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International Symposium on
Alternative in vitro methods to characterize the role of
Endocrine Active Substances (EASs)
in hormone-targeted tissues
December, 17th 2012
Istituto Superiore di Sanità — ISS
Aula Pocchiari
Rome, Italy
Welcome address
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Dear Colleagues,
it is a great pleasure to welcome you at the CAAT-IPAM-ISS Symposium on “Alternative in vitro methods to characterize the role of Endocrine Active Substances (EASs) in hormone-targeted tissues”, jointly
organised by the Department of Veterinary Public Health and Food Safety and the Department of Environment and Primary Prevention, whose activities include the use of alternative in vitro methods to investigate key issues of the role of EASs in endocrine-regulated targets.
The interest in alternative in vitro methods for toxicity testing has raised in the last years within the scientific community and has been moved forward also by the increasing societal demand on the reduction
of animal use coupled with the requirements in high quality evaluations of chemical safety. In particular,
the processes of screening and prioritization of chemicals may be strongly supported by an integrated
use of alternative (either in silico or in vitro) methods.
To this end, the European Commission recently adopted new legislative tools aimed to improve the use
of alternative methods as the Registration, Evaluation, Authorization and Restriction of Chemicals
(REACH, EC No 1907/2006), the Revised Directive on the Protection of Animals used for Scientific Purposes (EU 63/2010) and the new Cosmetics Regulation (EC No 1223/2009). In all of them, the 3Rs
(Refinement, Reduction, Replacement) principle is emphasized in many articles as part of the new toxicological requirements.
Therefore, we are grateful to the Center for Alternatives to Animal Testing (CAAT) Europe and the Italian Platform on Alternative Methods (IPAM) for their efforts to promote and disseminate alternative
methods and to contribute to the successful organization of this Symposium.
Along with a pleasant stay in Rome, we wish all participants a fruitful exchange of ideas.
Umberto Agrimi, Head of the Department of Veterinary Public Health and Food Safety
Loredana Musmeci, Head of the Department of Environment and Primary Prevention
Aims of the Symposium
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This Symposium is aimed to emphasize the role of alternative methods in search for potential Endocrine
Disrupting Chemicals (EDCs) or Endocrine Active Substances (EASs).
Under the REACH Regulation, EAS identification is one of the main concern since within the definition of
Substances of Very High Concern (SVHC, Article 57 of Regulation (EC) No 1907/2006 ) are included substances which are i) Carcinogenic, Mutagenic or toxic to Reproduction (CMR), ii) Persistent, Bioaccumulative and Toxic (PBT) or very Persistent and very Bioaccumulative (vPvB), and iii) identified, on a caseby-case basis, from scientific evidence as causing probable serious effects to human health or environment
of an equivalent level of concern as those above (e.g. endocrine disrupters).
Nowadays, EASs are challenging classical concepts in toxicology, due to their suggested “low dose effects” and/or “non monotonic dose responses”, leading, for these reasons, innovative approaches in risk
assessment.
The Symposium programme is divided in four sessions aimed to give an overview of the state-of-art in EAS
investigation by alternative methods, highlighting the academic, regulatory and industrial points of view as
well presenting critical issues in human targets of endocrine disrupters and their metabolic fate.
In particular, recent advances in the field of biokinetics as well as in the characterization of new molecular
and cellular biomarkers of different reproductive- and hormone-targeted tissues will be presented by invited international experts.
A general discussion among all participants at the end of the symposium is essential part of the program.
Isabella De Angelis, Department of Environment and Primary Prevention
Stefano Lorenzetti, Department of Veterinary Public Health and Food Safety
Acknowledgements
We acknowledge the Scientific Committee and the Scientific Secretariat for supporting the Symposium organization.
Scientific Committee
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Isabella De Angelis
Istituto Superiore di Sanità - ISS, Rome, Italy
Franca Fassio
MerckSerono, Ivrea, Italia
Simonetta Gemma
Istituto Superiore di Sanità - ISS, Rome, Italy
Stefano Lorenzetti
Istituto Superiore di Sanità - ISS, Rome, Italy
Laura Narciso
Istituto Superiore di Sanità - ISS, Rome, Italy
Costanza Rovida
Center for Alternatives on Animal Testing - CAAT Europe, Konstanz, Germany
Scientific Secretariat
Laura Narciso
Istituto Superiore di Sanità - ISS, Rome, Italy
This event has been supported by unrestricted grants of:
SCIENTIFIC PROGRAMME
INTRODUCTIVE SESSION
Chairpersons: Isabella de Angelis (ISS-Rome) and Stefano Lorenzetti (ISS-Rome)
8.50-9.10 Thomas Hartung (CAAT-Baltimore)
Endocrine disruption as the pilot of mapping the human toxome
9.10-9.30 Costanza Rovida (CAAT-Konstanz)
Implementation of regulatory issues
9.30-9.50 Alberto Mantovani (ISS-Rome)
Endocrine Active Substances / EASs: understanding modes of action for risk assessment
9.50-10.10 Johann Steinkellner (EFSA-Parma)
Exploration of alternative methods for toxicity assessment of pesticide metabolites
10.10-10.30 Serena Cinelli (RTC-Pomezia, Rome)
Improving test methods in the spirit of the 3Rs; the point of view of a contract research
organization
10.30-11.00 Coffee break
SESSION 1: EASs in reproductive-targeted tissues
Chairpersons: Simonetta Gemma (ISS-Rome) and Marcello Spanò (ENEA-Rome)
11.00-11.20 Stefano Lorenzetti (ISS-Rome)
A prostate perspective on male fertility and EASs: from toxicogenomics to phenotypic
anchoring
11.20-11.40 Marcello Spanò (ENEA-Rome)
Human sperm (epi)genetic biomarkers to assess the impact of EASs on male reproductive function
11.40-12.00 Luana Paulesu (Univ. Siena-Siena)
In vitro effects of EASs in human placenta
SESSION 2: EASs in different hormone-targeted tissues
Chairpersons: Alberto Mantovani (ISS-Rome) and Paolo Marzullo (Univ. Novara-Novara)
12.00-12.20 Igor Bendik-Falconnier (DSM-Basel)
Endocrine active nutrients explored in human bone cell cultures
12.20-12.40 Robert A Smith (Univ. Glasgow-Glasgow)
The use of cell models in determining neuronal responses to EASs
12.40-13.00 Arti Ahluwalia (Univ. Pisa-Pisa)
Dynamic in-vitro organ models of metabolism
13.00-13.30 General discussion
13.30-14.30 Lunch
SESSION 3: EASs and kinetics
Chairpersons: Thomas Hartung (CAAT-Baltimore) and Emanuela Testai (ISS-Rome)
14.30-14.50 Emanuela Testai (ISS-Rome)
The role of biokinetics in in vitro tests and in the interpretation of results
14.50-15.10 Frédéric Yves Bois (Univ. Compiegne-Compiegne)
Physiologically-based modeling of ovarian steroid hormones synthesis for EASs' health risk
assessment
15.10-15.30 Daniel R. Dietrich (CAAT-Konstanz)
EASs contra human & environmental health: relevant or playground for merchants of doom?
15.30-16.00 General discussion
INTRODUCTIVE SESSION
Thomas HARTUNG
CAAT, Johns Hopkins University, Baltimore, US & CAAT-Europe, University of Konstanz, Germany
[email protected]
Thomas Hartung, MD PhD, is professor and chair for toxicology, pharmacology, molecular microbiology and immunology at Johns Hopkins Bloomberg
School of Public Health, Baltimore MD, and University of Konstanz, Germany. He directs the Centers of Alternative to Animal Testing (CAAT) of
both universities. From 2002-2008 he led the European Center for the Validation of Alternative Methods (ECVAM) of the European Commission's Joint
Research Centre in Ispra, Italy. He has authored more than 350 articles.
ENDOCRINE DISRUPTION AS THE PILOTE OF MAPPING THE HUMAN TOXOME
The US National Academies / National Research Council report from 2007 "Toxicity Testing in the 21st Century: A vision and a strategy" has created an atmosphere of departure in the US. It suggests moving away from traditional (animal)
testing to modern technologies based on pathways of toxicity. These pathways of toxicity could be modeled in relatively
simple cell tests, which can be run by robots. The goal is to develop a public database for such pathways, the Human
Toxome, to enable scientific collaboration and exchange.
There is a continuously growing awareness about Tox-21c in all stakeholder groups. It was first embraced by scientists
and in the US. Most importantly, the US agencies followed fast on the 2007 NAS/NRC report: the Tox-21 alliance in
2008 (paper in Science first-authored by now NIH head Francis Collins, EPA made it their chemical testing paradigm in
2009, FDA followed most evidently with the Science article by FDA head Margret Hamburg in 2011). Chemical and consumer product industry got engaged, e.g. with the Human Toxicology Project Consortium. In Europe, all this is rather
delayed, with some adaptation of the vocabulary but not necessarily grasping the new approach. This is not alternative
methods under a new name. However, interest is lately increasing strongly in Europe.
Tox-21c suggests moving to a new resolution, i.e. pathways of toxicity. The problem is that the respective science is only
emerging. What will be needed is the Human Toxome as the comprehensive pathway list, an annotation of cell types, species, toxicant classes and hazards to these pathways, an integration of information in systems toxicology approaches, the
in-vitro-in-vivo-extrapolation by reversed dosimetry and finally making sense of the data, most probably in a probabilistic
way. The NIH is funding since September 2011 by a transformative research grant The Human Toxome project led by
CAAT. The project involves US EPA ToxCast, the Hamner Institute, Agilent and several members of the Tox-21c panel.
The new approach is shaped around pro-estrogenic endocrine disruption as a test case.
Early on, the need for quality assurance for the new approaches as a sparring partner for their development and implementation has been noted. Formal validation as developed for the first generation of alternative methods can only partially
serve this purpose. For this reason, the Evidence-based Toxicology Collaboration (EBTC) was created in the US and
Europe in 2011 and 2012, respectively. This collaboration of representatives from agencies, industry, academia and stakeholder groups aims to develop tools of Evidence-based Medicine for toxicology. The secretariat is run by CAAT, the first
conference was held in early 2012 hosted by US EPA and working groups have started to address pertinent issues and
methodologies. All together, Tox-21c and its implementation activities including the Human Toxome and the EBTC
promise a credible approach to revamp regulatory toxicology.
References
Hartung T, van Vliet E, Jaworska J, Bonilla L, Skinner N and Thomas R. Systems Toxicology. ALTEX 2012, 29: 119128.
Hartung T. From alternative methods to a new toxicology. Eur. J. Pharmaceutics Biopharmaceutics, 2011, 77:338–349.
Hartung T and McBride M. Food for thought… on mapping the human toxome. ALTEX 2011, 28, 83-93.
Leist M, Hasiwa M, Daneshian M and Hartung T. Validation and quality control of replacement alternatives – current
status and future challenges. Toxicological Research, DOI: 10.1039/C2TX20011B.
Hartung T. Evidence based-toxicology – the toolbox of validation for the 21st century? ALTEX 2010, 27:241-251.
Hartung T. Toxicology for the twenty-first century. Nature 2009, 460:208-212.
Hartung T. A toxicology for the 21st century: Mapping the road ahead. Toxicol Sci. 2009, 109(1):18-23.
Notes
Costanza ROVIDA
CAAT-Europe, University of Konstanz, Germany
[email protected]
Costanza Rovida is scientific Officer at the Center for Alternatives to Animal Testing in Europe
(CAAT-Europe) and project manager for REACH Mastery. She took a degree
in Chemistry with specialisation in Analytical Chemistry in 1989. After a period focussed on the optimisation of analytical techniques in the area of food
and environmental analyses, she moved to a pharmaceutical Industry where
she learned about method validation plus toxicology and drug efficacy. Convinced that in vivo methods are not the right scientific answer to our toxicological questions, in the period 2005-2008 she worked at ECVAM (European
Centre for Validation of Alternative Methods) where she gained experience of
alternative methods applied in the area of skin and respiratory sensitisation
and she had the opportunity to participate in the working groups organised by
the European Commission for the implementation of REACH, the latest
Regulation on the evaluation and authorisation of chemical substances. She
was one of the pioneer members of CAAT Europe and since 2009 she has strived for the application of
in vitro methods for regulatory purposes. Publications on this topic are many, including a publication
on Nature (Hartung, T. and Rovida, C. Opinion. Chemical regulators have overreached. 2009. Nature,
460). She is engaged in many scientific Committees that are active in the field of alternative methods,
by organizing symposia and workshops to disseminate the 3R strategies. She is also still very much
involved in the REACH process by following clients to be compliant with all the provisions of that
Regulation.
IMPLEMENTATION OF REGULATORY ISSUES
The term “endocrine disrupter” (ED), was introduced in the early 1990s and later defined as (WHO, 2002): “… an exogenous
substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact
organism, or its progeny, or (sub)populations.” Possibility of perturbation of the endocrine system has been considered for a
long time, since both the uterothophic bioassay (OECD TG 440) and the anti-androgenic screening test (OECD TG 441) were
originated in the 30’s.
In the latest 20 years, concern about endocrine disruptors is definitely increasing, and in the European Union there are at the
moment three important Regulation that specifically ask to address this effect. The first one was probably REACH
(Regulation 1907/2006) which includes the endocrine disruptor activity as a condition to include a substance in the authorization list. In addition, both Regulation 1107/2009 for Plant Protection Products and proposed new Regulation for Biocidal
products ask that approved substances should not have endocrine disruptor activity and for the definition both refers to Article
57(f) of REACH, which does not provide a clear definition for getting to a unique and generally approved decision. Regulation 1272/2008 (CLP) adds the endocrine disruptor activity in Annex II regarding the description of the procedure to assign
the classification as reproductive toxicant in the area where “weight of evidence” is described.
Even if endocrine disruptor activity is generally recognized as a serious effect, there is no agreement about the methods that
should be applied for the assessment and when the activity for a substance is universally recognized, as the case for Bisphenol
A, the discussion is still open for the definition of the no effect dose.
One of the problem is that endocrine disruption is not a toxicological endpoint in itself, but rather a class of modes and
mechanisms of action. There are a battery of in vitro tests available that must be used for initial screening, but in the end, confirmation in an intact organism is always required, i.e. in vivo in rats, even though it is well recognized that endocrine activity
is very different among species.
State of the art in this topic has been recently presented in a document issued by an expert panel charged by the European
Commission. Regardless the ambiguous conclusion of this report, there is an urgent need to define a set of methods for this
class of substances. Within the scope of REACH, 18 substances have been now selected due to their concern of being endocrine disruptors. Decision is expected in two years.
Notes
Alberto MANTOVANI
Food and Veterinary Toxicology Unit, Dept of Fodd Safety and Veterinary Public Health, Istituto
Superiore di Sanità , Rome, Italy
[email protected]
Alberto Mantovani (AM, 1956, DVM, MSc) is currently director of the Food
and Veterinary Toxicology Unit at the Italian National Health Institute (ISS).
The main research topics of the Unit are endocrine disrupters (ED), natural bioactive substances, trace elements and nanotoxicology; the research activities
pivot on translating mechanistic research into risk (and risk-to-benefit) assessment. AM co-authored about 50 scientific papers on toxicology and risk assessment in the 2006-12 period: he co-ordinated the pilot national project on
ED (2000-2003),the research area IV (cross-cutting technologies) of the EU
project ReProTect (2005-10) on non-animal testing strategies for reproductive
toxicants and the national PREVIENI project (2008-11) on the development
and use of biomarkers to assess the impact of ED exposure. Since 2003 AM is
an expert of the European Food Safety Authority (EFSA): member of
FEEDAP Panel (substances used in feeds) on 2003-12, member of PPR panel (pesticides) since July
2012 and expert collaborating to EFSA opinions on non-animal testing, bisphenol A and TTCthresholds of toxicological concerns.
ENDOCRINE ACTIVE SUBSTANCES: UNDERSTANDING PATHWAYS FOR RISK ASSESSMENT
Alberto Mantovani, Stefano Lorenzetti, Cinzia La Rocca, Laura Narciso, Sabrina Tait.
Endocrine disrupters (ED, see the dedicated ISS web area, http://www.iss.it/inte) are natural or man-made chemicals in
diet and/or environment able to cause adverse effects in exposed organisms or their progeny by altering endocrine homeostasis. In a broader sense, endocrine active substances (EAS) include all compounds modulating endocrine functions; thus,
EAS may also exert beneficial or protective effects. The so-called “phytoestrogens” are a recognized example of EAS that
may exert beneficial or adverse effects depending on the exposure level, lifestage, sex as well as on the interactions with
other EAS (see the EDID database at http://www.iss.it/inte). The endocrine system is a network regulating most body
functions: the pre- and post-natal lifestages are particularly sensitive, and long-term, persistent effects may result from
early exposures to EAS. From the toxicologist standpoint, rather than an effect per se endocrine disruption is a group of
modes of action highly relevant to risk assessment due to their complexity and serious long-term (even trans-generation)
impact. As a consequence, the assessment of ED (and EAS) would likely progress together with the development of new
testing approaches that exploit system biology and integrate pathways into the classical hazard characterization. Moreover, up to now the toxicology of ED has largely pivoted around the effects on fertility, reproductive development and
thyroid, whereas comparatively limited attention has been paid till now to other relevant aspects, such as the neuroendocrine and endocrine-immune interfaces and especially the impact on metabolic programming, which might represent a
field of considerable public health significance. The development of conceptual frameworks supported by robust scientific
is also needed to deal with long-debated issues in ED/EAS assessment, such as: i) the relevance of low-dose effects and
non-monotonic responses (an EAS eliciting different hits with partly overlapping dose-response relationships ?) or ii)
combined exposures to different EAS (is there something more than dose addition ?). We present two examples of published ISS studies providing mechanistic insights into risk assessment issues, namely, the use of transcriptomics to characterize components of mixtures and the use of clinically relevant biomarkers to characterize EAS potentially interfering
with the same pathways.
Acknowledgement: the abstract has been elaborated within the frame of the Ministry of Health grant “Reach: endocrine
disrupters”
Notes
Johann STEINKELLNER
European Food Safety Authority, EFSA - Pesticides Unit , Parma, Italy
[email protected]
Between 1994 and 2002 I have worked in the Department of Environmental Toxicology of the Institute for Cancer Research of
the University of Vienna in the field of genetic toxicology (e.g.
gene mutation assays with Salmonella typhimurium, chromosomal aberration and micronucleus tests with plant cells, cultivated
hepatic cells in rats and primary human lymphocytes, Comet Assays with a variety of cell types, spectrometric and biochemical
enzyme measurement methods and determination of GST genotyping of humans (e.g. with PCR). I have been teaching in the
field of genetic toxicology at the University of Vienna. I have
obtained a PhD in the field of anticarcinogensis/antimutagenesis
in humans. I hold a master degree in toxicology and am a Eurotox
registered member of the Austrian Society of Toxicology.
From 2002 to 2007 I have worked as a toxicologist in the field of health effects and environmental effects of “Existing Chemicals”, “New Chemicals” and Pesticides (Regulation 67/548/EEC and adaptations thereof, Directive 91/414/EC), Risk Assessment of “Existing Chemicals” (Regulation 793/93/
EEC) and have contributed to the implementation of REACH.
Between 2007 and 2009 I have worked at the European Food Safety Authority (EFSA) as a toxicologist responsible for human health risk assessment in the field of peer review of plant protection products under Directive 91/414/EC.
Since 2009 I am working as a toxicologist in EFSA’s Pesticide Unit in support of EFSAs Panel on
Plant Protection Products and their Residues (PPR). I have been involved, beyond many other activities in support of pesticide risk assessment, for instance in the drafting of scientific opinions/guidance
documents on neurotoxicity of deltamethrin, dermal absorption, exposure assessment for workers, operators, bystanders and residents, cumulative risk assessment of pesticides and the toxicological relevance of pesticide metabolites.
EXPLORATION OF ALTERNATIVE METHODS FOR TOXICITY ASSESSMENT OF PESTICIDE METABOLITES
The framework of evaluation and authorisation of chemical plant protection products and the active substances they contain
in the EU is laid down in Regulation (EC) No 1107/2009 and appertaining regulations. Assessment of the risk for the
consumer requiring also the identification of metabolites and of degradates of the active substances is a major part of this
process.
One of the outcomes of the evaluation of an application for use of an active substance on a crop is the establishment of two
residue definitions, one for monitoring and one for dietary risk assessment. While the residue definition for monitoring has
regulatory purposes for the enforcement of the MRLs (Maximum Residue Levels) and must reflect analytical practicalities,
the residue definition for dietary risk assessment may be wider, as its purpose is to assess consumer safety, and it should
therefore include all metabolites and degradates of toxicological relevance.
However, in practice only the toxicological properties of the active substance are investigated through the range of
toxicological studies while only very limited information about the toxicological properties of metabolites and degradates is
available in the majority of cases. In this context it is notable that subjecting all metabolites to the full testing scheme applied for active substances appears not feasible due to the sheer number of metabolites identified in many cases. In addition
this would lead to a significant increase in the use of test animals in the field of pesticide risk assessment.
Therefore EFSA’s PPR Panel has developed a scientific opinion in which alternative testing methods were explored in regard to their applicability for testing of pesticide metabolites.
The opinion identifies the Threshold of Toxicological Concern (TTC) concept as and appropriate screening tool for assessment of toxicity of metabolites and identified three critical steps in its application which are 1) estimation of the metabolite
level, 2) evaluation of genotoxity alerts and 3) detection of potentially neurotoxic metabolites. A TTC concept for acute
exposures was also established based on pesticide active substances to which an Acute Referenced Dose (ARfD) has been
allocated. Assessment schemes both for chronic and acute dietary risk assessment are presented in the opinion using a combination of the TTC approach with QSAR models/read across and targeted testing.
In addition to proposals for assessment of metabolites the opinion also describes how the proposed assessment tools could
be utilised for assessment of differential toxicity of pesticide isomers.
The results obtained from the work on this opinion will be the basis for the development of a guidance document for toxicity assessment of metabolites and isomers.
Notes
Serena CINELLI
RTC - Research Toxicology Centre, Rome – Italy
[email protected]
Dr. Serena Cinelli is Associate Scientific Director at Research Toxicology Centre (RTC), a Contract Research
Organisation specialised in non-clinical safety studies,
located in Pomezia (Rome), Italy. She has more than 25
years experience in genetic and in vitro toxicology testing
for industry and she is author of numerous toxicology
reports submitted to international regulatory authorities.
Dr. Cinelli also acts as Contract Professor in Environmental Mutagenesis and is author of numerous peer reviewed publications.
IMPROVING TEST METHODS IN THE SPIRIT OF THE 3RS; THE POINT OF VIEW OF A CONTRACT RESEARCH ORGANIZATION
Serena Cinelli, Germano Oberto, Isabella Andreini
A multiple-level tiered approach to identify and characterize the hazards of Endocrine Active Substances (EAS) is suggested
by the most important international organisations which provide guidance for industry for the safety assessment of chemicals. Both OECD and EPA indicate that non-clinical development should include a screening phase in which in silico, in
vitro and in vivo assays are performed to provide mechanistic data for hazard identification, followed by an in vivo testing
phase to better characterize the identified risk.
Based on current evidence, scientists and regulators acknowledge that stand alone in vitro methods are not sufficient to reliably predict in vivo effects due to the complicated nature of hormonal systems. Nevertheless several of the available in vitro
methods can provide extremely important data to clarify in vivo mechanisms of action of EAS and can be used as alternatives to in vivo assays traditionally employed for single endocrine mechanism and effect (e.g. Uterotrophic assay).
In order to obtain an effective reduction of animal use with the application of alternative in vitro methods, it is necessary to
satisfy different steps, such as a successful validation process, inclusion into regulatory requirements, acceptance by industry and wide application in non-clinical Contract Research Organizations (CROs).
The role of CRO is increasingly important in application of alternative methods since the general trend of industry is to contract out most of the non-clinical development. In this respect CRO is the ideal candidate not only to run but also to validate
new alternative methods since understanding the needs of industry and regulatory framework becomes a key factor.
However, one strong hurdle for the CRO is the balance between the immediate investments and the delayed validation project payback. The CRO must keep in mind that industry may have a conservative approach driven by the risk aversion: fear
that results from alternative in vitro approaches might be not readily accepted by regulatory authorities as the ones from in
vivo conventional studies.
Different scenarios will be presented by the author with possible situations and proposed solutions to manage safety assessment programs, aware that a complete fulfilment of the 3Rs philosophy is not possible, but a wise strategy of in vitro testing
selection and expert data interpretation can help to reduce and refine animal testing.
Notes
Notes
SESSION 1:
EASs IN REPRODUCTIVE TARGET TISSUES
Stefano LORENZETTI
Food and Veterinary Toxicology Unit, Dept of Fodd Safety and Veterinary Public Health, Istituto
Superiore di Sanità, Rome, Italy
[email protected]
Stefano Lorenzetti, graduated in Biological Sciences (1991) and in Human
Nutrition Sciences (2003), is employed since 2006 at the Dpt. of Food
Safety and Veterinary Public Health of the Italian National Health Institute
- ISS (Istituto Superiore di Sanità) in Rome. He has been lecturer (200609) in Molecular Biology and Toxicology at the Faculty of Medicine of
University Tor Vergata of Rome at the post-graduated course of Sciences
of Nutrition. Since January 2012, he has been appointed as a member of
the Italian national expert group of “Alternative methods to animal experimentation”.
He participated to different international project on the hazard and risk
characterization of environmental and dietary contaminants such as the EU
Integrated Project ReProTect (“Development of a novel approach in hazard and risk assessment or reproductive toxicity by a combination and application of in vitro, tissue and sensor technologies”) to develop
an integrated in vitro approach linking toxicogenomics to clinical biomarkers (phenotypic anchoring).
His research is focused on the set up of in vitro alternative methods to animal experimentation to study
the role of endocrine disruptors/EDCs or Endocrine Active Substances/EASs such as bioactive compounds of plant origin (e.g. polyphenols) and environmental and dietary contaminants (e.g., plasticizers,
pesticides and biocides).
A PROSTATE PERSPECTIVE ON MALE FERTILITY AND
COGENOMICS TO PHENOTYPIC ANCHORING
Endocrine Active Substances: FROM TOXI-
Stefano Lorenzetti
Although prostate function is critical for male fertility, in reproductive toxicology it is still an overlooked target (1). Indeed, LNCaP cell line may represent an alternative in vitro method of the human prostate epithelium to screen bioactive chemicals affecting male fertility (1-3): within the EU Integrated
Project ReProTect, LNCaP cell line has been used as a cellular model to investigate androgen receptor
(AR)-dependent signaling to perform toxicogenomic studies of (anti)androgen-like chemicals (1-5) and
a cell-based bioassay was employed to provide a phenotypic anchoring to gene expression profiling data (4). The selected cell-based, cell-specific, clinically used biomarker of effect has been the ProstateSpecific Antigen (PSA) monitored as secreted protein. Besides to be a supportive tool for the toxicogenomic approach, the PSA secretion assay has been thus implemented as an independent tool to investigate prostate-mediated effects on male reproduction (4).
References.
1. Lorenzetti S, Narciso L, Marcoccia D, Altieri I. 2012. J Biol Res, 1(LXXXIV):36-41.
2. Lorenzetti S, Altieri I, Arabi S, Balduzzi D, Bechi N, Cordelli E, Galli C, Ietta F, Modina SC, Narciso L, Pacchierotti F,
Villani P, Galli A, Lazzari G, Luciano AM, Paulesu L, Spanò M, Mantovani A. 2011. Annals Ist Super Sanità, 47(4):429444.
3. Lorenzetti S, Marcoccia D, Narciso L, Mantovani A. 2010. Reprod Toxicol, 30(1):25-35.
4. Lorenzetti S, Lagatta V, Marcoccia D, Aureli F, Cubadda F, Aricò E, Canini I, Castiello L, Parlato S, Gabriele L, Maranghi F, Mantovani A. 2008. Toxicol Lett 180S(S1):S123-S124.
5. Lorenzetti S, Narciso L. 2012. In: Computational approaches to nuclear receptors, edited by Pietro Cozzini and Glen E.
Kellogg, Drug Discovery series of Royal Society of Chemistry, Cambridge (UK). DOI: 10.1039/9781849735353.
Notes
Marcello SPANO’
Laboratory of Toxicology, ENEA Casaccia, Rome, Italy
[email protected]
Marcello Spanò (MS) is senior scientist at the Toxicology
lab, Unit of Radiobiology and Human Health, ENEA
(Italian National agency for New technologies, Energy and
sustainable economic development). His main interest is
represented by the study of sperm DNA/chromatin (epi)
genetic integrity after environmental exposures (endocrine
interferers, ionizing and not ionizing electromagnetic radiation). MS has more than 25 year experience in the field of
automated cytology development, use and application of
methods to detect damage in mammalian germ cells induced
by radiation and chemicals, also exploring the impact of
sperm DNA damage on human reproductive capabilities.
MS has been involved in EU projects on reproductive toxicology since 1991 and co-authored more than 130 peer review papers and book chapters.
HUMAN SPERM (EPI)GENETIC BIOMARKERS TO ASSESS THE IMPACT OF ENDOCRINE ACTIVE SUBSTANCES ON MALE REPRODUCTIVE FUNCTION
Marcello Spanò, Eugenia Cordelli and Francesca Pacchierotti
Infertility is a common disorder affecting about 15% of all couples trying to conceive and subfertility has become a relevant
growing problem in affluent countries. Temporal and spatial trends toward decreasing semen quality, increasing rates of reproductive tract abnormalities, and a well documented worldwide testicular cancer incidence increase pointed to possible environmental causes of male reproductive system impairment. Even if it is difficult to disentangle the responsibility of endocrine
stressors from other lifestyle factors potentially impairing human fertility, it is believed that a high level of environmental endocrine active substances (EASs), especially during fetal life, may disturb the hormone control of male urogenital tract organogenesis with lifelong consequences. From a toxicological point of view spermatogenesis is expected to be vulnerable to reproductive toxicants because of the large number of cell divisions and cell diffentiation processes continuously occurring throughout the whole reproductive life. In addition, the final stages of gamete differentiation in male mammals are sensitive targets of
DNA-reactive chemicals because they are repair deficient. Human biomonitoring studies of South African and Mexican populations living in endemic malarias areas have consistently indicated impaired semen quality associated with high levels of environmental DDT-exposure. There are limited but sound epidemiological data indicating adverse effects of EASs on human
sperm DNA. Low-level exposure to PCB-congeners apparently interferes with sperm chromatin integrity (and sperm motility
as well) in humans – findings that are consistent with experimental studies. The need to establish alternative methods modelling mammalian spermatogenesis is an issue of high priority. In this context, direct in vitro assays on spermatozoa addressing
motility and genetic damage are relevant since they can detect effects on terminally differentiated male gametes. It has recently
been proposed that EASs exposure can affect sperm chromatin integrity by a mechanism involving epigenetic changes to the
paternal genome. Rodents exposed in-utero to the fungicide vinclozolin or the insecticide methoxychlor showed abnormal
DNA methylation patterns in spermatozoa and impaired male fertility. Once established, these epigenetic changes may be permanent and thus paternally passed to subsequent generations. Following human population studies showing an association between EASs exposures and altered global methylation levels in blood lymphocytes, biomonitoring studies are in progress to
determine sperm DNA methylation patterns in relation to EASs exposures. It is probable that in the near future, reproductive
risk assessment from EASs exposure could benefit from more affordable high-throughput technologies providing a more complete evaluation of the possible genetic and epigenetic effects.
Notes
Luana PAULESU
Department of Physiology, University of Siena, Siena, Italy
[email protected]
Luana Ricci married Paulesu is Professor of Physiology and the Director of
the Center “Biology of Reproduction” at the University of Siena (Italy). She
carried out research on human placenta mainly focusing on the secretion and
action of cytokines. She also carried out studies on animal species with different types of placenta. In the last few years she has been a partner of ReProTect, an Integrated project Supported by the European Sixth framework,
studying the effect of estrogen-like compounds in in vitro models of human
placenta. She is author or co-author of 90 full-papers published in International Journals and of numerous chapters in scientific volumes. She is the Editor of the book “Signal molecules in animal and human gestation” Research
Signpost, Trivandum, Kerala, India. She has attended numerous congresses,
also as an invited speaker.
Bechi N, Ietta F, Romagnoli R, Focardi S, Corsi I, Buffi C, Paulesu L. Toxicol Sci. 2006;93(1):75-81.
Ietta F, Wu Y, Romagnoli R, Soleymanlou N, Orsini B, Zamudio S, Paulesu L, Caniggia I. Am J
Physiol Endocrinol Metab. 2007 Jan;292(1):E272-80.
Bremer S, Brittebo E, Dencker L, Knudsen LE, Mathisien L, Olovsson M, Pazos P, Pellizzer C, Paulesu LR, Schaefer W, Schwarz M, Staud F, Stavreus-Evers A, Vähänkangas K. Altern Lab Anim.
2007;35(4):421-39.
Ferro EA, Mineo JR, Ietta F, Bechi N, Romagnoli R, Silva DA, Sorda G, Bevilacqua E, Paulesu LR.
2008;172(1):50-8.
Ietta F, Bechi N, Romagnoli R, Bhattacharjee J, Realacci M, Di Vito M, Ferretti C, Paulesu L. Am J
Physiol Endocrinol Metab. 2010; 298 (3):E411-8.
Bechi N, Ietta F, Romagnoli R, Jantra S, Cencini M, Galassi G, Serchi T, Corsi I, Focardi S, Paulesu L.
Environ Health Perspect. 2010;118(3):427-31.
Mørck TJ, Sorda G, Bechi N, Rasmussen BS, Nielsen JB, Ietta F, Rytting E, Mathiesen L, Paulesu L,
Knudsen LE. Reprod Toxicol. 2010; 30(1):131-7.
Bhattacharjee J, Ietta F, Giacomello E, Bechi N, Romagnoli R, Fava A, Paulesu L. Placenta. 2010; 31
(5):423-30.
Paulesu L, Bhattacharjee J, Bechi N, Romagnoli R, Jantra S, Ietta F. Curr Pharm Des. 2010;16
(32):3601-15. Review.
de Oliveira Gomes A, de Oliveira Silva DA, Silva NM, de Freitas Barbosa B, Franco PS, Angeloni
MB, Fermino ML, Roque-Barreira MC, Bechi N, Paulesu LR, Dos Santos MC, Mineo JR, Ferro EA.
IN VITRO EFFECT OF EASs IN HUMAN PLACENTA
Luana Paulesu, Nicoletta Bechi, Roberta Romagnoli, Francesca Ietta
Endocrine disrupter chemicals (EDCs) are environmental pollutants of agricultural or industrial origin which may influence
human reproductive health. These compounds are able to interfere with the delicate balance of the endocrine system by mimicking the action of the steroid hormones. They can also be transferred from the mother to the embryo and cause reproductive and developmental toxicity. We investigated the effect of selected EDCs on in vitro models of human placenta. In particular we examined para-nonylphenol (p-NP) and Bisphenol A (BPA). The choriocarcinoma BeWo cell line and the HTR-8/
Sv-neo cells were used to identify chemical lethal concentration able to reduce 50% of cell viability (cell toxicity) and the
chorionic villous explants from fresh human placenta were used to perform functional studies of chemicals at non-toxic but
environmentally relevant concentrations. Vehicle-treated cultures were used as negative controls.
We found toxicity of the chemicals tested at concentrations in the order of µM. Lower non-toxic concentrations, in the order
of nM, were able to interfere with the hormone (β-human chorionic gonadotropin, β-hCG) secretion as well as inducing trophoblast differentiation and apoptosis.
These results raise concern about maternal exposure to environmental factors and the potential involvement of these chemicals in pregnancy disorders.
Notes
Notes
SESSION 2:
EASs IN DIFFERENT HORMONE TARGET TISSUES
Igor BENDIK-FALCONNIER
Igor Bendik-Falconnier, DSM Nutritional Products Ltd, NIC-RD-HN, CH-4002 Basel, Switzerland
[email protected]
Igor Bendik-Falconnier obtained his Bio II diploma in molecular genetics
and his Ph.D. in cell biology at the University of Basel, Switzerland. Subsequently he spent his post-doc and associate professor years at the Burnham
Institute, La Jolla and the Sidney Kimmel Cancer Center, La Jolla, California.
Back in Switzerland he was a deputy group leader at the department of research (ZLF) at the medical faculty of Basel, where he was awarded several
grants to explore cancer formation. In 1998, he started his career in industry
at Hoffmann-La Roche Ltd, later in Roche Vitamins, Basel, and explored the
efficacy of natural health–beneficial food ingredients. In the VHF department, he led the bone biology group. During his career, he has successfully
trained and supervised a number of diploma and Ph.D. students. Since the merger with DSM in October
2003, he has continued in his role as a senior scientist to support and lead different teams in nutritional
research with the focus on vitamins and nutraceuticals. During his industrial commitment he has led
different research teams and was responsible for different cellular and molecular research technologies.
Currently, he is heading the biostatistics and bioinformatics group at DSM Nutritional Products, Ltd.
ENDOCRINE ACTIVE NUTRIENTS EXPLORED IN HUMAN BONE CELL CULTURES
Many natural food-borne compound classes interact with the human body in an endocrine hormone-like manner. These nutrients fulfil important physiological roles mediated through healthy nutrition. An example is genistein, the major soy isoflavone, which is well known to be active in bone tissue, a common target for endocrine hormones. DSM has explored the
function of genistein in bone cell biology using available omics and primary cell culture technologies. DSM has develop
geniVida™, the nature-identical aglycone, as a product for postmenopausal bone health in the field of nutrition. This presentation will cover the use of cell culture technology for the characterization of an "endocrine active nutrient" in a given biological concept and the strategy to develop it as a nutraceutical for postmenopausal bone health. Besides efficacy, also the
industrial toolbox for testing of endocrine disruption properties and the regulatory framework will be discussed.
Notes
Robert A. SMITH
School of Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK.
[email protected]
Professor of Cellular Neuroanatomy (Personal Chair awarded January, 2007)
Honorary Senior Research Fellow (September 2011)
Degrees Held:
Master of Arts (1974): University of Oxford (Zoology Honours)
Master of Letters (2010): University of Glasgow
Doctor of Philosophy. (1978): University of Southampton (Cell
Biology)
Previous Posts: Postgraduate MRC studentship awarded by the MRC Toxicology
Unit, Carshalton; Postdoctoral Research Assistant, Southampton University ; Lecturer & Senior Lecturer
in Anatomy.
M a j o r Inte rna tio na l C o mmi t me nt s:
W HO T e mp o rar y Ad vi se r : Ge n e va, 1 9 8 9 . M e mb er o f Co n s ul ta tio n o n " In vi tro t ec h niq u e s fo r a s se ss me n t o f n e uro to x icc h e m ica ls" .
EuroTox 1990: Leipzig, Invited Speaker and Chairman of Symposium of In Vitro Neurotoxicology.
FRAME: London, 1990, Discussant of 2nd Working Party Report on Neurotoxicology.
INVITOX 92, 1992, De Haan, Invited Speaker.
ECVAM: Ispra. 1994, Member of In Vitro Neurotoxicology Working Party.
IPCS Consultation on "In vitro neurotoxicology": Research Triangle Park, USA, 1995
German Clinical Pharmacology & Experimental Toxicology Meeting 1996, Dresden. Plenary lecture
NIH, Bethesda, USA, September 1997. Invited Seminar.
Advisory Editorial Board of the International Review of Cell & Molecular Biology, 1998-present
University of South Bohemia, Czech Republic, 2000. Invited Seminar
NRC Laboratories, Ottawa, Canada, 2002, 2005, 2006. Visiting Scientist (funded by British Council.
EuroTox 2009: Dresden, Invited Speaker
Approximately 70 peer reviewed publications in cell biology, neurobiology, toxicology, gonadal differentiation and human
anatomy.
Major Reviews: SMITH, R.A, Ord, MJ (1983): Mitochondrial form and function relationships in vivo: Their potential in toxicology and pathology. Int. Rev.Cytol. 83, 63-134; SMITH, RA, Jiang, Z-G (1994): Neuronal modulation and plasticity in vitro. Int. Rev.Cytol. 153, 233-296; Fatokun, AA, Stone, TW, SMITH, RA (2008): Oxidative stress in neurodegeneration and
available means of protection. Frontiers in Bioscience 13, 3288-3311; Hou, ST, Jiang, SX, SMITH, RA (2008). Permissive
and repulsive cues and signalling pathways of axonal outgrowth and regeneration. Int. Rev. Cell Mol. Biol. 267, 125-181;
SMITH, RA (2009): Twenty-first century challenges for In Vitro Neurotoxicity. ATLA 37, 367-375; Roberts, RA, SMITH,
RA, Safe, S, Szabo, C, Tjalkens, RB, Robertson, FM (2010): Toxicological and patho-physiological roles of reactive oxygen
and nitrogen species. Toxicology 276, 85-94
DO CELL MODELS OFFER REALISTIC ALTERNATIVES FOR DETERMINING POTENTIAL NEURONAL RESPONSES TO ENDOCRINE ACTIVE SUBSTANCES?
The complexity of the human nervous system, comprised of a multitude of interacting neural and non-neuronal cell populations, not to mention the plasticity changes occurring during its development and throughout adult life, presents a difficult task
when attempting to determine perturbation following potential toxic insults. This is particularly so when employing in vitro
approaches. Emphasis has focussed on monitoring a number of neuronal-specific endpoints rather than merely reporting general cytotoxic responses leading to cell death, for example quantifying changes in neural and glial specific marker proteins,
including receptor expression, analysing effects on neurite outgrowth, functional competency and synaptogenesis. Primary neurons, mainly from rodent species, have been successfully cultured from numerous regions of the brain, spinal cord and peripheral systems, and have been widely applied to toxicological studies for many years (Smith, 2009). A number of transformed
neural lines, including those established from human tumours such as SH-SY5Y cells, have also been routinely used both in
immature and differentiated states with promising outcomes in screening programmes. During the last decade exciting developments in molecular biology have resulted in the generation of human embryonic neural stem cell lines, derived from umbilical
blood (Buzanska et al., 2010), and recently induced pluripotent stem cell (transduced from adult dermal fibroblasts) have become available (Kumar et al., 2012). Although issues of variablility and high costs may limit their current usefulness, such
technologies have potential as future in vitro model systems for toxicological assessments. The pros and cons of utilising these
in characterising the role of endocrine active substances relevant to neural tissues will be discussed.
References: Buzanska, L, Zychowicz, M, Ceriotti, L, Coecke, S, Rauscher, H, Sobanski, T, Whelan, M, Domanska, Janik, K, Colpo, P,
Rossi, F, (2010):Toxicology 270, 35-42; Kumar, KK, Aboud, AA, Bowman, AB, (2012): Neurotoxicology 33, 518-529; Smith, RA, (2009):
ATLA 37, 367-375
Notes
Arti AHLUWALIA
Interdepartmental Research Center "E.Piaggio", University of Pisa, Italy
[email protected]
Associate Professor of Bioengineering at the Department of Chemical Engineering, Faculty of Engineering, University of Pisa, and affiliated with the Interdepartmental Research Center “E. Piaggio”, where she is Vice Director and head
of
the
MCB
Group
(www.dionisio.centropiaggio.unipi.it
and
www.centropiaggio.unipi.it). Prof Ahluwalia is also an associate of the National
Council of Research Institute of Clinical Physiology (CNR-IFC), and head the
NanoBioscopy Lab at the Institute.
Although highly multidisciplinary in nature, her research has centered on the
interaction between biological systems and man-made devices or structures focused towards the creation of organ and system models in-vitro. She has coordinated and participated
in projects on tissue engineering, biosensing, bioreactors, neural cell culture, cardiovascular engineering, nanotoxicology and robots for autism. She has several papers published in international scientific
journals (over 90) and is author of 13 patents on microfabrication, and on microfabricated multicompartmental bioreactors. A total of 4 patents have been industrialized to date.
DYNAMIC IN-VITRO ORGAN MODELS OF METABOLISM
Unraveling the complexity of inter-organ and inter-tissue cross talk in vivo is a complex and challenging task. Intelligent in
vitro models able to recapitulate the physiological interactions between tissues in the body connected by the bloodstream have
enormous potential as they enable detailed studies on specific two-way or higher order organ-organ and tissue interactions.
These models are the first step towards building an integrated picture of systemic signaling in physiological or pathological
conditions. Using a scaled down fluidic system, we have developed in vitro models of endogenous metabolism and toxicity
through ingestion composed of 3-tissue connected cultures. The metabolic model represents the central abdominal region and
is composed of hepatocytes, endothelial cells and visceral adipose tissue. Closely paralleling in vivo nutrient balance, in normoglycemic media the model is able to maintain homeostatic glucose and lipid equilibrium. Moreover, it expresses systemic inflammation as well as endothelial specific stress in the presence of hyperglycemic and hypoinsulinemic conditions. A similar
model of nanomaterial ingestion, developed within the "InLiveTox" project, is composed of an epithelial barrier and downstream endothelial cells and hepatocytes. Exposure of the epithelial barrier causes downstream inflammation and stress even in
the absence of nanoparticles, indicating that the model can be used to study multiple pathways and systemic responses to metabolites or drugs.
Notes
Notes
SESSION 3:
EASs AND KINETICS
Emanuela Testai
Department of Environment and Primary Prevention - Istituto Superiore di Sanità – Rome, Italy
[email protected]
The expertise of dr. Testai is focused on mammalian toxicology, toxicokinetics and risk assessment. She has been involved not only in research but also
in regulatory activities in the area of risk assessment for human health associated to exposure to natural and synthetic chemicals with different field of application, as outlined below.
Education: Emanuela Testai received her degree in Biological Sciences from the University
of Pisa (Italy) in 1981. Her experimental training was carried out at the National Research
Council - Institute of Mutagenesis and Differentiation in Pisa (1979-1981). Her post-doctoral
training was completed at the Istituto Superiore di Sanità (National Institute for Health)
Rome (1982-1984).
Professional experience: Current Position Senior Scientist at Istituto Superiore di Sanità Rome, Italy- Department of Environment and Primary Prevention (since 2007). Head of the Mechanisms of Toxicity
Unit (since 2009). Young Researcher (1985-1994) and Researcher (1994-2007) at Istituto Superiore di Sanità
(Comparative Toxicology and Ecotoxicology Laboratory-Biochemical Toxicology Unit then Dept. Environment and
Primary Prevention). Between 2001-2004, responsible for the Biochemical Toxicology Unit.
Member of Scientific Committees within the European Community (Scientific Committee for Health and Environmental
Risks –SCHER since 2004), of WG of the European Food Safety Authority (EFSA), of National Committees for Plant Protection Products and for Biocides, National Inspector for Good Laboratory Practise, expert within the OECD Test Guidelines Program, consultant for the Italian Ministry of Health.
Scientific Leader of National/International Research Projects on xenobiotics metabolism and toxicity, granted by the Italian
Ministry of Health, the National Research Council, NATO, EU.Research Interests Molecular mechanisms of toxicity; biotransformation, toxicokinetics and toxicity of xenobiotics (environmental pollutants, pesticides, natural toxins); enzymology
of drug-metabolizing systems in hepatic and extrahepatic tissues; metabolic biomarkers of individual susceptibility to toxic
compounds; application of mechanistic and toxicokinetic data to risk assessment; molecular epidemiology (genotyping of
populations to identify metabolic biomarkers of individual susceptibility to toxic compounds); risk assessment associated to
exposure to cyanotoxins and study of cyanobacterial communities. Reviewer activity for: Drug Metabolism and Disposition,
Life Science, Toxicology, Environmental Toxicology and Pharmacology, Environmental Toxicology, Environmental Science and Technology, Toxicology in Vitro, Journal of Biochemical and Molecular Toxicology,Pharmacological Research,
Chemico-Biological Interactions, Reproductive Toxicology, Science of Total Environment, Food and Chemical Toxicology,
Analytica Chimica Acta; Annali ISS, Water Research, Critical Review in Toxicology, Archives of Toxicology.
Scientific Publications : Peer-reviewed international journals and book chapters 87; Other publications: 38; Congress
Communications:140
THE ROLE OF BIOKINETICS IN IN VITRO TESTS AND IN THE INTERPRETATION OF RESULTS
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. Indeed, following exposure to a chemical, its uptake, bioavailability and
biotransformation determine the actual in vivo target dose which is one of the most relevant parameter in quantitative risk assessment. A typical example is the oral pharmacokinetics for bisphenol A (BPA), showing <1% of total BPA in the unconjugated and biologically active form in serum from experimental animals and humans at peak levels, due to a relevant presystemic detoxication, when compared with the higher values attained after parenteral administration.
Despite the above consideration is even more relevant for alternative/non animal testing strategy, in vitro biokinetics is generally neglected: it is a matter of fact that the nominal applied concentration is generally associated to observed effects, rather
than the actual level of cell exposure. This causes a high level of uncertainty in the in vitro concentration-effect relationship, it
has been considered one of the major causes for in vitro/in vivo differences and make it difficult to translate an in vitro concentration into an in vivo dose (in vitro–in vivo extrapolation). The actual intracellular concentration may be affected both by
abiotic processes (i.e. interactions with medium/plate, chemical instability) or by interaction with cells (i.e. transport across the
membranes, biotransformation to reactive/inactive metabolites, bioaccumulation) altering in vitro bioavailability after acute
and even more after repeated treatments. Physiologically based toxicokinetic (PBTK) models can be developed for the integration of dynamic and kinetic data produced from in vitro methods into a biologically meaningful framework for the extrapolation to in vivo conditions. This approach could make possible to derive a NOEC in in vitro experimental models from which
extrapolate the corresponding in vivo dose. Examples will be discussed, evidencing the impact of kinetic behaviour on the effects of chemicals including endocrine active substances.
The work has been partially supported by the FP7-EU funded Project PredictIV Grant n° 202222
Notes
Frédéric Y. BOIS
Chair of Mathematical Modeling for Systems Toxicology, Bioegineering Department, Université de Technologie de Compiègne, Royallieu Research Center, Compiegne, France.
[email protected]
Frédéric Y. Bois is an internationally known expert in quantitative toxicology. He was
trained and worked at Harvard University, UCSF, UC Berkeley, the Lawrence Berkeley Laboratory, INSERM and INERIS. He is currently Professor at the Compiegne
Technology University and Research Director at the Institut National de l’Environnement Industriel et des Risques (INERIS). He has coordinated and participated to international research projects in the areas of statistics and mathematical modelling for
pharmacology, toxicology, epidemiology, and health risk assessment (funders: USFDA, US-NIH, US-EPA, US-OSHA, European Commission, INSERM, French Ministry of Research). Recipient of the American Statistical Association "Outstanding Statistical Application
Award" and of the French Epidaure Prize for Environmental Health Research.
EDUCATION: 1993: Habilitation, Université de Nancy, France.1988: Doctorat ès Sciences, Université de Metz, France.1981: Doctorat de
Pharmacie, Université de Nancy, France.
POSITIONS HELD: 2009-:Professor, Chair of Mathematical Modelling for Systems Toxicology, UTC and INERIS, France.1999-09: Research Scientist, Head of the Toxicology unit, then Scientific Officer, INERIS, France. 1991-99: Staff Scientist, Lawrence Berkeley Laboratory, Berkeley, USA, and INSERM, U444, Paris. 1987-90: Post-Doctoral Researcher Biologist, UC San Francisco and UC Berkeley, USA.
1986-87: Research Associate, Energy and Environmental Policy Center, Harvard University, USA.
SCIENTIFIC COMMUNICATIONS
Cheng S., Bois F., Environmental Health Perspectives, in press.
Adler S., Bois F., et al., 2011, 2010, Archives of Toxicology, 85:367-485.
Bois F., Jamei M., Clewell H.J., 2010, Toxicology, 278:256-267.
Bois F., 2010, Basic and Clinical Pharmacology and Toxicology, 106 :154-161, doi: 10.1111/j.1742-7843.2009.00488.x.
Bois F., 2009, GNU MCSim: Bioinformatics, 25:1453-1454, doi: 10.1093/bioinformatics/btp162.
Micallef S., Bois F., et al. 2007,Clinical Pharmacokinetics, 46:59-74.
Jonsson F, Jonsson E.N., Bois F., Marshall S., 2007, Journal of Biopharmaceutical Statistics, 17:65-92.
Amzal B., Bois F., et al. 2006, Journal of the American Statistical Association, 101:773-785.
Mezzetti M., Bois F., et al. 2003, Journal of the Royal Statistical Society, Series C, 52:291-305.
Bernillon P., Bois F., 2000, Environmental Health Perspectives, 108(suppl.5):883-893.
Bois F., Maszle D., 1997, Journal of Statistical Software, 2(9):http://www.stat.ucla.edu/journals/jss/v02/i09.
Gelman A., Bois F., Jiang J., 1996, Journal of the American Statistical Association, 91:1400-1412.
Bois F., Compton-Quintana P., 1992, Journal of Theoretical Biology, 159:361-375.
PHYSIOLOGICALLY-BASED MODELING OF OVARIAN STEROID HORMONES SYNTHESIS FOR ENDOCRINE ACTIVE SUBSTANCES' HEALTH RISK ASSESSMENT
Frédéric Y. Bois and Nadia Quignot
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 active substances (EASs). The effects of EASs on steroidogenic enzymes may
influence steroid output and thus lead to reproductive toxicity. We first review the most significant quantitative modeling efforts
aimed at predicting the EASs effects. Those examples clearly show that a tight coupling of systems toxicology with quantitative
in vitro to in vivo extrapolation through physiological pharmacokinetic modeling is required for improving predictive capacity.
Our most recent work is an illustration of that approach: to predict hormonal balance disruption on the basis of data on aromatase activity and mRNA levels modulation obtained in vitro on granulosa cells, we developed a mathematical model for the last
gonadal steps of the sex steroids 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 the ovarian estradiol concentrations in adult female rats
exposed for 6 hours to the parent compound and the metabolites of atrazine, bisphenol A, methoxychlor, and vinclozolin. Results are presented and discussed in the framework of the next generation of risk assessment for EASs.
Notes
Daniel R. DIETRICH
Human and Environmental Toxicology, University of Konstanz, Konstanz, Germany
[email protected]
Date of birth
July 27, 1959
Citizen of
Zurich, Switzerland
Profession
Registered Toxicologist (Dr. nat. sci., ETH), DGPT, SSTP, Eurotox, ATS. Aug.
2005 – Nov. 2006
adjunct judge, Court of Mediation and Appellation at the Swiss Supreme
Court, Bern, Switzerland (www.reko.admin.ch)
Jan 2002 Professor of Toxicology, Head of Human and Environmental Toxicology, University of Konstanz, Germany. Consultant toxicologist for SwissMedic (http://www.swissmedic.ch)
Jan. 1996 -2002 Professor of Toxicology, Head of Environmental Toxicology, University of Konstanz, Germany. Adjunct Professor, Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, USA. Adjunct Associate Professor, Swiss
Federal Institute of Technology (ETH), Zurich, Switzerland
May 1995 – Dec. 1995
Visiting Associate Professor, University of Pittsburgh, Department of
Environmental and Occupational Health and Toxicology, University of Pittsburgh, USA
April 1989 – July 1991
Post-doctoral fellow. University of North Carolina and Glaxo Research Laboratories, Research Tri
angle Park, North Carolina, USA.
Nov. 1985 – Oct. 1988
Ph.D. – Thesis Institute of Toxicology, Zurich, Switzerland.
Registered Board Member: Swiss Board of Toxicologists (SSPT), German Board of Toxicologists (GSPT), EUROTOX
Regitered Toxicologist, Fellow of the Academy of Toxicological Sciences (ATS)
Memberships: Academy of Toxicological Sciences (ATS), Society of Environmental Toxicology and Chemistry (SETAC),
Society of Toxicology (SOT), Swiss Society of Pharmacology and Toxicology (SGPT), German Society of Pharmacology and
Toxicology (DGPT), Society of Toxinology, Society for General and Applied Microbiology (VAAM), Federation of European
Microbiological Societies (FEMS), European Center for Ecotoxicology and Toxicology of Chemicals (ECETOC).
Publications: Peer-Reviewed: 120; Books and Bookchapters: 13; Book reviews, Reports and Laymen Communications: 14;
Abstracts of presentations at intl. meetings: >214; Invited lectures: >122.
Science Advisory Panels: (5 of 19). European Union, Institute of Consumer Health and Safety, ECVAM, JRC, Ispra, I (http://
www.jrc.cec.eu.int/default.asp@sidsz=who_we_are.htm), Chair of the Endocrine Disruption Task Force (ED-TF) “In-Vitro
Technologies for the Detection of Endocrine Effects”. Endocrine Disruption and Ecotoxicology Expert of the EDTAVMG non-animal, OECD (http://www.oecd.org/env/ehs), Paris, France, Co-Chair EDTA-VMG non-animal OECD April
2007 – December 2010. Expert, Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR) for the
European Commission DG-SANCO, Bruxelles, Belgium. Chair of the expert peer-review panel of US-EPA's draft Toxicological Reviews of Cyanobacterial Toxins: Anatoxin-a, Cylindrospermopsin, and Microcystins LR, RR, YR, and LA. January 10,
2007, Peer review meeting in Cincinnati, OH, USA. Expert IARC (WHO International Agency for Research on Cancer;
www.iarc.fr): Member of the Scientist team to develop IARC Monograph, Volume 94 'Ingested Nitrates and Nitrites, and
the Blue-green Algae Toxins Microcystin-LR and Nodularin' Lyon, 14-21 June 2006.
Editorships: Chemical Biological Interactions Editor-in-Chief (9/2012-); Toxicology in Vitro; Editor Asia and Asia-Pacific
(3/2004-8/2012), Reviews Editor ( 8/2003-8/2012).
EAS’S CONTRA HUMAN & ENVIRONMENTAL HEALTH: RELEVANT OR PLAYGROUND FOR MERCHANTS
OF DOOM?
Endocrine Active Substances (EAS) have been made responsible for increased incidences of nearly all human diseases imaginable and especially for the demise of populations of certain species. Moreover, unfounded suggestions such as the “low-dose
hypothesis” or “no-threshold hypothesis” for adverse effects have provided for a media and research hype that created an atmosphere of biased and emotional science that has all the ingredients that current “merchants of doom” in science need to thrive
without having to prove any of the hypotheses or suggestions put forth. The latter is exactly contrary to what is needed, namely
a through assessment of facts in a bigger context, a factual and critical assessment of what mechanistic toxicology, hazard characterization and risk assessment can achieve to properly estimate the dangers of EAS. Moreover, this assessment needs to be
compared to available epidemiological data for each of the diseases within the context of verified EAS exposure to ensure that
predictions from in vitro and in vivo mechanistic assessments are realistic and target the population(s) at highest risk. While the
environmental impact of EAS appears to restricted to locations of highest contamination, e.g. sewage treatment plant effluents
and thus can be remediated by technical improvement of sewage treatment, more generalized adverse effects of EAS in wildlife
has not been convincingly documented to allow causal relationships between presumed exposure and population effects. Similarly, although in vitro assays would predict a potential for EAS to interact with human endocrine receptors and associated signal transduction pathways and some in vivo experiments purport the existence of transgenerational effects at low doses of EAS,
or effects on development of endocrine organs, fertility or fecundity at high doses of EAS in surrogate animals, so far very little
of the latter evidence has been found to withstand scrutiny with regard to scientific quality. On the contrary, real-life exposures
with EAS, e.g. BPA in humans specifically dosed demonstrate that absence of biologically available and thus active form of
BPA and therefore the absence of potential endocrine activity. Corroborating the latter findings, an extremely thorough epidemiological study from Denmark annihilated the association of inhibited sperm counts in Danish men with exposure to EAS; despite that the latter was purported to be the case even in top journals for nearly two decades. These examples demonstrate 2 main
issues: 1.) only a rigorous scientific approach will provide analytical results that allow any predictions of risk in humans; 2.)
most epidemiological studies provide for wrong associations of exposure to EAS and adverse effects primarily due to faulty
design, too small cohorts and especially due to biased, politically motivated or financially driven approaches at the outset.
Notes
Notes
Notes
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