Elsevier

NeuroToxicology

Volume 29, Issue 6, November 2008, Pages 1107-1113
NeuroToxicology

Short communication
Effects of early gestational all-trans retinoic acid treatment on motor skills: A longitudinal study in the offspring of Sprague–Dawley rats

https://doi.org/10.1016/j.neuro.2008.09.003Get rights and content

Abstract

The purpose of the present study was to investigate the behavioral outcomes of all-trans retinoic acid (RA) treatment in the period spanning gestational day (GD) 8–10. A sublethal dose (2.5 mg/kg b.w.) compatible with high neonatal survival, sufficient to supply male offspring for later behavioral testing, was used. Indeed, the mortality rate at birth was 7.8%. Reproduction parameters (body weight gain of dams during gestation, number of dams giving birth, pregnancy length, litter size at birth), offspring body weight gain and the development of their somatic characteristics (ear unfolding, auditory conduit opening, eyes opening, hair growth) were not altered by RA. Instead, the onset of righting reflex and negative geotaxis were delayed by 2 days, suggesting vestibular involvement and abnormal functioning of the cerebellum. Then, the performance of RA-treated rats on open field and rotarod/accelerod tasks was assessed from postnatal day (PND) 21 to 90. Similar to the previously investigated GD 11–13 RA treatment, the GD 8–10 RA treatment impaired the open field activity and rotarod/accelerod performance in young adult rats, thus suggesting a task-specific rather than a stage-specific effect of low-dose retinoids during brain development. The delayed appearance of these outcomes underlines the relevance of longitudinal studies to sort out specific RA-targeted neurochemical-behavioral pathways that could be labelled as having no phenotype based on standard examination at birth.

Introduction

Retinoids are natural and synthetic compounds possessing a chemical structure or functional properties similar to vitamin A. Potential retinoid sources come from dietary intake, nutritional supplements, and some therapeutic drugs. Either an excess or a deficiency of vitamin A and related compounds causes an abnormal morphological development (for reviews, see Collins and Mao, 1999, Soprano and Soprano, 1995, Ross et al., 2000), mostly due to failure of mesenchimal and neural crest cells to form and migrate during early embryogenesis (Lee et al., 1995, Mulder et al., 2000). The evidence that, similar to laboratory animals, humans are also susceptible to retinoid teratogenesis (Lammer et al., 1985) is of great concern because retinoids and retinoic acid metabolism blocking agents (RAMBAs) are used as drugs for the treatment of skin diseases and various forms of cancer (Orfanos et al., 1997, Njar et al., 2006).

Although retinoids have few serious systemic side effects at therapeutic doses, however, because of their teratogenic properties women of childbearing age were advised, since the drugs were released, to use an adequate contraception at the time of treatment and to delay conception for at least 1 month after termination of therapy (Mitchell et al., 1995). Concerning the safe time for conception after therapy discontinuation, such measures were, and are still, based on the clinical pharmacokinetic profile of retinoids which seems to be a crucial determinant of foetal safety. For instance, the apparent half-life of elimination for isotretinoin in humans following a single oral dose ranges from 10 to 20 h, the drug level declining to safe levels after a period of 5–6 half-lives; however, after multiple doses the value for elimination half-life increases significantly, likely due to accumulation in a deep tissue compartment (Larsen et al., 1992, Nulman et al., 1998, Collins and Mao, 1999).

Despite the widespread dissemination of warnings by the manufacturer and government agencies, inadvertent foetal exposure has occurred, and cases of retinoid-associated embryopathy have been recorded even after topical exposure during early pregnancy (Honein et al., 2001, Loureiro et al., 2005). For isotretinoin, increasingly strenuous pregnancy risk management programs (RMP) have been implemented over the past 20 years, with further restrictions and requirements issued for preventing foetal exposure and retinoid-related birth defects. The limited success of previous-generation approaches to risk management, such as PPP® (Pregnancy Prevention Program) in 1988–2001 and SMART® (System to Manage Accutane Related Teratogenicity) in 2002–2005, led the Food and drug Administration (FDA) to request that isotretinoin manufacturers create a more rigorous performance-linked RMP. This new, and most stringent, program is an Internet-based, performance-linked system called iPLEDGE, which tries to ensure that the drug is dispensed only when there is documentary proof that the patient is not pregnant and is using two forms of birth control. Enrollment in the iPLEDGE national registry involving patients, physicians, pharmacies, and wholesalers, became mandatory in the U.S. on March 1, 2006 (Abroms et al., 2006).

Besides the severe malformations, there is also evidence that human infants who survive these frequently lethal malformations are often functionally impaired, and sometimes these impairments are seen in children who appear to be otherwise normal at birth (Adams and Lammer, 1991). The detection of functional deficits requires a variety of sophisticated postnatal examinations for sorting out the particular functions that are affected. Indeed, retinoids have been shown to induce behavioral teratogenesis in rats (Adams and Lammer, 1993). There is evidence from animal studies that gestational exposure to retinoic acid (RA) produces lethality and regional brain stunting that is dose and developmental stage specific, with a pronounced sensitive period on gestational days (GD) 11–13 (Holson et al., 1997). The GD 8–10 period is most sensitive for production of malformations, albeit at somewhat higher doses (Holson et al., 1997). Our previous study (Coluccia et al., 2008) has shown that the GD 11–13 all-trans RA treatment induces, in rat offspring, a delayed development of righting reflex, cliff aversion and pole grasping evaluated during the lactation period. Moreover, the offspring follow-up has shown strong and long-term impairment in the open field activity and rotarod/accelerod performance beginning to appear on postnatal day (PND) 40 and persisting until PND 90 (Coluccia et al., 2008).

The purpose of the present study was to investigate the behavioral outcomes of all-trans RA treatment at a sub-lethal dose in the early period spanning GD 8–10 that in rats is most sensitive for production of malformations (Holson et al., 1997). RA dosage (2.5 mg/kg b.w.) was previously identified as the level compatible with high neonatal survival, sufficient to supply offspring for later behavioral testing (Coluccia et al., 2008). Moreover, the offspring of treated dams was tested from birth through 90 days of age in order to assess the full temporal range of potentially adverse consequences of early gestational all-trans RA treatment on behavior. Finally, behavior was studied by using the same test paradigms as in the previously examined GD 11–13 spanning period (Coluccia et al., 2008), in order to identify replicable neurofunctional deficits.

Section snippets

Animals, animal husbandry, and dosing

The experiments were conducted in accordance with guidelines released by the Italian Ministry of Health (D.L. 116/92), the Declaration of Helsinki and the “Guide for the Care and Use of Laboratory Animals” as adopted and promulgated by the U.S.A. National Institutes of Health. The minimum number of animals was used. All animals were treated humanely according to institutional guidelines, with due consideration to the alleviation of distress and discomfort.

Primiparous Sprague–Dawley female rats

Reproduction data

Statistical analysis (Student's t-test) showed that dam weight gain, pregnancy length and litter size at birth were not significantly affected by the GD 8–10 all-trans RA treatment. Moreover, Fisher's-exact test showed that prenatal treatment with all-trans RA did not influence the number of dams giving birth, whereas significantly increased postnatal mortality of pups as compared to control animals (p < 0.05). The mortality rate at birth was 7.8% (9 out of 114 male and female pups). Overall

Discussion

The results of the present study show that the GD 8–10 all-trans RA treatment induces long-term behavioral outcomes in rat offspring. In particular, locomotion, motor coordination and motor learning were found to be significantly impaired in young adult animals. The RA treatment affected neither the development of somatic characteristics nor body weight gain, whereas produced a significant delay in the onset of both righting reflex and negative geotaxis. All the above alterations were observed

Conflict of interest statement

None.

Acknowledgement

This study was supported by grants from Bari University (“Fondo Ateneo” 2007, 2008).

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