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.

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.

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.

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.

Comparative sensitivity of 20 bioassays for soil quality.

Increasing evidence suggests that the use of a single bioassay will never provide a full picture of the quality of the environment. Only a test battery, composed of bioassays of different animal and plant species from different trophic levels will reduce uncertainty, allowing an accurate assessment of the quality of the environment. In the present study, a test battery composed of 20 bioassays of varying biological endpoints has been compared. Apart from lethality and reproductive failure in earthworms, springtails, nematoda, algae and vascular plants, these endpoints also included bioavailibility of metals (bacteria), heat-shock induction (nematodes, algae), DNA damage (bacteria, earthworm, vascular plants), beta-galactosidase (Daphnia) and esterase activity (algae) and a range of immunological parameters (earthworm). Four chemicals (cadmium, phenol, pentachlorophenol and trifluralin)--each representing a different toxic mode of action--were applied in a dilution series (from 1 mg/kg up to 1000 mg/kg) onto OECD standard soil. The tests have been performed both on these artificially contaminated soil samples and on aqueous extracts subsequently obtained from these soils. The results show that the immunological parameters and the loss of weight in the earthworms were among the most sensitive solid-phase assays. Esterase inhibition and heat-shock induction in algae were shown to be extremely sensitive when applied to soil extracts. As previously shown at the species level, no single biological endpoint was shown to be the most sensitive for all four modes of toxic action.

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.

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.

Polysulphone inhibits final differentiation steps of osteogenesis in vitro.

Biocompatibility is an important factor in the development of orthopedic implants as well as in the development of new tissue culture devices. Polysulphone has been used for orthopedic implants because of its mechanical properties, ease of sterilization, molding capacity, and biocompatibility. Therefore, polysulphone has been chosen as the prime material for the construction of tissue culture devices to be used for the cultivation of osteogenic cells (preosteoblast-like MN7 cells and primary bone marrow fragments), as well as complete fetal long bone explants under space flight conditions. Whereas polysulphone did not interfere with the proliferation in early stages of bone-forming cells, we show that leachable factors within the polysulphone polymer prevented the final steps of matrix formation as measured by collagen synthesis and matrix mineralization. These data argue against polysulphone as a material for orthopedic implants.

Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow.

In the presence of beta-glycerophosphate and vitamin C, cultures of normal mouse bone marrow cells form three-dimensional structures that stain positive with the Von Kossa technique and express alkaline phosphatase (ALP), collagen type I, and osteocalcin. Little is known about the characteristics and frequency of the cells that contribute to this phenomenon. Most likely, mature osteoblastic cells do not contribute to the nodule formation because no osteocalcin expressing cells are detected in the flushed marrow by in situ hybridization. Limiting dilution analysis shows that, in normal bone marrow, 1 of 2.2 x 10(5) cells has the potency to form a bone nodule and to express ALP, collagen, and osteocalcin in a temporal fashion. Upon in vivo treatment with 5-fluorouracil (5-FU), this frequency increases 12-fold, eg, 1 in 1.75 x 10(4) cells shows osteogenic activity. In comparison, fibroblast colony forming cells occur at a frequency of 1 of 2.5 x 10(4) or 1 of 5 x 10(3) plated cells in normal or 5-FU-treated marrow, respectively. Using density centrifugation, the majority of the osteoprogenitor cells in 5-FU marrow are found in the low-density (1.066 to 1.067 g/mL) fractions. In addition, these cells bind to nylon wool but not to plastic and aggregate in the presence of wheat germ agglutinin and soybean agglutinin. Scanning and transmission electron microscopy shows that the bone nodules in 5-FU marrow cultures are composed of fibroblastoid cells embedded in a mineralized collagen matrix. In conclusion, our results show that a quiescent cell population in the murine bone marrow with fibroblastoid characteristics contributes to the formation of bone-like nodules in vitro.

Recovery of the proliferative and functional integrity of mouse bone marrow in long-term cultures established after whole-body irradiation at different doses and dose rates.

Injury inflicted upon the bone marrow stroma following whole-body irradiation and its repair over a 1-year period has been assessed in murine long-term bone marrow cultures established at increasing time intervals after irradiation. Different doses at different dose rates (10 Gy at 0.05 cGy/min, 4.5 Gy and 10 Gy at 1.6 cGy/min, and 4 x 4.5 Gy [3 weeks between doses] at 60 cGy/min) were chosen so as to maximize differences in effect in the stroma. The cellularity of the adherent layer in long-term cultures established 1 month after irradiation was reduced by 40%-90% depending on the dose and dose rate. Simultaneous with the poor ability of the marrow to form adherent layers, the cumulative spleen colony-forming unit (CFU-S) and granulocyte-macrophage colony-forming cell (GM-CFC) production over a 7-week period was reduced to 0% and 30% of control cultures, respectively. The slow recovery of the adherent layer was paralleled by an increase in the numbers of CFU-S and GM-CFC in the supernatant. Cultures established from repeatedly irradiated mice performed poorly over the entire 1-year period. Whereas the regeneration of the stroma was near complete 1 year after irradiation, the CFU-S and GM-CFC levels reached only between 50% and 80% of control cultures, respectively. Also, the concentration of CFU-S and GM-CFC in the supernatant remained persistently lower in cultures established from irradiated mice as compared to control cultures. The levels of sulfated glycosaminoglycans, which have been implicated in the establishment of the functional integrity of the microenvironment, were not reduced in the adherent layers at any time after irradiation. These results indicate that the regeneration of the stroma is accompanied by an incomplete recovery of active hemopoiesis in vitro. However, no evidence was found for persistent functional defects in the stroma after irradiation, using the present endpoints.

The radiation response and recovery of bone marrow stroma with particular reference to long-term bone marrow cultures.

There is evidence for long-term haematopoietic dysfunction in some patients treated with radiotherapy. Although the underlying mechanisms are unclear, both stem cell and environmental defects have been implicated. In the present article we review the evidence concerning the role of stromal cells. According to the endpoints used, a wide range of radiosensitivities for the stroma have been reported. Long-term bone marrow cultures provide a system in which both functional and regenerative aspects of the stroma can be studied. A dose of 5 Gy applied prior to the establishment of long-term bone marrow cultures decreases both the formation of a confluent adherent stromal layer and its capacity to support haematopoiesis. In contrast, in its fully established phase, the adherent layer displays a high radioresistance due to the low proliferative stress applied to its stromal populations. A dose of 10 Gy given to a fully established adherent layer does not prevent haematopoietic engraftment and sustained haematopoiesis. At doses above 100 Gy a macrophage-like and epithelioid cell-type become dominant, which preserve their ability of producing growth regulatory molecules at doses as high as 500 Gy. These data suggest that the main effect on the stroma is a delayed expression of irradiation damage due to the slow rate of turnover of stromal cells. So far, there is little evidence for persistent deficiencies in the functional roles of stromal cell populations.