Volume 118, Issue 4, Pages 780-794 (April 2000) Does alcohol directly stimulate pancreatic fibrogenesis? Studies with rat pancreatic stellate cells  Minoti V. Apte*, Phoebe A. Phillips*, Roger G. Fahmy*, Samantha J. Darby*, Sally C. Rodgers*, Geoffrey W. McCaughan*, Mark A. Korsten‡, Romano C. Pirola*, Daya Naidoo§, Jeremy S. Wilson*  Gastroenterology  Volume 118, Issue 4, Pages 780-794 (April 2000) DOI: 10.1016/S0016-5085(00)70148-X Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Effect of ethanol and acetaldehyde on DNA synthesis (n = 5 separate cell preparations). DNA synthesis was estimated by measuring incorporation of [3H]thymidine into TCA-precipitable material. Results are expressed as a percent of control values (C) observed in cells incubated without ethanol or acetaldehyde. Ethanol at concentrations of 10 and 50 mmol/L (E10, E50) did not change the rate of DNA synthesis compared with controls. Acetaldehyde at both concentrations (150 μmol/L [A150] and 200 μmol/L [A200]) significantly decreased cell proliferation as indicated by reduced [3H]thymidine incorporation into DNA. *P < 0.005. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 αSMA expression. (A) Western blot analysis of cell lysates for αSMA expression (n = 5 separate cell preparations). A representative immunoblot for αSMA expression in cells incubated with either culture medium alone (controls; C), ethanol (10 and 50 mmol/L; E10 and E50, respectively), or acetaldehyde (150 and 200 μmol/L; A150 and A200, respectively). A single band was detected in each lane corresponding to the known molecular weight (42 kilodaltons) of αSMA. Compared with controls, there was a marked increase in band density in cells incubated with ethanol or acetaldehyde. (B) Densitometry of all Western blots (n = 5 separate cell preparations) showed a significant increase in αSMA levels (expressed in arbitrary densitometer units per microgram of protein loaded on the membrane) in ethanol-incubated (E10 and E50) and acetaldehyde-incubated (A150 and A200) cells compared with controls. *P < 0.05. (C) Western blot analysis for αSMA expression using lysates from cells in early culture (n = 3 separate cell preparations). A representative immunoblot for αSMA expression in cells used within 18 hours of isolation and culture (early culture cells) and incubated with either culture medium alone (controls) or ethanol (50 mmol/L; E50). A marked increase in band density was observed in cells incubated with ethanol compared with controls, in which αSMA expression was negligible. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 αSMA expression. (A) Western blot analysis of cell lysates for αSMA expression (n = 5 separate cell preparations). A representative immunoblot for αSMA expression in cells incubated with either culture medium alone (controls; C), ethanol (10 and 50 mmol/L; E10 and E50, respectively), or acetaldehyde (150 and 200 μmol/L; A150 and A200, respectively). A single band was detected in each lane corresponding to the known molecular weight (42 kilodaltons) of αSMA. Compared with controls, there was a marked increase in band density in cells incubated with ethanol or acetaldehyde. (B) Densitometry of all Western blots (n = 5 separate cell preparations) showed a significant increase in αSMA levels (expressed in arbitrary densitometer units per microgram of protein loaded on the membrane) in ethanol-incubated (E10 and E50) and acetaldehyde-incubated (A150 and A200) cells compared with controls. *P < 0.05. (C) Western blot analysis for αSMA expression using lysates from cells in early culture (n = 3 separate cell preparations). A representative immunoblot for αSMA expression in cells used within 18 hours of isolation and culture (early culture cells) and incubated with either culture medium alone (controls) or ethanol (50 mmol/L; E50). A marked increase in band density was observed in cells incubated with ethanol compared with controls, in which αSMA expression was negligible. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 αSMA expression. (A) Western blot analysis of cell lysates for αSMA expression (n = 5 separate cell preparations). A representative immunoblot for αSMA expression in cells incubated with either culture medium alone (controls; C), ethanol (10 and 50 mmol/L; E10 and E50, respectively), or acetaldehyde (150 and 200 μmol/L; A150 and A200, respectively). A single band was detected in each lane corresponding to the known molecular weight (42 kilodaltons) of αSMA. Compared with controls, there was a marked increase in band density in cells incubated with ethanol or acetaldehyde. (B) Densitometry of all Western blots (n = 5 separate cell preparations) showed a significant increase in αSMA levels (expressed in arbitrary densitometer units per microgram of protein loaded on the membrane) in ethanol-incubated (E10 and E50) and acetaldehyde-incubated (A150 and A200) cells compared with controls. *P < 0.05. (C) Western blot analysis for αSMA expression using lysates from cells in early culture (n = 3 separate cell preparations). A representative immunoblot for αSMA expression in cells used within 18 hours of isolation and culture (early culture cells) and incubated with either culture medium alone (controls) or ethanol (50 mmol/L; E50). A marked increase in band density was observed in cells incubated with ethanol compared with controls, in which αSMA expression was negligible. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Collagen synthesis. (A) Effect of ethanol and acetaldehyde on collagen synthesis (n = 5 separate cell preparations). Collagen synthesis was assessed by measuring incorporation of radiolabeled proline into collagenase-sensitive protein. Results are expressed as counts per minute in collagen per nanogram of DNA. Ethanol at 10 mmol/L (E10) induced a modest increase in collagen synthesis, whereas 50 mmol/L ethanol (E50) produced a marked, statistically significant increase in collagen synthesis. Acetaldehyde at both 150 μmol/L (A150) and 200 μmol/L (A200) significantly increased collagen synthesis in pancreatic stellate cells. *P < 0.05; **P < 0.03. (B) Northern blot analysis of pancreatic RNA from cells incubated with culture medium alone (C), 50 mmol/L ethanol (E50), or 200 μmol/L acetaldehyde (A200) (n = 5 separate cell preparations). A representative Northern blot shows the effect of ethanol and acetaldehyde on mRNA for procollagen α1(I) and GAPDH. An RNA ladder was run with each gel to ensure that the mRNA bands observed were of the expected size. Both ethanol and acetaldehyde increased the expression of procollagen α1(I) mRNA compared with controls. GAPDH expression was similar in the 3 lanes, indicating equal loading of RNA on the membrane. (C) Densitometry of dot blots for procollagen α1(I) mRNA (n = 5 separate cell preparations). Analysis of densitometric readings (expressed as percent of control) showed that both 50 mmol/L ethanol (E50) and 200 μmol/L acetaldehyde (A200) significantly increased mRNA levels for procollagen α1(I) compared with controls. *P < 0.03. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Collagen synthesis. (A) Effect of ethanol and acetaldehyde on collagen synthesis (n = 5 separate cell preparations). Collagen synthesis was assessed by measuring incorporation of radiolabeled proline into collagenase-sensitive protein. Results are expressed as counts per minute in collagen per nanogram of DNA. Ethanol at 10 mmol/L (E10) induced a modest increase in collagen synthesis, whereas 50 mmol/L ethanol (E50) produced a marked, statistically significant increase in collagen synthesis. Acetaldehyde at both 150 μmol/L (A150) and 200 μmol/L (A200) significantly increased collagen synthesis in pancreatic stellate cells. *P < 0.05; **P < 0.03. (B) Northern blot analysis of pancreatic RNA from cells incubated with culture medium alone (C), 50 mmol/L ethanol (E50), or 200 μmol/L acetaldehyde (A200) (n = 5 separate cell preparations). A representative Northern blot shows the effect of ethanol and acetaldehyde on mRNA for procollagen α1(I) and GAPDH. An RNA ladder was run with each gel to ensure that the mRNA bands observed were of the expected size. Both ethanol and acetaldehyde increased the expression of procollagen α1(I) mRNA compared with controls. GAPDH expression was similar in the 3 lanes, indicating equal loading of RNA on the membrane. (C) Densitometry of dot blots for procollagen α1(I) mRNA (n = 5 separate cell preparations). Analysis of densitometric readings (expressed as percent of control) showed that both 50 mmol/L ethanol (E50) and 200 μmol/L acetaldehyde (A200) significantly increased mRNA levels for procollagen α1(I) compared with controls. *P < 0.03. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Collagen synthesis. (A) Effect of ethanol and acetaldehyde on collagen synthesis (n = 5 separate cell preparations). Collagen synthesis was assessed by measuring incorporation of radiolabeled proline into collagenase-sensitive protein. Results are expressed as counts per minute in collagen per nanogram of DNA. Ethanol at 10 mmol/L (E10) induced a modest increase in collagen synthesis, whereas 50 mmol/L ethanol (E50) produced a marked, statistically significant increase in collagen synthesis. Acetaldehyde at both 150 μmol/L (A150) and 200 μmol/L (A200) significantly increased collagen synthesis in pancreatic stellate cells. *P < 0.05; **P < 0.03. (B) Northern blot analysis of pancreatic RNA from cells incubated with culture medium alone (C), 50 mmol/L ethanol (E50), or 200 μmol/L acetaldehyde (A200) (n = 5 separate cell preparations). A representative Northern blot shows the effect of ethanol and acetaldehyde on mRNA for procollagen α1(I) and GAPDH. An RNA ladder was run with each gel to ensure that the mRNA bands observed were of the expected size. Both ethanol and acetaldehyde increased the expression of procollagen α1(I) mRNA compared with controls. GAPDH expression was similar in the 3 lanes, indicating equal loading of RNA on the membrane. (C) Densitometry of dot blots for procollagen α1(I) mRNA (n = 5 separate cell preparations). Analysis of densitometric readings (expressed as percent of control) showed that both 50 mmol/L ethanol (E50) and 200 μmol/L acetaldehyde (A200) significantly increased mRNA levels for procollagen α1(I) compared with controls. *P < 0.03. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) ADH activity in pancreatic stellate cells (n = 5 separate cell preparations). Cells incubated with culture medium alone (C) exhibited ADH activity. Moreover, incubation of pancreatic stellate cells with 50 mmol/L ethanol (E50) for 48 hours resulted in a statistically significant increase in ADH activity over control levels. *P < 0.04. (B) Effect of the ADH inhibitor 4MP on ethanol-stimulated collagen synthesis (n = 5 separate cell preparations). Collagen synthesis was assessed by measuring incorporation of radiolabeled proline into collagenase-sensitive protein. Data are expressed as percent of control values. Incubation of pancreatic stellate cells with 50 mmol/L ethanol in the presence of 4MP (E50 + 4MP) prevented the stimulation of collagen synthesis observed in stellate cells incubated with ethanol alone (E50). Collagen synthesis in cells incubated with 4MP (4MP) alone was similar to that in control cells (C). *P < 0.02. (C) Effect of cyanamide (an inhibitor of acetaldehyde metabolism) on collagen synthesis (n = 5 separate cell preparations). Collagen synthesis was assessed by measuring incorporation of radiolabeled proline into collagenase-sensitive protein. Data are expressed as percent of control values. Incubation with 200 μmol/L acetaldehyde (A200) significantly increased collagen synthesis in pancreatic stellate cells. Cyanamide alone (Cy) also increased collagen synthesis in stellate cells. Inhibition of acetaldehyde metabolism by incubating cells with acetaldehyde in the presence of cyanamide (A200 + Cy) failed to augment the synthesis of collagen any further. *P < 0.04; **P < 0.015. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) ADH activity in pancreatic stellate cells (n = 5 separate cell preparations). Cells incubated with culture medium alone (C) exhibited ADH activity. Moreover, incubation of pancreatic stellate cells with 50 mmol/L ethanol (E50) for 48 hours resulted in a statistically significant increase in ADH activity over control levels. *P < 0.04. (B) Effect of the ADH inhibitor 4MP on ethanol-stimulated collagen synthesis (n = 5 separate cell preparations). Collagen synthesis was assessed by measuring incorporation of radiolabeled proline into collagenase-sensitive protein. Data are expressed as percent of control values. Incubation of pancreatic stellate cells with 50 mmol/L ethanol in the presence of 4MP (E50 + 4MP) prevented the stimulation of collagen synthesis observed in stellate cells incubated with ethanol alone (E50). Collagen synthesis in cells incubated with 4MP (4MP) alone was similar to that in control cells (C). *P < 0.02. (C) Effect of cyanamide (an inhibitor of acetaldehyde metabolism) on collagen synthesis (n = 5 separate cell preparations). Collagen synthesis was assessed by measuring incorporation of radiolabeled proline into collagenase-sensitive protein. Data are expressed as percent of control values. Incubation with 200 μmol/L acetaldehyde (A200) significantly increased collagen synthesis in pancreatic stellate cells. Cyanamide alone (Cy) also increased collagen synthesis in stellate cells. Inhibition of acetaldehyde metabolism by incubating cells with acetaldehyde in the presence of cyanamide (A200 + Cy) failed to augment the synthesis of collagen any further. *P < 0.04; **P < 0.015. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 (A) ADH activity in pancreatic stellate cells (n = 5 separate cell preparations). Cells incubated with culture medium alone (C) exhibited ADH activity. Moreover, incubation of pancreatic stellate cells with 50 mmol/L ethanol (E50) for 48 hours resulted in a statistically significant increase in ADH activity over control levels. *P < 0.04. (B) Effect of the ADH inhibitor 4MP on ethanol-stimulated collagen synthesis (n = 5 separate cell preparations). Collagen synthesis was assessed by measuring incorporation of radiolabeled proline into collagenase-sensitive protein. Data are expressed as percent of control values. Incubation of pancreatic stellate cells with 50 mmol/L ethanol in the presence of 4MP (E50 + 4MP) prevented the stimulation of collagen synthesis observed in stellate cells incubated with ethanol alone (E50). Collagen synthesis in cells incubated with 4MP (4MP) alone was similar to that in control cells (C). *P < 0.02. (C) Effect of cyanamide (an inhibitor of acetaldehyde metabolism) on collagen synthesis (n = 5 separate cell preparations). Collagen synthesis was assessed by measuring incorporation of radiolabeled proline into collagenase-sensitive protein. Data are expressed as percent of control values. Incubation with 200 μmol/L acetaldehyde (A200) significantly increased collagen synthesis in pancreatic stellate cells. Cyanamide alone (Cy) also increased collagen synthesis in stellate cells. Inhibition of acetaldehyde metabolism by incubating cells with acetaldehyde in the presence of cyanamide (A200 + Cy) failed to augment the synthesis of collagen any further. *P < 0.04; **P < 0.015. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of oxidant stress on stellate cells. (A) Effect of the pro-oxidant complex (10 μmol/L FeSO4/200 μmol/L ascorbic acid) on MDA production (n = 5 separate cell preparations). Cells incubated with the pro-oxidant complex exhibited significantly increased MDA production (expressed as nanomoles of MDA per milligram of cellular protein) compared with control cells (■) at 3 hours of incubation. At 24 hours of incubation, MDA levels in FeSO4/ascorbic acid-treated cells (□) were lower than at 3 hours but remained higher than control levels. *P < 0.03. (B) Western blot for αSMA expression in cells subjected to oxidant stress. A representative immunoblot shows increased αSMA expression in cells incubated with the pro-oxidant complex 10 μmol/L FeSO4/200 μmol/L ascorbic acid (Oxidant Stress) compared with control cells incubated with culture medium alone (Control). Densitometry of all immunoblots (n = 5 separate cell preparations) showed significantly increased αSMA levels in cells subjected to oxidant stress compared with controls (see Results). (C) Effect of oxidant stress on procollagen α1(I) mRNA. A representative Northern blot shows increased procollagen α1(I) mRNA expression in cells subjected to oxidant stress than in cells incubated with culture medium alone (Control). Densitometric analysis of dot blots for procollagen α1(I) mRNA (n = 5 separate cell preparations) confirmed significantly higher levels in cells subjected to oxidant stress compared with controls (see Results). (D) Effect of the antioxidant vitamin E on MDA production (n = 5 separate cell preparations). Pancreatic stellate cells incubated with the pro-oxidant complex exhibited significantly increased MDA production compared with controls. This increase was prevented by the presence of 100 μmol/L vitamin E (OS + VE) in the incubation medium. *P < 0.025. (E) Effect of 100 μmol/L vitamin E (VE) on oxidant stress–induced collagen synthesis (n = 5 separate cell preparations). The increase in collagen synthesis observed in cells subjected to oxidant stress (OS) was prevented by the antioxidant vitamin E (OS + VE). *P < 0.005. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of oxidant stress on stellate cells. (A) Effect of the pro-oxidant complex (10 μmol/L FeSO4/200 μmol/L ascorbic acid) on MDA production (n = 5 separate cell preparations). Cells incubated with the pro-oxidant complex exhibited significantly increased MDA production (expressed as nanomoles of MDA per milligram of cellular protein) compared with control cells (■) at 3 hours of incubation. At 24 hours of incubation, MDA levels in FeSO4/ascorbic acid-treated cells (□) were lower than at 3 hours but remained higher than control levels. *P < 0.03. (B) Western blot for αSMA expression in cells subjected to oxidant stress. A representative immunoblot shows increased αSMA expression in cells incubated with the pro-oxidant complex 10 μmol/L FeSO4/200 μmol/L ascorbic acid (Oxidant Stress) compared with control cells incubated with culture medium alone (Control). Densitometry of all immunoblots (n = 5 separate cell preparations) showed significantly increased αSMA levels in cells subjected to oxidant stress compared with controls (see Results). (C) Effect of oxidant stress on procollagen α1(I) mRNA. A representative Northern blot shows increased procollagen α1(I) mRNA expression in cells subjected to oxidant stress than in cells incubated with culture medium alone (Control). Densitometric analysis of dot blots for procollagen α1(I) mRNA (n = 5 separate cell preparations) confirmed significantly higher levels in cells subjected to oxidant stress compared with controls (see Results). (D) Effect of the antioxidant vitamin E on MDA production (n = 5 separate cell preparations). Pancreatic stellate cells incubated with the pro-oxidant complex exhibited significantly increased MDA production compared with controls. This increase was prevented by the presence of 100 μmol/L vitamin E (OS + VE) in the incubation medium. *P < 0.025. (E) Effect of 100 μmol/L vitamin E (VE) on oxidant stress–induced collagen synthesis (n = 5 separate cell preparations). The increase in collagen synthesis observed in cells subjected to oxidant stress (OS) was prevented by the antioxidant vitamin E (OS + VE). *P < 0.005. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of oxidant stress on stellate cells. (A) Effect of the pro-oxidant complex (10 μmol/L FeSO4/200 μmol/L ascorbic acid) on MDA production (n = 5 separate cell preparations). Cells incubated with the pro-oxidant complex exhibited significantly increased MDA production (expressed as nanomoles of MDA per milligram of cellular protein) compared with control cells (■) at 3 hours of incubation. At 24 hours of incubation, MDA levels in FeSO4/ascorbic acid-treated cells (□) were lower than at 3 hours but remained higher than control levels. *P < 0.03. (B) Western blot for αSMA expression in cells subjected to oxidant stress. A representative immunoblot shows increased αSMA expression in cells incubated with the pro-oxidant complex 10 μmol/L FeSO4/200 μmol/L ascorbic acid (Oxidant Stress) compared with control cells incubated with culture medium alone (Control). Densitometry of all immunoblots (n = 5 separate cell preparations) showed significantly increased αSMA levels in cells subjected to oxidant stress compared with controls (see Results). (C) Effect of oxidant stress on procollagen α1(I) mRNA. A representative Northern blot shows increased procollagen α1(I) mRNA expression in cells subjected to oxidant stress than in cells incubated with culture medium alone (Control). Densitometric analysis of dot blots for procollagen α1(I) mRNA (n = 5 separate cell preparations) confirmed significantly higher levels in cells subjected to oxidant stress compared with controls (see Results). (D) Effect of the antioxidant vitamin E on MDA production (n = 5 separate cell preparations). Pancreatic stellate cells incubated with the pro-oxidant complex exhibited significantly increased MDA production compared with controls. This increase was prevented by the presence of 100 μmol/L vitamin E (OS + VE) in the incubation medium. *P < 0.025. (E) Effect of 100 μmol/L vitamin E (VE) on oxidant stress–induced collagen synthesis (n = 5 separate cell preparations). The increase in collagen synthesis observed in cells subjected to oxidant stress (OS) was prevented by the antioxidant vitamin E (OS + VE). *P < 0.005. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of oxidant stress on stellate cells. (A) Effect of the pro-oxidant complex (10 μmol/L FeSO4/200 μmol/L ascorbic acid) on MDA production (n = 5 separate cell preparations). Cells incubated with the pro-oxidant complex exhibited significantly increased MDA production (expressed as nanomoles of MDA per milligram of cellular protein) compared with control cells (■) at 3 hours of incubation. At 24 hours of incubation, MDA levels in FeSO4/ascorbic acid-treated cells (□) were lower than at 3 hours but remained higher than control levels. *P < 0.03. (B) Western blot for αSMA expression in cells subjected to oxidant stress. A representative immunoblot shows increased αSMA expression in cells incubated with the pro-oxidant complex 10 μmol/L FeSO4/200 μmol/L ascorbic acid (Oxidant Stress) compared with control cells incubated with culture medium alone (Control). Densitometry of all immunoblots (n = 5 separate cell preparations) showed significantly increased αSMA levels in cells subjected to oxidant stress compared with controls (see Results). (C) Effect of oxidant stress on procollagen α1(I) mRNA. A representative Northern blot shows increased procollagen α1(I) mRNA expression in cells subjected to oxidant stress than in cells incubated with culture medium alone (Control). Densitometric analysis of dot blots for procollagen α1(I) mRNA (n = 5 separate cell preparations) confirmed significantly higher levels in cells subjected to oxidant stress compared with controls (see Results). (D) Effect of the antioxidant vitamin E on MDA production (n = 5 separate cell preparations). Pancreatic stellate cells incubated with the pro-oxidant complex exhibited significantly increased MDA production compared with controls. This increase was prevented by the presence of 100 μmol/L vitamin E (OS + VE) in the incubation medium. *P < 0.025. (E) Effect of 100 μmol/L vitamin E (VE) on oxidant stress–induced collagen synthesis (n = 5 separate cell preparations). The increase in collagen synthesis observed in cells subjected to oxidant stress (OS) was prevented by the antioxidant vitamin E (OS + VE). *P < 0.005. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Effect of oxidant stress on stellate cells. (A) Effect of the pro-oxidant complex (10 μmol/L FeSO4/200 μmol/L ascorbic acid) on MDA production (n = 5 separate cell preparations). Cells incubated with the pro-oxidant complex exhibited significantly increased MDA production (expressed as nanomoles of MDA per milligram of cellular protein) compared with control cells (■) at 3 hours of incubation. At 24 hours of incubation, MDA levels in FeSO4/ascorbic acid-treated cells (□) were lower than at 3 hours but remained higher than control levels. *P < 0.03. (B) Western blot for αSMA expression in cells subjected to oxidant stress. A representative immunoblot shows increased αSMA expression in cells incubated with the pro-oxidant complex 10 μmol/L FeSO4/200 μmol/L ascorbic acid (Oxidant Stress) compared with control cells incubated with culture medium alone (Control). Densitometry of all immunoblots (n = 5 separate cell preparations) showed significantly increased αSMA levels in cells subjected to oxidant stress compared with controls (see Results). (C) Effect of oxidant stress on procollagen α1(I) mRNA. A representative Northern blot shows increased procollagen α1(I) mRNA expression in cells subjected to oxidant stress than in cells incubated with culture medium alone (Control). Densitometric analysis of dot blots for procollagen α1(I) mRNA (n = 5 separate cell preparations) confirmed significantly higher levels in cells subjected to oxidant stress compared with controls (see Results). (D) Effect of the antioxidant vitamin E on MDA production (n = 5 separate cell preparations). Pancreatic stellate cells incubated with the pro-oxidant complex exhibited significantly increased MDA production compared with controls. This increase was prevented by the presence of 100 μmol/L vitamin E (OS + VE) in the incubation medium. *P < 0.025. (E) Effect of 100 μmol/L vitamin E (VE) on oxidant stress–induced collagen synthesis (n = 5 separate cell preparations). The increase in collagen synthesis observed in cells subjected to oxidant stress (OS) was prevented by the antioxidant vitamin E (OS + VE). *P < 0.005. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) Effect of 100 μmol/L vitamin E (VE) on MDA production in cells exposed to ethanol or acetaldehyde (n = 5 separate cell preparations). Cells incubated with 50 mmol/L ethanol (E50) or 200 μmol/L acetaldehyde (A200) for 3 hours showed increased MDA levels (indicating increased lipid peroxidation) compared with controls. This increase was abolished in the presence of the antioxidant vitamin E (E50 + VE and A200 + VE). *P < 0.02. (B) Effect of 100 μmol/L vitamin E (VE) on ethanol-stimulated collagen synthesis (n = 5 separate cell preparations). The observed increase in collagen synthesis in cells exposed to 50 mmol/L ethanol (E50) was prevented when vitamin E was added to the incubation medium (E50 + VE). *P < 0.04. (C) Effect of 100 μmol/L vitamin E (VE) on acetaldehyde-stimulated collagen synthesis (n = 5 separate cell preparations). Cells exposed to 200 μmol/L acetaldehyde (A200) showed higher collagen synthesis than controls (C). In the presence of vitamin E (A200 + VE), the acetaldehyde-induced increase in collagen synthesis was abolished. *P < 0.05. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) Effect of 100 μmol/L vitamin E (VE) on MDA production in cells exposed to ethanol or acetaldehyde (n = 5 separate cell preparations). Cells incubated with 50 mmol/L ethanol (E50) or 200 μmol/L acetaldehyde (A200) for 3 hours showed increased MDA levels (indicating increased lipid peroxidation) compared with controls. This increase was abolished in the presence of the antioxidant vitamin E (E50 + VE and A200 + VE). *P < 0.02. (B) Effect of 100 μmol/L vitamin E (VE) on ethanol-stimulated collagen synthesis (n = 5 separate cell preparations). The observed increase in collagen synthesis in cells exposed to 50 mmol/L ethanol (E50) was prevented when vitamin E was added to the incubation medium (E50 + VE). *P < 0.04. (C) Effect of 100 μmol/L vitamin E (VE) on acetaldehyde-stimulated collagen synthesis (n = 5 separate cell preparations). Cells exposed to 200 μmol/L acetaldehyde (A200) showed higher collagen synthesis than controls (C). In the presence of vitamin E (A200 + VE), the acetaldehyde-induced increase in collagen synthesis was abolished. *P < 0.05. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) Effect of 100 μmol/L vitamin E (VE) on MDA production in cells exposed to ethanol or acetaldehyde (n = 5 separate cell preparations). Cells incubated with 50 mmol/L ethanol (E50) or 200 μmol/L acetaldehyde (A200) for 3 hours showed increased MDA levels (indicating increased lipid peroxidation) compared with controls. This increase was abolished in the presence of the antioxidant vitamin E (E50 + VE and A200 + VE). *P < 0.02. (B) Effect of 100 μmol/L vitamin E (VE) on ethanol-stimulated collagen synthesis (n = 5 separate cell preparations). The observed increase in collagen synthesis in cells exposed to 50 mmol/L ethanol (E50) was prevented when vitamin E was added to the incubation medium (E50 + VE). *P < 0.04. (C) Effect of 100 μmol/L vitamin E (VE) on acetaldehyde-stimulated collagen synthesis (n = 5 separate cell preparations). Cells exposed to 200 μmol/L acetaldehyde (A200) showed higher collagen synthesis than controls (C). In the presence of vitamin E (A200 + VE), the acetaldehyde-induced increase in collagen synthesis was abolished. *P < 0.05. Gastroenterology 2000 118, 780-794DOI: (10.1016/S0016-5085(00)70148-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions