Attenuation of thioacetamide-induced hepatocellular injury by short-term repeated injections associated with down-regulation of metabolic enzymes and relationship with MHC class II-presenting cells.

The liver is the primary organ participating in the metabolism of xenobiotics and is therefore an important target in the safety assessment of drugs, chemicals and environmental toxins. Drug-induced liver injury (DILI) has recently become widely recognized in human medicine as an adverse event. The progression of DILI often involves "damage-associated molecular patterns" (DAMPs) of gene and protein expression such as high-mobility group boxes (HMGBs), S100 proteins and heat shock proteins (Hsp). DAMPs are released from injured or necrotic cells and are bound to Toll-like receptors (TLRs) and modulate inflammatory reactions. Previously, in thioacetamide (TAA; 300mg/kg body weight, single injection)-induced rat liver, we demonstrated that the expressions of DAMPs, TLR4 and major histocompatibility complex (MHC) class II were simultaneously increased, accompanied with progression of hepatocellular injury and inflammation. Here we investigated the association of DILI and DAMPs, TLRs and MHC class II by using rat livers repeated injections with TAA (100mg/kg body weight, once, three times). Two days after TAA single injection, centrilobular hepatocellular necrosis with infiltration of mononuclear cells was observed, being paralleled with increase in serum levels of aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP). However, two days after duplicate and triplicate injections, only mild degenerative change of hepatocytes and slight infiltration of mononuclear cells were seen in the affected centrilobular area. Serum levels of AST, ALT and ALP were also decreased to the same levels of control. mRNA expressions of DAMPs (HMGBs, S100A4 and Hsp 70-2), TLR4 and MHC class II tended to be increased only on single injection, although the number of MHC class II-positive cells in the centrilobular area was still increased on each examination point. The analysis of enzymes (CYP2E1 and Flavin monooxygenase (FMO) 3), which metabolize TAA in hepatocytes, showed a significant decrease in FMO3 on the duplicate and triplicate injections. Autophagy and regulatory T cells were not significantly changed for the attenuation of hepatocyte injury. Collectively, these results suggest that hepatocytes may adapt accumulation of the toxicant by changing their enzyme functions; furthermore, MHC class II cells, which still showed increased number in the duplicate and triplicate injections, may be related with protection from the toxicant.

Attenuation of alpha-naphthylisothiocyanate (ANIT)-induced biliary fibrosis by depletion of hepatic macrophages in rats.

Biliary fibrosis is a complex process in which macrophages and myofibroblasts may play central roles. We investigated biliary fibrosis lesions induced in the Glisson's sheath in rats by alpha-naphthylisothiocyanate (ANIT) administration under macrophage depletion. Hepatic macrophages were depleted in F344 rats with liposome-encapsulated clodronate (CLD) (10mL/kg body weight, i.v) followed by bile duct injury with ANIT (75mg/kg body weight, i.p) (ANIT+CLD group). Rats received empty-liposomes (Lipo) followed by ANIT, and served as control (ANIT+Lipo group). In both ANIT+Lipo and ANIT+CLD groups, ANIT-induced bile duct injury with inflammatory cell infiltration was seen on days 1-3, and subsequently reparative fibrosis occurred on days 5 and 7. In comparisons between the two groups, macrophages reacting to CD68, CD163, MHC class II and CD204 were less in numbers in ANIT+CLD group; the most sensitive immunophenotype was of CD163-positive. Furthermore, in ANIT+CLD group interstitial mesenchymal cells/myofibroblasts reacting to vimentin, desmin and α-smooth muscle actin were also less in grades and tended to be delayed in appearance. Interestingly, MCP-1, IFN-γ, IL-10, and TGF-ß1 mRNAs were significantly increased mainly on day 2 in ANIT+Lipo group, while the levels of these factors were prominently lower in ANIT+CLD group. Collectively, depletion of hepatic macrophages plays roles in attenuating biliary fibrogenesis by production of inflammatory factors. The present results indicated clearly importance of macrophage functions in the pathogenesis of biliary fibrosis.

The kinetics of damage-associated molecular patterns (DAMPs) and toll-like receptors during thioacetamide-induced acute liver injury in rats.

Drug-induced liver injury (DILI) is a common problem in human medicine and it is a major reason to withdraw marketed drugs. However, the mechanism of DILI is still less known. Damage-associated molecular patterns (DAMPs), such as high-mobility group boxes (HMGBs), S100 proteins and heat shock proteins (HSPs), are released from injured or necrotic cells, bind to toll-like receptors (TLRs) and modulate inflammatory reactions. Here we investigated the kinetics of DAMPs, TLRs and MHC class II in a rat model of DILI with thioacetamide (TAA). After TAA administration, extensive necrosis was observed on days 1 and 2, followed by infiltration of inflammatory cells on day 3. The levels of serum liver enzymes also peaked on day 1. Expression of HMGB-1, -2 and S100A4 peaked on day 2. TLR-4 was up-regulated on day 3. The number of MHC class II-positive macrophages increased until day 2. These results suggest that HMGB-1, -2 and S100A4 are associated with hepatocellular necrosis and that DAMPs may activate TLR-4 and MHC class II during TAA-induced liver injury. Our data would contribute to the elucidation of the mechanism of DILI.

Transient effects of empty liposomes on hepatic macrophage populations in rats.

Liposomes have been used as a vehicle for encapsulating chemicals or toxins in toxicological studies. We investigated the transient effects of empty liposomes on hepatic macrophages by applying a single intravenous injection at a dose of 10 ml/kg body weight in 6-week-old male F344 rats. One day after injection, the numbers of hepatic macrophages reacting to CD163, CD68, Iba-1, MHC class II, Gal-3 and CD204 were significantly increased in liposome-treated rats. CD163(+) Kupffer cells and CD68(+) macrophages with increased phagocytic activity in hepatic lobules were most sensitive. The histological architecture of the liver was not changed following liposome injection; however, hepatocytes showed increased proliferating activity, demonstrable with proliferation marker immunostaining and by an increase in gene profiles related to the cell cycle. In the liposome-treated rats, interestingly, AST and ALT values were significantly decreased, and MCP-1, IL-1ß and TGF-ß1 mRNAs were significantly increased. Collectively, the present study found that hepatic macrophages activated by liposomes can influence liver homeostasis. This information would be useful for background studies on liposomes.

Establishment and characterization of a transplantable tumor line (RMM) and cell line (RMM-C) from a malignant amelanotic melanoma in the F344 rat, with particular reference to galectin-3 expression in vivo and in vitro.

To investigate characteristics of malignant melanomas with various pathobiological features, a homotransplantable tumor line (RMM) was established from a spontaneous amelanotic melanoma found in the pinna of an aged F344 rat. RMM tumors were transplanted in syngeneic rats by serial subcutaneous implantation with 100% intake. The original and RMM tumors consisted of spindle-shaped cells arranged mainly in interlacing bundles. Immunohistochemically, the neoplastic cells were positive to PNL-2 (melanocytes), nestin (neuroectodermal stem cells), S-100 (neurogenic cells) and vimentin (mesenchymal cells). Electron microscopically, tumor cells possessed single membrane-bound pre-melanosomes. Further, a cell line (RMM-C) was induced from an RMM tumor. RMM-C cells and the induced tumors in syngeneic rats showed immunohistochemical reactions similar to the original and RMM tumors. Interestingly, serum level of galectin-3 expression was increased with growing RMM tumors, and the expression was influenced by TNF-α (increase) or TGF-ß1 (decrease), indicating a possible biomarker of amelanotic melanomas. The RMM tumors and RMM-C cell line could become useful tools for studies on the pathobiology, including tumor immunity, and development of therapeutic strategies against this malignancy. These tools are the first tumor lines of amelanotic melanomas in the rat.

Depletion of Hepatic Macrophages Aggravates Liver Lesions Induced in Rats by Thioacetamide (TAA).

Hepatic macrophages play crucial roles in hepatotoxicity. We investigated immunophenotypes of macrophages in liver injury induced in rats by thioacetamide (TAA; 300 mg/kg, intraperitoneal) after hepatic macrophage depletion; hepatic macrophages were depleted by liposomal clodronate (CLD; 10 ml/kg, i.v.) one day before TAA injection. Samples were obtained on post-TAA injection days 0, 1, 2, 3, 5, and 7. TAA injection induced coagulation necrosis of hepatocytes on days 1 through 3 and subsequent reparative fibrosis on days 5 and 7 in the centrilobular area, accompanied by increased numbers of M1 macrophages (expressing cluster of differentiation [CD]68 and major histocompatibility complex class II) and M2 macrophages (expressing CD163 and CD204) mainly on days 1 through 3. TAA + CLD treatment markedly decreased the numbers of M1 and M2 macrophages mainly on days 1 through 3; CD163(+) Kupffer cells were most sensitive to CLD depletion. In TAA + CLD-treated rats, interestingly, coagulation necrosis of hepatocytes was prolonged with more increased levels of hepatic enzymes (aspartate transaminase, alanine transaminase, and alkaline phosphatase) to TAA-treated rats; reparative fibrosis was incomplete and replaced by dystrophic calcification in the injured area, indicating the aggravated damage. Furthermore, in TAA + CLD-treated rats, inflammatory factors (monocyte chemoattractant protein [MCP]-1, interferon-γ, tumor necrosis factor-α, and interleukin-10) and fibrosis-related factors (transforming growth factor-ß1, matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1) were decreased at messenger RNA levels, indicating abnormal macrophage functions. It was clearly demonstrated that hepatic macrophages have important roles in tissue damage and remodeling in hepatotoxicity.

Immunophenotypical characterization and influence on liver homeostasis of depleting and repopulating hepatic macrophages in rats injected with clodronate.

Hepatic macrophages (including Kupffer cells) play a crucial role in the homeostasis and act as mediators of inflammatory response in the liver. Hepatic macrophages were depleted in male F344 rats by a single intravenous injection of liposomal clodronate (CLD; 50mg/kg body weight), and immunophenotypical characteristics of depleting and repopulating macrophages were analyzed by different antibodies specific for macrophages. CD163(+) Kupffer cells were almost completely depleted on post-injection (PI) days 1-12. Macrophages reacting to CD68, Iba-1, and Gal-3 were drastically reduced in number on PI day 1 and then recovered gradually until PI day 12. MHC class II(+) and CD204(+) macrophages were moderately decreased during the observation period. Although hepatic macrophages detectable by different antibodies were reduced in varying degrees, Kupffer cells were the most susceptible to CLD. Liver situation influenced by depleted hepatic macrophages was also investigated. No marked histological changes were seen in the liver, but the proliferating activity of hepatocytes was significantly increased, supported by changes of gene profiles relating to cell proliferation on microarray analysis on PI day 1; the values of AST and ALT were significantly elevated; macrophage induction/activation factors (such as MCP-1, CSF-1, IL-6 and IL-4) were increased exclusively on PI day 1, whereas anti-inflammatory factors such as IL-10 and TGF-ß1 remained significantly decreased after macrophage depletion. The present study confirmed importance of hepatic macrophages in liver homeostasis. The condition of hepatic macrophages should be taken into consideration when chemicals capable of inhibiting macrophage functions are evaluated.