated disease, predict disease risk, and/or serve as biomarkers of iAs exposure in humans.

Transcriptional ontogeny of the developing liver.

BACKGROUND: During embryogenesis the liver is derived from endodermal cells lining the digestive tract. These endodermal progenitor cells contribute to forming the parenchyma of a number of organs including the liver and pancreas. Early in organogenesis the fetal liver is populated by hematopoietic stem cells, the source for a number of blood cells including nucleated erythrocytes. A comprehensive analysis of the transcriptional changes that occur during the early stages of development to adulthood in the liver was carried out. RESULTS: We characterized gene expression changes in the developing mouse liver at gestational days (GD) 11.5, 12.5, 13.5, 14.5, 16.5, and 19 and in the neonate (postnatal day (PND) 7 and 32) compared to that in the adult liver (PND67) using full-genome microarrays. The fetal liver, and to a lesser extent the neonatal liver, exhibited dramatic differences in gene expression compared to adults. Canonical pathway analysis of the fetal liver signature demonstrated increases in functions important in cell replication and DNA fidelity whereas most metabolic pathways of intermediary metabolism were under expressed. Comparison of the dataset to a number of previously published microarray datasets revealed 1) a striking similarity between the fetal liver and that of the pancreas in both mice and humans, 2) a nucleated erythrocyte signature in the fetus and 3) under expression of most xenobiotic metabolism genes throughout development, with the exception of a number of transporters associated with either hematopoietic cells or cell proliferation in hepatocytes. CONCLUSIONS: Overall, these findings reveal the complexity of gene expression changes during liver development and maturation, and provide a foundation to predict responses to chemical and drug exposure as a function of early life-stages.

Transcriptional characterization of Salmonella TA100 in log and stationary phase: influence of growth phase on mutagenicity of MX.

The Salmonella mutagenicity assay can be performed using cells that are in different growth phases. Thus, the plate-incorporation assay involves plating stationary-phase cells with the mutagen, after which the cells undergo a brief lag phase and, consequently, are exposed to the mutagen and undergo mutagenesis while in the logarithmic (log) phase. In contrast, a liquid-suspension assay involves exposure of either log- or stationary-phase cells to the mutagen for a specified period of time, sometimes followed by a wash, resulting in the cells growing in medium in the absence of the mutagen. To explore global gene expression in Salmonella, and to test for possible effects of growth phase and transcriptional status on mutagenesis, we performed microarray analysis on cells of Salmonella strain TA100 exposed to the drinking-water mutagen MX in either the log or stationary phase. The genes in functional pathways involving amino acid transport and metabolism and energy metabolism were expressed differentially in log-phase cells, whereas genes in functional pathways involving protein trafficking, cell envelope, and two-component systems using common signal transduction were expressed differentially in stationary-phase cells. More than 90% of the ribosomal-protein biosynthesis genes were up-regulated in stationary- versus log-phase cells. MX was equally mutagenic to cells in log- and stationary-phase growth when the results were expressed as mutant frequencies (revertants/survivors/µM), but it was twice as mutagenic in stationary-phase cells when the results were expressed as mutant yields (revertants/nmole or revertants/µM). There was a complex transcriptional response underlying these results, with mucA/B being greatly up-regulated in log-phase cells but umuC/D up-regulated in stationary-phase cells. The transcriptional state of TA100 cells at the time of mutagen treatment may influence the outcome of mutagen treatment.

Discovering collectively informative descriptors from high-throughput experiments.

BACKGROUND: Improvements in high-throughput technology and its increasing use have led to the generation of many highly complex datasets that often address similar biological questions. Combining information from these studies can increase the reliability and generalizability of results and also yield new insights that guide future research. RESULTS: This paper describes a novel algorithm called BLANKET for symmetric analysis of two experiments that assess informativeness of descriptors. The experiments are required to be related only in that their descriptor sets intersect substantially and their definitions of case and control are consistent. From resulting lists of n descriptors ranked by informativeness, BLANKET determines shortlists of descriptors from each experiment, generally of different lengths p and q. For any pair of shortlists, four numbers are evident: the number of descriptors appearing in both shortlists, in exactly one shortlist, or in neither shortlist. From the associated contingency table, BLANKET computes Right Fisher Exact Test (RFET) values used as scores over a plane of possible pairs of shortlist lengths 12. BLANKET then chooses a pair or pairs with RFET score less than a threshold; the threshold depends upon n and shortlist length limits and represents a quality of intersection achieved by less than 5% of random lists. CONCLUSIONS: Researchers seek within a universe of descriptors some minimal subset that collectively and efficiently predicts experimental outcomes. Ideally, any smaller subset should be insufficient for reliable prediction and any larger subset should have little additional accuracy. As a method, BLANKET is easy to conceptualize and presents only moderate computational complexity. Many existing databases could be mined using BLANKET to suggest optimal sets of predictive descriptors.

Global gene expression profiling of hyperkeratotic skin lesions from inner Mongolians chronically exposed to arsenic.

The skin is an organ that is highly sensitive to chronic arsenic (As) exposure. Skin lesions such as hyperkeratoses (HKs) are common early manifestations of arsenicosis in humans. HKs can be precursor lesions of nonmelanoma skin cancers (NMSCs), but the driving forces behind their formation and how they may ultimately progress to NMSCs are unknown. The goal of this study was to examine the global gene expression profiles of As-related HKs in an effort to better understand gene expression changes that are potentially associated with early stages of As carcinogenesis. HK biopsies were removed from individuals living in an arsenicosis-endemic region in Inner Mongolia who had been exposed to high As levels in their drinking water for >20 years. Gene expression profiling was performed on RNA isolated from 7 individuals in this group and from 4 lesion-free skin samples from healthy individuals. Consistent with the pathological characteristics of the HK lesions, major functional categories and known canonical pathways represented by altered transcripts include those involved in development, differentiation, apoptosis, proliferation, and stress response. The results of this study may help define a signature profile of gene expression changes associated with long-term As exposure in the skin.

Oncogene expression profiles in K6/ODC mouse skin and papillomas following a chronic exposure to monomethylarsonous acid.

We have previously observed that a chronic drinking water exposure to monomethylarsonous acid [MMA(III)], a cellular metabolite of inorganic arsenic, increases tumor frequency in the skin of keratin VI/ornithine decarboxylase (K6/ODC) transgenic mice. To characterize gene expression profiles predictive of MMA(III) exposure and mode of action of carcinogenesis, skin and papilloma RNA was isolated from K6/ODC mice administered 0, 10, 50, and 100 ppm MMA(III) in their drinking water for 26 weeks. Following RNA processing, the resulting cRNA samples were hybridized to Affymetrix Mouse Genome 430A 2.0 GeneChips(R). Micoarray data were normalized using MAS 5.0 software, and statistically significant genes were determined using a regularized t-test. Significant changes in bZIP transcription factors, MAP kinase signaling, chromatin remodeling, and lipid metabolism gene transcripts were observed following MMA(III) exposure as determined using the Database for Annotation, Visualization and Integrated Discovery 2.1 (DAVID) (Dennis et al., Genome Biol 2003;4(5):P3). MMA(III) also caused dose-dependent changes in multiple Rho guanine nucleotide triphosphatase (GTPase) and cell cycle related genes as determined by linear regression analyses. Observed increases in transcript abundance of Fosl1, Myc, and Rac1 oncogenes in mouse skin support previous reports on the inducibility of these oncogenes in response to arsenic and support the relevance of these genomic changes in skin tumor induction in the K6/ODC mouse model.

Differential Effects of Nano TiO2 and CeO2 on Normal Human Lung Epithelial Cells In Vitro.

Nano-TiO2 and nano-CeO2 are among the most widely used engineered nanoparticles (NPs). We investigated a variety of endpoints to assess the toxicity of eight of these NPs to induce potentially adverse health effects in an In Vitro human respiratory epithelial cell model. These endpoints include cytotoxicity, reactive oxygen species (ROS)/reactive nitrogen species (RNS) production, 8-hydroxy-2_-deoxyguanosine (8-oxo-dG), endogenous DNA adducts, Apurinic/apyrimidinic (AP) sites, 4-Hrdoxynonenal (4-HNE) protein adducts, Malondialdehyde (MDA) protein adducts, and genomics analysis on altered signaling pathways. Our results indicated that cytotoxicity assays are relatively insensitive, and we detected changes in other endpoints at concentrations much lower than those inducing cytotoxicity. Among the ROS-related endpoints, 8-oxo-dG is relatively more sensitive than other assays, and nano-TiO2 induced more 8-oxo-dG formation than nano-CeO2. Finally, there are many signaling pathways changes at concentrations at which no cytotoxicity was observed. These alterations in signaling pathways correlated well with In Vitro toxicity that was observed at higher concentrations, and with in vivo adverse outcome pathways caused by nano-TiO2 and nano-CeO2 in experimental animals.

Integrated disinfection by-products (DBP) mixtures research: gene expression alterations in primary rat hepatocyte cultures exposed to DBP mixtures formed by chlorination and ozonation/postchlorination.

Large-scale differential gene expression analysis was used to examine the biological effects of disinfected surface waters on cultured rat hepatocytes. Source water from East Fork Lake (Harsha Lake), a reservoir on the Little Miami River in Ohio, was spiked with iodide and bromide and disinfected by chlorination or ozonation/postchlorination. The chlorinated and ozonated/postchlorinated waters were concentrated, respectively, 136- and 124-fold (full strength) by reverse-osmosis membrane techniques. Volatile disinfection by-products (DBP) lost during concentration were restored to the extent possible. Primary rat hepatocytes were exposed to either full-strength or 1:10 or 1:20 dilutions of the concentrates for 24 h and assayed for cytotoxicity and gene expression alterations. The full-strength concentrates were cytotoxic, whereas the diluted samples exhibited no detectable cytotoxicity. Differential gene expression analysis provided evidence for the underlying causes of the severe cytotoxicity observed in rat hepatocytes treated with the full-strength ozonation/postchlorination concentrate (e.g., cell cycle arrest, metabolic stasis, oxidative stress). Many gene expression responses were shared among the hepatocyte cultures treated with dilutions of the ozonation/ postchlorination and chlorination concentrates. The shift in the character of the response between the full-strength concentrates and the diluted samples indicated a threshold for toxicity. A small subset of gene expression changes was identified that was observed in the response of hepatocytes to peroxisome proliferators, phthalate esters, and haloacetic acids, suggesting a peroxisome proliferative response.

Toxicogenomic analysis incorporating operon-transcriptional coupling and toxicant concentration-expression response: analysis of MX-treated Salmonella.

BACKGROUND: Deficiencies in microarray technology cause unwanted variation in the hybridization signal, obscuring the true measurements of intracellular transcript levels. Here we describe a general method that can improve microarray analysis of toxicant-exposed cells that uses the intrinsic power of transcriptional coupling and toxicant concentration-expression response data. To illustrate this approach, we characterized changes in global gene expression induced in Salmonella typhimurium TA100 by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), the primary mutagen in chlorinated drinking water. We used the co-expression of genes within an operon and the monotonic increases or decreases in gene expression relative to increasing toxicant concentration to augment our identification of differentially expressed genes beyond Bayesian-t analysis. RESULTS: Operon analysis increased the number of altered genes by 95% from the list identified by a Bayesian t-test of control to the highest concentration of MX. Monotonic analysis added 46% more genes. A functional analysis of the resulting 448 differentially expressed genes yielded functional changes beyond what would be expected from only the mutagenic properties of MX. In addition to gene-expression changes in DNA-damage response, MX induced changes in expression of genes involved in membrane transport and porphyrin metabolism, among other biological processes. The disruption of porphyrin metabolism might be attributable to the structural similarity of MX, which is a chlorinated furanone, to ligands indigenous to the porphyrin metabolism pathway. Interestingly, our results indicate that the lexA regulon in Salmonella, which partially mediates the response to DNA damage, may contain only 60% of the genes present in this regulon in E. coli. In addition, nanH was found to be highly induced by MX and contains a putative lexA regulatory motif in its regulatory region, suggesting that it may be regulated by lexA. CONCLUSION: Operon and monotonic analyses improved the determination of differentially expressed genes beyond that of Bayesian-t analysis, showing that MX alters cellular metabolism involving pathways other than DNA damage. Because co-expression of similarly functioning genes also occurs in eukaryotes, this method has general applicability for improving analysis of toxicogenomic data.

Enhanced mutagenesis of Salmonella tester strains due to deletion of genes other than uvrB.

The standard Salmonella mutagenicity (Ames) tester strains are missing 15-119 genes due to the extended Delta(gal-bio-uvrB) mutations that render the strains excision-repair deficient (DeltauvrB). We constructed strains of Salmonella that are homologous to tester strains TA98 and TA100 except that in place of the uvrB deletion, they contain single-gene defects in either uvrB, moaA, moeA, or both uvrB and moeA. We then tested the following mutagens in these strains: 2-acetylaminofluorene, Glu-P-1, 4-aminobiphenyl, benzo[a]pyrene, MX, 1-nitropyrene, 6-hydroxylaminopurine (HAP), and 2-amino-6-hydroxylaminopurine (AHAP). We confirmed in Salmonella a previous finding in Escherichia coli that the enhanced mutagenicity of the purine analogues HAP and AHAP is not due to the deletion of the uvrB gene but due to the deletion of moeA and/or moaA, which are involved in molybdenum cofactor biosynthesis. The spontaneous mutant frequency and induced mutagenic potency of mutagens due to the extended DeltauvrB mutation are due largely to the deletion of uvrB and to some extent of moeA/moaA at the frameshift hisD3052 allele of TA98 but involve other genes in addition to uvrB and moeA/moaA at the base-substitution hisG46 allele of TA100. The extended DeltauvrB mutation does not prevent the detection of mutagens that would have been detected in a strain containing a single uvrB defect. Because of the deletion of moeA/moaA, the extended uvrB deletion generally enhanced spontaneous and induced mutagenicity, especially at the base-substitution allele. This enhanced sensitivity may underlay the severe health effects in humans who have mutations in molybdenum cofactor biosynthesis genes.

Transcriptional repressor functions of Drosophila E2F1 and E2F2 cooperate to inhibit genomic DNA synthesis in ovarian follicle cells.

Individual members of the E2F/DP protein family control cell cycle progression by acting predominantly as an activator or repressor of transcription. In Drosophila melanogaster the E2f1, E2f2, Dp, and Rbf1 genes all contribute to replication control in ovarian follicle cells, which become 16C polyploid and subsequently undergo chorion gene amplification late in oogenesis. Mutation of E2f2, Dp, or Rbf1 causes ectopic DNA replication throughout the follicle cell genome during gene amplification cycles. Here we show by both reverse transcription-PCR and DNA microarray analysis that the transcripts of prereplication complex (pre-RC) genes are elevated compared to the wild type in E2f2, Dp, and Rbf1 mutant follicle cells. For some genes the magnitude of this transcriptional derepression is greater in Rbf1 than in E2f2 mutants. These differences correlate with differences in the magnitude of the replication defects in follicle cells, which attain an inappropriate 32C DNA content in both Rbf1 and Dp mutants but not in E2f2 mutants. The ectopic genomic replication of E2f2 mutant follicle cells can be suppressed by reducing the Orc2, Orc5, or Mcm2 gene dose by half, indicating that small changes in pre-RC gene expression can affect DNA synthesis in these cells. We conclude that RBF1 forms complexes with both E2F1/DP and E2F2/DP that cooperate to repress the expression of pre-RC genes, which helps confine DNA synthesis to sites of gene amplification. In contrast, E2F1 and E2F2 repressors function redundantly for some genes in the embryo. Thus, the relative functional contributions of E2F1 and E2F2 to gene expression and cell cycle control depends on the developmental context.

Folate deficiency enhances arsenic effects on expression of genes involved in epidermal differentiation in transgenic K6/ODC mouse skin.

Chronic arsenic exposure in humans is associated with cancers of the skin, lung, bladder and other tissues. There is evidence that folate deficiency may increase susceptibility to arsenic effects, including skin lesions. K6/ODC mice develop skin tumors when exposed to 10ppm sodium arsenite for 5 months. In the current study, K6/ODC mice maintained on either a folate deficient or folate sufficient diet were exposed to 0, 1, or 10ppm sodium arsenite in the drinking water for 30 days. Total RNA was isolated from skin samples and gene expression analyzed using Affymetrix Mouse 430 2.0 GeneChips. Data from 24 samples, with 4 mice in each of the 6 treatment groups, were RMA normalized and analyzed by two-way ANOVA using GeneSpring. Top gene ontology (GO) categories for genes responding significantly to both arsenic treatment and folate deficiency include nucleotide metabolism and cell organization and biogenesis. For many of these genes, folate deficiency magnifies the response to arsenic treatment. In particular, expression of markers of epidermal differentiation, e.g., loricrin, small proline rich proteins and involucrin, was significantly reduced by arsenic in the folate sufficient animals, and reduced further or at a lower arsenic dose in the folate deficient animals. In addition, expression of a number of epidermal cell growth/proliferation genes and cellular movement genes was altered. These results indicate that arsenic disrupts the normal balance of cell proliferation and differentiation, and that folate deficiency exacerbates these effects, consistent with the view that folate deficiency is a nutritional susceptibility factor for arsenic-induced skin tumorigenesis.

Antimutagenicity of cinnamaldehyde and vanillin in human cells: Global gene expression and possible role of DNA damage and repair.

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary flavorings that exhibit antimutagenic activity against mutagen-induced and spontaneous mutations in bacteria. Although these compounds were antimutagenic against chromosomal mutations in mammalian cells, they have not been studied for antimutagenesis against spontaneous gene mutations in mammalian cells. Thus, we initiated studies with VAN and CIN in human mismatch repair-deficient (hMLH1(-)) HCT116 colon cancer cells, which exhibit high spontaneous mutation rates (mutations/cell/generation) at the HPRT locus, permitting analysis of antimutagenic effects of agents against spontaneous mutation. Long-term (1-3 weeks) treatment of HCT116 cells with VAN at minimally toxic concentrations (0.5-2.5mM) reduced the spontaneous HPRT mutant fraction (MF, mutants/10(6) survivors) in a concentration-related manner by 19-73%. A similar treatment with CIN at 2.5-7.5microM yielded a 13-56% reduction of the spontaneous MF. Short-term (4-h) treatments also reduced the spontaneous MF by 64% (VAN) and 31% (CIN). To investigate the mechanisms of antimutagenesis, we evaluated the ability of VAN and CIN to induce DNA damage (comet assay) and to alter global gene expression (Affymetrix GeneChip) after 4-h treatments. Both VAN and CIN induced DNA damage in both mismatch repair-proficient (HCT116+chr3) and deficient (HCT116) cells at concentrations that were antimutagenic in HCT116 cells. There were 64 genes whose expression was changed similarly by both VAN and CIN; these included genes related to DNA damage, stress responses, oxidative damage, apoptosis, and cell growth. RT-PCR results paralleled the Affymetrix results for four selected genes (HMOX1, DDIT4, GCLM, and CLK4). Our results show for the first time that VAN and CIN are antimutagenic against spontaneous mutations in mammalian (human) cells. These and other data lead us to propose that VAN and CIN may induce DNA damage that elicits recombinational DNA repair, which reduces spontaneous mutations.