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Chimia 51 (1997) 915-921 standing of underlying molecular mecha- © Neue Schweizerische Chemische Gesellschaft nisms and modes of toxic action on differ- lSSN 0009-4293 ent levels of biological organization, while the other trend is aimed at understanding the complex interactions and feedback Molecular Mechanisms in mechanisms in ecosystems disturbed by pollutants. Taken together, these two ap- : An Interplay proaches complement each other and will finally enhance the understanding of the between Environmental effects of pollutants on living systems. In this paper, we focus on the mode-of-ac- Chemistry and Biology tion-based molecular approach.

C 3 Beate I. Escher3)*, Renata Behrab), Rik I.L. Eggen ), and Karl Fent ) 2. Ecotoxicology at EA WAG

Fig. 1 shows an overview of the pro- Abstract. A close collaboration between environmental chemistry and biological cesses considered in the molecular ap- sciences is required for a complete understanding of ecotoxicological effects. Bioavail- proach to ecotoxicology. Emphasis is ability and uptake of pollutants cannot be regarded as isolated chemical or biological placed on the importance of understand- questions. Knowledge of the effective in the organism or at the target ing the interplay between environmental site(s) is essential to link the fate and effects of a chemical and is a prerequisite for chemistry and , thereby linking quantitative investigation of the modes of toxic action. These modes of action need to the concepts of bioavailability, effective be unraveled using whole-organism or in vitro systems in order to be able to develop in the organisms or at the specific biomarkers and biosensors that can be applied as early warning systems. Our target sites, and the mechanism of ecotox- mode-of-action-based approaches, in which chemical and biological analytical tools icity. are combined, should improve the understanding of ecotoxicological effects and should Evaluation of the adverse effects of be implemented in the future in risk assessment. pollutants in aquatic ecosystems requires discrimination between the total concen- tration of a chemical, the bioavailable 1. Introduction has led to an accumulation of valuable fraction, and the final concentration at the empirical data on the effects of specific target site(s) (Fig. 1, left). Environmental Ecotoxicology is the science of the pollutants on selected species that are cur- chemistry plays a major role in assessing impact of toxic substances on living or- rently used for regulatory purposes. Much the influence of environmental processes ganisms, encompassing all levels of bio- less emphasis has so far been placed on the on the fate of a substance in the environ- logical organization from single organ- development of general concepts that al- ment [2]. The fate of a chemical is affected isms to ecosystems [1]. Ecotoxicology low assessments of effects on the basis of by its physicochemical properties, the char- integrates environmental chemistry, bio- explanatory principles. acteristics of the environment, and by chemistry, toxicology, and ecology in a In this more conceptually oriented ap- biological processes. As a result, only a multidisciplinary manner. The unifying proach to ecotoxicoJogy, essentially two fraction of the total input into the ecosys- theme of ecotoxicological research is to trends have evolved in the last decade. tem will be available for uptake by organ- provide general concepts to evaluate the One trend is directed towards the under- isms. potential harmfulness of pollutants. This research is the basis for the development of tools that can be used in environmental regulation. bioavailability and uptake toxicity and response Despite major advances in the last de- cade, descriptive studies still make up the majority of ecotoxicological research con- cerned with the effects of chemicals. This

*Correspondence: Dr. B.I. Escher Swiss Federal Institute for Environmental Science and Technology (EA WAG) and £'Ie Institute for Aquatic Sciences of the Swiss Federal Institute of Technology (IGW /ETHZ) enVIron· effecti effects at alfeCls al b,oavallable a) Department of Chemistry mental concenlr Ion molecular Inl'v,du(li concenlration b) Department of Limnology fale inorgan m level r vel C) and Department of Microbiology Oberlandstrasse ]33 CH-8600 Diibendorf Fig. 1. The effective concentration of a pollutant in an organism (e.g. fish, daphnia, algae) or at the Tel.: +41 18235068, Fax: +4] 18235471 target site inside the organism is the link between the environmental fate of a pollutant and its toxic E-Mail: [email protected] effect. CHEMISTRY ATEAWAG 916

CHIMIA 51 (1997) Nr. 12 (Dclcmhcr)

Properties of a compound that affect of sensitive ecotoxicological endpoints. are used to examine the stress response of its fate include its speciation and the hy- In vitro assays can, to a certain extent, an organism. In the biosensor approach, drophobicity of the different chemical spe- replace toxicity tests on whole organisms the expression of affected genes can be cies. Speciation is important for metals and can be used for the assessment of the linked to artificially introduced marker (see Sect. 3), organometallic compounds toxic potential of chemicals and environ- genes whose products can easily be mea- [3][4] and hydrophobic ionizable organic mental samples. Since they are simplified sured (e.g., luciferase, {J-galactosidase, compounds (HIOC) (see Sect. 4). In case model systems, in vitro systems offer the green-fluorescence ; 'or arylsulfa- of organotin compounds, charged species opportunity to focus in detail on specific tase) [7]. Biosensors work in both prokary- usually show a lower but still significant modes of actions [3]. From an ethical otic and eukaryotic organisms and may be bioaccumu]ation as compared to the cor- point of view, they are less problematic used for on-line monitoring of the envi- responding neutral species [5]. The freely than animal testing. However, a major ronment. dissolved fraction of a compound is pri- problem of in vitro systems is the difficul- marily available for uptake by organisms. ty to extrapolate the results to whole or- Sorption to minerals and organic matter ganisms. Another disadvantage is shared 3. Uptake and Effects of Metals in Algae reduces the bioavailable concentration. with anima] testing on single species in Even the presence of dissolved organic that they typically cannot account for spe- Most studies on the ecotoxicology of carbon reduces the bioavailability of or- cies-specific differences in sensitivity. metals in the aquatic environment try to ganic compounds [5]. Therefore, species-specific systems have relate biological responses to nominal con- The uptake and effect of chemicals is to be developed. In Sect. 5, different appli- centrations [8]. In aquatic systems, metals not solely determined by the bioavailable cations of a fish-specific in vitro system occur in various chemical forms, the for- concentration of a chemical, but is also are shown, employing a permanent fish mation of which is influenced by the local- influenced by biological factors, particu- hepatoma cell line (PLHC-I). ly prevailing physicochemical conditions, larly differences in lipid content and sen- Further processes whose importance e.g., acidity, salinity, inorganic and organ- sitivity of organisms. Differences in sen- cannot be unraveled by looking only at ic ligands, and the presence of particles sitivity among species or populations of lethality or other observable effect end- [9]. Chemical speciation is an important the same species are related to differences points include defense and repair mecha- determinant of metal uptake and toxicity in morphology, developmental stage, sex, nisms. Organisms have developed protec- although its characterization is not trivial genotype, metabolic activity, and individ- tive mechanisms (such as mobilization of considering the di versity of chemica] con- ual history. Moreover, organisms can various cellular constituents and stituents of aquatic ecosystems. Moreover, evolve protection mechanisms in response that in a highly coordinated way minimize in many cases, the concentrations of the to continuous exposure to elevated con- disturbances of cellular homeostasis) that free and other bioavailable forms cannot centrations of a chemical. This results in allow them, within certain limits, to resist be directly measured but have to be calcu- an increased tolerance to the chemica], adverse conditions, including the negative lated. Studies of metal-algae interactions which further obscures concentration-ef- effects of anthropogenic chemicals. If the in chemically defined culture media, in fect relationships. The link between total, disturbances become too large or are chron- combination with thermodynamic calcu- bioavailable, and effecti ve concentrations ic, however, organisms react with stress lations of the equilibrium speciation, are is further illustrated in the following sec- responses, which are accompanied by an an approach to gain insights into the influ- tion using the example of uptake and ef- increased production of enzymes that ence of the speciation of a metal on its fects of metals in algae. dampen the deleterious effects of the stres- biological availability [10]. Once inside the organism, the pollut- SOl'S, or by repairing damaged cellular At EA WAG, evaluation of the effects ant may initiate a variety of effects, rang- components. When protective mechanisms of chemical speciation on growth, metal ing from cellular impairment to lethality. are overridden, toxic effects can follow, uptake, and accumulation in algae is car- Any observable effect ultimately has a resulting in deleterious effects on individ- ried out at metal concentrations relevant molecular cause (Fig. 1, right). This para- ual organisms and eventually on popula- for freshwater phytoplankton. Consider- digm is supposed to be valid not only for tions. Examples of these effects are de- ation of the kinetic properties of a copper effects on individual organisms, such as tailed in Sect. 6. uptake system indicates thatScenedesmus lethality or behavioral changes, but also In the final section of this paper, the subspicatus has a high ability to accumu- for the reproductive capability of popula- focus is on how the results offundamental late copper, reflecting its adaptation to the tions and the functioning of ecosystems. research on the impact of pollutants from bioavai]able copper concentration [11]. To gain insight into the toxic effects of a molecular to organism level can be used According to the free-ion activity model environmental chemicals, we look at the to develop tools and methods for environ- for metal-organism interactions [12], cop- mode(s) of toxic action of a given chemi- mental regulation and risk management. per and zinc uptake in S. subspicatus is calor mixture of chemicals and correlate The majority of the assays presented here related to the free and not to the total the responses to effective concentrations can be used as biomarkers and biosensors. concentration in the culture medium [13]. at the target site(s). Particularly for com- A biomarker is defined as a measurable This was indicated by experiments carried pounds that are present in severa] chemi- response at any level of biological organi- out in the presence of two different con- cal forms and/or act concomitantly ac- zation that can be related to an impact of centrations of the synthetic ligand EDT A. cording to different modes of action, the contaminants [6]. Biomarkers serve as Because EDT A acts as a metal buffer, understanding of the overall toxic effect screening tools for environmental con- manipulations of the total meta] concen- requires methods for distinguishing and tamination of mixtures of unknown com- tration allow regulation of the free metal quantifying the different modes of action. position and are used as an early warning concentration. As shown in Fig. 2 for This approach is illustrated in Sect. 4. system of exposure and effects. Just as copper, when plotted against the free Cu2+ Understanding the mode of action of biomarkers are an important tool for as- concentration in the media containing pollutants is relevant in the development sessing biochemical effects, biosensors 10-4 und 10-5 M EDT A, the copper con- CHEMISTRY AT EA WAG 917

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tents in the cells are comparable. Under environmental conditions, the influence 10-4-.------, of metal speciation on biological avail- ability may, however, be more complicat- ed, A comparison of the cellular copper 10-5- contents of S. subspicatus grown in a syn- o thetic culture medium or in lake water CD CO o indicated that although grown at the same 0) CO 10 -6- 6 free Cu2+ concentration, the metal content Q'> was higher in cells grown in lake water. o This observation was substantiated by the o 7- isolation of algae from the field which -S1O- showed a higher copper content than algae <3 6~ grown in a synthetic culture medium with 10-8+---.----,.---r----r---.------.,.----.-----/ similar free Cu2+ concentrations. Possible -14 -12 -10 -8 -6 explanations include the releaseofligands log Cu2+ (M) by the algae, which may bind and there- fore reduce the free ion concentration in the medium. It is also possible that other Fig. 2. The uptake of copper by the green alga Scenedesmus subspicatus after 5 d of growth as a copper species such as lipophilic metal- function of the free Cu2+ concentration. Two different EDT A concentrations were used: 10-4 M (0), organic complexes are available for up- and \0-5 M ([:,). take. To what extent some of the copper- organic complexes are bioavailable is not yet fully understood. metals are accumulated in organisms, the these different modes of membrane tox- The role of biological variables in the most effective approach for predicting tox- icities, using the simple cyclic photosyn- control of metal availability is evidenced icity may be to relate it to the concentra- thetic system of the purple bacterium by comparati ve studies in which the growth tion of toxicant taken up. Studies on the Rhodobacter sphaeroides. Biomembrane rate of various species, including field molecular mechanisms of protection and vesicles isolated from this bacterium con- isolates, was related to the free copper on the toxic action of metals may allow tain the complete functioning photosyn- concentration. With regard to the range of tests of this hypothesis. thetic system. The decay kinetics of the free metal concentration in which growth membrane potential that is induced by a was optimal, each species had a distinct short flash of light are used to evaluate the tolerance range for metals [13]. Tolerance 4. Inhibition of Energy Metabolism uncoupling activity and the narcotic ef- to metals may result from intracellular fect, whereas the redox kinetics of several immobilization through metal-binding A variety of environmental pollutants components of the electron-transfer chain and peptides, reduced metal up- interfere with energy metabolism through are used as indicators of specific inhibi- take and enhanced exclusion, extracellu- different modes of action, particularly, tion [16]. lar binding through organic chelators re- inhibiton, uncoupling, and narcosis. In Besides their use for assessing toxic leased by algae, or metal transformations fact, the targets of certain are effects and modes of action, the mem- [14]. Our current research is directed to- energy-transducing membranes, which are brane vesicles of R. sphaeroides may also wards an understanding of the tolerance a common feature in unicellular organ- serve as a model membrane for uptake mechanisms. isms, mitochondria, and chloroplasts. studies of pollutants. This allows one to Intracellular regulation of metals was Here, energy in the form of redox equiva- correlate uptake and speciation at the tar- indicated by the fact that S. subspicatus lents in oxidative phosphorylation or light get site with the actual toxic effect. It is retained an optimal growth rate over a in photosynthesis is transformed into an generally assumed that charged organic broad range of intracellular metal con- electrochemical proton gradient. This pro- molecules cannot penetrate biological tents. Interspecific difference in sensitivi- ton gradient drives ATPase, an membranes and are consequently not bio- ty to metals was also indicated by experi- that synthesizes ATP from ADP and inor- logically active. This assumption is based ments in which natural algal communities ganic phosphate. Pesticides and a variety on the partitioning of charged organic were subjected to a long-term exposure to of other environmental pollutants can di- molecules into the organic bulk solvent copper in river water [15]. At high copper rectly inhibit membrane energetization by octanol, which is more than three orders of concentrations, the community was re- blocking the electron-transfer chain. Hy- magnitude smaJler than that of the corre- duced to a few species. This reduced com- drophobic ionizable organic compounds sponding neutral compound [17]. Sorp- munity showed a remarkably high toler- such as weak organic acids (e.g., the phe- tion studies of substituted phenols and ance to copper, but also a co-tolerance to nolic pesticides dinoseb orpentachlorophe- substituted anilines to the membrane ves- other metals such as zinc, nickel, and sil- nol) can in addition destroy the electro- icles of R. sphaeroides or to pure phospho- ver. These results again raise the question chemical proton gradient thereby short- lipid model membrane vesicles have of the strategies employed by organisms circuiting the energy cycle. Finally, any shown, however, that a significant amount for controlling the cellular speciation of a hydrophobic compound can disturb mem- of charged organic molecules is incorpo- metal. We are currently examining this brane energetization by nonspecific mem- rated into the membrane [18]. Only the question. Preliminary results are indica- brane perturbation, the so-called narcotic uptake of neutral molecules into mem- tive of a reduction ofbioavailable metal by effect or baseline toxicity. branes can be satisfactorily modeled by extracellular complexation, and of cellu- In our work, we apply time-resolved octanol-water partitioning, whereas the lar immobilization. In similar cases where spectroscopy to distinguish and quantify uptake of charged molecules is underesti- CHEMISTRY ATEAWAG 918

CHI MIA 5/ (1997) Nr. 12 (Delelnber) mated by several orders of magnitude. In though there are differences in the intrin- ing interactions of pollutants with cells, the membrane-water systems, the distri- sic activity of the various substituted phe- and for deriving structure-activity rela- bution ratio of the neutral species is at nols, due to differences in the substitution tionships. The in vitro cytotoxicity on fish most only one order of magnitude higher pattern on the phenol ring, the major deter- cells of more than 50 environmental chem- than that of the corresponding anionic or minant of the overall effect is the mem- icals with different modes of toxic action, cationic species. brane burden of the chemical together including organotins, chi oro- and nitro- By comparing the effective concentra- with its speciation. phenols, sulfonic acids, and alkylphenols tions of substituted phenols in the mem- It is well accepted that the narcotic have been shown to correlate with the in brane with the inhibitory effect on the effect of environmental pollutants is di- vivo acute toxicity to fish [21]. electron transport, we have shown that the rectly related to the membrane burden of One well-established and important dissociated phenolate species is a more the chemical [20]. The results obtained biomarker in the exposure assessment of potent inhibitor of electron transport than from the uncoupling study of substituted aquatic systems is the induction of cyto- the corresponding neutral phenol species phenols show that this concept of mem- chrome P450-dependent monooxygenas- [16]. brane burdens can be extended to specifi- es (CYP) [6]. These enzymes are impor- The effective concentration and speci- cally acting compounds, if speciation is tant in the metabolism and detoxification ation in the membrane play an even more taken into consideration. This is a promis- of pollutants, but may also cause toxicity important role for the uncoupling effect of ing starting point for developing predic- via bioactivation of xenobiotics or by al- substituted phenols [19]. The mechanism tive methods for toxic effects of specifi- teration of steroid-hormone balance. At underlying the uncoupling is a protono- cally acting compounds and for assessing least 50 different genes are known in mam- phoric shuttle: a proton is taken up by a the toxicity of mixtures. mals, and they are also present in lower phenolate from the aqueous side with the animals and plants [22] [23]. In vertebrates higher proton concentration. The neutral including fish, they are localized mai nlyin phenol thus produced diffuses across the S. Fish-specific in vitro Systems: the endoplasmatic reticulum of the liver membrane, discharges the proton in the Interactions with Enzymes and and several other organs. The monooxy- aqueous phase of the opposite side, and Proteins genase system consists of two enzymes, the residual phenolate diffuses back across CYP and NADPH-cytochrome P450 re- the membrane to close the cycle. Experi- As most environmental chemicals act ductase, which are interrelated via the mental findings show that both species are species-specifically, toxicity towards fish transfer of electrons resulting in the oxida- required to cause the uncoupling effect. If can only be assessed in fish-specific sys- tion of a variety of substrates. the hydroxy function is blocked by meth- tems. With the use of a permanent fish Two distinctly different reactions are ylation, or if only one species is present at hepatoma cell line such as PLHC-I we elicited in the fish cell line after exposure physiological pH, then only non specific want i) to develop novel in vitro assays for to environmental chemicals: induction and narcotic effects are observed. The results the assessment of the environmental tox- inhibition of CYP. In our studies, we have obtained so far suggest that substituted icity to fish and to reduce and replace addressed both aspects by focusing on the phenols with acidity constants in the range animal testing, and ii) to demonstrate and isoform CYPIA, which is important in of 4-8 act according to the uncoupling validate the usefulness of such concepts in ecotoxicology. We use an enzyme-linked mechanism and exert their maximum ac- basic and applied ecotoxicological research immunosorbent assay (ELISA) technique tivity at a pH at which there is an approx- and practice. Our studies show that fish that has been established in the permanent imately equal ratio of neutral and charged cell-culture systems are suited for assess- fish cell line PLHC-l for the quantifica- species in the membrane (Fig. 3). AI- ing the cytotoxicity of chemicals, for study- tion ofCYP 1A protein with a fish-specific

4 4

>. 3.5 0 3.5 o +-' :> 3 0 3 +-' x • o x 0 • • «S 2.5 2.5 o Fig. 3. Uncoupling activity of some • • substituted phenols (0, dinoseb, pKa o x 0 D = 4.62; ., 3,4-dinitrophenol, pK, = ~ 2 x 2 o x :;:; x • .x X 5.48; X, 2,3,4,5-tetrachlorophenol, «S ~ X • pK, = 6.35) as afunction of pH (left) Q) 1.5 1.5 and as a function of the chemical ~ x x speciation (right), expressed as ra- 1 1 0 x tioofneutralphenol [HAJ to charged phenolate [A-J in the biological mem- 0.5 0.5 brane (m). The relative activity cor- 4 responds to the effect that is elicited 5 6 7 8 9 10- by a certain total concentration of pH phenols in the membrane (0.0015 mol·kg-') (adapted from [19], see for detai 1s). CHEMISTRY ATEAWAG 919

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monoclonal antibody [24] as well as mea- surements of the enzyme activity with an 100 ethoxyresorufin-O-deethylase (EROO) assay. c Polychlorinated biphenyls (PCBs), polychlorinated dibenzodioxins and furans, and polycyclic aromatic hydrocar- bons (PAHs) are potentinducers ofCYP lA in fish [23], in fish cell lines [24], and in other aquatic animals. The induction of CYPIA can be regarded as a process re- sulting in an altered metabolism and in disturbances of hormone balance, but also ref as an adaptative process to chronic expo- sure to such pollutants. The induction of CYPI A can be used as a biomarker for the o ecotoxicological assessment of environ- 1 0-3 10.2 10.1 10° mental samples. This is illustrated in Fig. 4, where a landfill leachate contaminated landfill leachate equivalent by PAHs and additional compounds is (ml / ml medium) analyzed for its CYPI A induction poten- tial. Comparison with known model com- Fig. 4. Assessment of the CYP IA induction potential of different landfilileachates (a,b,c are different pounds such as chrysene allows the devel- samples, ref refers to uncontaminated reference sample) that are contaminated by PAHs and other opment of toxic equivalency factors, and compounds. PLHC-] cells were exposed to extracts of the leachates in DMSO in minimal essential medium (containing ~I % DMSO) for 24 h (n = 3 ± SEM). P450 enzyme activity was determined by thus the employment in ecotoxicological its EROD activity. At low concentration of leachate extracts, given as landfill leachate equivalents, risk assessment. CYPIA induction occurs, whereas at higher concentrations enzyme activity is inhibited. On the other side, we focus on chemi- cals such as organotin compounds and heavy metals that cause an inhibition and compete with the substrate on the binding trogenic chemicals often exert their action destruction of CYPIA [25], and on mix- site of the enzyme. These findings gave by binding to the estrogen receptor or by tures of both inducing and inhibiting com- evidence that organotins do not interfere regulating the activity of estrogen-respon- pounds [26][27]. Tributyltin (TBT) and with the binding of an inducer with the sive genes. In recent years, various sys- triphenyltin (TPT) chloride, both of which arylhydrocarbon receptor (Ab receptor) of tems have been developed for detecting predominated as the hydroxide species at the cell, and do not interfere wi th CYP 1A the estrogenic potential of such com- the experimental pH, strongly inhibited protein synthesis. However, they act at the pounds. However, established permanent the hepatic CYP system both in vivo [28] level of the CYP protein (destruction of fish cell culture systems are missing. Be- [29] and in vitro [25). The CYP protein, its apoprotein) and inhibit the catalytic activ- fore using fish cell cultures for estrogenic- activity, and the reductases were affected. ity. Heavy metals including CdlI, COIl, ity assays, these cells had to be adapted to Of the P450 system, the isoform CYPIA CUll, Nill, PbIl, and ZnJ[ have been shown hormone-free medium [33). We are pres- was selectively affected in various fish to act similarly [27). Organotins seem to ently focusing on the induction of vitello- species by TBT and TPT in vivo [28][29]. inhibit the catalytic activity by binding to genin and are searching for other markers At high concentrations, however, addi- amino acids such as cysteine and histidine for estrogenicity. Vitellogenin is one of tional CYP forms were affected as well. at the active site, or on other sites of the the most important biomarkers of estroge- The mechanisms responsible for the loss enzyme [26][29). The lipophilic TBT and nicity, as it is the precursor of the yolk and inactivation of cytochrome P450forms TPT most likely penetrate hydrophobic protein in the eggs of fish, amphibians, in vivo are based on the direct destruction membranes, in which cytochromes P450 reptiles, and birds, and is specifically syn- of P450 and formation of P420 with sub- are embedded, thereby gaining access to thesized in females under the control of sequent rapid degradation and breakdown these enzymes. The action is directed to estradiol [34]. Hence, estrogenic chemi- of the apoprotein by proteases [29). CYP, as cytochrome bs is unaffected.and cals that act via binding to the estrogen The in vitro studies have shown that in the reductases are affected differently by receptor may be detected with this bio- fish microsomes [25] and in a fish cell line TBT and TPT pointing to a specific mode marker [34). [26] TBT and TPT strongly interact with of action [28-30). microsomal monooxygenase systems, re- Presently, we are adapting the perma- sulting in the inhibition of CYPIA activ- nent fish hepatoma cell line PLHC-l to be 6. Oxidative Stress ity, inhibition of NAO(P)H cytochrome c able to assess chemicals with estrogenic reductase activity, and thus the loss of an activity. Environmental chemicals that Oxidative stress has been recognized enzyme system responsible for the detox- negatively affect the endocrine system of to be associated with the toxicity of nu- ification of environmental pollutants and organisms are of particular importance in merous chemicals and with the pathogen- the metabolism of endogenous substanc- ecotoxicology, as they limit the fitness of esis of many diseases, e.g., carcinogene- es. It was shown that the activity of orga- populations to a significant degree by in- sis, Altzheimer' s disease, or atherosclero- notins was due to the parent compound hibiting reproductive success [31]. An in- sis. It has been defined as a situation in and not to metabolites. CYPIA activity is creasing number of widely used industrial which an overabundance of oxidants or inhi bited by a noncompetiti ve mechanism, and agricultural chemicals mimic the ef- free radicals damage or destroy a cell. In which means that the inhibitor does not fects of natural estrogens [32). These es- aerobic organisms, a major threat is caused CHEMISTRY ATEAWAG 920 CHIMIA 5/ (1997) Nr. 12 (Dcl.cmber) by the partial reduction of oxygen, leading as a model system by answering the fol- edge about the components, genes, and to the production of highly toxic reactive lowing questions: i) by what chemicals enzymatic activities that are involved in oxygen species (ROS) such as superoxide and at what concentration is the oxidative these defense and repair mechanisms. radicals, hydrogen peroxide, or hydroxyl stress response activated, ii) which indi- Upon oxidative stress in prokaryotes, radicals [35]. These are produced during vidual components are involved in the the expression of ca. 60 genes is induced; normal enzymatic or metabolic processes, oxidative stress response and what is their some of the regulatory genes have been and they can also be produced upon trans- function, iii) how is the stress response identified [42][43]. The current knowl- fer of electrons from organic radicals to regulated, iv) what are the limits of the edge of the oxidative stress response in the oxygen. Transition metals playa key role adaptive response, and v) which of the bacterium Escherichia coli has been suc- in ROS formation since they may catalyze components could be used as sensiti ve and cessfully applied in a biosensor approach the formation of organic radicals, but also specific monitors for the detection of ox- in which DNA damage could be specifi- catalyze the Fenton reaction in which the idative stress or oxidative-stress-inducing cally measured using luciferase as the most reactive hydroxyl radical is produced compounds? marker gene. In the eukaryotic organism from hydrogen peroxide [36][37]. This Oxidative stress can be assessed Saccharomyces cerevisiae, the number of metal-ion-catalyzed reaction is most dam- through the detection ofROS themselves. reacting genes has been determined to be aging when it occurs site-specifically with As a result of the high reacti vities and as a at least 16 [44]. The regulation of the metals in proteins, DNA, or lipids. As a consequence of the short half-lives of rad- response is, however, complex as a result consequence, in cells that are exposed to icals, however, determination of organic of compartmentalization and the higher single environmental pollutants like ni- radicals and reactive oxygen has been and structural organization of eukaryotic or- troaromatics, azo compounds, halogenat- still is a major challenge for analytical ganisms. In our research, we focus on the ed compounds, pyridinyl compounds, chemists. Detection is difficult, particu- antioxidant response in the green alga quinones, metalloids and metals [38], or to larly in biological systems, since, once Chlamydomonas reinhardtii [45]. Recent- mixtures of pollutants and transition met- produced, the radicals may react instanta- ly, we have isolated a glutathione-peroxi- als [39J, an increased production of ROS neously. However, such measurements are dase-homologous gene (gpxh) whose ex- is observed and an increased oxidative required to unequivocally link biological pression is strongly induced upon expo- threat is created. Fortunately, cells main- effects with a specific toxic molecule. sure to pollutants that cause an oxidative tain a variety of defenses to protect against Analytical methods to quantify radical stress, as shown by Northern blot (Fig. 5) these ROS [40]. Essentially, three general production include infrared phosphores- and RNAse protection assays [46]. We are antioxidant defense systems exist, water- cence, pulse radiolysis, ESR spin trap- currently investigating the specificity, the soluble reductants (e.g., glutathione, ascor- ping, or the use of quenchers [41]. sensitivity, and the limits of the induction bate), lipophilic reductants (e.g., a-toco- Alternatively, biological measurements of the gpxh gene by studying its expres- pherol, or f3-carotene), and enzymes (e.g., are used to indirectly assess oxidative sion after exposure to various stressors. catalases, peroxidases or superoxide dis- stress. This approach takes advantage of The regulatory sequences of the gpxh gene mutases). In addition, cells have the ca- the fact that damaged biomolecules are have been coupled with an arylsulfatase pacity to remove or repair damaged mol- more stable and more easily detectable gene, a marker gene that can be easily ecules, e.g. by DNA repair systems, nu- than the radicals that caused them. It should measured in C. reinhardtii. Arylsulfatase cleases, or proteases. however be kept in mind that as a result of activity will be measured under various The major goal of our research is to defense and repair mechanisms, the mag- growth conditions and in the presence of understand the protective systems of or- nitude of oxidative stress is underestimat- various pollutants. These experiments will ganisms using the oxidati ve stress response ed. This points to the importance of know 1- give insights aboutthe physiological func- tion of gpxh and about its potential use as biosensor for oxidative stress.

7. Concluding Remarks and Outlook

Prediction of the toxicity of chemicals and assessment of their impact in the en vi- ronment are major goals of ecotoxicolog- ical research. The number of modifying 1.3 kb factors that determine ecotoxicological gpxh effects is almost countless and requires a systematic organization and stepwise de- mRNA duction of principles for a holistic under- standing. The fate and effect of a pollutant are influenced both by the biophysico- chemical properties of the chemical and the properties of the abiotic and biotic Fig. 5. Northern blot analysis of environment. Consequently, chemical, total RNA (10 !Jg) from Chlamy- physical, and biological analytical tools domonas reinhardtii cells that were need to be developed and combined. exposed to 2 mM H202for 0, 30, and ]80 min. Using the gpxh gene as a o 30 180 Minutes Since ecotoxicological evaluations are probe, its mRNA levels were deter- based on concentration-effect relation- mined. ships, the significance of measures of con- CHEMISTRY AT EA WAG 921

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