Microplastic identification and quantification from organic rich sediments: A validated laboratory protocol.

Plastic pollution presents a global environmental concern with potentially widespread ecological, socio-economic and health implications. Methodological advances in microplastic extraction, quantification and identification from sediments have been made. However, integrating these fragmentary advances into a holistic, cost-effective protocol and applying it to organic rich sediments with fine grain size remains a challenge. Nonetheless, many hot spots of microplastic contamination such as harbour and estuarine sediments are characterised by such sediments. We conducted a series of experiments to integrate methodological advances, and clarify their applicability to organic rich sediments with fine grain size. The resulting protocol consisted of three stages. First, pre-treatment with Fenton's reagent was found to be efficient in reducing organic matter content, compatible with later Fourier Transform-Infrared Spectroscopy (FT-IR) for polymer identification, although it did affect the size of polyethylene (PE) and polyethylene terephthalate (PET). Secondly, a novel density separation column with a top overflow (the OC-T) obtained recovery rates above 90% for microplastics present in a ZnCL2 solution. Finally, automated epifluorescence microscopic image analysis of Nile Red stained filters with selected validation of polymer identities using FT-IR revealed 91.7% of stained particles to be plastics. A case study on estuarine sediments demonstrated a high extraction efficiency with quantification possible down to 125 µm and detection possible down to 62.5 µm. This makes this protocol suitable for large scale monitoring of microplastics in sediments of estuarine origin provided polymer specific recovery rates, background contamination and uncertainty in Nile Red identification is accounted for. Subject to further validation, the protocol could also offer a solution to similar organic rich sediments with fine grain size, such as some soils and sludge, to improve our ability to conduct cost-effective, large scale monitoring of microplastic contamination.

Evidence-based clinical practice guidelines for nonalcoholic fatty liver disease/nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is currently the most common cause of chronic liver disease in industrialized countries worldwide, and has become a serious public health issue not only in Western countries but also in many Asian countries including Japan. Within the wide spectrum of NAFLD, nonalcoholic steatohepatitis (NASH) is a progressive form of disease, which often develops into liver cirrhosis and increases the risk of hepatocellular carcinoma. In turn, a large proportion of NAFLD/NASH is the liver manifestation of metabolic syndrome, suggesting that NAFLD/NASH plays a key role in the pathogenesis of systemic atherosclerotic diseases. Currently, a definite diagnosis of NASH requires liver biopsy, though various noninvasive measures are under development. The mainstays of prevention and treatment of NAFLD/NASH include dietary restriction and exercise; however, pharmacological approaches are often necessary. Currently, vitamin E and thiazolidinedione derivatives are the most evidence-based therapeutic options, although the clinical evidence for long-term efficacy and safety is limited. This practice guideline for NAFLD/NASH, established by the Japanese Society of Gastroenterology in cooperation with The Japan Society of Hepatology, covers lines of clinical evidence reported internationally in the period starting from 1983 to January 2012, and each clinical question was evaluated using the GRADE system. Based on the primary release of the full version in Japanese, this English summary provides the core essentials of this clinical practice guideline comprising the definition, diagnosis, and current therapeutic recommendations for NAFLD/NASH in Japan.(AU)

Cyclosporin A causes a hypermetabolic state and hypoxia in the liver: prevention by dietary glycine.

Acute cyclosporin A (CsA) treatment inhibits mitochondrial respiration, yet effects of chronic treatment remain unclear. Accordingly, the effects of chronic CsA on oxygen metabolism in perfused rat liver and isolated mitochondria were investigated. Basal rates of oxygen uptake of around 120 micromol/g/h in isolated perfused livers from vehicle-treated controls were elevated about 1.6-fold by chronic CsA treatment. In the presence of ammonium chloride, a substrate for urea synthesis, oxygen uptake was about 150 micromol/g/h and was increased about 1.7-fold by CsA, indicating that chronic CsA treatment causes a robust hypermetabolic state in the liver. In isolated mitochondria, state 3 rates of oxygen uptake were increased about 1.6-fold by chronic CsA treatment. Since significant increases in oxygen consumption could cause hypoxia, the hypoxia marker pimonidazole was given. Pimonidazole binding in the liver was increased about 3-fold by chronic CsA. Moreover, intracellular calcium in Kupffer cells isolated from vehicle-treated rats was not altered by CsA addition; however, in cells isolated from chronic CsA-treated rats, CsA increased intracellular calcium about 15-fold and prostaglandin E(2) (PGE(2)) production 3.5-fold. Importantly, dietary glycine (5%) largely blocked chronic CsA-induced activation of Kupffer cells, blunted production of PGE(2), prevented the hypermetabolic state, and minimized tissue hypoxia. Taken together, it is concluded that chronic CsA treatment causes a hypermetabolic state leading to hypoxia and injury to the liver. It is hypothesized that CsA activates Kupffer cells and increases production of PGE(2), which alters mitochondria leading to a hypermetabolic state. Glycine inhibits activation of Kupffer cells thus preventing liver injury.

Long-term alcohol exposure changes sensitivity of rat Kupffer cells to lipopolysaccharide.

BACKGROUND: Chronic ethanol treatment enhances Kupffer cell sensitivity to lipopolysaccharide (LPS). In this model, CD14 in Kupffer cells was increased significantly 4 weeks after ethanol. Moreover, it was shown that prostaglandin E2 produced by activated Kupffer cells participated in the mechanism of ethanol-induced fatty liver. This study was designed to elucidate the temporal effect of chronic ethanol exposure on Kupffer cell sensitization to LPS. METHODS: Rats were given ethanol every 24 hr intragastrically for up to 12 weeks, and Kupffer cells were isolated 24 hr after the final ethanol administration and cultured in RPMI 1640 with 10% fetal bovine serum. After addition of LPS to Kupffer cells, intracellular calcium ([Ca2+]i) was measured. RESULTS: CD14 in Kupffer cells was increased approximately 2-fold, and then it decreased and returned to control levels. The LPS-induced increases in [Ca2+]i and tumor necrosis factor-alpha by Kupffer cells were also increased approximately 3-fold over control values, but they also returned to control levels. Triglyceride content increased with the duration of chronic ethanol treatment. At 8 weeks, prostaglandin E2 produced by Kupffer cells increased approximately 3-fold over control values and triglycerides by approximately 4-fold before gradually decreasing to basal levels. After 12 weeks of ethanol exposure, LPS-induced increases in [Ca2+]i and tumor necrosis factor-alpha production were only approximately 50% as high as peak levels at 4 weeks. Liver triglyceride content at 12 weeks was reduced significantly compared with values at 8 weeks. CONCLUSIONS: Kupffer cells at the early stage of chronic ethanol exposure exhibited sensitization to LPS, but this sensitivity was blunted later. This correlated with triglyceride accumulation in the liver. These data indicate that long-term alcohol exposure changes the sensitivity of rat Kupffer cells to LPS but that the magnitude of the effect is time dependent.

Leptin augments inflammatory and profibrogenic responses in the murine liver induced by hepatotoxic chemicals.

Lines of evidence suggested a possible link between leptin and hepatic fibrosis; however, whether leptin modulates the fibrogenesis in the liver remains unclear. The purpose of this study, therefore, was to evaluate the effect of leptin on inflammatory and profibrogenic responses in the liver caused by hepatotoxic chemicals. Male C57Bl/6 mice were given carbon tetrachloride (CCl(4)) (0.1 microL/g body weight [BW], intraperitoneally [IP]) and/or recombinant murine leptin (1 microg/g BW, IP) simultaneously, and sacrificed up to 72 hours later. Further, some mice were given thioacetamide (TAA; 200 microg/g BW, IP) and leptin 3 times per week for 4 weeks to evaluate the effect of leptin on chronic fibrogenic responses. A simultaneous injection of leptin enhanced acute CCl(4)-induced necroinflammatory and subsequent fibrotic changes in the hepatic lobules. The steady-state messenger RNA (mRNA) levels of alpha1(I) procollagen and heat shock protein 47 (HSP47) in the liver were potentiated when leptin was injected together with CCl(4). Expression of alpha smooth muscle actin (alpha-SMA) in the liver after CCl(4) treatment was also augmented markedly in combination with leptin. Further, leptin increased transforming growth factor beta1 (TGF-beta1) mRNA in the liver 24 hours after acute CCl(4) about 4-fold higher than CCl(4) alone. Moreover, leptin enhanced hepatic fibrosis and induction of alpha1(I) procollagen mRNA caused by chronic TAA administration. Collectively, these findings indicated that leptin augments both inflammatory and profibrogenic responses in the liver caused by hepatotoxic chemicals. It is postulated that the increase in systemic leptin levels enhances up-regulation of TGF-beta1, leading to activation of stellate cells, thereby augmenting the fibrogenic response in the liver.

Kupffer cell sensitization by alcohol involves increased permeability to gut-derived endotoxin.

BACKGROUND: Studies with gut sterilization and Kupffer cell inactivation support the hypothesis that endotoxin and Kupffer cells are involved in mechanisms of alcohol-induced liver injury. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hr later. Moreover, we established a new, simple animal model of ethanol hepatotoxicity based on Kupffer cell sensitization. The purpose of this study was to determine the mechanisms by which alcohol sensitizes Kupffer cells to lipopolysaccharide (LPS). METHODS: Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hr intragastrically, and ethanol concentration, ethanol elimination, and portal vein endotoxin were measured. Gut permeability was measured in isolated segments of ileum by translocation of horseradish peroxidase. Kupffer cells were isolated 24 hr after ethanol administration in vivo and were cultured in RPMI 1640 with 10% fetal bovine serum. After the addition of LPS, intracellular Ca2+ was measured by using a microspectrofluorometer with the fluorescent indicator fura-2, and tumor necrosis factor (TNF)-alpha was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western analysis. RESULTS: Ethanol levels exhibited a cyclic pattern in ethanol-treated rats. Similar results were obtained in groups given ethanol and antibiotics for 4 weeks. Rates of alcohol elimination were around 3.5 mmol/kg/hr in control rats. After 4 weeks of ethanol treatment with or without antibiotics, elimination rates were not changed. Translocation of horseradish peroxidase was increased about 3-fold in gut segments by treatment with ethanol. This increase was not altered by treatment with antibiotics. Moreover, portal vein endotoxin levels were increased from nearly undetectable levels to 80 pg/ml in plasma of rats treated with ethanol. As expected, this increase was prevented (<20 pg/ml) by antibiotics. In isolated Kupffer cells from rats treated with ethanol for 4 weeks, CD14, LPS-induced intracellular Ca2+, and TNF-alpha all were increased. These phenomena were blocked by antibiotics. CONCLUSIONS: Kupffer cells isolated from rats treated with ethanol for 4 weeks exhibit sensitization to LPS. It is likely that increased permeability of the gut is a prominent event that leads to alcoholic liver injury.

Sex difference in alcohol-related organ injury.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Nobuhiro Sato and Kai O. Lindros. The presentations were (1) Sex differences in ethanol pharmacokinetics, by E. Baraona; (2) Estrogen regulates the sensitivity to endotoxin in hepatic Kupffer cells, by K. Ikejima; (3) Sex difference in alcohol-related organ injury, by E. Mezey; (4) Aggravated ethanol-induced liver injury in female rats: Protection by the antiestrogen toremifene, by Harri A. Järveläinen; and (5) Alcohol metabolism in Asian subjects: Sex differences and flushing response, by V. A. Ramchandani.

How is the liver primed or sensitized for alcoholic liver disease?

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Hidekazu Tsukamoto and Yoshiyuki Takei. The presentations were (1) Tribute to Professor Rajendar K. Chawla, by Craig J. McClain; (2) Dysregulated TNF signaling in alcoholic liver disease, by Craig J. McClain, S. Joshi-Barve, D. Hill, J Schmidt, I. Deaciuc, and S. Barve; (3) The role of mitochondria in ethanol-mediated sensitization of the liver, by Anna Colell, Carmen Garcia-Ruiz, Neil Kaplowitz, and Jose C. Fernandez-Checa; (4) A peroxisome proliferator (bezafibrate) can prevent superoxide anion release into hepatic sinusoid after acute ethanol administration, by Hirokazu Yokoyama, Yukishige Okamura, Yuji Nakamura, and Hiromasa Ishii; (5) S-adenosylmethionine affects tumor necrosis factor-alpha gene expression in macrophages, by Rajendar K. Chawla, S. Barve, S. Joshi-Barve, W. Watson, W. Nelson, and C. McClain; (6) Iron, retinoic acid and hepatic macrophage TNFalpha gene expression in ALD, by Hidekazu Tsukamoto, Min Lin, Mitsuru Ohata, and Kenta Motomura; and (7) Role of Kupffer cells and gut-derived endotoxin in alcoholic liver injury, by N. Enomoto, K. Ikejima, T. Kitamura, H. Oide, Y. Takei, M. Hirose, B. U. Bradford, C. A. Rivera, H. Kono, S. Peter, S. Yamashina, A. Konno, M. Ishikawa, H. Shimizu, N. Sato, and R. Thurman.

Tolerance and sensitization to endotoxin in Kupffer cells caused by acute ethanol involve interleukin-1 receptor-associated kinase.

Ethanol changes sensitivity of Kupffer cells to endotoxin. Here, the hypothesis that interleukin-1 receptor-associated kinase (IRAK), a downstream signaling molecule of toll-like receptors, regulates the response to LPS in Kupffer cells after ethanol treatment was evaluated. C57BL/6 mice were given ethanol intragastrically, and LPS was injected 1 or 21 h later. One hour after ethanol treatment, serum transaminases after LPS were 60% of control, while ethanol increased these parameters about 3-fold 21 h after ethanol. Pretreatment with antibiotics blocked these effects of ethanol. In Kupffer cells from mice treated with ethanol 1 h earlier, LPS-induced TNFalpha production, and IRAK expression and activity and NFkappaB were decreased 50-60% of control. In contrast, in Kupffer cells from mice treated with ethanol 21 h earlier, LPS-induced TNFalpha production, expression and activity of IRAK were increased 1.5-fold over controls, while NFkappaB was elevated 3-fold. These data indicate that ethanol-induced tolerance and sensitization of Kupffer cells to endotoxin in mice involve IRAK.

Glycine prevents apoptosis of rat sinusoidal endothelial cells caused by deprivation of vascular endothelial growth factor.

Apoptosis of sinusoidal endothelial cells (SECs) is one of the initial events in the development of ischemia-reperfusion injury of the liver. Glycine has been shown to diminish ischemia-reperfusion injury in the liver and improve graft survival in the rat liver transplantation model. Here, we investigated the effect of glycine on apoptosis of primary cultured rat SECs induced by vascular endothelial growth factor (VEGF) deprivation. Isolated rat SECs were cultured in EBM-2 medium supplemented with 10% fetal bovine serum (FBS) and growth factors including 20 ng/mL VEGF for 3 days. SECs at 3 days of culture showed spindle-like shapes; however, cells started shrinking and detaching from dishes by VEGF deprivation. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated d-uridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) staining in these conditions. Control SECs contained only a few percent of TUNEL-positive cells; however, they started increasing 4 hours after VEGF deprivation, and the percentage of TUNEL-positive cells reached about 50% at 8 hours and almost 100% at 16 hours after VEGF deprivation. Interestingly, this increase in TUNEL-positive cells after VEGF deprivation was prevented significantly when glycine (1-10 mmol/L) was added to the medium, the levels being around 60% of VEGF deprivation without glycine. Furthermore, strychnine (1 micromol/L), a glycine receptor antagonist, inhibited this effect of glycine, suggesting the possible involvement of the glycine receptor/chloride channel in the mechanism. Moreover, Bcl-2 protein levels in SECs were decreased 8 hours after VEGF deprivation, which was prevented almost completely by glycine. It is concluded that glycine prevents apoptosis of primary cultured SECs under VEGF deprivation.

Kupffer cell-derived prostaglandin E(2) is involved in alcohol-induced fat accumulation in rat liver.

Destruction of Kupffer cells with gadolinium chloride (GdCl(3)) and intestinal sterilization with antibiotics diminished ethanol-induced steatosis in the enteral ethanol feeding model. However, mechanisms of ethanol-induced fatty liver remain unclear. Accordingly, the role of Kupffer cells in ethanol-induced fat accumulation was studied. Rats were given ethanol (5 g/kg body wt) intragastrically, and tissue triglycerides were measured enzymatically. Kupffer cells were isolated 0-24 h after ethanol, and PGE(2) production was measured by ELISA, whereas inducible cyclooxygenase (COX-2) mRNA was detected by RT-PCR. As expected, ethanol increased liver triglycerides about threefold. This increase was blunted by antibiotics, GdCl(3), the dihydropyridine-type Ca(2+) channel blocker nimodipine, and the COX inhibitor indomethacin. Ethanol also increased PGE(2) production by Kupffer cells about threefold. This increase was also blunted significantly by antibiotics, nimodipine, and indomethacin. Furthermore, tissue triglycerides were increased about threefold by PGE(2) treatment in vivo as well as by a PGE(2) EP(2)/EP(4) receptor agonist, whereas an EP(1)/EP(3) agonist had no effect. Moreover, permeable cAMP analogs also increased triglyceride content in the liver significantly. We conclude that PGE(2) derived from Kupffer cells, which are activated by ethanol, interacts with prostanoid receptors on hepatocytes to increase cAMP, which causes triglyceride accumulation in the liver. This mechanism is one of many involved in fatty liver caused by ethanol.

Taurine preserves gap junctional intercellular communication in rat hepatocytes under oxidative stress.

Gap junctional intercellular communication (GJIC) between hepatocytes is important for the maintenance of differentiated liver functions. Taurine is known to be cytoprotective, and is used clinically to improve liver functions. We evaluated the effect of taurine on GJIC in hepatocyte doublets under oxidative stress. Hepatocyte doublets were isolated from female Wistar rats, using a collagenase perfusion technique, and cultured in Leibovitz-15 medium containing fetal bovine serum (10%). H2O2 (2 mM) and/or taurine (0.1-1 mM) were added 2 h after inoculation, and the culture was incubated for 3 h. Fluorescent dye (Lucifer Yellow CH) coupling between adjacent cells was evaluated by microinjection. The distribution and quantity of connexin 32 (Cx32) in hepatocytes were detected using indirect immunofluorescence analysis and Western blotting. Steady state mRNA levels of Cx32 were detected by Northern blotting. The percentage of dye coupling 5 h after inoculation was 88 +/- 6.3% in the control. however, this was decreased to almost half the control value by H2O2. Taurine prevented the decrease caused by H2O2 in a dose-dependent manner. Immunofluorescence analysis for Cx32 demonstrated numerous punctate fluorescent spots along the intercellular plasma membrane in controls, which were significantly decreased by H2O2. Taurine prevented the decrease of Cx32. Western blot analysis also showed the decrease of Cx32 protein levels by H2O2 treatment, which decrease was prevented by taurine. Interestingly, H2O2 and/or taurine treatments did not affect Cx32 mRNA levels. Our findings indicated that H2O2 treatment decreased GJIC between hepatocytes, most likely due to augmenting the degradation of Cx32 proteins, whereas taurine prevented this process. This effect of taurine is beneficial for the preservation of differentiated functions in the liver under oxidative stress.

Alcohol enhances lipopolysaccharide-induced increases in nitric oxide production by Kupffer cells via mechanisms dependent on endotoxin.

BACKGROUND: Ethanol causes both tolerance and sensitization of Kupffer cells. Accordingly, this study examines the effect of acute ethanol consumption on nitric oxide (NO) production from Kupffer cells with or without lipopolysaccharide (LPS) treatment. METHODS: Rats were given ethanol (4 g/kg body weight) intragastrically, and Kupffer cells were isolated 2 and 24 hr later. Some rats were treated for 4 days with 150 mg/kg/day of polymyxin B and 450 mg/kg/day of neomycin to prevent growth of intestinal bacteria, the primary source of endotoxin in the gastrointestinal tract. After addition of LPS, NO was measured by the Griess reaction. RESULTS: Two hours after ethanol administration, LPS-induced NO production by Kupffer cells was diminished by 50% but was enhanced 2-fold at 24 hr. Sterilization of the gut with antibiotics blocked this enhancement. CONCLUSIONS: Kupffer cells isolated from rats early after ethanol exhibited tolerance to LPS, whereas sensitization was observed later. It is likely that sensitization to Kupffer cell is caused by gut-derived endotoxin.

Role of Kupffer cells and gut-derived endotoxins in alcoholic liver injury.

The hepatotoxic effects of alcohol have been described in detail, but factors responsible for its hepatotoxicity have only partially been characterized. For example, it is known that chronic ethanol ingestion increases hepatotoxicity and produces fatty liver, hepatitis and cirrhosis. However, acute ethanol consumption reduces endotoxin hepatotoxicity. It now appears that Kupffer cells participate in several aspects of these phenomena. Previously, most studies on the effects of alcohol on liver function have focused chiefly on the hepatocyte. Recently, attention has been directed towards the effect of ethanol ingestion on Kupffer cell function, which is stimulated by gut-derived endotoxins (lipopolysaccharides) via mechanisms dependent on increased gut permeability and the possible relationship between Kupffer cells and alcohol-induced liver injury. Here we will review new evidence for the proposal that Kupffer cells and endotoxins play a pivotal role in hepatotoxicity following alcohol exposure, based on studies using the continuous intragastric enteral feeding model developed by Tsukamoto and French and an acute model developed by us.

Glycine-gated chloride channels in neutrophils attenuate calcium influx and superoxide production.

Recently, it was demonstrated that liver injury and TNF-alpha production as a result of endotoxin (lipopolysaccharide, LPS) were attenuated by feeding animals a diet enriched with glycine. This phenomenon was shown to be a result of, at least in part, activation of a chloride channel in Kupffer cells by glycine, which hyperpolarizes the cell membrane and blunts increases in intracellular calcium concentrations ([Ca(2+)](i)) similar to its action in the neuron. It is well known that hepatotoxicity due to LPS has a neutrophil-mediated component and that activation of neutrophils is dependent on increases in [Ca(2+)](i). Therefore, the purpose of this study was to determine if glycine affected agonist-induced increases in [Ca(2+)](i) in rat neutrophils. The effect of glycine on increases in [Ca(2+)](i) elicited either by the bacterial-derived peptide formyl-methionine-leucine-phenylalanine (FMLP) or LPS was studied in individual neutrophils using Fura-2 and fluorescence microscopy. Both FMLP and LPS caused dose-dependent increases in [Ca(2+)](i), which were maximal at 1 microM FMLP and 100 microgram/ml LPS, respectively. LPS increased intracellular calcium in the presence and absence of extracellular calcium. Glycine blunted increases in [Ca(2+)](i) in a dose-dependent manner with an IC(50) of approximately 0.3 mM, values only slightly higher than plasma levels. Glycine was unable to prevent agonist-induced increases in [Ca(2+)](i) in chloride-free buffer. Moreover, strychnine (1 microM), an antagonist of the glycine-gated chloride channel in the central nervous system, reversed the effects of glycine (1 mM) on FMLP- or LPS-stimulated increases in [Ca(2+)](i). To provide hard evidence for a glycine-gated chloride channel in the neutrophil, the effect of glycine on radioactive chloride uptake was determined. Glycine caused a dose-dependent increase in chloride uptake into neutrophils with an ED(50) of approximately 0.4 mM, an effect also prevented by 1 microM strychnine. Glycine also significantly reduced the production of superoxide anion from FMLP-stimulated neutrophils. Taken together, these data provide clear evidence that neutrophils contain a glycine-gated chloride channel that can attenuate increases in [Ca(2+)](i) and diminish oxidant production by this important leukocyte.

Estrogen is involved in early alcohol-induced liver injury in a rat enteral feeding model.

The aim of this study was to investigate whether reduction in blood estrogen by removal of the ovaries would decrease the sensitivity of female rats to early alcohol-induced liver injury using an enteral ethanol feeding model, and if so, whether estrogen replacement would compensate. Livers from ovariectomized rats with or without estrogen replacement after 4 weeks of continuous ethanol exposure were compared with nonovariectomized rats in the presence or absence of ethanol. Ethanol increased serum alanine transaminase (ALT) levels from 30 +/- 6 to 64 +/- 7 U/L. This effect was blocked by ovariectomy (31 +/- 7) and totally reversed by estrogen replacement (110 +/- 23). Ethanol increased liver weight and fat accumulation, an effect that was minimized by ovariectomy and reversed partially by estrogen replacement. Infiltrating leukocytes were increased 6. 7-fold by ethanol, an effect that was blunted significantly by ovariectomy and reversed by estrogen replacement. Likewise, a similar pattern of changes was observed in the number of necrotic hepatocytes. Blood endotoxin and hepatic levels of CD14 messenger RNA (mRNA) and protein were increased by ethanol. This effect was blocked in ovariectomized rats and elevated by estrogen replacement. Moreover, Kupffer cells isolated from ethanol-treated rats with estrogen replacement produced more tumor necrosis factor alpha (TNF-alpha) than those from control and ovariectomized rats. It is concluded, therefore, that the sensitivity of rat liver to alcohol-induced injury is directly related to estrogen, which increases endotoxin in the blood and CD14 expression in the liver, leading to increased TNF-alpha production.

Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells.

CD14 is a lipopolysaccharide (LPS) receptor distributed largely in macrophages, monocytes, and neutrophils; however, the role of CD14 in activation of Kupffer cells by LPS remains controversial. The purpose of this study was to determine if different methods used to isolate Kupffer cells affect CD14. Kupffer cells were isolated by collagenase (0.025%) or collagenase-Pronase (0.02%) perfusion and differential centrifugation using Percoll gradients and cultured for 24 h before experiments. CD14 mRNA was detected by RT-PCR from Kupffer cell total RNA as well as from peritoneal macrophages. Western blotting showed that Kupffer cells prepared with collagenase possess CD14; however, it was absent in cells obtained by collagenase-Pronase perfusion. Intracellular calcium in Kupffer cells prepared with collagenase was increased transiently to levels around 300 nM by addition of LPS with 5% rat serum, which contains LPS binding protein. This increase in intracellular calcium was totally serum dependent. Moreover, LPS-induced increases in intracellular calcium in Kupffer cells were blunted significantly (40% of controls) when cells were treated with phosphatidylinositol-specific phospholipase C, which cleaves CD14 from the plasma membrane. However, intracellular calcium did not increase when LPS was added to cells prepared by collagenase-Pronase perfusion even in the presence of serum. These cells were viable, however, because ATP increased intracellular calcium to the same levels as cells prepared with collagenase perfusion. Tumor necrosis factor-alpha (TNF-alpha) mRNA was increased in Kupffer cells prepared with collagenase perfusion 1 h after addition of LPS, an effect potentiated over twofold by serum; however, serum did not increase TNF-alpha mRNA in cells isolated via collagenase-Pronase perfusion. Moreover, treatment with Pronase rapidly decreased CD14 on mouse macrophages (RAW 264.7 cells) and Kupffer cells. These findings indicate that Pronase cleaves CD14 from Kupffer cells, whereas collagenase perfusion does not, providing an explanation for why Kupffer cells do not exhibit a CD14-mediated pathway when prepared with procedures using Pronase. It is concluded that Kupffer cells indeed contain a functional CD14 LPS receptor when prepared gently.

Peroxisomes are involved in the swift increase in alcohol metabolism.

The purpose of this study was to determine whether catalase-dependent alcohol metabolism is activated by alcohol (i.e., swift increase in alcohol metabolism). When ethanol or the selective substrate for catalase, methanol, was given (5.0 g/kg) in vivo 2 to 3 h before liver perfusion, methanol and oxygen metabolism were increased significantly. This increase was blocked when the specific Kupffer cell toxicant GdCl3 was administered 24 h before perfusion. These data support the hypothesis that catalase-dependent alcohol metabolism is activated by acute alcohol and that Kupffer cells are involved. Ethanol treatment in vivo increased ketogenesis from endogenous fatty acids nearly 3-fold and increased plasma triglycerides and hepatic acyl CoA synthetase activity; all increases were blocked by GdCl3. These findings support the hypothesis that ethanol increases H2O2 supply for catalase-dependent alcohol metabolism by increasing fatty acid supply. Infusion of oleate stimulated oxygen uptake 1.5-fold and methanol metabolism 4-fold, but these parameters were not altered by GdCl3. Moreover, the effects of ethanol treatment were blocked by the cyclooxygenase inhibitor indomethacin, and prostaglandin E2 (PGE2) was increased more than 200% in media from cultured Kupffer cells from rats treated with ethanol in vivo. Furthermore, lipoprotein lipase activity in retroperitoneal fat pads, which is known to be inhibited by PGE2, was reduced 70% by ethanol. These data are consistent with the hypothesis that Kupffer cells play a key role in activation of catalase-dependent alcohol metabolism, most likely by producing mediators (e.g., PGE2) that inhibit lipoprotein lipase, increase the supply of fatty acids to the liver, and increase generation of H2O2 via peroxisomal beta-oxidation.

A novel hepatic stellate (Ito) cell-derived protein, epimorphin, plays a key role in the late stages of liver regeneration.

Limited data exist regarding morphogenesis and differentiation during liver regeneration. We examined the role of epimorphin on liver regeneration. After 70% partial hepatectomy, mouse liver was collected on days 1, 3, 7, and 14 for immunohistochemistry and the detection of epimorphin mRNA and connexin 32. Using primary cultured rat hepatocytes, morphogenesis and differentiation of cells were tested with or without epimorphin. Seven days after cell inoculation, the expression of connexin 32 and the cell-cell communication was tested as a marker of differentiation. Epimorphin was detected exclusively in hepatic stellate cells. Connexin 32 was detected only in hepatocytes. After partial hepatectomy, epimorphin mRNA was detected on day 3 and peaked at day 7, followed by protein expression. Connexin 32 expression showed a similar time course. Cultured hepatocytes formed multicellular spheroids in an active epimorphin-coated culture dish and showed positive dye coupling, whereas the cell-cell communication was lost without active epimorphin. Because epimorphin was expressed late in liver regeneration, it might play a role in morphogenesis and differentiation.