Increase in mitochondrial mass and paradoxical decrease in mitochondrial transmembrane potential in a troglitazone-treated immortalized human hepatocyte cell line

454A

AASLD ABSTRACTS

HEPATOLOGY October 2001

1127

1128

IDENTIFICATION OF D R U G - I N D U C E D LIVER INJURY A M O N G 3 0 7 CASES OF SEVERE LIVER INJURY: A RETROSPECTIVE MULTI-CENTER STUDY. George Ostapowicz, Grace Samuel, University of Texas Southwestern Medical Center, Dallas, TX; Anne M Larson, University of Washington, Seattle, WA; Timothy Davern, University of California, San Francisco, San Francisco, CA; Robert J Fontana, University of Michigan, Ann Arbor, MI; Timothy McCashland, University of Nebraska, Omaha, NE; Edwyn Harrison, Mayo Clinic, Scottsdale, AZ; Linda Hynan, Joe Webster, Prem Shukla, William M Lee, University of Texas Southwestern Medical Center, Dallas, TX

LYMPHOCYTE TOXICITY ASSAY: A TOOL TO ASSESS TRIMETHOPRIM-

Drug4nduced liver injury (DILl) remains a threat in light of two recent drugs withdrawn by FDA for hepatotoxicity. Acute liver failure (ALF) definitions (coagulopathy [INR > 1.5 ] plus encephalopathy) yield a limited number of cases, since many with severe liver injury are not encephalopathic. There are no absolute criteria available to confirm drug-related injury. Causality assessment methods (CAMs) are used, but their validityhas been difficult to determine. The current pilot study aimed to further define the role of DILI in the US, employing a broader definition of severe liver injury (SLI), and to test the utility of a CAM. We compared the assessments of clinician/investigators with the most popular CAM (RUCAM/CIOMS) in 307 patients with SLI, defined as an acute hepatic illness of < 26 weeks with either hilirubin >10 mg/dl or coagulopathy (INR > 1.5) with bifirubin > 4 mg/dl. Retrospective medical record analysis was performed at 6 academic sites. Appropriate codes and laboratory logs for high ALT and INR were scanned to identify possible cases. From 964 medical records reviewed, 307 met study definitions, and were entered on a case report form, then into a central Access® database. Investigators provided case summaries, includinga specific etiology attribution. Of the 307 cases, 194 (63%) were female. ALF was present in 66% (the lower threshold yielded an additional 50% patients). The etiologies were: acetaminophen n~ 110, other drugs/toxins 65, viral hepatitis 57, indeterminate 24, other 51. Of the 65 drug- or toxin-induced liver injury, 4 cases were due to mushrooms. The most common drugs implicatedwere: herbs n=9, NSAIDs9, antibiotics/antifungals 9, busulfan 5, trimethoprim/sulfa 4, isoniazid 3, carbamazepine 3, troglitazone 3, disulfiram 2, and one each for terbinafine, niacin, ecstasy, BCNU, cytarabine, desflurane, IL-2, pemoline, valspodar, zidovudine, zafirlukast, pravastatin, lisinopril, albendazole. The RUCAM was then applied to 61 cases of DILl and 24 indeterminate cases. An accurate CAM (all data available) could be determined for 85% of the drug-induced cases--in only two was a rechallenge performed. The median DILl score was 5.7 (range -2 to 11); for the indeterminate cases 2.4 (range -1 to 7), but there was considerable overlap. Among 24 indeterminate cases, drugs (including acetaminophen, n=6) might have been implicated in 18 if more temporal data had been available. In addition to 36% acetaminophen cases, DILI comprises 20% of cases of severe liver injury in this retrospective study: herbs and NSAIDs are most frequently implicated. Attribution of etiology is easier for viral than for drugrelated cases which require specific temporal data. The use of multiple drugs or herbs, unspecified doses and the lack of specific stop and start dates limits the use of CAMs, leading to indeterminate classifications in an additional 30% of SLI cases. Many considered indeterminate were likelyDILl if more information had been available. A prospective study for both the incidence and causality assessment of drug-induced SLI is warranted to aid in the earlier identification of toxic reactions to new therapeutic agents.

SULFAMETHOXAZOLE HYPERSENSITMTY SYNDROME REACTIONS IN HUMAN IMMUNODEFICIENCY VIRUS - INFECTED I N D M D U A L S . Manuela Neuman, Izabella Malkiewicz, Elizabeth Phillips, Anita Rachlis, Elaine Yeung, Debra Ong, Nell H Shear, Sunnybrook & Women's College Health Sciences Centre, Toronto, ON Canada Hypersensitivity syndrome reactions are a complex symptom defined by the triad of fever, rash and internal organ (mainly liver) involvement. Drug hypersensitivity syndrome is a severe potentially life-threatening reaction especially in patients infected with human immunodeficiency virus (HIV). Trimethoprim-sulfamethoxazole (TMP-SMX) is still a very important agent particularly in the HIV-infected host, who require lifelong preventive therapy against Pneumocystis carinii pneumonia in w h o m there is an adverse reaction rate to TMP-SMX of 40%. Objectives: To assess drug-induced mitochondrial damage as shown by the lymphocyte toxicity assay (LTA) and to correlate the LTA values with their significance based on clinical hypersensitivity to TMPSMX in HIV-infected individuals. Design a n d Methods: HIV-positive individuals were identified based on their exposure to TMP-SMX. Patient lymphocytes were analyzed for toxicity to SMX and TMP. Results: Twemy-five subjects participated in the study. Of the patients exposed to TMP-SMX, 19 had positive histories for TMP-SMX hypersensitivity, while the other 6 patients tolerated these drugs. For SMX testing, mean cytotoxicity values were higher among those with positive histories (31.7 -+ 11.6%) than those with negative histories (22.4 - 13.8%) (p<0.05). Similarly, TMP testing indicated higher mean eytotoxicity values for those with TMP hypersensitivity (30.0 --- 14.5%) than those who tolerated TMP (16.4% ~ 6.7%). Five of the patients that have high LTA for SMX (32.5 - 5.6%) presented low toxicity for TMP (10.7 ± 1.6%). Conclusions: The LTA has potential for use as a diagnostic tool to assess TMP-SMX hypersensitivity in HIV-infected individuals. If used prospectively, the test may suggest the drug of choice for therapeutic interventions in a population at risk such as HIV patients.

1129

1130

DECREASED BIOACTIVATION A N D / O R DETOXIFICATION DOES NOT

INCREASE IN MITOCHONDRIAL MASS A N D PARADOXICAL DECREASE IN MITOCHONDRIAL TRANSMEMBRANE POTENTIAL IN A TROGLITAZONE-TREATED IMMORTALIZED HUMAN HEPATOCYTE CELL LINE. Shoichiro Shishido, Hironori Koga, Masaru Harada, Hiroto Kumemura, Shinichiro Hanada, Eitaro Taniguchi, Kurume University, Kurume Japan; Masayoshi Namba, Okayama University, Okayama Japan; Hiromasa Oohira, Ynkio Sato, Fukushima Medical University, Fukushima Japan; Ryukichi Kumashiro, Takato Ueno, Michio Sata, Kurume University, Knrume Japan

EXPLAIN RESILIENCY OF DIABETIC MICE TO ACETAMINOPItEN HEPATOTOXlCITY A N D LETHALITY. Kartik Shankar, Vishal S Vaidya, Tao Wang, Department of Toxicology, The University of Louisiana at Monroe, Monroe, LA; Jose E Manantou, Department of Pharmaceutical Sciences, University of Connecticut, Stross, CT; Thomas J Bucci, Pathology Associates Intl, National Center for Toxicological Research, Jefferson, AR; Harihara M Mehendale, Department of Toxicology, The University of Louisiana at Monroe, Monroe, LA Incidence of hepatotoxicity is increasing among diabetic patients. It is well known that hepatotoxicity of several chemicals and drugs is potentiated in diabetic rats. In contrast to these data, we have shown that diabetic mice are resistant to thioacetamide (TA) hepatotoxicity and lethality. In the present study we have examined whether diabetic mice are protected against hepatotoxicity of acetaminophen (AP), carbon tetrachloride (CC14) and bromobenzene (BB). Streptozotocin (STZ)-induced male Swiss Webster diabetic mice (27~33 g) challenged with ordinarily lethal doses of AP, CC14 or BB were considerably resilient against toxicity of all three hepatotoxicants. Maximum protection was observed with AP; a LDT0 dose of AP in normal mice yielded no mortality in their diabetic counterparts. Measurement of liver injury (alanine aminotransferase and aspartate aminotransferase) and liver function (bilirnbin) after AP administration revealed that diabetic mice had significantly lower liver injury. This was also evident from histopathological examination of H & E stained liver sections. Liver tissue repair measured via 3H-thymidine pulse labeling indicated earlier onset of S-phase in the diabetic mice. Immunohistochemical staining of liver sections for proliferating ceU nnclear antigen (PCNA) showed greater number of ceils in cell cycle in the diabetic mice. In a separate study PCNA analysis of liver sections from STZ-induced diabetic mice not treated with AP revealed greater number of cells in G2 phase. Apoptosis evaluated by immunohistochemical staining (Apotag) did not show any changes due to diabetes alone. Immunochemical analysis of covalently bound AP to liver revealed no difference between normal and diabetic mice. Covalent binding of reactive metabolites of AP to liver macromolecules (58, 56 and 44 kD target proteins) represents the net effect of all bioactivation and detoxification mechanisms. Our results indicate that changes in bioactivation a n d / o r detoxification do not account for the resiliency of diabetic mice. It appears that up-regulation of liver regeneration mechanisms in the diabetic mice confers protection against AP toxicity.

Peroxisome proliferator-activated receptor 2/(PPART), a member of the nuclear hormone receptor superfamily, regulates cellular differentiation (Nat Med 1998). Recent evidence has suggested that mitochondrial proliferation and paradoxical membrane depolarization were involved in cellular differentiation in colon cancer cells (J Cell Biol 1997). The AIMS of this study were to assess i) potential of troglitazone, a PPARy ligand, to induce hepatocellnlar differentiation, and ii) alterations in hepatocellular mitochondrial mass and mitochondrial transmembrane potential (MTP) through PPAR'y activation by troglitazone. Methods: An immortalized human hepatocyte cell line (OUMS29) was used in this study. Induction of hepatoceUular differentiation was assessed by Western blotting for both p27Kipl protein, a differentiation-associated cycfin-dependent kinase inhibitor, and hepatocyte-specific proteins, such as albumin, alpha-l-antitrypsin (AAT), and alpha-fetoprotein (AFP). Mitochondrial mass and MTP were examined by flow cytometry using the mitochondrial probes, nonyl-acridine orange (NAO) and rhodamine 123, respectively. Ultrastructural examination of troglitazone-treated OUMS-29 cells was performed by transmission electron microscopy. Results: Increased expressions of p27, albumin, and AAT were demonstrated in troglitazone-treated OUMS-29 cells, without any significant decrease in AFP expression, indicating a partial hepatoceUular differentiation. In concert with these protein expressions, an increase in mitochondrial mass in the troglitazone-treated cells was found by NAO analysis. However, these cells also demonstrated a decline in unit MTP as determined by rhodamine 123. Ultrastructural examination of troglitazone-treated cells confirmed enlarged mitochondria. Conclusion: These results suggest that troglitazone has potential to induce hepatocellular differentiation, involving increase in mitochondrial mass and paradoxical decrease in MTP.