Effects of 28-day nose-only inhalation of PCB52 (2,2',5,5'-Tetrachlorobiphenyl) on the brain transcriptome

Abstract

A semi-volatile polychlorinated biphenyl (PCB) congener, PCB52, is present in the indoor air of schools; however, the effects of inhaled PCB52 on the brain have not been investigated. This study exposed male Sprague-Dawley rats at 39 days of age and female rats at 42 days of age to PCB52 for 4 hours per day over 28 consecutive days through nose-only inhalation. Neurobehavioral tests were conducted during the last 5 days of exposure. The total estimated PCB52 exposures after 28 days were 1080±20 µg/kg BW for male rats and 1140±10 µg/kg BW for female rats. PCB52 and its metabolites were detected by gas chromatography-tandem mass spectrometry in the brain, lung, and serum, with the lung showing the highest concentrations. PCB52 levels were higher in the brains of females than males. Males showed increased exploratory behavior compared to controls, whereas females exhibited decreased exploratory behavior compared to controls in the same tests. PCB52 exposure did not impact locomotor activity or working memory. Gene expression and pathway analysis in the striatum and cerebellum suggest that PCB52 inhalation causes mitochondrial dysfunction. No significant differences were observed by immunohistochemical evaluation in the density and percent area of total cells, astrocytes, or microglia in the striatum and cerebellar cortex. Our results indicate multilevel effects of inhaled PCB52 on the rat brain, from gene expression to behavioral effects.

Keywords: Exploratory behavior; Inhalation toxicity; Neurotoxicity; Polychlorinated biphenyls; Transcriptomics.

Fig 1.

Adolescent rats were exposed for 4 h daily to inhaled PCB52 for 28 days, and neurobehavioral testing was performed on days 24 to 28. (A) Exposure paradigm of the 28 day exposure of rats to PCB52 and the timeline of the Y-maze (day 24), Open Field Test (OFT, days 25 and 26), Hole Board Test (HBT, day 27), and Elevated Plus Maze (EPM, day 28). (B) Inhalation apparatus used to generate PCB52-laden air and expose rats by nose-only breathing to PCB52. Arrows indicate the direction of airflow. (C) Estimated exposure levels to PCB52 throughout the experiment, and (D) the total estimated PCB52 exposures of PCB52 and sham-exposed male and female rats. Violin plots represent the frequency distribution with median and quartiles indicated by dotted lines (bold and fine lines, respectively) (N=6). Values represent means ± standard deviation.

Fig 2.

Day 1 (A-C) and day 2 (D-F) open field results of male and female rats during the 28 day exposure to inhaled PCB52, including (A) the habitation index derived from the ratio of first 5 min and last 5 min epoch of distance traveled on day 1, (B) the number of entries to the center zone, and (C) the time in the center zone over the first 5 min period of the trial revealed an increased anxiety-like behavior in PCB52 exposed female rats, with a reduced time and number of entries into the center zone. (D) The habituation index derived from the ratio of first 5 min and last 5 min epoch of distance traveled on day 2, (E) the number of entries, and (F) the time spent in the center over the first 5 min period, showed significant interactions between sex and exposure for several endpoints. The violin plots represent the frequency distribution with median and quartiles indicated by dotted lines (bold and fine lines, respectively) (N=6). Effects of sex and exposure were assessed by 2-way ANOVA followed by multiple comparisons uncorrected Fisher’s LSD posthoc test.

Fig 3.

PCB52 and hydroxylated metabolites were detected in the brain, liver, and serum of rats exposed to PCB52 by inhalation. The analysis of serum samples revealed the presence of additional PCB52 metabolites, suggesting that inhaled PCB52 is metabolized to hydroxylated and sulfated metabolites. (A) Target analysis of PCB52 and OH-PCB52 levels (ng/g wet tissue) in brain, lung, and serum using GC-MS/MS. (B) Representative chromatograms, (C) mass spectra, and (D) the relative intensity (%) of OH-PCB52, PCB52 sulfate, OH-PCB52 sulfate and MeO-OH-PCB52 metabolites identified using LC-HRMS. (E) The proposed metabolic pathway of PCB52. #: peak represented impurity; CYP: cytochrome P450; SULT: sulfotransferase; COMT: catechol-O-methyltransferase.

Fig 4.

Inhaled PCB52 alters the transcriptome of the striatum in female (panels A1-A3) and male (panels B1-B2) rats. Association of genes with the top six KEGG pathways and diseases in the striatum of female rats following PCB52 inhalation, identified by an iPathwayGuide analysis, are shown in panels A1 and A2, respectively. The normalized counts of selected genes associated with these KEGG pathways and diseases are plotted in panel A3. The violin plots represent the frequency distribution with median and quartiles indicated by dotted lines (bold and fine lines, respectively). Changes in the top 5 gene sets and KEGG pathways activated and suppressed in the striatum of male rats exposed to inhaled PCB52 compared to sham-exposed animals, identified using a gene set analysis, are shown in panels B1 and B2, respectively. For the corresponding gene set analysis of data from female rats, see Fig. S13. Male RNA sequencing data were not analyzed using the iPathwayGuide software because of the small number of DEGs, see Materials and Methods. The dot size indicates the number of differentially affected genes in the gene set or pathway, and the color indicates the adjusted p-values of the estimated significance of the corresponding enrichment analysis. The GeneRatio indicates the number of genes enriched in a particular gene set over the total number of genes.

Fig 5.

RNA sequencing reveals significant changes in the gene expression in the cerebellum of female (A-C) and male rats (D-F) exposed to PCB52 for 28 days via inhalation compared to the sham animals. (A) Gene set and (B) KEGG pathway enrichment dot plots of the top 5 activated and suppressed genes, and (C) the KEGG “oxidative phosphorylation” pathway in the mitochondria plotted based on fold change in female rats. (D) Gene set and (E) KEGG pathway enrichment dot plots of the top 5 activated and suppressed genes, and (F) the KEGG “oxidative phosphorylation” pathway in the mitochondria plotted based on fold change in male rats. The dot size indicates the number of differentially affected genes, and the color indicates the adjusted p-values of the estimated significance of the corresponding enrichment analysis. The GeneRatio indicates the number of genes enriched in a particular gene set over the total number of genes.