Effects of bile salts on cholestan-3β,5α,6β-triol-induced apoptosis in dog gallbladder epithelial cells

Biochimica et Biophysica Acta 1530 (2001) 199^208 www.elsevier.com/locate/bba

E¡ects of bile salts on cholestan-3L,5K,6L-triol-induced apoptosis in dog gallbladder epithelial cells Tadashi Yoshida a , J. Henrie«tte Klinkspoor a , Rahul Kuver a; *, Martin Poot b , Peter S. Rabinovitch b , Steven P. Wrenn c , Eric W. Kaler c , Sum P. Lee a a

c

Division of Gastroenterology, Department of Medicine, Box 356424, University of Washington School of Medicine, 1959 NE Paci¢c St., and the Veterans A¡airs Medical Center, Seattle, WA 98195, USA b Department of Pathology, University of Washington School of Medicine, Seattle, WA, USA Department of Chemical Engineering, Center for Molecular and Engineering Thermodynamics, University of Delaware, Newark, DE, USA Received 29 September 2000; received in revised form 28 November 2000; accepted 1 December 2000

Abstract Oxysterols are cytotoxic agents. The gallbladder epithelium is exposed to high concentrations of oxysterols, and so elucidating the mechanisms of cytotoxicity in this organ may enhance our understanding of the pathogenesis of biliary tract disorders. We investigated the cytotoxic effects of the oxysterol cholestan-3L,5K,6L-triol (TriolC) on dog gallbladder epithelial cells. Apoptosis was the major form of cytotoxicity, as determined by analysis of nuclear morphologic changes and by multiparameter flow cytometry. Hydrophobic bile salts are known to have cytotoxic effects, whereas hydrophilic bile salts have cytoprotective effects. We therefore examined whether the hydrophobic bile acid taurodeoxycholic acid (TDC) and the hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had modifying effects on oxysterol-induced cytotoxicity. TriolC caused an increase in the number of apoptotic cells from 14 þ 11% (control) to 48 þ 12% of total cells (P 6 0.01). After combining TriolC with TDC, cell apoptosis increased to 63 þ 16% (P 6 0.05), whereas after addition of TUDC, the number of apoptotic cells decreased to 31 þ 12% (P 6 0.05) of total cells. In summary, oxysterols such as TriolC induce apoptosis. Hydrophobic bile salts enhance TriolC-induced apoptosis, whereas hydrophilic bile salts diminish TriolC-induced apoptosis. These results suggest that interactions between oxysterols and bile salts play a role in the pathophysiology of biliary tract disorders. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Oxysterol; Bile salt; Gall bladder; Biliary tract ; Apoptosis

1. Introduction Cholesterol is present ubiquitously in mammalian Abbreviations: DGBE cell, dog gallbladder epithelial cell ; LDH, lactate dehydrogenase; TC, taurocholic acid; TCDC, taurochenodeoxycholic acid; TDC, taurodeoxycholic acid; TriolC, cholestan-3L,5K,6L-triol; TUDC, tauroursodeoxycholic acid * Corresponding author. Fax: +1-206-768-5200; E-mail: [email protected]

tissues, and is considered essential for the formation and function of cellular membranes. Oxysterols are derived in vitro as autoxidation products of cholesterol [1]. Cholesterol is also metabolized to various cholesterol oxides (oxysterols) in vivo during the production of bile acids and hormones. Therefore, many species of oxysterols have been identi¢ed in animal and human tissues [1,2] and in plasma [3]. Oxysterols are also found in high concentrations in the atheromatous plaque [4]. In animal experiments oxysterols

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are much more atherogenic than cholesterol itself [5^ 7]. In humans, oxysterols might be involved in the initiation and progression of atherosclerosis. While various oxysterol species have been recently described in human bile [8], little is known about the e¡ects of oxysterols on the biliary tract. The gallbladder is often regarded as a passive reservoir that stores and concentrates bile secreted by the liver. To achieve this function, during the storage-concentration period the gallbladder epithelium is exposed to high concentrations of bile acids, phospholipids, and cholesterol. We hypothesize that oxysterols in bile are involved in the pathogenesis of biliary tract disease, such as chronic in£ammation and gallstone formation. This is analogous to the possible involvement of oxysterols in blood in the pathogenesis of atherosclerosis. It has been suggested that oxysterols can induce apoptosis, although the mechanism remains unclear. Recently, we described the e¡ects on cell proliferation of two oxysterols, cholestan-3L,5K,6L-triol (TriolC) and 7-ketocholesterol on biliary epithelial cells from the dog [9]. We found that dog gallbladder epithelial (DGBE) cells are more resistant to oxysterol-induced growth inhibition than human pulmonary artery endothelial cells. Moreover, TriolC was much more cytotoxic than 7-ketocholesterol in both cell types. Bile salts are the major lipid components in bile. Hydrophobic bile salts (chenodeoxycholic acid, deoxycholic acid and their conjugates) have cytotoxic e¡ects on hepatocytes and biliary tract epithelial cells, whereas hydrophilic bile salts (ursodeoxycholic acid and its conjugates) have cytoprotective e¡ects [10]. However, there is no information about the interaction between oxysterols and bile salts and their combined cytotoxic e¡ects on biliary epithelial cells. In this study, we have investigated the cytotoxic effects of TriolC in the absence and presence of bile salts on dog gallbladder epithelial cells. 2. Materials and methods 2.1. Materials Vitrogen was purchased from Celtrix Laboratories (Palo Alto, CA, USA). Tissue culture plates were from Falcon (Lincoln Park, NJ, USA). TriolC was

obtained from Steraloids (Wilton, NH, USA). Taurocholic acid (TC), taurochenodeoxycholic acid (TCDC), taurodeoxycholic acid (TDC), and tauroursodeoxycholic acid (TUDC) were obtained from Calbiochem-Novabiochem (La Jolla, CA, USA) or Sigma (St. Louis, MO, USA). Cell culture medium and reagents were obtained from Sigma. Hoechst 33342, SYTO 11, and CMXRos dyes were purchased from Molecular Probes (Eugene, OR, USA). 2.2. Cell culture Gallbladder epithelial cells were isolated from dog gallbladder by trypsinization, as previously described [11]. Stock cultures were grown on 60 mm petri dishes coated with 1 ml Vitrogen gel (1:1 mixture of Vitrogen and medium) in Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 20 mM HEPES, 100 IU/ml penicillin, and 100 Wg/ml streptomycin. Medium was changed twice a week and the cells were maintained in a 37³C incubator with 5% CO2 . The cells were passaged when con£uent (every 7^10 days), using trypsin (2.5 g/l) and EDTA (1 g/l) treatment. 2.3. TriolC and bile salt treatments DGBE cells were plated onto Vitrogen-coated plates in culture medium 24 h before treatment. TriolC was dissolved in pure ethanol at di¡erent concentrations and added to the culture medium at a ¢nal ethanol concentration of 0.5%. Bile salts were dissolved in serum free culture medium at a concentration of 100 mM and subsequently diluted in culture medium to their ¢nal concentrations. The cells were returned to the incubator until harvesting of the samples. 2.4. Counting of viable cells DGBE cells were plated onto Vitrogen-coated 24well plates at 20 000 cells per well and cultured for 24 h. TriolC and/or bile salts were then added to the cells as described above and subsequently the cells were returned to the incubator for 24^72 h. After treatment the cells were harvested by trypsinization. Viable cells that excluded Trypan blue were then counted using a hemocytometer. Untreated cells

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DGBE cells were plated onto Vitrogen-coated 24well plates at 50 000 cells per well and cultured for 24 h. TriolC and/or bile salts were then added to the cells as described above and subsequently the cells were returned to the incubator for 4 h. After treatment the cell culture medium was harvested and spun down at 500Ug for 5 min to obtain cell free supernatant. Activity of the endogenous enzyme LDH in the medium was assayed by the method of Amador et al. [12]. Untreated cells were used as a control. Results are expressed as a percentage of control in units of LDH activity per mg cell protein.

culture medium. The cell suspensions were treated with 15 Wl of 1 mM Hoechst 33342 dye, 15 Wl of 10 WM SYTO 11 dye, and 1.5 Wl of 200 nM CMXRos dye, and incubated at 37³C for 30 min. For each sample, 40 000 events were analyzed with a Coulter Epics Elite Flow Cytometer (Coulter, Hialeah, FL, USA) equipped with 10 mW UV excitation and 15 mW 488 nm excitation. The SYTO 11 and CMXRos dyes were excited with the 488 nm line of the argon laser, SYTO 11 £uorescence was collected with a 525/40 nm bandpass ¢lter and CMXRos was collected with a 645 nm longpass ¢lter. Cell cycle distributions were obtained from the Hoechst 33342 data. Cell cycle analysis was performed using the MultiCycle software package (Phoenix Flow Systems, San Diego, CA, USA). Results are expressed as a percentage of total cell counts in each assay.

2.6. Cell morphology determination

2.8. Statistical analysis

DGBE cells were plated onto 24-well plates at 60 000 cells per well and cultured for 24 h. Cells were treated with bile salts with or without TriolC as outlined above for 24 h. The experiment was done in quadruplicate on three separate occasions. Cells were ¢xed and stained using the Di¡-Quik Stain Kit (Dade Behring, Dudingen, Switzerland), and examined using an Olympus IMT-2 inverted microscope with an attached Sony CCD/RGB digital camera. Images were captured using MCID-M5 software (Imaging Research, St. Catherines, Ont., Canada).

Data are expressed as the mean þ S.D. and Student's t-test was used to assess the signi¢cance of di¡erences; P 6 0.05 was considered signi¢cant.

were used as control. Results are expressed as a percentage of control. 2.5. Lactate dehydrogenase (LDH) release assay

2.7. Flow cytometry Cell cycle status and the number of apoptotic cells were determined using £ow cytometry by simultaneous staining with Hoechst 33342, SYTO 11 and CMXRos dyes. DGBE cells were plated onto 60 mm petri dishes coated with Vitrogen at 200 000 cells per dish and cultured for 24 h. TriolC and/or bile salts were then added to the cells as described above, and the cells were returned to the incubator for 24 h. After treatment, £oating cells in the medium were collected. Cells that remained attached to the Vitrogen coating were harvested with trypsin/EDTA, and mixed with £oating cells. The cells were spun down at 500Ug for 5 min and resuspended in 1.5 ml of

3. Results 3.1. The e¡ect of TriolC on dog gallbladder epithelial cell viability We examined the cytotoxic e¡ects of TriolC on DGBE cell viability. DGBE cells were treated with TriolC at concentrations ranging from 10 WM to 40 WM for up to 72 h and the number of viable cells was counted every 24 h by Trypan blue exclusion (Fig. 1). No signi¢cant inhibition of cell growth was observed in the ¢rst 24 h after treatment. However, at higher concentrations of TriolC (30 WM and 40 WM) cell growth was inhibited. After 48 h of incubation, both lower and higher concentrations of TriolC inhibited cell growth. The inhibitory e¡ect was dose dependent (Fig. 1). At a concentration of 30 WM TriolC, and 24 h of incubation, an inhibition of cell growth to approx. 60% of control was observed. Therefore, in our subsequent experiments, treatment with 30 WM TriolC for 24 h was used as a standard treatment.

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we next investigated the e¡ects of four di¡erent bile salts on TriolC-induced cytotoxicity. Twenty-four hours after plating DGBE cells onto culture plates, bile salts at concentrations ranging from 0.25 mM to 1.0 mM were added to the cells in combination with 30 WM TriolC. After 24 h of treatment, the number of viable cells was counted by Trypan blue exclusion (Table 1). As described above, treatment with 30 WM TriolC in the absence of bile salts resulted in a decrease in the number of viable cells to approx. 60% of the control in each group. When the cells were treated with bile salts alone, only the hydrophobic bile salts TDC and TCDC inhibited cell growth (P 6 0.01), whereas the more hydrophilic bile salts TC and TUDC did not inhibit cell growth. The inhibition of cell growth varied with the hydrophobicity of the bile salts, and only occurred at the highest concentration (1 mM). Next, we investigated the combined e¡ects of bile salts and TriolC on cell growth. When the cells were co-incubated with either TDC or TCDC and TriolC, these bile salts further decreased the number of viable cells compared to treatment with TriolC alone. This e¡ect depended on the concentration of the bile salts. TC enhanced TriolC-induced cytotoxicity only at a concentration of 1 mM. Addition of TUDC, however, increased the number of viable cells compared to treatment with TriolC alone (P 6 0.05). The

Fig. 1. The e¡ect of TriolC on DGBE cells. DGBE cells were treated with TriolC at concentrations ranging from 10 WM to 40 WM for 72 h. Cells were harvested every 24 h and the number of viable cells counted by Trypan blue exclusion. The number of viable cells was calculated as a percentage of control after each treatment. Data are expressed as mean þ S.D. for three individual experiments in triplicate wells (n = 9). a, control ; b, 10 WM TriolC; O, 20 WM TriolC; R, 30 WM TriolC ; E, 40 WM TriolC.

3.2. The e¡ect of bile salts on TriolC-induced cytotoxicity Having established the conditions in which TriolC has an intermediate cytotoxic e¡ect on DGBE cells,

Table 1 The e¡ects of bile salts on cell growth of TriolC-treated DGBE cells Bile salts (mM) TUDC 0.00 0.25 0.50 0.75 1.00

TC

3TriolC

+TriolC

100 þ 13 100 þ 18 100 þ 20 96 þ 21 92 þ 15

b

62 þ 13 62 þ 18b 76 þ 18a 85 þ 23c 83 þ 17c

TCDC

3TriolC

+TriolC

100 þ 7 102 þ 17 94 þ 11 106 þ 11 96 þ 17

b

54 þ 12 50 þ 23b 44 þ 9b 45 þ 10b 30 þ 11b;d

3TriolC 100 þ 8 100 þ 16 95 þ 16 97 þ 14 77 þ 18d

TDC +TriolC

3TriolC

+TriolC

b

100 þ 9 99 þ 10 94 þ 11 94 þ 14 87 þ 11c

62 þ 141b 41 þ 25b;c 33 þ 19b;d 15 þ 16b;d 12 þ 15b;d

60 þ 17 39 þ 24b;c 17 þ 7b;d 13 þ 7b;d 3 þ 3b;d

Bile salts at concentrations ranging from 0.25 mM to 1.0 mM were added to DGBE cells in the presence (+) or absence (3) of 30 WM TriolC. After 24 h, the number of viable cells was counted by Trypan blue exclusion. Untreated cells were used as controls. The number of cells was calculated as a percentage of control after each bile salt treatment. Data are expressed as mean þ S.D. for three individual experiments in triplicate wells (n = 9). Statistical signi¢cances were evaluated between treatments with and without TriolC at the same concentration of bile salts, and between the di¡erent concentrations of bile salt in each series. Bile salts are shown in order of increasing hydrophobicity. a P 6 0.05 compared with treatment with bile acid only at the same concentration. b P 6 0.01 compared with treatment with bile acid only at the same concentration. c P 6 0.05 compared with treatment with 0 mM of bile acids with or without TriolC. d P 6 0.01 compared with treatment with 0 mM of bile acids with or without TriolC.

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number of viable cells increased with increasing TUDC concentration (Table 1). At TUDC concentrations of s 0.75 mM, the cytotoxicity of TriolC was reduced such that there was no signi¢cant di¡erence in cell viability between cells in the absence and presence of TriolC. Therefore, the combined e¡ects of bile salts and TriolC depend on the hydrophobicity of the bile salt used. Hydrophobic bile salts, such as TDC and TCDC, enhance TriolC-induced cytotoxicity. TC, which is of intermediate hydrophobicity, has an intermediate e¡ect, whereas the hydrophilic bile salt TUDC prevents TriolC-induced cytotoxicity. 3.3. Combined e¡ects of bile salts and TriolC on LDH release by DGBE cells We next investigated whether the e¡ects of bile salts on TriolC-induced cytotoxicity could be ascribed to bile salt-induced lysis of DGBE cells. Cells were plated out and cultured for 24 h before treatment. Cells were treated for 4 h with either TDC or TUDC at concentrations ranging from 0.25 mM to 1.0 mM in the presence or absence of 30 WM TriolC. Subsequently, the release of the endogenous enzyme LDH into the culture medium of the cells was measured (Table 2). TriolC by itself did not cause an increase in LDH release from the cells. Neither TDC nor TUDC increased the LDH activity in the medium in the presence of TriolC. This suggests that the e¡ect of bile salts on TriolC-induced cytotoxicity cannot be explained by an increase or decrease in cell lysis.

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3.4. The e¡ects of bile salts and TriolC on the morphology of DGBE cells We examined the morphology of treated and untreated cells using light microscopy. As shown in Fig. 2, cells that were untreated were indistinguishable morphologically from cells treated with TDC or TUDC (Fig. 2A compared to Fig. 2B,C). DGBE cells were con£uent in each scenario, with prominent nuclei. However, cells treated with TriolC were found to have su¡ered cytotoxic damage, with most cells no longer evident over the course of the experiment. Microscopic examination showed the remaining pyknotic nuclei, with a paucity of cytoplasmic staining (Fig. 2D). Cell treated with both TDC and TriolC also showed changes suggestive of widespread cell death, with pyknotic nuclei, reminiscent of apoptotic bodies (Fig. 2E). However, when cells were treated with both TUDC and TriolC, fewer cells were found to be dead, although pyknotic nuclei suggestive of apoptotic bodies were still present (as shown by the arrow in Fig. 2F). These morphologic data suggested that the TriolC-induced cellular effects were due to the induction of apoptosis, and that these TriolC e¡ects were partially prevented when TUDC, but not TDC, was added together with the oxysterol. 3.5. The e¡ects of bile salts and TriolC on cytokinetics and apoptosis in DGBE cells Since both oxysterols and bile salts are known to a¡ect cell apoptosis, we next examined the e¡ects of

Table 2 The e¡ects of bile salts on LDH release from TriolC-treated DGBE cells Bile salts (mM) 0.00 0.25 0.50 1.00

TDC

TUDC

3TriolC

+TriolC

3TriolC

100 þ 5.5 109 þ 13.3 105 þ 18.9 98 þ 14.5

92 þ 17.5 99 þ 19.3 91 þ 16.5 105 þ 38.4

100 þ 10.5 113 þ 19.6 115 þ 23.0 114 þ 26.8

+TriolC 108 þ 17.8 102 þ 16.6 106 þ 24.5 96.9 þ 18.5

Bile salts at concentrations ranging from 0.25 mM to 1.0 mM were added to DGBE cells in the presence (+) or absence (3) of 30 WM TriolC. After 4 h, the LDH activity in the medium was assayed as described in Section 2. Untreated cells were used as controls. The LDH activities were calculated as a percentage of control after each bile salt treatment. Data are expressed as mean þ S.D. for three individual experiments in triplicate wells (n = 9). No statistically signi¢cant di¡erences were found between treatments with and without TriolC at the same concentration of bile salts, and between 0 mM bile salt and the indicated concentration of bile salt in each series.

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Fig. 2. Cellular morphology e¡ects of bile salts and TriolC on DGBE cells. DGBE cells were treated with 0.5 mM TDC or TUDC in the presence or absence of TriolC (30 WM) for 24 h. Following treatment, cells were ¢xed and stained using the Di¡-Quik Staining Kit, and cellular morphology examined with light microscopy at 40U magni¢cation. (A) Control. (B) TDC. (C) TUDC. (D) TriolC. (E) TriolC and TDC. (F) TriolC and TUDC. Arrow in F shows an apoptotic body.

bile salts and TriolC on the cell cycle and apoptosis in DGBE cells. DGBE cells were co-incubated with 30 WM TriolC and 0.5 mM TDC or TUDC. Twentyfour hours after treatment, cells released into the culture medium and cells attached to the culture vessel were harvested and treated simultaneously with Hoechst 33342, CMXRos, and SYTO 11 dyes. Stained cells were then analyzed by £ow cytometry [13]. The ratios between normal and apoptotic cells were determined by double staining with CMXRos and SYTO 11 dyes. This method is highly sensitive in distinguishing apoptotic cells from non-apoptotic cells, compared with morphological examination [13]. CMXRos and CYTO 11 dye show decreased £uorescence with apoptotic cells in comparison to

the normal cells in the same sample. Both dyes identify the same cells as apoptotic based on CMXRoslow or SYTO-low £uorescence. CMXRos £uorescence is sensitive to changes in the mitochondrial membrane potential [13] which occur during apoptosis [14,15]. The biochemical mechanism by which SYTO 11 £uorescence reports apoptosis is unknown. SYTO 11 £uorescence showed little RNase sensitivity or stoichiometry with cellular DNA content [13]. The coincident changes in SYTO 11 and CMXRos £uorescence during apoptosis suggest that both dyes respond to two probably separate events that take place in concert during this form of cell demise. TriolC increased the number of apoptotic cells to 48 þ 12.3% from control (14 þ 11.0%) (Fig. 3 and

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Table 3). No induction of apoptosis was observed after treatment with TDC and TUDC in the absence of TriolC. However, co-incubation with TDC and TriolC resulted in a further increase in the number of apoptotic cells to 63 þ 16.0% compared with controls (P 6 0.05). In contrast, co-incubation with TUDC and TriolC reduced the number of apoptotic cells to 31 þ 12.3% compared with controls (P 6 0.05). Although the number of apoptotic cells was still higher than after treatment with TUDC only, TUDC dramatically inhibited the apoptotic e¡ect of TriolC. We also analyzed the phase of the cell cycle in

Fig. 3. Fluorescent intensity distributions after co-staining with CMXRos and SYTO 11 dyes of DGBE cells treated with TriolC and bile salts. DGBE cells were treated with 0.5 mM TDC or TUDC in the presence or absence of TriolC for 24 h. Floating cells in the medium and harvested cells were stained with CMXRos dye and SYTO 11 dye. Stained cells were assayed with £ow cytometry. Normal cells (N) and apoptotic cells (A) were distinguished by their £uorescent intensity distribution. Two individual experiments in triplicate wells were performed. Typical results for each treatment are shown.

Table 3 The e¡ects of bile salts on cell apoptosis in TriolC-treated DGBE cells Bile acids

3TriolC

+TriolC

None TDC TUDC

14 þ 11.0 12 þ 9.8 14 þ 10.6

48 þ 12.3a 63 þ 16.0a;b 31 þ 12.3a;c

Bile salts (0.5 mM) were added to DGBE cells in the presence (+) or absence (3) of 30 WM TriolC. After 24 h, the apoptotic cells were analyzed by £ow cytometry using CMXRos dye and SYTO 11 dye. The number of apoptotic cells was evaluated as a percentage of total cell counts in each assay. Data are expressed as mean þ S.D. for two individual experiments in triplicate wells (n = 6). a Signi¢cantly higher than without TriolC (P 6 0.01). b Signi¢cantly higher than TriolC without bile salts (P 6 0.05). c Signi¢cantly lower than TriolC without bile salts (P 6 0.05).

Fig. 4. Cell cycle analysis of DGBE cells after treatment with TriolC and bile salts. DGBE cells were treated with 0.5 mM TDC or TUDC in the presence or absence of TriolC for 24 h. Floating cells in the medium and attached cells were harvested and stained with Hoechst 33342 dye for measurement of DNA content of the cells. Stained cells were analyzed by £ow cytometry. The cell cycle populations were determined from DNA contents. Two individual experiments in triplicate wells were performed. Typical results for each treatment are shown.

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Table 4 The e¡ects of bile salts on cell cycle status in TriolC-treated DGBE cells Bile salt treatment

3TriolC G1

None TDC TUDC

46 þ 4.8 46 þ 6.6 44 þ 7.7

+TriolC G2 16 þ 8.0 15 þ 2.1 14 þ 6.8

S

G1 a

38 þ 12.6 40 þ 4.2 42 þ 4.5

71 þ 6.8 69 þ 5.3a 72 þ 2.3a

G2

S

13 þ 4.0 12 þ 7.7 14 þ 2.2

16 þ 9.8a 17 þ 8.6a 14 þ 2.3a

Bile salts (0.5 mM) were added to DGBE cells in the presence (+) or absence (3) of 30 WM TriolC. Twenty-four hours after treatment, cell cycle status was analyzed by £ow cytometry using Hoechst 33342 dye. The number of cells in each phase was evaluated by the percentage of total cell counts in each assay. Data are expressed as mean þ S.D. for two individual experiments in triplicate wells (n = 6). a P 6 0.01 compared with treatment with bile salts alone.

DGBE cells by means of the DNA content. Treatment with TriolC resulted in a higher proportion of cells arrested at the G1 phase and a decreased proportion of cells in the S phase (Fig. 4 and Table 4). Co-incubating with bile salts did not a¡ect the changes in cell cycle as caused by TriolC. These results suggest that TDC and TUDC modify the e¡ects of TriolC on cell apoptosis, but not its e¡ects on the cell cycle. 4. Discussion There have been a number of studies examining the cytotoxic e¡ects of oxysterols on di¡erent cell lines [9,16^21]. Other studies have demonstrated the induction of apoptosis by oxysterols [22^28]. However, little is known about the mechanism of oxysterol-induced injury and virtually nothing is known about the e¡ects of oxysterols on the biliary tract. Since oxysterols are synthesized in the liver during the conversion of cholesterol to bile acids [29], it is likely that they are secreted into the biliary tract. Recently, we have demonstrated that 4,6-cholestadiene-3-one, cholest-4-ene-3-one and other unde¢ned oxysterols are indeed present in human bile and gallstones [8]. Oxysterol contents as high as 70% of total sterol were found in pigment gallstones [8]. We have also described the cytotoxic e¡ects of 7-ketocholesterol and TriolC on cultured dog gallbladder epithelial cells [9]. Therefore, we hypothesize that oxysterols may a¡ect biliary epithelial cell biological function in ways not explored previously. Gallbladder epithelial cells are exposed to high concentrations of lipids in bile on their apical surfa-

ces. Bile salts are major components in bile and have multiple physiological functions. For example, bile salts can induce mucin secretion by the gallbladder epithelium [30^32], which serves to protect the cell membrane surface from cytotoxic agents. In addition, bile salts themselves can have cytotoxic or cytoprotective e¡ects. Hydrophobic bile salts (deoxycholic acid and chenodeoxycholic acid and their conjugates) have cytotoxic e¡ects and induce apoptosis, whereas hydrophilic bile salts (ursodeoxycholic acid and its conjugates) have cytoprotective e¡ects and prevent apoptosis [10,33^42]. Since the gallbladder is not only continually exposed to bile salts, but at the same time to oxysterols, we decided to investigate the interactive e¡ects of bile salts and oxysterols on gallbladder epithelial cells. Our previous experiments showed that TriolC was more cytotoxic to DGBE cells than 7-ketocholesterol [9], so TriolC is a potent agent for testing. The major ¢ndings of this study are that the hydrophobic bile salts TDC and TCDC enhanced TriolC-induced cytotoxicity, whereas the hydrophilic bile salt TUDC prevented TriolC-induced inhibition of cell growth. Interestingly, both the enhancing and preventive e¡ects were more pronounced with increasing bile salt concentration. In addition, using analysis of nuclear morphology and £ow cytometry, we determined that TriolC-induced cytotoxicity proceeded via apoptosis. The mechanism by which oxysterols and hydrophobic bile salts induce apoptosis, and hydrophilic bile salts prevent apoptosis, is not clear. Bcl-2 protein [22], Ca2‡ channel blocker [25] and mevalonate [24] partially inhibit oxysterol-induced apoptosis. Apoptosis induced by hydrophobic bile salts is possibly

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mediated by protein kinase C [37,38], or cAMP [40]. Mitochondrial membrane potential might be involved in the prevention of apoptosis by ursodeoxycholic acid [41,42]. In the present study, we have not investigated the mechanism by which TriolC causes apoptosis or the mechanism of interaction between bile salts and TriolC. However, our detection method for apoptotic cells is based on the measurement of mitochondrial demise. Therefore, we speculate that TUDC prevents TriolC-induced mitochondrial damage and TDC enhances it. Furthermore, both TDC and TUDC at the concentrations tested did not alter the number of apoptotic cells or a¡ect the cell cycle by themselves, while they modi¢ed the e¡ects of TriolC. These ¢ndings indicate that both TDC and TUDC a¡ect the sensitivity of the cells for the e¡ects of TriolC. In a previous study [43], we demonstrated that bile salts can alter the cholesterol concentration in gallbladder epithelial cell membranes. Hydrophobic bile salts increased cholesterol exchange between model biles and cell membranes, while hydrophilic bile salts increased the stability of the membrane cholesterol composition [43]. Since oxysterols can insert themselves into cell membranes, we speculate that TDC accelerates the membrane insertion of TriolC, while TUDC decreases it. Further studies will be required to determine the mechanism of interaction between TriolC and bile salts. The concentrations of TriolC and of the bile salts used in this study are physiologically relevant. Oxysterol concentrations in the micromolar range in gallbladder bile have been surmised based on measurements of oxysterol concentrations in hepatic bile [9]. Furthermore, TriolC in the concentration range used in the present study a¡ects mucin secretion, an important physiologic function of gallbladder epithelial cells [9]. Similarly, bile salts in the millimolar range are typically found in gallbladder bile. These studies were performed on gallbladder epithelial cells cultured in a monolayer, with TriolC and bile salts applied to the apical surface of cells. Due to direct contact with bile, higher concentrations of oxysterols and bile salts are postulated to interact with the apical plasma membrane of gallbladder epithelial cells in vivo as compared to the basolateral surface. Therefore, application of these agents to the apical side makes sense physiologically. Whether similar e¡ects occur when these agents are applied

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to the basolateral surface of the cells was not investigated in the present study. In addition, the presence of the mucus layer on the apical surface of the cells did not hinder the e¡ects observed. This is consistent with our earlier observations showing that bile salts stimulate mucin secretion when applied to the apical surface of cultured gallbladder epithelial cells [31,32]. In the present study, we have limited our investigations to only one of several oxysterols that are found in the biliary tract. However, our study clearly demonstrates that di¡erent bile salts, at concentrations at which they do not a¡ect cell physiology by themselves, can interact with other cytotoxic agents, such as oxysterols, and modify their toxicity. TriolC induces apoptosis in dog gallbladder epithelial cells. Hydrophobic bile salts enhance TriolC-induced apoptosis, whereas hydrophilic bile salts prevent apoptosis. In the biliary tract, the ability of hydrophobic bile salts and oxysterols to combine their strengths in inducing apoptosis might play an important role in the pathogenesis of a number of biliary tract and other gastrointestinal diseases. Therefore, when investigating the adverse and bene¢cial e¡ects of individual compounds on the biliary epithelium, their possible combined e¡ects should also be taken into consideration. Acknowledgements This work was supported by the Medical Research Service of the Department of Veterans A¡airs. References [1] L.L. Smith, Chem. Phys. Lipids 44 (1987) 87^125. [2] L.L. Smith, B.H. John, Free Radic. Biol. Med. 7 (1989) 285^ 332. [3] C.J.W. Brooks, R.M. McKenna, W.J. Cole, J. MacLachlan, T.D.V. Lawrie, Biochem. Soc. Trans. 11 (1983) 700^701. [4] A.J. Brown, S.L. Leong, R.T. Dean, W. Jessup, J. Lipid Res. 38 (1997) 1730^1745. [5] Y.V. Yuan, D.D. Kitts, D.V. Godin, Br. J. Nutr. 78 (1997) 993^1014. [6] H.N. Hodis, D.W. Crawford, A. Sevanian, Atherosclerosis 89 (1991) 117^126. [7] M.M. Mahfouz, H. Kawano, F.A. Kummerow, Am. J. Clin. Nutr. 66 (1997) 1240^1249. [8] G. Haigh, S.P. Lee, Hepatology 30 (1999) 429A.

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