Pathogenic role of Kupffer cell activation in the reperfusion injury of cold-preserved liver.

The present study was designed to investigate the possible participation of Kupffer cells in the development of reperfusion injury of the cold-stored liver graft. In the cold preservation of Kupffer cells with Euro-Collins solution, the proportion of asialo-GM1-positive cells was significantly increased at 12 and 24 hr of storage, and the TNF alpha-producing activity in these cells was approximately fivefold greater than control. Northern blot analysis demonstrated that TNF alpha mRNA was remarkably elevated in the reperfusion of the cold-preserved liver, although that of the prereperfused graft was only slightly induced. The reperfusion experiments of the cold-stored liver graft showed that addition of anti-TNF alpha antibody to the perfusate suppressed the elevation of the effluent levels of GOT and LDH significantly, and that pretreatment with a Kupffer cell inhibitor, gadolinium chloride, inhibited the increase of these enzymes in the effluents almost completely. Histological study revealed deposition of a fibrinlike substance in the sinusoid and the central veins extensively in the reperfused liver graft, whereas no apparent deposition was observed in the gadolinium-pretreated liver. Thus, the present study showed that Kupffer cells were primed by cold preservation with Euro-Collins solution, and then activated when the reperfusion was done. It seems likely that the Kupffer cell activation induced by cold preservation/reperfusion plays a major role in reperfusion injury with sinusoidal microcirculatory disturbance, and that TNF alpha is responsible for the impairment of the reperfused liver graft.

Decreased expression and rare somatic mutation of the CIP1/WAF1 gene in human hepatocellular carcinoma.

CIP1/WAF1, a critical downstream effector of tumor suppressor p53, encodes a cyclin-dependent kinase inhibitor. By Northern blot analysis, the CIP1/WAF1 mRNA level in the tumor was significantly lower than that in the corresponding normal liver from 19 Japanese patients with hepatocellular carcinoma (P < 0.05). In the tumor from only one out of 19 patients (5%), somatic mutations of the CIP1/WAF1 as well as that of p53 gene were identified by RT-PCR/SSCP analysis. These results suggest that the decreased CIP1/WAF1 expression is involved in the carcinogenesis or the progression of hepatocellular carcinoma.

Expression of membrane-type matrix metalloproteinase-1 in human pancreatic adenocarcinomas.

The expression of a new type of matrix metalloproteinase, membrane-type matrix metalloproteinase-1 (MT-MMP-1), was examined in 24 cases of primary pancreatic adenocarcinomas and 9 cases of secondary liver tumors derived from pancreatic adenocarcinomas, using a non-radioactive in situ hybridization and immunohistochemical methods. Out of 24 cases of primary pancreatic adenocarcinomas, 18 showed positive expression of MT-MMP-1 transcripts in cancer cells and 20 of 24 showed positive expression in the tumor stromal cells. The immunoreactivity of the gene products for MT-MMP-1 was demonstrated to be almost the same, as shown by in situ hybridization in these 24 cases. In particular, both the staining intensity for MT-MMP-1 transcripts and the immunoreactivity of the gene products in the tumor stromal cells of mucinous cystadenocarcinomas were significantly weaker than those of common-type ductal adenocarcinomas among the 24 cases. All of the 9 cases of secondary liver tumors derived from pancreatic adenocarcinomas showed positive expression for MT-MMP- transcripts but less immunoreactivity for the gene products. These results suggest that MT-MMP-1 is transcribed and translated in both cancer cells and the tumor stromal cells in human pancreatic adenocarcinomas. Furthermore, considering that common-type ductal adenocarcinoma of the pancreas usually shows a strong desmoplastic reaction, while mucinous cystadenocarcinoma typically does not, MT-MMP-1 expressed in the tumor stromal cells of common-type adenocarcinomas may be involved in processes leading to the desmoplastic reaction.

Tumoricidal activity of Kupffer cells augmented by anticancer drugs.

The effect of Mitomycin-C (MMC) and Adriamycin (ADM) on the antitumor-associated function of Kupffer cells was examined. MMC and ADM enhanced the production of superoxide by Kupffer cells in cultures at low concentrations likely to occur in clinical use. The expression of interleukin-2 receptor, Ia antigen and asialoGM1 antigen, measured by flowcytometry, was increased by contact with MMC. Growth inhibition of AH130, rat ascites hepatoma, and P815, murine mastocytoma, by Kupffer cells treated with anticancer drugs was greater than that by Kupffer cells alone or anticancer agent alone. These results show that MMC and ADM activate Kupffer cells, leading to synergistic antitumor activity. The results suggest that some anticancer agents act through immunological mechanisms as well as through direct antineoplastic activity.

Effect of PGE2 on interleukin-1 and superoxide release from primary-cultured human hepatic macrophages.

In order to learn more about how human hepatic macrophages function, we analyzed the effect of exogenous PGE2 on the amounts of interleukin-1 (IL-1) and superoxide (O2-) released from primary-cultured human hepatic macrophages (HHM phi). When endogenous PGE2 production was blocked by indomethacin, exogenous PGE2 reduced IL-1 release from HHM phi in a dose-dependent manner, whereas it tended to increase O2- release from HHM phi. These results may suggest the probable contribution of PGE2 in regulating HHM phi mediator release in vivo.

[Studies on PCB toxicity involving 2C subfamily cytochrome P450].

It has been demonstrated that PCB metabolism is mainly catalyzed by 1A and 2B subfamily cytochrome P450s, CYP1A1/2 and CYP2B1/2. These studies were conducted mostly with hepatic enzymes in rodents. The 1A and 2B subfamily P450 s are constitutively expressed little, but markedly induced by xenobiotics such as 3-methylcholanthrene and phenobarbital in rodents. On the other hand, the recent studies showed that cytochrome P450s in human liver are remarkably different from isoform of rodents in constitution and enzyme activities. In the present study, we first tried to metabolize some PCBs with 2C subfamily cytochrome P450 (CYP2C) purified from dog liver microsomes. The data suggested that CYP2C may not be involved in PCB metabolism. Since CYP2C is the same most abundant enzyme as 3A subfamily P450 in human liver and plays a major role for metabolism of many drugs used clinically, and may also play an important role for metabolism of some steroid hormones, we further studied the inhibition of CYP2C-catalyzed steroid metabolism by typical PCB congeners. CYP2C-mediated steroid metabolism is greatly inhibited by 2, 4, 5, 2', 4', 5'-hexachlorbiphenyl, but not by 3, 4, 5, 3', 4'-pentachlorobiphenyl. On the contrary, 3, 4, 5, 3', 4'-pentachlorobiphenyl markedly suppressed CYP2C expression in the dog liver. These results suggest that residual PCBs may affect the current situation of steroid hormones in Yusho patients, and may cause PCB drug interactions.

Strychnine and brucine as the potent inducers of drug metabolizing enzymes in rat liver: different profiles from phenobarbital on the induction of cytochrome P450 and UDP-glucuronosyltransferase.

The inducing activities of two alkaloids, strychnine and brucine, on the hepatic drug metabolizing enzymes were studied in rats. Administration of strychnine in the drinking water to rats significantly increased the hepatic microsomal activities of benzphetamine N-demethylation, strychnine 2-hydroxylation and testosterone hydroxylations at positions 16 alpha and 16 beta. These results together with that of immunostaining of microsomal proteins revealed that strychnine is a potent inducer of CYP2B1 and 2B2. The comparable induction of CYP2B1/2 was observed by brucine treatment with less toxic effect. Although this inducer increased CYP2B cytochrome P450s (P450s) to the maximum levels after 4 consecutive days of administration, the maximal increase by strychnine was attained after 3 days of administration. Immunoblotting experiment suggested that significant proteolysis of CYP2B1 occurs during treatment by strychnine and brucine. These alkaloids exhibited no ability to induce the activities of testosterone hydroxylations at positions 2 alpha, 6 beta and 7 alpha, benzo[a]pyrene 3-hydroxylation and aniline hydroxylation. In addition to the CYP2B P450, strychnine and brucine induced glutathione S-transferase toward 1-chloro-2,4-dinitrobenzene and UDP-glucuronosyltransferase toward 4-nitrophenol. On the other hand, the glucuronidations of 4-hydroxybiphenyl and morphine were not enhanced by alkaloid treatments. These results indicated that strychnine and brucine cause phenobarbital-like induction of the P450 enzyme, but show a different profile from phenobarbital in the induction of UDP-glucuronosyltransferase.

N-Dealkylation and hydroxylation of ebastine by human liver cytochrome P450.

Ebastine [4'-tert-butyl-4-[4-(diphenylmethoxy)piperidino]butyro phe- none] is a new-generation, nonsedative, H1 antihistamine. The present study was performed to characterize the cytochrome P450 (CYP) isoforms responsible for ebastine N-dealkylation and hydroxylation. Human liver microsomes metabolized ebastine to two major metabolites, i.e. a desbutyrophenone metabolite (des-BP) and hydroxyebastine (M-OH), and the ratio of Vmax values was 3:1. N-Dealkylation yielded des-BP, whereas M-OH, an hydroxylation product, could be further oxidized to the pharmacologically active carebastine. In a panel of 14 human liver microsomal preparations, the rate of dealkylation showed a highly significant correlation with CYP3A-mediated testosterone 6beta-hydroxylation but not with reactions of seven other CYP isoforms. However, there was no correlation between the two pathways for ebastine (dealkylation and hydroxylation). Differential chemical inhibition in liver microsomes, in which dealkylation was more sensitive than hydroxylation, was demonstrated with ketoconazole, troleandomycin, cyclosporin A, and midazolam. Anti-CYP3A antibodies markedly reduced the dealkylation rate (>95%) in liver microsomes but exhibited insignificant effects on hydroxylation (<5%). Among 12 cDNA-expressed human CYP isoforms, which account for up to 70% of the total CYP enzyme content in human liver, CYP3A4 alone metabolized ebastine; the ratio of des-BP to M-OH formation was 12:1. This ratio for metabolism by the pure enzyme was much larger than the ratio (3:1) observed for the microsomal reaction mixture. This change in ratio, which is attributed to a decrease in M-OH formation, indicates that, although ebastine is metabolized to two major metabolites, N-dealkylation to des-BP is mediated by CYP3A, whereas hydroxylation to M-OH appears to be mediated mainly by unidentified enzymes other than CYP3A.

Brucine as a potent inducer of CYP2B3, the third member of the CYP2B subfamily P450 in rats.

We have indicated that strychnine and brucine induce concurrently an unknown P450 (2B-UP) which cross-reacts with anti-CYP2B1 antibody (Fujisaki et al., J. Pharmacol. Exp. Ther., 268, 1024-1031, 1994). We purified 2B-UP from brucine-treated rats and characterized it in this study. The purification was achieved by solubilization with sodium cholate followed by successive chromatographic steps with omega-aminooctyl-Sepharose 4B, DEAE-Sephacel and hydroxyapatite. The minimum molecular weight of purified 2B-UP was calculated to be 48000 by sodium dodecyl sulfate-gel electrophoresis. This preparation showed no Soret peak in the ultraviolet absorption spectrum indicating absence of heme. The amino terminal sequence of 2B-UP up to the 10th residue was consistent with the deduced amino acid sequence of CYP2B3 cDNA, but did not agree with the sequences of CYP2B1/2. The result strongly suggests that 2B-UP is CYP2B3. Thus, we indicated here that Strychnos alkaloid, brucine, is a potent inducer of the CYP2B3 or the closely related P450.

Reduced expression of kan-1 (encoding putative bile acid-CoA-amino acid N-acyltransferase) mRNA in livers of rats after partial hepatectomy and during sepsis.

We isolated a cDNA clone, kan-1, from a rat liver cDNA library using a reverse transcriptase PCR cloning method. The kan-1 cDNA encoded a polypeptide of 420 amino acids, and was 70 and 69% identical in nucleotide and amino acid sequences respectively with human liver bile acid-CoA-amino acid N-acyltransferase (BAT). Thus Kan-1 is probably a rat homologue of human BAT (rBAT). Kan-1/rBAT mRNA was mainly expressed in the livers of adult rats and rats immediately after, but not before, birth. It was expressed in the hepatocytes, the sinusoidal endothelial cells and the Kupffer cells of the liver. An anti-Kan-1/rBAT polyclonal antibody detected a protein of molecular mass 46 kDa in the liver. After partial hepatectomy, the levels of Kan-1/rBAT mRNA decreased at 6 and 12 h in the regenerating liver. In a sepsis model, hepatic expression of Kan-1/rBAT mRNA decreased at 6 and 12 h after caecal ligation and puncture. The kinetics of Kan-1/rBAT mRNA expression suggests that it may play a role in acute-phase reactions.

Augmented local immunity in the liver by a streptococcal preparation, OK432, related to antitumor activity of hepatic macrophages.

The aim of this study was to investigate the augmentative effect of a streptococcal preparation, OK432, on the immunological competence of hepatic macrophages. We found that OK432 was distributed predominantly to hepatic macrophages after intravenous injection, and Northern blot analysis revealed that OK432 induced the gene expression of IL-1 alpha, beta, and TNF alpha in the liver. The induction of mRNAs was evident 1 h after the intravenous injection of OK432 and their accumulation reached a maximal level at 3 h. TNF production of hepatic macrophages was also increased by the intravenous injection of OK432. Furthermore, OK432 significantly increased the proportion of IL-2 receptor-positive hepatic macrophages. As for antitumor activity in the liver being augmented by OK432, the cytotoxic and cytostatic activity of hepatic macrophages from OK432-treated rats against tumor cells was significantly increased and OK432 markedly reduced the number of tumor nodules in the liver after the inoculation of tumor cells via the portal vein. These findings, which indicate that OK432 has various immuno-stimulating actions on hepatic macrophages, leading to the augmentation of antitumor activity in the liver, suggest that OK432 may be of some benefit in helping to prevent hepatic metastasis, at least in part, via its activation of hepatic macrophages.

Inhibition of tumor growth and microvascular angiogenesis by the potent angiogenesis inhibitor, TNP-470, in rats.

The antiangiogenic effects of TNP-470 on the neovascularization of tumors were studied by examining ultrastructural alterations in the vasculature and interstitial fluid pressure (IFP) of tumors. Wistar rats were first inoculated subcutaneously (s.c.) with the Walker 256 carcinosarcoma cell line, then either vehicle medium or TNP-470, 30 mg/kg, was injected s.c. on day 1. A tumor growth assay, the necrotic area, and the IFP in the tumor were all measured on day 12. The antiangiogenic effects of TNP-470 were studied by scanning electron microscopic images of tumor vascular casts. TNP-470 was observed to inhibit tumor growth and increase the necrotic area significantly. In the TNP-470-treated group, the IFP in the superficial layer, defined as 2-3 mm from the tumor capsule, and in the deep layer, defined as 8-10 mm from the tumor capsule, were significantly higher than the corresponding values in the control. Moreover, vascular casts showed a significant reduction in the budding of sprouts in the superficial layer, and a decrease in the maximum diameter of the tumor vessels in the deep layer. It is possible that the higher IFP in the TNP-470-treated tumors might have prevented tumor vessel dilation. The findings of this study demonstrated that TNP-470 inhibited the budding of tumor vessel sprouts, and increased the IFP. These processes seem to act synergistically to suppress tumor angiogenesis.

Overexpression of matrix metalloproteinase 9 gene in hepatocellular carcinoma with invasive potential.

The prognosis for hepatocellular carcinoma (HCC) depends mainly on the clinicopathological characteristic regarding invasion and metastasis. In addition, another distinguishing feature of HCC is the high incidence of concomitant liver cirrhosis, in which the extracellular matrix proliferates markedly. Therefore, the present study was designed to investigate the molecules responsible for the invasion potential of HCC by focusing on matrix metalloproteinase (MMP) in particular, MMP-2 and MMP-9 and the corresponding tissue inhibitor of metalloproteinase (TIMP-2 and TIMP-1), because these enzymes participate in the degradation of the extracellular matrix including the basement membrane. Tumorous and adjacent nontumorous liver samples were obtained from 23 HCC patients who underwent a partial hepatectomy. In 16 of the 23 HCC samples, transcripts for MMP-9 were detected in the tumorous tissues, and 15 of 16 of these samples showed stronger expression in the tumorous tissues than in the nontumorous tissues. On the other hand, MMP-2 messenger RNA (mRNA) was detected in 18 of the 23 cases. Eight of these 18 cases showed more intense expression in the tumorous tissues than in the nontumorous tissues, whereas the expression levels were lower in the tumorous tissues than in the nontumorous tissues in 7 of 18 samples. With respect to the correlation between the clinicopathological features and mRNAs expression, it was found that the expression of MMP-9 mRNA in HCC with capsular infiltration was significantly higher than in HCC without capsular infiltration. HCC with capsular infiltration also tended to have a higher ratio of MMP-9 mRNA expression to TIMP-1 mRNA expression. In addition, the expression of MMP-2 mRNA in nontumorous cirrhotic tissues was significantly higher than in nontumorous tissues from patients with chronic hepatitis. Immunohistochemical examinations revealed that MMP-9 immunoreactivity was the most intense in the HCC cells, particularly in those cells in the marginal areas of the tumorous tissues. In conclusion, the present study shows that MMP-9 is closely participated in capsular infiltration in HCC.

Chemical mediators released by primary-cultured human hepatic macrophages in patients with and without cirrhosis: a study in tumor-bearing patients.

To elucidate the possible role of chemical mediators in modulating the host-defense activity of patients with cirrhosis, primary-cultured human hepatic macrophages (HHMphi) were obtained from cirrhotic and noncirrhotic patients who received liver resections because of the presence of malignant liver tumors. The cirrhotic and noncirrhotic groups consisted of patients with similar malignancies: noncirrhotic patients had normal liver function and normal liver histology for nontumorous portions. The cultured HHMphi were analyzed for their ability to release chemical mediators with specific activities in the host defense system. Dose-dependent increases in superoxide release, interleukin-1 (IL-1) release, and, within a relatively narrow range, prostaglandin-E2 (PGE2) release were observed in opsonized zymosan (oz)-stimulated HHMphi derived from both cirrhotic and noncirrhotic patients. The release of O2- and PGE2 from HHMphi derived from cirrhotic patients was significantly less than HHMphi derived from noncirrhotic patients, whereas the release of IL-1 was significantly greater. Although, because of the limited sample availability, only tumor-bearing patients were studied, the mediator-releasing ability of HHMphi derived from cirrhotic patients was significantly different from the ability of HHMphi derived from noncirrhotic patients with similar malignancies. This phenomenon may be related to altered host defenses in patients with cirrhosis.

A novel cytochrome P450 enzyme responsible for the metabolism of ebastine in monkey small intestine.

Small intestinal microsomes of cynomolgus monkeys were found to catalyze hydroxylation and dealkylation of an H(1)-antihistamine prodrug, ebastine. To identify the main enzyme responsible for ebastine hydroxylation, which has been hitherto unknown, we purified two cytochrome P450 isoforms, named P450 MI-2 and P450 MI-3, from the intestinal microsomes on the basis of the hydroxylation activity. P450 MI-2 and P450 MI-3 showed the respective apparent molecular weights of 56,000 and 53,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The internal amino acid sequence of P450 MI-2 had high similarity with those of human CYP4F2, CYP4F3, and CYP4F8. The first 27 amino acid residues of P450 MI-3 were highly homologous with those of monkey CYP3A8 and human CYP3A4/5/7. Furthermore, P450 MI-2 and P450 MI-3 were recognized by anti-CYP4F and anti-CYP3A antibodies, respectively, in immunoblot analysis and catalyzed leukotriene B(4) omega-hydroxylation and testosterone 6beta-hydroxylation, which are known to be mediated by CYP4F and CYP3A, respectively. Although both enzymes had ebastine hydroxylation activity, the V(max) value of P450 MI-2 was much higher than that of P450 MI-3 (37.0 versus 0.406 nmol/min/nmol of P450), and the former K(M) (5.1 microM) was smaller than the latter K(M) (10 microM). Anti-CYP4F antibody inhibited the hydroxylation in small intestinal microsomes strongly (70%), but anti-CYP3A antibody did not. These results indicate that P450 MI-2 belongs to the CYP4F subfamily and is mainly responsible for hydroxylation of ebastine in monkey small intestinal microsomes. This suggests that the small intestinal CYP4F enzyme, P450 MI-2, can play an important role in the metabolism of drugs given orally.

Hepatic macrophage malfunction in rats with obstructive jaundice and its biological significance.

The present study was designed to investigate the pathophysiology of obstructive jaundice by analyzing the function of hepatic macrophages and their role in immune responses and homeostasis in rats. The phagocytic index, determined by the rate of disappearance of 51Cr-endotoxin from the peripheral blood after intravenous injection, was increased in obstructive jaundice 2 weeks after bile duct ligation. The superoxide production of isolated hepatic macrophages and peripheral blood monocytes, measured by the superoxide dismutase inhibitable ferricytochrome c reduction method, was increased. Prostaglandin E2 release, measured by RIA, was markedly increased in rats with obstructive jaundice, but there was no significant difference in interleukin-1 release between jaundiced and control rats. The flow-cytometric analysis of surface molecules of hepatic macrophages showed decreased expression of interleukin-2 receptor in rats with obstructive jaundice. Thus, the functions of hepatic macrophages in rats with obstructive jaundice were impaired. This malfunction may disturb the immunoregulatory network and metabolism, although the exact implications of the altered function of hepatic macrophages have not yet been clarified.

kan-1 (bile acid CoA:amino acid N-acyltransferase) messenger RNA as a novel predictive indicator for prognosis of hepatocellular carcinoma patients after partial hepatectomy.

We identified kan-1 complementary DNA (cDNA), the sequence of which is identical to bile acid CoA:amino acid N-acyltransferase (BAT), a liver enzyme that catalyzes the conjugation of bile acids with glycine or taurine. Kan-1(BAT) messenger RNA (mRNA) levels of the resected specimens obtained from 37 hepatocellular carcinoma (HCC) patients were studied in an attempt to evaluate prognostic significance in HCC patients after partial hepatectomy. Using Northern blot hybridization, kan-1(BAT) mRNA levels were quantified in tumorous and nontumorous tissues, and the ratio of the former to the latter was defined as the kan-1(BAT) ratio. Twelve patients had a kan-1(BAT) ratio < 0.5 (low kan-1[BAT] ratio), and 25 patients had a ratio >0.5 (high kan-1[BAT] ratio). The patients with a low kan-1(BAT) ratio demonstrated poorer survival than the patients with a high kan-1(BAT) ratio (P = .0013). The overall estimated hazard ratio for death in patients with a low kan-1(BAT) ratio was 68.05 according to a multivariate model (P = .0005). Thus, the kan-1(BAT) ratio may serve as a new molecular prognostic marker in HCC patients, following hepatic resection.

Expression of cytokine genes during liver regeneration after partial hepatectomy in rats.

In the present study to demonstrate the relationship between cytokines and liver regeneration we investigated by Northern blot hybridization the cytokine gene induction in the regenerating liver and several other organs (spleen, lung, and kidney) in the rat after partial hepatectomy (PH). We also examined whether Kupffer cells and the spleen are involved in the induction of cytokine mRNA in the regenerating liver. Both IL-1 alpha and beta mRNA increased transiently 1/2 to 1 hr after PH in nonparenchymal cells (NPC) of the regenerating liver; they reached a maximum before the peak of hepatocyte DNA synthesis. PH also induced a slight, but significant, gene expression of IL-1 in lung and kidney in the early postoperative period. TNF-alpha mRNA increased gradually in the spleen, but not the liver, of partially hepatectomized rats at 3 to 12 hr and then reached a peak at 24 hr after PH. IL-6 transcripts were not detected in the regenerating liver, spleen, lung, or kidney during liver regeneration. In contrast, no cytokine gene expression was induced in any of these four organs during the first 3 days after sham operation or unilateral nephrectomy. When Kupffer cell activity was suppressed by gadolinium chloride pretreatment, or when splenectomy was performed 24 hr before PH, the constitutive IL-1 alpha and beta mRNA expressions in NPC of the normal rat liver were completely suppressed. In conclusion, the present study demonstrates, for the first time, the specific kinetics of cytokine gene expression in the liver, spleen, lung, and kidney after PH.(ABSTRACT TRUNCATED AT 250 WORDS)