- Email: [email protected]
Plasma selenoproteins in patients with cirrhosis
A1060
AASLD ABSTRACTS
Q ALTERATIONS IN SEX STEROID RECEPTOR S T A T U S AND M E T A B O L I S M IN PEROXISOME PROLIFERATOR-INDUCED CARCINOGENESIS. P.K. Eagon, M.S. Elm, M.J. Epley, H. Shinozuka, K.N. Rao. VA Medical Center and Depts. of Medicine and Pathology, University of Pittsburgh School of Medicine, Pittsburgh PA.
Introduction: Both androgenic and estrogenic steroids have been shown to influence the development and course of several liver diseases. Estrogens are well known as hyperplastic agents in liver. In addition, hepatoeellular carcinoma (HCC) displays a predominantly male distribution worldwide, suggesting that androgens might play a role in its etiology. Because estrogen (ER) and androgen (AR) receptors are putative mediators of hormone action, several studies have assessed whether receptor status differs in normal and malignant human liver specimens; results are mixed, but in general, the evidence suggests that HCCs have less ER and more AR. In the current work. a mt model of hepatocarcinogenesis was used to determine temporal changes in sex hormone receptors and metabolism during carcinogenesis. Methods: Male rats were fed peroxisome proliferator (PP) agents (diethylhexylphthalate [DEI-IP], clofibrate [CLO] and BR931 for various time periods up to > 1 year. Livers were examined for proliferation markers, and activity of cytosolic (c) and nuclear (n) ER and AR as well as two enzymes important in hormone homeostasis, E 2-hydroxylase (E2-OHase) and aromatase (AROM). Results: At all time points, the liver/body ratio and DNA content significantly increased over coutrol values. During early exposure to DEHP and CLO (3, 7 & 60 days), nER and nAR increased significantly in PP-treated rats. E2-OHase activity decreased by ~60% (p<.05) and serum E relative to testosterone (T) increased. Long-term (> I yr) BR931-treated rats developed tumors, which were analyzed separately from the histologically normal surrounding liver. In the tumors, eER decreased 97% and nER 59%; however, only eER in the surrounding liver decreased. In contrast, AR activity increased in the tumor (cAR, 177% and .nAR, 184% of control) and also in the normal liver surrounding the tumor (cAR, 147% and nAR, 191% of control). Both tumor and surrounding liver demonstrated drastic reductions in steroid metabolism, with aromatase 15% and E2-OHase 11% of that of nondiseased liver. A likely cousequence of this reduction in enzyme activity is the observed elevation of both serum E and T in the tumor-bearing animals. Conclusions: Early in carcinogenesis but prior to tumor formation, both ER and AR are elevated, as is circulating E relative to T. In tumor-bearing livers, only the AR elevation persists, and is accompanied by an increased serum T level, most likely as a result of decreased aromatase. This study supports the thesis that estrogens act as proliferative agents early in carcinogenesis, although androgens may provide an additional stimulus. Once HCC has developed, however, ER and thus E responsiveness is lost. and the tumor retains only its androgen responsiveness. These observations also provide a basis for the observed failure of tamoxifen as a useful therapeutic agent in HCC,
• HUMAN HEPATOMA CELL GROWTH IS REDUCED BY ANTI-SENSE MYC AND ITS RELATION TO ALPHA-FETOPROTEIN GENE EXPRESSION. H. Ebinuma, H. Saito, T. Watanabe ,S. Tada, S. Tsunematsu, T. Masuda, K. Atsukawa, M. Tsuchiya, H. Ishii. Dept. of I n t e r n a l Medicine, School of Medicine, Keio University, Tokyo, J a p a n We h a v e p r e v i o u s l y r e p o r t e d t h a t s o d i u m b u t y r a t e (SB) is a p o t e n t i n d u c e r of t h e d i f f e r e n t i a t i o n in h u m a n h e p a t o m a cell lines, PLC/PRF/5, HCC-M a n d HCC-T. C - m y c g e n e e x p r e s s i o n is d e c r e a s e d s e v e r a l h o u r s a f t e r t h e a d d i t i o n o f SB w i t h a d e c l i n e in the proliferative rate with significant morphological change (Int J C a n c e r 48, 291, 1991). In PLC/PRF/5 cells, a l p h a - f e t o p r o r e i n (AFP) g e n e e x p r e s s i o n w a s also r e d u c e d b y SB t r e a t m e n t . T h e o b i e c t i v e of t h i s s t u d y w a s to see t h e r e l a t i o n s h i p b e t w e e n g e n e e x p r e s s i o n of c - m y c a n d AFP, a n d m o r p h o l o g i c a l c h a n g e i n h u m a n h e p a t o m a cell lines. M a t e r i a l s a n d M e t h o d s : One t h o u s a n d of HCC-M (Int J C a n c e r 3 2 , 1 4 1 , 1 9 8 3 ) , HCC-T ( C a n c e r 64, 1054, 1 9 8 9 ) o r PLC/PRF/5 ceils w e r e c u l t u r e d in a 9 6 - w e l l m i c r o t i t e r p l a t e , a n d v a r i o u s c o n c e n t r a t i o n s of a n t i - s e n s e m y c (5'- AACGTTGAGGGGCAT-3', for e x o n 1) o r s e n s e m y c ( 5 ' ATGCCCTCAACGTT-3') w a s a d d e d a n d cell g r o w t h w a s m e a sured by a colorimetric method. The morphological change was o b s e r v e d b y a p h a s e - c o n t r a s t m i c r o s c o p e . T o t a l RNA w a s ext r a c t e d f r o m ceils b e f o r e a n d a f t e r t h e a d d i t i o n of a n t i - s e n s e o r s e n s e m y c a n d c h a n g e s of c - m y c a n d AFP g e n e e x p r e s s i o n w e r e a n a l y z e d b y N o r t h e r n b l o t t i n g . Results : A n t i - s e n s e m y c s u p p r e s s e d cell g r o w t h of t h e s e cell l i n e s d o s e - d e p e n d e n t l y , alt h o u g h s e n s e m y c d i d not. No s i g n i f i c a n t m o r p h o l o g i c a l C h a n g e w a s o b s e r v e d b y t h e t r e a t m e n t w i t h a n t i - s e n s e o r s e n s e myc. E x p r e s s i o n of c - m y c d e c r e a s e d t i m e - d e p e n d e n t l y a s s o c i a t e d w i t h d e c r e a s e d AFP e x p r e s s i o n , t h o u g h a t i m e - l a g w a s o b s e r v e d b e t w e e n t h e s e two g e n e e x p r e s s i o n s . S e n s e m y c g e n e d i d n o t affect gene expressions. Conclusion : These results suggest that c - m y c a n d AFP g e n e e x p r e s s i o n are c l o s e l y l i n k e d e a c h o t h e r c o n t r i b u t i n g to t h e m o d u l a t i o n of HCC cell g r o w t h , b u t n o t to the morphological change.
GASTROENTEROLOGY, Vol. 108, No. 4
• PLASMA SELENOPROTEINS IN PATIENTS WITH CIRRHOSIS. Dayna S. Early, Kristina E. Hill, Martha E. Boeglin, Ivan S. Palmer, and Raymond F. Burk. Department of Medicine, Vanderbilt University, Nashville, TN 37232 and Department of Chemistry, SDSU, Brookings, S.D. 57007. Two selenoproteins are present in plasma. Selenoprotein P (Se-P) is a selenium-rich protein secreted by the fiver, and extracellular glutathione peroxidase (eGSH-Px) is a selanoprotein secreted largely by the kidneys. In addition to the selenium in these selenoproteins, a significant amount of selenium is present in plasma as plant-derived selenomethionine substituted randomiy for methionine in proteins. We measured Se-P by RIA, eGSHPx activity with H202, and selenium in plasma. Subjects with cirrhosis of Child's classes A, B, and C were studied as well as normal controls. controls n 13 Se-Pt 0.92 -+ 0,2 eGSH-Px* 1 3 6 + 2 7 seleniumf 14-+1.4
Child's A 14 0,80 + 0.3 124+40 11"1-2.2"
Child's B 16 0.62 + 0.3* 166+61 9.8-+2.3*
Child's C 10 0.45 + 0.2* 204+97* 8.4-+1.8"
tSe-P, mU/ml; eGSH-Px, nmol NADPH ord(min-ml); selenium p.g/dl *Different from control values by Fisher's PLSD Plasma selenium decreased with severity of cirrhosis. This has been reported previously and is related at least partly to a decrease in albumin that contains selenium as selenomethionine. The true selenopmteins responded differently from one another to liver disease. Se-P decreased with increasing severity of cirrhosis and eGSH-Px increased. Thus, the decrease in plasma selenium concentration in cirrhosis does not represent a sirhple decrease in all plasma selenium compartments. The fall in Se-P is consistent with its origin being the liver. Se-P is postulated to function in oxidant defense so ks depression might contribute to the systemic effects of cirrhosis. Supported by NIH ES 06093.
DUPLEX-(ULTRA)SONOGRAPHY: A NON-INVASIVE PREDICTOR OF HEPATIC FIBROSIS. M.Ecker, ILFleisebmann, T.Eberl, M.Wienbeek. Dept. Me,d. llI, Zentralldinikum A~burg. Germany.
Objecaves: It is difficult m make the diagnosis of hepatic fibrosis on the basis of clinical and laboratorydata. This study was designed to measure portal and hepatic vein blood-flow velocities (PBF and I-IBF) by duplex-sonography in patients with different stages of liver disease and test its usefulness in making the diagnosis of hepatic fibrosis. Patients and methods: 80 c,onse~ative m-patients and I0 healthy volunteerswith normal liver function (physical examination, rotaJne laboratory data including serum bilirubin, prothrombin time and ammonia, ICG-test, number cormection test)were investigatedby duplex-sonography and real-time sonography. All examinations were performed with a computer instrument CS 9600/9500 (Picker International, Espelkamp) and a 3.5 MHz sector mmsducer. The Doppler-gate width was 6 to 10 rsm. Each value was averaged from 3 readings. Fatty liver was staged by rceltime sonography into 3 degrees (FL 1-3). The diagnosis of liver fibrosis and fatty liver was made by liver histolo~. Differences were tested for statistical significance by the two tailed t-test. Results: There was no significant difference in portal blood flow between the different groups of patients and controls. But hepatic vein blood-flow velocity (Vmean) was significantly increased in patients with hepatic fibrosis and FL 3 (i)<0.05) (Tab,) as compared to volunteers and patients with normal and mild to moderate fatty liver. The sensitivityof inoreased Vmean for the diagnosis of hepatic fibrosis is 79%, the specificity 85°/*. The positive predictive value is 79'/,, the negative n'edictive value 88%. Cases ControlGroup HealthySubjects °FLI °FL2 °FL3 Fibrosisl Fibrosis2
n
Age
Vmean*
C195%
Table: 10 25.8 10.3crn/s
08.93
37 6 6 5 7 19
09.34_
51.6 38.4 55.0 54.1 40.0 43.8
10.0en4s 12.0cm/s 12.3cm/s 14.1cm/s 16.4~m/s 17.3em/s
* Mean-Blood-FlowVelocity in Hepatic Veins ° Fatty Liver 1 Dia~osis by Histology 2 Diagnosis by Histology and Sunography
Conclusions: The duplex measusement of hepatic blood-flow velocity seems to be
useful in detecting hepatic fibrosis.
AASLD ABSTRACTS
Q ALTERATIONS IN SEX STEROID RECEPTOR S T A T U S AND M E T A B O L I S M IN PEROXISOME PROLIFERATOR-INDUCED CARCINOGENESIS. P.K. Eagon, M.S. Elm, M.J. Epley, H. Shinozuka, K.N. Rao. VA Medical Center and Depts. of Medicine and Pathology, University of Pittsburgh School of Medicine, Pittsburgh PA.
Introduction: Both androgenic and estrogenic steroids have been shown to influence the development and course of several liver diseases. Estrogens are well known as hyperplastic agents in liver. In addition, hepatoeellular carcinoma (HCC) displays a predominantly male distribution worldwide, suggesting that androgens might play a role in its etiology. Because estrogen (ER) and androgen (AR) receptors are putative mediators of hormone action, several studies have assessed whether receptor status differs in normal and malignant human liver specimens; results are mixed, but in general, the evidence suggests that HCCs have less ER and more AR. In the current work. a mt model of hepatocarcinogenesis was used to determine temporal changes in sex hormone receptors and metabolism during carcinogenesis. Methods: Male rats were fed peroxisome proliferator (PP) agents (diethylhexylphthalate [DEI-IP], clofibrate [CLO] and BR931 for various time periods up to > 1 year. Livers were examined for proliferation markers, and activity of cytosolic (c) and nuclear (n) ER and AR as well as two enzymes important in hormone homeostasis, E 2-hydroxylase (E2-OHase) and aromatase (AROM). Results: At all time points, the liver/body ratio and DNA content significantly increased over coutrol values. During early exposure to DEHP and CLO (3, 7 & 60 days), nER and nAR increased significantly in PP-treated rats. E2-OHase activity decreased by ~60% (p<.05) and serum E relative to testosterone (T) increased. Long-term (> I yr) BR931-treated rats developed tumors, which were analyzed separately from the histologically normal surrounding liver. In the tumors, eER decreased 97% and nER 59%; however, only eER in the surrounding liver decreased. In contrast, AR activity increased in the tumor (cAR, 177% and .nAR, 184% of control) and also in the normal liver surrounding the tumor (cAR, 147% and nAR, 191% of control). Both tumor and surrounding liver demonstrated drastic reductions in steroid metabolism, with aromatase 15% and E2-OHase 11% of that of nondiseased liver. A likely cousequence of this reduction in enzyme activity is the observed elevation of both serum E and T in the tumor-bearing animals. Conclusions: Early in carcinogenesis but prior to tumor formation, both ER and AR are elevated, as is circulating E relative to T. In tumor-bearing livers, only the AR elevation persists, and is accompanied by an increased serum T level, most likely as a result of decreased aromatase. This study supports the thesis that estrogens act as proliferative agents early in carcinogenesis, although androgens may provide an additional stimulus. Once HCC has developed, however, ER and thus E responsiveness is lost. and the tumor retains only its androgen responsiveness. These observations also provide a basis for the observed failure of tamoxifen as a useful therapeutic agent in HCC,
• HUMAN HEPATOMA CELL GROWTH IS REDUCED BY ANTI-SENSE MYC AND ITS RELATION TO ALPHA-FETOPROTEIN GENE EXPRESSION. H. Ebinuma, H. Saito, T. Watanabe ,S. Tada, S. Tsunematsu, T. Masuda, K. Atsukawa, M. Tsuchiya, H. Ishii. Dept. of I n t e r n a l Medicine, School of Medicine, Keio University, Tokyo, J a p a n We h a v e p r e v i o u s l y r e p o r t e d t h a t s o d i u m b u t y r a t e (SB) is a p o t e n t i n d u c e r of t h e d i f f e r e n t i a t i o n in h u m a n h e p a t o m a cell lines, PLC/PRF/5, HCC-M a n d HCC-T. C - m y c g e n e e x p r e s s i o n is d e c r e a s e d s e v e r a l h o u r s a f t e r t h e a d d i t i o n o f SB w i t h a d e c l i n e in the proliferative rate with significant morphological change (Int J C a n c e r 48, 291, 1991). In PLC/PRF/5 cells, a l p h a - f e t o p r o r e i n (AFP) g e n e e x p r e s s i o n w a s also r e d u c e d b y SB t r e a t m e n t . T h e o b i e c t i v e of t h i s s t u d y w a s to see t h e r e l a t i o n s h i p b e t w e e n g e n e e x p r e s s i o n of c - m y c a n d AFP, a n d m o r p h o l o g i c a l c h a n g e i n h u m a n h e p a t o m a cell lines. M a t e r i a l s a n d M e t h o d s : One t h o u s a n d of HCC-M (Int J C a n c e r 3 2 , 1 4 1 , 1 9 8 3 ) , HCC-T ( C a n c e r 64, 1054, 1 9 8 9 ) o r PLC/PRF/5 ceils w e r e c u l t u r e d in a 9 6 - w e l l m i c r o t i t e r p l a t e , a n d v a r i o u s c o n c e n t r a t i o n s of a n t i - s e n s e m y c (5'- AACGTTGAGGGGCAT-3', for e x o n 1) o r s e n s e m y c ( 5 ' ATGCCCTCAACGTT-3') w a s a d d e d a n d cell g r o w t h w a s m e a sured by a colorimetric method. The morphological change was o b s e r v e d b y a p h a s e - c o n t r a s t m i c r o s c o p e . T o t a l RNA w a s ext r a c t e d f r o m ceils b e f o r e a n d a f t e r t h e a d d i t i o n of a n t i - s e n s e o r s e n s e m y c a n d c h a n g e s of c - m y c a n d AFP g e n e e x p r e s s i o n w e r e a n a l y z e d b y N o r t h e r n b l o t t i n g . Results : A n t i - s e n s e m y c s u p p r e s s e d cell g r o w t h of t h e s e cell l i n e s d o s e - d e p e n d e n t l y , alt h o u g h s e n s e m y c d i d not. No s i g n i f i c a n t m o r p h o l o g i c a l C h a n g e w a s o b s e r v e d b y t h e t r e a t m e n t w i t h a n t i - s e n s e o r s e n s e myc. E x p r e s s i o n of c - m y c d e c r e a s e d t i m e - d e p e n d e n t l y a s s o c i a t e d w i t h d e c r e a s e d AFP e x p r e s s i o n , t h o u g h a t i m e - l a g w a s o b s e r v e d b e t w e e n t h e s e two g e n e e x p r e s s i o n s . S e n s e m y c g e n e d i d n o t affect gene expressions. Conclusion : These results suggest that c - m y c a n d AFP g e n e e x p r e s s i o n are c l o s e l y l i n k e d e a c h o t h e r c o n t r i b u t i n g to t h e m o d u l a t i o n of HCC cell g r o w t h , b u t n o t to the morphological change.
GASTROENTEROLOGY, Vol. 108, No. 4
• PLASMA SELENOPROTEINS IN PATIENTS WITH CIRRHOSIS. Dayna S. Early, Kristina E. Hill, Martha E. Boeglin, Ivan S. Palmer, and Raymond F. Burk. Department of Medicine, Vanderbilt University, Nashville, TN 37232 and Department of Chemistry, SDSU, Brookings, S.D. 57007. Two selenoproteins are present in plasma. Selenoprotein P (Se-P) is a selenium-rich protein secreted by the fiver, and extracellular glutathione peroxidase (eGSH-Px) is a selanoprotein secreted largely by the kidneys. In addition to the selenium in these selenoproteins, a significant amount of selenium is present in plasma as plant-derived selenomethionine substituted randomiy for methionine in proteins. We measured Se-P by RIA, eGSHPx activity with H202, and selenium in plasma. Subjects with cirrhosis of Child's classes A, B, and C were studied as well as normal controls. controls n 13 Se-Pt 0.92 -+ 0,2 eGSH-Px* 1 3 6 + 2 7 seleniumf 14-+1.4
Child's A 14 0,80 + 0.3 124+40 11"1-2.2"
Child's B 16 0.62 + 0.3* 166+61 9.8-+2.3*
Child's C 10 0.45 + 0.2* 204+97* 8.4-+1.8"
tSe-P, mU/ml; eGSH-Px, nmol NADPH ord(min-ml); selenium p.g/dl *Different from control values by Fisher's PLSD Plasma selenium decreased with severity of cirrhosis. This has been reported previously and is related at least partly to a decrease in albumin that contains selenium as selenomethionine. The true selenopmteins responded differently from one another to liver disease. Se-P decreased with increasing severity of cirrhosis and eGSH-Px increased. Thus, the decrease in plasma selenium concentration in cirrhosis does not represent a sirhple decrease in all plasma selenium compartments. The fall in Se-P is consistent with its origin being the liver. Se-P is postulated to function in oxidant defense so ks depression might contribute to the systemic effects of cirrhosis. Supported by NIH ES 06093.
DUPLEX-(ULTRA)SONOGRAPHY: A NON-INVASIVE PREDICTOR OF HEPATIC FIBROSIS. M.Ecker, ILFleisebmann, T.Eberl, M.Wienbeek. Dept. Me,d. llI, Zentralldinikum A~burg. Germany.
Objecaves: It is difficult m make the diagnosis of hepatic fibrosis on the basis of clinical and laboratorydata. This study was designed to measure portal and hepatic vein blood-flow velocities (PBF and I-IBF) by duplex-sonography in patients with different stages of liver disease and test its usefulness in making the diagnosis of hepatic fibrosis. Patients and methods: 80 c,onse~ative m-patients and I0 healthy volunteerswith normal liver function (physical examination, rotaJne laboratory data including serum bilirubin, prothrombin time and ammonia, ICG-test, number cormection test)were investigatedby duplex-sonography and real-time sonography. All examinations were performed with a computer instrument CS 9600/9500 (Picker International, Espelkamp) and a 3.5 MHz sector mmsducer. The Doppler-gate width was 6 to 10 rsm. Each value was averaged from 3 readings. Fatty liver was staged by rceltime sonography into 3 degrees (FL 1-3). The diagnosis of liver fibrosis and fatty liver was made by liver histolo~. Differences were tested for statistical significance by the two tailed t-test. Results: There was no significant difference in portal blood flow between the different groups of patients and controls. But hepatic vein blood-flow velocity (Vmean) was significantly increased in patients with hepatic fibrosis and FL 3 (i)<0.05) (Tab,) as compared to volunteers and patients with normal and mild to moderate fatty liver. The sensitivityof inoreased Vmean for the diagnosis of hepatic fibrosis is 79%, the specificity 85°/*. The positive predictive value is 79'/,, the negative n'edictive value 88%. Cases ControlGroup HealthySubjects °FLI °FL2 °FL3 Fibrosisl Fibrosis2
n
Age
Vmean*
C195%
Table: 10 25.8 10.3crn/s
08.93
37 6 6 5 7 19
09.34_
51.6 38.4 55.0 54.1 40.0 43.8
10.0en4s 12.0cm/s 12.3cm/s 14.1cm/s 16.4~m/s 17.3em/s
* Mean-Blood-FlowVelocity in Hepatic Veins ° Fatty Liver 1 Dia~osis by Histology 2 Diagnosis by Histology and Sunography
Conclusions: The duplex measusement of hepatic blood-flow velocity seems to be
useful in detecting hepatic fibrosis.