1 Abstract
Ecotoxicology aims to understand toxic effects of chemicals in the environment. Effects can be observed at different levels of the biological organization, from molecular to ecosystem level.
Biomarkers on molecular and biochemical levels are used in ecotoxicology as early warning signals of chemical exposure, possible toxic effects and underlying mechanisms. As methods and technologies improve, more biomarkers are being implemented in ecotoxicological studies, due to the general interest in early detection and thus efficient prevention of environmental risks. However, to be of value in ecotoxicological assessment, a connection between biomarker response and effects at higher levels of biological organization should be established. Also, baseline variability for the biomarker in question as well as response to natural fluctuations of environmental factors should be evaluated.
The aim of this thesis was to increase value and understanding of biomarkers in ecotoxicological assessment by (1) linking responses across different levels of biological organization, and (2) gaining better understanding of the relative importance of ecological and physiological factors affecting oxidative biomarkers.
Paper I is focused on evolutionary conserved drug targets and the toxicity of pharmaceuticals for non-target organisms. The main conclusion from this study is that pharmaceuticals with conserved drug targets in non-target organisms have a higher toxicity than pharmaceuticals for which drug- targets have not been identified in the species. The effects were evaluated using end points at molecular, biochemical and individual levels. Consistent with the expected higher sensitivity of molecular and biochemical end points, the effects on the low-level biomarkers were observed at lower concentrations than at the individual level.
Paper II is focused on delineating effects of feeding and toxic exposure on oxidative biomarkers commonly used in ecotoxicology. The results are in agreement with the theory of caloric restriction that links enhanced caloric intake to increased pro-oxidative processes in animals. In our experiments with the cladoceran Daphnia magna, we observed positive effects for both antioxidant capacity and oxidized lipids in response to enhanced feeding rates. This have implications for the use of oxidative stress biomarkers in ecotoxicology as many substances have inhibitory effects on feeding rate and thus, changes in oxidative biomarkers can result from the altered feeding rate rather than other toxic mechanisms. Therefore, possible changes in feeding rate should be assessed when conducting exposure experiments or interpreting field data in studies employing oxidative stress biomarkers.
However, it was concluded that the ratio between antioxidative capacity and protein content was independent of feeding rate. Thus, this biomarker is suitable for xenobiotic exposure in D. magna.
This thesis have contributed to better understanding of molecular and biochemical biomarkers in ecotoxicological studies in regard to the connections between effects at different biological levels and confounding factors in biomarker response.
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Contents
Abstract ...1
Abbreviations ...3
List of papers ...4
1. Introduction ...5
1.1 Aim and objectives of the thesis……..……….………6
2. Background ………7
2.1 Oxidative stress ……… 7
2.1.1 Generation of ROS and antioxidative defense ……….7
2.1.2 Oxidative stress in ecology and ecotoxicology ……….8
2.2 Model species – Daphnia magna ………..9
2.3 Biomarkers ……….10
2.3.1 RNA content ………..10
2.3.2 Gene expression of specific proteins ………10
2.3.3 Antioxidative defense ……….11
2.3.4 Lipid peroxidation ……….11
3. Summary of papers ………11
3.1 Paper I ………..11
3.2 Paper II ……….12
4. Discussion ………12
5. Conclusions and future perspectives ……..……….13
6. Acknowledgements ………..14
7. References ………..15
3 Abbreviations
ASTM American Society for Testing and Materials ATP Adenosine triphosphate
CAT Catalase
CYP Cytochrome
DNA Deoxyribonucleic acid EC Effect concentration
EPA (US) Environmental Protection Agency GPX Glutathione peroxidase
hsp Heat shock proteins
LOEC Lowest observed effect concentration MDA Malondialdehyde
NAD(P)H Nicotinamide adenine dinucleotide phosphate-oxidase NOEC No observed effects concentration
OECD Organisation for Economic Co-operation and Development ORAC Oxygen radical absorbance capacity
PUFA Polyunsaturated fatty acid
qPCR Quantitative real-time polymerase chain reaction RNA Ribonucleic acid
ROS Reactive oxygen species rRNA Ribosomal ribonucleic acid SOD Superoxide dismutase
4 List of papers
Paper I S. Furuhagen, A. Fuchs, E. Lundström, M. Breitholtz and E. Gorokhova Do pharmaceuticals with evolutionary conserved molecular drug targets pose a greater environmental threat? Manuscript
My contributions:
(1) conducted the exposures for biomarker analyses and carried out DNA and RNA measurements;
(2) participated in the acute and reproduction tests and qPCR analysis;
(3) conducted statistical analysis for RNA data;
(4) was the responsible author of the manuscript.
Paper II S. Furuhagen, B. Liewenborg, M. Breitholtz and E. Gorokhova The effects of feeding and xenobiotics on oxidative stress in Daphnia magna: implications for eco- toxicological testing Manuscript
My contributions:
(1) planned and performed all laboratory work;
(2) conducted most of the data processing;
(3) was the responsible author of the manuscript.
5 1. Introduction
Increasing amounts of chemicals are found in the environment due to anthropogenic activities. The main aim within the field of ecotoxicology is to understand the possible adverse effects of these chemicals on biota and the underlying processes, both biochemical and physiological (Chapman 2002). Toxicity of chemical substances (xenobiotics) is commonly assessed by conducting tests with individual organisms, where acute and chronic effects are observed. Effect concentrations are often expressed either as ECx values, which is the concentration where X% effect is observed, or as NOEC (no observed effect concentration) and LOEC (lowest observed effect concentration) values. Acute effects are often assayed by exposure to high concentrations of xenobiotics during a specific part of an organism´s life cycle, whereas chronic effects are assayed by exposure during a longer time period, covering multiple stages of the life cycle. Acute tests, in which mortality is often used as an end point, are cheap and fast to perform, whereas chronic tests are more time consuming and thus more expensive, but on the other hand use sublethal end points with higher ecological relevance.
Effects from both acute and chronic exposures are often assayed on the individual level, by performing single species studies under controlled conditions. Ecotoxicological studies using multiple species, such as microcosm and mesocosm studies are more ecologically relevant since they incorporate interactions between co-occurring species and their abiotic environment. However, the results from such studies can be difficult to interpret since more factors affecting the observed responses are involved, making interpretation more complex. Therefore, if the aim of an eco- toxicological assay is to gain a better understanding of the underlying physiological and biological mechanisms of toxic effects, it is necessary to study model species in a controlled environment.
Model species often have a well-studied physiology and nowadays even mapped genome. Examples of common model animal species in ecotoxicological assays are fish (OECD 229), earthworm (OECD 222) and water flea (OECD 202, OECD 211). Using standardized organisms and test conditions (light, temperature and feeding regimes, test medium, etc.) facilitate inter-laboratory comparisons and data interpretation.
The toxicity of chemical substances may be assessed across all biological organization levels, from molecules to ecosystems (fig 1). These different organization levels are not isolated from each other, but are connected through multiple processes and interactions. Effects observed at one level may propagate to both higher and lower levels of the biological hierarchy. Therefore, to provide a holistic view of the possible consequences of xenobiotic exposure, toxic effects need to be studied at different organization levels.
There are many definitions of biomarkers and what should be included in this term. Peakall (1994) defined a biomarker as a biological response to chemical exposure that gives a measurement of exposure and sometimes toxic effect. According to this definition, a biomarker can be a response at any level of biological organization. However, some want to restrict the definition to include only end points at sub-organismal levels (van Gestel and van Brummelen 1996). The focus in this thesis will be on biomarkers on molecular and biochemical levels. Many biochemical and molecular biomarkers used in ecotoxicology were first developed within the field of medicine and human toxicology. In humans, many of the biomarkers have known connections with alterations in physiology and health.
However, this is not always true for biomarkers in ecotoxicology. In particular, only for a few biomarkers commonly used in animal models, e.g. vitellogenin in fish (Jobling et al. 1998, Larsson et al. 1999, Lange et al. 2001), functional relationships are established, which complicates the use and
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interpretation of these measurements for the health status of the organisms in question. Therefore, to understand responses of molecular and biochemical biomarkers and to predict consequences for the wellbeing of organisms and populations, it is vital to gain more knowledge about the linkages between the responses at different biological organization levels.
1.1. Aim and objectives of the thesis
The overall aim of this thesis was to increase the value and understanding of biomarkers in ecotoxicological research. All studies included in the thesis were conducted using the water flea Daphnia magna as a model species. The first objective was to link toxic effects at different biological organization levels by assaying end points at multiple levels (paper I). The second objective was to integrate ecological (food abundance) and physiological factors (feeding rate) when evaluating oxidative biomarker response in ecotoxicological studies (paper II).
Ecosystem Community Population Individual
Organ Tissue Cellular Biochemical
Molecular
Lower complexityHigher complexity
Later effectsEarlier effects
Figure 1. The different levels within the biological organization.
Earliest toxic effects can often be observed at the lower levels as xenobiotics triggers molecular and cellular responses that may have consequences higher up in the organization. The complexity is increasing in the higher levels making it more difficult to understand toxic mechanisms and responses (modified from van der Oost et al.
(2003)).
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7 2. Background
2.1. Oxidative stress
2.1.1. Generation of ROS and antioxidative defense
All aerobic organisms use oxygen to generate energy in form of ATP. This process takes place in the mitochondria, where the electron transport chain transports electrons through complexes in the inner mitochondrial membrane by a series of redox reactions and thus generating a potential gradient to produce ATP. However, this system is not perfect as some components in the chain leaks electrons. These electrons can react with oxygen present in their surroundings and generate superoxide (O2•-
) that further reacts forming hydrogen peroxide (H2O2) and hydroxyl radicals (OH•).
These extremely reactive molecules, that are collectively called reactive oxygen species (ROS), can cause oxidative damages on biomolecules, such as DNA, proteins and lipids (Halliwell and Gutteridge 2007). Even though ROS can be produced by several processes, the mitochondria are the main cellular production sites (Liu et al. 2002). Approximately 2-3 % of the oxygen consumed in an organism is converted to ROS and these molecules have an important function in the cells redox regulation and as signaling substances (Halliwell and Gutteridge 2007). However, overproduction of ROS may lead to oxidative stress and this condition can be harmful for the cell. Fortunately, all cells have a defense system against ROS, the antioxidative defense. The antioxidant defense constitutes of an exogenous and an endogenous part with antioxidative enzymes and molecules that help detoxification of ROS by catalyzing a number of reactions that ultimately convert ROS into water. The exogenous defense constitutes of low-molecular-mass agents; some, like bilirubin and uric acid, are produced in vivo while others, like vitamin C and E, are obtained through dietary intake (Halliwell and Gutteridge 2007). The endogenous defense is inducible and increases in response to enhanced ROS production and is summarized in table 1. The most common antioxidative enzymes are superoxide dismutase (SOD) that converts O2•-
to H2O2 while catalase (CAT) and glutathione peroxidase (GPX) catalyze the metabolism of H2O2 to water. Iron (Fe) catalyzes the conversion of O2•-
and H2O2 to OH• and OH-. Under normal physiological conditions, the production of ROS and the activity of the antioxidative defense are in balance with each other, but this balance can shift in response to a number of factors (see section 2.1.2). When the balance is shifted towards an increased ROS production, the cell is experiencing oxidative stress (fig 2). Oxidative stress may result in increased levels of damaged biomolecules and is linked to a deteriorated health (Valko et al. 2007) and accelerating aging processes (Balaban et al. 2005). However, transient experiences of oxidative stress can be beneficial in terms of physiological adaptation (Naviaux 2012).
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Table 1. The cellular generation and metabolism of reactive oxygen species (ROS). Each step in the metabolism chain is catalyzed by a specific enzyme.
Reaction Enzyme
O2 + e- → O2•-
2H+ + 2O2•-
→ H2O2 + O2 SOD
H2O2 + O2•-
→ OH• + OH- + O2 Fe 2GSH + H2O2→ 2GSSG + 2H2O GPX
2H2O2 → 2H2O + O2 CAT
OH• + e- + H+ → H2O
SOD – Superoxide dismutase, GPX - Glutathione peroxidase, CAT - Catalase
2.1.2. Oxidative stress in ecology and ecotoxicology
In the last two decades, the role of antioxidants and oxidative stress have got increasing attention in ecology (Costantini 2008) and ecotoxicology (Livingstone 2001). In ecology, oxidative status have been related to aging (Barata et al. 2005), reproductive success (Pagano et al. 2001, Alonso-Alvarez et al. 2004) and fitness (D'Autreaux and Toledano 2007). There are a number of environmental stressors, such as temperature, salinity, UV radiation and xenobiotics, which can enhance the cellular production of ROS in aquatic organisms (Valavanidis et al. 2006). Oxidative damages on biomolecules may have adverse effects on crucial life-history traits and increased investments in defense and repair mechanisms can have trade-off effects, resulting in decreased fitness as energy is allocated from other functions (Monaghan et al. 2009). Xenobiotics can cause increased ROS production through many mechanisms, e.g. activation of enzymes, such as CYP and NAD(P)H oxidases, inactivation or depletion of antioxidative enzymes, interference with the mitochondrial electron transport or redox cycling (Livingstone 2001, Halliwell and Gutteridge 2007). The increasing number of chemical substances and mixtures in the environment makes it important to study the impact of these chemicals on oxidative biomarkers and the consequences this may have on different physiological and ecological functions.
9 2.2. Model organism - Daphnia magna
The test species D. magna is a fresh water cladoceran that has a large geographic distribution. When living in optimal conditions, D. magna reproduces asexually via parthenogenesis, whereas males and resting eggs are produced when the environmental conditions are less favorable (Kleiven et al. 1992);
this form of reproduction is known as cyclic parthenogenesis. Because of the short generation time and small size, D. magna is easy to culture under controlled conditions in laboratory environment.
Ecotoxicological assays with D. magna have been conducted for more than a century, with the first study performed as early as in 1900 (Warren 1900, Shaw et al. 2008). Due to its well-studied physiology and morphology, D. magna is a suitable model species in ecological, evolutionary and ecotoxicological studies. Several standard tests with the species have been developed by different organizations (OECD, EPA, ASTM) and they are now mandatory in environmental risk assessment of chemical substances. In addition to its use in tests assaying individual end points, such as mortality, reproduction, growth and feeding, several methods for assaying molecular and biochemical end points have been developed. The genome of a close congener, Daphnia pulex, has been mapped (Colbourne et al. 2005) making it possible to study genetic effects also in D. magna.
In-control Out of
control
ROSOxidized
macromolecules
AOX
Oxidative stress
Stress level
R espons ee
Point of no return max AOX
Figure 2. A simplified graph of the oxidative system. The production on reactive oxygen species (ROS) (black line) is fluctuating during normal physiological conditions. The amount of oxidized macromolecules (red line) is kept to a minimum by the induction of the antioxidative defense (AOX) (green line). The production of ROS can be induced by a stressor and in turn enhance the antioxidative activity up to a certain level. After that the anti-oxidant defense cannot quench the high concentration of ROS and the levels of oxidized macromolecules are increasing. When activity of the defense is ceasing, there are nothing to protect the cell against oxidative attacks.
10 2.3. Biomarkers
Biomarkers can be divided in to three groups depending on how they are being used: exposure biomarkers, effects biomarkers and susceptibility biomarkers (van der Oost et al. 2003). The focus in this thesis will be on the two first classes.
In stressed organisms, the earliest responses occur at sub-organismal levels. Xenobiotics trigger early response by inducing enzymes responsible for metabolizing and detoxifying harmful substances. The exposure can also induce other protection mechanisms, e.g. production of heat shock proteins (hsp) that has a function as chaperones and protect cellular and protein structures (Feder and Hofmann 1999). If these mechanisms are not adequate, it may result in adverse effects on cellular structures and functions. By measuring and quantifying these responses, biomarkers can be used as indicators of exposure and experience of stress. However, for a biomarker to be indicative of effects there must be a connection to adverse effects at higher levels. van der Oost et al. (2003) have proposed six criteria for evaluation of new biomarkers. According to these, biomarkers should have/be (i) reliable, cheap and easy to perform, (ii) sensitive to pollutants, (iii) defined baseline levels and known response to natural variations and toxic exposure, (iv) established response to confounding factors, (v) known toxic mechanisms, and (vi) established relationship between biomarker alterations and effects at higher biological levels. A completely validated biomarker should fulfill all these criteria.
2.3.1. RNA content
The RNA content of an organism can be used as both a proxy of fitness (Dahlhoff 2004) and a toxicological end point (Ibiam and Grant 2005). The majority of the cellular RNA is ribosomal RNA (rRNA) that is correlated with the protein synthesis activity (Millward et al. 1973); hence, the organismal RNA content is an indirect measurement of protein production. That is why RNA concentration has been used to estimate growth, metabolism and feeding of zooplankton (Bamstedt and Skjoldal 1980, Holmborn et al. 2009). In addition to direct damaging effects and loss of protein functions, xenobiotics may induce a number of protective proteins for both repair and detoxifying mechanisms (Monsinjon and Knigge 2007), which could result in increased RNA content. Thus, altered RNA content in response to xenobiotics indicate an effect on protein production and maybe also on growth rate.
2.3.2. Gene expression of specific proteins
Snape et al (2004) introduced the term ecotoxicogenomics to describe the use of genomic techniques in ecotoxicology. In response to stress, organisms may regulate expression and transcription of specific genes to counteract adverse effects. To understand toxic mechanisms, these responses are of primarily importance. Thanks to the ever increasing number of mapped genomes, particularly for model species, there are now unprecedented opportunities for detecting these genotoxic effects. Microarrays are widely used for screening of gene expression alterations in response to stressors as multiple genes can be assayed simultaneously (Poynton and Vulpe 2009).
While microarray provides information about up- and down- regulation of genes and limited quantitative information, real-time qPCR technique gives full quantitative information of the gene
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expression changes for a specific gene (VanGuilder et al. 2008). This is a more powerful tool if the objective is to test a hypothesis regarding involvement of a few specific genes in the response in question. If gene expressions of specific genes are to be used as effect biomarkers, the hypothesis of their predictive power of later effects has to be addressed.
2.3.3. Antioxidative defense
As it is difficult to measure the cellular concentration of short-lived ROS, induction of the antioxidative defense is used as a proxy of enhanced ROS production. This is commonly measured as the activity of individual antioxidative enzymes (Wilhelm et al. 2001, Morales et al. 2004). However, it may be complicated to quantify and interpret the integrated response of the antioxidative defense system with this approach as individual enzymes can respond differently catalyzing specific reactions in the detoxification chain of ROS. There are a number of assays that measure the total antioxidative capacity (Huang et al. 2005) and gives a value of the combined activity of endogenous enzymes and co-factors as well as the exogenous defense. The assay used in this thesis is oxygen radical absorbance capacity (ORAC) that has been applied to a variety of biological materials, such as plants and food (Wu et al. 2004, Prior et al. 2005), human tissue (Cao and Prior 1998, Gheldof et al. 2003), and invertebrates, including crustaceans (Gorokhova et al. 2013). However, I have found no previous studies where ORAC has been measured or used as a biomarker in Daphnia spp.
2.3.4. Lipid peroxidation
When the balance of ROS production and antioxidative defense is disrupted, ROS react with a variety of biomolecules, including polyunsaturated fatty acids (PUFA), which have two or more carbon- carbon double bonds. Damages on these lipids are often measured by assaying their end-products, of which the most commonly assayed marker in D. magna is malonic dialdehyde (MDA). Lipid peroxidation may cause DNA adducts (Marnett 1999) and damages on membrane associated proteins that alter the fluidity of membranes (Ohyashiki et al. 1986). Lipid peroxidation is a very common end point in oxidative stress assessment in mammals (Banerjee et al. 1999) as well as in D.
magna (Barata et al. 2005) and is in this thesis assayed to assess the levels of oxidative damage.
3. Summary of papers
3.1. Paper I Do pharmaceuticals with evolutionary conserved molecular drug targets pose a greater environmental risk?
The objective of this paper was to test the hypothesis that the presence of human drug target in non- target organisms will influence the toxicity of pharmaceuticals. The effects of pharmaceutical exposure were assayed using D. magna as a model species and end points at three organization levels: individual (immobility and reproduction tests), molecular (gene expression analysis) and biochemical (RNA measurement). Using data from Gunnarsson et al. (2008), three pharmaceuticals were selected for this study. Two of these, miconazole and promethazine, have a drug target
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ortholog in D. magna, whereas no drug target for the third pharmaceutical, levonorgestrel, has been identified in this species.
The results from this study show that the presence of drug target orthologs does influence the toxicity of pharmaceuticals. The effects of miconazole and promethazine could be observed at all three organization levels, whereas no effects of levonorgestrel exposure were observed at the tested concentration range. By assaying end points at different organization levels, a more comprehensive view of the toxic effects was provided. Moreover, our results underline the importance of considering drug target conservation in pharmaceutical risk assessments.
3.2. Paper II The effects of feeding and xenobiotics on oxidative stress in Daphnia magna:
implications for ecotoxicological testing
This study aimed to delineate the effects of feeding activity and toxic exposure on biomarkers of oxidative stress in D. magna. The positive effects of calorie restricted diets on oxidative status have been known for a long time and are believed to also affect aging and health. Feeding rate in D.
magna is a sensitive end point of general toxic response to xenobiotics. As biomarkers of oxidative stress are becoming widely used to assess toxic exposure, it is important to separate the effects of altered feeding rates and those of toxic exposure on antioxidant levels and oxidative damage. This was done in this study by manipulating feeding rate in D. magna using different food concentrations and by exposure to two model contaminants, the pesticide lindane and the pharmaceutical haloperidol. To assess antioxidative capacity and oxidative damage, individual protein content, ORAC and lipid peroxidation were measured in the test animals.
The results confirm the positive effect of feeding rate on all three biochemical variables measured.
Moreover, the xenobiotics altered the relationships between feeding and oxidative biomarkers. We also found that ORAC/protein ratio is a suitable biomarker for toxic exposure as it was independent of feeding rate within the tested range of food concentration. The results from this study provide knowledge about the effects of feeding activity on oxidative biomarkers and stress the importance of understanding the baseline variability of antioxidant and lipid peroxidation biomarkers.
4. Discussion
In contrast to human health risk assessment, where the health of individuals is at focus, environmental risk assessment addresses effects on population level and above. As the ultimate aim is to protect the ecosystem function and integrity, ecotoxicological end points and biomarkers have to be predictive of consequences at higher biological levels to have any value in risk assessments. The connection between effects at different biological levels must be understood to ensure that information obtained through ecotoxicological assays is relevant and interpretable (Forbes et al.
2006). An integrative approach has been taken in the studies included in this thesis to evaluate whether biomarkers commonly used in ecotoxicological studies are predictive of effects at higher biological levels (paper I) and how they are related to ecological (food abundance) and physiological (feeding activity) factors (paper II). This approach is crucial for validating biomarkers as effect biomarkers and to understand their alterations to natural occurring changes (e.g., food abundance).
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The connection between different biological organization levels was clearly demonstrated in paper I, were effects on RNA content and gene expression for cuticle protein and vitellogenin was observed for the same pharmaceuticals that also had effects on individual level. It is important to demonstrate this connection to be able to make assumptions, based on biomarker response, about delayed effects at higher biological levels. This connection is vital for mechanistic biomarkers as well as for those connected to energy allocation since both may have consequences on individual and population levels. Paper II showed xenobiotic impact on ORAC – protein relationship which indicates effects on metabolism and energy allocation as a result of toxic exposure. A connection between this biomarker response and life-history traits would strengthen antioxidative capacity as an effect biomarker in ecotoxicological studies.
Sensitivity to stress is one of the 6 criteria for validation of biomarkers as it is desirable to detect warning signals before the environmental stress is too severe for recovering to take place. Recovery from stress may be more difficult and energy demanding higher up in the biological organization as molecular and biochemical alterations often are more easily reversed than ecosystem changes. The sensitivity of biomarkers was shown in paper I, where molecular and biochemical markers were the most sensitive end points when assessing the toxicity of pharmaceuticals with conserved drug targets. Individual end points are often of more general toxicity and even though our results show that these can detect potent xenobiotics, molecular and biochemical biomarkers detected effects at concentrations several fold lower.
To be of value in risk and hazard assessments, biomarkers should fulfill the criteria mentioned above and should thus be evaluated for their relevance, sensitivity, mechanistic function and alterations to confounding factors, both physiological and ecological. Even though a lot is known about the impact of such factors, e.g. food quality, age and fitness, on oxidative stress there have only been a few studies concerning the influence of these factors in combination with toxic exposure. Feeding activity has a major impact on oxidative processes in D. magna (paper II), highlighting the importance to understand biomarker response to natural alterations in food availability in ecotoxicological assays.
Our results also stress the importance of knowing what is measured, functional or toxic response or a combination of these, and hence, how the results should be interpreted. Single species laboratory studies are of great importance in this work, due to the possibility of controlling and measuring confounding factors. The knowledge from such studies can then be implemented in studies where more complex interactions are included, e.g. in mesocosm and field studies.
5. Conclusions and future perspectives
Integrating individual end points and biomarkers in ecotoxicological studies is a powerful approach for linking effects at multiple levels of biological organization. This provides an understanding of the mechanisms involved and a tool for predicting higher-level effects based on the biomarker responses.
To the best of our knowledge, ORAC has never previously been assayed in D. magna and we showed that ORAC normalized to protein content can be used as a biomarker of xenobiotic exposure since it is independent of feeding rate and sensitive to toxic exposure. Hence, ORAC is a promising assay to
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study further in ecological and ecotoxicological studies as a quantitative measurement of antioxidative capacity.
The findings from this thesis have contributed to an increasing understanding of biomarkers in eco- toxicological studies in regard to the connections between effects at different biological levels and confounding factors in biomarker response. Nevertheless, there are still questions that have to be addressed for biomarkers of oxidative stress to obtain full potential as effect biomarkers. One of these questions concerns delayed effects on organismal fitness and life-history traits as results of biochemical alterations. More research is also needed on possible synergistic effects of multiple stressors, both anthropogenic and natural, on oxidative stress. However, with the growing interest and research about mechanisms and consequences of oxidative stress, it is likely that the value of oxidative biomarkers in ecotoxicological studies will continue to increase.
6. Acknowledgements
First of all I would like to thank my supervisor Elena Gorokhova for sharing your great knowledge and always making me think one step further. I also want to thank my co-supervisor Magnus Breitholtz for the support and for always coming with encouraging comments when I need it. A big, big thanks to Karin Ek and Birgitta Liewenborg for all the support in the lab. I would be lost without you two!
All the wonderful colleagues here at ITM make it fun to come to work even on dark and cloudy days!
Thanks! Especially thanks to Sara S, Martin, Elin, Karin S, Jon and Erik for all coffee breaks, talks and jokes. Thank you Carro for always being there listening when life is great and when life is less great.
Our roommates, Henrik and Maria, deserve a big thanks for having to listen to our never-ending talks. Thanks Alfred for all the badminton games that gives me a well-needed break from work. I would also like to thank PhD students and post docs at ITMo for great after works and dinners, and especially Seth, Marko and Dimpan for being excellent travel company!
Tack till alla vänner som antagligen får höra mer om små vattenlevande organismer än dom önskar och tack Josefin för du gör måndagar till bästa dagen i veckan! Stort tack till mamma, pappa och Christin!
15 7. References
Alonso-Alvarez, C., S. Bertrand, G. Devevey, M. Gaillard, J. Prost, B. Faivre and G. Sorci (2004). "An experimental test of the dose-dependent effect of carotenoids and immune activation on sexual signals and antioxidant activity." American Naturalist 164(5): 651-659.
Balaban, R. S., S. Nemoto and T. Finkel (2005). "Mitochondria, oxidants, and aging." Cell 120(4): 483- 495.
Bamstedt, U. and H. R. Skjoldal (1980). "RNA concentration of zooplankton - relationship with size and growth." Limnology and Oceanography 25(2): 304-316.
Banerjee, B. D., V. Seth, A. Bhattacharya, S. T. Pasha and A. K. Chakraborty (1999). "Biochemical effects of some pesticides on lipid peroxidation and free-radical scavengers." Toxicology Letters 107(1-3): 33-47.
Barata, C., J. C. Navarro, I. Varo, M. C. Riva, S. Arun and C. Porte (2005). "Changes in antioxidant enzyme activities, fatty acid composition and lipid peroxidation in Daphnia magna during the aging process." Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology 140(1): 81- 90.
Barata, C., I. Varo, J. C. Navarro, S. Arun and C. Porte (2005). "Antioxidant enzyme activities and lipid peroxidation in the freshwater cladoceran Daphnia magna exposed to redox cycling compounds."
Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 140(2): 175-186.
Cao, G. H. and R. L. Prior (1998). "Comparison of different analytical methods for assessing total antioxidant capacity of human serum." Clinical Chemistry 44(6): 1309-1315.
Chapman, P. M. (2002). "Integrating toxicology and ecology: putting the "eco" into ecotoxicology."
Marine Pollution Bulletin 44(1): 7-15.
Colbourne, J. K., V. R. Singan and D. G. Gilbert (2005). "wFleaBase: The Daphnia genome database."
Bmc Bioinformatics 6.
Costantini, D. (2008). "Oxidative stress in ecology and evolution: lessons from avian studies." Ecology Letters 11(11): 1238-1251.
D'Autreaux, B. and M. B. Toledano (2007). "ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis." Nature Reviews Molecular Cell Biology 8(10): 813-824.
Dahlhoff, E. P. (2004). "Biochemical indicators of stress and metabolism: Applications for marine ecological studies." Annual Review of Physiology 66: 183-207.
Feder, M. E. and G. E. Hofmann (1999). "Heat-shock proteins, molecular chaperones, and the stress response: Evolutionary and ecological physiology." Annual Review of Physiology 61: 243-282.
Forbes, V. E., A. Palmqvist and L. Bach (2006). "The use and misuse of biomarkers in ecotoxicology."
Environmental Toxicology and Chemistry 25(1): 272-280.
Gheldof, N., X. H. Wang and N. J. Engeseth (2003). "Buckwheat honey increases serum antioxidant capacity in humans." Journal of Agricultural and Food Chemistry 51(5): 1500-1505.
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Gorokhova, E., M. Lof, M. Reutgard, M. Lindstrom and B. Sundelin (2013). "Exposure to contaminants exacerbates oxidative stress in amphipod Monoporeia affinis subjected to fluctuating hypoxia."
Aquatic Toxicology 127: 46-53.
Gunnarsson, L., A. Jauhiainen, E. Kristiansson, O. Nerman and D. G. J. Larsson (2008). "Evolutionary conservation of human drug targets in organisms used for environmental risk assessments."
Environmental Science & Technology 42(15): 5807-5813.
Halliwell, B. and J. M. C. Gutteridge (2007). Free radicals in biology and medicine. United States, Oxford University press.
Holmborn, T., K. Dahlgren, C. Holeton, H. Hogfors and E. Gorokhova (2009). "Biochemical proxies for growth and metabolism in Acartia bifilosa (Copepoda, Calanoida)." Limnology and Oceanography- Methods 7: 785-794.
Huang, D. J., B. X. Ou and R. L. Prior (2005). "The chemistry behind antioxidant capacity assays."
Journal of Agricultural and Food Chemistry 53(6): 1841-1856.
Ibiam, U. and A. Grant (2005). "RNA/DNA ratios as a sublethal endpoint for large-scale toxicity tests with the nematode Caenorhabditis elegans." Environmental Toxicology and Chemistry 24(5): 1155- 1159.
Jobling, S., M. Nolan, C. R. Tyler, G. Brighty and J. P. Sumpter (1998). "Widespread sexual disruption in wild fish." Environmental Science & Technology 32(17): 2498-2506.
Kleiven, O. T., P. Larsson and A. Hobaek (1992). "Sexual reproduction in Daphnia magna requires 3 stimuli." Oikos 65(2): 197-206.
Lange, R., T. H. Hutchinson, C. P. Croudace, F. Siegmund, H. Schweinfurth, P. Hampe, G. H. Panter and J. P. Sumpter (2001). "Effects of the synthetic estrogen 17 alpha-ethinylestradiol on the life-cycle of the fathead minnow (Pimephales promelas)." Environmental Toxicology and Chemistry 20(6): 1216- 1227.
Larsson, D. G. J., M. Adolfsson-Erici, J. Parkkonen, M. Pettersson, A. H. Berg, P. E. Olsson and L. Forlin (1999). "Ethinyloestradiol - an undesired fish contraceptive?" Aquatic Toxicology 45(2-3): 91-97.
Liu, Y. B., G. Fiskum and D. Schubert (2002). "Generation of reactive oxygen species by the mitochondrial electron transport chain." Journal of Neurochemistry 80(5): 780-787.
Livingstone, D. R. (2001). "Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms." Marine Pollution Bulletin 42(8): 656-666.
Marnett, L. J. (1999). "Lipid peroxidation - DNA damage by malondialdehyde." Mutation Research- Fundamental and Molecular Mechanisms of Mutagenesis 424(1-2): 83-95.
Millward, D. J., P. J. Garlick, W. P. T. James, Nnanyelu.Do and J. S. Ryatt (1973). "Relationship between protein-synthesis and RNA content in skeletal-muscle." Nature 241(5386): 204-205.
Monaghan, P., N. B. Metcalfe and R. Torres (2009). "Oxidative stress as a mediator of life history trade-offs: mechanisms, measurements and interpretation." Ecology Letters 12(1): 75-92.
Monsinjon, T. and T. Knigge (2007). "Proteomic applications in ecotoxicology." Proteomics 7(16):
2997-3009.
17
Morales, A. E., A. Perez-Jimenez, M. C. Hidalgo, E. Abellan and G. Cardenete (2004). "Oxidative stress and antioxidant defenses after prolonged starvation in Dentex dentex liver." Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 139(1-3): 153-161.
Naviaux, R. K. (2012). "Oxidative Shielding or Oxidative Stress?" Journal of Pharmacology and Experimental Therapeutics 342(3): 608-618.
OECD (2004). OECD 202 Guideline for testing of chemicals. Daphnia sp., acute immobilisation test.
OECD (2004). OECD 222 Guideline for the testing of chemicals. Earthworm reproduction test.
OECD (2009). OECD 229 Guideline for the testing of chemicals. Fish short term reproduction assay.
OECD (2012). OECD 211 Guideline for the testing of chemicals. Daphnia magna reproduction test Ohyashiki, T., T. Ohtsuka and T. Mohri (1986). "A change in the lipid fluidity of the porcine intestinal brush-border membranes by lipid-peroxidation - studies using pyrene and fluorescent stearic-acdi derivatives." Biochimica Et Biophysica Acta 861(2): 311-318.
Pagano, G., A. de Biase, I. B. Deeva, P. Degan, Y. K. Doronin, M. Iaccarino, R. Oral, N. M. Trieff, M.
Warnau and L. G. Korkina (2001). "The role of oxidative stress in developmental and reproductive toxicity of tamoxifen." Life Sciences 68(15): 1735-1749.
Peakall, D. B. (1994). "The role of biomarkers in environmental assessment. 1. Introduction."
Ecotoxicology 3(3): 157-160.
Poynton, H. C. and C. D. Vulpe (2009). "Ecotoxicogenomics: Emerging technologies for emerging contaminants." Journal of the American Water Resources Association 45(1): 83-96.
Prior, R. L., X. L. Wu and K. Schaich (2005). "Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements." Journal of Agricultural and Food Chemistry 53(10): 4290-4302.
Shaw, J. R., M. E. Pfrender, B. D. Eads, R. Klaper, A. Callaghan, R. M. Sibly, I. Colson, B. Jansen, D.
Gilbert and J. K. Colbourne (2008). Daphnia as an emerging model for toxicological genomics.
Comparative Toxicogenomics. C. Hogstrand and P. Kille. Amsterdam, Elsevier North Holland. 2: 165- 219.
Snape, J. R., S. J. Maund, D. B. Pickford and T. H. Hutchinson (2004). "Ecotoxicogenomics: the challenge of integrating genomics into aquatic and terrestrial ecotoxicology." Aquat Toxicol 67(2):
143-154.
Valavanidis, A., T. Vlahogianni, M. Dassenakis and M. Scoullos (2006). "Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants." Ecotoxicology and Environmental Safety 64(2): 178-189.
Valko, M., D. Leibfritz, J. Moncol, M. T. D. Cronin, M. Mazur and J. Telser (2007). "Free radicals and antioxidants in normal physiological functions and human disease." International Journal of Biochemistry & Cell Biology 39(1): 44-84.
van der Oost, R., J. Beyer and N. P. E. Vermeulen (2003). "Fish bioaccumulation and biomarkers in environmental risk assessment: a review." Environmental Toxicology and Pharmacology 13(2): 57- 149.
18
van Gestel, C. A. M. and T. C. van Brummelen (1996). "Incorporation of the biomarker concept in ecotoxicology calls for a redefinition of terms." Ecotoxicology 5(4): 217-225.
VanGuilder, H. D., K. E. Vrana and W. M. Freeman (2008). "Twenty-five years of quantitative PCR for gene expression analysis." Biotechniques 44(5): 619-626.
Warren, E. (1900). "On the reaction on Daphnia magna to certain changes in its environment."
Quarterly Journal of Microscopical Science 43: 119-224.
Wilhelm, D., T. Tribess, C. Gaspari, F. D. Claudio, M. A. Torres and A. R. M. Magalhaes (2001).
"Seasonal changes in antioxidant defenses of the digestive gland of the brown mussel (Perna perna)."
Aquaculture 203(1-2): 149-158.
Wu, X. L., G. R. Beecher, J. M. Holden, D. B. Haytowitz, S. E. Gebhardt and R. L. Prior (2004).
"Lipophilic and hydrophilic antioxidant capacities of common foods in the United States." Journal of Agricultural and Food Chemistry 52(12): 4026-4037.
