HEPATOLOGY Vol. 22, No. 4, P t . 2, 1995 A A S L D A B S T R A C T S 5 1 1 A

1617 EFFECTS OF VITAMIN E, ASCORBIC ACID AND CATALASE ON BROMOBENZENE- AND HYDROGEN PEROXIDE-INDUCED INTRA- CELLULAR OXIDATION AND SINGLE STRAND DNA BREAKAGE IN HEP G2 CELLS. J Wu t,3,4, K Karlsson 2 and A Danielsson 3. Depts. of 1Histology and Cell Biology, 2Clinical Chemistry, and 3Medicine, University of Umeft, Ume~t, Sweden and 4Dept. of Medicine, Jeffexson Medical College, Philadelphia, PA 19107

Background: Water soluble vitamin E (Trolox C, VE), aseorbic acid (VC) and catalase have been shown to protect isolated rat hepatocytes against bromobenzene (BB)-iodueed toxicity in our previous study.

Methods: In order to study the mechanisms of this protection and pathogenesis of BB-induced hepatocelhiiar injury, a fluorometrical assay using multiple well plates for investigation of ini~acellular oxidation, indicated by c o n v e r s i o n of d ichlorof luorescln diacetate to dichiorofluorescein (DCF), was developed. Single strand DNA breakage (SSDB) was also evaluated in Hep G2 cells by a radiolabellng method.

Results: BB (2.4 and 4.8 raM) may induce a significant increase in DCF fluorescence intensity compared to the controls (111.9"&1.8, 115.7+1.3 vs 100.0-2:2.0%, p<0.01). VE and VC completely inhibited BB-induced enhancement of fluorescence intensity (76.4+9.0, 66.4:t:1.2 vs 109.0"/:1.5%, p<0.001), as well as reduced intracellular oxidation in untreated Hep G2 cells. Hydrogen peroxide (H202, 200 and 400 $tM) evoked a dose- dependent increase of DCF fluorescence intensity in Hep G2 cells (127.4+4.1 and 150.1:1:8.5%) compared to the controls, and the effect was completely blocked by VE (2.0 raM, 98.5+9.9%, p<0.01) and cataiase (4800 unit/ml, 100.9+9.7%, p<0.01). BB caused significant SSDB in Hep G2 cells during 2 h suspension Incubation (2.4 and 4.8 raM; 25.6+1.3, 46.1+3.7 vs 17.3+6.1%, p<0.01) and long term (24 h) incubation (4.8 raM, 65.2+3.6 vs 20.4+9.0%, 13<0.01). H202 (400 pM) led to marked SSDB in 15 rain (49.5+3.8%), and the effect was attenuated by VE (30.9!-0.8% vs 41.7+1.8%, p<0.01). Unexpectedly, VC (0.5 raM) alone elicited a great amount of SSDB during both suspension (82.0+__2.3%) and long term incubations (72.8+1.7%) in Hep G2 cells.

Conluslons: Metabolism of BB in Hep G2 ceils induces production of H202 or other free radicals and leads to SSDB in the ceils. VE, VC and catalase display strong intracelhilar anti-oxidative effects. VE could partially inhibit H202-induced SSDB in the cells. However, VC caused marked DNA damage in our system. One possible explanation for this finding is the conversion of ascorbic acid to ascorbate.

1618 MODELS FOR THE DOMINANT NEGATIVE EFFECT OF THE ALDH2 *2 ALLELE RESPONSIBLE FOR MITOCHONDRIAL ALDEHYDE DEHYDROGENASE DEFICIENCY IN HUMANS. Q. Xia_o and D.W. Crabb, Department of Medicine and Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN

Individuals heterozygous or homozygous for the variant aldehyde dehydrogenase (ALDH2) allele (ALDH2*2), which encodes a protein differing only at residue 487 from the normal protein, have decreased liver ALDH2 activity and experience cutaneous flushing when they drink alcohol. The mechanism by which this allele exerts its dominant effect is unknown. To study this effect, the human ALDH2*I eDNA was cloned and the ALDH2*2 allele was generated by site-directed mutagenesis. These cDNAs were transduced using retroviral vectors into HeLa cells, which do not normally express ALDH2~ The normal allele directed synthesis of immunorcactive ALDH2 protein (ALDH2E). Extracts of these cells contained increased aldehyde dehydrogenase activity with low K~ for the aldehyde substrate. The ALDH2*2 allele directed synthesis of mRNA and immanoreactive protein (ALDH2K), but the protein lacked enzymatic activity. When ALDH2*I- expressing cells were transduced with ALDH2*2 vectors, both mRNAs were expressed and immunorcactive proteins with isoelectric points ranging between those of ALDH2E and ALDH2K were present, indicating that the subunits formedheteromers. ALDH2 activity in these cells was reduced below that of the parental ALDH2*l-expressing cells. Cells expressing higher levels of ALDH2K had lower residual ALDH2 activity. Thus, the ALDH2*2 allele is sufficient to cause ALDH2 deficiency in vitro. The degree of reduction in ALDH2 activity was correlated with the relative expression of the ALDH2*I and ALDH2*2 alleles. The data best fit a model in which (ALDH2E)4 and (ALDH2E)3(ALDH2K)I tetramers have enzymatic activity, but heterotetramers with more than one ALDH2K subunit are inactive. This model predicts that heterozygous livers should exhibit perhaps 18% of usual ALDH2 activity. It remains uncertain why no ALDH2 activity can be detected on gel electrophoresis of the heterozygous liver extracts. (Supported by NIH 006434 and a grant from the American Liver Foundation).

1619 HEPATIC CYST COMPUTED TOMOGRAPHY OF HU,~YN FAS- CIOLIASIS OBSTRUCTIVE PHASE. Ouyang Xinzhong. Hospital Library,First Affiliated Hospital,di- angxi Medical College,Nanchang, 330OO6,China.

Introduction : Hepatic computed tomography of human fascioliasis in international had 10s cases report.:~thods : After the fasciola hepa- tica in hepatic cyst of one human fasciolia- sis drive out,respectively examine by computed tomography. Results: The hepatic cysts of many similar round and diverse composed ~otal con- struction of tree branching cysts: the large cyst of right front leaf is 62x48x40 mm , had cross from right hepatica to below diaphragm, CT value 15 Hu,edge distinct : the t,'o cysts of right behind leaf is 30x26 mm, a cyst of left leaf is 30×20 mm,the ten cysts of left and ri- ght leaf is 10-4 mm,CT value 5-19 }~. A part more large hepatic cysts had more dense cystic wall than hepatic tissue, had focal fibrosis, to show characteristic no equal arc line dense photograph in cysts periphery, has different with hepatic tumour.The paroxysmal pain in he- patic area. In s cyst had one adult fasciola hepatica.After drive out the fasciola hepatica and cysts content discharge,~fter ten months, amount of the cysts and the cysts size not change.The hepatic area painless. Conclusions : The hepatic cysts computed tomography of human fascioliasis obstructive phase has specific diagnostic characteristics: (I) Many hepatic cysts similar tree branching.(2)Varied density photograph of cystic wall.(3) Diverse hepatic density photograph around few bile @uctule.

1620 MICROTUBULE-DEPENDENT BILE ACID TRANSPORT IS IMPAIRED IN HEPATOCYTE DOUBLETS IN REGENERATING LIVER. T. Yamada. T. Kitamura. M. Hirose. N. Enomoto, A. Yumoto. S. Suzuki. S. Watanabe and N. Sato. Department of Gastroenterology, Juntendo University School of Medicine, Tokyo, Japan.

It has been believed that bile acid transport is in part dependent on microtubular system. However, its role regarding cellular polarity has been poorly understood in bepatocytes proliferation. To clarify the relation between cellular polarity and proliferation, we investigated fluorescein isothiocyanate-glycocholate (FITC-GC) transport and microtubular distribution in hepatocyte doublets in normal rats and those after partial hepatectomy. M E T H O D S : FITC-GC was synthesized as previously reported. All experimentswere performed using hepatocytes derived from normal rats (NR), rats 18hr after two-thirds hepatectomy (PHR-18hr) and 10days after one (PHR-10days). Following incubation of doublets with 2$tM FITC-GC for 1 min, bile canalicular secretion of FITC-GC was investigated in fluorescence microscope. Distribution of microtubular network in doublets was examined by immunofluorescence staining using anti-[btubulin antibody. DNA synthesis was determined by BrdU incorporation. RESULTS: BrdU labeling index of hepatocytes was significantly higher in PHR-18hr than NR and PHR-10days. Percent of canalicular secreion of FITC-GC was significantly decreased in PHR-18hr, as compared with NR, but recovered in PHR-10days.

NR PHR- 18hr PHR- 10days BrdU labeling index 0.8+_0.02% 34.5+_2.8% 3.0+_0.97%

(z~3) (.--3) (~3) % of canalicular 55.8+_1.8% 3 5 . 2 + 6 . 5 % 54.7+_7.4%

~ o n (n=5) (n=4) (n=-3)

Immunofluorescence study in doublets demonstrated fine reticular network radiating from the pericanalicular region to the cell periphery in NR. This disappeared in PHR-18hr, and came out again in PHR-10days. CONCLUSIONS: These results indicate that the decreased cellular polarity may be caused by the impaired microtubnies, which are possibly depolymerized in cellular proliferation.