Johan Bierkens

Applications and pitfalls of stress-proteins in biomonitoring

The vast number of potentially hazardous chemicals and the complex interactions that can occur between them in environmental mixtures, call for inexpensive, early and sensitive endpoints that reflect their biological effect. The existing validated bioassays, mostly based on lethality or reproduction, have been shown to be inadequate in respect of their sensitivity, the duration and expense of the test. In contrast, changes at biochemical level are usually the first detectable responses to environmental perturbation. Because these alterations underlie all effects at higher organisational level, they have often been shown to be very sensitive indicators of pollution. Stress-proteins (also referred to as heat-shock proteins or hsp) have recently been recognised as being one of the primary defence mechanisms that are activated by the occurrence of denatured proteins in the cell. Four major stress-protein families of 90,70,60 and 16-24 kDa are the most prominent and are frequently referred to as hsp90, hsp70, hsp60 and low molecular weight (LMW) stress-proteins. Three aspects of stress-proteins have been characterised that are essential if they want to be used as biomarkers of pollution: (1) they are part of the cellular protective response; (2) their synthesis is likely to be induced by a large number of chemicals; and (3) they are highly conserved in all organisms from bacteria to plants and man. Also, field studies have shown (be it for a limited number of stressors) that the stress response can occur even at the minute concentrations of pollutants that are usually found in the environment. However, increasing knowledge on the kinetics and persistence of the stress response to complex environmental mixtures, on the influence of both physiological and environmental parameters (pH, eutrophication, ellipsis), on the constitutive levels of stress-proteins and on the acquisition of tolerance, is required before one could safely apply stress-proteins to assess on-site pollution. Still, included in a test battery of complementary bioassays, stress protein may be very valuable as tier I biomarkers, i.e. broad response biomarkers that are used for preliminary screening of the environment.

Dose-dependent induction of heat shock protein 70 synthesis in Raphidocelis subcapitata following exposure to different classes of environmental pollutants.

In this study an enzyme-linked immunosorbent assay (ELISA) was developed to detect the stress protein Hsp70 in the green alga Raphidocelis subcapitata. Using this ELISA, the response to a variety of pollutants, including ZnCl2, SeO2 (heavy metals), lindane (organochlorine pesticide), pentachlorophenol (PCP, chlorinated hydrocarbon insecticide and fungicide), carbaryl (carbamate pesticide) and sodium dodecyl sulphate (SDS; surfactant) was tested. Our results show that Hsp 70 is produced in a dose-dependent way in response to most chemicals investigated (except PCP) and at concentrations below the range of classical cytotoxicity testing (i.e. growth inhibition, lethality). Still, the potential to induce Hsp70 varied among the pollutants tested, the heavy metals ZnCl2 and SeO2 being the strongest inducers of Hsp70. Combined with the existing literature, these results indicate that Hsp70 in R. subcapitata is a sensitive biomarker for a wide range of environmental pollutants.

Predicting blood lead levels from current and past environmental data in Europe.

The present case study on lead in Europe illustrates the use of the Integrated Monitoring Framework Strategy to assess the health outcome of environmental pollution by evaluating the associations between lead in various environmental compartments (air, soil, dust, drinking water and diet) and lead concentrations in blood (B-Pb) for various age-related sub-populations. The case study was aimed to investigate whether environmental, exposure and biomonitoring data at general population level, covering all EU member states, could be integrated. Although blood lead has been monitored extensively in Europe, consistent datasets are not yet available. Data diverge with regard to objectives, regional scale, sampling years, gender, age groups and sample size. Significant correlations were found between B-Pb and the concentrations of Pb in air and diet. The significant decrease of the Pb in air over time from 0.31 μg/m(3) (P95: 0.94; n=98) prior to 1990 to 0.045 μg/m(3) (P95: 0.11; n=256) in 2007 (latest observations included) (Δ=-85%) corresponds to a decline in B-Pb by 48% and 57% in adult women and adult men, respectively. For pre-school children a more shallow decline in B-Pb of 16% was calculated over the same period. Similarly, the reduction in Pb-dietary intake from on average 68.7 μg/d (P95: 161.6; n=19) in 1978 to 35.7 μg/d (P95: 82.3; n=33) in the years post 2000 (Δ=-48%) is paralleled by a decline in B-Pb of 32, 33 and 19% in adult women, primary- and pre-school children, respectively. Insufficient data exist for other age groups to calculate statistically significant correlations. Although regression models have been derived to predict B-Pb for different sub-populations in Europe based on Pb concentrations in air and soil as well as dietary intake, it is concluded that the available data are insufficient to accurately predict actual and future simultaneous exposure to Pb from various environmental compartments, and as a consequence the health impact of Pb for various target populations at EU scale. At least due to data availability, air Pb remains the best predictor of B-Pb in the population. However, lead emission sources have largely been reduced and inhalation of lead in air is not causal to B-Pb levels. Therefore, there is a need of adequate data for Pb in soil and house dust, and in diet and drinking water as these are causal exposure sources with a longer Pb half-life than air. An extended and more harmonized surveillance system monitoring B-Pb, especially in children, is urgently required in order to identify, quantify and reduce still remaining sources of Pb exposure.

Effect of different environmental variables on the synthesis of Hsp70 in Raphidocelis subcapitata

Heat-shock proteins (Hsp) or stress proteins are strong candidates for biomarkers of environmental pollution since they are activated very early in the cascade of cellular events that follow toxic exposure and at concentrations below the lethal dose. Included in a test battery comprised of different bioassays, Hsp induction could provide a general purpose tier I indicator of pollution. Still, little is known on the induction of Hsp under different environmental conditions. In the present study we have made use of an Enzyme Linked Immunosorbent Assay (ELISA) to detect the synthesis of Hsp70 in Raphidocelis subcapitata in response to changes in pH, temperature, humic acids, nitrates and phosphates. The results show that algae respond to these changes in the environment by a transient increase in Hsp70 levels, the extend of which is dependent on the actual parameter under investigation. Out of these five parameters studied, only temperature and possibly pH were able to induce acquired tolerance, i.e. algae grown at a pH or at a temperature different from control conditions were shown to have acquired resistance to a subsequent challenge with Zn (10(-5) M). Adjustment of the pH and temperature in two physico-chemically different natural surface waters was demonstrated to be sufficient to obtain similar induction patterns of Hsp70 upon exposure to zinc. These results qualify Hsp70 as a good biomonitor for environmental pollution provided essential environmental parameters such as pH and temperature are kept constant.

Health impact assessment and monetary valuation of IQ loss in pre-school children due to lead exposure through locally produced food.

A case study has been performed which involved the full chain assessment from policy drivers to health effect quantification of lead exposure through locally produced food on loss of IQ in pre-school children at the population level across the EU-27, including monetary valuation of the estimated health impact. Main policy scenarios cover the period from 2000 to 2020 and include the most important Community policy developments expected to affect the environmental release of lead (Pb) and corresponding human exposure patterns. Three distinct scenarios were explored: the emission situation based on 2000 data, a business-as-usual scenario (BAU) up to 2010 and 2020 and a scenario incorporating the most likely technological change expected (Most Feasible Technical Reductions, MFTR) in response to current and future legislation. Consecutive model calculations (MSCE-HM, WATSON, XtraFOOD, IEUBK) were performed by different partners on the project as part of the full chain approach to derive estimates of blood lead (B-Pb) levels in children as a consequence of the consumption of local produce. The estimated B-Pb levels were translated into an average loss of IQ points/child using an empirical relationship based on a meta-analysis performed by Schwartz (1994). The calculated losses in IQ points were subsequently further translated into the average cost/child using a cost estimate of €10.000 per loss of IQ point based on data from a literature review. The estimated average reduction of cost/child (%) for all countries considered in 2010 under BAU and MFTR are 12.16 and 18.08% as compared to base line conditions, respectively. In 2020 the percentages amount to 20.19 and 23.39%. The case study provides an example of the full-chain impact pathway approach taking into account all foreseeable pathways both for assessing the environmental fate and the associated human exposure and the mode of toxic action to arrive at quantitative estimates of health impacts at the individual and the population risk levels alike at EU scale. As the estimated B-Pb levels fall below the range of observed biomonitoring data collected for pre-school children in 6 different EU countries, results presented in this paper are only a first approximation of the costs entailed in the health effects of exposure to lead and the potential benefits that may arise from MFTR measures inscribed in Commission policies.

Modelling the exposure to chemicals for risk assessment: a comprehensive library of multimedia and PBPK models for integration, prediction, uncertainty and sensitivity analysis - the MERLIN-Expo tool

MERLIN-Expo is a library of models that was developed in the frame of the FP7 EU project 4FUN in order to provide an integrated assessment tool for state-of-the-art exposure assessment for environment, biota and humans, allowing the detection of scientific uncertainties at each step of the exposure process. This paper describes the main features of the MERLIN-Expo tool. The main challenges in exposure modelling that MERLIN-Expo has tackled are: (i) the integration of multimedia (MM) models simulating the fate of chemicals in environmental media, and of physiologically based pharmacokinetic (PBPK) models simulating the fate of chemicals in human body. MERLIN-Expo thus allows the determination of internal effective chemical concentrations; (ii) the incorporation of a set of functionalities for uncertainty/sensitivity analysis, from screening to variance-based approaches. The availability of such tools for uncertainty and sensitivity analysis aimed to facilitate the incorporation of such issues in future decision making; (iii) the integration of human and wildlife biota targets with common fate modelling in the environment. MERLIN-Expo is composed of a library of fate models dedicated to non biological receptor media (surface waters, soils, outdoor air), biological media of concern for humans (several cultivated crops, mammals, milk, fish), as well as wildlife biota (primary producers in rivers, invertebrates, fish) and humans. These models can be linked together to create flexible scenarios relevant for both human and wildlife biota exposure. Standardized documentation for each model and training material were prepared to support an accurate use of the tool by end-users. One of the objectives of the 4FUN project was also to increase the confidence in the applicability of the MERLIN-Expo tool through targeted realistic case studies. In particular, we aimed at demonstrating the feasibility of building complex realistic exposure scenarios and the accuracy of the modelling predictions through a comparison with actual measurements.

Assessing multimedia/multipathway exposures to inorganic arsenic at population and individual level using MERLIN-Expo

In this study, we report on model simulations performed using the newly developed exposure tool, MERLIN-Expo, in order to assess inorganic arsenic (iAs) exposure to adults resulting from past emissions by non-ferrous smelters in Belgium (Northern Campine area). Exposure scenarios were constructed to estimate external iAs exposure as well as the toxicologically relevant As (tAs, i.e., iAs, MMA and DMA) body burden in adults living in the vicinity of the former industrial sites as compared to adults living in adjacent areas and a reference area. Two scenarios are discussed: a first scenario studying exposure to iAs at the aggregated population level and a second scenario studying exposure at the individual level for a random sub-sample of subjects in each of the three different study areas. These two scenarios only differ in the type of human related input data (i.e., time-activity data, ingestion rates and consumption patterns) that were used, namely averages (incl. probability density functions, PDFs) in the simulation at population level and subject-specific values in the simulation at individual level. The model predictions are shown to be lower than the corresponding biomonitoring data from the monitoring campaign. Urinary tAs levels in adults, irrespective of the area they lived in, were under-predicted by MERLIN-Expo by 40% on average. The model predictions for individual adults, by contrast, under-predict the biomonitoring data by 7% on average, but with more important under-predictions for subjects at the upper end of exposure. Still, average predicted urinary tAs levels from the simulations at population level and at individual level overlap, and, at least for the current case, lead to similar conclusions. These results constitute a first and partial verification of the model performance of MERLIN-Expo when dealing with iAs in a complex site-specific exposure scenario, and demonstrate the robustness of the modelling tool for these situations.