An Integrated Systems Approach to Decode the Impact of Adolescent Nicotine Exposure in Utero and Postnatally Oxycodone Exposed Offspring

Abstract

Perinatal exposure to prescription opioids pose a critical public health risk. Notably, research has found significant neurodevelopmental and behavioral deficits between in utero (IUO) and postnatal (PNO) oxycodone-exposed offspring but there is a notable gap in knowledge regarding the interaction of these groups to other drug exposure, particularly nicotine exposure. Nicotine's widespread use represents a ubiquitous clinical interaction that current research does not address. Children often experiment with drugs and risky behavior; therefore, adolescence is a key timepoint to characterize. This study employed an integrated systems approach to investigate escalating nicotine exposure in adolescence and subsequent nicotine withdrawal in the IUO- and PNO-offspring. Western blot analysis found alterations of the blood-brain barrier (B.B.B.) and synaptic proteins. RT-qPCR further validated immune dysfunction in the central nervous system (CNS) consistent with compromised B.B.B. Peripheral nicotine metabolism was consistent with increased catabolism of nicotine concerning PNO & IUO, a predictor of greater addiction risk. Lastly, behavioral assays found subtle deficits to withdrawal in nociception and anxiety-like behavior. This study showed, for the first time, the vulnerabilities of PNO- and IUO-exposed groups concerning nicotine use during early adolescence and withdrawal.

Keywords: adolescent; behavior; inflammation; nicotine; oxycodone; synaptic biology.

Figure 1

Nicotine and oxycodone administration paradigm. This paradigm details the nicotine administration timing, dose escalation, withdrawal condition used and termination. Generated using Biorender.com

Figure 2

Body weight analysis. A. Mass of animals during nicotine treatment (P28-P43) n=37–60. B. Average weight gain during nicotine treatment. IUO-nicotine group displayed decreased weight gain during nicotine treatment when compared to IUO-sham. Two-way ANOVA followed by Tukey’s test: p-value: ≤.05 Error bars represent SEM: # ≤.05 when compared to sham counterpart. C-D. Weight gain observed during nicotine treatment is sexually dimorphic and significantly affected by the addition of nicotine. n=16–34. Two-way ANOVA followed by Tukey’s test: p-value: ≤.05 Error bars represent S.E.M. Represent significance when compared to Saline-counterpart: **** ≤.00001. # Represents significance when compared to PNO-counterpart: #### ≤.00001. S: Saline, SN: Saline-nicotine, PS: Postnatal oxycodone-saline, P.N.: Postnatal oxycodone-nicotine, I.S.: In utero oxycodone-saline, and IN: In utero oxycodone-nicotine. n=6–16.

Figure 3

Enzymatic evaluation of CYP2A6. A. General overview of the rate-limiting step in nicotine metabolism. B. Western blot analysis on CYP2A6 expression experimental groups revealed the greater potential for metabolism nicotine in PNO and IUO groups. IUO observed an inverse stepwise trend as compared to saline expression of CYP2A6. C. PNO animals displayed greater nicotine metabolism following treatment. Saline-, PNO-, and IUO -withdrawal groups displayed a stepwise trend toward lower serum cotinine. Two-way ANOVA followed by Tukey’s test: p-value: ≤.05 Error bars represent SEM: **(##) ≤ .001, ### ≤.001 N=6/group/treatment. Created with Bio-Render.com

Figure 4

Blood-brain barrier expression analysis. Western blot analysis on homogenate samples isolated from PNO, IUO and saline showing possible compensatory regulation of junctional proteins. Two-way ANOVA followed by Tukey’s test when appropriate: p-value: ≤ .05. Error bars represent SEM: *≤.05. N=6/group/treatment.

Figure 5

Synaptic protein and neurotransmission analysis. Western blot analysis on synaptosomal samples isolated from PNO, IUO, and saline demonstrating unique regulation. Synaptophysin is upregulated in IUO-withdrawal group. Two-way ANOVA followed by Tukey’s test when appropriate: p-value: ≤ .05. Error bars represent SEM: *≤.05. N=6/group/treatment.

Figure 6

Cytokine production and microglia analysis. Interluekin-6 was significantly downregulated in nicotinetreated and withdrawal groups, with a less pronounced effect between PNO and IUO. Two-way ANOVA followed by Dunnett’s test: p-value ≤.05. Error bars represent SEM: *≤.05, ** ≤.001; IBA-1 N=4; IL-6, IL-1β, TNF-α N=5. B. Astrocyte and microglia counting. No significant differences were found. Two-way ANOVA followed by Tukey’s test when appropriate: p-value: ≤.05 Error bars represent SEM: *≤.05, ** ≤.001, n=5–6/group/treatment.

Figure 7

Behavioral analysis. A. Hot Plate analysis at P43 or P44. Overall, no significant differences were observed. PNO-withdrawal animals’ trend toward lower pain threshold when compared to PNO-saline. B. Marble burying assay revealed no significant difference in anxiety-like behavior between groups. Two-way ANOVA followed by Tukey’s test: p-value: ≤.05 Error bars represent S.E.M. n-value displayed in graphs.