Hepatocyte-specific deletion of small heterodimer partner protects mice against acetaminophen-induced hepatotoxicity via activation of Nrf2.

Acetaminophen (APAP) overdose stands as the primary cause of acute liver failure in the United States. APAP hepatotoxicity involves hepatic glutathione (GSH) depletion and mitochondrial damage. To counteract the toxicity of APAP, the nuclear factor erythroid 2 like 2 (Nrf2) activates the expression of genes responsible for drug detoxification and GSH synthesis. In this study, we present evidence that the elimination of hepatocyte small heterodimer partner, a critical transcriptional repressor for liver metabolism, results in Nrf2 activation and protects mice from APAP-induced acute liver injury. Initial investigations conducted on wildtype (WT) mice revealed a swift downregulation of Shp mRNA within the first 24 h after APAP administration. Subsequent treatment of hepatocyte-specific Shp knockout (ShpHep-/-) mice with 300 mg/kg APAP for 2 h exhibited comparable bioactivation of APAP with that observed in the WT controls. However, a significant reduction in liver injury was observed in ShpHep-/- after APAP treatment for 6 and 24 h. The decreased liver injury correlated with a faster recovery of GSH, attributable to heightened expression of Nrf2 target genes involved in APAP detoxification and GSH synthesis. Moreover, in vitro studies revealed that SHP protein interacted with NRF2 protein, inhibiting the transcription of Nrf2 target genes. These findings hold relevance for humans, as overexpression of SHP hindered APAP-induced NRF2 activation in primary human hepatocytes. In conclusion, our studies have unveiled a novel regulatory axis involving SHP and NRF2 in APAP-induced acute liver injury, emphasizing SHP as a promising therapeutic target in APAP overdose-induced hepatotoxicity.

Role of HNF4alpha-cMyc interaction in liver regeneration and recovery after acetaminophen-induced acute liver injury.

BACKGROUND AND AIMS: Overdose of acetaminophen (APAP) is the major cause of acute liver failure in the western world. We report a novel signaling interaction between hepatocyte nuclear factor 4 alpha (HNF4α) cMyc and nuclear factor erythroid 2-related factor 2 (Nrf2) during liver injury and regeneration after APAP overdose. APPROACH AND RESULTS: APAP-induced liver injury and regeneration were studied in male C57BL/6J (WT) mice, hepatocyte-specific HNF4α knockout mice (HNF4α-KO), and HNF4α-cMyc double knockout mice (DKO). C57BL/6J mice treated with 300 mg/kg maintained nuclear HNF4α expression and exhibited liver regeneration, resulting in recovery. However, treatment with 600-mg/kg APAP, where liver regeneration was inhibited and recovery was delayed, showed a rapid decline in HNF4α expression. HNF4α-KO mice developed significantly higher liver injury due to delayed glutathione recovery after APAP overdose. HNF4α-KO mice also exhibited significant induction of cMyc, and the deletion of cMyc in HNF4α-KO mice (DKO mice) reduced the APAP-induced liver injury. The DKO mice had significantly faster glutathione replenishment due to rapid induction in Gclc and Gclm genes. Coimmunoprecipitation and ChIP analyses revealed that HNF4α interacts with Nrf2 and affects its DNA binding. Furthermore, DKO mice showed significantly faster initiation of cell proliferation resulting in rapid liver regeneration and recovery. CONCLUSIONS: These data show that HNF4α interacts with Nrf2 and promotes glutathione replenishment aiding in recovery from APAP-induced liver injury, a process inhibited by cMyc. These studies indicate that maintaining the HNF4α function is critical for regeneration and recovery after APAP overdose.

Modulation of O-GlcNAc Levels in the Liver Impacts Acetaminophen-Induced Liver Injury by Affecting Protein Adduct Formation and Glutathione Synthesis.

Overdose of acetaminophen (APAP) results in acute liver failure. We have investigated the role of a posttranslational modification of proteins called O-GlcNAcylation, where the O-GlcNAc transferase (OGT) adds and O-GlcNAcase (OGA) removes a single ß-D-N-acetylglucosamine (O-GlcNAc) moiety, in the pathogenesis of APAP-induced liver injury. Hepatocyte-specific OGT knockout mice (OGT KO), which have reduced O-GlcNAcylation, and wild-type (WT) controls were treated with 300 mg/kg APAP and the development of injury was studied over a time course from 0 to 24 h. OGT KO mice developed significantly lower liver injury as compared with WT mice. Hepatic CYP2E1 activity and glutathione (GSH) depletion following APAP treatment were not different between WT and OGT KO mice. However, replenishment of GSH and induction of GSH biosynthesis genes were significantly faster in the OGT KO mice. Next, male C57BL/6 J mice were treated Thiamet-G (TMG), a specific inhibitor of OGA to induce O-GlcNAcylation, 1.5 h after APAP administration and the development of liver injury was studied over a time course of 0-24 h. TMG-treated mice exhibited significantly higher APAP-induced liver injury. Treatment with TMG did not affect hepatic CYP2E1 levels, GSH depletion, APAP-protein adducts, and APAP-induced mitochondrial damage. However, GSH replenishment and GSH biosynthesis genes were lower in TMG-treated mice after APAP overdose. Taken together, these data indicate that induction in cellular O-GlcNAcylation exacerbates APAP-induced liver injury via dysregulation of hepatic GSH replenishment response.

Connexin hemichannel inhibition reduces acetaminophen-induced liver injury in mice.

Historically, connexin hemichannels have been considered as structural precursors of gap junctions. However, accumulating evidence points to independent roles for connexin hemichannels in cellular signaling by connecting the intracellular compartment with the extracellular environment. Unlike gap junctions, connexin hemichannels seem to be mainly activated in pathological processes. The present study was set up to test the potential involvement of hemichannels composed of connexin32 and connexin43 in acute hepatotoxicity induced by acetaminophen. Prior to this, in vitro testing was performed to confirm the specificity and efficacy of TAT-Gap24 and TAT-Gap19 in blocking connexin32 and connexin43 hemichannels, respectively. Subsequently, mice were overdosed with acetaminophen followed by treatment with TAT-Gap24 or TAT-Gap19 or a combination of both after 1.5h. Sampling was performed 3, 6, 24 and 48h following acetaminophen administration. Evaluation of the effects of connexin hemichannel inhibition was based on a series of clinically relevant read-outs, measurement of inflammatory cytokines and oxidative stress. Subsequent treatment of acetaminophen-overdosed mice with TAT-Gap19 only marginally affected liver injury. In contrast, a significant reduction in serum alanine aminotransferase activity was found upon administration of TAT-Gap24 to intoxicated animals. Furthermore, co-treatment of acetaminophen-overdosed mice with both peptides revealed an additive effect as even lower serum alanine aminotransferase activity was observed. Blocking of connexin32 or connexin43 hemichannels individually was found to decrease serum quantities of pro-inflammatory cytokines, while no effects were observed on the occurrence of hepatic oxidative stress. This study shows for the first time a role for connexin hemichannels in acetaminophen-induced acute liver failure.

Dual Role of Epidermal Growth Factor Receptor in Liver Injury and Regeneration after Acetaminophen Overdose in Mice.

Epidermal growth factor receptor (EGFR) plays a crucial role in hepatocyte proliferation. Its role in acetaminophen (APAP)-mediated hepatotoxicity and subsequent liver regeneration is completely unknown. Role of EGFR after APAP-overdose in mice was studied using pharmacological inhibition strategy. Rapid, sustained and dose-dependent activation of EGFR was noted after APAP-treatment in mice, which was triggered by glutathione depletion. EGFR-activation was also observed in primary human hepatocytes after APAP-treatment, preceding elevation of toxicity markers. Treatment of mice with an EGFR-inhibitor (EGFRi), Canertinib, 1h post-APAP resulted in robust inhibition of EGFR-activation and a striking reduction in APAP-induced liver injury. Metabolic activation of APAP, formation of APAP-protein adducts, APAP-mediated JNK-activation and its mitochondrial translocation were not altered by EGFRi. Interestingly, EGFR rapidly translocated to mitochondria after APAP-treatment. EGFRi-treatment abolished mitochondrial EGFR activity, prevented APAP-mediated mitochondrial dysfunction/oxidative-stress and release of endonucleases from mitochondria, which are responsible for DNA-damage/necrosis. Treatment with N-acetylcysteine (NAC), 4h post-APAP in mice did not show any protection but treatment of EGFRi in combination with NAC showed decrease in liver injury. Finally, delayed treatment with EGFRi, 12-h post-APAP, did not alter peak injury but caused impairment of liver regeneration resulting in sustained injury and decreased survival after APAP overdose in mice. Impairment of regeneration was due to inhibition of cyclinD1 induction and cell cycle arrest. Our study has revealed a new dual role of EGFR both in initiation of APAP-injury and in stimulation of subsequent compensatory regeneration after APAP-overdose.

Inhibitor of apoptosis signal-regulating kinase 1 protects against acetaminophen-induced liver injury.

Metabolic activation and oxidant stress are key events in the pathophysiology of acetaminophen (APAP) hepatotoxicity. The initial mitochondrial oxidative stress triggered by protein adduct formation is amplified by c-jun-N-terminal kinase (JNK), resulting in mitochondrial dysfunction and ultimately cell necrosis. Apoptosis signal-regulating kinase 1 (ASK1) is considered the link between oxidant stress and JNK activation. The objective of the current study was to assess the efficacy and mechanism of action of the small-molecule ASK1 inhibitor GS-459679 in a murine model of APAP hepatotoxicity. APAP (300 mg/kg) caused extensive glutathione depletion, JNK activation and translocation to the mitochondria, oxidant stress and liver injury as indicated by plasma ALT activities and area of necrosis over a 24h observation period. Pretreatment with 30 mg/kg of GS-459679 almost completely prevented JNK activation, oxidant stress and injury without affecting the metabolic activation of APAP. To evaluate the therapeutic potential of GS-459679, mice were treated with APAP and then with the inhibitor. Given 1.5h after APAP, GS-459679 was still protective, which was paralleled by reduced JNK activation and p-JNK translocation to mitochondria. However, GS-459679 treatment was not more effective than N-acetylcysteine, and the combination of GS-459679 and N-acetylcysteine exhibited similar efficacy as N-acetylcysteine monotherapy, suggesting that GS-459769 and N-acetylcysteine affect the same pathway. Importantly, inhibition of ASK1 did not impair liver regeneration as indicated by PCNA staining. In conclusion, the ASK1 inhibitor GS-459679 protected against APAP toxicity by attenuating JNK activation and oxidant stress in mice and may have therapeutic potential for APAP overdose patients.

Hepatitis C virus structural proteins can exacerbate or ameliorate acetaminophen-induced liver injury in mice.

Chronic hepatitis C virus (HCV) infection predisposes patients to develop liver failure after acetaminophen (APAP) overdose. Mechanisms involved in this were explored using transgenic mice expressing the HCV structural proteins core, E1 and E2. Treatment of C57BL/6J mice with 200 mg/kg body weight APAP resulted in significant liver injury at 6 h as indicated by elevated ALT levels, focal centrilobular necrosis and nuclear DNA fragmentation. HCV transgenic mice showed a variable response, with approximately half the animals showing exacerbation of all parameters of liver injury, while the other half was protected. HCV transgenic mice with higher liver injury had lower liver glutathione levels, elevated mitochondrial oxidative stress and enhanced release of apoptosis-inducing factor (AIF) from the mitochondria. This was accompanied by induction of a higher ER stress response and induction of autophagy. Transgenic animals showing protection against liver injury had a robust recovery of liver glutathione content at 6 h when compared to wild-type animals, accompanied by reduction in mitochondrial oxidative stress and AIF release. This was accompanied by an elevation in glutathione S-transferase mRNA levels and activity, which suggests that an efficient clearance of the reactive intermediate may contribute to the protection against APAP hepatotoxicity in these mice. These results demonstrate that while HCV infection could exacerbate APAP-induced liver injury due to induction and amplification of mitochondrial oxidant stress, it could also protect against injury by activation of APAP scavenging mechanisms.

Interactions Between Nuclear Receptor SHP and FOXA1 Maintain Oscillatory Homocysteine Homeostasis in Mice.

BACKGROUND & AIMS: Hyperhomocysteinemia is often associated with liver and metabolic diseases. We studied nuclear receptors that mediate oscillatory control of homocysteine homeostasis in mice. METHODS: We studied mice with disruptions in Nr0b2 (called small heterodimer partner [SHP]-null mice), betaine-homocysteine S-methyltransferase (Bhmt), or both genes (BHMT-null/SHP-null mice), along with mice with wild-type copies of these genes (controls). Hyperhomocysteinemia was induced by feeding mice alcohol (National Institute on Alcohol Abuse and Alcoholism binge model) or chow diets along with water containing 0.18% DL-homocysteine. Some mice were placed on diets containing cholic acid (1%) or cholestyramine (2%) or high-fat diets (60%). Serum and livers were collected during a 24-hour light-dark cycle and analyzed by RNA-seq, metabolomic, and quantitative polymerase chain reaction, immunoblot, and chromatin immunoprecipitation assays. RESULTS: SHP-null mice had altered timing in expression of genes that regulate homocysteine metabolism compared with control mice. Oscillatory production of S-adenosylmethionine, betaine, choline, phosphocholine, glyceophosphocholine, cystathionine, cysteine, hydrogen sulfide, glutathione disulfide, and glutathione, differed between SHP-null mice and control mice. SHP inhibited transcriptional activation of Bhmt and cystathionine γ-lyase by FOXA1. Expression of Bhmt and cystathionine γ-lyase was decreased when mice were fed cholic acid but increased when they were placed on diets containing cholestyramine or high-fat content. Diets containing ethanol or homocysteine induced hyperhomocysteinemia and glucose intolerance in control, but not SHP-null, mice. In BHMT-null and BHMT-null/SHP-null mice fed a control liquid, lipid vacuoles were observed in livers. Ethanol feeding induced accumulation of macrovesicular lipid vacuoles to the greatest extent in BHMT-null and BHMT-null/SHP-null mice. CONCLUSIONS: Disruption of Shp in mice alters timing of expression of genes that regulate homocysteine metabolism and the liver responses to ethanol and homocysteine. SHP inhibits the transcriptional activation of Bhmt and cystathionine γ-lyase by FOXA1.

Protection against acetaminophen-induced liver injury by allopurinol is dependent on aldehyde oxidase-mediated liver preconditioning.

Acetaminophen (APAP) overdose causes severe and occasionally fatal liver injury. Numerous drugs that attenuate APAP toxicity have been described. However these compounds frequently protect by cytochrome P450 inhibition, thereby preventing the initiating step of toxicity. We have previously shown that pretreatment with allopurinol can effectively protect against APAP toxicity, but the mechanism remains unclear. In the current study, C3HeB/FeJ mice were administered allopurinol 18h or 1h prior to an APAP overdose. Administration of allopurinol 18h prior to APAP overdose resulted in an 88% reduction in liver injury (serum ALT) 6h after APAP; however, 1h pretreatment offered no protection. APAP-cysteine adducts and glutathione depletion kinetics were similar with or without allopurinol pretreatment. The phosphorylation and mitochondrial translocation of c-jun-N-terminal-kinase (JNK) have been implicated in the progression of APAP toxicity. In our study we showed equivalent early JNK activation (2h) however late JNK activation (6h) was attenuated in allopurinol treated mice, which suggests that later JNK activation is more critical for the toxicity. Additional mice were administered oxypurinol (primary metabolite of allopurinol) 18h or 1h pre-APAP, but neither treatment protected. This finding implicated an aldehyde oxidase (AO)-mediated metabolism of allopurinol, so mice were treated with hydralazine to inhibit AO prior to allopurinol/APAP administration, which eliminated the protective effects of allopurinol. We evaluated potential targets of AO-mediated preconditioning and found increased hepatic metallothionein 18h post-allopurinol. These data show metabolism of allopurinol occurring independent of P450 isoenzymes preconditions the liver and renders the animal less susceptible to an APAP overdose.

Plasma and liver acetaminophen-protein adduct levels in mice after acetaminophen treatment: dose-response, mechanisms, and clinical implications.

At therapeutic doses, acetaminophen (APAP) is a safe and effective analgesic. However, overdose of APAP is the principal cause of acute liver failure in the West. Binding of the reactive metabolite of APAP (NAPQI) to proteins is thought to be the initiating event in the mechanism of hepatotoxicity. Early work suggested that APAP-protein binding could not occur without glutathione (GSH) depletion, and likely only at toxic doses. Moreover, it was found that protein-derived APAP-cysteine could only be detected in serum after the onset of liver injury. On this basis, it was recently proposed that serum APAP-cysteine could be used as diagnostic marker of APAP overdose. However, comprehensive dose-response and time course studies have not yet been done. Furthermore, the effects of co-morbidities on this parameter have not been investigated. We treated groups of mice with APAP at multiple doses and measured liver GSH and both liver and plasma APAP-protein adducts at various timepoints. Our results show that protein binding can occur without much loss of GSH. Importantly, the data confirm earlier work that showed that protein-derived APAP-cysteine can appear in plasma without liver injury. Experiments performed in vitro suggest that this may involve multiple mechanisms, including secretion of adducted proteins and diffusion of NAPQI directly into plasma. Induction of liver necrosis through ischemia-reperfusion significantly increased the plasma concentration of protein-derived APAP-cysteine after a subtoxic dose of APAP. While our data generally support the measurement of serum APAP-protein adducts in the clinic, caution is suggested in the interpretation of this parameter.

Liver-specific loss of Atg5 causes persistent activation of Nrf2 and protects against acetaminophen-induced liver injury.

Autophagy is an evolutionarily conserved biological process that degrades intracellular proteins and organelles including damaged mitochondria through the formation of autophagosome. We have previously demonstrated that pharmacological induction of autophagy by rapamycin protects against acetaminophen (APAP)-induced liver injury in mice. In contrast, in the present study, we found that mice with the liver-specific loss of Atg5, an essential autophagy gene, were resistant to APAP-induced liver injury. Hepatocyte-specific deletion of Atg5 resulted in mild liver injury characterized by increased apoptosis and compensatory hepatocyte proliferation. The lack of autophagy in the Atg5-deficient mouse livers was confirmed by increased p62 protein levels and the absence of LC3-lipidation as well as autophagosome formation. Analysis of histological and clinical chemistry parameters indicated that the Atg5 liver-specific knockout mice are resistant to APAP overdose (500 mg/kg). Further investigations revealed that the bioactivation of APAP is normal in Atg5 liver-specific knockout mice although they had lower CYP2E1 expression. There was an increased basal hepatic glutathione (GSH) content and a faster recovery of GSH after APAP treatment due to persistent activation of Nrf2, a transcriptional factor regulating drug detoxification and GSH synthesis gene expression. In addition, we found significantly higher hepatocyte proliferation in the livers of Atg5 liver-specific knockout mice. Taken together, our data suggest that persistent activation of Nrf2 and increased hepatocyte proliferation protect against APAP-induced liver injury in Atg5 liver-specific knockout mice.

The impact of partial manganese superoxide dismutase (SOD2)-deficiency on mitochondrial oxidant stress, DNA fragmentation and liver injury during acetaminophen hepatotoxicity.

UNLABELLED: Acetaminophen (APAP) hepatotoxicity is the most frequent cause of acute liver failure in many countries. The mechanism of cell death is initiated by formation of a reactive metabolite that binds to mitochondrial proteins and promotes mitochondrial dysfunction and oxidant stress. Manganese superoxide dismutase (SOD2) is a critical defense enzyme located in the mitochondrial matrix. The objective of this investigation was to evaluate the functional consequences of partial SOD2-deficiency (SOD2+/-) on intracellular signaling mechanisms of necrotic cell death after APAP overdose. Treatment of C57Bl/6J wild type animals with 200mg/kg APAP resulted in liver injury as indicated by elevated plasma alanine aminotransferase activities (2870±180U/L) and centrilobular necrosis at 6h. In addition, increased tissue glutathione disulfide (GSSG) levels and GSSG-to-GSH ratios, delayed mitochondrial GSH recovery, and increased mitochondrial protein carbonyls and nitrotyrosine protein adducts indicated mitochondrial oxidant stress. In addition, nuclear DNA fragmentation (TUNEL assay) correlated with translocation of Bax to the mitochondria and release of apoptosis-inducing factor (AIF). Furthermore, activation of c-jun-N-terminal kinase (JNK) was documented by the mitochondrial translocation of phospho-JNK. SOD2+/- mice showed 4-fold higher ALT activities and necrosis, an enhancement of all parameters of the mitochondrial oxidant stress, more AIF release and more extensive DNA fragmentation and more prolonged JNK activation. CONCLUSIONS: the impaired defense against mitochondrial superoxide formation in SOD2+/- mice prolongs JNK activation after APAP overdose and consequently further enhances the mitochondrial oxidant stress leading to exaggerated mitochondrial dysfunction, release of intermembrane proteins with nuclear DNA fragmentation and more necrosis.

Cyclophilin D deficiency protects against acetaminophen-induced oxidant stress and liver injury.

Acetaminophen (APAP) hepatotoxicity is the main cause of acute liver failure in humans. Although mitochondrial oxidant stress and induction of the mitochondrial permeability transition (MPT) have been implicated in APAP-induced hepatotoxicity, the link between these events is unclear. To investigate this, this study evaluated APAP hepatotoxicity in mice deficient of cyclophilin D, a protein component of the MPT. Treatment of wild type mice with APAP resulted in focal centrilobular necrosis, nuclear DNA fragmentation and formation of reactive oxygen (elevated glutathione disulphide levels) and peroxynitrite (nitrotyrosine immunostaining) in the liver. CypD-deficient (Ppif(-/-)) mice were completely protected against APAP-induced liver injury and DNA fragmentation. Oxidant stress and peroxynitrite formation were blunted but not eliminated in CypD-deficient mice. Thus, mitochondrial oxidative stress and induction of the MPT are critical events in APAP hepatotoxicity in vivo and at least part of the APAP-induced oxidant stress and peroxynitrite formation occurs downstream of the MPT.

The role of osteopontin and tumor necrosis factor alpha receptor-1 in xenobiotic-induced cholangitis and biliary fibrosis in mice.

Proinflammatory and profibrotic cytokines such as osteopontin (OPN) and tumor necrosis factor-alpha receptor-1 (TNFR(1)) may be critically involved in the pathogenesis of cholangiopathies and biliary fibrosis. We therefore aimed to determine the role of genetic loss of either OPN or TNFR(1) in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice as a model of xenobiotic-induced sclerosing cholangitis with biliary-type liver fibrosis using respective knock-out mice. OPN and TNFR(1) knock-out mice were fed a 0.1% DDC-supplemented diet for 4 weeks and compared with corresponding wild-type (WT) controls. Liver morphology (H&E staining), serum markers of liver injury and cholestasis (ALT, AP, bilirubin), markers of inflammation in liver (CD11b and F4/80 immunostaining, mRNA expression of iNOS, MCP-1, IL-1beta, INF-gamma, TNF-alpha and OPN), degree of ductular reaction (immunohistochemistry with morphometric analysis and western blotting for cholangiocyte-specific marker keratin 19) and degree of liver fibrosis (Sirius-red staining, hepatic hydroxyproline content for quantification) were compared between groups. DDC feeding in OPN and TNFR(1) knock-out mice and respective WT controls resulted in comparable extent of liver injury, inflammatory response, ductular reaction and liver fibrosis. Our data indicate that genetic loss of neither OPN nor TNFR(1) significantly effects on the pathogenesis of DDC-induced sclerosing cholangitis, ductular reaction and resulting biliary fibrosis.

The strength of the Fas ligand signal determines whether hepatocytes act as type 1 or type 2 cells in murine livers.

UNLABELLED: The BH3-interacting domain death agonist Bid has been shown to be critical for Fas-induced hepatocellular apoptosis. Furthermore, some studies have suggested that phosphorylation of Bid may determine its apoptotic function and may act as a switch to nonapoptotic functions. The aim of this study was to evaluate the role of Bid and phosphorylated Bid for Fas ligand (FasL)-induced apoptosis in murine livers. The monoclonal antibody Jo2 and a hexameric form of sFasL (MegaFasL) were used to induce apoptosis in wild-type, Bid-deficient (Bid(-/-)), Bid transgenic mice expressing a nonphosphorable form of Bid and Fas receptor-deficient lpr mice. Apoptosis sensitivity was determined in healthy mice and in mice following bile duct ligation, partial hepatectomy, or suramin pretreatment. As previously reported, loss of Bid protects mice against Jo2-induced liver failure. Remarkably however, Bid(-/-) mice are highly sensitive to MegaFasL-induced apoptosis. MegaFasL-treated Bid(-/-) mice showed a typical type I cell signaling behavior with activation of caspase-3 without Bax translocation to the mitochondria and no cytochrome C/Smac release into the cytosol. In contrast to previous in vitro findings, phosphorylation of Bid does not affect the sensitivity of hepatocytes to Fas receptor-mediated apoptosis in vivo. CONCLUSION: Our data suggest that Bid mainly amplifies a weak death receptor signal in quiescent and nonquiescent hepatocytes rendering the liver more sensitive to FasL-induced apoptosis. Thus, depending on the efficacy of Fas receptor activation, hepatocytes and nonparenchymal cells can either behave as type I or type II cells.

Chronic toxicity and carcinogenicity of 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin displays a distinct dose/time toxicity threshold (c x t = k) and a life-prolonging subthreshold effect.

Chronic toxicity of 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD) including its carcinogenicity was studied in female Sprague-Dawley rats in lifetime experiments. Six single dose and three multiple dose rate experiments were conducted with a single dose corn oil control group and a multiple dose rate corn oil control group, respectively. The lowest dose (1.0 mg/kg) of HpCDD and multiple dose rates of corn oil (4.0 ml/kg every other week) both prolonged the life of rats by about 2 months over that of single dose corn oil controls. Higher doses resulted in a predictable shortening of the life of rats after single dose administrations as well as after multiple dose rate administrations. The c x t = k paradigm previously validated for acute toxicity [Toxicol. Sci. 49 (1999) 102] was confirmed for chronic toxicity including carcinogenicity of HpCDD. The c x t = k product was independent of dosing regimen. Anemia and squamous cell carcinoma of the lungs were the earliest and most prevalent endpoints of toxicity. A dose of 2.1 mg/kg and 3.1 mg/kg of HpCDD caused 16.6% and 73.3% lung cancer, respectively. Liver cancer had a low prevalence and was a very late effect occurring only at doses lethal acutely for most rats in the three highest dosage groups. There was no correlation in the dose-dependence of non-malignant hepatic lesions and liver cancer.