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

Abstract

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.

Fig. 1

25(OH)D₃ blood serum and lung tissue levels in 8-week ethanol- and diallyl disulfide (DADS)-fed mice. a Ethanol exposure resulted in statistically unchanged levels of calcidiol in the serum. b Lung tissue levels of calcidiol were reduced by ~50 % in the ethanol-exposed mice (N = 7). *p < 0.05 ethanol compared to the control

Fig. 2

1,25(OH)₂D₃ levels in lung tissue and BALF of ethanol- and DADS-fed mice. Levels of 1,25(OH)₂D₃ in lung tissue and BALF of ethanol, 25(OH)D₃, and DADS-fed mice. a Chronic dietary ethanol exposure reduced lung tissue levels of 1,25(OH)₂D₃ by ~50 %, and dietary consumption of DADS recovered 1,25(OH)₂D₃ by ~85 %. b Chronic dietary ethanol reduced BALF 1,25(OH)₂D₃ levels by ~46 %, and the co-exposure of dietary DADS recovered 1,25(OH)2D3 to control levels. Dietary exposure to higher concentrations of vitamin D3/cholecalciferol (0.25 μg/day) induces the production of 1,25(OH)₂D₃ to more than three times control levels. Ethanol consumption in the higher vitamin D3 reduced 1,25(OH)₂D₃ by ~50 %. N = 7 *p < 0.05 compared to control, #p < 0.05 compared to ethanol, ~p < 0.05 compared to D3 group using one-way ANOVA

Fig. 3

Vitamin D receptor (VDR) levels in 8-week ethanol- and DADS-fed mice. VDR levels were ~49 % higher in the lung tissue of 8-week ethanol-fed mice. The dietary supplementation of DADS completely ablated this ethanol-induced response (N = 7). *p < 0.05 compared to control, #p < 0.05 compared to ethanol group

Fig. 4

CYP27B1 and CYP2E1 enzyme levels in lung tissue of ethanol-fed mice with dietary supplementation of DADS. a CYP27B1 levels in ethanol-fed mice were ~35 % less than the levels in the ethanol group compared to the control. The dietary supplementation of DADS recovered this ethanol-induced reduction by 65 %. b CYP2E1 levels in ethanol-fed mice were statistically significantly increased by ~32 %. This upregulation of CYP2E1 protein was completely ablated by the dietary supplementation of DADS (N = 7). *p < 0.05 compared to control, ~p < 0.05 compared to ethanol group

Fig. 5

Gene expression in tracheal epithelial cells of ethanol and diallyl disulfide-fed mice. Gene expression was quantified in tracheal epithelial cells of 6-week ethanol- and diallyl disulfide (DADS)-fed mice. a An ethanol-mediated threefold upregulation of gene expression was observed in calcidiol-converting enzyme CYP27B1 gene expression. This upregulation was attenuated by DADS co-exposure. b A non-statistical ethanol-mediated sevenfold upregulation of ethanol-induced VDR gene expression was observed, and the co-exposure of DADS attenuated this upregulation. c A statistically significant increase in gene expression was observed in acetaldehyde-producing CYP2E1 in ethanol-fed mice that was ablated by co-exposure to DADS. d A slight non-statistically significant reduction in CYP24A1 gene expression was observed in the tracheal epithelial cells of chronic ethanol and diallyl disulfide-fed mice (N = 7; *p < 0.05 as compared to the control group, ~p < 0.05 as compared to the ethanol group)