[PDF] Innovative non-animal testing strategies for reproductive toxicology

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429ANN IST SUPER SANITÀ 2011

| V

OL. 47, NO. 4: 429-444

D O

I: 10.4415/A

NN _11_04_16 R ESE A RCH A N D M ETHO D OLOG I ES

summary. Reproductive toxicity, with its many targets and mechanisms, is a complex area of toxicology;

thus, the screening and identi?cation of reproductive toxicants is a main scienti?c challenge for the safety assessment of chemicals, including the European Regulation on Chemicals (REACH). Regulatory agen-

cies recommend the implementation of the 3Rs principle (re?nement, reduction, replacement) as well as

of intelligent testing strategies, through the development of invitro methods and the use of mechanistic

information in the hazard identi?cation and characterization steps of the risk assessment process. The EU

Integrated Project ReProTect (6

th Framework Programme) implemented an array of invitro tests to study

different building blocks of the mammalian reproductive cycle: methodological developments and results

on male and female germ cells, prostate and placenta are presented. Keywords: alternative methods, male and female germ cells, prostate, placenta, REACH.

riassunto (Strategieinnovativeperimetodialternativiintossicologiariproduttiva:ilcontributodeipartners

italianinelprogettoeuropeoReProTect). La tossicità riproduttiva, con i suoi bersagli e meccanismi di azione, è una branca complessa della tossicologia: lo screening e l'identi?cazione di sostanze tossiche per

la riproduzione è una delle maggiori s?de scienti?che per la valutazione della sicurezza delle sostanze chi-

miche, previsto anche dal Regolamento Europeo REACH. Le autorità regolatorie raccomandano l'appli-

cazione del principio delle 3R e lo sviluppo di strategie sperimentali razionali per mettere a punto metodi

invitro e l'uso, nell'ambito del processo di valutazione, di un approccio meccanicistico nell'identi?cazione e caratterizzazione del rischio. Il progetto europeo ReProTect ha sviluppato un insieme di saggi invitro per

studiare le diverse fasi del ciclo riproduttivo dei mammiferi: sviluppi metodologici e risultati ottenuti su

spermatozoi, ovociti, prostata e placenta sono presentati in questa rassegna. Parolechiave: metodi alternativi, cellule germinali maschili e femminili, prostata, placenta, REACH.

Innovative non-animal testing strategies

for reproductive toxicology: the contribution of Italian partners within the EU project ReProTect stefano lorenzetti(a) , Ilaria Altieri (a) , sabina Arabi (b) , Donatella Balduzzi (b) , nicoletta Bechi (c) eugenia cordelli (d) , cesare galli (e) , Francesca Ietta (c) , silvia c. Modina (f) , laura narciso (a)

Francesca pacchierotti

(d) , paola Villani (d) , Andrea galli (b) , giovanna lazzari (e)

Alberto Maria luciano

(f) , luana paulesu (c) , Marcello spanò (d) and Alberto Mantovani (a) (a) (c) (d)

CentroRicercheENEACasaccia,Rome,Italy

(e) (f)

IntroDuctIon

The ReProTect Project: conceptual framework

and outcomes

The identi?cation of reproductive toxicants and

their mechanisms of action is a major scienti?c chal- lenge during safety assessment of chemicals; indeed, reproductive toxicology is one of the most compli-

cated ?elds of toxicology, due to multiple organs and tissues involved, potentially different modes of

toxicant action and dependence on the endocrine system. In particular, endocrine disrupters (EDs) represent a big challenge for experimental toxicol- ogy, due to their complex effects on signal networks and programming [1]. Besides the scienti?c chal-

Addressforcorrespondence: Stefano Lorenzetti, Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare, Istituto

Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. E-mail: stefano.lorenzetti@iss.it.

430Stefano Lorenzetti, Ilaria Altieri, Sabina Arabi, et al.

r ese A rch A n D M etho D olog I es lenge, the assessment of the impact of chemicals on the reproductive cycle is also a major ethical and social issue and needs to be considered since it deals with life stages that are potentially more suscepti- ble and are pivotal for the protection of new gen- erations (see also the new paradigm of "sustainable food safety") [2]. For the determination of reproductive toxicity, only in vivo studies are currently accepted by the reg- ulatory authorities. Indeed, there is a major concern that reproductive toxicity testing will signi?cantly increase due to the requirements of the new EU reg- ulatory framework for the Registration, Evaluation and Authorization of Chemicals (REACH) [3]. Several thousands of existing and new chemicals are expected to require testing under new and more stringent safety provisions; in turn, this will require using a high number of experimental animals in time-consuming and expensive tests, leading to a slow assessment and management of many, poten- tially noxious chemicals. A number of useful and promising in vitro models are already available but they need to be converted into tests with a predic- tive power for toxicological safety testing [4]. Then, it is not just a matter of making available ef?cient test methods by developing or validating in vitro as- says but it is necessary to develop integrated and/ or intelligent testing strategies that are advocated within REACH [5] as well as in other risk assess- ment frameworks. For instance, the European Food Safety Authority (EFSA) points out reproductive toxicity studies as one area where the development of alternative methods is more dif?cult; whereas a number of in vitro methods are under development and some are undergoing validation, at present they cannot yet provide the information that can be de- rived from currently used in vivo methods. In recent years several bodies (e.g., the Scienti?c Committee on Health and Environmental Risks, the European Food Safety Authority, the International Forum

Towards Evidence-Based Toxicology) recommend-

ed that risk assessment should more and more rely on the capacity to integrate in vitro assays in a test- ing strategy covering a number of critical targets as well as exploiting the new molecular biology tools [6-8]. Therefore, besides bringing about a decrease in the number of animal tests, a new testing strategy for reproductive toxicology should provide more detailed information on mechanisms of toxicity in the different target tissues, leading to a new, more science-based risk assessment paradigm. Indeed, mechanistic toxicology evolves by relying, to a large extent, on methodologies that substitute or comple- ment traditional animal tests. To date, the impres- sive developments of biotechnology and informatics of the last decades have been embraced by regula- tory toxicology in a possibly too slow and cautious way; however, several agencies have initiated a de- bate about how to create a novel, evidence-based toxicological paradigm. Such paradigm would fully exploit information on identi?ed toxicity pathways by making the best possible use of human cell cul- tures, system biology and in silico modelling [9].

The EU Integrated Project ReProTect ("Development

of a novel approach in hazard and risk assessment of reproductive toxicity by combination and appli- cation of in vitro, tissue and sensor technologies", 6 th

Framework Programme) has represented the ?rst

and most relevant response of the European research system to the many questions posed by the new devel- opments of reproductive toxicology. ReProTect was committed to provide an array of in vitro tests for the investigation of the different aspects and phases of the reproductive cycle as well as to contribute to the development of intelligent testing strategies for the compilation of reliable and valid safety informa- tion [10]. Thus, the short-term project achievement has been the development and optimization of a broad array of already available in vitro tests in or- der to make them amenable to subsequent formal validation studies. Indeed, within ReProTect all in vitro tests have been optimised following the proce- dure of the modular approach for test development [11] recommended by the European Centre for the

Validation of Alternative Methods (ECVAM): this

approach is based on a stepwise accomplishment starting with the de?nition of the test (mechanistic basis, standard operation procedure, identi?cation of relevant toxicological endpoints). The perform- ance of the assays is then assessed on a panel of recognized reproductive toxicants, in order to ob- tain dose-response curves of the selected endpoints and to demonstrate their repeatability. To date, the ensuing step (transferability of the test to another laboratory to determine the reproducibility of the results) has been completed for one test (see below "Effects of reproductive toxicants on the processes of oocyte in vitro maturation and fertilization in a bovine model") [12].

In addition, the ReProTect project has been much

more ambitious, pivoting on two innovative con- cepts [10]: a) to create a battery of test for reproductive toxic ity targeting the building blocks of the reproduc- tive cycle; b) to integrate different approaches based on tar get cells/tissues and on mechanisms/pathways.

Overall, the complexity of organs and events in-

volved in the reproductive cycle simply prevents to ?nd "the" alternative test for reproductive toxicology [13]. However, the reproductive cycle can be broken up in building blocks (components and/or pathways), so to set up a comprehensive battery of tests, each of them addressing one component. This led to a project structure with three major research areas dealing with cell/tissue speci?c approaches and with the ambition of providing an array of test targeting the essential steps of reproductive cycle: fertility; implantation; prenatal development. Whereas the developmental toxicity tests were closer to optimization phase, the test batteries dealing with male and female fertility and implantation were more complex to develop en-

431NOVEL STRATEGIES IN REPRODUCTIVE TOXICOLOGY

R ESE A RCH A N D M ETHO D OLOG I ES compassing a number of cells/tissues (sperm, leydig cells, oocytes, ovary granulosa cells, trophoblasts, etc.) and of targeted parameters (DnA integrity, ster- oidogenesis, etc.). noticeably, during reprotect some new targets were identied; one main example is prostate, a key functional gland for male fertility somehow over- looked in toxicity testing [14]. A fourth research area was represented by cross-cutting technologies, i.e., those approaches focussed primarily on toxicant modes of action and potentially relevant to many or all components of the reproductive cycle; in this area, immediate success was obtained with assays on the transactivation of human estrogen and an- drogen receptors [15, 16] as well as with the use of structure-activity relationship modelling to improve predictivity of toxicology data sets [17].

During the nal year of the reprotect project, a

ring trial, named “Feasibility study", was conduct- ed, in which blinded chemicals with toxicologically well-documented proles were tested by employ- ing a battery of 14 in vitro assays developed within the project itself [18]. this comparative analysis together with a weight of evidence approach al- lowed a robust prediction of adverse effects on fertility and embryonic development caused by the

10 test chemicals in vivo: the vast majority of the

predictions made on the basis of the in vitro results turned out to be correct when compared to the whole animal data [18]. the core feasibility study was anked and supported by satellite studies, such as those conducted with the newly developed psA secretion assay to screen substances targeting the prostate [14]. At the end of project, the reprotect test battery holds promise for use as a screening ap- proach for reproductive toxicity testing according to the eu reAch legislation. overall, reprotect has involved 32 european part- ners from academia, industry and governmental insti- tutes, with a major participation of Italian partners: eneA, (Italian national Agency for new technologies, energy and sustainable economic Development, rome, Italy), Istituto sperimentale Italiano lazzaro spallanzani (rivolta d"Adda, cremona, Italy), Istituto superiore di sanità (the Italian national Institute of health, rome, Italy, led by A. Mantovani, coordinator of the Wp IV on cross-cutting technologies), Avantea (cremona, led by g. lazzari, coordinator of the Wp I on fertility); university of siena and university of Milano were involved contributing with innovative approaches covering main components of the repro- ductive system, namely the male and female gametes (Workpackage/Wp I, Fertility), the prostate (Wp IV, cross cutting technologies) and the placenta (Wp II, Implantation). Functional as well as molecular pa- rameters were exploited both as potential predictors of in vivo effects and as building blocks in a modular testing battery [10, 19]. the outcomes of specic ac- tivities and their relevance for a new testing strategy on reproductive toxicity are discussed in the ensuing paragraphs.

TESTSONMATURESPERMATOZOA

Methods used to evaluate reproductive toxicity, in- cluding male-mediated developmental toxicity, require a large number of animals and validated in vitro alter- natives are not yet available [20]. the strategy pursued by the reprotect project was to break down the mam- malian reproductive cycle into several in vitro work- able tiers. In this context, within the framework of the fertilization tier, the tests proposed and described be- low focus on two crucial features of the mammalian sperm, which are acquired extratesticularly during its transit through the epididymus. Actually, it is during the epididymal maturation that sperm gain the capac- ity for progressive motility and the newly reorganized sperm nuclear DnA around the protamine core reach- es its zenith of compactation when the inter- and intra- protamine disulde bridges between cysteine residues are formed to provide a highly rigid structure. It is spec- ulated that the generation of a more protected genome (sperm has practically no DnA repair system) and of a more hydrodynamic sperm head can speed transit through the female reproductive tract. therefore, both processes, namely nucleus remodeling and acquisition of motility, seem essential to fulll the primary goals of the male gamete genome, that is a successful fecun- dation and a fully sustained pregnancy. consequently, it is reasonable to assume that any impairment to these concurrent, apparently non correlated, differentiation pathways should hamper the proper expression of the sperm reproductive capability.

Direct assays on spermatozoa, which represent the

functional end-product of the whole intra-testicular spermatogenesis process and its maturation during epididymal transit, have the main advantage of be- ing able to detect effects on the terminally-differen- tiated male gametes ready to undergo fertilization. For participating in fertilization spermatozoa must be alive and motile, able to undergo acrosome re- action and deliver an intact male genome into the oocyte to create an embryo with a high chance of completing a full-term healthy pregnancy. Any dam- age to these properties (motility, plasma membrane integrity, genetic integrity) could hamper its repro- ductive capacities before or after the fertilization process. notably, both motility and genetic integrity have been demonstrated to be predictors of human fertility in the general population [21, 22]. Ideally, an in vitro sperm assay should rely on a read- ily accessible source of sperm obtainable from animals without invasive procedures. Furthemore, in order to ensure the repeatability of the test and the possibility of easy inter-laboratory exchange an in vitro sperm assay should involve sperm which are homogeneous, constant over time and disposed to be stored for long period storage at low temperature without losing their functional/structural properties. the use of bovine sperm, easily collected in sufciently large amount, matches many of these ideals [20] and has been se- lected in the reprotect trials. the use of frozen semen represents an innovative approach of great interest for testing the toxic effect of chemicals on fertility, because

432Stefano Lorenzetti, Ilaria Altieri, Sabina Arabi, et al.

r ese A rch A n D M etho D olog I es it does not involve invasive procedures which compro- mise animal welfare. Frozen bovine semen is readily accessible and represents a material that can be stored for long periods in liquid nitrogen, without losing its biological properties, in order to ensure the repeatabil- ity of the tests and to afford the possibility of exchange among different laboratories.

Mammalian

sperm motility: a tool for in vitro evaluationofchemicaltoxicity One of the most important laboratory tests to evaluate sperm quality has historically been the visual estimation of sperm motility. The introduction of a computerized measurement by image analysers improved the accuracy of the method with the advantage of eliminating the hu- man factor and led to a more objective analysis. Within the ReProTect project sperm motility parameters meas- ured on mature bovine sperm were used to determine the toxicity of chemical compounds [23]. Sperm motil- ity has been chosen as endpoint, since this parameter is considered to be a sensitive marker for epididymal toxi- cants [21]. Straws of 500 μl containing about 15 x 10 6 spermatozoa were thawed and subsequently incubated for one hour with different test compounds. After in- cubation, Total Motility/TM (%), Progressive Motility/ PM (%) and Average Path Velocity/VAP (μm/sec) were measured by a Computer Assisted Semen Analysis sys- tem (HTM-IVOS, Hamilton Thorne) [24].

This approach was tested on 36 compounds, some

of which were recognized or suspected as reproductive toxicants with different mechanisms of action while others were known to have a toxic effect on cells. Each compound was dissolved in an appropriate vehicle de- pending on its physical and chemical properties. The con- centration of vehicle was determined in a preliminary test (0.2% for Ethanol and to 1% for Acetone, Demi water and DMSO). The compounds were the following: Acetone (vehicle), Antimycin A, Benzalkonium chlo- ride, Benzene, Butyl-4-hydroxybenzoate, Cadmium chloride, 3-Chloro-1,2,-propanediol, Colchicine, Cycloheximide, Demi water (vehicle), 1,2-Dibromo-3- chloropropane, 2,4-Dichlorophenol, 2,4-Dinitrophenol,

Diethylstilbestrol, Diethyldithiocarbamate, DMSO

(vehicle), Epichlorohydrin, Ethanol (vehicle), 5-quotesdbs_dbs26.pdfusesText_32

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