HEPATOLOGY Vol. 22, No. 4, Pt . 2, 1995 A A S L D A B S T R A C T S 3 7 3 A

1065 CONDITIONALLY IMMORTALIZED HEPATOCYTES FROM LONG-EVANS CINNAMON (LEC) RATS: AN IN VITRO MODEL FOR GENE REPLACEMENT THERAPY FOR WILSON'S DISEASE. ML Schilsky, S Kabischer. P Manehikalaoudi. LH Pr im~ . Marion Bessin Liver Res. Ctr., Albert Einstein Coil. of Med., Bronx, N'Y.

The LEC rat is a rodent model for Wilson's disease. Ahn: to estab- fish an in vitro model for gene therapy for Wilson's disease by condi- tional immortalization of LEC rat bepatocytes. Criteria for the LEC phenotype evaluated included the expression of specific differentiated hepatocellular proteins, Cu transport, Cu content and subceUular dis- tribufion. Methods: Immortalized cell lines (tsLEC) were obtained by retroviral mediated transformation of primary LEC hepatoeytes with the temperature sensitive SV40 T-Ag. Immortalized cells were grown and maintained at 33°C or transferred to 37 or 39°C for 24 h. Cu uptake and efflux was determined using 67Cu; Cu content by atomic absorption spectrophotometry following incubation of ceils in media containing I00 tzM Cu for 24 h at 370C. Expression of SV40 T-Ag, asialoglycoprotein receptor (ASGR), protein disulfide isomer- ase (PDI) was estimated by Western blot analysis. Results: Cu content of tsLEC were 1.05/zg/mg protein compared to 0.46 for immortalized control rat hepatocytes. Cu content of lysosomal/mito- chondrlal, microsomal and cytosol subfractions of tsLEC contained 21, 4, and 65% of cellular Cu content as compared to 8, 4, and 83 in a control. Initial rate of Cu uptake in medium with 10 ttM Cu for tsLEC grown at 33°C was 5.08 pmoles/min/mg protein, and in- creased to 20.60 for ceils at 37°C. Net Cu efflux from tsLEC main- tained at 37°C was 1.8, 1.7 and L2% of the total cell associated Cu over 5, 15 and 30 rain. Expression of T-Ag decreased by 46 and 58% for tsLEC at 37 and 39°C, respectively, while ASGR increased by 42 and 72%. Steady state levels of PDI were unchanged. Conclu- sions: 1) Cell surface Cu transport is modulated by the state of hepatocellular differentiation; 2) Normal Cu transport in conjunction with altered Cu content and subcellular distribution define the LEC phenotype in tsLEC ceils; 3) Immortalized LEC hepatocytes provide a model for the study of gene replacement therapy for Wilson's disease. Supported by NIH CA61259.

1066 MITOCHONDRIAL ABNORMALITIES IN LONG-EVANS CINNAMON (LEC) RATS: I Sternlieb. N Ouintana, I Volenberg and ML Schilsky*. National Center for Study of Wilson's Disease, St. Lukes's/Roosevelt Med. Center, Columbia University, New York, and *Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY.

Aim: To identify cellular targets of copper toxicity in the hepatocytes of LEC rats, an animal model of Wilson's disease. Methods: Fine sections of glutaraldehyde/osmium fixed, Epon embedded samples of liver from 23 LEC rats ranging in age from 10 to 89 weeks, were examined by electron microscopy. The hepatic copper concentration ranged from 325 to 2126 meg/g dry weight (mean, 930 meg/g) Thirteen rats displayed mild to severe jaundice. Portions of liver from 3 rats, aged 26, 43 and 89 weeks, respectively, unaffected by cholangiofibrosis, were included in this study. Results: Pleomorphism with changes in size, shape, contents and electron density was conspicuous in many mitochondria. In certain hepatocytes mitochondrial matrical density was enhanced due to condensa- tion; or decreased, due to swelling, with increased electron lucency of the matrix. Cristal changes included dilatations, rearrangements, elongation with parallel stacking or absence of cristae. Mitochondrial granules were absent in certain organdies. Dense matrical inclusions, or flocculent, electron opaque densities, indicating mitochonddal degeneration, were also seen. Some of these alterations were similar to those identified as human Wilsonian Types II and III mitochondrial changes. The severity of the mitochondrial changes did not correlate with the hepatic copper concentra- tion, but did correlate with worsening icterus. The specificity of the observed changes for the LEC rat and for Wilsonian hepatocytes is evident since neither experimental copper loading, nor naturally occurring animal models of hepatic copper toxicosis, namely the Bedlington terrier and the toxic milk mouse, exhibit similar changes even though their hepatic copper concentrations often exceed those of the LEC rats.

1067 PROLIFERATION OF HEPATIC LYSOSOMES AND AUTOPHAGIC VACUOLES IN THE LONG EVANS CINNAMON (LEC) RAT. BM Myers, BA Hamilton, ML Schilsky*. Department of Medicine, University of Florida, Gainesville, FL and *Albert Einstein College of Medicine, Bronx, NY,

The Long-Evans Cinnamon (LEC) rat spontaneously develops hepatic copper overload similar to Wilson's disease. Fulminant hepatitis, chronic hepatitis, and hepatocellular carcinoma (HCC) eventually occur in the LEC rat. In addition to significant inflammation, the histological changes in the LEC rat include pleomorphic nuclei and mitochondrial changes. The AIM of our study was to quantitatively assess the subcellular morphological changes of the liver in the LEC rat. METHODS: Control (Con) (n=3) and eight month old LEC rats (n=4) were sacrificed and liver was fixed in 2.0% glutaraldehyde, washed in PBS, and postfixed in 1% OsO,. Samples were dehydrated, embedded in Spurrs and thin sectioned for transmission electron microscopy. Quantitative morphometry of lysosomes, peroxisomes, and autophagic vacuoles was performed on approximately 80 to 120 hepatocytes for each rat using the Sigma Scan image analysis program. Hepatic copper concentrations were measured by atomic absorption spectrophotometry. RESULTS: Hepatic Cu concentrations were increased 33-fold in the LEC rats (Con 33.98+7 #g/g, LEC 1114.25+202 ttg/g, p<0.02). Quantitative morphometry demonstrated significant increases in the area ratios of lysosomes (Con 0.387 +0.042, LEC 1.518+0.060, p<0.001) and autophagic vacuoles (Con 0.524+0.125, LEC 1.930-+0.261, p<0.001). Peroxisomes were slightly increased (Con 0.501-+0.031, LEC 0.628+0.044, p<0.03). SUMMARY: Spontaneous hepatic copper overload in the LEC rat results in a significant proliferation of lysosomes and autophagic vacuoles and a slight increase in peroxisomes. The increases in autophagy and proliferation of lysosomes may be in response to organellar injury (particularly mitochondria) due to excess copper.

1 0 6 ~ IDENTIFICATION OF HETEROZYGOUS CARRIERS OF THE WILSON'S DISEASE (WD) GENE. Th. M a i e r - ~ . C. PoliL S. Rack. E. Cauza. Ch. Mannhahcr, P. Ferfnci. Dept.Internal Medicine IV, Univ.of Vierma, Austria To identify carriers of the WD gene 11 fanfilies of index cases with a total of 71 subjects were examined by RFLPs using the flanking genes D13S31, D13S59 and D13S26. If RFLP analysis was not informative linkage analysis by the PCR based microsatellite markers D13S295, D13S301, D13S296, D13S314 and D13S133 was performed, By this approach a correct classification of all tested subjects was possible. Heterozygous caniers of the W'dson's disease gene were examined clinically and by routine hiboratory testing including determination of ALT, AST, GGTP, cemloplasmin (ep) and bifimbin. R~UIts:

N Relation to index case parent siblin~ child ~and~eat other second de~ree TOTAL

N heterozygous obligate detected by carriers RFLP/PCR

22 22 17 17 5 5 4 4 7 7

55 27 28

heterozyg low ep

2 4

ous with abnormal

LFT 9 4

3 2

18 In 3 carriers with low ep hepatic copper determined in liver biopsy specimens by atom absorption spectroscopy was 3 fold increased (130, 172, 233) but below the diagnostic level of 250 pg/g Thus, heterozygous carriers of the WD gene cannot be reliably detected by routine laboratory testing. Surprisingly, the carrier rate among siblings was higher than expected (17 were heterozygous~ 3 were normal). No heterozygote had evidence of liver disease (only GGTP values were elevated in 18 heterozygous family members).