Dataset Information

The long amplicon quantitative PCR for DNA damage assay as a sensitive method of assessing DNA damage in the environmental model, Atlantic killifish (Fundulus heteroclitus).

ABSTRACT: DNA damage is an important mechanism of toxicity for a variety of pollutants, and therefore, is often used as an indicator of pollutant effects in ecotoxicological studies. Here, we adapted a PCR-based assay for nuclear and mitochondrial DNA damage for use in an important environmental model, the Atlantic killifish (Fundulus heteroclitus). We refer to this assay as the long amplicon quantitative PCR (LA-QPCR) assay. To validate this method in killifish, DNA damage was measured in liver, brain, and muscle of fish dosed with 10 mg/kg benzo[a]pyrene. This exposure caused 0.4-0.8 lesions/10 kb. We also measured DNA damage in liver and muscle tissues from killifish inhabiting a Superfund site, confirming the utility of this method for biomonitoring. In both cases, damage levels were comparable in nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Since extensive nDNA sequence data are not readily available for many environmentally relevant species, but mitochondrial genomes are frequently fully sequenced, this assay can be adapted to examine mtDNA damage in virtually any species with little development. Therefore, we argue that this assay will be a valuable tool in assessing DNA damage in ecotoxicological studies.

SUBMITTER: Jung D

PROVIDER: S-EPMC2676791 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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The long amplicon quantitative PCR for DNA damage assay as a sensitive method of assessing DNA damage in the environmental model, Atlantic killifish (Fundulus heteroclitus).

Jung Dawoon D Cho Youngeun Y Meyer Joel N JN Di Giulio Richard T RT

Comparative biochemistry and physiology. Toxicology & pharmacology : CBP 20080724 2

DNA damage is an important mechanism of toxicity for a variety of pollutants, and therefore, is often used as an indicator of pollutant effects in ecotoxicological studies. Here, we adapted a PCR-based assay for nuclear and mitochondrial DNA damage for use in an important environmental model, the Atlantic killifish (Fundulus heteroclitus). We refer to this assay as the long amplicon quantitative PCR (LA-QPCR) assay. To validate this method in killifish, DNA damage was measured in liver, brain, a ...[more]

PMID: 18706522

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Project description:Understanding molecular mechanisms of toxicity is facilitated by experimental manipulations, such as disruption of function by gene targeting, that are especially challenging in non-standard model species with limited genomic resources. While loss-of-function approaches have included gene knock-down using morpholino-modified oligonucleotides and random mutagenesis using mutagens or retroviruses, more recent approaches include targeted mutagenesis using zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. These latter methods provide more accessible opportunities to explore gene function in non-traditional model species. To facilitate evaluation of toxic mechanisms for important categories of aryl hydrocarbon pollutants, whose actions are known to be receptor mediated, we used ZFN and CRISPR-Cas9 approaches to generate aryl hydrocarbon receptor 2a (AHR2a) and AHR2b gene mutations in Atlantic killifish (Fundulus heteroclitus) embryos. This killifish is a particularly valuable non-traditional model, with multiple paralogs of AHR whose functions are not well characterized. In addition, some populations of this species have evolved resistance to toxicants such as halogenated aromatic hydrocarbons. AHR-null killifish will be valuable for characterizing the role of the individual AHR paralogs in evolved resistance, as well as in normal development. We first used five-finger ZFNs targeting exons 1 and 3 of AHR2a. Subsequently, CRISPR-Cas9 guide RNAs were designed to target regions in exon 2 and 3 of AHR2a and AHR2b. We successfully induced frameshift mutations in AHR2a exon 3 with ZFN and CRISPR-Cas9 guide RNAs, with mutation frequencies of 10% and 16%, respectively. In AHR2b, mutations were induced using CRISPR-Cas9 guide RNAs targeting sites in both exon 2 (17%) and exon 3 (63%). We screened AHR2b exon 2 CRISPR-Cas9-injected embryos for off-target effects in AHR paralogs. No mutations were observed in closely related AHR genes (AHR1a, AHR1b, AHR2a, AHRR) in the CRISPR-Cas9-injected embryos. Overall, our results demonstrate that targeted genome-editing methods are efficient in inducing mutations at specific loci in embryos of a non-traditional model species, without detectable off-target effects in paralogous genes.

Project description:The Atlantic killifish (Fundulus heteroclitus), native to estuarine areas of the Atlantic coast of the United States, has become a valuable ecotoxicological model as a result of its ability to acclimate to rapid environmental changes and adapt to polluted habitats. MicroRNAs (miRNAs) are highly conserved small RNAs that regulate gene expression and play critical roles in stress responses in a variety of organisms. Global miRNA expression in killifish and the potential roles miRNA have in environmental acclimation have yet to be characterized. Accordingly, we profiled miRNA expression in killifish gill for the first time and identified a small group of highly expressed, well-conserved miRNAs as well as 16 novel miRNAs not yet identified in other organisms. Killifish respond to large fluctuations in salinity with rapid changes in gene expression and protein trafficking to maintain osmotic balance, followed by a secondary phase of gene and protein expression changes that enable remodeling of the gills. Arsenic, a major environmental toxicant, was previously shown to inhibit gene expression responses in killifish gill, as well the ability of killifish to acclimate to a rapid increase in salinity. Thus, we examined the individual and combined effects of salinity and arsenic on miRNA expression in killifish gill. Using small RNA sequencing, we identified 270 miRNAs expressed in killifish, and found that miR-135b was differentially expressed in response to arsenic and at 24?h following transfer to salt water. Predicted targets of miR-135b are involved in ion transport, cell motility and migration, GTPase mediated signal transduction and organelle assembly. Consistent with previous studies of these two environmental stressors, we found a significant interaction (i.e., arsenic dependent salinity effect), whereby killifish exposed to arsenic exhibited an opposite response in miR-135b expression at 24?h post hyperosmotic challenge compared to controls. By examining mRNA expression of predicted miRNA targets during salinity acclimation and arsenic exposure, we found that miR-135b targets were significantly more likely to decrease during salinity acclimation than non-targets. Our identification of a significant interaction effect of arsenic and salinity on miR-135b expression supports the hypothesis that arsenic alters upstream regulators of stress response networks, which may adversely affect the killifish response to osmotic stress. The characterization of miRNAs in this ecotoxicological model will be a valuable resource for future studies investigating the role of miRNAs in response to environmental stress.

Project description:BackgroundThe most toxic aromatic hydrocarbon pollutants are categorized as dioxin-like compounds (DLCs) to which extreme tolerance has evolved independently and contemporaneously in (at least) four populations of Atlantic killifish (Fundulus heteroclitus). Surprisingly, the magnitude and phenotype of DLC tolerance is similar among these killifish populations that have adapted to varied, but highly aromatic hydrocarbon-contaminated urban/industrialized estuaries of the US Atlantic coast. Multiple tolerant and neighboring sensitive killifish populations were compared with the expectation that genetic loci associated with DLC tolerance would be revealed.ResultsSince the aryl hydrocarbon receptor (AHR) pathway partly or fully mediates DLC toxicity in vertebrates, single nucleotide polymorphisms (SNPs) from 42 genes associated with the AHR pathway were identified to serve as targeted markers. Wild fish (N = 36/37) from four highly tolerant killifish populations and four nearby sensitive populations were genotyped using 59 SNP markers. Similar to other killifish population genetic analyses, strong genetic differentiation among populations was detected, consistent with isolation by distance models. When DLC-sensitive populations were pooled and compared to pooled DLC-tolerant populations, multi-locus analyses did not distinguish the two groups. However, pairwise comparisons of nearby tolerant and sensitive populations revealed high differentiation among sensitive and tolerant populations at these specific loci: AHR 1 and 2, cathepsin Z, the cytochrome P450s (CYP1A and 3A30), and the NADH dehydrogenase subunits. In addition, significant shifts in minor allele frequency were observed at AHR2 and CYP1A loci across most sensitive/tolerant pairs, but only AHR2 exhibited shifts in the same direction across all pairs.ConclusionsThe observed differences in allelic composition at the AHR2 and CYP1A SNP loci were identified as significant among paired sensitive/tolerant populations of Atlantic killifish with multiple statistical tests. The genetic patterns reported here lend support to the argument that AHR2 and CYP1A play a role in the adaptive response to extreme DLC contamination. Additional functional assays are required to isolate the exact mechanism of DLC tolerance.

Dataset Information

The long amplicon quantitative PCR for DNA damage assay as a sensitive method of assessing DNA damage in the environmental model, Atlantic killifish (Fundulus heteroclitus).

Publications

The long amplicon quantitative PCR for DNA damage assay as a sensitive method of assessing DNA damage in the environmental model, Atlantic killifish (Fundulus heteroclitus).

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