Elsevier

NeuroToxicology

Volume 30, Issue 4, July 2009, Pages 647-657
NeuroToxicology

Vinblastine and doxorubicin administration to pregnant mice affects brain development and behaviour in the offspring

https://doi.org/10.1016/j.neuro.2009.04.009Get rights and content

Abstract

Background

Administration of chemotherapy during the fetal phase of pregnancy may put late-developing organs like the central nervous system at risk.

Methods

Transplacental transfer of doxorubicin and vinblastine was measured in C57/BJ mice by high-performance liquid chromatographic detection of the drugs in maternal and fetal plasma, 90 min after intravenous injection. Further, doxorubicin, vinblastine or saline were administered to pregnant C57/6J mouse dams on gestational day 17.5. Effects on brain morphology of the offspring were examined at 24 h p.i. (immediate phase) and at 4–5 months p.i. (residual phase), using light- and electron microscopy. At the age of 3 months, offspring performed a behavioural test battery addressing neuromotor performance, exploration and anxiety, and learning and memory.

Results

Fetal plasma levels of doxorubicin and vinblastine reached respectively 5.0 ± 0.2% and 13.9 ± 2.4% of the maternal plasma levels. In the immediate phase, pathological examination revealed endothelial and perivascular parenchymal damage to the neocortical subventricular zone and a less constant thickening of the leptomeninx, in some cases also cortical lamination defects were noted. Brain histology was within normal limits in the mice of the residual phase group. Behavioural testing revealed subtle differences between drug-exposed and control mice. Grip strength was reduced in drug-exposed mice, but other tests for motor performance were normal. Several exploratory measures were altered, and there were some indications of increased anxiety in the drug-exposed mice. In the passive avoidance task, step-through latency was shorter in the drug-exposed mice, but their normal performance in the Morris water maze indicated that this was probably not due to impaired memory.

Conclusion

The current preclinical data reveal subtle changes in behaviour and transiently also in brain morphology in the mice that were prenatally exposed to vinblastine or doxorubicin.

Introduction

Yearly, more than 5 million children are born in Europe and it is estimated that 2500–5000 of these pregnancies are complicated by maternal cancer. The most frequently encountered malignancies are breast cancer and haematological malignancies, for which combination chemotherapy including doxorubicin and/or vinblastine, is the standard first line treatment (Cardonick and Iacobucci, 2004).

Cytotoxic drugs are designed to eradicate rapidly dividing cells, and thus, additionally to their effect on tumor cells, they may interfere with organogenesis and fetal growth (Xing et al., 1992, Wiebe and Sipila, 1994, Dawrant et al., 2007). Case reports are rare and mainly describe fetal losses, mutations and congenital malformations at birth, like skeletal and cardiac malformations, after the application of cytostatic drugs during early pregnancy (Zemlickis et al., 1993, Wiebe and Sipila, 1994, Ebert et al., 1997, Arnon et al., 2001, Cardonick and Iacobucci, 2004). Available data suggest a normal short-term outcome when chemotherapy is administered during the second or third trimester (Cardonick and Iacobucci, 2004, Ring et al., 2005, Hahn et al., 2006). However, the use of chemotherapeutics during the fetal phase of pregnancy might affect tissues like the nervous system, in which organogenesis proceeds well into the early postnatal period and defects of which will only become clear at a later age. Application of other toxins like heavy metals, ethanol and cocaine are known to adversely affect development during late pregnancy (Koren and Nulman, 1994). As chemotherapeutic drugs have a significant teratogenic potential far beyond the toxins listed above, their transplacental transport and impact on the developing fetus need to be characterized in detail. The more so since anatomical aspects of neural development of children prenatally exposed to chemotherapy have remained largely unstudied, and while data on functional aspects seem reassuring, analysis has been superficial so far (Ring et al., 2005, Hahn et al., 2006).

In adult rodents, doxorubicin reaches and damages neurohemal regions (neurohypophysis, median eminence, postremal area), but is largely excluded from brain regions with a functional blood–brain barrier (BBB) (Bigotte et al., 1982, Bigotte and Olsson, 1983, Hsieh et al., 2008). Circumventing the BBB by direct microinjection into the cerebral ventricles led to accumulation of the drug in neuronal and glial cell nuclei of the surrounding parenchyma (Bigotte and Olsson, 1984). As the BBB is not yet functional in the fetal and perinatal CNS, any drug that effectively passes the placenta may also reach the fetal CNS. Moreover, the fetal CNS offers an expanded “target list” for cytostatic drugs, as events like cell proliferation and migration, axon outgrowth and synaptogenesis are still well in progress in the late-fetal CNS. An exception may be the oligodendral myelin sheaths, which largely form after birth. In summary, the use of anthracyclins and vinca-alkaloids during pregnancy may cause a unique set of nervous system damage different from neurological side effects seen in children or adults.

Here we aimed to relate transplacental passage of vinblastine and doxorubicin to brain pathology and behavioural impairments in the offspring in a mouse model. Preliminary experiments revealed a potential impact on layer formation during fetal cortical development. Therefore, we have focused on cortical brain regions, i.e. cerebral (neo)cortex, hippocampus and cerebellum.

Drugs were administered on gestational day (GD) 17.5, after ending the embryonic development (Carnegie stage 23 is reached at GD 16.5) and corresponding to the peak of cell proliferation and migration in the mouse brain. Key events of neuron production, migration and settling occur during region-specific periods in mice, e.g. between GD 13 and P 2 in the murine neocortex (Hatten, 1999) or between GD 13 and about P 20 in cerebellum (Altman and Bayer, 1978) and hippocampal formation (Altman and Bayer, 1990, Stanfield and Cowan, 1979), when in the two latter cases the protracted proliferation of interneurons is taken into account. At GD 17.5, in all three major cortical brain regions cited above both the positioning of the respective macroneurons (hippocampal pyramidal neurons and subgranular neocortical pyramidal neurons, cerebellar Purkinje cells) and the proliferation of the microneurons is well underway. The use of the maximum tolerated dose at this stage should reveal effects both related to the final positioning of the cells defining the local architecture as well as effects related to cell proliferation and migration.

Both immediate (24 h p.i.) and residual (4–5 months p.i.) brain damage were investigated by light- and electron microscopy, and a battery of behavioural tests was performed in 3-month-old offspring.

Section snippets

Drug dosages

In the clinical setting, the most frequently applied dose of vinblastine is 6 mg/m2 per cycle, and of doxorubicin 25–60 mg/m2 corresponding to 0.2 mg/kg for vinblastine and 2 mg/kg for doxorubicin. Considering the inter-species differences in the metabolism and effect of drugs, for most drug dosages applied in mice should be much higher than in humans to reach the same effect (Collins, 2001). Compared to humans, mice are much less sensitive to vinca-alkaloids (van Tellingen et al., 1993). In

Transplacental transfer

Doxorubicin levels in fetal plasma reached 5.0 ± 0.2% of the maternal levels. For vinblastine a transplacental transfer of 13.9 ± 2.4% was measured (Table 1). In 1 out of 7 vinblastine-exposed mice, transfer was measured as 47%, which was much higher than the values obtained in the other 6 mice, despite an identical experimental procedure and sample processing and analysis. Therefore, we considered this value as an outlier.

General appearance

At GD 18.5, the frequency of visible fetal resorptions was comparable

Discussion

Currently, chemotherapy in pregnant women is postponed to the second and third trimester to avoid severe teratogenic effects on the fetus (Wiebe and Sipila, 1994, Ebert et al., 1997, Cardonick and Iacobucci, 2004, Dawrant et al., 2007). As this will basically shift the risk of damage to late-developing tissues like the nervous system, detailed assessment of brain development and resulting neurological performance is crucial for fine-tuning treatment regimens and to provide targeted diagnostic

Conclusions

Application of high dosages of doxorubicin and vinblastine during late gestation in mice causes transient regionally limited lesions to brain microvasculature and surrounding parenchyma and an inconsistent development of cortical ectopias resembling microgyri and isolated lissencephaly type 2-like overmigrations. The immediate lesions do not seem to leave permanent traces. Functional testing of the drug-exposed offspring indicated a few subtle, but specific alterations in their neurobehavioural

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

The research is financially supported by Research Foundation-Flanders (F.W.O.) Project G. 0358.06, ‘Stichting tegen kanker Project SCIE2006-17’, Research Fund-K.U. Leuven. Frédéric Amant is clinical researcher for Research Foundation-Flanders (F.W.O.); Kristel Van Calsteren is aspirant for Research Foundation-Flanders (F.W.O.).

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