Determinants of vitamin D status among Black and White low-income pregnant and non-pregnant reproductive-aged women from Southeast Louisiana.

BACKGROUND: Vitamin D deficiency is a growing public health problem, with pregnant women being particularly vulnerable due to its influences on maternal and neonatal outcomes. However, there are limited data published about mediators of vitamin D status in Louisiana women. We aimed to assess the vitamin D status and its determinants among low-income pregnant and non-pregnant reproductive-aged women from southeast Louisiana. METHODS: This study was conducted using data from the Gulf Resilience on Women's Health (GROWH) research consortium cohort of pregnant and non-pregnant women which contained sociodemographic and dietary variables as well as blood and salivary element concentrations. Serum 25-hydroxy vitamin D was measured using an enzyme-linked immunosorbent assay in 86 pregnant and 98 non-pregnant women with an even distribution of race in both groups. RESULTS: The prevalence of deficient vitamin D levels in the total cohort (184 women) was 67% and the mean 25(OH) vitamin D3 was 24.1 ng/mL (SD 10.7). Self-identifying as White, being pregnant, autumn season, young age and high exposure to tobacco smoke measured by cotinine were significantly associated with higher serum levels of vitamin D. Visiting Women and Infant clinics (WIC) was an important determinant in improving 25(OH) vitamin D3 levels for Black women but not for White women and concentrations varied more among Black women across seasons compared to White women. CONCLUSIONS: Serum vitamin D levels are inadequate among a high proportion of Black and White low-income pregnant and reproductive-aged women living in Southeast Louisiana who were enrolled in the GROWH study. Black women who are over 35 years old and non-WIC participants constitute the subpopulation most at risk for vitamin D deficiency, especially during the winter. As an overall higher level of deficiency exists in Black women, if even small behavioral and dietary modifications are produced by WIC, this can lead to a comparatively greater improvement in vitamin D status in women from Southeast Louisiana who self-identify as Black.

Protective role of CYP2E1 inhibitor diallyl disulfide (DADS) on alcohol-induced malondialdehyde-deoxyguanosine (M1dG) adduct formation.

BACKGROUND: Alcohol use disorders are often associated with lung disease. Alcohol exposure leads to the production of reactive oxygen species, lipid peroxidation, and formation of malondialdehyde (MDA) as well as to induce the expression of cytochrome p450 2E1 (CYP2E1). Likewise, cigarette smoking can lead to lung lipid peroxidation and formation of MDA. MDA can bind to DNA forming MDA-deoxyguanosine (M1dG) adducts, which have been implicated in alcohol-related cancers and cardiovascular disease. Because CYP2E1 regulates MDA production, and our previous studies have shown that alcohol and cigarette smoke can lead to MDA formation, we hypothesized that CYP2E1 would modulate M1dG adduct formation and single-strand DNA damage in alcohol- and cigarette smoke-exposed lung cells and tissue. METHODS: Normal human bronchial epithelial cells (HBECs) were pretreated with 10 µM diallyl disulfide (DADS) for 1 hour and treated with 80 mM ethanol (EtOH) ± 5% cigarette smoke extract (CSE) for 3 hours for comet assay and 6 hours for CYP2E1, MDA, and M1dG adduct assays. C57BL/6 mice were administered 20% EtOH ad libitum in drinking water for 8 weeks and exposed to whole-body cigarette smoke for 5 weeks. Mice were also fed a CYP2E1 inhibitor, DADS, at 1 µM/g of feed in their daily diet for 7 weeks. Whole lung tissue homogenate was used for CYP2E1, MDA, and M1dG adduct assays. RESULTS: EtOH exposure significantly increased HBEC olive tail moment. DADS pretreatment of HBECs attenuated this EtOH effect. EtOH also induced MDA and M1dG adduct formation, which was also significantly reduced by DADS treatment. CSE ± EtOH did not enhance these effects. In lung tissue homogenate of 8-week alcohol-fed mice, MDA and M1dG adduct levels were significantly elevated in comparison with control mice and mice fed DADS while consuming alcohol. No increase in MDA and M1dG adduct formation was observed in 5-week cigarette smoke-exposed mice. CONCLUSIONS: These findings suggest that CYP2E1 plays a pivotal role in alcohol-induced M1dG adducts, and the use of DADS as dietary supplement can reverse the effects of alcohol on M1dG formation.

Co-exposure to cigarette smoke and alcohol decreases airway epithelial cell cilia beating in a protein kinase Cε-dependent manner.

Alcohol use disorders are associated with increased lung infections and exacerbations of chronic lung diseases. Whereas the effects of cigarette smoke are well recognized, the interplay of smoke and alcohol in modulating lung diseases is not clear. Because innate lung defense is mechanically maintained by airway cilia action and protein kinase C (PKC)-activating agents slow ciliary beat frequency (CBF), we hypothesized that the combination of smoke and alcohol would decrease CBF in a PKC-dependent manner. Primary ciliated bronchial epithelial cells were exposed to 5% cigarette smoke extract plus100 mmol/L ethanol for up to 24 hours and assayed for CBF and PKCε. Smoke and alcohol co-exposure activated PKCε by 1 hour and decreased both CBF and total number of beating cilia by 6 hours. A specific activator of PKCε, DCP-LA, slowed CBF after maximal PKCε activation. Interestingly, activation of PKCε by smoke and alcohol was only observed in ciliated cells, not basal bronchial epithelium. In precision-cut mouse lung slices treated with smoke and alcohol, PKCε activation preceded CBF slowing. Correspondingly, increased PKCε activity and cilia slowing were only observed in mice co-exposed to smoke and alcohol, regardless of the sequence of the combination exposure. No decreases in CBF were observed in PKCε knockout mice co-exposed to smoke and alcohol. These data identify PKCε as a key regulator of cilia slowing in response to combined smoke and alcohol-induced lung injury.

Hybrid malondialdehyde and acetaldehyde protein adducts form in the lungs of mice exposed to alcohol and cigarette smoke.

BACKGROUND: Most alcohol abusers smoke cigarettes and approximately half of all cigarette smokers consume alcohol. However, no animal models of cigarette and alcohol co-exposure exist to examine reactive aldehydes in the lungs. Cigarette smoking results in elevated lung acetaldehyde (AA) and malondialdehyde (MDA) levels. Likewise, alcohol metabolism produces AA via the action of alcohol dehydrogenase and MDA via lipid peroxidation. A high concentration of AA and MDA form stable hybrid protein adducts known as malondialdehyde-acetaldehyde (MAA) adducts. We hypothesized that chronic cigarette smoke and alcohol exposure in an in vivo mouse model would result in the in vivo formation of MAA adducts. METHODS: We fed C57BL/6 mice ad libitum ethanol (20%) in drinking water and exposed them to whole-body cigarette smoke 2 h/d, 5 d/wk for 6 weeks. Bronchoalveolar lavage fluid and lung homogenates were assayed for AA, MDA, and MAA adduct concentrations. MAA-adducted proteins were identified by Western blot and ELISA. RESULTS: Smoke and alcohol exposure alone elevated both AA and MDA, but only the combination of smoke+alcohol generated protein-adducting concentrations of AA and MDA. MAA-adducted protein (~500 ng/ml) was significantly elevated in the smoke+alcohol-exposed mice. Of the 5 MAA-adducted proteins identified by Western blot, 1 protein band immunoprecipitated with antibodies to surfactant protein D. Similar to in vitro PKC stimulation by purified MAA-adducted protein, protein kinase C (PKC) epsilon was activated only in tracheal epithelial extracts from smoke- and alcohol-exposed mice. CONCLUSIONS: These data demonstrate that only the combination of cigarette smoke exposure and alcohol feeding in mice results in the generation of significant AA and MDA concentrations, the formation of MAA-adducted protein, and the activation of airway epithelial PKC epsilon in the lung.

Serum 25-Hydroxyvitamin D and Diet Mediates Vaso-Occlusive Related Hospitalizations in Sickle-Cell Disease Patients.

Sickle cell disease (SCD) is a genetic disorder resulting from the presence of mutated hemoglobin S (HbS). Homozygous carriers will present with early manifestations of painful vaso-occlusive crises. SCD patients have been reported to be severely deficient in vitamin D (<20 ng/mL). Four years (2010⁻2014) of individual de-identified Sickle Cell Clinic of Southern Louisiana (SCCSL-SCD) patient records were analyzed for vitamin D status and the level of crisis-related ER/hospital utilization. To determine the dietary, and behavioral mediators of SCD-crisis in our study population, a cohort of 102 SCCSL-SCD patients were administered a survey that evaluated sun exposure, dietary behaviors, and pain frequency and severity. Patients with circulating levels of 25(OH)D3 less than 14.1 ng/mL reported having more crisis-related hospital visits per year (10) than patients with 25(OH)D3 serum levels >34 ng/mL. The result of the dietary survey detailed a relationship between patients who reported to have "Almost Never" consumed fish or milk in their diets and more frequent hospital stays and ER visits than those who reported consuming these products on a daily basis. Those who consumed these foods in their diet several times a month also had fewer ER visits when compared to the "Almost Never" category.

Dietary diallyl disulfide supplementation attenuates ethanol-mediated pulmonary vitamin D speciate depletion in C57Bl/6 mice.

BACKGROUND: Slightly more than 5 % of the United States population heavily consumes ethanol, i.e., more than 14 drinks for men and 7 drinks for women a week. Chronic ethanol consumption can result in increased liver disease, reduced recovery from burn injury, and more frequent and severe respiratory infections. Chronic ethanol over-consumption also leads to vitamin D dysmetabolism and depletion. Vitamin D is a fat-soluble pro-hormone that regulates musculoskeletal health, cellular proliferation/differentiation, and innate and adaptive immune response. METHODS: In this study, C57BL/6 mice were fed 20 % ethanol in their water ad libitum for 7 weeks. Some mice were fed either a standard chow or a modified diet containing 0.15 µg/day of diallyl disulfide (DADS). Whole blood, lung tissue, and bronchial alveolar lavage fluid (BALF) were collected at sacrifice and analyzed for 25(OH) D3, 1,25 (OH)2D3, vitamin D receptor VDR, CYP2E1, and CYP27B1 levels. RESULTS: Ethanol reduced 25(OH) D3 and 1,25 (OH)2D3 in lung tissue and BALF on average 31 %. The largest ethanol-mediated reduction was in the 1,25 (OH)2D3 (42 %) measured in the BALF. Dietary supplementation of DADS restored BALF and lung tissue protein of 25(OH) D3 and 1,25(OH)2D3 to control levels. Chronic ethanol consumption also resulted in tissue increases of vitamin D response (VDR) protein, Cyp2E1, and reductions in vitamin D-activating enzyme CYP27B1. All three of these effects were attenuated by dietary supplementation of DADS. CONCLUSIONS: In conclusion, the pulmonary metabolic disturbances mediated by chronic ethanol consumption as measured by 1,25(OH)2D3 protein levels, epithelial lining fluid, and lung tissue can be ameliorated by dietary supplementation of DADS in C57BL/6 mice.

Diallyl sulfide inhibits diethylstilbestrol induced DNA damage in human breast epithelial cells (MCF-10A).

Breast cancer is the second leading cause of cancer deaths in women in the United States. Diethylstilbestrol (DES) is a synthetic estrogen that has been shown to cause cancer in animals and humans, altering cell viability as well as inducing DNA damage. Diallyl sulfide (DAS) is a garlic organosulfide that has been shown to inhibit both the initiation and promotion phases of cancer in vivo and in vitro, as well as reduce the risk of cancer in epidemiological studies. MCF-10A cells, regarded as a normal breast epithelial cell line, were treated with varying concentrations of DES, DAS or various dose combinations of DES and DAS concomitantly, and assessed for cell viability, DNA strand breaks, and lipid peroxidation. DES (10µM) in combination with 1, 10, or 100µM DAS resulted in a 31%, 34%, or 36% respective increase in cell viability compared to the DES treatment alone, after 24h. At the same time point, 1, 10, and 100µM DAS were all effective in significantly reducing DES (100µM)-induced strand breaks to near that of the vehicle control. Additionally, 1µM DAS was effective in significantly reducing DES (100µM)-induced lipid peroxidation after 3h. The results of this research suggest that DAS is effective in recovering cell viability, attenuating DNA strand breaks, and decreasing lipid peroxidation in MCF-10A cells.

Malondialdehyde-acetaldehyde-adducted protein inhalation causes lung injury.

In addition to cigarette smoking, alcohol exposure is also associated with increased lung infections and decreased mucociliary clearance. However, little research has been conducted on the combination effects of alcohol and cigarette smoke on lungs. Previously, we have demonstrated in a mouse model that the combination of cigarette smoke and alcohol exposure results in the formation of a very stable hybrid malondialdehyde-acetaldehyde (MAA)-adducted protein in the lung. In in vitro studies, MAA-adducted protein stimulates bronchial epithelial cell interleukin-8 (IL-8) via the activation of protein kinase C epsilon (PKCɛ). We hypothesized that direct MAA-adducted protein exposure in the lungs would mimic such a combination of smoke and alcohol exposure leading to airway inflammation. To test this hypothesis, C57BL/6J female mice were intranasally instilled with either saline, 30µL of 50µg/mL bovine serum albumin (BSA)-MAA, or unadducted BSA for up to 3 weeks. Likewise, human lung surfactant proteins A and D (SPA and SPD) were purified from human pulmonary proteinosis lung lavage fluid and successfully MAA-adducted in vitro. Similar to BSA-MAA, SPD-MAA was instilled into mouse lungs. Lungs were necropsied and assayed for histopathology, PKCɛ activation, and lung lavage chemokines. In control mice instilled with saline, normal lungs had few inflammatory cells. No significant effects were observed in unadducted BSA- or SPD-instilled mice. However, when mice were instilled with BSA-MAA or SPD-MAA for 3 weeks, a significant peribronchiolar localization of inflammatory cells was observed. Both BSA-MAA and SPD-MAA stimulated increased lung lavage neutrophils and caused a significant elevation in the chemokine, keratinocyte chemokine, which is a functional homologue to human IL-8. Likewise, MAA-adducted protein stimulated the activation of airway and lung slice PKCɛ. These data support that the MAA-adducted protein induces a proinflammatory response in the lungs and that the lung surfactant protein is a biologically relevant target for malondialdehyde and acetaldehyde adduction. These data further implicate MAA-adduct formation as a potential mechanism for smoke- and alcohol-induced lung injury.

Alcohol exposure alters mouse lung inflammation in response to inhaled dust.

Alcohol exposure is associated with increased lung infections and decreased mucociliary clearance. Occupational workers exposed to dusts from concentrated animal feeding operations (CAFOs) are at risk for developing chronic inflammatory lung diseases. Agricultural worker co-exposure to alcohol and organic dust has been established, although little research has been conducted on the combination effects of alcohol and organic dusts on the lung. Previously, we have shown in a mouse model that exposure to hog dust extract (HDE) collected from a CAFO results in the activation of protein kinase C (PKC), elevated lavage fluid cytokines/chemokines including interleukin-6 (IL-6), and the development of significant lung pathology. Because alcohol blocks airway epithelial cell release of IL-6 in vitro, we hypothesized that alcohol exposure would alter mouse lung inflammatory responses to HDE. To test this hypothesis, C57BL/6 mice were fed 20% alcohol or water ad libitum for 6 weeks and treated with 12.5% HDE by intranasal inhalation method daily during the final three weeks. Bronchoalveolar lavage fluid (BALF), tracheas and lungs were collected. HDE stimulated a 2-4 fold increase in lung and tracheal PKCε (epsilon) activity in mice, but no such increase in PKCε activity was observed in dust-exposed mice fed alcohol. Similarly, alcohol-fed mice demonstrated significantly less IL-6 in lung lavage in response to dust than that observed in control mice instilled with HDE. TNFα levels were also inhibited in the alcohol and HDE-exposed mouse lung tissue as compared to the HDE only exposed group. HDE-induced lung inflammatory aggregates clearly present in the tissue from HDE only exposed animals were not visually detectable in the HDE/alcohol co-exposure group. Statistically significant weight reductions and 20% mortality were also observed in the mice co-exposed to HDE and alcohol. These data suggest that alcohol exposure depresses the ability of the lung to activate PKCε-dependent inflammatory pathways to environmental dust exposure. These data also define alcohol as an important co-exposure agent to consider in the study of inhalation injury responses.