Serotonin and corticosterone rhythms in mice exposed to cigarette smoke and in patients with COPD:implication for COPD-associated neuropathogenesis

The circadian timing system controls daily rhythms of physiology and behavior, and disruption of clock function can trigger stressful life events. Daily exposure to cigarette smoke (CS) can lead to alteration in diverse biological and physiological processes. Smoking is associated with mood disorders, including depression and anxiety. Patients with chronic obstructive pulmonary disease (COPD) have abnormal circadian rhythms, reflected by daily changes in respiratory symptoms and lung function. Corticosterone (CORT) is an adrenal steroid that plays a considerable role in stress and anti-inflammatory responses. Serotonin (5-hydroxytryptamine; 5HT) is a neurohormone, which plays a role in sleep/wake regulation and affective disorders. Secretion of stress hormones (CORT and 5HT) is under the control of the circadian clock in the suprachiasmatic nucleus. Since smoking is a contributing factor in the development of COPD, we hypothesize that CS can affect circadian rhythms of CORT and 5HT secretion leading to sleep and mood disorders in smokers and patients with COPD. We measured the daily rhythms of plasma CORT and 5HT in mice following acute (3 d), sub-chronic (10 d) or chronic (6 mo) CS exposure and in plasma from non-smokers, smokers and patients with COPD. Acute and chronic CS exposure affected both the timing (peak phase) and amplitude of the daily rhythm of plasma CORT and 5HT in mice. Acute CS appeared to have subtle time-dependent effects on CORT levels but more pronounced effects on 5HT. As compared with CORT, plasma 5HT was slightly elevated in smokers but was reduced in patients with COPD. Thus, the effects of CS on plasma 5HT were consistent between mice and patients with COPD. Together, these data reveal a significant impact of CS exposure on rhythms of stress hormone secretion and subsequent detrimental effects on cognitive function, depression-like behavior, mood/anxiety and sleep quality in smokers and patients with COPD

Disorder of G2-M Checkpoint Control in Aniline-Induced Cell Proliferation in Rat Spleen.

Aniline, a toxic aromatic amine, is known to cause hemopoietic toxicity both in humans and animals. Aniline exposure also leads to toxic response in spleen which is characterized by splenomegaly, hyperplasia, fibrosis and the eventual formation of tumors on chronic in vivo exposure. Previously, we have shown that aniline exposure leads to iron overload, oxidative DNA damage, and increased cell proliferation, which could eventually contribute to a tumorigenic response in the spleen. Despite our demonstration that cell proliferation was associated with deregulation of G1 phase cyclins and increased expression of G1 phase cyclin-dependent kinases (CDKs), molecular mechanisms, especially the regulation of G2 phase and contribution of epigenetic mechanisms in aniline-induced splenic cellular proliferation remain largely unclear. This study therefore, mainly focused on the regulation of G2 phase in an animal model preceding a tumorigenic response. Male Sprague-Dawley rats were given aniline (0.5 mmol/kg/day) in drinking water or drinking water only (controls) for 30 days, and expression of G2 phase cyclins, CDK1, CDK inhibitors and miRNAs were measured in the spleen. Aniline treatment resulted in significant increases in cell cycle regulatory proteins, including cyclins A, B and CDK1, particularly phosphor-CDK1, and decreases in CDK inhibitors p21 and p27, which could promote the splenocytes to go through G2/M transition. Our data also showed upregulation of tumor markers Trx-1 and Ref-1 in rats treated with aniline. More importantly, we observed lower expression of miRNAs including Let-7a, miR-15b, miR24, miR-100 and miR-125, and greater expression of CDK inhibitor regulatory miRNAs such as miR-181a, miR-221 and miR-222 in the spleens of aniline-treated animals. Our findings suggest that significant increases in the expression of cyclins, CDK1 and aberrant regulation of miRNAs could lead to an accelerated G2/M transition of the splenocytes, and potentially to a tumorigenic response on chronic aniline exposure

Protein expression of Trx-1 and Ref-1 in rat spleens.

<p>(A) Western blot determination of Trx-1 and Ref-1 in the control and aniline-treated rats. (B) Densitometric measurement of the protein bands. Values are mean ± SD; *<i>p</i> < 0.05.</p

Primer sequences of cyclins A, B1, p21 and p27 for real-time PCR analysis.

<p>Primer sequences of cyclins A, B1, p21 and p27 for real-time PCR analysis.</p

Trichloroethene metabolite dichloroacetyl chloride induces apoptosis and compromises phagocytosis in Kupffer Cells: Activation of inflammasome and MAPKs.

Exposure to trichloroethene (TCE), an occupational and ubiquitous environmental contaminant, is associated with the development of several autoimmune diseases, including autoimmune hepatitis (AIH). However, mechanisms contributing to TCE-mediated AIH are not known. Earlier, we have shown that dichloroacetyl chloride (DCAC), one of the reactive metabolites of TCE with strong acylating capability, can elicit an autoimmune response at much lower dose than TCE in female MRL+/+ mice. Furthermore, Kupffer cells (KCs), the liver resident macrophages, are crucial for hepatic homeostasis, but can also participate in the immunopathogenesis of AIH. However, contribution of KCs in TCE-mediated AIH and the underlying mechanisms are not understood. We hypothesized that increased apoptosis and delayed clearance of apoptotic bodies, due to compromised KC function, will result in the breakdown of self-tolerance, autoimmunity, and ultimately AIH. Therefore, using an in vitro model of immortalized mouse KCs, we investigated the contribution of DCAC in TCE-mediated AIH. KCs were treated with different concentrations of DCAC and apoptosis was measured by Annexin V and PI staining. Also, the impact of DCAC on phagocytic potential of KCs was evaluated. Furthermore, markers of inflammasome (NLRP3 and caspase1) were analyzed by real-time PCR and Western blot analysis. DCAC treatment resulted in significantly increased early and late-stage apoptosis, accompanied with inflammasome activation (NLRP3 increases). DCAC treatment resulted in decreased phagocytic function of KCs in a dose-dependent manner, with reduced MFG-E8 levels (phagocytotic function). Furthermore, DCAC exposure led to induction of phos-ERK and phos-AKT signaling. These findings suggest that DCAC induces apoptosis and inflammasome activation, while compromising the phagocytic function of KCs. Our data support that increased apoptosis and impaired KC function by DCAC could be contributory to TCE-mediated AIH

p27 and p21 gene expression in the rat spleens following aniline exposure.

<p>The fold change in mRNA expression (2^−ΔΔCT) was determined by real-time PCR analysis. Values are means ± SD (<i>n</i> = 3). *<i>p</i> < 0.05 vs. respective controls.</p

Cyclins A and B1 gene expression in rat spleens following aniline exposure.

<p>The fold change in mRNA expression (2^−ΔΔCT) was determined by real-time PCR analysis. Values are means ± SD (<i>n</i> = 3). *<i>p</i> < 0.05 vs. respective controls.</p

Real-time PCR analysis of miRNAs Let-7a, miR-15b, miR24, miR-100 and miR-125 (A), and miRNAs miR-181a, miR-221 and miR-222 (B) expression in rat spleens following aniline exposure.

<p>Values are means ± SD (<i>n</i> = 3). *<i>p</i> < 0.05 vs. respective controls.</p

Protein expression of CDK1 and p-CDK1 in rat spleens.

<p>(A) Western blot determination of CDK1 and p-CDK1 protein expression in control and aniline-treated rats. (B) Densitometric analysis of protein expressions. Values are mean ± SD; *<i>p</i> < 0.05.</p