Global Gene Expression Analysis Reveals Dynamic and Developmental Stage-Dependent Enrichment of Lead-Induced Neurological Gene Alterations

Peterson, Samuel M., Zhang, Jun, Weber, Gregory, Freeman, Jennifer L., Environmental Health Perspectives

BACKGROUND: The underlying genetic mechanisms specific to subtle neurological alterations associated with environmental lead (Pb) exposures have not been clearly elucidated.

OBJECTIVES: The goal of this study was to identify novel gene targets and the underlying genetic mechanisms associated with developmental Pb neurotoxicity.

METHODS: We first exposed zebrafish embryos to a range of Pb concentrations throughout early development to establish relative toxicity. Using the data from that experiment, we exposed another group of zebrafish embryos to a subledial dose of Pb (100 ppb) immediately after fertilization through 72 or 120 hr postfertilization (hpf). Global gene expression was then analyzed for molecular pathways and gene ontology enrichment, and Western blot analysis was performed to investigate the translation of gene expression changes to protein levels.

RESULTS: After 72 hpf, we identified 231 probes representing 90 nonredundant genes with well-established function or orthology to human genes as being altered by Pb exposure. This gene set was both confirmatory and novel in nature and was highly enriched for neurological development, function, and disease. Moreover, gene changes at this time point were correlated to altered protein levels. Alternatively, the gene set at 120 hpf did not share association with neurological development.

CONCLUSIONS: Global gene expression alterations associated with developmental Pb exposure were dynamic and dependent on developmental stage. Gene expression alterations at the 72-hpf time point were highly enriched with genes and molecular pathways associated with neurological development and disease. Moreover, we identified a number of novel targets for future exploration into their role in the genetic mechanisms underlying Pb-induced neurological alterations.

KEY WORDS: Danio rerio, gene expression, genomics, lead, microarray, Pb, toxicogenomics, zebrafish. Environ Health Perspect 119:615-621 (2011). doi:10.1289/ehp.1002590 [Online 8 December 2010]

The heavy metal lead (Pb) is a well-studied toxicant known to cause a wide array of adverse health effects. The developing nervous system is especially susceptible to Pb-induced alterations, and the Centers for Disease Control and Prevention (CDC) currently recommends that primary prevention strategies be implemented when a child's blood Pb level (BLL) exceeds 10 [micro]g/dL (CDC 2005). However, increasingly stringent regulatory actions have failed to limit exposures to a level documented as safe and without neurological impacts (Canfield et al. 2003; Min et al. 2007), including recent epidemiological studies that linked subinterventional BLLs to decreased IQ (intelligence quotient) and attention span and increased behavioral problems and odds ratio for the development of attention deficit hyperactivity disorder (Eubig et al. 2010, Surkan et al. 2007). Moreover, although the mechanisms of Pb toxicity have been studied for many years, the molecular mechanisms underlying the subtle neurological impacts are not understood.

The developing nervous system is particularly susceptible to Pb toxicity because of increased levels of cell proliferation, migration, and differentiation and the complexity of cell interactions (Rodier 1994, 2004). In addition, children absorb a greater percentage of ingested Pb, and circulating Pb has greater access to the brain because the blood-brain barrier is not yet fully established (Cory-Slechta and Schaumburg 2000; Leggett 1993). Also, Pb can be released from maternal bone stores and transferred to a developing fetus (Gardella 2001). As a result, Pb exposure can be initiated early in prenatal development, and studies have indicated that umbilical BLLs [less than or equal to] 10 [micro]g/dL are linked with deleterious effects on cognitive development (Gomaa et al. 2002). Thus, it is important to study Pb exposure in vivo during embryo-genesis at a relevant concentration.

The zebrafish presents a complementary model to rodents to assess impacts of prenatal chemical exposure. To specifically assess adverse effects associated with chemical exposure during embryonic development in rodents, dam dosing and sacrifice are generally required. Alternatively, the zebrafish model has gained popularity in developmental and toxicological studies because of rapid ex utero embryonic development in which embryos can be dosed in a petri dish. Additionally, a near-transparent chorion permits easy visualization of the developing organism throughout the entire embryonic period. Moreover, a single breeding pair can produce hundreds of embryos weekly, providing a large pool of individuals for experimental testing. The application of the zebrafish model in developmental biology research has resulted in a plethora of background literature, as well as the incorporation of the species into genetic and toxicology research. Moreover, a finished genome sequence and the homology between zebrafish and human genes permit translation of molecular mechanisms of toxicity observed in the zebrafish model system to humans (Barbazuk et al. 2000). Thus, the zebrafish provides an ideal model organism for defining molecular alterations associated with early developmental Pb exposure.

Although numerous studies have investigated Pb toxicity (see "Discussion" for examples), the molecular mechanisms underlying the subtle neurological effects on the developing nervous system are not thoroughly understood. The use of microarray technology allows for the simultaneous measurement of the expression level of genes on a genome-wide scale. When used with proper statistical approaches and genetic pathway analyses, a list of statistically significant, differentially expressed genes can be combined with an extensive database of gene and gene product interactions to provide a robust determination of commonly affected gene networks and pathways (Mattingly et al. 2009; Yang et al. 2007). Based on the results of these calculations, the ability to predict and make hypotheses about specific mechanisms of toxicity is strongly improved. In this study we aimed to identify key genes and regulatory networks perturbed by sublethal developmental Pb exposure. Our goal was to achieve enrichment of genes and molecular pathways associated with neurological development and disease in order to identify novel gene targets for future investigation into the specific genetic mechanisms underlying developmental Pb toxicity at sublethal doses. Using zebrafish ex utero development, we initiated exposure conditions immediately after fertilization to correspond to the mammalian in utero prenatal developmental period. We combined genomewide expression analysis with pathway analysis to uncover novel genes of interest and to highlight critical time points in development. Further, we investigated translation …

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Publication information: Article title: Global Gene Expression Analysis Reveals Dynamic and Developmental Stage-Dependent Enrichment of Lead-Induced Neurological Gene Alterations. Contributors: Peterson, Samuel M. - Author, Zhang, Jun - Author, Weber, Gregory - Author, Freeman, Jennifer L. - Author. Journal title: Environmental Health Perspectives. Volume: 119. Issue: 5 Publication date: May 2011. Page number: 615+. © 2006 National Institute of Environmental Health Sciences. COPYRIGHT 2011 Gale Group.

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