Effects of Dietary Fish Oil on the Depletion of Carcinogenic PAH-DNA Adduct Levels in the Liver of B6C3F1 Mouse

Many carcinogenic polycyclic aromatic hydrocarbons (PAHs) and their metabolites can bind covalently to DNA. Carcinogen-DNA adducts may lead to mutations in critical genes, eventually leading to cancer. In this study we report that fish oil (FO) blocks the formation of DNA adducts by detoxification of PAHs. B6C3F1 male mice were fed a FO or corn oil (CO) diet for 30 days. The animals were then treated with seven carcinogenic PAHs including benzo(a)pyrene (BaP) with one of two doses via a single intraperitoneal injection. Animals were terminated at 1, 3, or 7 d after treatment. The levels of DNA adducts were analyzed by the 32P-postlabeling assay. Our results showed that the levels of total hepatic DNA adducts were significantly decreased in FO groups compared to CO groups with an exception of low PAH dose at 3 d (P = 0.067). Total adduct levels in the high dose PAH groups were 41.36±6.48 (Mean±SEM) and 78.72±8.03 in 109 nucleotides (P = 0.011), respectively, for the FO and CO groups at 7 d. Animals treated with the low dose (2.5 fold lower) PAHs displayed similar trends. Total adduct levels were 12.21±2.33 in the FO group and 24.07±1.99 in the CO group, P = 0.008. BPDE-dG adduct values at 7 d after treatment of high dose PAHs were 32.34±1.94 (CO group) and 21.82±3.37 (FO group) in 109 nucleotides with P value being 0.035. Low dose groups showed similar trends for BPDE-dG adduct in the two diet groups. FO significantly enhanced gene expression of Cyp1a1 in both the high and low dose PAH groups. Gstt1 at low dose of PAHs showed high levels in FO compared to CO groups with P values being 0.014. Histological observations indicated that FO played a hepatoprotective role during the early stages. Our results suggest that FO has a potential to be developed as a cancer chemopreventive agent

Analysis of the transcriptome in hyperoxic lung injury and sex-specific alterations in gene expression.

Exposure to high concentration of oxygen (hyperoxia) leads to lung injury in experimental animal models and plays a role in the pathogenesis of diseases such as Acute Respiratory Distress Syndrome (ARDS) and Bronchopulmonary dysplasia (BPD) in humans. The mechanisms responsible for sex differences in the susceptibility towards hyperoxic lung injury remain largely unknown. The major goal of this study was to characterize the changes in the pulmonary transcriptome following hyperoxia exposure and further elucidate the sex-specific changes. Male and female (8-10 wk) wild type (WT) (C57BL/6J) mice were exposed to hyperoxia (FiO2>0.95) and gene expression in lung tissues was studied at 48 h. A combination of fold change ≥1.4 and false discovery rate (FDR)<5% was used to define differentially expressed genes (DEGs). Overrepresentation of gene ontology terms representing biological processes and signaling pathway impact analysis (SPIA) was performed. Comparison of DEG profiles identified 327 genes unique to females, 585 unique to males and 1882 common genes. The major new findings of this study are the identification of new candidate genes of interest and the sex-specific transcriptomic changes in hyperoxic lung injury. We also identified DEGs involved in signaling pathways like MAP kinase and NF-kappa B which may explain the differences in sex-specific susceptibility to hyperoxic lung injury. These findings highlight changes in the pulmonary transcriptome and sex-specific differences in hyperoxic lung injury, and suggest new pathways, whose components could serve as sex-specific biomarkers and possible therapeutic targets for acute lung injury (ALI)/acute respiratory distress (ARDS) in humans

Fold change for the selected genes in the microarray and qPCR experiment.

<p><i>Notes: </i><b><i>C</i></b><i>: Common to both male and female animals; </i><b><i>F</i></b><i>: In females only; </i><b><i>M</i></b><i>: In males only.</i></p

Venn diagram showing the comparison of upregulated and downregulated DEGs in the lung between male and female mice after hyperoxia exposure (FiO₂>95%) for 48 hours.

<p>Venn diagram showing the comparison of upregulated and downregulated DEGs in the lung between male and female mice after hyperoxia exposure (FiO₂>95%) for 48 hours.</p

List of genes selected for validation by qPCR analysis.

<p>List of genes selected for validation by qPCR analysis.</p

Real time RT-PCR analysis of mRNA from the lungs of male and female mice exposed to room air or hyperoxia for 48 h.

<p>Values are means ± SEM from n = 3 groups. Each group consisted of pooled RNA from four animals. Figure 5a: Real time RT-PCR analysis of differentially expressed genes common to both male and female mice. Significant upregulation over room air levels are indicated by * p<0.05 and *** p<0.001(one-way ANOVA). Figure 5b: Real time RT-PCR analysis of genes showing sex-specific changes. Fold change over room air levels are represented on the y-axis. Significant up or downregulation over room air levels are indicated by *p<0.05 and **p<0.01 (one-way ANOVA).</p

List of pulmonary DEGs after hyperoxia exposure.

<p>List of pulmonary DEGs after hyperoxia exposure.</p

Increased Slc7a11 expression in the lungs (male) following hyperoxia exposure.

<p><b>8A:</b> Western blot assay for Slc7a11 expression in lungs following hyperoxia exposure. Twenty µg of whole lung protein from male animals at room air and exposed to 48 h of hyperoxia was subjected to western blotting using antibodies against Slc7a11. Under each sample lane is the corresponding β-actin blot to assess for protein loading. <b>8B:</b> Densitometry analysis of pulmonary Slc7a11 immunoblots at room air and after 48 h of hyperoxia exposure. Significant differences from room air controls are indicated by **, p<0.01.</p

Pathway analysis by ORA (over representation analysis).

<p>Analysis of enrichment of biological processes in the three groups; 4a: analysis of DEGs common in both male and female animals after hyperoxia exposure 4b: analysis of DEGs in male animals 4c: analysis of DEGs in female animals. Overrepresentation of gene ontology terms representing biological processes among the DEGs was tested using a conditional hypergeometric test (p-value <0.01).</p