Investigating combined toxicity of binary mixtures in bees: meta-analysis of laboratory tests, modelling, mechanistic basis and implications for risk assessment

Bees are exposed to a wide range of multiple chemicals “chemical mixtures” from anthropogenic (e.g. plant protection products or veterinary products) or natural origin (e.g. mycotoxins, plant toxins). Quantifying the relative impact of multiple chemicals on bee health compared with other environmental stressors (e.g. varroa, viruses, and nutrition) has been identified as a priority to support the development of holistic risk assessment methods. Here, extensive literature searches and data collection of available laboratory studies on combined toxicity data for binary mixtures of pesticides and non-chemical stressors has been performed for honey bees (Apis mellifera), wild bees (Bombus spp.) and solitary bee species (Osmia spp.). From 957 screened publications, 14 publications provided 218 binary mixture toxicity data mostly for acute mortality (lethal dose: LD50) after contact exposure (61%), with fewer studies reporting chronic oral toxicity (20%) and acute oral LC50 values (19%). From the data collection, available dose response data for 92 binary mixtures were modelled using a Toxic Unit (TU) approach and the MIXTOX modelling tool to test assumptions of combined toxicity i.e. concentration addition (CA), and interactions (i.e. synergism, antagonism). The magnitude of interactions was quantified as the Model Deviation Ratio (MDR). The CA model applied to 17% of cases while synergism and antagonism were observed for 72% (MDR > 1.25) and 11% (MDR < 0.83) respectively. Most synergistic effects (55%) were observed as interactions between sterol-biosynthesis-inhibiting (SBI) fungicides and insecticide/acaricide. The mechanisms behind such synergistic effects of binary mixtures in bees are known to involve direct cytochrome P450 (CYP) inhibition, resulting in an increase in internal dose and toxicity of the binary mixture. Moreover, bees are known to have the lowest number of CYP copies and other detoxification enzymes in the insect kingdom. In the light of these findings, occurrence of these binary mixtures in relevant crops (frequency and concentrations) would need to be investigated. Addressing this exposure dimension remains critical to characterise the likelihood and plausibility of such interactions to occur under field realistic conditions. Finally, data gaps and further work for the development of risk assessment methods to assess multiple stressors in bees including chemicals and non-chemical stressors in bees are discussed

A computational model to predict rat ovarian steroid secretion from in vitro experiments with endocrine disruptors.

A finely tuned balance between estrogens and androgens controls reproductive functions, and the last step of steroidogenesis plays a key role in maintaining that balance. Environmental toxicants are a serious health concern, and numerous studies have been devoted to studying the effects of endocrine disrupting chemicals (EDCs). The effects of EDCs on steroidogenic enzymes may influence steroid secretion and thus lead to reproductive toxicity. To predict hormonal balance disruption on the basis of data on aromatase activity and mRNA level modulation obtained in vitro on granulosa cells, we developed a mathematical model for the last gonadal steps of the sex steroid synthesis pathway. The model can simulate the ovarian synthesis and secretion of estrone, estradiol, androstenedione, and testosterone, and their response to endocrine disruption. The model is able to predict ovarian sex steroid concentrations under normal estrous cycle in female rat, and ovarian estradiol concentrations in adult female rats exposed to atrazine, bisphenol A, metabolites of methoxychlor or vinclozolin, and letrozole

Physiologically based toxicokinetic models for prediction of complex metabolic interactions between chemical in mixtures

The emergence of metabolic interactions during in vivo co-exposures depends on the concentrations of mixture components actually delivered to the metabolizing cells. For simulation and prediction purposes, physiologically based pharmacokinetic (PBPK) models have been used successfully for quite a while to predict such concurrent internal exposures. However, a classical PBPK framework alone cannot address the complexity of metabolism and its receptor mediated modulations for more than a handful of interacting chemicals. A solution to that problem is to integrate PBPK and systems biology modeling. We demonstrate here such an approach and present the software tools we have developed. Our software (an extension of GNU MCSim) automatically merges metabolic pathways models for individual chemicals and couples them to a template PBPK model. The transport and metabolism models generated are very efficient and can simulate interactions between a theoretically unlimited number of substances at tissue levels. We use a fine-grain description of reactions, so that development and computation time increases only linearly with the number of substances considered, even though the number of possible interactions increases exponentially. Several examples of application to the prediction of the joint kinetics of mixtures up to a hundred of chemicals are given. The efficient parameterization of such models remains an issue which we discuss. Our integrative approach can be extended beyond metabolic interactions, into the realm of cellular effects

Perturbation endocrinienne et évaluation du risque pour la reproduction humaine : entre défis scientifiques d'aujourd'hui et enjeux de demain

International audienceFor many years, the issue of endocrine disruptors (ED) has persisted as an unresolved question for research, the public and regulatory bodies. As the mechanisms of action of ED are progressively elucidated, other issues have arisen, in connection with new challenges. The strategy of evaluation of these substances has been supported by multidisciplinary research intended to characterise the targets and modes of action of ED ; by the development of highly specific tools ; and by the development of an appropriate regulatory framework. Despite promising technologies, assessment of ED toxicity still has inherent limitations. Thus, researchers seeking to optimise the characterisation of hazards are moving in new directions, with lower doses, mixtures of substances, targeted exposure windows and ever more predictive tools.Depuis de nombreuses années, la problématique des perturbateurs endocriniens (PE) est un questionnement permanent pour la recherche, le grand public et les organismes réglementaires. Au fur et à mesure que les mécanismes d'action des PE sont élucidés, d'autres questions apparaissent, en lien avec de nouveaux défis. La stratégie d'évaluation de ces substances a été étayée par des recherches multidisciplinaires dans le but de caractériser les cibles et les modes d'action des PE ; par la mise au point d'outils hautement spécifiques ; ainsi que par le développement de cadres réglementaires propres. Malgré des technologies prometteuses, des limites sont encore aujourd'hui inhérentes à l'évaluation de la toxicité des PE. Aussi, les chercheurs, dans l'optique de caractériser au mieux les dangers, s'orientent vers de nouvelles directions, avec des doses plus faibles, des mélanges de substances, des fenêtres d'exposition ciblées et des outils toujours plus prédictifs

Flux analyses of <i>in vitro</i> and <i>in vivo</i> experiments.

<p>Graphs A and B represent the <i>in vitro</i> flux analysis of steroid hormones conversion at 48 h after addition of 200 nM A into the medium, without or with FSH 20 ng/ml. Graphs C, D, and E illustrate the <i>in vivo</i> flux analysis of steroid hormones conversion at several times of the estrus cycle (corresponding to diestrus, proestrus, and estrus stages). The aromatization reaction of A into E1 is taken as the reference reaction for each condition. The flux values for that reference were 7.29×10⁻⁹ pmoles/min/cell <i>in vitro</i> without FSH, 8.72×10⁻⁸ pmoles/min/cell <i>in vitro</i> with FSH, 6.09×10⁻⁹ pmoles/min/cell <i>in vivo</i> in the diestrus stage, 6.17×10⁻⁹ pmoles/min/cell in the proestrus stage, and 5.10×10⁻⁹ pmoles/min/cell in the estrus stage of the estrous cycle. Values for the other reactions in each condition are relative to the corresponding reference. Arrow thicknesses are proportional to the flux absolute values.</p

Model parameter distributions used to describe <i>in vivo</i> variability (in addition to those of Table 4).

<p>LN (geometric mean, geometric SD): lognormal distribution.</p

A comparison of two human cell lines and two rat gonadal cell primary cultures as in vitro screening tools for aromatase modulation

International audienceEnvironmental toxicants are a serious health concern, and numerous studies have been devoted to studying the effects of environmental Endocrine Disrupting Chemicals (EDCs). The balance between androgens and estrogens controls the function of many EDC-sensitive organs, and the aromatase enzyme plays a key role in maintaining this balance. In vitro studies have suggested that aromatase expression and activity is a promising biomarker for initial screenings of putative hormonal disrupting compounds. To further validate the aromatase biomarker, we tested several EDCs (atrazine, bisphenol A. methoxychlor, methoxychlor metabolite HPTE, vinclozolin, vinclozolin metabolite M2) in four different models (human cell lines H295R and JEG-3, rat primary cultures of granulosa and leydig cells). We evaluated the similarities/differences in the chemical impact on aromatase mRNA levels and enzymatic activity for the different species and cell types. Aromatase gene expression was assessed by q-RT-PCR, and enzymatic activity was assessed via a tritiated water method with either intact cells or isolated microsomes. The aromatase gene mRNA levels and cellular enzymatic activity varied between the four different models tested, which suggests that the EDC effect varies among different cell types. However, regulation of microsomal aromatase activity appeared to be conserved across all the species and cell types tested. These results suggest that several well characterized complementary cellular models are required to fully characterize the effects of putative EDCs and predict the in vivo effects

Model parameter values (for one cell) obtained from direct measurements on granulosa cells <i>in vitro</i> or from the published literature values.

<p>A, androstenedione; T, testosterone; E₁, estrone; E₂, estradiol.</p>a<p>Hargrove, 1993a <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053891#pone.0053891-Hargrove1" target="_blank">[47]</a>; Hargrove, 1993b <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053891#pone.0053891-Hargrove2" target="_blank">[48]</a>.</p>b<p>mRNA and protein synthesis rates were calculated under steady-state assumption with data from direct measurements on granulosa cells <i>in vitro</i> (see text).</p>c<p>Renwick <i>et al.</i>, 1981 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053891#pone.0053891-Renwick1" target="_blank">[49]</a>.</p>d<p>Breen <i>et al.</i>, 2009 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053891#pone.0053891-Breen1" target="_blank">[26]</a>.</p>e<p>Data were arbitrately fixed.</p>f<p>Plowchalk and Teeguarden, 2002 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053891#pone.0053891-Plowchalk1" target="_blank">[50]</a>.</p>g<p>Direct <i>in vitro</i> measurement.</p

Overview of the computational model for steroidogenesis last metabolic steps in a rat granulosa cell.

<p>The transcription and translation events for the three last major enzymes involved in estradiol synthesis, and sex steroid synthesis itself, are modeled, with relevant FSH control, endocrine disrupting chemical (EDC) modulation, or methoxychlor (MXC) aromatase competitive inhibition. Steroids can be transported in and out of cell. <i>In vitro</i>, the exterior compartment corresponds to the culture medium; <i>in vivo</i> it corresponds to the ovary tissue (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053891#pone-0053891-g002" target="_blank">Figure 2</a>). Aliases (repeated species labels) are used for clarity but correspond in fact to a unique species.</p