Ethacrynic-acid-induced glutathione depletion and oxidative stress in normal and Mrp2-deficient rat liver

Oxidative stress in the liver is sometimes accompanied by cholestasis. We investigated the localization and role of multidrug-resistance-associated protein (Mrp) 2, a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress. Normal Sprague-Dawley rat (SDR) and Mrp2-deficient Eisai hyperbilirubinemic rat (EHBR) livers were perfused with 500 microM EA. The release of glutamic pyruvic transaminase (GPT) and thiobarbituric-acid-reactive substances (TBARS) from EHBR liver was markedly delayed compared with that from SDR liver. This is mainly due to the higher basal level of glutathione (GSH) in EHBR liver (59.1 +/- 0.3 nmol/mg protein) compared with SDR liver (39.7 +/- 1.5 nmol/mg protein). EA similarly induced a rapid reduction in GSH followed by mitochondrial permeability transition in the isolated mitochondria from both SDR and EHBR. Internalization of Mrp2 was detected before nonspecific disruption of the canalicular membrane and GPT release in SDR liver perfused with 100 microM EA. SDR liver preperfused with hyperosmolar buffer (405 mosmol/L) for 30 min induced internalization of Mrp2 without changing the basal GSH level, while elimination of hepatic GSH by 300 microM EA perfusion was significantly delayed thereafter. Concomitantly, hepatotoxicity assessed by the release of GPT and TBARS was also significantly attenuated under hyperosmolar conditions. In conclusion, preserved cytosolic and intramitochondrial GSH is the key factor involved in the acute hepatotoxicity induced by EA and its susceptibility could be altered by the presence of Mrp2.     

Mrp2 is involved in benzylpenicillin-induced choleresis

Benzylpenicillin (PCG; 180 micromol/kg), a classic beta-lactam antibiotic, was intravenously given to Sprague-Dawley (SD) rats and multidrug resistance-associated protein 2 (Mrp2)-deficient Eisai hyperbilirubinemic rats (EHBR). A percentage of the [(3)H]PCG was excreted into the bile of the rats within 60 min (SD rats: 31.7% and EHBR: 4.3%). Remarkably, a transient increase in the bile flow ( approximately 2-fold) and a slight increase in the total biliary bilirubin excretion were observed in SD rats but not in the EHBR after PCG administration. This suggests that the biliary excretion of PCG and its choleretic effect are Mrp2-dependent. Positive correlations were observed between the biliary excretion rate of PCG and bile flow (r(2) = 0.768) and more remarkably between the biliary excretion rate of GSH and bile flow (r(2) = 0.968). No ATP-dependent uptake of [(3)H]PCG was observed in Mrp2-expressing Sf9 membrane vesicles, whereas other forms of Mrp2-substrate transport were stimulated in the presence of PCG. GSH efflux mediated by human MRP2 expressed in Madin-Darby canine kidney II cells was enhanced in the presence of PCG in a concentration-dependent manner. In conclusion, the choleretic effect of PCG is caused by the stimulation of biliary GSH efflux as well as the concentrative biliary excretion of PCG itself, both of which were Mrp2 dependent.     

Determination of phase I metabolic enzyme activities in liver microsomes of Mrp2 deficient TR- and EHBR rats

The canalicular multispecific organic anion transporter/multidrug resistance protein 2 (cMOAT/Mrp2) plays a major role in the transport of anionic xenobiotics across the bile canalicular membrane. Transport deficient rats (TR-) and Eisai-hyperbilirubinemic rats (EHBR), defective in Mrp2, are mutants of Wistar and Sprague Dawley (SD) rats, respectively. In this study, Phase I metabolic enzyme activities in liver microsomes prepared from these mutant male and female rats were compared to their corresponding non-mutant rats. The total cytochrome P450 contents and NADPH-cytochrome P450 reductase activity in male and female TR- rats were significantly higher than in Wistar rats. In male TR- rats, ethoxyresorufin O-deethylation (EROD), pentoxyresorufin O-deethylation (PROD), testosterone 2alpha, 7alpha and 16 alpha-hydroxylase activities were higher, but testosterone 6beta-hydroxylase activity and the rate of androstenedione formation were lower than in Wistar rats. Female TR- rats had higher 7alpha-hydroxylase activity, but EROD activity was lower in female Wistar rats. Similar studies conducted in EHBR versus SD rats demonstrated increased total cytochrome P450 content in male and female EHBR rats; NADPH-cytochrome P450 reductase activity was not significantly affected. Decreased PROD activity and the rate of androstenedione formation were observed in male and female EHBR rats. Furthermore, testosterone 6beta-hydroxylase activity was lower in male EHBR rats than in male SD rats while testosterone 7alpha-hydroxylase activity was significantly higher in male and female EHBR rats. Thus, in addition to Mrp2 deficiency, differential expression of CYP isoforms and their potential impact on the metabolism and pharmacokinetics of compounds should be considered when interpreting data from these rat strains.     

A probable relationship between characteristic accumulation of doxorubicin and P-glycoprotein transporter in rat liver

Correlations between the distribution of anthracycline antibiotics doxorubicin (DX) and daunorubicin (DR) in the liver of rats injected with a single i.v. injection of each drug and the reported distribution of P-glycoprotein transporter for the drug was histochemically examined. Immunocytochemical studies for DX or DR using monoclonal antibody that equally detects both drugs, as well as conventional electron microscopy were employed. DX persisted for more than 5 days in the cytoplasm and nuclei of the hepatocytes in a characteristic granular morphology on the bile capillaries. Meanwhile, DR distributed in almost the same pattern, but more rapidly decreased to a level that almost no granular morphology in the hepatocytes was visible 24 h after the injection. Also, unknown large cells that strongly reacted with the antibody appeared in the hepatic sinusoids near the interlobular triads rather than the central vein. The accumulation sites of DX or DR on the bile capillaries seems to correspond to specific sites where the ATP-binding cassette transport protein P-glycoprotein for the anthracycline occurs, suggesting a possibility that DX or DR is actually and actively excreted at these sites, possibly through P-glycoprotein. This is supported by conventional electron microscopic studies, since no specific subcellular organelles such as lysosomes assemble in the vicinity of the bile capillaries of the hepatocytes.     

Enhanced expression of basolateral multidrug resistance protein isoforms Mrp3 and Mrp5 in rat liver by LPS

Lipopolysaccharide (LPS) induces hepatocellular down-regulation and endocytic retrieval of multidrug resistance protein 2 (Mrp2, Abcc2). Basolateral Mrp isoforms may compensate for the intracellular metabolic changes in cholestasis. Therefore, the effect of LPS on the zonal localization of Mrp2 and Mrp3 and the expression of Mrp3, Mrp4, Mrp5, and Mrp6 mRNA were investigated in rat liver. In normal rat liver Mrp3 was found in pericentral hepatocytes also expressing glutamine synthetase. In LPS-treated rat liver the decrease in Mrp2 protein was most pronounced in pericentral hepatocytes, with only minor down-regulation in periportal hepatocytes. Conversely, induction of Mrp3 was found in pericentral hepatocytes with a low expression of Mrp2. Furthermore, we found a strong induction of Mrp5 mRNA. Likewise, Mrp6 mRNA was up-regulated, however Mrp6 protein expression was not significantly altered. It is concluded that Mrp3 is inversely regulated to Mrp2 in a zonal pattern and may compensate for the LPS-induced loss of Mrp2 in the perivenous area. Induction of pericentral Mrp3 and up-regulation of Mrp5 mRNA may play an important role in the hepatocellular clearance of cholephilic substances and cyclic nucleotides accumulating after LPS treatment.     

Subcellular distribution of titanium in the liver after treatment with the antitumor agent titanocene dichloride. A study using electron spectroscopic imaging

In the present study, the subcellular distribution of titanium in the liver of mice was determined 24 and 48 h after application of a therapeutic (ED100; ED = effective dose) and a toxic (LD25; LD = lethal dose) dose (60 and 80 mg/kg, respectively) of the antitumor agent titanocene dichloride by electron spectroscopic imaging at the ultrastructural level. At 24 h, titanium was mainly accumulated in the cytoplasm of endothelial and Kupffer cells, lining the hepatic sinusoids. Titanium was detected in the nucleoli and the euchromatin of liver cells, packaged as granules together with phosphorus and oxygen. One day later titanium was still present in cytoplasmic inclusions within endothelial and Kupffer cells, whereas in hepatocyte nucleoli only a few deposits of titanium were observed at 48 h. At this time titanium was mainly accumulated in the form of highly condensed granules in the euchromatin and the perinucleolar heterochromatin. It was found in the cytoplasm of liver cells, incorporated into cytoplasmic inclusion bodies which probably represent lysosomes. Sometimes these inclusions were situated near bile canaliculi and occasionally extruded their content into the lumen of bile capillaries. This observation suggests a mainly biliary elimination of titanium-containing metabolites. These results confirm electron spectroscopic imaging to be an appropriate method for determining the subcellular distribution of light and medium-weight elements within biological tissues. Insights into the cellular mode of action of titanocene complexes or titanocene metabolites can be deduced from the findings of the present study.     

Interactions between hepatic Mrp4 and Sult2a as revealed by the constitutive androstane receptor and Mrp4 knockout mice

The ABC transporter, Mrp4, transports the sulfated steroid DHEA-s, and sulfated bile acids interact with Mrp4 with high affinity. Hepatic Mrp4 levels are low, but increase under cholestatic conditions. We therefore inferred that up-regulation of Mrp4 during cholestasis is a compensatory mechanism to protect the liver from accumulation of hydrophobic bile acids. We determined that the nuclear receptor CAR is required to coordinately up-regulate hepatic expression of Mrp4 and an enzyme known to sulfate hydroxy-bile acids and steroids, Sult2a1. CAR activators increased Mrp4 and Sult2a1 expression in primary human hepatocytes and HepG2, a human liver cell line. Sult2a1 was down-regulated in Mrp4-null mice, further indicating an inter-relation between Mrp4 and Sult2a1 gene expression. Based on the hydrophilic nature of sulfated bile acids and the Mrp4 capability to transport sulfated steroids, our findings suggest that Mrp4 and Sult2a1 participate in an integrated pathway mediating elimination of sulfated steroid and bile acid metabolites from the liver.     

Subcellular distribution of titanium in the liver after treatment with the antitumor agent titanocene dichloride

In the present study, the subcellular distribution of titanium in the liver of mice was determined 24 and 48 h after application of a therapeutic (ED₁₀₀; ED = effective dose) and a toxic (LD₂₅; LD = lethal dose) dose (60 and 80 mg/kg, respectively) of the antitumor agent titanocene dichloride by electron spectroscopic imaging at the ultrastructural level. At 24 h, titanium was mainly accumulated in the cytoplasm of endothelial and Kupffer cells, lining the hepatic sinusoids. Titanium was detected in the nucleoli and the euchromatin of liver cells, packaged as granules together with phosphorus and oxygen. One day later titanium was still present in cytoplasmic inclusions within endothelial and Kupffer cells, whereas in hepatocyte nucleoli only a few deposits of titanium were observed at 48 h. At this time titanium was mainly accumulated in the form of highly condensed granules in the euchromatin and the perinucleolar heterochromatin. It was found in the cytoplasm of liver cells, incorporated into cytoplasmic inclusion bodies which probably represent lysosomes. Sometimes these inclusions were situated near bile canaliculi and occasionally extruded their content into the lumen of bile capillaries. This observation suggests a mainly biliary elimination of titanium-containing metabolites. These results confirm electron spectroscopic imaging to be an appropriate method for determining the subcellular distribution of light and medium-weight elements within biological tissues. Insights into the cellular mode of action of titanocene complexes or titanocene metabolites can be deduced from the findings of the present study.     

Characterization of bile acid transport mediated by multidrug resistance associated protein 2 and bile salt export pump

Biliary excretion of certain bile acids is mediated by multidrug resistance associated protein 2 (Mrp2) and the bile salt export pump (Bsep). In the present study, the transport properties of several bile acids were characterized in canalicular membrane vesicles (CMVs) isolated from Sprague--Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR) whose Mrp2 function is hereditarily defective and in membrane vesicles isolated from Sf9 cells infected with recombinant baculovirus containing cDNAs encoding Mrp2 and Bsep. ATP-dependent uptake of [(3)H]taurochenodeoxycholate sulfate (TCDC-S) (K(m)=8.8 microM) and [(3)H]taurolithocholate sulfate (TLC-S) (K(m)=1.5 microM) was observed in CMVs from SD rats, but not from EHBR. In addition, ATP-dependent uptake of [(3)H]TLC-S (K(m)=3.9 microM) and [(3)H]taurocholate (TC) (K(m)=7.5 microM) was also observed in Mrp2- and Bsep-expressing Sf9 membrane vesicles, respectively. TCDC-S and TLC-S inhibited the ATP-dependent TC uptake into CMVs from SD rats with IC(50) values of 4.6 microM and 1.2 microM, respectively. In contrast, the corresponding values for Sf9 cells expressing Bsep were 59 and 62 microM, respectively, which were similar to those determined in CMVs from EHBR (68 and 33 microM, respectively). By co-expressing Mrp2 with Bsep in Sf9 cells, IC(50) values for membrane vesicles from these cells shifted to values comparable with those in CMVs from SD rats (4.6 and 1.2 microM). Moreover, in membrane vesicles where both Mrp2 and Bsep are co-expressed, preincubation with the sulfated bile acids potentiated their inhibitory effect on Bsep-mediated TC transport. These results can be accounted for by assuming that the sulfated bile acids trans-inhibit the Bsep-mediated transport of TC.     

Regulation of hepatic glucose metabolism by translocation of glucokinase between the nucleus and the cytoplasm in hepatocytes.

We studied the role of glucokinase translocation between the nucleus and the cytoplasm in hepatocytes. In cultured hepatocytes, both the translocation of glucokinase from the nucleus to the cytoplasm and the rate of glucose phosphorylation were increased when cells were incubated with high concentrations of glucose. The addition of low concentrations of fructose, which is known to stimulate glucose phosphorylation, stimulated both glucokinase translocation and glucose phosphorylation. There was a good correlation between the increase in cytoplasmic glucokinase induced by fructose and that in the glucose phosphorylation rate induced by fructose. Furthermore, we observed a linear relationship between cytoplasmic glucokinase activity and rate of glucose phosphorylation over various glucose concentrations in the absence or presence of fructose. These results indicate that glucose phosphorylation in hepatocytes depended on glucokinase in the cytoplasmic compartment--that is, the increase in the rate of glucose phosphorylation was due to the increase in translocation of glucokinase out of the nucleus. Also, oral administration of glucose, fructose, or glucose plus fructose to 24-h fasted rats induced translocation of glucokinase in the liver. All of these results indicate that hepatic glucose metabolism is regulated by the translocation of glucokinase.     

Ultrastructure of the liver of the fingerling rainbow trout Salmo gairdneri Richardson

Portions of the livers of fingerling rainbow trout were studied by light and electron microscopy. The histology, cytology and ultrastructure of mesothelial cells, serosal fibroblasts, hepatocytes, sinusoidal endothelial cells, endothelial cells of central veins and blood cells were described. Mesothelial cells and fibroblasts constituted a very thin capsule. Hepatocytes contained extensive areas of rough surfaced endoplasmic reticulum, consisting mainly of parallel cisternae and pools of glycogen. One or two nuclei and numerous mitochondria occurred in the areas of endoplasmic reticulum, but never in the pools of glycogen. Hepatocyte surface possibilities included hepatocyte to hepatocyte, hepatocyte to bile canaliculus, hepatocyte to space of Disse and hepatocyte to serosa. The trout liver was compared compared to channel catfish liver and to rat liver. Functional implications of the structural features were discussed.     

Tissue and subcellular distribution of glucokinase in rat liver and their changes during fasting-refeeding

The distribution of glucokinase in rat liver under both normal feeding and fasting-refeeding conditions was investigated immunohistochemically. Under normal feeding conditions, glucokinase immunoreactivity was observed in both nuclei and cytoplasm of parenchymal cells. The nuclei were stained intensely and evenly, whereas the cytoplasm showed weak immunoreactivity of different degrees of staining intensity depending on the location of the cells. The cytoplasm of perivenous hepatocytes was stained more intensely, though not so much more, than that of periportal hepatocytes. The cytoplasm of hepatocytes surrounding the terminal hepatic venule (THV), of hepatocytes surrounding the portal triad, and of some other hepatocytes showed a stronger immunoreactivity than that of residual hepatocytes. The nuclear immunoreactivity in hepatocytes surrounding the portal triad and in some other hepatocytes was weak or absent, and positive immunoreactivity was detected at the plasma membrane of some of these cells. After 72 h of fasting, glucokinase immunoreactivity was markedly decreased in all hepatocytes. After the start of refeeding, the cytoplasmic immunoreactivity began to increase first in the parenchymal cells surrounding the THV and extended to those in the intermediate zone followed by those in the periportal zone. In contrast, the increase in nuclear immunoreactivity started in hepatocytes situated in the intermediate zone adjacent to the perivenous zone and then extended to those in the perivenous zone followed by those in the periportal zone. Hepatocytes surrounding either THV or portal triad showed a distinctive change in immunoreactivity during the refeeding period. After 10 h of refeeding, strong immunoreactivity was observed in both the cytoplasm and the nuclei of all hepatocytes, and appreciable glucokinase immunoreactivity was detected at the plasma membrane of some hepatocytes. These findings are discussed from the standpoint of a functional role of glucokinase in hepatic glucose metabolism.     

Immunohistochemical localization of glutathione S-transferase-T1 in murine kidney, liver, and lung

 Glutathione S-transferase-mediated metabolism of exogenous compounds usually leads to detoxification, but there are some exceptions. For example, glutathione S-transferase-T1 (GSTT1) can also generate genotoxic metabolites. Studies on the biology of GSTT1 are limited by the lack of specific antibodies recognizing GSTT1 in animal tissues. We localized GSTT1 immunohistochemically in mouse kidney, liver, and lung using a novel antibody targeted against the C-terminus of rat GSTT1 (rGSTT1). The antibody was characterized using immunoblot and shown to specifically recognize rGSTT1 and mouse GSTT1, but not human GSTT1. In kidney, GSTT1 staining was detected only in collecting duct epithelium. In liver, pericentral hepatocytes showed cytoplasmic and nuclear staining. Nuclear staining was also observed in several other hepatocytes without relation to liver zonation. Nuclei and supranuclear cytoplasm of bile duct epithelium and endothelium of interlobular arterioles also reacted strongly. In lung, staining was observed in bronchiolar epithelium and in surrounding muscle cells. Type II pneumocytes and endothelial cells of intrapulmonary capillaries also showed strong positive staining. This report describes the first immunohistochemical localization of GSTT1 in mammalian tissues. The reported location of GSTT1 is consistent with its known metabolic activity toward compounds such as dichloromethane and their metabolism into genotoxic products. Accepted: 11 May 1998     

Short-term regulation of multidrug resistance-associated protein 3 in rat and human hepatocytes

The short-term regulation of multidrug resistance-associated protein 3 (Mrp3/MRP3) by cAMP and PKC was investigated in sandwich-cultured rat and human hepatocytes and isolated perfused rat livers. The modulator glucagon (500 nM) and the phorbol ester PMA (0.1 muM) were utilized to increase intracellular cAMP and PKC levels, respectively. In glucagon-treated rat hepatocytes, efflux of the Mrp3 substrate 5-(6)-carboxy-2',7'-dichlorofluorescein (CDF) increased approximately 1.5-fold, even in hepatocytes treated with the organic anion transporter (Oatp) inhibitor sulfobromophthalein (BSP). Confocal microscopy revealed more concentrated Mrp3 fluorescence in the basolateral membrane (less diffuse staining pattern) with glucagon treatment. PMA had no effect on Mrp3 activity or localization in sandwich-cultured rat hepatocytes. Glucagon and PMA treatment in isolated perfused rat livers resulted in a threefold increase (14 +/- 4.6 mul.min(-1).g liver(-1)) and a fourfold decrease (1.3 +/- 0.3 mul.min(-1).g liver(-1)) in CDF basolateral clearance compared with control livers (4.7 +/- 2.3 mul.min(-1).g liver(-1)), whereas CDF biliary clearance was not statistically different. In sandwich-cultured human hepatocytes, glucagon treatment resulted in a 1.3-fold increase in CDF efflux and a concomitant increase in MRP3 fluorescence in the basolateral membrane. In summary, cAMP and PKC appear to be involved in the short-term regulation of Mrp3/MRP3, as demonstrated by alterations in activity and localization in rat and human hepatocytes.     

Immunocytochemistry for bestatin and its application to drug accumulation studies in rat intestine and kidney

The in vivo role of transporters in drug disposition, in the context of other transporters, and metabolism has not been established. We prepared an anti-bestatin serum against bestatin conjugated to albumin with glutaraldehyde (GA). The antiserum was specific for GA-conjugated bestatin and weakly reacted with free bestatin, but no reaction occurred with structurally unrelated compounds according to both the inhibition and binding ELISAs. The antiserum allowed us to develop an immunocytochemical (ICC) method for detecting the uptake of bestatin in the rat intestine and kidney. Three hours after a single oral administration of bestatin, the ICC method revealed that the drug distributed in the microvilli, cytoplasm and nuclei of the absorptive epithelial cells at much larger amounts than in all other cell types in the small intestine. However, no drug was detected in the mucin goblets in the epithelium. In the kidney, the drug distributed to a greater extent in the S3 segment than in the S1 and S2 segments of the proximal tubule, and also was detected in the microvilli of the proximal tubule cells (S1, S2 and S3). These findings that bestatin accumulated in large amounts, especially in the cells and/or at the sites where the transporters PEPT1 and PEPT2 occur, corresponded well to those observed with β-lactam amoxicillin in the previous ICC studies. Thus, this may indicate a possibility that both the transporters might be involved, at least in part, in the distribution of bestatin in the small intestine and kidney under the conditions examined.     

Expression of HBsAg and HBcAg in liver tissue: correlation with disease activity.

The patterns of Hepatitis B surface antigen (HBsAg) and Hepatitis B core antigen (HBcAg) expression were studied in liver biopsies taken from 41 patients with chronic HBV disease. Immunohistochemical methods were used on deparaffinized sections for the identification of HBsAg and HBcAg in liver tissue. Twenty-one of the 41 cases (51.2%) were classified as inactive liver disease and 20 (48.8%) as active liver disease. In liver biopsies with inactive disease, HBsAg demonstrated varying types of cytoplasmic expression in a rather high number of hepatocytes distributed mainly in clusters, while HBcAg was rarely expressed in liver nuclei. On the other hand, in liver biopsies with active disease HBsAg was characterized by a diffuse cytoplasmic expression in a few discrete hepatocytes, while HBcAg was expressed in the nuclei of the hepatocytes in 70% of the cases and in half of the positive cases it was also detected in the cytoplasm. In conclusion, HBsAg expression in a few scattered hepatocytes correlates with active liver disease and positive HBcAg, while varying HBsAg cytoplasmic expression in a rather high number of clustered hepatocytes is related to chronic inactive liver disease and negative expression of HBcAg.     

Expression of the insulinoma gene rig during liver regeneration and in primary cultured hepatocytes

We have isolated a novel gene, rig (rat insulinoma gene) from rat insulinomas. In the present study, rig was found to be expressed in rat regenerating liver and in primary cultured rat hepatocytes. The level of rig mRNA was increased at the proliferative phase of liver regeneration. In synchronously cultured hepatocytes, the rig mRNA level was elevated at the G1 phase of the cell cycle and the rig-protein was accumulated in the nuclei during the S phase. These results indicated that rig could be involved in a more general way in growth or cell replication.     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration.

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36-40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G2/M phase of the cell cycle was significantly higher than that in G0/G1 phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G2/M phase.