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f the several antioxidants assessed, only N-acetylcysteine (NAC)as been shown to reduce MDA levels to control values in thed-treated chick embryo. However, the molecular mechanisms byhich NAC acts to alter oxidative stress in the Cd-induced VBWD

hick model remain unclear. We therefore designed this study tonvestigate the hypothesis that pre-treatment with NAC upregu-ates the expression levels of genes encoding antioxidant enzymesn the normal embryos exposed to Cd compared with malformedmbryos.

Methods: After 60 h incubation, chick embryos were pre-reated with NAC exposed to either chick saline, Cd or Cd withAC pre-treatment. Chick embryos were then harvested 24 h

24H) post-treatment and divided into five groups: control, Cdroup without malformation (Cd(−)), Cd group with malformationCd(+)), NAC + Cd(+) and NAC + Cd(−). Real-time RT-PCR (reverseranscription polymerase chain reaction) was performed to evalu-te the relative mRNA expression levels of superoxide dismutaseSOD), catalase (CAT) and glutathione peroxidase 4 (GPX4) in eachroup. Differences between five groups at each gestational dayere tested by tested by Tukey–Kramer post hoc test following

ne-way ANOVA. Statistical significance was accepted at p-values0.05.

Results: The mRNA expression levels of SOD2 and CAT at 24Host-treatment were significantly decreased in Cd(+) (0.26 ± 0.11nd 0.59 ± 0.22, respectively) compared to controls (0.48 ± 0.11nd 1.01 ± 0.21, respectively) (p < 0.05), whereas there was no sig-ificant difference between controls and Cd(−). In addition, genexpression levels of SOD2 and CAT were significantly increased inAC + Cd(−) (0.50 ± 0.15 and 1.01 ± 0.31, respectively) compared

o NAC + Cd(+) (0.20 ± 0.19 and 0.62 ± 0.19, respectively) (p < 0.05).owever, there were no significant differences in the expression

evels of SOD1 and GPX4 among any groups.Conclusion: Our results provide evidence, for the first time, that

d decreased gene expression levels of SOD2 and CAT in malformedmbryos compared to controls. In addition, we also clearly demon-trated that pre-treatment with NAC upregulates SOD2 and CATene expression levels in NAC + Cd(−) compared to NAC + Cd(+) inhe chick embryo. Our findings suggest that SOD2 and CAT may playmore important role in preventing Cd-induced teratogenesis than

he other antioxidant enzymes.

oi:10.1016/j.reprotox.2010.05.040

athogenesis of head malformations in CD1 mice exposed intero to VPA

lena Menegola ∗, Maria L. Broccia, Francesca Di Renzo

Università degli Studi di Milano, Department of Biology, Milan, Italy

In a recent preliminary study, the i.p. injection on day 8 p.c. with00 mg/kg bw of valproic acid (VPA) resulted at term of gestation

n nearly 25% postimplantation loss, axial skeletal defects (nearly00%) and head abnormalities, described as exencephaly (nearly0%) or abnormalities of the frontal region (in almost all non exen-ephalic fetuses). After analysis of embryos, VPA-related apoptosisas detected at the level of somites and at the head mesenchyme

rom 8 to 24 h after treatment, while at 48 h after treatment noathological apoptosis was detected. Interestingly, apoptosis wasever detected at the level of neural epithelium or placodes. Toetter describe the pathogenesis of VPA-related head abnormal-

ties, in the present work CD1 mice were i.p. injected on day 8

.c. with VPA 0–600 mg/kg bw and conceptuses were examined at4–48–72–96 h after treatment or at term of gestation. Consideringhat head mesenchyme has a double origin (from paraxial meso-erm and from migrating neural crest cells that condense to formhe ectomesenchyme), ectomesenchyme was stained by whole

Toxicology 30 (2010) 227–232

mount and immunohistochemistry procedures. 50% of fetuses weredouble stained for bone and cartilage, the rest processed for vis-ceral analysis. The analysis of uterine content at term of gestationrevealed that, after VPA exposure, 4% of fetuses died nearly at termof gestation and 22% were reabsorbed. The same analysis at midges-tation showed a peak of mortality nearly at 25–30 somites and thatat 96 h after treatment the postimplantation loss index was 11%. Byconsequence, nearly 11% of conceptuses should die in later stages.Embryos examined at midgestation showed delays in the closureof neural folds at 24 h after treatment (37% in VPA group embryosversus 7% in controls). In later stages neural fold closure occurredand 100% of embryos showed at 96 h after treatment a closed neuraltube. Exencephaly was evident respectively in 25–21–37% of VPAexposed embryos at 48–72–96 h after treatment. As 31% of exen-cephaly was recorded at term, this kind of malformation seemsto be determined during the examined embryonic period. Due tothe fact that the percentage of exencephaly observed at midgesta-tion and at term was unchanged, exencephaly per se is not directlyrelated to embryolethality. The delay in neural tube closure couldbe the pathogenic pathway involved in VPA-related exencephalybut, as cell death was not detected in neuroepithelium nor pre-viously or in the present study, the cause of this delay shouldbe related to different mechanisms. No other abnormalities weredetected at the morphological examination of the embryos. On thecontrary nearly 20% of fetuses showed cardiovascular defects andalmost the totality of not exencephalic fetuses showed abnormalmorphology and fusion between frontal bones or extra intrafrontalbone. Finally, the immunodetection of ectomesenchyme showedthat, in the head, this tissue was abnormally distributed and itscells were suffering. As ectomesenchyme is the source for a numberof cranial vault elements (including frontal bones), a relationshipbetween VPA-related mesenchyme abnormalities and head mal-formations is proposed.

doi:10.1016/j.reprotox.2010.05.041

Developmental exposure to paracetamol causesanti-androgenic effects in male rat offspring

Ulla Hass a,∗, Pernille Rosenskjold Jacobsen a, Julie Boberg a, SofieChristiansen a, Christine Nellemann a, David Møbjerg Kristensen b,Henrik Leffers b

a Division of Toxicology and Risk Assessment, National Food Institute,Technical University of Denmark, Denmarkb Department of Growth & Reproduction, Rigshospitalet, University ofCopenhagen, Denmark

Paracetamol (acetaminophen) and non-steroidal anti-inflammatory drugs are widely used mild analgesics. In bothEurope and The United States more than 50% of pregnant womenhave reported intake of mild analgesics during pregnancy withthe majority using paracetamol. This raises concern becausestudies from the 1980s indicate that prenatal exposure to mildanalgesics may affect masculinisation in animals. The present workinvestigated the effects of paracetamol on foetal and postnataldevelopment of the male reproductive system. Initially, pregnantWistar rats (n = 4–5) were gavaged from gestation day (GD) 13to 21 with vehicle, 150, 250, or 350 mg/kg bw/day of paraceta-mol. Caesarean section was performed on GD 21 and anogenitaldistance was recorded in all foetuses. No clinical signs of generaltoxicity were observed during the daily observations, and no signs

of liver toxicity were detected in the pregnant dams. Maternalbody weight gain, litter sizes, numbers of live foetuses, resorptions,implantation, and sex ratios in the litters were not affected in thedosed groups as compared to controls. Intrauterine exposure toparacetamol resulted in reduced AGD index (AGD adjusted for

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ody weight) in male foetuses in all three dose groups withoutffecting the body weight, indicating a reduced masculinisationf the male offspring. To verify these results, we performed andditional study with 6–7 pregnant animals per group, usingnly the top dose of 350 mg/kg/day paracetamol. Consistent withhe previous data, this study showed a significant reduction inGDi in the GD 21 male foetuses. When the data from the twotudies were combined, including study number as a factor inhe statistical analysis, the reductions in AGD and AGDi weretatistically significant for all three dose groups.

In a postnatal study, dams were exposed from GD 10 to GD 19nd from the day after birth to postnatal day (PND) 22 to vehicle or50 mg/kg bw/day (n = 8 and 10), and all offspring were examinedor anogenital distance on PND 1 and number of retained nipples onND 13. There were no signs of maternal toxicity and no effects onarturition, litter size and birth weight. In the male pups exposedo paracetamol, the AGD index was significantly reduced and theumber of retained nipples was significantly increased. In conclu-ion, exposure to paracetamol during development at dose levelsrom 150 to 350 mg/kg/day resulted in anti-androgenic effects in

ale rat offspring comparable with the effects seen after exposureo known anti-androgenic compounds such as phthalates. Sincehis dose level is only three to seven times the dose recommendedor humans of 50 mg/kg/day, further work is needed to determinehe no adverse effect level (NOAEL). In addition, these results sug-est that exposure to paractamol is a risk factor for development ofale reproductive disorders.

oi:10.1016/j.reprotox.2010.05.042

o mate or not to mate: A retrospective analysis of fourteen two-eneration studies for evaluation of the added value of a secondeneration. An update

anon Beekhuijzen ∗, Birgit Peter, Amy Zmarowski, Harry Emmen

NOTOX B.V., ‘s-Hertogenbosch, The Netherlands

An extended one-generation study is proposed to replace theurrent two-generation study, and would eliminate breeding for aecond generation unless triggered by effects on parental reproduc-ion or pup development. Fourteen two-generation studies in ratsfive studies added to the already published nine [1]) were reviewedo determine whether reproductive or developmental toxicity was

ore pronounced in the second generation. For thirteen out of theourteen studies reviewed, mating for a second generation had nodded value as similar findings at the same dose levels were notedor both generations (twelve studies) or less severe findings wereoted for the second generation (one study). The remaining studyperformed according to the OECD416 guideline prior to the 2001pdate) showed very slight developmental findings (only 8% lowerup body weights compared to controls) in the first generationhough very severe developmental effects were seen during theecond generation. As this study was performed according to theld guideline, it was potentially missing crucial data important forvaluation. Based on the assessment of these fourteen studies, itan be acknowledged that criteria to trigger additional mating asuggested by Cooper et al. [2] under the proposed extended one-eneration design are of very limited utility, since additional matingould have been triggered in seven of these studies of which allere unnecessary. In the one case where additional mating waseeded, no triggers were apparent. Moreover, in thirteen out of

ourteen studies the second generation did not reveal additionalnformation. Based on this investigation, together with contempo-ary publications, it can be concluded that the second generationas no added value for toxicological risk assessment and shouldot be included in the extended one-generation study. Further-

Toxicology 30 (2010) 227–232 231

more, with its use of more animals/litter together with additionalexaminations, the extended one-generation study is recommendedto replace the current two-generation study because it offers amore extensive evaluation of the F1 generation during develop-ment while using less animals overall.

References

1] Beekhuijzen M, Zmarowski A, Emmen H, Frieling W. To mate or not to mate:a retrospective analysis of two-generation studies for evaluation of criteria totrigger additional mating in the extended one-generation design. ReproductiveToxicology 2009;28:203–8.

2] Cooper RL, Lamb IV JC, Barlow SM, Bently K, Brady AM, Doerrer NG, et al. Atiered approach to life stages testing for agricultural chemical safety assessment.Critical Reviews in Toxicology 2006;36:69–98.

doi:10.1016/j.reprotox.2010.05.043

Towards 21st Century Toxicology: Ultrasonic vocalizations(USVs) as animal-friendly marker to test developmental(neuro)toxicity

Didima de Groot a,∗, Luke Blauw a, Ronald Bulthuis c,Lianne Damsteegt a, Linda van der Horst a, Marlies Otto a,Jasper Swierstra a, Herma van de Wiel b, Carina Rubingh a

a TNO Quality of Life, Zeist, The Netherlandsb TNO Defence, Security and Safety, Rijswijk, The Netherlandsc METRIS BV, Hoofddorp, The Netherlands

Twenty-first Century Toxicology focuses on sustainable test-ing approaches that are more predictive for man, less laborious,preferably faster than current methods and contributing to ani-mal 3Rs (Replacement, Reduction, Refinement). Here, we exploredthe usefulness, applicability and reliability of utrasonic vocaliza-tions (USVs) of rat pups, i.e. calls of 35–65 kHz emitted by the pupsto their mothers, as an animal-friendly marker to test develop-mental (neuro)toxicity. The questions posted were: (1) can USVsserve as a marker to study normal development and (2) as amarker to study developmental (neuro)toxicity. To this end, the ratswere exposed perinatally via daily diet to different organotin com-pounds, i.e. dioctyltinchloride (DOTC (primarily immunotoxic): 3,10, and 30 mg/kg bw) or tributylinoxide (TBTO (primarily neu-rotoxic): 8 mg/kg bw). USVs were studied during 30s each day,from postnatal day (PN) 4 to 18, while the pup was placed inan open arena. For comparison with this new endpoint, conven-tional sensory and physical landmarks as proposed in guidelinesfor developmental toxicity were studied as well. The results showedthat under standard diet conditions, a typical pattern of develop-ment of USV calling was observed from PN4 to 18 with a maximumat about PN11. TBTO reduced the maximum number of calls, whichin turn was reached at a later postnatal day, resulting in differentslope of the USV-curve over time and reduction of mean numberof calls/treatment. TBTO also affected the frequency of the USVcalls: tendency to an increase of frequency at early days (PN4)and reduced frequency towards PN18; mean frequency per treat-ment (PN4–18) was increased. DOTC did not affect the developmentof the USVs. The findings were supported by the results of con-ventional physical and sensory landmarks, pointing at delayeddevelopment by TBTO but not by DOTC. Together, the results sup-

doi:10.1016/j.reprotox.2010.05.044