Date of Degree

2-2016

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor(s)

Abdeslem El Idrissi

Committee Members

William L'Amoreaux

Mohammed Junaid

Bassem F. El-Khodor

Miklos Toth

Subject Categories

Behavioral Neurobiology | Molecular and Cellular Neuroscience

Keywords

Autism, Dibutylphthalate, Fragile X, Endocrine, Steroidogenesis, GABA A Receptor

Abstract

The etiology of autism is thought to involve the complex interplay among genetic and environmental factors. Patterns of inheritance suggest an epigenetic component to the development of autism. A variety of environmental risk factors are known to induce epigenetic changes in DNA, affecting many genes including those autism-associated genes (AAG). The plasticizer dibutyl phthalate (DBP; CAS 84-74-2) is a developmental and reproductive toxin that causes a broad range of birth defects resulting in neurological impairments.

To date, although the effect of DBP as an endocrine and a reproductive disruptor are established, there are only few studies that address the effects of low levels of DBP. In addition, there are limited reports on the neurotoxic effects of this phthalate on the developing and mature brain. Additional research analyzing the low-dose effects of this endocrine disruptor need to be further investigated, since it is most un likely for humans to be exposed to high concentrations of DBP. This study aimed to investigate the neurotoxic effects of gestational exposure to low levels of DBP in the offspring of mice.

Our current findings suggest that gestational exposure to low doses of DBP causes gender-specific neurobehavioral abnormalities in the offspring, consistent with those found in humans on the autism spectrum. These neurobehavioral alterations elicited by DBP were consistent with altered inhibitory (GABA) function in the brain. We hypothesized that gestational exposure to DBP altered the maturation of the GABAergic system, which lead to developmental delay observed. In addition, we used the fragile x mouse model (fmr1 ko) and found that all DBP-induced phenotypes were reproducible and exaggerated in the fmr1 ko. Since fmr1 ko mice have diminished GABAergic function, show abnormal expression of many genes implicated in the etiology of autism, and show many of DBP-induced behaviors (e.g heightened anxiety, and hyperactivity) this further confirmed the strong link between the genetic makeup and the susceptibility to environmental risk factors. Our data showed that adult male mice at postnatal day 60 (PND 60) injected with DBP (1 mg/kg BW i.p) showed significant neurobehavioral alterations characterized by reduced locomotive activity in the open field test. These effects were observed 15 min post DBP injection, indicating a fast-acting mechanism. These findings suggested that DBP targeted key proteins regulating neurobehavioral activity and that gestational exposure may alter the biochemistry of the brain to induce long lasting neurobehavioral deficits. Thus, in order to determine the effects of DBP on early brain development, we injected pregnant mice with DBP on gestational day 10 in order to target critical windows of brain development, mainly neurogenesis (embryonic day 10 (E10) and E20). This critical window precedes dendritic growth and spinogenesis that spans from E20 and continues to postnatal day 40 (PND 40). Recent findings in the past decade have introduced the ideology that the brain generates neurosteroids de novo using steroidogenic key proteins similar to those found in periphery glands. These neurosteroids are produced in high concentrations early in development to promote the protection and development of the central nervous system. DBP interferes with steroidogenesis by down-regulating the expression of key steroidogenic proteins during embryogenesis. Interference during this time period can cause developmental delay and alterations in the organization of the GABAergic system critical for the formation and connection of neuronal networks. Our findings demonstrated that some of the neurobehavioral phenotype elicited by a single injection to the adult mice could be reproduced in the offspring of DBP-injected pregnant mice. The prenatal exposure of DBP in the offspring mice at PND 60 exhibited increase in locomotive activity and anxiety, heightened fear-potentiated freezing response and a significant decrease in learning as measured by Morris water maze and the acquisition and retention of a passive avoidance task. In the social interaction paradigm, DBP offspring exposed mice exhibited a reduction in the number of social interactions when presented with a stranger mouse compared to non-DBP treated mice. Interestingly, there was a significant decrease observed in fmr1 ko male mice. Our findings for the passive avoidance learning task were also male-specific. These neurobehavioral effects elicited by exposure to DBP were consistent with altered inhibitory function in the brain of these mice. Since all the behavioral tests results were heightened in males much more than females; we sought to focus our efforts on understanding the molecular mechanisms underlying the effects of DBP on neurodevelopment in males.

To elucidate the molecular and neuronal basis mediating DBP-induced neurobehavioral alterations, we mapped the temporal expression of GABAergic markers (GABAA receptor and glutamic acid decarboxylase (GAD67)). Since the GABAergic system plays a crucial role early in development by establishing neuronal formation and networks; we investigated the expression of two essential co-transporters that aid the system in orchestrating these events - The sodium potassium chloride co-transporter (NKCC1) and potassium chloride co-transporter (KCC2). These pumps are responsible for establishing the chloride electrochemical gradient switch early in development. The early gradient generates the excitatory actions of GABA and results in the driving force behind the formation of these neuronal networks. Finally, we analyzed key proteins in the steroidogenic pathway (steroidogenic acute regulatory protein (StAR) and the mitochondrial enzyme P450 side chain cleavage (P450scc) in the brain. These proteins are rate limiting and essential in the synthesis of neurosteroids like progesterone and estradiol. These critical neurosteroids are essential in the regulation of dendritic growth, spine generation and synaptogenesis.

Our results showed that gestational exposure of DBP significantly increased mRNA levels of StAR and P450scc. Additionally, mRNA levels for the β3 subunit of the GABAA receptor were significantly upregulated in early development (PND 1 and 7). Although embryogenesis marks the time of increased growth, development and increased peak production of neurosteroids, these findings are consistent with studies showing that insult to the brain also elevates mRNA levels of StAR and the activity of P450scc as a neuroprotective mechanism. Later in development at PND 60, the levels of mRNA were significantly decreased. Consistent with this reduction in gene expression of StAR and P450scc, GAD67 and the GABAA receptor protein expression were also reduced.

Since the GABAergic system is the major inhibitory system in the CNS and steroid hormones regulate GABAA receptors, we tested the threshold for seizure induction as an index for the potency of the GABAergic system. We found that the offspring of DBP-injected mice had a significant decrease in the threshold of limbic seizures as measured by reduced latency, increased duration and mortality rate in response to kainic acid injection (15 mg/kg BW). Additionally, in vivo electrophysiological recordings from the cortex showed that in utero DBP-injected mice experienced higher frequency discharge and lower threshold of seizure activity when injected with low doses of kainic acid (5mg/kg BW) compared to controls. Furthermore, the fmr1 ko, with intrinsic seizure susceptibility, showed a further reduction in seizure threshold and increased excitability when exposed to DBP in utero.

Our findings indicated that gestational exposure of DBP induced neurotoxicity in the brain by disrupting the expression of key proteins in the steroidogenic pathway. Furthermore, disruption of this pathway early in development may have caused a delay in the developmental maturation of the GABAergic system. These results may provide supporting evidence in the understanding of the neurotoxicity of DBP in the developing and mature brain.

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