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Transcriptional biomarkers of toxicity – powerful tools or random noise?: An applied perspective from studies on bivalves
University of Skövde, School of Bioscience. University of Skövde, Systems Biology Research Environment. Lund University. (Ekologisk modellering, Ecological Modeling)ORCID iD: 0000-0002-2418-1163
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Aquatic organisms are constantly at risk of being exposed to potentially harmful chemical compounds of natural or anthropogenic origin. Biological life can for instance respond to chemical stressors by changes in gene expression, and thus, certain gene transcripts can potentially function as biomarkers, i.e. early warnings, of toxicity and chemical stress. A major challenge for biomarker application is the extrapolation of transcriptional data to potential effects at the organism level or above. Importantly, successful biomarker use also requires basal understanding of how to distinguish actual responses from background noise. The aim of this thesis is, based on response magnitude and variation, to evaluate the biomarker potential in a set of putative transcriptional biomarkers of general toxicity and chemical stress.

Specifically, I addressed a selection of six transcripts involved in cytoprotection and oxidative stress: catalase (cat), glutathione-S-transferase (gst), heat shock proteins 70 and 90 (hsp70, hsp90), metallothionein (mt) and superoxide dismutase (sod). Moreover, I used metal exposures to serve as a proxy for general chemical stress, and due to their ecological relevance and nature as sedentary filter-feeders, I used bivalves as study organisms.

In a series of experiments, I tested transcriptional responses in the freshwater duck mussel, Anodonta anatina, exposed to copper or an industrial wastewater effluent, to address response robustness and sensitivity, and potential controlled (e.g. exposure concentration) and random (e.g. gravidness) sources of variation. In addition, I performed a systematic review and meta-analysis on transcriptional responses in metal exposed bivalves to (1) evaluate what responses to expect from arbitrary metal exposures, (2) assess the influence from metal concentration (expressed as toxic unit), exposure time and analyzed tissue, and (3) address potential impacts from publication bias in the scientific literature.

Response magnitudes were generally small in relationship to the observed variation, both for A. anatina and bivalves in general. The expected response to an arbitrary metal exposure would generally be close to zero, based on both experimental observations and on the estimated impact from publication bias. Although many of the transcripts demonstrated concentration-response relationships, large background noise might in practice obscure the small responses even at relatively high exposures. As demonstrated in A. anatina under copper exposure, this can be the case already for single species under high resolution exposures to single pollutants. As demonstrated by the meta-regression, this problem can only be expected to increase further upon extrapolation between different species and exposure scenarios, due to increasing heterogeneity and random variation. Similar patterns can also be expected for time-dependent response variation, although the meta-regression revealed a general trend of slightly increasing response magnitude with increasing exposure times.

In A. anatina, gravidness was identified as a source of random variability that can potentially affect the baseline of most assessed biomarkers, particularly when quantified in gills. Response magnitudes and variability in this species were generally similar for selected transcripts as for two biochemical biomarkers included for comparison (AChE, GST), suggesting that the transcripts might not capture early warnings more efficiently than other molecular endpoints that are more toxicologically relevant. Overall, high concentrations and long exposure durations presumably increase the likelihood of a detectable transcriptional response, but not to an extent that justifies universal application as biomarkers of general toxicity and chemical stress. Consequently, without a strictly defined and validated application, this approach on its own appears unlikely to be successful for future environmental risk assessment and monitoring. Ultimately, efficient use of transcriptional biomarkers might require additional implementation of complementary approaches offered by current molecular techniques.

Place, publisher, year, edition, pages
Lund: Lund University (Media-Tryck) , 2023. , p. 199
Keywords [en]
Biomarkers, Bivalves, Bivalvia, Ecotoxicology, Environmental toxicology, Environmental risk assessment, RT-qPCR, qPCR, Quantitative PCR, Systematic review, Meta-analysis, Dose-response relationships, Concentration-response relationships, Heavy metals, Metals, General toxicity, Copper, Duck mussel, Anodonta anatina, Freshwater, Gene transcription
Keywords [sv]
Biomarkörer, Musslor, Bivalvia, Ekotoxikologi, Miljötoxikologi, Miljöriskbedömning, RT-qPCR, qPCR, Kvantitativ PCR, Systematic review, Metaanalys, Dos-responssamband, Koncentration-responssamband, Tungmetaller, Metaller, Allmän toxicitet, Koppar, Allmän dammussla, Anodonta anatina, Sötvatten, Gentranskription
National Category
Biochemistry Molecular Biology Pharmacology and Toxicology Environmental Sciences Genetics and Genomics Ecology
Research subject
Ecological Modelling Group
Identifiers
URN: urn:nbn:se:his:diva-22180ISBN: 978-91-8039-496-3 (print)ISBN: 978-91-8039-495-6 (electronic)OAI: oai:DiVA.org:his-22180DiVA, id: diva2:1726010
Public defence
2023-02-10, Blå hallen, Sölvegatan 37, Lund, 09:00 (English)
Opponent
Supervisors
Available from: 2023-01-12 Created: 2023-01-12 Last updated: 2025-02-20Bibliographically approved
List of papers
1. Transcriptional and biochemical biomarker responses in a freshwater mussel (Anodonta anatina) under environmentally relevant Cu exposure
Open this publication in new window or tab >>Transcriptional and biochemical biomarker responses in a freshwater mussel (Anodonta anatina) under environmentally relevant Cu exposure
Show others...
2020 (English)In: Environmental Science and Pollution Research, ISSN 0944-1344, E-ISSN 1614-7499, Vol. 27, no 9, p. 9999-10010Article in journal (Refereed) Published
Abstract [en]

Molecular biomarkers, like gene transcripts or enzyme activities, are potentially powerful tools for early warning assessment of pollution. However, a thorough understanding of response and baseline variation is required to distinguish actual effects from pollution. Here, we assess the freshwater mussel Anodonta anatina as a biomarker model species for freshwater ecosystems, by testing responses of six transcriptional (cat, gst, hsp70, hsp90, mt, and sod) and two biochemical (AChE and GST) biomarkers to environmentally relevant Cu water concentrations. Mussels (n = 20), collected from a stream free from point source pollution, were exposed in the laboratory, for 96 h, to Cu treatments (< 0.2 mu g/L, 0.77 +/- 0.87 mu g/L, and 6.3 +/- 5.4 mu g/L). Gills and digestive glands were extracted and analyzed for transcriptional and biochemical responses. Biological and statistical effect sizes from Cu treatments were in general small (mean log(2) fold-change <= 0.80 and Cohen's f <= 0.69, respectively), and no significant treatment effects were observed. In contrast, four out of eight biomarkers (cat, gst, hsp70, and GST) showed a significant sex:tissue interaction, and additionally one (sod) showed significant overall effects from sex. Specifically, three markers in gills (cat, mt, GST) and one in digestive gland (AChE) displayed significant sex differences, independent of treatment. Results suggest that sex or tissue effects might obscure low-magnitude biomarker responses and potential early warnings. Thus, variation in biomarker baselines and response patterns needs to be further addressed for the future use of A. anatina as a biomarker model species.

Place, publisher, year, edition, pages
Springer Berlin/Heidelberg, 2020
Keywords
Bivalve, Gene expression, Response variability, Sex effects, Effect size, RT-qPCR
National Category
Other Biological Topics
Research subject
INF502 Biomarkers; Ecological Modelling Group; Bioinformatics
Identifiers
urn:nbn:se:his:diva-18132 (URN)10.1007/s11356-020-07660-4 (DOI)000524949600099 ()31933076 (PubMedID)2-s2.0-85077999744 (Scopus ID)
Funder
Knowledge Foundation
Note

CC BY 4.0

Environmental Science and Pollution Research. ISSN: 0944-1344 (Print) 1614-7499 (Online)

Available from: 2020-01-14 Created: 2020-01-14 Last updated: 2023-01-12Bibliographically approved
2. Molecular biomarker responses in the freshwater mussel Anodonta anatina exposed to an industrial wastewater effluent
Open this publication in new window or tab >>Molecular biomarker responses in the freshwater mussel Anodonta anatina exposed to an industrial wastewater effluent
2022 (English)In: Environmental Science and Pollution Research, ISSN 0944-1344, E-ISSN 1614-7499, Vol. 29, no 2, p. 2158-2170Article in journal (Refereed) Published
Abstract [en]

Using a selection of molecular biomarkers, we evaluated responses in freshwater mussels (Anodonta anatina) exposed to effluent from an industrial wastewater treatment facility. The aims of this work were to (1) assess biomarkers of general toxicity under sublethal exposure to an anthropogenic mixture of chemicals, represented by an arbitrary effluent, and (2) evaluate the potential of A. anatina as a bioindicator of pollution. Adult mussels (n = in total 32; 24 males and 8 females) were exposed (96 h) in the laboratory to a fixed dilution of effluent or to a control treatment of standardized freshwater. Metal concentrations were in general higher in the effluent, by an order of magnitude or more, compared to the control. Toxic unit estimates were used as proxies of chemical stress, and Cu, Ni, and Zn were identified as potential major contributors (Cu> Ni > Zn). Six transcriptional (cat, gst, hsp70, hsp90, mt, sod) and two biochemical (AChE, GST) biomarkers were analyzed in two tissues, gills, and digestive glands. Out of the 16 responses (eight biomarkers x two tissues), 14 effect sizes were small (within +/- 28 % of control) and differences non-significant (p > 0.05). Results did however show that (1) AChE activity increased by 40% in gills of exposed mussels compared to control, (2) hsp90 expression was 100% higher in exposed female gills compared to control, and (3) three marker signals (AChE in both tissues, and hsp70 in gills) differed between sexes, independent of treatment. Results highlight a need for further investigation of molecular biomarker variability and robustness in A. anatina.

Place, publisher, year, edition, pages
Springer, 2022
Keywords
Bivalve, Effect size, Mixture toxicity, RT-qPCR, Sex effects, Wastewater
National Category
Environmental Sciences Ecology
Research subject
Ecological Modelling Group
Identifiers
urn:nbn:se:his:diva-20490 (URN)10.1007/s11356-021-15633-4 (DOI)000682415400003 ()34363176 (PubMedID)2-s2.0-85112648604 (Scopus ID)
Note

CC BY 4.0

Correspondence to Gustaf MO Ekelund Ugge. gustaf.ekelund_ugge@biol.lu.se

Correction in: Environmental Science and Pollution Research, September 2021. doi:10.1007/s11356-021-16224-z - "The layout of Table 2 contains missing line [breaks] that make the table confusing to read. The original article has been corrected."

Available from: 2021-08-20 Created: 2021-08-20 Last updated: 2023-01-12Bibliographically approved
3. Evaluation of transcriptional biomarkers using a high-resolution regression approach: Concentration-dependence of selected transcripts in copper-exposed freshwater mussels (Anodonta anatina)
Open this publication in new window or tab >>Evaluation of transcriptional biomarkers using a high-resolution regression approach: Concentration-dependence of selected transcripts in copper-exposed freshwater mussels (Anodonta anatina)
2022 (English)In: Environmental Toxicology and Pharmacology, ISSN 1382-6689, E-ISSN 1872-7077, Vol. 90, article id 103795Article in journal (Refereed) Published
Abstract [en]

We tested concentration-dependence of selected gene transcripts (cat, gst, hsp70, hsp90, mt and sod) for evaluation as biomarkers of chemical stress. Contrary to the common approach of factorial designs and few exposure concentrations, we used regression across a high-resolution concentration series. Specifically, freshwater mussels (Anodonta anatina) were acutely (96 h) exposed to Cu (13 nominal concentrations, measuring 0.13–1 600 µg/L), and transcripts were measured by RT-qPCR. In digestive glands, cat, hsp90 and mt decreased with water Cu (p < 0.05), but response magnitudes saturated at < 2-fold decreases. In gills, gst, hsp70, hsp90 and mt increased with water Cu (p < 0.05). While hsp70, hsp90 and mt exceeded 2-fold increases within the exposure range, high Cu concentrations were required (38–160 µg/L). Although gill responses were generally more robust compared to digestive glands, overall small response magnitudes and moderate sensitivity may set limit for potential application as general biomarkers of chemical stress.

Place, publisher, year, edition, pages
Elsevier, 2022
Keywords
Bivalves, Dose-response, Molecular biomarkers, Response magnitude, RT-qPCR, Unionida
National Category
Environmental Sciences Other Biological Topics Biochemistry Molecular Biology
Research subject
Ecological Modelling Group
Identifiers
urn:nbn:se:his:diva-20863 (URN)10.1016/j.etap.2021.103795 (DOI)000787964100006 ()34971800 (PubMedID)2-s2.0-85122497764 (Scopus ID)
Note

CC BY 4.0

© 2022 The Authors

Correspondence to: Department of Biology, Lund University, Sölvegatan 37, 223 62 Lund, Sweden. E-mail address: gustaf.ekelund_ugge@biol.lu.se (G.M.O. Ekelund Ugge).

Available from: 2022-01-20 Created: 2022-01-20 Last updated: 2025-02-20Bibliographically approved
4. Transcriptional Responses as Biomarkers of General Toxicity: A Systematic Review and Meta-Analysis on Metal-Exposed Bivalves
Open this publication in new window or tab >>Transcriptional Responses as Biomarkers of General Toxicity: A Systematic Review and Meta-Analysis on Metal-Exposed Bivalves
2023 (English)In: Environmental Toxicology and Chemistry, ISSN 0730-7268, E-ISSN 1552-8618, Vol. 42, no 3, p. 628-641Article, review/survey (Refereed) Published
Abstract [en]

Through a systematic review and a series of meta-analyses, we evaluated the general responsiveness of putative transcriptional biomarkers of general toxicity and chemical stress. We targeted metal exposures performed on bivalves under controlled laboratory conditions, and selected six transcripts associated with general toxicity for evaluation: catalase (cat), glutathione-S-transferase (gst), heat shock proteins 70 and 90 (hsp70, hsp90), metallothionein (mt) and superoxide dismutase (sod). Transcriptional responses (n = 396) were extracted from published scientific articles (k = 22) and converted to log response ratios (lnRRs). By estimating toxic units (TUs), we normalized different metal exposures to a common scale, as a proxy of concentration. Using Bayesian hierarchical random effect models, we then tested the effects of metal exposure on lnRR, both for metal exposure in general and in meta-regressions using TU and exposure time as independent variables. Corresponding analyses were also repeated with transcript and tissue as additional moderators. Observed patterns were similar for general as for transcript- and tissue-specific responses. The expected overall response to arbitrary metal exposure was a lnRR of 0.50, corresponding to a 65 % increase relative a non-exposed control. However, when accounting for publication bias, the estimated ‘true’ response showed no such effect. Furthermore, expected response magnitude increased slightly with exposure time, but there was little support for general monotonic concentration-dependence with regards to TU. Altogether, this work reveals potential limitations that need consideration prior to applying the selected transcripts as biomarkers in environmental risk assessment. This article is protected by copyright. All rights reserved. Environ Toxicol Chem 2022;00:0–0. 

Place, publisher, year, edition, pages
John Wiley & Sons, 2023
Keywords
Chemical analysis, Metals, Molluscs, Random processes, Risk assessment, Superoxide dismutase, Tissue, Toxicity, Aquatic toxicology, Bayesian statistics, Eco-toxicology, Meta-regression, Metal exposures, Mollusk toxicology, Publication bias, Risks assessments, Toxic unit, Transcriptional response, Biomarkers, ecotoxicology
National Category
Environmental Sciences Pharmacology and Toxicology Biochemistry Molecular Biology Other Biological Topics
Research subject
Ecological Modelling Group
Identifiers
urn:nbn:se:his:diva-22097 (URN)10.1002/etc.5494 (DOI)000928969800001 ()36200657 (PubMedID)2-s2.0-85142195831 (Scopus ID)
Note

CC BY 4.0

© 2022 SETAC. This article is protected by copyright. All rights reserved.

First published: 06 October 2022

Correspondence gustaf.ekelund_ugge@biol.lu.se

Available from: 2022-12-01 Created: 2022-12-01 Last updated: 2025-02-20Bibliographically approved

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