Transcriptome-Wide Prediction and Measurement of Combined Effects Induced by Chemical Mixture Exposure in Zebrafish Embryos.

BACKGROUND: Humans and environmental organisms are constantly exposed to complex mixtures of chemicals. Extending our knowledge about the combined effects of chemicals is thus essential for assessing the potential consequences of these exposures. In this context, comprehensive molecular readouts as retrieved by omics techniques are advancing our understanding of the diversity of effects upon chemical exposure. This is especially true for effects induced by chemical concentrations that do not instantaneously lead to mortality, as is commonly the case for environmental exposures. However, omics profiles induced by chemical exposures have rarely been systematically considered in mixture contexts. OBJECTIVES: In this study, we aimed to investigate the predictability of chemical mixture effects on the whole-transcriptome scale. METHODS: We predicted and measured the toxicogenomic effects of a synthetic mixture on zebrafish embryos. The mixture contained the compounds diuron, diclofenac, and naproxen. To predict concentration- and time-resolved whole-transcriptome responses to the mixture exposure, we adopted the mixture concept of concentration addition. Predictions were based on the transcriptome profiles obtained for the individual mixture components in a previous study. Finally, concentration- and time-resolved mixture exposures and subsequent toxicogenomic measurements were performed and the results were compared with the predictions. RESULTS: This comparison of the predictions with the observations showed that the concept of concentration addition provided reasonable estimates for the effects induced by the mixture exposure on the whole transcriptome. Although nonadditive effects were observed only occasionally, combined, that is, multicomponent-driven, effects were found for mixture components with anticipated similar, as well as dissimilar, modes of action. DISCUSSION: Overall, this study demonstrates that using a concentration- and time-resolved approach, the occurrence and size of combined effects of chemicals may be predicted at the whole-transcriptome scale. This allows improving effect assessment of mixture exposures on the molecular scale that might not only be of relevance in terms of risk assessment but also for pharmacological applications. https://doi.org/10.1289/EHP7773.

Comparison of cholin- and carboxylesterase enzyme inhibition and visible effects in the zebra fish embryo bioassay under short-term paraoxon-methyl exposure.

The acute zebra fish embryo test (Danio rerio Hamilton-Buchanan, 1822) is an accepted bioassay to assess the toxicity of waste water that may be used for the replacement of testing with adult fish. It is also suggested for chemical hazard characterization and assessment, although only a few groups of substances have yet been studied. Specifically acting substances such as neurotoxic insecticides pose a potentially hazard for non-target fish. To establish whether the proposed zebra fish embryo test protocol and the inhibition of cholinesterases (acetylcholinesterase EC 3.1.1.7, propionylcholinesterase EC 3.1.1.8) and carboxylesterase (EC 3.1.1.1) enzymes can be used in a similar fashion for hazard characterization and risk assessment of chemicals and environmental samples, two types of experiments were conducted. Visual effects of exposure to the organophosphate metabolite paraoxon-methyl after 24 and 48 h in the zebra fish embryo test system were analysed with the use of an inverse microscope (rate of mortality, developmental disturbances, heart rate and others). The inhibition to cholinesterases and carboxylesterase was also measured. Enzyme inhibition as a biomarker of exposure was about 70 times more sensitive than the effects in the zebra fish embryo test with an IC50 below 1.2 micromol compared with an EC50 of 91 micromol. The dose-response relationships showed different curve characteristics with a linear increase of enzyme inhibition compared with a sigmoidal curve for the overt effects. Significant overt effects could only be seen at concentrations at which already 80% of the activities of the different esterases were inhibited.

Joint algal toxicity of 16 dissimilarly acting chemicals is predictable by the concept of independent action.

For a predictive assessment of the aquatic toxicity of chemical mixtures, two competing concepts are available: concentration addition and independent action. Concentration addition is generally regarded as a reasonable expectation for the joint toxicity of similarly acting substances. In the opposite case of dissimilarly acting toxicants the choice of the most appropriate concept is a controversial issue. In tests with freshwater algae we therefore studied the extreme situation of multiple exposure to chemicals with strictly different specific mechanisms of action. Concentration response analyses were performed for 16 different biocides, and for mixtures containing all 16 substances in two different concentration ratios. Observed mixture toxicity was compared with predictions, calculated from the concentration response functions of individual toxicants by alternatively applying both concepts. The assumption of independent action yielded accurate predictions, irrespective of the mixture ratio or the effect level under consideration. Moreover, results even demonstrate that dissimilarly acting chemicals can show significant joint effects, predictable by independent action, when combined in concentrations below individual NOEC values, statistically estimated to elicit insignificant individual effects of only 1%. The alternative hypothesis of concentration addition resulted in overestimation of mixture toxicity, but differences between observed and predicted effect concentrations did not exceed a factor of 3.2. This finding complies with previous studies, which indicated near concentration-additive action of mixtures of dissimilarly acting substances. Nevertheless, with the scientific objective to predict multi-component mixture toxicity with the highest possible accuracy, concentration addition obviously is no universal solution. Independent action proves to be superior where components are well known to interact specifically with different molecular target sites, and provided that reliable statistical estimates of low toxic effects of individual mixture constituents can be given. With a regulatory perspective, however, fulfilment of both conditions may be regarded as an extraordinary situation, and hence concentration addition may be defendable as a pragmatic and precautionary default assumption.

Water quality objectives for mixtures of toxic chemicals: problems and perspectives.

The need to develop water quality objectives not only for single substances but also for mixtures of chemicals seems evident. For that purpose, the conceptual basis could be the use of the two existing biometric models: concentration addition (CA) and independent action (IA), which is also called response addition. Both may allow calculation of the toxicity of mixtures of chemicals with similar modes of action (CA) or dissimilar modes of action (IA), respectively. The joint research project Prediction and Assessment of the Aquatic Toxicity of Mixtures of Chemicals (PREDICT) within the framework of the IVth Environment and Climate Programme of the European Commission, provided the opportunity to address (a) chemometric and QSAR criteria to classify substances as supposedly similarly or dissimilarly acting; (b) the predictive values of both models for the toxicity of mixtures at low, statistically nonsignificant effect concentrations of the individual components; and (c) the predictability of mixture toxicity at higher levels of biological complexity. In this article, the general outline, methodological approach, and some preliminary findings of PREDICT are presented. A procedure for classifying chemicals in relation to their structural and toxicological similarities has been developed. The predictive capabilities of CA and IA models have been demonstrated for single species and, to some extent, for multispecies testing. The role of very low effect concentrations in multiple mixtures has been evaluated. Problems and perspectives concerning the development of water quality objectives for mixtures are discussed.

Predicting the joint algal toxicity of multi-component s-triazine mixtures at low-effect concentrations of individual toxicants.

Herbicidal s-triazines are widespread contaminants of surface waters. They are highly toxic to algae and other primary producers in aquatic systems. This results from their specific interference with photosynthetic electron transport. Risk assessment for aquatic biota has to consider situations of simultaneous exposure to various of these toxicants. In tests with freshwater algae we predicted and determined the toxicity of multiple mixtures of 18 different s-triazines. The toxicity parameter was the inhibition of reproduction of Scenedesmus vacuolatus. Concentration-response analyses were performed for single toxicants and for mixtures containing all 18 s-triazines in two different concentration ratios. Experiments were designed to allow a valid statistical description of the entire concentration-response relationships, including the low concentration range down to EC1. Observed effects and effect concentrations of mixtures were compared to predictions of mixture toxicity. Predictions were calculated from the concentration-response functions of individual s-triazines by applying the concepts of concentration addition and independent action (response addition) alternatively. Predictions based on independent action tend to underestimate the overall toxicity of s-triazine mixtures. In contrast, the concept of concentration addition provides highly accurate predictions of s-triazine mixture toxicity, irrespective of the effect level under consideration and the concentration ratio of the mixture components. This also holds true when the mixture components are present in concentrations below their individual NOEC values. Concentrations statistically estimated to elicit non-significant effects of only 1% still contribute to the overall toxicity. When present in a multi-component mixture they can co-operate to give a severe joint effect. Applicability of the findings obtained with s-triazines to mixtures of other contaminants in aquatic systems and consequences for risk assessment procedures are discussed.

A general best-fit method for concentration-response curves and the estimation of low-effect concentrations.

Risk assessments of toxic chemicals currently rely heavily on the use of no-observed-effect concentrations (NOECs). Due to several crucial flaws in this concept, however, discussion of replacing NOECs with statistically estimated low-effect concentrations continues. This paper describes a general best-fit method for the estimation of effects and effect concentrations by the use of a pool of 10 different sigmoidal regression functions for continuous toxicity data. Due to heterogeneous variabilities in replicated data (i.e., heteroscedasticity), the concept of generalized least squares is used for the estimation of the model parameters, whereas a nonparametric variance model based on smoothing spline functions is used to describe the heteroscedasticity. To protect the estimates against outliers, the generalized least-squares method is improved by winsorization. On the basis of statistical selection criteria, the best-fit model is chosen individually for each set of data. Furthermore, the bootstrap methodology is applied for constructing confidence intervals for the estimated effect concentrations. The best-fit method for the estimation of low-effect concentrations is validated by a simulation study, and its applicability is demonstrated with toxicity data for 64 chemicals tested in an algal and a bacterial bioassay. In comparison with common methods of concentration-response analysis, a clear improvement is achieved.

Alterations of physiological energetics, growth and reproduction of Daphnia magna under toxicant stress.

The study investigates the relationship between changes in physiological energetics of organisms and alterations of growth, development and reproduction of Daphnia magna. Groups of primiparous daphnids were subjected to 8-day exposures to the heavy metals cadmium and copper or to the cationic surfactant, cetyltrimethylammonium bromide (CTAB). Energetic alterations were estimated from the measurement of oxygen consumption and feeding activity which was performed during the last 3 days of the exposure period and from the calculation of simplified carbon balances. The physiological effects were compared to effects on organismal growth and reproduction as obtained from 17-day exposure experiments. Toxicant exposure reduced weight and body length of daphnids indicating an impaired growth rate, but effects on total metabolic costs measured as weight-specific oxygen consumption could not be detected. Net carbon gain of individuals decreased in a concentration-dependent way for the tested chemicals reflecting effects on biomass of daphnids. In the case of cadmium and copper, reproduction ( summation operatormx: number of offspring per female of age x born during the time interval x-1 to x, summarised over the entire exposure period) and the estimate for the intrinsic rate of natural increase, derived from the 17-day exposure-experiment, were affected at concentrations comparable to the effect levels as observed for growth. In the case of copper, the concentrations affecting growth and reproduction were close to the 17-day LC(50) value. CTAB caused a reduction in body length of primiparous daphnids whereas a decrease in the reproductive performance was not apparent. In conclusion, the chemicals did not change metabolic costs of exposed daphnids as it would be expected as a consequence of resistance or repair mechanisms, however, they induced alterations of SFG, growth, reproduction and intrinsic rate of natural increase. These alterations were chemical-specific. The fact that toxicant-related effects on growth and reproduction could not be linked to an elevated metabolic rate of daphnids may indicate that demand side effects occurred early during exposure - before the start of respirometric measurements - or that effects on growth were caused by an altered energy uptake. The results illustrate the importance of trade-off processes in regulating the distribution of energy among growth and reproduction of daphnids.

Quantitative structure-activity analysis of the algae toxicity of nitroaromatic compounds.

Proliferation toxicity toward the algae Scenedesmus vacuolatus in a 24 h one-generation reproduction assay was determined for nitrobenzene and 18 derivatives, including two phenols. The resultant EC(50) values covering more than 4 orders of magnitude were subjected to a quantitative structure-activity analysis (QSAR) using hydrophobicity in terms of the octanol/water partition coefficient in logarithmic form, log K(ow), and 16 quantum chemical descriptors of molecular reactivity that were calculated with the AM1 scheme. For 13 mononitro derivatives and the highly hydrophobic trifluralin, a narcotic-type mode of action can explain most of the toxicity variation. Correction of log K(ow) for ionization for the phenols and quantification of the molecular susceptibility for one-electron reduction as apparently rate-determining biotransformation step by the energy of the lowest unoccupied molecular orbital, E(LUMO), yields a highly significant QSAR for all 19 compounds (r(adj)(2) = 0.90), which can be further improved when adding the maximum net atomic charge at the nitro nitrogen, q(nitro)(-)(N), as the third descriptor (r(adj)(2) = 0.93). Comparison of the energy of the singly occupied molecular orbital, E(SOMO), of the radical anions as initial metabolites with the E(SOMO) of known redox cyclers suggests that dinitrobenzenes and TFM as well as multiply chlorinated nitrobenzenes may also exert oxidative stress. This is based on an E(SOMO) window of -0.30 to 0. 55 eV as a tentative criterion for molecular structures to have the potential for redox cycling, derived from a set of eight known redox cyclers. The discussion includes a detailed analysis of apparently relevant metabolic pathways and associated modes of toxic action of nitroaromatics.

Approaches to assessing combination effects of oestrogenic environmental pollutants.

Concerns about possible combination effects of environmental chemicals with estrogenic activity have motivated the search for synergisms between such agents. However, much published work has taken no account of the concepts and methods for analysing combination effects, which were developed in toxicology and pharmacology. In the present communication, we draw attention to conceptual frameworks relevant for a sound analysis of the effects of mixtures of oestrogenic compounds. A model calculation is presented demonstrating that it is conceivable that weakly oestrogenic compounds may be able to act together to produce significant effects, even when they are present at concentrations below their individual effect thresholds. Our results suggest that it may not be necessary to invoke synergisms in order to explain the discrepancy between the high concentrations of these agents required to produce effects in in vitro assay systems and their low concentrations in the environment.

Bioassay-directed identification of organic toxicants in river sediment in the industrial region of bitterfeld (Germany)-A contribution to hazard assessment

Bioassay-directed identification of toxicants in an acetonic extract of a sediment of the riverine Spittelwasser in the industrial region of Bitterfeld (Germany) was conducted. For this purpose, a combination of chromatographical fractionation, chemical analysis, and a biotest battery including Vibrio fischeri (inhibition of bioluminescence), Daphnia magna (immobilization), and Scenedesmus vacuolatus (inhibition of cell multiplication) was applied. Major toxicants identified and confirmed were methyl parathion (D. magna), prometryn, N-phenyl-beta-naphthalene amine, PAHs (S. vacuolatus), and tributyltin (all biotests). Toxicity to V. fischeri was dominated by elemental sulfur. Results indicate high toxicant loads in the sediment about 7 years after closedown of a majority of chemical production sites at Bitterfeld. Comparison of potential exposure and toxicity data indicate a severe hazard potential to aquatic organisms due to organic toxicants. The results illustrate the potency of a biotest battery for identification of toxicants in contaminated sediment within the frame of toxicity identification procedures.http://link.springer-ny. com/link/service/journals/00244/bibs/37n2p164.html

Biotransformation of estradiol in the human keratinocyte cell line HaCaT: metabolism kinetics and the inhibitory effect of ethanol.

PURPOSE: The aim of our study was to investigate the kinetics of beta-estradiol (E2) metabolism in the human keratinocyte cell line HaCaT and to estimate the effect of the potential inhibitor ethanol on the biotransformation reaction. METHODS: The formation rates of estrone (E1) in dependence on substrate concentrations were determined in HaCaT cells using tritium labelled E2. Experiments were conducted with and without addition of dehydroepiandrosterone (DHEA) and ethanol. Possible toxic effects on the cells due to ethanol were investigated by cytotoxicity tests. RESULTS: The metabolism of E2 in HaCaT cells exhibited Michaelis-Menten kinetics with Km and Vmax values of 3.5 microM and 216 pmol x mg(-1) protein x h(-1), respectively. The reaction was inhibited by DHEA and ethanol. The alcohol showed a reversible competitive inhibition mechanism for concentrations of 4 to 8% (v/v). Lower ethanol concentrations had no effect, whereas levels > or =10% significantly decreased cell viability leading to a different inhibition mechanism. CONCLUSIONS: The HaCaT cell line seems to be a suitable model for studying enzyme kinetics equivalent to the human skin. The concentration dependent inhibitory effect of ethanol observed in this cell line may be relevant for the transdermal E2 application in patients.

Synergisms with mixtures of xenoestrogens: a reevaluation using the method of isoboles.

There are concerns about possible combination effects of environmental chemicals with oestrogenic activity and their implications for human health. Such chemicals are present in complex mixtures in our environment. A number of studies searching for possible synergistic interactions between xenoestrogens have appeared in the literature. However, in these studies no account was taken of established concepts and methods for analysing combination effects. In the present review, we highlight conceptual issues which may be useful for a sound analysis of the effects of mixtures of xenoestrogens. We find that much published work suffers from an undue focus on measuring effects of mixtures at only one dose level. Assessments of combination effects are frequently complicated by a lack of information on dose-response relationships. Some studies which purportedly show absence of synergy have in fact overlooked synergisms.

Rate control in transdermal beta-estradiol reservoir membrane systems: the role of membrane and adhesive layer.

PURPOSE: The aim of our study was to clarify the kinetic performance of a membrane controlled reservoir system (MCRS) for beta-estradiol (E2) under in vitro conditions by determination of the role of membrane and adhesive layer on E2 flux control. METHODS: E2 and ethanol fluxes across EVA membrane or membrane coated with adhesive from saturated solutions in defined ethanol/PBS mixtures were measured in the symmetric and asymmetric configuration. Physicochemical parameters of the EVA membrane were determined. RESULTS: The E2 flux across the 9% EVA membrane steadily increased with increasing ethanol concentrations in both configurations, due to enhanced uptake of E2 by the polymer and increasing membrane diffusivity. Permeation across the EVA membrane coated with an adhesive layer in symmetric and asymmetric configuration increased up to maximum values of 0.80+/-0.14 micrograms X cm-2 X h-1 and 0.37+/-0.02 micrograms X cm-2 X h-1, respectively, at 62.5% (v/v) ethanol. The fluxes then decreased with further increase in the volume fraction of ethanol due to a dramatically reduced permeability of the adhesive layer. For the asymmetric case, a linear dependence of E2 on the ethanol fluxes was observed. CONCLUSIONS: The E2 flux from MCRS is strictly dependent on reservoir ethanol concentrations, whereas the adhesive layer represents the rate controlling barrier at high ethanol levels (> 70% v/v).

Pharmacokinetics of the transdermal reservoir membrane system delivering beta-estradiol: in vitro/in vivo-correlation.

PURPOSE: The aim of our study was to investigate the high fluctuations of Estradiol (E2) plasma levels transdermally delivered in postmenopausal women by a commercially available membrane controlled reservoir system (MCRS). METHODS: The transdermal E2 flux either out of a complete MCRS or across its membrane out of defined ethanol water mixtures was determined, as well as E2 plasma profiles in 6 postmenopausal women produced by a MCRS. RESULTS: The transdermal in vitro E2 flux rate out of a complete MCRS, claimed to deliver 25 microg/day, increased steadily, reaching a maximum value of 2.06 +/- 0.58 microg/h at 30 to 40 hours and decreased to a rate of about 0.5 microg/h from 60 to 90 hours. No statistically significant differences between plasma profiles calculated from the in vitro investigation and derived from a clinical study could be identified. The E2 flux in defined ethanol/water mixtures across MCRS-membrane, adhesive and skin layer increased with increasing ethanol concentrations up to a maximum of 227 +/- 34 ng/cm2/h at an ethanol concentration of 62.5% (V/V) and decreased with further increase in the volume fraction of ethanol. CONCLUSIONS: In vitro as well as in vivo investigations showed high fluctuation of E2 plasma profiles in postmenopausal women produced by the MCRS. These fluctuations are caused by a non-constant input rate of E2 which may be due to changing ethanol concentrations in the reservoir of the MCRS.

Regulations for combined effects of pollutants: consequences from risk assessment in aquatic toxicology.

In the analysis of combined effects two reference concepts are currently considered as equally valid for the assessment of mixture toxicities: these are LOEWE additivity (concentration addition) and BLISS independence (response addition) (Greco et al., 1995). The aim of this study of 137 binary mixtures of pesticides and surfactants using an algal biotest was to find rational procedures for the assessment of mixture toxicities in the aquatic environment. By introducing an index on prediction quality the quantitative relationships between predicted and observed effects are evaluated for each concept. It is shown that LOEWE additivity leads to good predictions of mixture toxicities for most combinations, whereas BLISS independence tends to underestimate mixture toxicities. By this it is reaffirmed that there is a solid basis for forthcoming regulatory activities on mixtures of chemicals.

Drinking water: for human consumption only? The amendment of directive 80/778/EEC parameter 55 in the light of aquatic toxicology.

In the current debate on the amendment of the European Drinking Water Directive (80/778/EEC) it is proposed that maximum admissible concentrations for pesticides and related products intended for human consumption shall be based on toxicological evidence. This paper compares limit values for human drinking water, based on mammal toxicology with aquatic toxicity data and water quality objectives for several pesticides proposed for the EC positive list. As aquatic organisms show higher sensitivities to pesticides it is concluded that sustainable water management practises require acknowledgement of ecotoxicological evidence.

In situ nuclear magnetic resonance of N pulse labels monitors different routes for nitrogen assimilation.

Nuclear magnetic resonance offers the possibility of noninvasive in situ observation of (15)N pulse labeling in the presence of light. In vivo, exclusively the delta-nitrogen of Gln is labeled in the cyanobacterium Microcystis firma when glutamate synthase is inhibited by azaserine. In contrast, the green alga Chlorella fusca is additionally capable of incorporating nitrogen into Glu, thus providing evidence for an anabolic function of glutamate dehydrogenase in this organism.