Garlic Extract Diallyl Sulfide (DAS) Activates Nuclear Receptor CAR to Induce the Sult1e1 Gene in Mouse Liver

Constituent chemicals in garlic extract are known to induce phase I and phase II enzymes in rodent livers. Here we have utilized Car+/+ and Car−/− mice to demonstrate that the nuclear xenobiotic receptor CAR regulated the induction of the estrogen sulfotransferase Sult1e1 gene by diallyl sulfide (DAS) treatment in mouse liver. DAS treatment caused CAR accumulation in the nucleus, resulting in a remarkable increase of SULT1E1 mRNA (3,200 fold) and protein in the livers of Car+/+ females but not of Car−/− female mice. DAS also induced other CAR-regulated genes such as Cyp2b10, Cyp3a11 and Gadd45β. Compared with the rapid increase of these mRNA levels, which began as early as 6 hourrs after DAS treatment, the levels of SULT1E1 mRNA began increasing after 24 hours. This slow response to DAS suggested that CAR required an additional factor to activate the Sult1e1 gene or that this activation was indirect. Despite the remarkable induction of SULT1E1, there was no decrease in the serum levels of endogenous E2 or increase of estrone sulfate while the clearance of exogenously administrated E2 was accelerated in DAS treated mice

CAR dependent <i>Sult1e1</i> gene induction.

<p>(A) Wild type and <i>Car</i> null mice were given DAS (80 mg/100 g body weight) or DADS (8 mg/100 g body weight) by gastric gavage. 24 hrs after chemical administration, <i>Sult1e1</i> and <i>Car</i> gene expression was analyzed by quantitative PCR as described in the methods section. The relative mRNA amount of each gene was calculated so that wild type vehicle treated samples have one unit of expression. (B) Left panel; Liver cytosol extracts from the same mice used in (A) were prepared and SULT1E1 protein content was analyzed by western blotting. Recombinant mouse SULT1E1 protein was used as positive control and indicated with R, while V represents vehicle treatment. Anti Hsp70 western blotting is shown as a loading control. Right panel; Estrogen sulfotransferase activities were measured for liver extracts from the same mice used in (A). n.d. stands for no activity detected. (C) Liver nuclear extracts from the same mice used in (A) were prepared and CAR protein content was analyzed by western blotting. Anti Laminβ western blotting is shown as a loading control.</p

The effect of DAS treatment on serum estrogen.

<p>(A) E2 levels in sera from 4 week old mice treated with vehicle or DAS for 48 hrs are shown. * p<0.05 for vehicle-injected group versus DAS-injected group. (B) Estrone sulfate levels in sera from 8 week old female mice treated with vehicle or DAS for 48 hrs are shown. (C) The clearance of exogenously administered E2 from mice sera was analyzed using DAS treated and vehicle treated ovariectomized mice. Ovariectomized mice were given DAS via gavage and E2 (0.5 mg/kg) was injected subcutaneously 42 hrs after the DAS administration. The E2 content in serum was determined by radioimmunoassay and plotted against time after E2 treatment. **, p<0.01 for vehicle-injected group versus DAS-injected group at 3 hrs after E2 treatement.</p

Gene activation by DAS.

<p>The expression of <i>Cyp2b10</i>, <i>Cyp3a11</i>, <i>Gadd45b</i> and <i>Cyp1a1</i> genes was analyzed in DAS treated and vehicle treated mice livers. The same RNA samples treated with vehicle or DAS from wild type and <i>Car</i> null mice liver used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021229#pone-0021229-g001" target="_blank">FIG. 1A</a> were analyzed to determine the expression of these genes.</p

Gene induction time course.

<p>(A) Wild type and <i>Car</i> null mice were given DAS (80 mg/100 g body weight) by gastric gavage. B. Following 6, 24, 48, 72, and 96 hrs after chemical administration, the expression of <i>Sult1e1</i>, <i>Cyp2b10</i>, <i>Cyp3a11</i>, and <i>Gadd45b</i> genes was analyzed by quantitative PCR as described in the methods section. The relative mRNA amount of each gene was calculated so that wild type vehicle treated samples have one unit of expression. (B) Liver nuclear extracts from DAS treated mice at indicated time after the chemical administration were prepared and CAR protein content was analyzed by western blotting. Anti TATA binding protein (TBP) western blotting is shown as a loading control.</p