Browsing by Author "Freedman, Jonathan H"

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    Molecular characterization of numr-1 and numr-2: genes that increase both resistance to metal-induced stress and lifespan in Caenorhabditis elegans
    (2010) Tvermoes, Brooke E; Boyd, Windy A; Freedman, Jonathan H
    To define the mechanisms involved in the molecular response to the carcinogenic metal cadmium, two novel metal-inducible genes from C. elegans were characterized: numr-1 and numr-2 (nuclear localized metal responsive). numr-1 and numr-2 sequences and cellular patterns of expression are identical, indicating that these are functionally equivalent genes. Constitutive transcription of numr-1 and numr-2 is developmentally regulated and occurs in the intestine, in head and tail neurons, and vulva muscles. Exposure to metals induces numr-1 and numr-2 transcription in pharyngeal and intestinal cells. Other environmental stressors do not affect transcription, indicating that these are metal-specific, stress-responsive genes. NUMR-1 and NUMR-2 target to nuclei and colocalize with HSF-1, suggesting that they may be components of nuclear stress granules. Nematodes overexpressing NUMR-1 and NUMR-2 are resistant to stress and live longer than control animals; likewise reducing expression increases sensitivity to metals and decreases neuromuscular functions. Upstream regulatory regions of both genes contain potential binding sites for DAF-16 and SKN-1, which are components of the insulin-IGF-like signaling pathway. This pathway regulates longevity and stress responses in C. elegans. NUMR-1 and NUMR-2 may function to promote resistance to environmental stressors and longevity, which is mediated by the insulin-IGF-like signaling pathway.
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    Persisting neurobehavioral effects of developmental copper exposure in wildtype and metallothionein 1 and 2 knockout mice.
    (BMC pharmacology & toxicology, 2016-11) Petro, Ann; Sexton, Hannah G; Miranda, Caroline; Rastogi, Anit; Freedman, Jonathan H; Levin, Edward D

    Background

    Metallothioneins (MT) are small proteins, which are crucial for the distribution of heavy and transition metals. Previously, we found in mice that knockout of MT 1 and 2 genes (MTKO) impaired spatial learning and potentiated the learning impairment caused by developmental mercury exposure. The current study examined the neurocognitive and neurochemical effects of MTKO with the developmental copper (Cu) supplementation.

    Methods

    Wildtype (WT) and MTKO mice were given supplemental Cu (0, 10 or 50 mg/l) in their drinking water during gestation and until weaning. When the mice were young adults they were trained on the win-shift 8-arm radial maze test of spatial learning and memory. After cognitive testing, their brains were analyzed for norepinepherine, dopamine and serotonin levels.

    Results

    In the spatial learning test, wildtype mice showed the normal sex difference with males performing more accurately than the females. This effect was eliminated by MTKO and restored by moderate Cu supplementation during development. In neurochemical studies, MTKO caused a significant overall increase in serotonin in all of the regions studied: the frontal cortex, posterior cortex, hippocampus, striatum, midbrain, and brainstem. MTKO also caused a significant increase in norepinepherine in the brainstem and hippocampus. In wildtype mice, Cu supplementation during development caused a significant decline in dopamine and norepinepherine in the midbrain and dopamine in the frontal cortex. These effects were blocked by MTKO.

    Conclusions

    The normal sex difference in spatial working memory accuracy, which was eliminated by MTKO, was restored by moderate copper supplementation. MTKO increased serotonin across all brain areas studied and increased norepinepherine only in the hippocampus and brainstem. MTKO blocked copper-induced decreases in dopamine and norepinepherine in the midbrain and dopamine in the frontal cortex.
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    The use of comparative genomics to investigate mechanisms of cadmium induced transcription
    (2009) Tvermoes, Brooke Erin

    Cadmium is a human carcinogen and a persistent environmental pollutant of increasing concern. Yet, the exact molecular targets of cadmium toxicity and the molecular mechanisms by which cadmium influences gene expression have not been fully elucidated. Therefore, the characterization of cadmium-inducible genes will provide a better understanding of the underlying mechanism involved in sensing cadmium-stress and the subsequent signaling pathways important for cellular defense against cadmium toxicity. To this end, we characterized two cadmium-responsive genes of no known biological function from the nematode Caenorhabditis elegans (C. elegans), numr-1 and numr-2.

    Expression analysis of numr-1 and numr-2 revealed the same temporal and spatial expression patterns of both genes in the absence and presence of metal treatment. In the absence of metal, constitutive expression of numr-1/-2 was developmentally regulated. When adult animals were exposed to metal, numr-1/-2 expression dramatically increased. We show that worms overexpressing numr-1/-2 were more resistant to metal stress and longer lived than control animals; whereas reducing numr-1/-2 activity resulted in increased sensitivity to metal exposure. Furthermore, in the absence of metal, the two numr-1 mutant alleles, tm2775 and ok2239, exhibited decreased muscular functions. The molecular characterization of numr-1 and numr-2 also revealed that the expression of these two genes, at least in part, was regulated by changes in intracellular calcium concentrations ([Ca2+]i). This finding lead us to reevaluate the role of calcium mobilization in cadmium-induced transcription.

    While several studies have indicated that exposure to cadmium resulted in increased [Ca2+]i, the mechanism by which cadmium can effect [Ca2+]i and concurrent effects on gene expression remain poorly understood. Therefore, we investigated the effects of low-level cadmium exposure, sufficient to induce transcription of cadmium-responsive genes, on the regulation of [Ca2+]i. In these studies, we utilized the protein-based calcium sensor YC 3.60 stably expressed in a HEK293 cell line. YC 3.60 is insensitive to cadmium ions, and thus is useful to monitor changes in [Ca2+]i following cadmium treatment. Exposing HEK293 cells to 1-30 µM cadmium was sufficient to induce transcription of cadmium-responsive genes such as metallothionein. Cadmium exposure from 1-10 µM had no effect on cell viability, [Ca2+]i mobilization, or increased transcriptional activity of calcium-responsive genes. In contrast, exposure to 30 µM cadmium significantly decreased cell viability, reduced intracellular calcium stores, and significantly altered the transcriptional activity of calcium-responsive genes. Taken together, these data indicate that low-level cadmium exposures (1-10 µM) can induce transcription of cadmium-responsive genes such as metallothionein independent of [Ca2+]i mobilization.

    To gain further insight into the mechanistic relationship between cadmium and calcium we investigated the effects of cadmium exposure on the defecation cycle of C. elegans. Defecation is a highly rhythmic behavior that is regulated by calcium oscillations. We found that low-level cadmium exposures, sufficient to induce expression of cadmium-responsive genes such as numr-1/-2, significantly shortened the defecation cycle but did not alter the rhythm of the cycle or the magnitude of the intestinal calcium oscillations. Modulation of lipid metabolism in C. elegans results in a similar shortened defecation cycle, whereas modulation of [Ca2+]i results in lengthened and arrhythmic defection cycles, suggesting that the mechanism by which cadmium alters defecation is independent of [Ca2+]i mobilization.

    In summary, the data in this work demonstrates that low-level cadmium exposure induces expression of cadmium-responsive genes independent of calcium mobilization. Thus, modulation of intracellular calcium is unlikely the primary mechanism by which cadmium regulates transcription at low-levels of exposure.

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    Toxicogenomic Responses to Inorganic and Organic Mercury in Caenorhabditis elegans
    (2010) McElwee, Matt

    Mercury is a toxic metal that can exist in multiple chemical forms, all of which are toxic to humans. Despite years of research, only a fragmented understanding of the molecular mechanisms of toxicity exists. Furthermore, it is not known to what extent different mercury species act similarly or dissimilarly at the molecular level. The objective of this study was to investigate the extent to which inorganic and methylmercury act differently at the molecular level.

    The relative toxicity of mercuric chloride (HgCl2) and methylmercury chloride (MeHg) in C. elegans was determined by testing the effect of mercurial exposure on growth, reproduction and induction of stress-response genes. MeHg was more toxic than HgCl2, though the difference in toxicity between the two mercurials varied by assay. Using approximately sub-, low- and high toxic exposures to both mercurials, microarrays were performed to determine the effects of the HgCl2 and MeHg on transcription. A total of 473 genes were differentially expressed in the three HgCl2 treatments, while a total of 2865 genes were differentially expressed in the three MeHg treatments. Analysis of the microarray data by hierarchical clustering, principal components analysis and a self-organizing map indicated that the transcriptional effects of the two mercurials were vastly different. Gene Ontology analysis and pathway mapping indicated that the two mercurials had very different effects on biological processes as well.

    The biological function of genes up-regulated by mercurials was tested using RNA interference (RNAi). The effect of RNAi and mercury co-exposure on C. elegans growth was tested for 599 genes. Knock-down of 18 of these genes was found to significantly affect growth of C. elegans exposed to mercury. Of these 18 genes, only 2 were found to significantly affect growth in response to both mercurials.

    ABCG2, BACE1, BACE2, CHKA, CHKB, ELOVL3, ELOVL6, GCLC and PARG are human homologs of the genes found to significantly affect growth of C. elegans in mercury exposure. The effect of sub-, low-, and high-toxic treatments of both mercurials on expression of these genes was tested using three cell lines: SK-N-SH, HepG2 and HEK293. Of these 162 cell-gene-mercury-toxicity combinations, there were 36 in which the gene was differentially expressed. In 24 of these, the gene was significantly differentially expressed by only one of the mercurials. The evolutionary conservation of function of these genes in mercury exposure was tested using RNAi. A total of 11 significant gene-mercury interactions were found between the three cell lines, but there was not a cell type-gene combination in which exposure to both mercurials was found to significantly affect cytotoxicity.

    In whole organism and cell culture studies, inorganic and methylmercury were found to have different effects on transcription. In both systems, there was very little overlap in the genes involved in mercury resistance and susceptibility. These data indicate that molecular mechanisms of toxicity differ by mercurial.