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Method: Using standardised procedures, NTIS has provided fetalrisk assessment and collected prospective outcome data on 69 ther-apeutic exposures to zopiclone during pregnancy.

Results: Thirty (43.5%) fetuses were exposed in the firsttrimester; two (2.9%) in the first and second trimester; one (1.4%) inthe second trimester; seven (10.1%) throughout pregnancy and 29(42.0%) pregnancies in which exposure time was not stated. Therewere 50 live borns, nine elective terminations of pregnancy, eightspontaneous abortions and two intrauterine deaths. The incidenceof spontaneous abortions and ETOPs were within the expectedrange of 10–20% and 23%, respectively. The overall malformationrate was 3/50 live born infants (6.0%, 95% CI 1.6–17.5) which is notsignificantly higher than expected (background incidence of 2–3%).Malformations comprised amitrypsin deficiency (a hereditary con-dition), a case of tricuspid regurgitation with valve dysplasia anda live born with asymmetric ears and low birth weight. Causalitycould not be established in any of these cases due to polyther-apy. Seven of the otherwise healthy live born infants were reportedto exhibit signs of neonatal withdrawal; a finding consistent withexperience for other centrally acting drugs.

Conclusions: The limited published data alongside the follow-up data provided by NTIS indicate that therapeutic exposure tozopiclone in pregnancy does not appear to be associated with anincreased risk of congenital malformations. However, there are stilltoo few data to state that there is no risk. The data provided byNTIS greatly increase the total number of outcomes of therapeuticzopiclone exposure during pregnancy but further data are requiredbefore any firm conclusions can be drawn regarding the safety ofzopiclone in pregnancy.

References

1] Diav-Citrin O, Okotore B, Lucarelli K, Koren G. Pregnancy outcome following first-trimester exposure to zopiclone: a prospective controlled cohort study. Am JPerinatol 1999;16(4):157–60.

2] Wilton LV, Pearce GL, Martin RM, Mackay FJ, Mann RD. The outcomes of preg-nancy in women exposed to newly marketed drugs in general practice inEngland. Br J Obstet Gynaecol 1998;105(8):882–9.

3] Inman W, Kubota K, Pearce G, Wilton L. PEM report number 10: zopiclone. Phar-macoepidemiol Drug Saf 1993;2:499–521.

doi:10.1016/j.reprotox.2008.05.046

Polycyclic aromatic hydrocarbons and occupational risk factor

Elena Szabova 1, Dagmar Zeljenkova 1, Eva Nescakova 2, MilanSimko 1, Ladislav Turecky 3

1 Slovak Medical University, Bratislava, Slovakia2 Department of Anthropology CU, 84215 Bratislava, Slovakia3 Department of Biochemistry-Faculty of Medicine CU, 81000Bratislava, Slovakia

Introduction: Polycyclic aromatic hydrocarbons (PAH) have beenidentified as major source of carcinogenic risk in urban areas andclassified as cancerogens according to the IARC categorization. Oursamples for analysis were taken at the Matadoroll-Slovakia plantwhere rubber semiproducts were produced. In our study we fol-lowed the effect of polluted working environment regarding thealpha-1-antitrypsin (AAT) values and presence of DNA damage bycytogenetic analysis of employees. The AAT values can increasein acute or activated chronic inflammations, systemic diseases,malignant tumours, myocardial infarction, and during postoper-ative status. AAT is also found in lung alveoli where it is formed.When lungs are exposed to, e.g. tobacco smoke the amount of typ-ical lung phagocytotic cells have been increased.

oxicology xxx (2008) xxx–xxx 19

Methods: In our study 62 persons were involved for evalua-tion of AAT values. 40 and 22 employees were involved in theexposed and control groups. For cytogenetic analysis a groups of38 exposed subjects, and 19 unexposed persons were involved.Kruskal-Wallis one-way analysis and one-way analysis of variancewere performed for evaluation of cytogenetic results. Statisticalfunctions of Excel Program were used for mathematic-statisticaldata processing. Modified t-test for standardized deviation wasused for statistical evaluation of differences in the followed groups.

Results: Exposed employees showed statistically significanthigher AAT values compared to the control group (t = 4.732,P < 0.001). Regarding to the fact that there exists information aboutcorrelation between AAT values and smoking habits at the litera-ture those effects were also studied. Statistically significant lowervalues were found in non-smokers compared to smokers (t = −2,4217, P < 0.018). The exposed group showed a statistical signifi-cant greater proportion of chromatid and chromosome breaks withchromosome exchanges occurred rarely.

Conclusion: As the occurrence of aberrant cells was in the allow-able range of the average aberration occurrence in Slovakia (<2%)we do not evaluate this risk status from this point of view as clas-togene destruction of somatic cells in workers. In the immunologicprofile of persons working at the chemical part of the plant and thatof clerical employees of the same plant no considerable changesin the followed parameters of humoral profile were found. Never-theless, in both groups a markedly increased number of personswere found with positive markers of atopy compared to the controlgroup. Considering that only trace amounts of the chemical agentscan cause sensitization in “sensitive” individuals, and based also onour results, we suppose that employees of the plant were exposed tothe highly sensitizing chemicals. The effects of these agents did notoccur only at the plant itself but probably in its round atmosphereas well.

doi:10.1016/j.reprotox.2008.05.047

Effects of ethylenethiourea (ETU) on thyroid and repro-ductive tract after pre- and post-natal administration inSprague–Dawley rat

Roberta Tassinari 1,2, Gabriele Moracci 1, Antonietta D’Ambrosio 1,Daniele Marcoccia 1, Simona De Angelis 2, Antonella Olivieri 2,Agostino Eusepi 3, Antonio Di Virgilio 3, Stefano Lorenzetti 1,Alberto Mantovani 1, Francesca Maranghi 1,∗

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1 Department of Food Safety and Veterinary Public Health, Rome, Italy2 Department of Cellular Biology and Neurosciences, Rome, Italy3 Service for Biotechnology and Animal Welfare, Istituto Superiore diSanita, Rome, Italy

Introduction: Ethylenebisdithiocarbamates are fungicideswhose metabolite ETU is an endocrine disrupter (ED) with thyre-ostatic effects [1,2]. Few toxicological data are available on therole of ETU during the development and differentiation of thyroidand reproductive tract in both sexes. The aim of the present studywas to evaluate the potential for thyroid and reproductive systemdisruption of ETU following pre- and post-natal exposure.

Methods: Pregnant rats (15/group) were treated with 0 and1.0 mg/kg bw/day of ETU on gestational day 7–20 and post-natal day(PND) 1–22 to cover development and morphogenesis of both thy-roid and reproductive systems. Dams were checked daily for generaltoxicity and sacrificed at weaning (PND23). Pups were examineddaily for timing of vaginal patency (from PND35, females) andpreputial separation (from PND40, males). Vaginal smears werecollected for evaluation of oestrus cycle (from PND55). At sexualmaturity (males PND62, females PND71) blood samples were col-

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lected for thyroid hormones (TH) evaluation and F1 pups weresacrificed. Thyroids and reproductive organs were excised andweighted.

Results: Females. No signs of general toxicity were observed.No significant differences in body weight gain, timing of vaginalopening, thyroid relative weight and TH were recorded betweengroups. Ovary (left −11%, right −3%) and uterus relative weight(−38%, P ≤ 0.05) were decreased in the treated group. Changesoccurred in the days of estrus cycles (4.8 ± 0.4 vs. 8.4 ± 3.9, P ≤ 0.01)with a concomitant increased duration of estrus and metaestrusstages. Males. No signs of general toxicity were observed. Bodyweight gain was significantly increased (+5%, P ≤ 0.05) in the treatedgroup. No significant differences in timing of preputial separa-tion were observed. Thyroid relative weight was increased (+7%),whereas testis (left −15% right −14%, P ≤ 0.05) and prostate relativeweight (−23%, P ≤ 0.01) were significantly decreased in the treatedgroup than in the control mice. TSH serum level was significantlyincreased (8.3 ± 3.4 ng/ml vs. 5.9 ± 2.0 ng/ml, P = 0.04) in the treatedmice; no changes in TH levels were observed.

Conclusions: ETU administration during the critical period ofdevelopment and differentiation exerted reproductive effects withapparent different gender mechanism. In F1 treated females,altered sexual parameters were not accompanied by concurrentaltered thyroid functionality, while in males unbalanced thyroidhomeostasis was related to decreased relative weight of reproduc-tive organs. This extended one-generation study can represent avaluable, time- and cost-saving tool for evaluating ED effects. Fur-ther studies on the potential involvement of thyroid-gonads axis inETU developmental toxicity are suggested.

This study was supported by: Project of the Italian Health Ministryn. M6B and NoE CASCADE EU contract no. FOOD-CT-2004-506319.

References

1] Lentza-Rizos C. Ethylenethiourea (ETU) in relation to use of ethylenebisdithio-carbamate (EBDC) fungicides. Rev Environ Contam Toxicol 1990;115:1–37.

2] Freyberger A, Ahr HJ. Studies on the goitrogenic mechanism of action of N,N,N′ ,N′-tetramethylthiourea. Toxicology 2006;217:169–75.

doi:10.1016/j.reprotox.2008.05.048

Gene expression changes in differentiating embryonic stem cellsinduced by monobutyl phthalate

Dorien A.M. Verhallen 1,2, Peter J. Hendriksen 2, Jeroen L.Pennings 1, Frederik-Jan van Schooten 2, Aldert H. Piersma 1,∗

1 National Institute for Public Health and the Environment (RIVM),Bilthoven, The Netherlands2 University Maastricht, Maastricht, The Netherlands

Since the increasing demand for in vitro toxicity screening tests,a lot of work has been performed in this field. Especially in repro-ductive toxicology the need for alternative toxicity tests is high,because in this field the highest numbers of animals are used. Thedevelopment of the embryonic stem cell test, validated in 2002,has been a significant step forward for the prediction of embry-otoxicity based on in vitro data. This validated method predictsembryotoxicity due to compound exposure, based on inhibition ofdifferentiation of embryonic stem cells (ESC) towards cardiomy-ocytes. We hypothesize that the sensitivity of this alternative testmay be increased by analysis of differentiation at the level oftranscriptomics. Therefore, we studied the gene expression pro-file of ESC in the early phase of differentiation with and withoutcompound exposure. Hanging drops containing ESC single cell sus-pensions were allowed to aggregate. At day 3, these aggregates

oxicology xxx (2008) xxx–xxx

were transferred to bacterial dishes, which allows suspension cul-ture. After 2 days of incubation, the aggregates were seeded ontissue culture plastics, and the differentiation was assessed bycounting contracting cardiomyocyte foci at day 10. The cells wereexposed from day 3 onwards to monobutyl phthalate (MBP) or sol-vent control. Samples for RNA extraction were collected after 6, 12and 24 h of suspension culture. Samples were analyzed on spottedRNA microarrays (22k) from MAD (Amsterdam, NL) and differen-tial gene expression patterns were analyzed and compared withexisting data. Since very few developmental-related gene sets wereavailable from open sources, gene sets were collected from litera-ture. GSEA comparative analysis of this gene set collection clearlyshowed differentiation-related gene expression changes with time.Furthermore, exposure of the differentiating cells to MBP resultedin increased expression of pluripotency-related gene sets, whichindicates inhibition differentiation due to MBP exposure. Also, genesets which describe the differentiation of ESC towards cardiomy-ocytes were downregulated in MBP exposed samples. In addition,the expression of proliferation-related gene sets was induced afterMBP exposure, which indirectly indicates inhibition of differen-tiation. These results show that we are able to demonstrate thedifferentiation of ESC towards cardiomyocytes at a very early stage.Furthermore, inhibition of cardiomyocyte differentiation by MBPcould be demonstrated in this early phase, based on the use ofrelevant gene sets.

doi:10.1016/j.reprotox.2008.05.049

Monoclonal antibody therapy during pregnancy—Experience ofthe Berlin teratology information service

Corinna Weber-Schoendorfer ∗, Christof Schaefer

Pharmakovilgilanz- und Beratungszentrum Embryonaltoxikologie,Berlin, Germany

Introduction: Therapeutic use of monoclonal antibodies forinflammatory bowel diseases, rheumatic diseases and malig-nancies has been steadily increasing. Infliximab, adalimumab,rituximab, trastuzumab are Ig G1 monoclonal antibodies. Mono-clonal antibodies are large molecules (>140,000 g/mol) that canhardly pass the placenta during embryogenesis. Later in pregnancya transplacental transfer is well documented [1], similar to phys-iological IgG1 or maternal autoantibodies that pass the placentathrough an active process. Infliximab and adalimumab are TNF-�

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antagonists. As TNF-� is known to be important for the developingimmune system, TNF-� antagonists may harm the fetus. Further-more, infliximab was associated with a three-fold higher incidenceof lymphoma in treated patients. The majority of published casesconcern infliximab use (more than 100 pregnancies) during the firsttrimester. So far, there is no support for teratogenicity. However,diminishing of the child’s B cells was observed after rituximab [2]and oligohydramnios after trastuzumab therapy during the secondhalf of pregnancy [3].

Material: We have had 134 consultations concerning mono-clonal antibodies since 1999. There were 50 consultations in 2007and, if the trend continues, we expect 100 inquiries this year. Amongall consultations up to now 25 were dealing with lactation, 25 withpaternal exposure, and 84 with maternal exposure of a planned orongoing pregnancy. 24 of the maternal exposed pregnancies havebeen evaluated (1 abciximab; 5 adalimumab; 11 infliximab; 1 oma-lizumab; 4 rituximab; 2 trastuzumab) and 14 pregnancies are stillongoing. Apart from 1 retrospective case report on infliximab allpregnancies have been ascertained prospectively. Ten pregnancieswere exposed to monoclonal antibody-therapy only during the 1sttrimester, 5 were treated (3–10 weeks) prior to conception (4 inflix-