# Neurotoxicologically Outcomes of Perinatal Chlordiazepoxide Exposure on the Fetal Prefrontal Cortex Cells in Rat Pup: Chlordiazepoxide Effects on Fetal Prefrontal Cortex

## TL;DR

This study shows that prenatal exposure to chlordiazepoxide harms the fetal prefrontal cortex in rats by causing mitochondrial dysfunction and increased cell death.

## Contribution

The study provides new evidence on the neurotoxic effects of prenatal chlordiazepoxide exposure on fetal prefrontal cortex development in rats.

## Key findings

- Chlordiazepoxide exposure increased mitochondrial ROS and decreased GSH in fetal prefrontal cortex cells.
- Prenatal chlordiazepoxide caused mitochondrial swelling, reduced ATP, and increased cytochrome c release.
- Exposure led to higher apoptosis markers (Bax, p53, caspase 8) and reduced Nissl-stained neurons in rat pups.

## Abstract

Chlordiazepoxide is a benzodiazepine which is widely used as an anxiolytic,
sedative and muscle-relaxant and its effects on neurodevelopment is yet to
be identified. The aim of the current experimental study was to determine
the effects of prenatal exposure to chlordiazepoxide on development of the
prefrontal cortex (PFC).

A total number of 9 pregnant Wister rats that were randomly assigned to three
groups receiving standard rat food and drinking water ad libitum (n=3) or
chlordiazepoxide (10 mg/kg) (n=3) and an equal volume of vehicle (0.9% NaCl)
(n=3) intraperitoneal (i.p.) injection once daily from first to 21st day of
gestation, respectively. At the end of the experiment, 14-day-old neonatal
rat pups (n=8 per each group) were sacrificed and their PFC cells were
extracted. Mitochondria were extracted from the PFC cells and their level of
reactive oxygen species (ROS), protein density, Glutathione (GSH) content,
mitochondrial membrane potential (MMP), swelling, cytochrome c release and
ATP level was identified. We also performed the Nissl staining, DNA
fragmentation assay and RNA extraction and real-time polymerase chain
reaction (PCR) on PFC cells.

We found that isolated mitochondria from rat pups receiving chlordiazepoxide
(E), had significantly higher ROS formation (P0.001), decreased GSH
(P0.001), lower MMP (P0.001), higher mitochondrial swelling (P0.001),
decreased ATP level (P0.001), increased cytochrome c release (P0.001) and
higher Bax (P0.001), p53 (P0.001), cytochrome c (P0.001) and caspase 8 mRNAs
(P0.001). The Nissle-stained neurons decreased while the apoptosis
significantly increased (P0.001).

The results of this in vivo study provide evidence regarding negative effects
of prenatal exposure to chlordiazepoxide on PFC.