retrorsine and Chemical-and-Drug-Induced-Liver-Injury

Retrosine (RTS) is a pyrrolozidine alkaloid and a known hepatotoxin that widely exist in nature. The mechanisms involved in toxic action of pyrrolizidine alkaloids need further investigation. The objective of the present study was to evaluate the correlation of RTS hepatotoxicity with hepatic RTS concentration and pyrrole-protein adduction. Mice were intragastrically treated with RTS alone or RTS and ketoconazole (KTZ) simultaneously. Sera and liver tissues were collected at various time points after administration, followed by the determination of changes in serum transaminase activity, hepatic RTS concentration and pyrrole-protein adduction. The correlation of RTS hepatotoxicity with hepatic RTS concentration and hepatic pyrrole-protein adduction were examined by use of Sigmoid-Emax PK/PD models. Dose-dependent hepatotoxicity, hepatic RTS concentration and pyrrole-protein adduction were observed in the animals, which could be modulated by co-treatment with KTZ. The fit parameters indicated pyrrole-protein adduction was more closely related with liver injury than hepatic RTS concentration. Similar correlation was observed in mice given low-dose of RTS for 4 consecutive days. RTS hepatotoxicity is correlated with hepatic pyrrole-protein adduction derived from RTS rather than hepatic RTS concentration. The observed protein modification would be a good indicator to predict the hepatoxicity of RTS at low dose.

Topics: Animals; Chemical and Drug Induced Liver Injury; Liver; Mice; Proteins; Pyrroles; Pyrrolizidine Alkaloids

Pyrrolizidine alkaloids (PAs) are phytotoxins widely present in various natural products and foodstuffs. The present study aims to investigate the effects of fasting on PA-induced hepatotoxicity and the underlying biochemical mechanisms. The results of hepatotoxic study showed that 15-h overnight fasting significantly exacerbated the hepatotoxicity of retrorsine (RTS, a representative toxic PA) in fasted rats compared to fed rats, as indicated by remarkably elevated plasma ALT and bilirubin levels and obvious liver histological changes. Further toxicokinetic studies revealed that fasting significantly enhanced cytochromes P450 enzymes (CYPs)-mediated metabolic activation of RTS leading to increased formation of pyrrole-protein adducts and thus decreased the in vivo exposure and excretion of both parent RTS and its N-oxide metabolite. Metabolic studies demonstrated that fasting induced enzyme activities of CYP1A2, CYP2B6 and CYP2E1 that participated in catalyzing RTS to its reactive pyrrolic metabolites. Moreover, fasting also dramatically decreased hepatic glutathione (GSH) content, which restricted the detoxification of GSH by neutralizing the reactive pyrrolic metabolite of RTS, further contributing to the enhanced hepatotoxicity. The present findings may have an impact on future PA toxicity tests with different dietary styles and/or risk assessment of metabolite-mediated toxins by considering the profound effects of fasting.

Topics: Alanine Transaminase; Animals; Bilirubin; Chemical and Drug Induced Liver Injury; Cytochrome P-450 Enzyme System; Fasting; Glutathione; Liver; Male; Pyrrolizidine Alkaloids; Rats; Rats, Sprague-Dawley

1,2-unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites that are found in many plant species throughout the world. They are of concern for risk assessment as consumption of contaminated foodstuff can cause severe liver damage. Of late, transporter-mediated uptake and transport has advanced as a vital determinant of PA toxicity. In this study, the authors investigate a transporter-mediated uptake of PAs and its implications in PA toxicity.. Our results confirm previous findings of active transport mechanisms of PAs into hepatocytes and highlight the importance of toxicokinetic studies for the risk assessment of PAs.

Topics: Cations; Chemical and Drug Induced Liver Injury; Hepatocytes; Humans; Peptides; Pyrrolizidine Alkaloids; Taurocholic Acid

Pyrrolizidine alkaloids (PAs) are a type of natural phytotoxin that contaminate food and feed and become an environmental health risk to humans and livestock. PAs exert toxicity that requires metabolic activation by cytochrome P450 (CYP) 3A, and case reports showed that fetuses are quite susceptible to PAs toxicity. The aim of this study was to explore the characteristics of developmental toxicity and fetal hepatotoxicity induced by retrorsine (RTS, a typcial toxic PA) and the underlying mechanism. Pregnant Wistar rats were intragastrically administered with 20 mg/(kg·day) RTS from gestation day (GD) 9 to 20. Results showed that prenatal RTS exposure lowered fetal bodyweights, reduced hepatocyte numbers, and potentiated hepatic apoptosis in fetuses, particularly females. Simutaneously, RTS increased CYP3A expression and pregnane X receptor (PXR) activation in female fetal liver. We further confirmed that RTS was a PXR agonist in LO2 and HepG2 cell lines. Furthermore, agonism or antagonism of androgen receptor (AR) either induced or blocked RTS-mediated PXR activation, respectively. As a PXR agonist, RTS toxicity was exacerbated in female fetus due to the increased CYP3A induction and self-metabolism, while the inhibitory effect of AR on PXR activation reduced the susceptibility of male fetus to RTS. Our findings indicated that PXR may be a potential therapeutic target for PA toxicity.

Topics: Animals; Chemical and Drug Induced Liver Injury; Cytochrome P-450 CYP3A; Female; Fetus; Liver; Male; Pregnancy; Pregnane X Receptor; Prenatal Exposure Delayed Effects; Pyrrolizidine Alkaloids; Rats; Rats, Wistar

Pyrrolizidine alkaloids (PAs) have been found in over 6000 plants worldwide and represent the most common hepatotoxic phytotoxins. Currently, a definitive diagnostic method for PA-induced liver injury (PA-ILI) is lacking. In the present study, using a newly developed analytical method, we identified four pyrrole-amino acid adducts (PAAAs), namely pyrrole-7-cysteine, pyrrole-9-cysteine, pyrrole-9-histidine, and pyrrole-7-acetylcysteine, which are generated from reactive pyrrolic metabolites of PAs, in the urine of PA-treated male Sprague Dawley rats and PA-ILI patients. The elimination profiles, abundance, and persistence of PAAAs were systematically investigated first in PA-treated rat models via oral administration of retrorsine at a single dose of 40 mg/kg and multiple doses of 5 mg/kg/day for 14 consecutive days, confirming that these urinary excreted PAAAs were derived specifically from PA exposure. Moreover, we determined that these PAAAs were detected in ~ 82% (129/158) of urine samples collected from ~ 91% (58/64) of PA-ILI patients with pyrrole-7-cysteine and pyrrole-9-histidine detectable in urine samples collected at 3 months or longer times after hospital admission, indicating adequate persistence time for use as a clinical test. As direct evidence of PA exposure, we propose that PAAAs can be used as a biomarker of PA exposure and the measurement of urinary PAAAs could be used as a non-invasive test assisting the definitive diagnosis of PA-ILI in patients.

Topics: Adult; Aged; Aged, 80 and over; Amino Acids; Animals; Biomarkers; Chemical and Drug Induced Liver Injury; Female; Humans; Male; Middle Aged; Pyrroles; Pyrrolizidine Alkaloids; Rats; Rats, Sprague-Dawley; Time Factors

Pyrrolizidine alkaloids (PAs) are extensively distributed in plants and are known to damage hepatic sinusoidal endothelial cells (HSECs) via metabolic activation mediated by hepatic cytochrome P450 enzymes (CYPs), particularly the CYP3A4 isozyme. Different PAs have distinct toxic potencies and their toxic effects on HSECs are difficult to be determined in cultured cells, because HSECs lack the key CYP3A4 isozyme for metabolic activation. This study aims to establish a novel, convenient and reliable CYP3A4-expressing HSEC model using human HSECs transduced with lentivirus carrying CYP3A4-ires-eGFP, for evaluating the hepatotoxicity of different PAs on their target HSECs. The developed CYP3A4-expressing HSEC (HSEC-CYP3A4) model was verified by the expression of GFP and CYP3A4 and by the ability to metabolize nifedipine, a classic CYP3A4 substrate. Treated with retrorsine, a representative toxic PA, HSEC-CYP3A4 cells showed significantly reduced cell viability, depletion of GSH, and increased formation of pyrrole-protein adducts. Furthermore, this newly developed cell model successfully discriminated the cytotoxic potency of different PAs evidenced by their IC

Topics: Acinar Cells; Activation, Metabolic; Cell Line; Cell Survival; Cells, Cultured; Chemical and Drug Induced Liver Injury; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Endothelial Cells; Hepatic Veins; Humans; Liver; Mononuclear Phagocyte System; Pyrrolizidine Alkaloids

In this study, we reveal that liver organoid transplantation through the portal vein is a safe and effective method for the treatment of chronic liver damage. The liver organoids significantly reconstituted the hepatocytes; hence, the liver was significantly enlarged in this group, compared to the monolayer cell transplantation group in the retrorsine/partial hepatectomy (RS/PH) model. In the liver organoid transplantation group, the bile ducts were located in the donor area and connected to the recipient bile ducts. Thus, the rate of bile reconstruction in the liver was significantly higher compared to that in the monolayer group. By transplanting liver organoids, we saw a level of 70% replacement of the damaged liver. Consequently, in the transplantation group, diminished ductular reaction and a decrease of placental glutathione S-transferase (GST-p) precancerous lesions were observed. After trans-portal injection, the human induced pluripotent stem cell (hiPSC)-derived liver organoids revealed no translocation outside the liver; in contrast, the monolayer cells had spread to the lungs. The hiPSC-derived liver organoids were attached to the liver in the immunodeficient RS/PH rats. This study clearly demonstrates that liver organoid transplantation through the portal vein is a safe and effective method for the treatment of chronic liver damage in rats.

Topics: Animals; Cells, Cultured; Chemical and Drug Induced Liver Injury; Female; Glutathione Transferase; Hepatectomy; Humans; Induced Pluripotent Stem Cells; Liver Regeneration; Liver Transplantation; Organ Culture Techniques; Organoids; Portal Vein; Pyrrolizidine Alkaloids; Rats; Treatment Outcome

Hepatic and pulmonary toxicity in fetal rats induced by pyrrolizidine alkaloids (PAs) was investigated. Retrorsine (RTS) or monocrotaline (MCT) was intragastrically administered during pregnancy. The reduction of body and tail lengths was consistent with body weight loss in PA-exposed fetuses, and pathological lesions in liver and lung were observed only in fetuses. Both PAs reduced fetal serum transaminase activities. The GSH/GSSG ratio, GSH peroxidase and superoxide dismutase activities also decreased but glutathione S-transferase activity increased in fetal lung, especially for MCT. The pyrrole-protein adducts in fetal liver and lung could be detected, and those adducts in RTS fetal lungs were about 65% of those in MCT group. In conclusion, prenatal PAs exposure induced fetal hepatic and pulmonary toxicities through the generation of pyrrole metabolites and oxidative injury. The difference on fetal pulmonary redox homeostasis between two PAs groups might be associated with the content of PAs migrated to fetal lungs.

Topics: Animals; Chemical and Drug Induced Liver Injury; Female; Fetal Growth Retardation; Fetus; Liver; Lung; Lung Injury; Maternal-Fetal Exchange; Monocrotaline; Pregnancy; Prenatal Injuries; Pyrrolizidine Alkaloids; Rats, Wistar

Retrorsine (RTS) is a hepatotoxic pyrrolizidine alkaloid present in plants of the Senecio genus. The present study is aimed at clarifying the role of organic cation transporters (OCTs) in the liver disposition of RTS, and the coupling of OCT1 and cytochrome P450 (CYP) 3A4 in the hepatotoxicity of RTS. MDCK or LLC-PK1 cells stably expressing liver uptake or efflux transporters were used to investigate the interaction of RTS with these transporters. Primary cultured rat hepatocytes (PCRH) and double-transfected MDCK-hOCT1-CYP3A4 cells were used to determine the contribution of OCT1 and CYP3A4 to the toxicity of RTS. The results showed that RTS inhibited the OCT1-mediated 1-methyl-4-phenylpyridinium (MPP(+)) uptake in MDCK-hOCT1 cells with the IC50 of 2.25±0.30μM. The uptake of RTS in MDCK-hOCT1 cells and PCRH was significantly inhibited by OCT1 inhibitors, while hOCT3, human multidrug and toxin extrusion (hMATE) transporter 1, multidrug resistance 1 (MDR1), and breast cancer resistance protein (BCRP) showed weak or no obvious interaction with RTS. The toxic effect of RTS on the PCRH was attenuated by OCT1 inhibitors, quinidine and (+)-tetrahydropalmatine ((+)-THP). Compared to mock cells, MDCK-CYP3A4 cells showed a decrease in viability after being treated with RTS. Furthermore, RTS showed a more severe toxicity in the OCT1/CYP3A4 double-transfected cells compared to all other cells. Our data suggests that OCT1 mediates the liver-specific uptake of RTS, and plays an important role in RTS-induced hepatotoxicity together with CYP3A4. Consequently, the OCT1 inhibitors could be applied to protect the liver from the toxicity of RTS.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Line; Cell Survival; Chemical and Drug Induced Liver Injury; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Dogs; Hepatocytes; Humans; Hydrogen-Ion Concentration; Madin Darby Canine Kidney Cells; Mass Spectrometry; Neoplasm Proteins; Organic Cation Transporter 1; Polymerase Chain Reaction; Primary Cell Culture; Pyrrolizidine Alkaloids; Rats; Rats, Sprague-Dawley; Transfection

Cell-based therapies as an alternative to liver transplantation have been anticipated for the treatment of potentially fatal liver diseases. Not only mature hepatocytes (MHs) but also hepatic stem/progenitor cells are considered as candidate cell sources. However, whether the stem/progenitor cells have an advantage to engraft and repopulate the recipient liver compared with MHs has not been comprehensively assessed. Therefore, we used Thy1(+) (oval) and CD44(+) (small hepatocytes) cells isolated from GalN-treated rat livers as hepatic stem and progenitor cells, respectively. Cells from dipeptidylpeptidase IV (DPPIV)(+) rat livers were transplanted into DPPIV(-) livers treated with retrorsine following partial hepatectomy. Both stem and progenitor cells could differentiate into hepatocytes in host livers. In addition, the growth of the progenitor cells was faster than that of MHs until days 14. However, their repopulation efficiency in the long term was very low, since the survival period of the progenitor cells was much shorter than that of MHs. Most foci derived from Thy1(+) cells disappeared within 2 months. Many cells expressed senescence-associated β-galactosidase in 33% of CD44-derived foci at day 60, whereas the expression was observed in 13% of MH-derived ones. The short life of the cells may be due to their cellular senescence. On the other hand, the incorporation of sinusoidal endothelial cells into foci and sinusoid formation, which might be correlated to hepatic maturation, was completed faster in MH-derived foci than in CD44-derived ones. The survival of donor cells may have a close relation to not only early integration into hepatic plates but also the differentiated state of the cells at the time of transplantation.

Topics: Animals; beta-Galactosidase; Cell Differentiation; Cell Proliferation; Cell Survival; Cellular Senescence; Chemical and Drug Induced Liver Injury; Dipeptidyl Peptidase 4; Female; Hepatectomy; Hepatocytes; Hyaluronan Receptors; Liver; Male; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; Stem Cell Transplantation; Stem Cells; Thy-1 Antigens

Oval cells and hepatocytes rarely proliferate simultaneously. This study aimed to determine the impacts of hepatocyte transplantation on the response and fate of oval cells that are activated to proliferate in acute severe hepatic injury. Retrorsine + D-galactosamine (R+D-gal) treatment was used to induce acute hepatic injury and to elicit extensive activation of oval cells in male dipeptidyl peptidase IV-deficient F344 rats. These rats were then randomized to receive wild-type hepatocyte transplantation or vehicle intraportally. The kinetics of oval cell response and their differentiation fate were analyzed. Results showed that oval cells were activated early and differentiated into hepatocytes in R+D-gal-treated rats without hepatocyte transplantation. With hepatocyte transplantation, the oval cells were recruited later and continued to proliferate in parallel with the massive proliferation of transplanted hepatocytes. They formed ductules and differentiated into biliary cells. When hepatocytes were transplanted at the day when oval cells were at their peak response, the numerous activated oval cells ceased to differentiate into hepatocytes and remained in ductular form. The ductular oval cells were capable of differentiating into hepatocytes again when the donor hepatocytes were inhibited to proliferate. We conclude that hepatocyte transplantation changes the mechanism of liver reconstitution and affects the differentiation fate of host oval cells in acute severe hepatic injury.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Differentiation; Chemical and Drug Induced Liver Injury; Dipeptidyl Peptidase 4; Galactosamine; Hepatocytes; Humans; Liver; Male; Pyrrolizidine Alkaloids; Random Allocation; Rats; Rats, Inbred F344

Liver regeneration after partial hepatectomy (PH) in rats exposed to the pyrrolizidine alkaloid retrorsine is accomplished through the proliferation and differentiation of a population of small hepatocyte-like progenitor cells (SHPCs). The activation, emergence, and outgrowth of SHPCs in response to the liver deficit generated through surgical PH have been well characterized. However, the participation of these cells in the restoration of hepatocyte numbers and regeneration of liver tissue mass following necrotic injury has not been investigated. To investigate the capacity of SHPCs to respond to necrotizing liver injury, we combined retrorsine treatment with the centrilobular-specific toxin carbon tetrachloride (CCl(4)). Male Fischer 344 rats were treated with retrorsine (30 mg/kg ip) at 6 and 8 weeks of age, followed by CCl(4) treatment (1500 mg/kg ip) 5 weeks later. Liver tissues were harvested at 3, 7, 14, 21, and 30-days post-injection. The dose of CCl(4) employed resulted in the necrotic destruction of 59±2% of liver mass and elicited a regenerative response equivalent to that of surgical PH. Livers from retrorsine-exposed CCl(4)-treated rats exhibit SHPC proliferation similar to retrorsine-exposed rats subjected to PH (RP). SHPCs appear at 3-days post-injection, continue to expand at 7-days and 14-days post-injection, and completely regenerate/restore the liver mass and structure in these animals by 30-days post-injection. The magnitude of SHPC response observed in the undamaged periportal zone of the liver in these animals is unaffected (versus RP rats) by the loss of the centrilobular region. The results of this study show that SHPCs are capable of regenerating liver after exposure to necrotizing agents and suggest that the progenitor cell of origin of the SHPCs is not restricted to the centrilobular zone of the liver parenchyma.

Topics: Animals; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Hepatectomy; Hepatocytes; Liver Regeneration; Male; Necrosis; Pyrrolizidine Alkaloids; Random Allocation; Rats; Rats, Inbred F344; Stem Cells

The therapeutic effects of bone marrow and hepatocyte transplantation were investigated regarding the treatment of retrorsine-partial hepatectomy-induced liver injury.. Analbuminemic F344alb rats were given two doses of retrorsine 2 wk apart, followed 4 wk later by transplantation with F344 rat bone marrow cells or hepatocytes immediately after a two-thirds hepatectomy. The survival rate, liver regeneration rate, liver functions, albumin-positive hepatocytes, and normal albumin gene sequences in the liver and serum albumin levels were investigated in the recipients.. Although 65% retrorsine/partial hepatectomy-treated F344alb died between 1 and 11 d after the partial hepatectomy, only 27.5% of the animals died following bone marrow transplantation, and 50% with hepatocyte transplantation. Both bone marrow and hepatocyte transplantation ameliorated acute liver injury after a partial hepatectomy. Bone marrow transplantation yielded a very small increase in the number of albumin-positive hepatocytes in the liver, while hepatocyte transplantation resulted in massive replacement of the liver tissues by the donor hepatocytes associated with an elevation of serum albumin after an extended time.. Both bone marrow and hepatocyte transplantation could prevent acute hepatic injury, conceivably due to a paracrine mechanism.

Topics: Acute Disease; Animals; Antineoplastic Agents, Phytogenic; Bone Marrow Transplantation; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Hepatectomy; Hepatocytes; Liver Diseases; Liver Regeneration; Male; Paracrine Communication; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; Serum Albumin; Survival Rate

Although the appearance of hepatic foci in the pancreas has been described in animal experiments and in human pathology, evidence for the conversion of human pancreatic cells to liver cells is still lacking. We therefore investigated the developmental plasticity between human embryonic pancreatic cells and liver cells. Cells were isolated and expanded from 7-8-week-old human fetal pancreata (HFP) and were characterized for the absence and presence of pancreatic and hepatic markers. In vitro expanded HFP were treated with fibroblast growth factor 2 (FGF2) and dexamethasone (DX) to induce a liver phenotye in the cells. These treated cells in various passages were further studied for their capacity to be functional in hepatic parenchyma following retrorsine-induced injury in nude C57 black mice. Amylase- and EPCAM-positive-enriched cells isolated from HFP and treated with FGF2 and DX lost expression of pancreatic markers and gained a liver phenotype. Hepatic differentiation was based on the expression (both at the mRNA and protein level) of liver markers albumin and cytokeratin 19. When transplanted in vivo into nude mice treated with retrorsine, both cell types successfully engrafted and functionally differentiated into hepatic cells expressing human albumin, glycogen, dipeptidyl peptidase, and gamma-glutamyltranspeptidase. These data indicate that human fetal pancreatic cells have a capacity to alter their gene expression profile in response to exogenous treatment with FGF2 and DX. It may be possible to generate an unlimited supply of hepatocytes in vitro for cell therapy.

Topics: Animals; Antigens, Differentiation; Cell Differentiation; Cell Transplantation; Cells, Cultured; Chemical and Drug Induced Liver Injury; Dexamethasone; Female; Fetus; Fibroblast Growth Factor 2; Hepatocytes; Humans; Mice; Mice, Nude; Pancreas; Pyrrolizidine Alkaloids

Efficient repopulation by transplanted hepatocytes in the severely injured liver is essential for their clinical application in the treatment of acute hepatic failure. We studied here whether and how the transplanted hepatocytes are able to efficiently repopulate the toxin-induced acute injured liver. Male dipeptidyl peptidase IV-deficient F344 rats were randomized to receive retrorsine plus D-galactosamine (R+D-gal) treatment or D-galactosamine-alone (D-gal) to induce acute hepatic injury, and retrorsine-alone. In these models, retrorsine was used to inhibit the proliferation of endogenous hepatocytes while D-galactosamine induced acute hepatocyte damage. Wild-type hepatocytes (1 x 10(7)/ml) were transplanted intraportally 24 h after D-galactosamine or saline injection. The kinetics of proliferation and repopulation of transplanted cells and the kinetics of cytokine response, hepatic stellate cell (HSC) activation, and matrix metalloproteinase (MMP2) expression were analyzed. We observed that early entry of transplanted hepatocytes into the hepatic plates and massive repopulation of the liver by transplanted hepatocytes occurred in acute hepatic injury induced by R+D-gal treatment but not by D-gal-alone or retrorsine-alone. The expressions of transforming growth factor-alpha and hepatocyte growth factor genes in the R+D-gal injured liver were significantly upregulated and prolonged up to 4 weeks after hepatocyte transplantation. The expression kinetics were parallel with the efficient proliferation and repopulation of transplanted hepatocytes. HSC was activated rapidly, markedly, and prolongedly up to 4 weeks after hepatocyte transplantation, when the expression of HGF gene and repopulation of transplanted hepatocytes were reduced afterward. Furthermore, the expression kinetics of MMP2 and its specific distribution in the host areas surrounding the expanding clusters of transplanted hepatocytes are consistent with those of activated HSC. Impaired hepatocyte regeneration after acute severe hepatic injury may initiate serial compensatory repair mechanisms that facilitate the extensive repopulation by transplanted hepatocytes that enter early the hepatic plates.

Topics: Animals; Chemical and Drug Induced Liver Injury; Galactosamine; Hepatic Stellate Cells; Hepatocyte Growth Factor; Hepatocytes; Liver Failure, Acute; Liver Regeneration; Male; Matrix Metalloproteinase 2; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; Transforming Growth Factor alpha; Up-Regulation

After liver injury, parenchymal regeneration occurs through hepatocyte replication. However, during regenerative stress, oval cells (OCs) and small hepatocyte like progenitor cells (SHPCs) contribute to the process. We systematically studied the intra-hepatic and extra-hepatic sources of liver cell replacement in the hepatitis B surface antigen (HBsAg-tg) mouse model of chronic liver injury. Female HBsAg-tg mice received a bone marrow (BM) transplant from male HBsAg-negative mice, and half of these animals received retrorsine to block indigenous hepatocyte proliferation. Livers were examined 3 and 6 months post-BM transplantation for evidence of BM-derived hepatocytes, OCs, and SHPCs. In animals that did not receive retrorsine, parenchymal regeneration occurred through hepatocyte replication, and the BM very rarely contributed to hepatocyte regeneration. In mice receiving retrorsine, 4.8% of hepatocytes were Y chromosome positive at 3 months, but this was frequently attributable to cell fusion between indigenous hepatocytes and donor BM, and their frequency decreased to 1.6% by 6 months, as florid OC reactions and nodules of SHPCs developed. By analyzing serial sections and reconstructing a 3-dimensional map, continuous streams of OCs could be seen that surrounded and entered deep into the nodules of SHPCs, connecting directly with SHPCs, suggesting a conversion of OCs into SHPCs. In conclusion, during regenerative stress, the contribution to parenchymal regeneration from the BM is minor and frequently attributable to cell fusion. OCs and SHPCs are of intrinsic hepatic origin, and OCs can form SHPC nodules.

Topics: Animals; Bone Marrow Transplantation; Chemical and Drug Induced Liver Injury; Female; Hepatitis B Surface Antigens; Liver; Liver Diseases; Liver Regeneration; Male; Mice; Mice, Transgenic; Pyrrolizidine Alkaloids; Stem Cells

The aim of this study was to determine the potential of bone marrow derived cells to participate in liver repopulation. In this model, the injected cells had a "selective growth advantage" compared to the native hepatocytes whose proliferation was blocked by retrorsine.. Total bone marrow cells were isolated from male Fisher 344 rats not deficient in dipeptidyl peptidase activity (F344, DPP IV+). The animals were given an injection of retrorsine and were divided in 2 groups: 1/group R (N=13): female F344 rats received 4.106 male cells at day 0 (labeled by chromosome Y). 2/group RH (N=19): Male F344 DPP IV- rats received 4.106 male DPP IV+ cells after hepatectomy at day 0 (labelled by DPP IV activity).. Group R: no male cell was detected by PCR at day 14, 28, 56 and 84. Group RH: isolated DPP IV+ transplanted cells were observed at days 14 and 28 in the periportal areas. Later, these cells were no longer visible. Liver regeneration occurred by proliferation of small clusters of hepatocytes.. In this experimental model the capacity of transplanted bone marrow cells to repopulate the liver was tested against the same capacity of native liver stem cells. Liver regeneration occurred via native liver cells seen as small hepatocytes. In this model the small hepatocytes may be considered as hepatic stem cells.

Topics: Animals; Bone Marrow Transplantation; Cell Proliferation; Chemical and Drug Induced Liver Injury; Chronic Disease; Female; Liver; Liver Diseases; Liver Regeneration; Male; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344

Mature hepatocytes divide to restore liver mass after injury. However, when hepatocyte division is impaired by retrorsine poisoning, regeneration proceeds from another cell type: the small hepatocyte-like progenitor cells (SHPCs). Our aim was to test whether SHPCs could originate from mature hepatocytes.. Mature hepatocytes were genetically labeled using retroviral vectors harboring the beta-galactosidase gene. After labeling, retrorsine was administered to rats followed by a partial hepatectomy to trigger regeneration. A liver biopsy was performed one month after surgery and rats were sacrificed one month later.. We observed the proliferation of small hepatocytes arranged in clusters in liver biopsies. These cells expressed Ki67 antigen and displayed a high mitotic index. At sacrifice, regeneration was completed and clusters had merged. A significant proportion of clusters also expressed beta-galactosidase demonstrating their origin from labeled mature hepatocytes. Finally, the overall proportion of beta-galactosidase positive cells was identical at the time of hepatectomy as well as in liver biopsy and at sacrifice.. The constant proportion of beta-galactosidase positive cells during the regeneration process demonstrates that mature hepatocytes are randomly recruited to proliferate and compensate parenchyma loss in this model. Furthermore, mature hepatocytes are the source of SHPC after retrorsine injury.

Topics: Animals; Animals, Genetically Modified; Antineoplastic Agents, Phytogenic; beta-Galactosidase; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Hepatocytes; Lac Operon; Liver Diseases; Liver Regeneration; Male; Pyrrolizidine Alkaloids; Rats; Rats, Sprague-Dawley; Stem Cells

The adult rodent liver contains at least two recognized populations of cells with stem-like properties that contribute to liver repair/regeneration under different pathophysiological circumstances: (i) unipotential committed progenitor cells (differentiated hepatocytes and biliary epithelial cells) and (ii) multipotential nonparenchymal progenitor cells (oval cells). In retrorsine-induced hepatocellular injury the capacity of fully differentiated rat hepatocytes to replicate is severely impaired and massive proliferation of oval cells does not occur. Nevertheless, retrorsine-exposed rats can replace their entire liver mass after 2/3 surgical partial hepatectomy through the emergence and expansion of a population of small hepatocyte-like progenitor cells that expresses phenotypic characteristics of fetal hepatoblasts, oval cells, and fully differentiated hepatocytes, but differ distinctly from each type of cell. The activation, proliferation, and complete regeneration of normal liver structure from small hepatocyte-like progenitor cells have not been recognized in other models of liver injury characterized by impaired hepatocyte replication. We suggest that the selective emergence and expansion of small hepatocyte-like progenitor cells observed in the retrorsine model reflect a novel mechanism of complete liver regeneration in the adult rat. Furthermore, we suggest that these cells may represent a novel progenitor cell population that (i) responds to liver deficit when the replication capacity of differentiated hepatocytes is impaired, (ii) expresses an extensive proliferative capacity, (iii) can give rise to large numbers of progeny hepatocytes, and (iv) can restore tissue mass.

Topics: Animals; Cell Line; Chemical and Drug Induced Liver Injury; Hepatectomy; Liver Diseases; Liver Regeneration; Male; Phenotype; Pyrrolizidine Alkaloids; Rats; Rats, Inbred F344; Stem Cells

A wide variety of medicinal herbs contain hepatotoxic pyrrolizidine alkaloids (PAs), and often cause acute and chronic liver damages in man. Liquorice, a known antihepatitis, is commonly used with PA-containing herbs concurrently, and hepatotoxicity induced by such combined uses was not pronounced. The present study is to investigate effects of glycyrrhizin (GL) and 18beta-glycyrrhetinic acid (GA), the major biologically active ingredients of liquorice, against PA-induced hepatotoxicity in rats. Single dose (35 mg/kg, i.p.) of retrorsine (RET), a typical potent hepatotoxic PA, was given to rats to induce liver injury. A single dose pretreatment with GL or GA prior to retrorsine challenge did not show hepatoprotection. However, when rats were pretreated with either GL (200 mg/kg/day, i.p.) or GA (10 mg/kg/day, i.p.) for three consecutive days prior to retrorsine exposure, the elevated serum GOT and GPT levels induced by retrorsine were significantly reduced. Serum levels of transaminases almost returned to normal (GOT: 56+/-2 (control), 104+/-5 (RET), 64+/-3 (GL + RET) and 59+/-3 (GA + RET). GPT: 40+/-2 (control), 90+/-7 (RET), 45+/-2 (GL + RET) and 45+/-4 (GA + RET) SF units/ml). Furthermore, no extensive hepatocellular damages were observed. The results demonstrated that a three-day pretreatment with either GL or GA exhibited protective effect on retrorsine-induced liver damage in rats.

Topics: Administration, Topical; Alanine Transaminase; Animals; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents, Phytogenic; Aspartate Aminotransferases; Chemical and Drug Induced Liver Injury; Drug Administration Schedule; Glutathione; Glycyrrhetinic Acid; Glycyrrhizic Acid; Liver; Liver Diseases; Male; Pyrrolizidine Alkaloids; Rats; Rats, Sprague-Dawley; Time Factors

To explore the hypothesis that a second xenobiotic agent is required with excess copper to produce Indian Childhood Cirrhosis, this study investigated the effect of the pyrrolizidine alkaloid retrorsine fed to the mother during the suckling period upon the serial changes in neonatal copper status.. Female Wistar rats with new-born litters were fed either a control or a retrorsine (50 mg/kg) diet. At 0, 4, 8, 11, 15, 18 and 21 days, pups from each litter were weighed, sacrificed and their livers removed for copper, DNA and metallothionein analysis. Serum samples were assayed for caeruloplasmin oxidase activity and albumin.. 1) Higher than adult level of hepatic copper in normal rats which rose post-natally before declining from day 11 after birth, 2) raised hepatic copper concentrations and total copper in the retrorsine group from day 15; levels were higher than adult at birth, 3) reduced serum caeruloplasmin oxidase activity and albumin levels in retrorsine group, but both groups lower than adult, 4) lower hepatic metallothionein levels in retrorsine group, but both groups higher than adult, and 5) reduced liver DNA in the retrorsine group when expressed as total DNA and per gram of tissue. These changes were not secondary to under-nutrition as a small study on under-nourished rat neonates showed that copper handling is not significantly altered when compared to well-nourished rats.. Retrorsine passing to rat neonates via breast milk causes: 1) the accumulation of hepatic copper, 2) impairment of the rise in serum caeruloplasmin, which could indicate a decline in synthesis or failure of copper incorporation into the apo-protein, 3) a decrease in hepatic metallothionein and serum albumin levels, again suggesting diminished protein synthesis, and 4) reduced hepatic DNA indicative of decreased cell number but increased cell size. Accumulation of liver copper but reduction of copper-binding proteins could result in free copper and explain the synergistic hepatotoxicity of copper and retrorsine.

Topics: Animals; Animals, Suckling; Body Weight; Ceruloplasmin; Chemical and Drug Induced Liver Injury; Copper; DNA; Drug Evaluation, Preclinical; Female; Liver; Liver Diseases; Metallothionein; Milk; Nutrition Disorders; Organ Size; Pyrrolizidine Alkaloids; Rats; Rats, Wistar; Serum Albumin

Topics: Alkaloids; Carcinogens; Carcinoma, Hepatocellular; Chemical and Drug Induced Liver Injury; Hepatectomy; Hepatitis; Liver Neoplasms; Neoplasms; Neoplasms, Experimental; Neoplasms, Radiation-Induced; Pathology; Pyrrolizidine Alkaloids; Radiation Injuries, Experimental; Rats; Research; Senecio; Toxicology; Whole-Body Irradiation