Expression of target and reference genes in Daphnia magna exposed to ibuprofen

Heckmann, Lars-Henrik; Connon, Richard E.; Hutchinson, Thomas H.; Maund, Stephen J.; Sibly, Richard M.; Callaghan, Amanda

doi:10.1186/1471-2164-7-175

Cited by 118 publications

(76 citation statements)

References 21 publications

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“…cDNA was synthesized from the RNA samples (2 μg RNA) with MMLV reverse transcriptase. qPCR was performed according to MIQE standards (Bustin et al, 2009) to determine the expression of HR96 in the daphnids with forward primer 5′-TCT-GCG-ACA-AGG-CTT-TAG-GTT-3′ and reverse primer 5′-AGG-GCA-TTC-CGT-CTA-AAG-AAG-GCT-3′ at an annealing temperature of 58° C. β-actin was used as the housekeeping gene with forward primer 5′-CCA-CAC-TGT-CCC-CAT-TTA-TGA-AG-3′and reverse primer 5′-CGC-GAC-CAG-CCA-AAT-CC-3′ at an annealing temperature of 52.2° C as described previously (Heckmann et al, 2006). Efficiencies of the reactions were determined based on standard curves from 1:1, 1:5, 1:25, 1:125, 1:625 and 1:3125 dilutions of cDNA mixtures taken from all samples.…”

Section: Methodsmentioning

confidence: 99%

The HR96 activator, atrazine, reduces sensitivity of D. magna to triclosan and DHA

2015

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HR96 is a CAR/PXR/VDR ortholog in invertebrates, and a promiscuous endo- and xenobiotic nuclear receptor involved in acclimation to toxicants. Daphnia HR96 is activated by chemicals such as atrazine and linoleic acid (LA) (n-6 fatty acid), and inhibited by triclosan and docosahexaenoic acid (DHA)(n-3 fatty acid). We hypothesized that inhibitors of HR96 may block the protective responses of HR96 based on previously performed luciferase assays. Therefore, we performed acute toxicity tests with two-chemical mixtures containing a HR96 inhibitor (DHA or triclosan) and a HR96 activator (LA or atrazine). Surprisingly, results demonstrate that triclosan and DHA are less toxic when co-treated with 20–80 μM atrazine. Atrazine provides concentration-dependent protection as lower concentrations have no effect and higher concentrations cause toxicity. LA, a weaker HR96 activator, did not provide protection from triclosan or DHA. Atrazine’s protective effects are presumably due to its ability to activate HR96 or other toxicologically relevant transcription factors and induce protective enzymes. Atrazine did not significantly induce glucosyltransferase, a crucial enzyme in triclosan detoxification. However, atrazine did increase antioxidant activities, crucial pathways in triclosan’s toxicity, as measured through GST activity and the TROLOX equivalence assay. The increase in antioxidant capacity is consistent with atrazine providing protection from a wide range of toxicants that induce ROS, including triclosan and unsaturated fatty acids predisposed to lipid peroxidation.

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Section: Methodsmentioning

confidence: 99%

The HR96 activator, atrazine, reduces sensitivity of D. magna to triclosan and DHA

2015

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“…Nine different housekeeping genes recently introduced for QPCR in D. magna by Heckmann et al, 2006 [51] were used in QPCR analysis: actin, alpha-tubulin, cyclophilin, glyceraldehyde-3-phosphate dehydrogenase (GapDH), succinate dehydrogenase (SucDH), TATA-box binding protein (TBP), ubiquitin conjugating enzyme (UBC), 18S ribosomal RNA (18S), and 28S ribosomal RNA (28S) . A normalisation factor was calculated based on the endogenous controls assessed by geNorm [52] according to Schwarzenberger et al, 2009 [53].…”

Section: Methodsmentioning

confidence: 99%

Gene expression and activity of digestive proteases in Daphnia: effects of cyanobacterial protease inhibitors

et al. 2010

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BackgroundThe frequency of cyanobacterial blooms has increased worldwide, and these blooms have been claimed to be a major factor leading to the decline of the most important freshwater herbivores, i.e. representatives of the genus Daphnia. This suppression of Daphnia is partly attributed to the presence of biologically active secondary metabolites in cyanobacteria. Among these metabolites, protease inhibitors are found in almost every natural cyanobacterial bloom and have been shown to specifically inhibit Daphnia's digestive proteases in vitro, but to date no physiological responses of these serine proteases to cyanobacterial protease inhibitors in Daphnia have been reported in situ at the protein and genetic levels.ResultsNine digestive proteases were detected in D. magna using activity-stained SDS-PAGE. Subsequent analyses by LC-MS/MS and database search led to the identification of respective protease genes. D. magna responded to dietary protease inhibitors by up-regulation of the expression of these respective proteases at the RNA-level and by the induction of new and less sensitive protease isoforms at the protein level. The up-regulation in response to dietary trypsin- and chymotrypsin-inhibitors ranged from 1.4-fold to 25.6-fold. These physiological responses of Daphnia, i.e. up-regulation of protease expression and the induction of isoforms, took place even after feeding on 20% cyanobacterial food for only 24 h. These physiological responses proved to be independent from microcystin effects.ConclusionHere for the first time it was shown in situ that a D. magna clone responds physiologically to dietary cyanobacterial protease inhibitors by phenotypic plasticity of the targets of these specific inhibitors, i.e. Daphnia gut proteases. These regulatory responses are adaptive for D. magna, as they increase the capacity for protein digestion in the presence of dietary protease inhibitors. The type and extent of these responses in protease expression might determine the degree of growth reduction in D. magna in the presence of cyanobacterial protease inhibitors. The rapid response of Daphnia to cyanobacterial protease inhibitors supports the assumption that dietary cyanobacterial protease inhibitors exert a strong selection pressure on Daphnia proteases themselves.

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“…For example, atrazine and its metabolites affected eicosanoids in the isopode Hyalella azteca suggesting possible perturbations in neuropeptide hormonal systems [55]. Similarly, ibuprofen inhibits reproduction in the crustacean Daphnia magna due to the de-regulation of the ecosanoid signaling pathway, and hence of prostaglandins, which in crustaceans control reproduction [62,65]. A typical vertebrate estrogenic EDC, ethinylestradiol, alters metabolite pathways related to energy reserves, signal transduction, immune response, and neuromodulation in the unionid mussel Lampsilis fasciola.…”

Section: Evolutionary Perspective Of Endocrine Disruption: Edcs In Inmentioning

confidence: 99%

Endocrine Disruption in the Omics Era: New Views, New Hazards, New Approaches

Oliveira¹,

Barata²,

Piña³

2016

TOBIOTJ

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Abstract:The genome revolution has brought about a complete change on our view of biological systems. The quantitative determination of changes in all the major molecular components of the living cells, the "omics" approach, opened whole new fields for all health sciences, including toxicology. Endocrine disruption, i.e., the capacity of anthropogenic pollutants to alter the hormonal balance of the organisms, is one of the fields of Ecotoxicology in which omics has a relevant role. In the first place, the discovery of scores of potential targets in the genome of almost any Metazoan species studied so far, each of them being a putative candidate for interaction with endocrine disruptors. In addition, the understanding that ligands, receptors, and their physiological functions suffered fundamental variations during animal evolution makes it necessary to assess disruption effects separately for each major taxon. Fortunately, the same deal of knowledge on genes and genomes powered the development of new high-throughput techniques and holistic approaches. Genomics, transcriptomics, proteomics, metabolomics, and others, together with appropriate prediction and modeling tools, will mark the future of endocrine disruption assessment both for wildlife and humans.

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Expression of target and reference genes in Daphnia magna exposed to ibuprofen

Cited by 118 publications

References 21 publications

The HR96 activator, atrazine, reduces sensitivity of D. magna to triclosan and DHA

The HR96 activator, atrazine, reduces sensitivity of D. magna to triclosan and DHA

Gene expression and activity of digestive proteases in Daphnia: effects of cyanobacterial protease inhibitors

Endocrine Disruption in the Omics Era: New Views, New Hazards, New Approaches

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