Supplemental Methods
Materials. GCDC, t-BOOH, H2O2, PhASO and bovine heart CL were obtained from Sigma
Aldrich Chemical Co. (St. Louis, MO). CsA was purchased from Alexis Biochemicals (San
Diego, CA)., 2’, 7’-Dichloroflorescin diacetate (DCF-DA) from Calbiochem (San Diego, CA),
and Vital E-300 from Schering-Plough Animal Health (Omaha, NE). Primary antibodies against
Bax, Bad, Bcl-2, Bcl-xL and cytochrome oxidase IV (COX IV) were obtained from Cell
Signaling Technology (Beverly, MA); antibodies toward CpD and VDAC from Calbiochem (San
Diego, CA); antibodies toward Bak, ANT, Trx, and TrxR from Santa Cruz Biotechnology (Santa
Cruz, CA). Anti-mouse cytochrome c was obtained from BD Biosciences (San Jose, CA) and
MnSOD antibodies from Stressgen Bioreagents (Victoria, British Columbia). Antibodies against
α-TTP were a gift from R. Farese and prepared according to (1). 1, 1’, 2, 2’-tetramyristoyl CL
was purchased from Avanti Polar Lipids (Alabaster, AL). All other chemicals were reagent grade
or better.
Isolation of Rat Liver Mitochondria. Male and female Sprague-Dawley rats were maintained on
diets containing 101mg α-TH/kg diet (Harlan Teklad Global 18% Protein Rodent Diet-Madison,
WI) and liver mitochondria were isolated from adult (9 week-old) rats and from pooled livers of 6
day-old, 10 day-old, 21 day-old, or 36 day-old rats (30-35, 15-20, 6-10 and 2-4 rats per
experiment, respectively) by differential centrifugation (2). Mitochondrial purity and recovery
were assessed by measuring marker enzyme activities specific for mitochondria (citrate synthase),
microsomes (cytochrome c reductase), and lysosomes (N-acetylglucosaminidase) (3).
Enrichment was calculated by dividing the specific activity of each organelle marker enzyme by
that of the liver homogenate; percentage recovery was calculated by dividing the total activity of
each organelle marker enzyme by that of the homogenate. Excellent recovery and purity of both
young and adult liver mitochondria was observed (Table 1S). Mitochondrial fractions were used
fresh for MPT and oxidative injury analyses or stored at -70°C prior to analysis of proteins by
immunoblotting, or antioxidants (GSH, α-TH) and CL, as detailed below.
MPT Measurements: Mitochondria were diluted in 10mM 4-morpholinopropanesulfonic acid
(MOPS), 100mM NaCl, 200mM sucrose and 2mM potassium phosphate pH 7.4, treated with 1%
Chelex-100 (MPT buffer), at 25°C for 5 minutes in the absence or presence of 5µΜ CsA, an
MPT blocker. Next, 5mM sodium succinate, 100µΜ CaCl2 and 5µM rotenone were added for an
additional 5 minutes before addition of MPT inducers: 25-100µΜ GCDC. For determining the
effect of other MPT inducers, 100µΜ PhASO, 100µΜ t-BOOH, or 10mM H2O2 were added in
place of GCDC. Absorbance of mitochondria in suspension was monitored at 540nm for 5
minutes and expressed as the ∆O.D./5 minutes. A decrease in absorbance (i.e., swelling) was
used to indicate induction of the MPT. The effect of bile acid exposure on mitochondrial
morphology was also evaluated by electron microscopy. Briefly, aliquots of mitochondria from
young and adult rat liver were incubated in the absence or presence of 100µM GCDC (as above),
removed, fixed immediately in 2.5% glutaraldehyde, and post-fixed in 2% OsO4 prior to
transmission electron microscopy, as previously described (4). There were no differences at
baseline in mitochondrial morphology among the rats of different ages (Figure 1c).
SDS-PAGE and Immunoblotting. Proteins were subject to SDS-PAGE and immunoblotting as
previously described (5). With some immunoblots, COX II and β-actin were probed to confirm
equal loading in mitochondria and soluble fractions, respectively. Immunoreactive bands were
developed by chemiluminescence using a LumiGLO Reagent and Peroxide kit (Cell Signaling
Technology, Beverley, MA) and quantified by densitometry using Un-SCAN-IT gel 6.1
automated digitizing systems (Orem, UT). To determine the effects of 25-100µM GCDC on
mitochondrial cytochrome c levels, mitochondria were removed at the end of the MPT
experiment, repelleted, and immunoblotted as described above. Basal levels of α-TTP in liver
homogenate were analyzed by immunoblot.
Protein Determination. Protein was determined by the Bradford assay (6) using the Quick-Start
Bradford Dye Reagent (Bio-Rad Laboratories, Hercules, CA) and BSA as standard.
References
1. Terasawa Y, Ladha Z, Leonard SW, Morrow JD, Newland D, Sanan D, Packer L, Traber MG,
Farese RV Jr 2000 Increased atherosclerosis in hyperlipidemic mice deficient in alpha-tocopherol
transfer protein and vitamin E. Proc Natl Acad Sci 97:13830-13834
2. Sokol RJ, Straka MS, Dahl R, Devereaux MW, Yerushalmi B, Gumpricht E, Elkins N, Everson
G 2001 Role of oxidant stress in the permeability transition induced in rat hepatic mitochondria
by hydrophobic bile acids. Pediatr Res 49:519-531
3. Sokol RJ, Devereaux MW, Mierau GW, Hambidge KM, Shikes RS 1990 Oxidant injury to
hepatic mitochondrial lipids in rats with dietary copper overload. Modification by vitamin E
deficiency. Gastroenterology 99:1061-1071
4. Sokol RJ, Dahl R, Devereaux MW, Yerushalmi B, Kobak GE, Gumpricht E 2005 Human
hepatic mitochondria generate reactive oxygen species and undergo the permeability transition in
response to hydrophobic bile acids. J Pediatr Gastroenterol Nutr 41:235-243
5. Gumpricht E, Dahl R, Devereaux MW, Sokol RJ 2005 Licorice compounds glycyrrhizin and
18beta-glycyrrhetinic acid are potent modulators of bile acid-induced cytotoxicity in rat
hepatocytes. J Biol Chem 280:10556-10563
6. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities
of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254
Table 1S. Enrichment and Recovery of Hepatic Marker Enzymes in Mitochondrial Preparations
Enzyme Marker
10-Day Old
Adult
p-Value
Citrate Synthase (mitochondrial)
Enrichment (fold)
Recovery (%)
7.24±0.6
82.2±3.0
6.34±0.7
85.4±3.1
0.36
0.48
0.03±0.01
0.50±0.28
0.42±0.20
0.60±0.41
0.06
1.0
0.61±0.15
4.60±0.60
1.07±0.15
4.00±0.78
0.06
0.56
Cytochrome c Reductase (microsomal)
Enrichment (fold)
Recovery (%)
N-acetylglucosaminidase (lysosomal)
Enrichment (fold)
Recovery (%)
Enzyme activities were measured in hepatic mitochondria isolated from 10-day old and
adult rats, and enrichment and recovery were calculated as described in Methods. Results
were obtained from 4-5 separate experiments and expressed as mean ± SEM.
Figure 1S
Protein expression of (A) MPT pore proteins and (B) antioxidant enzymes in 10-day old and adult
rat hepatic mitochondria. No differences were observed in mitochondrial levels of MPT pore
proteins or antioxidant enzymes between the adult () and 10-day old () rat.