Behavioral and Cognitive Performance Following Exposure to Second-Hand Smoke (SHS) from Tobacco Products Associated with Oxidative-Stress-Induced DNA Damage and Repair and Disruption of the Gut Microbiome

Abstract

Exposure to second-hand Smoke (SHS) remains prevalent. The underlying mechanisms of how SHS affects the brain require elucidation. We tested the hypothesis that SHS inhalation drives changes in the gut microbiome, impacting behavioral and cognitive performance as well as neuropathology in two-month-old wild-type (WT) mice and mice expressing wild-type human tau, a genetic model pertinent to Alzheimer's disease mice, following chronic SHS exposure (10 months to ~30 mg/m³). SHS exposure impacted the composition of the gut microbiome as well as the biodiversity and evenness of the gut microbiome in a sex-dependent fashion. This variation in the composition and biodiversity of the gut microbiome is also associated with several measures of cognitive performance. These results support the hypothesis that the gut microbiome contributes to the effect of SHS exposure on cognition. The percentage of 8-OHdG-labeled cells in the CA1 region of the hippocampus was also associated with performance in the novel object recognition test, consistent with urine and serum levels of 8-OHdG serving as a biomarker of cognitive performance in humans. We also assessed the effects of SHS on the percentage of p21-labeled cells, an early cellular marker of senescence that is upregulated in bronchial cells after exposure to cigarette smoke. Nuclear staining of p21-labeled cells was more prominent in larger cells of the prefrontal cortex and CA1 hippocampal neurons of SHS-exposed mice than in sham-exposed mice, and there was a significantly greater percentage of labelled cells in the prefrontal cortex and CA1 region of the hippocampus of SHS than air-exposed mice, suggesting that exposure to SHS may result in accelerated brain aging through oxidative-stress-induced injury.

Keywords: 8-OH-dG; APE1; cognitive performance; gut microbiome; novel object recognition; p21; second-hand smoke.

Figure 1

Microbiome richness (i.e., number of taxa in a sample) (Left) and Shannon entropy (i.e., number of taxa in a sample weighted by taxon abundance) (Right) were increased in male mice exposed to SHS. In contrast, SHS had no detectable effect on measures of α-diversity in female mice. Points represent an α-diversity measure from a single mouse in the study. Horizontal lines represent 95% confident intervals based on a bootstrapping analysis. Samples from SHS-exposed mice are in orange, while samples from air controls are in green. The results are shown with genotype collapsed.

Figure 2

Scatterplots illustrating the relationship between measures of mouse microbiome α-diversity as well as measures of cognitive performance. The top row of plots illustrates results based on microbiome richness (i.e., number of taxa in a sample), while the bottom row illustrates results based on Shannon entropy (i.e., number of taxa weighted by taxon abundance). Each column represents one or more plots based on a particular measure of cognition (Y.maze_PctSponAlt: percentage of spontaneous alterations in a Y maze; Probe1_CumDistSEPlat.cm: cumulative distance to the target location in the first water maze probe trial; Probe2_CumDistSEPlat.cm: cumulative distance to the target location in the second water maze probe trial). Points represent individual microbiome samples. Blue lines illustrate the line of best fit as determined by linear regression. Only significant associations after multiple test correction are shown here.

Figure 3

Ordinations of microbiome β-diversity. The Bray–Curtis dissimilarity metric quantified the dissimilarity in community composition across samples while weighting these differences based on the abundance of taxa. These Bray–Curtis dissimilarities were used to generate the distance based redundancy analysis ordination illustrated here, wherein samples are represented by points in the ordination. This ordination is color coded in three forms representing three different analyses: (A) the relationship between β-diversity and SHS exposure as well as mouse sex; (B) the relationship between β-diversity and distance moved in the open field test; (C) the relationship between β-diversity and cumulative distance to the target location in the second water maze probe trial. The main text provides specific information on how to interpret each of the above plots.

Figure 4

Senescence-associated β-galactosidase (SA-β-gal) staining in the prefrontal cortex (A) and the hippocampus (B) of mice chronically exposed to SHS. Higher magnifications of the prefrontal cortex (boxes: 34.7x). Stained sections from air- and SHS-exposed mice (n = 3/tx) were manually counted for β-Gal staining, as previously described [30]. There was no effect of SHS on β-Gal staining in either brain region.

Figure 5

p21 staining in the prefrontal cortex (A) and CA1 region of the hippocampus (B) of mice chronically exposed to SHS. These brain areas were stained for the senescence marker p21. Note that there was prominent staining of cells within the prefrontal cortex and the hippocampal CA1 region of SHS- than air-exposed mice (“arrows”). Higher magnifications (boxes) of the prefrontal cortex (middle and right images in (A) indicate that the nuclear staining was more prominent in larger cells. (C) Stained sections from both air- and SHS-exposed mice were manually counted for p21 staining, as previously described [42]. The percentages of labelled cells in the prefrontal cortex and hippocampal CA region of SHS-exposed mice were significantly greater than air-exposed mice. Values are expressed as percentages of labeled cells. * p < 0.05, ** p < 0.01, 2-tailed t-tests.

Figure 6

(A) The percentage of 8-OH-dG-labeled cells in the CA1 region of the hippocampus was negatively correlated with the frequency of exploring the novel object (r = −0.8482, p = 0.0326, Pearson. (B) The percentage of 8-OH-dG-labeled cells in the CA1 region of the hippocampus was negatively correlated with the time spent exploring the novel object (r = −0.9182, p = 0.0098, Pearson). (C) The percentage of APE1-labeled cells in the CA1 region of the hippocampus was negatively correlated with the time spent exploring the familiar object (r = −0.8176, p = 0.0469, Pearson).