The production of oxysterols in bile by activated human leukocytes

Abstract

Oxysterols are naturally occurring intermediates in the conversion of cholesterol to bile acids, the major route for elimination of cholesterol. Additionally, they are important signaling agents, particularly in control of cholesterol synthesis; however, some species also are cytotoxic and carcinogenic. Oxysterols in plasma, contained in oxidized low-density lipoprotein, are strongly correlated with atherosclerosis. Oxysterols are found in infected human bile and the oxysterol content in gallstones correlates with bacterial DNA in the stones. Here we demonstrate that human leukocytes, activated by the presence of bacterial lipopolysaccharide, are able to oxidize cholesterol to a variety of oxysterols, including species known to be carcinogenic. The occurrence of oxidized forms of cholesterol (oxysterols) in living tissues has now been known for more than six decades [1] and these compounds have been associated with atherogenicity for forty years [2]. It is now well established that oxysterols are closely associated with the genesis of atherosclerosis [3][4][5] and most probably with various forms of cancer [6][7][8]. Cancer of the biliary system has recently shown an alarming upsurge in incidence and mortality in both the UK and US [9,10] in addition to continuing increases in countries with already high incidence, such as Japan [11]. Little is known of the etiology of cholangiocarcinoma and gallbladder cancer. However, we have shown that bacterial infection is involved in the pathogenesis of mixed cholesterol stones and brown pigment stones [12] and that the content of oxysterols is negatively correlated with the cholesterol content of gallstones [13]. We have demonstrated the presence of bacteria in gallstones [12] and of oxysterols in human gallstones and bile [13]. We also have shown that hepatic bile from patients with biliary infection contains significantly higher levels of oxysterols than does such bile from patients lacking infection of the biliary system [18]. We hypothesize that the oxidation of cholesterol in bile occurs as a result of the action of activated leukocytes on invading bacteria. Here we demonstrate that the incubation of activated human peripheral leukocytes with normal human bile, or with model bile, can result in the production of a wide range of oxysterols. Keywords Materials and methods Model bileModel bile was made by dissolving 108 mg of sodium taurocholate (Calbiochem, La Jolla, CA) in 10 ml of methanol and adding 38 mg of phosphatidylcholine (Type XVIE, Sigma, St. Louis, MO). Ten milligrams of three-times recrystallized cholesterol was added with suffcient chloroform to dissolve all components with gentle warming. The solvent was removed on a rotary evaporator at 40 °C, the flask filled with argon, sealed, and stored at -70 °C until used. At that time, 10 ml of DMEM (Gibco, Grand Island, NY) was added with warming to make a clear solution. LeukocytesOne hundred milliliters of blood, drawn from volunteers, was mixed with 100 ml Hanks" BSS (Mediatech, Fisher Scientific, Los...

Gallbladder cancer: Can newer insights improve the outcome?

“…Progressive physiological concentration of the bile, leads to their increased levels, promoting chronic injury and mutagenesis. This has been shown experimentally for certain oxysterols 9 . The fundus of the GB being most dependent in the erect position is most prone to stagnation.…”

“…This has been shown experimentally for certain oxysterols. 9 The fundus of the GB being most dependent in the erect position is most prone to stagnation. It is thus not surprising to note that the fundus is the commonest site for GBC.…”

Mechanisms of oxysterol-induced disease: insights from the biliary system

“…Model or human bile incubated with lipopolysaccharide produced a variety of oxysterols, including 3-ketocholest-4-ene, 3-keto-cholesta-4, 6-diene, 7β-hydroxycholesterol, 7-ketocholesterol and 7-ketocholesta-3, 5-diene [53]. Taken together, the results from this series of studies suggest that during bacterial infection of the biliary tract, biliary cholesterol can be converted to a variety of oxysterols.…”

“…In a subsequent in vitro study, incubation of human leukocytes with model bile in the presence of bacterial lipopolysaccharide resulted in increased concentrations of oxysterols [53]. Model or human bile incubated with lipopolysaccharide produced a variety of oxysterols, including 3-ketocholest-4-ene, 3-keto-cholesta-4, 6-diene, 7β-hydroxycholesterol, 7-ketocholesterol and 7-ketocholesta-3, 5-diene [53].…”

The Role of Oxysterols in Human Cancer

“…Oxysterols were found in fresh bile samples, as well as in gallstones, and the occurrence of these oxysterols correlated with the presence of bacteria in the same samples [54]. It has been hypothesized that leukocytes are responsible for oxysterol formation in this case [55], connecting these oxysterols with carcinogenesis of gallbladder or bile duct cells. Moreover, the increased level of several oxysterol-binding proteins (OSBPs) was found in cholangiocarcinoma patients, suggesting that oxysterols take part in progression of this cancer via OSBPs and that OSBPs play a potential role as molecular markers of cholangiocarcinoma [56].…”