International Symposium on Alternative in vitro methods to characterize the role of EAS in hormone-targeted tissues Rome 17.12.2012 The role of biokinetics in in vitro tests and the interpretation of results Emanuela Testai Istituto Superiore di Sanità Department of Environment and Primary Prevention Mechanisms of Toxicity Unit Rome-Italy emanuela.testai@iss.it 1
Alternative in vitro methods to characterize the role of EAS The International Programme for Chemical Safety (2002) has established the following definition for endocrine disrupters: Endocrine disrupters are exogenous substances or mixtures that alter function(s) of the endocrine system and consequently cause adverse health effects in an intact organism, or its progeny, or (sub)populations. US EPA and IPCS do not consider endocrine disruption to be an adverse effect per se , but rather to be a mode or mechanism of action potentially leading to other outcomes, i.e. carcinogenic, reproductive/developmental effects, routinely considered in reaching regulatory decisions. 2
Alternative in vitro methods to characterize the role of EAS Difference between endocrine modulation and endocrine disruption . Many adaptive, compensatory, and physiologically normal/necessary processes result in measurable endocrine changes. It is only when these natural mechanisms are affected to such a degree that adverse effects are induced that ED occurs. Exogenous chemicals in order to affect the endocrine system must act against the background of circulating levels of endogenous hormones, which are usually much more potent than any ED (the potency issue ). There are at least two clear requirements for a substance to be defined as an ED: the demonstration of an adverse effect and of an endocrine disruption mode-of-action (biological plausibility). 3
Alternative in vitro methods to characterize the role of EAS In vitro studies can be used to study the MoA and for priority testing based on hazard. For the time being their use for risk assessment purposes is limited due to difficulties in carrying out quantitative in vitro to in vivo extrapolation. Need of translating information from the cell level, to organs and subsequently to organisms and to distinguish between adaption vs. adversity , likely identifying actual in vitro marker of adversity . ‘ Omics ’ techniques are producing a bulk of information, but before we can quantitatively use it more knowledge is need for a correct interpretation. Lack of information on actual exposure of cell: in this respect in vitro biokinetics data providing the actual level of cell exposure producing an in vitro observed effects can improve the in vitro-in vivo extrapolation. 4
Alternative in vitro methods to characterize the role of EAS The knowledge of the bioavailability of a given compound by the relevant uptake routes should represent the starting point for any toxicological testing. In vivo the actual internal dose reaching the target is the more relevant parameter in evaluating exposure and in the quantitative risk assessment. When developing testing strategies, kinetics is considered the crucial body of information for the design and performance of toxicological tests and for toxicity data interpretation. This consideration applies also to alternative/non animal testing strategy Biokinetics processes have been evoked to explain the in vitro/in vivo differences 5 5
Alternative in vitro methods to characterize the role of EAS In vitro biokinetics Identification of actual cellular exposure (peak concentrations, AUC, parent vs metabolites) The actual intracellular concentration may greatly differ from the nominal applied concentrations due to altered bioavailability : interactions with the medium components, the plate, the cell itself, evaporation, chemical instability (abiotic processes). physiological cellular processes : mechanism of transport across the membranes, biotransformation, bioaccumulation. In vitro the nominal applied concentration rather than the actual level of cell exposure is usually associated to the observed effects. 6 6
Alternative in vitro methods to characterize the role of EAS In vitro biokinetics Test Item Evaporation Plastic Protein binding binding Passive/Active Free Concentration in (Transporters) the medium Cells Uptake Characterization Target of the cell model Metabolism Free Concentration 7
Alternative in vitro methods to characterize the role of EAS In repeated treatments for prolonged time of exposure the uncertainty about the actual level of exposure of cells in vitro is greatly enhanced (possibility of induction/inhibition phenomena, cumulative toxicity). Predict-IV — Profiling the toxicity of new drugs: a non animal-based approach integrating toxicodynamics and biokinetics WP3: Non animal-based models for in vitro kinetics and human kinetic prediction The ultimate goal is to contribute to the derivation of NOEC values (relatively to drug safety) in model systems based on human cells representative of in vivo target organs, from which it would be possible to extrapolate the corresponding in vivo dose. 8 8
Alternative in vitro methods to characterize the role of EAS Primary human Primary rat HepaRG Human renal cells hepatocytes(PHH) hepatocytes (PRH) (RPTEC/TERT1) Cellule Cellule 50 50 Concentrazione Concentrazione 40 40 30 30 comparison 20 20 10 10 0 0 0 1 2 3 4 20 24 0 1 2 3 4 20 24 Tempo Tempo 5 time points 5 time points Cellule Cellule Seeding D13 Daily treatment D0 Last dose First day/first dose) Sample collection and transport according to specific SOP Extration from biological matrix according to specific SOP Quantitative analysis LD= 1/10 di TC 10 Concentrations HD= TC 10 9
Alternative in vitro methods to characterize the role of EAS Human kidney cells (RPTEC/TERT1): CSA LD - HD: 5-15 µM Adsorption to the plastic in this system not relevant LD HD High potential for cells bioaccumulation cells Kinetic of intracellular conc medium and in the medium during 24 hrs very low metabolic competence Wilmes A., et al. J. Proteomics 79, 180-194 (2013) 10
Alternative in vitro methods to characterize the role of EAS Biokinetic model: ⃝ LD ∆ HD CsA (A) CsA supernatant concentration (B)CSA Intracellular concentration Wilmes A., et al. J. Proteomics 79, 180-194 (2013) 11
Alternative in vitro methods to characterize the role of EAS Can kinetics information help in explaining controversial aspects in the area of ED ? In the case of endocrine active substances a strong debate is going on about the ‘ low dose hypothesis ’, according to which “low dose effects”, which are not present at higher doses may display a non-monotonic dose-response (NMDR) . Therefore for those given effect, a simple monotonic extrapolation from high to low doses during risk assessment of those substances is no more valid. Which is the actual definition of low doses? environmentally-relevant doses doses in the range of typical human exposure doses below those used in traditional toxicological studies doses below the presumed NO(A)EL or BMDL derived by testing 12
Alternative in vitro methods to characterize the role of EAS Starting from Paracelsus statement All substances are poisons. It’s the dose that makes the poison the paradigm in toxicology and risk assessment is that the individual response of an organism to a chemical increases/decreases proportionally to the exposure (dose). This gives rise to a monotonic dose-response relationship In monotonic responses the effect either increases or decreases over the full dose range tested. It is generally accepted that for most chemicals (with no genotoxic potential) there is a threshold dose below which there is no adverse effect. 13
Alternative in vitro methods to characterize the role of EAS The possibility exists that non monotonic dose-response relationship occurs (NMDR) with U-shaped or inverted U-shaped profile. The case of ETE is well known. As an example the effects due to copper deficiency are much more severe than the ones due to the its excess. A dose-response curve is non-monotonic when the slope of the curve changes sign somewhere within the range of doses examined. Non monotonicity is not synonymous with low dose , because there are low dose effects that follow monotonic dose-response curves. 14
Alternative in vitro methods to characterize the role of EAS Non-linearities in the toxicokinetics may be the cause of non monotonic dose-response relationship NMDR if the MoA is concentration dependent : two receptors with different actions and different KDs. two enzymes involved in the biotransformation with different affinity (Km) producing different metabolites with different effects. saturation, induction/inhibition of metabolizing enzymes of the unique metabolic pathway High doses: saturation of metabolites formation- 4.0 accumulation of the parent – possible different effect or counteracting 3.0 effects due to metabolites 2.0 Low doses: the effect due 1.0 to metabolites increases with the dose 0.0 0.0 0.5 1.0 1.5 2.0 2.5 2C19 1A2 2B6 15
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