1. Volume 121, Issue 3, Pages 685-698 (September 2001)
Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances the resolution of liver fibrosis in rats 
Matthew C. Wright, Razo Issa, David E. Smart, Nathan Trim, Graeme I. Murray, John N. Primrose, Michael J.P. Arthur, John P. Iredale, Derek A. Mann 
Gastroenterology 
Volume 121, Issue 3, Pages (September 2001)
DOI: /gast
Copyright © 2001 American Gastroenterological Association Terms and Conditions

2. Fig. 1 Structure of gliotoxin and mt-glio.
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
Light micrographs of rat and human HSCs treated with gliotoxin. Culture-activated (14-day) rat HSCs were treated for 4 hours with (A) DMSO solvent vehicle control or (B) 1.5 μmol/L gliotoxin. Culture-activated (24-day) human (H2) HSCs cells were treated for 4 hours with (C) DMSO solvent vehicle control or (D) 1.5 μmol/L gliotoxin. Results typical of at least 6 separate preparations.
Gastroenterology  , DOI: ( /gast )
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4. Fig. 3
Effect of gliotoxin on caspase 3 activity and DNA integrity in rat HSCs. (A) Culture-activated (14-day) rat HSCs in 100-mm diameter plates were treated with 0.05% (vol/vol) DMSO (control), 1.5 μmol/L gliotoxin (gliotoxin), 20 μmol/L Z-VAD-FMK, or 200 μmol/L chlorpromazine for 3 hours and cells harvested for examination of caspase 3 activity as outlined in Materials and Methods. Results are the mean and standard deviation of caspase activities determined from 3 separate experiments. *Significantly different (P > 95%) activity vs. control cells using the Student t test (two-tailed). (B) Culture-activated (14-day) rat HSCs in 6-well plates were treated with either 0.05% (vol/vol) DMSO (control) for 4 hours or 1.5 μmol/L gliotoxin. At each time point, both detached and attached cells were harvested together and low molecular weight DNA (20,000 g supernatant) fragmentation determined as outlined in Materials and Methods. 1 kb, 1 kilobase DNA ladder (Promega). Results typical of 3 separate experiments. (C) Culture-activated (14-day) rat HSCs in 6-well plates were treated with either 0.05% (vol/vol) DMSO (control), 1.5 μmol/L gliotoxin (gt), or 1.5 μmol/L mt-glio. HSCs were also treated as indicated with between 100 nmol/L and 100 μmol/l Z-VAD-FMK or 300 μmol/L PDTC. After 4 hours of treatment, DNA fragmentation was determined as outlined in Materials and Methods. Results are typical of at least 4 separate experiments. (D) Culture-activated (14-day) rat HSCs were treated with 0.05% (vol/vol) DMSO vehicle control or 1.5 μmol/L gliotoxin for 2 hours, harvested, and stained with propidium iodide as outlined in Materials and Methods. Before staining, both control and gliotoxin-treated HSCs excluded trypan blue indicating that the cell membranes were intact. Events of control cells (1 × 104; clear) are compared with events from gliotoxin-treated HSCs (1 × 104; shaded). Results typical of 6 separate experiments. (E) Culture-activated (14-day) rat HSCs were treated with 0.05% (vol/vol) DMSO (CONTROL) or 1.5 μmol/L gliotoxin (GLIOTOXIN) for 2 hours and DNA strand breaks examined by TUNEL staining as outlined in Materials and Methods. TUNEL staining fidelity was determined by staining control and gliotoxin-treated cells without the incorporation of dUTP in the protocol (not shown) and resulted in staining similar to CONTROL panel.
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
Cell death in response to TNF-α plus cycloheximide, gliotoxin, and other compounds that cause necrosis; comparison between rat hepatocytes and rat HSCs. (A) Culture-activated (14-day) rat hepatic stellate cells (■) or rat hepatocytes (○) were treated with either 0.05% (vol/vol) DMSO or 1.5 μmol/L gliotoxin and cell attachment determined by direct assay of protein in each well after 4 hours as outlined in Materials and Methods. Results expressed as the mean and standard deviation percentage attachment vs. control of 3 separate experiments. Longer incubation of gliotoxin with hepatocytes did not result in significantly different levels of cell death. (B) Viability of rat hepatocytes as judged by attachment (clear bars) and 0.1% (wt/vol) trypan blue exclusion (shaded bars) after 4 hours treatment with 0.05% (vol/vol) DMSO (CONTROL), 50 μmol/L gliotoxin (GLIOTOXIN), 200 μmol/L chlorpromazine (CHLORPROMAZINE), 10 ng/mL TNF-α + 10 μmol/L cycloheximide (TNF + CYCLO), or 200 μmol/L methapyrilene (METHAPYRILENE). (C) DNA cleavage in rat HSCs or rat hepatocytes. Rat HSCs were treated for 4 hours with 0.05% (vol/vol) DMSO (CONTROL), 10 ng/mL TNF-α + 10 μmol/L cycloheximide (TNF + CYCLO), or 1.5 μmol/L gliotoxin (GLIOTOXIN). Rat hepatocytes were treated for 4 hours with 0.05% (vol/vol) DMSO (CONTROL), 50 μmol/L gliotoxin (GLIOTOXIN), 200 μmol/L chlorpromazine (CHLORPROMAZINE), 10 ng/mL TNF-α + 10 μmol/L cycloheximide (TNF + CYCLO), or 200 μmol/L methapyrilene (METHAPYRILENE).
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6. Fig. 5
Mechanism of action of gliotoxin in rat HSCs; effects on NF-κB DNA binding activity, the MPT, intracellular Ca2+, and DNA cleavage. (A) Culture-activated (14-day) rat HSCs were treated with (+TNF-α) or without (−TNF-α) 10 ng/mL TNF-α 15 minutes after addition of 0.05% (vol/vol) DMSO vehicle control (control) or the indicated concentration of NF-κB inhibitor. After a further 30 minutes and before any significant morphologic changes, cells were washed with ice-cooled PBS, nuclear extracts prepared, and NF-κB DNA binding activity assessed by gel shift analysis. Each lane contains 6 μg protein, CONTROL + EX, control nuclear extract containing 50-fold molar excess cold NF-κB oligonucleotide to demonstrate specific saturable binding. Results are typical of at least 3 separate experiments. (B) Culture-activated rat HSCs were loaded and calcein (green) and TMRM (red) fluorescence imaged as outlined in Materials and Methods. Results presented as calcein (left), TMRM (middle), and combined (right) fluorescence determined under identical conditions after 2 hours of treatment with 1.5 μmol/L gliotoxin (top panels), 4 hours treatment with 1.5 μmol/L gliotoxin (middle panels), or DMSO vehicle control (lower panels). Results typical of 3 separate experiments. (C) Culture-activated (14-day) rat HSCs in 6-well plates were treated with either 0.05% (vol/vol) DMSO (no additions), 1.5 μmol/L (gliotoxin GT), and/or the following: Q-2AM, 50 μmol/L quin-2-am; TAM, 100 μmol/L tamoxifen; mIBG, 250 μmol/L m-iodobenzylguanidine; CsA, 10 μmol/L cyclosporin A; act-d, 1 μg/mL actinomycin d; and CHX, 10 μmol/L cycloheximide. After 4 hours of treatment, DNA fragmentation was determined as outlined in Materials and Methods. Results are typical of at least 3 separate experiments. Note that in all cases in which HSCs were treated with gliotoxin irrespective of other additions to the medium, cellular detachment and other morphologic alterations still occurred. (D) Culture-activated rat HSCs were loaded and fluo-3 and fluorescence imaged as outlined in Materials and Methods. Results presented as time zero (bottom left), 4 hours of DMSO control (bottom right), 2 hours of treatment with 1.5 μmol/L gliotoxin (top left), and 4 hours of treatment with 1.5 μmol/L gliotoxin (top right). Results typical of 3 separate experiments.
Gastroenterology  , DOI: ( /gast )
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7. Fig. 6
Effect of a single injection of gliotoxin on α-smooth muscle actin liver immunostaining after treatment for 7 weeks with carbon tetrachloride. One day after the final injection of carbon tetrachloride, rats were administered gliotoxin and killed after a further day. (A) Control: liver section from a rat treated with vehicle (olive oil) for 7 weeks and DMSO; (B) gliotoxin only: liver section from a rat treated with the vehicle (olive oil) for 7 weeks and 3 mg gliotoxin/kg body wt; (C) carbon tetrachloride only: liver section from a rat treated with carbon tetrachloride for 7 weeks and DMSO vehicle; and (D) carbon tetrachloride and gliotoxin: liver section from a rat treated with carbon tetrachloride for 7 weeks and 3 mg gliotoxin/kg body wt. Results are typical of 5 separate animals.
Gastroenterology  , DOI: ( /gast )
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8. Fig. 7
Effect of a single injection of gliotoxin on liver TUNEL staining after treatment for 7 weeks with carbon tetrachloride. Rats were treated for 7 weeks with carbon tetrachloride. One day after the final injection of carbon tetrachloride, rats were administered gliotoxin and killed after a further day. (A) Liver sections from rats treated with CCl4 and gliotoxin were TUNEL stained with or without (−dUTP) the incorporation of dUTP in the staining protocol. Sections were then counterstained with hematoxylin. (B) Dual staining demonstrating that TUNEL-positive cells (red) co-stain cells positive for α-smooth muscle actin (blue).
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

9. Fig. 8
Effect of a single injection of gliotoxin on liver sirius red staining after treatment for 7 weeks with carbon tetrachloride. Rats were treated for 7 weeks with carbon tetrachloride. One day after the final injection of carbon tetrachloride, rats were administered gliotoxin and killed after a further day. (A) Control: liver section from a rat treated with vehicle (olive oil) for 7 weeks and DMSO; (B) gliotoxin only: liver section from a rat treated with the vehicle (olive oil) for 7 weeks and 3 mg gliotoxin/kg body wt; (C) carbon tetrachloride only: liver section from a rat treated with carbon tetrachloride for 7 weeks and DMSO vehicle; and (D) carbon tetrachloride and gliotoxin: liver section from a rat treated with carbon tetrachloride for 7 weeks and 3 mg gliotoxin/kg body wt. Results are typical of 5 separate animals.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions