Linda Pluta

Genome-wide analysis of human HSF1 signaling reveals a transcriptional program linked to cellular adaptation and survival

Although HSF1 plays an important role in the cellular response to proteotoxic stressors, little is known about the structure and function of the human HSF1 signaling network under both stressed and unstressed conditions. In this study, we used a combination of chromatin immunoprecipitation microarray analysis and time course gene expression microarray analysis with and without siRNA-mediated inhibition of HSF1 to comprehensively identify genes regulated directly and indirectly by HSF1. The correlation between promoter binding and gene expression was not significant for all genes bound by HSF1, suggesting that HSF1 binding per se is not sufficient for expression. However, the correlation with promoter binding was significant for genes identified as HSF1-regulated following siRNA knockdown. Among promoters bound by HSF1 following heat shock, a gene ontology analysis showed significant enrichment only in categories related to protein folding. In contrast, analysis of the extended HSF1 signaling network following siRNA knockdown showed enrichment in a variety of categories related to protein folding, anti-apoptosis, RNA splicing, ubiquitinylation and others, highlighting a complex transcriptional program regulated directly and indirectly by HSF1.

Estrogenicity of bisphenol A in a human endometrial carcinoma cell line

The ability of bisphenol A (BPA) to affect human estrogen receptor (ER) binding, expression of progesterone receptor (PR) mRNA and protein, and cell proliferation has been measured in the human endometrial cell line, ECC-1. Although less potent than 17beta-estradiol, BPA was able to bind to the human uterine ER. BPA also induced both mRNA and protein to levels similar to E2. BPA-mediated PR mRNA induction was antagonized by ICI, suggesting an ER-mediated pathway. Finally, E2 produced a 2-fold increase in cell number, while BPA showed no difference compared with vehicle control. The increase by E2 was inhibited by treatment with the either ICI 182,780 (ICI) or BPA, suggesting similar binding sites. Although ER binding is similar, E2 affected both proliferation and PR expression, while BPA only affected PR gene expression. The results of this study provide evidence that two ER agonists can act differentially in vitro to affect the expression of genes involved in regulating cellular growth and development, though the human risk potential remains to be determined.

Dose-dependent transitions in Nrf2-mediated adaptive response and related stress responses to hypochlorous acid in mouse macrophages.

Hypochlorous acid (HOCl) is potentially an important source of cellular oxidative stress. Human HOCl exposure can occur from chlorine gas inhalation or from endogenous sources of HOCl, such as respiratory burst by phagocytes. Transcription factor Nrf2 is a key regulator of cellular redox status and serves as a primary source of defense against oxidative stress. We recently demonstrated that HOCl activates Nrf2-mediated antioxidant response in cultured mouse macrophages in a biphasic manner. In an effort to determine whether Nrf2 pathways overlap with other stress pathways, gene expression profiling was performed in RAW 264.7 macrophages exposed to HOCl using whole genome mouse microarrays. Benchmark dose (BMD) analysis on gene expression data revealed that Nrf2-mediated antioxidant response and protein ubiquitination were the most sensitive biological pathways that were activated in response to low concentrations of HOCl (<0.35 mM). Genes involved in chromatin architecture maintenance and DNA-dependent transcription were also sensitive to very low doses. Moderate concentrations of HOCl (0.35 to 1.4 mM) caused maximal activation of the Nrf2 pathway and innate immune response genes, such as IL-1beta, IL-6, IL-10 and chemokines. At even higher concentrations of HOCl (2.8 to 3.5 mM) there was a loss of Nrf2-target gene expression with increased expression of numerous heat shock and histone cluster genes, AP-1-family genes, cFos and Fra1 and DNA damage-inducible Gadd45 genes. These findings confirm an Nrf2-centric mechanism of action of HOCl in mouse macrophages and provide evidence of interactions between Nrf2, inflammatory, and other stress pathways.

Mutational spectrum of 1,3-butadiene and metabolites 1,2-epoxybutene and 1,2,3,4-diepoxybutane to assess mutagenic mechanisms

1,3-Butadiene (BD) is a multisite carcinogen and is mutagenic in multiple tissues of B6C3F1 mice. BD is bioactivated to at least three directly mutagenic metabolites: 1,2-epoxybutene (EB), 1,2-epoxy-3,4-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB). However, the contribution of these individual metabolites to the carcinogenicity and in vivo mutatidnal spectrum of BD is uncertain. To assess the role of two BD metabolites EB and DEB in the in vivo mutagenicity of the parent compound BD, we examined the in vitro mutational spectra of EB and DEB in human and rodent cells. We also examined the in vivo mutagenicity and mutational spectrum of inhaled EB in the lung. In the bone marrow and spleen of B6C3F1 laci transgenic mice, BD-induced an increased frequency of the identical class of point mutations at A:T base pairs: AT-->GC transitions and AT-->TA transversions. BD exposure also induced an increased frequency of GC-->AT transitions in the spleen that was not observed in bone marrow, demonstrating tissue-specific differences in mutation spectrum. Exposure of Rat2 laci transgenic cells and human TK6 lymphoblasts to EB-induced an increased frequency of AT-->TA transversions. DEB exposure induced an increased frequency of AT-->TA transversions and partial deletions at hprt in human cells. In Rat laci transgenic cells, DEB was not mutagenic at laci but induced an increased frequency of micronuclei. In contrast to inhaled BD, inhaled DEB and EB were not mutagenic in the bone marrow or spleen. However, EB was mutagenic in the lungs. In the lung of mice, EB-induced specific increases in GC-->AT transitions, AT-->TA transversions, and deletion events. AT-->TA transversions are the most consistent mutation observed across biological systems following in vivo exposure to BD or in vitro exposures to EB and DEB. Although, BD exposure in mice induces chromosomal alterations and single base substitutions, the specific BD metabolite that induces the genetic events leading to tumors is uncertain. At present, it appears that only DEB can effectively induce this range of mutagenic events at levels of this metabolite that occur in the blood of mice exposed to BD. Detailed investigations to identify relevant biomarkers of BD exposure and response, particularly DNA adducts or lesions, that can be biologically linked to the range of genotoxic events known to occur in mice exposed to BD are needed.

Assessment of the in vivo mutagenicity of ethylene oxide in the tissues of B6C3F1 lacI transgenic mice following inhalation exposure

In the present study, the lacI mutant frequency was determined in the tissues of B6C3F1 lacI transgenic mice exposed by inhalation to ethylene oxide (EO). Groups of 15 male transgenic lacI B6C3F1 mice were exposed to either 0, 50, 100, or 200 ppm EO for 4 weeks (6 h/day, 5 days/week) and were sacrificed at 0, 2, or 8 weeks after the last EO exposure. The lacI transgene was recovered from lung, bone marrow, spleen, and germ cells for determination of the lacI mutant frequency. The tissues selected for analysis were tumor target site tissues in chronic bioassays (lung tumors and lymphomas) and germ cells. The lacI mutant frequency in lung was significantly increased at 8 weeks post exposure to 200 ppm EO (6.2 +/- 2.2 vs. 9.1 +/- 1.5. p = 0.046). In contrast, the lacI mutant frequency in spleen and bone marrow at 2 and 8 weeks was not significantly increased in mice exposed to 200 ppm EO. The lacI mutant frequencies in male germ cells for 200 ppm EO-exposed mice were not increased compared to air controls at 2 and 8 weeks post-exposure. In a spleen cell fraction two of three EO-exposed mice at the 200 ppm exposure level demonstrated an elevated lacI mutant frequency. The increased lacI mutant frequency in these animals was likely due to mutant siblings that contained background G:C --> A:T transitions at CpG sites. These results demonstrate that a 4-week inhalation exposure to EO is mutagenic in lung. However, EO did not increase the frequency of mutations recovered at the lacI transgene in other tissues examined under the conditions used in the present studies. Since the mutational spectrum for EO in other systems consists of an increased proportion of large deletions, the lack of a mutagenic response in the tissues examined is likely due to the lack of recovery of large deletions in lambda-based shuttle vector systems. These data indicate that a primary mechanism of EO-induced mutagenicity in vivo is likely through the induction of deletions, not specific point mutations.

Exposure of hematopoietic stem cells to benzene or 1,4-benzoquinone induces gender-specific gene expression.

Chronic exposure to benzene results in progressive decline of hematopoietic function and may lead to the onset of various disorders, including aplastic anemia, myelodysplastic syndrome, and leukemia. Damage to macromolecules resulting from benzene metabolites and misrepair of DNA lesions may lead to changes in hematopoietic stem cells (HSCs) that give rise to leukemic clones. We have shown previously that male mice exposed to benzene by inhalation were significantly more susceptible to benzene‐induced toxicities than females. Because HSCs are targets for benzene‐induced cytotoxicity and genotoxicity, we investigated DNA damage responses in HSC from both genders of 129/SvJ mice after exposure to 1,4‐benzoquinone (BQ) in vitro or benzene in vivo. 1,4‐BQ is a highly reactive metabolite of benzene that can cause cellular damage by forming protein and DNA adducts and producing reactive oxygen species. HSCs cultured in the presence of 1,4‐BQ for 24 hours showed a gender‐independent, dose‐dependent cytotoxic response. RNA isolated from 1,4‐BQ–treated HSCs and HSCs from mice exposed to 100 ppm benzene by inhalation showed altered expression of apoptosis, DNA repair, cell cycle, and growth control genes compared with unexposed HSCs. Rad51, xpc, and mdm‐2 transcript levels were increased in male but not female HSCs exposed to 1,4‐BQ. Males exposed to benzene exhibited higher mRNA levels for xpc, ku80, ccng, and wig1. These gene expression differences may partially explain the gender disparity in benzene susceptibility. HSC culture systems such as the one used here will be useful for testing the hematotoxicity of various substances, including other benzene metabolites.

Comparison of Microarrays and RNA-Seq for Gene Expression Analyses of Dose-Response Experiments

Relative to microarrays, RNA-seq has been reported to offer higher precision estimates of transcript abundance, a greater dynamic range, and detection of novel transcripts. However, previous comparisons of the 2 technologies have not covered dose-response experiments that are relevant to toxicology. Male F344 rats were exposed for 13 weeks to 5 doses of bromobenzene, and liver gene expression was measured using both microarrays and RNA-seq. Multiple normalization methods were evaluated for each technology, and gene expression changes were statistically analyzed using both analysis of variance and benchmark dose (BMD). Fold-change values were highly correlated between the 2 technologies, whereas the -log p values showed lower correlation. RNA-seq detected fewer statistically significant genes at lower doses, but more significant genes based on fold change except when a negative binomial transformation was applied. Overlap in genes significant by both p value and fold change was approximately 30%-40%. Random sampling of the RNA-seq data showed an equivalent number of differentially expressed genes compared with microarrays at ~5 million reads. Quantitative RT-PCR of differentially expressed genes uniquely identified by each technology showed a high degree of confirmation when both fold change and p value were considered. The mean dose-response expression of each gene was highly correlated between technologies, whereas estimates of sample variability and gene-based BMD values showed lower correlation. Differences in BMD estimates and statistical significance may be due, in part, to differences in the dynamic range of each technology and the degree to which normalization corrects genes at either end of the scale.

Long-term mutagenicity studies with chloroform and dimethylnitrosamine in female lacI transgenic B6C3F1 mice

The weight of evidence indicates that chloroform induces cancer in the female B6C3F1 mouse liver via a nongenotoxic‐cytotoxic mode of action. However, it is probable that DNA damage occurs secondary to events associated with cytolethality and regenerative cell proliferation. The purpose of the present study was to evaluate the potential mutagenic activity of chloroform in the B6C3F1 lacI transgenic mouse liver mutagenesis assay including mutagenic events that might occur secondary to cytolethality. The positive control, dimethylnitrosamine (DMN) is a DNA‐reactive mutagen and carcinogen. DMN‐induced mutations were anticipated to require only a brief exposure and without further treatment were predicted to remain unchanged over time at those frequencies. Chloroform‐induced mutations secondary to toxicity were anticipated to require longer exposure periods and to occur only under conditions that produced sustained cytolethality and regenerative cell proliferation. Female B6C3F1 lacI transgenic mice were treated with daily doses of 2, 4, or 8 mg/kg of DMN by gavage for 4 days and then held until analysis 10, 30, 90, and 180 days postexposure. Livers from DMN‐treated mice exhibited a dose‐related 2‐ to 5‐fold increase over control mutant frequencies and remained at those levels for 10 through 180 days postexposure. Thus, following the initial induction by DMN no selective mutation amplification or loss was seen for this extended period of time. Female B6C3F1 lacI mice were exposed daily for 6 hr/day 7 days/week to 0, 10, 30, or 90 ppm chloroform by inhalation, representing nonhepatotoxic, borderline, or overtly hepatotoxic chloroform exposures. Timepoints for determination of lacI mutant frequency were 10, 30, 90, and 180 days of exposure. No increase in lacI mutant frequency in the liver was observed at any dose or timepoint with chloroform, indicating a lack of DNA reactivity. DNA alterations secondaryto toxicity either did not occur or were of a typenot detectable by lacI mutant frequency analysis,such as large deletions. Environ. Mol. Mutagen. 31:248–256, 1998

Genomic analysis of human lung fibroblasts exposed to vanadium pentoxide to identify candidate genes for occupational bronchitis

BackgroundExposure to vanadium pentoxide (V2O5) is a cause of occupational bronchitis. We evaluated gene expression profiles in cultured human lung fibroblasts exposed to V2O5in vitro in order to identify candidate genes that could play a role in inflammation, fibrosis, and repair during the pathogenesis of V2O5-induced bronchitis.MethodsNormal human lung fibroblasts were exposed to V2O5 in a time course experiment. Gene expression was measured at various time points over a 24 hr period using the Affymetrix Human Genome U133A 2.0 Array. Selected genes that were significantly changed in the microarray experiment were validated by RT-PCR.ResultsV2O5 altered more than 1,400 genes, of which ~300 were induced while >1,100 genes were suppressed. Gene ontology categories (GO) categories unique to induced genes included inflammatory response and immune response, while GO catogories unique to suppressed genes included ubiquitin cycle and cell cycle. A dozen genes were validated by RT-PCR, including growth factors (HBEGF, VEGF, CTGF), chemokines (IL8, CXCL9, CXCL10), oxidative stress response genes (SOD2, PIPOX, OXR1), and DNA-binding proteins (GAS1, STAT1).ConclusionOur study identified a variety of genes that could play pivotal roles in inflammation, fibrosis and repair during V2O5-induced bronchitis. The induction of genes that mediate inflammation and immune responses, as well as suppression of genes involved in growth arrest appear to be important to the lung fibrotic reaction to V2O5.

Evaluation of gene expression changes in human primary uroepithelial cells following 24-hr exposures to inorganic arsenic and its methylated metabolites.

Gene expression changes in primary human uroepithelial cells exposed to arsenite and its methylated metabolites were evaluated to identify cell signaling pathway perturbations potentially associated with bladder carcinogenicity. Cells were treated with mixtures of inorganic arsenic and its pentavalent or trivalent metabolites for 24 hr at total arsenic concentrations ranging from 0.06 μM to 18 μM. One series (five samples) was conducted with arsenite and pentavalent metabolites and a second (10 samples) with arsenite and trivalent metabolites. Similar gene expression responses were obtained for pentavalent or trivalent metabolites. A suite of eight gene changes was consistently identified across individuals that reflect effects on key signaling pathways: oxidative stress, protein folding, growth regulation, metallothionine regulation, DNA damage sensing, thioredoxin regulation, and immune response. No statistical significance of trend (NOSTASOT) analysis of these common genes identified lowest observed effect levels (LOELs) from 0.6 to 6.0 μM total arsenic and no observed effect levels (NOELs) from 0.18 to 1.8 μM total arsenic. For the trivalent arsenical mixture, benchmark doses (BMDs) ranged from 0.13 to 0.92 μM total arsenic; benchmark dose lower 95% confidence limits (BMDLs) ranged from 0.09 to 0.58 μM total arsenic. BMDs ranged from 0.53 to 2.7 μM and BMDLs from 0.35 to 1.7 μM for the pentavalent arsenical mixture. Both endpoints varied by a factor of 3 across individuals. Thisstudy is the first to examine gene expression response in primary uroepithelial cells from multiple individuals and to identify no effect levels for arsenical‐induced cell signaling perturbations in normal human cells exposed to a biologically plausible concentration range.