Platelet activating factor receptor blockade alone or in combination with leukotriene synthesis inhibition does not ameliorate α-naphthylisothiocyanate-induced hepatotoxicity

Alpha-naphthylisothiocyanate (ANIT) is a cholangiolitic hepatotoxicant that causes periportal edema, hepatic parenchymal and biliary epithelial cell necrosis, and cholestasis in the rat. A hallmark of ANIT hepatotoxicity is periportal inflammation that includes neutrophil infiltration. Neutrophils are requisite for the expression of ANIT-induced liver injury; however, the mechanism(s) of neutrophil accumulation in the liver and the role of these cells in ANIT hepatotoxicity is incompletely understood. Platelet activating factor (PAF) is a proinflammatory agent that is both a chemoattractant for and an activator of neutrophils. Therefore, we evaluated the role of PAF in ANIT-induced liver injury. Rats were treated with the PAF receptor antagonist, WEB 2086 (WEB), to determine if it afforded protection from ANIT hepatotoxicity. In a separate study, a combination of WEB and the leukotriene synthesis inhibitor, Zileuton (ZIL), was used to address the possible interaction of PAF and leukotrienes in ANIT-induced liver injury. Treatment of rats with WEB, alone or in combination with Zileuton, did not attenuate ANIT-induced liver injury as assessed by increases in plasma alanine aminotransferase or γ-glutamyl transferase activities. In addition, neither treatment ameliorated ANIT-induced cholestasis assessed as increased plasma bilirubin concentration. These results suggest that PAF, alone or in combination with products of 5-lipoxygenase, does not contribute to ANIT-induced liver injury.     

Structural comparison of monoclonal antibody immunopurified pulmonary and hepatic cytochrome P-450 from 3-methylcholanthrene-treated rats

A pulmonary cytochrome P-450 was purified from lung microsomes of 3-methylcholanthrene (MC)-treated rats by immunoaffinity chromatography using a monoclonal antibody to MC-induced rat liver cytochrome P-450. The isolated pulmonary cytochrome P-450 was MC-inducible and had an apparent molecular weight of 57 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight, as well as the NH2-terminal sequence of the first nine amino acids of the pulmonary cytochrome P-450, was identical to that of an epitopically related MC-induced rat liver cytochrome P-450. In addition, partial proteolysis of both cytochromes P-450 yielded indistinguishable peptide patterns on SDS-Page. Treatment of rats with MC, therefore, induces a pulmonary cytochrome P-450 which is structurally identical to the MC-induced hepatic enzyme by several criteria.     

Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans

Following acetaminophen (APAP) overdose there is an inflammatory response triggered by the release of cellular contents from necrotic hepatocytes into the systemic circulation which initiates the recruitment of neutrophils into the liver. It has been demonstrated that neutrophils do not contribute to APAP-induced liver injury, but their role and the role of NADPH oxidase in injury resolution are controversial. C57BL/6 mice were subjected to APAP overdose and neutrophil activation status was determined during liver injury and liver regeneration. Additionally, human APAP overdose patients (ALT: > 800 U/L) had serial blood draws during the injury and recovery phases for the determination of neutrophil activation. Neutrophils in the peripheral blood of mice showed an increasing activation status (CD11b expression and ROS priming) during and after the peak of injury but returned to baseline levels prior to complete injury resolution. Hepatic sequestered neutrophils showed an increased and sustained CD11b expression, but no ROS priming was observed. Confirming that NADPH oxidase is not critical to injury resolution, gp91^phox−/− mice following APAP overdose displayed no alteration in injury resolution. Peripheral blood from APAP overdose patients also showed increased neutrophil activation status after the peak of liver injury and remained elevated until discharge from the hospital. In mice and humans, markers of activation, like ROS priming, were increased and sustained well after active liver injury had subsided. The similar findings between surviving patients and mice indicate that neutrophil activation may be a critical event for host defense or injury resolution following APAP overdose, but not a contributing factor to APAP-induced injury.     

Modulation of 3-methylcholanthrene-induced rat hepatic and renal cytochrome P450 and phase II enzymes by plant phenols: protocatechuic and tannic acids

Naturally occurring phenolics, protocatechuic and tannic acids have been reported to be inhibitors of chemical mutagenesis and carcinogenesis in experimental models. Here, we have studied the effect of pretreatment with these compounds on MC-induced cytochrome P450 and phase II enzymes in rats. The male Wistar rats were treated intraperitoneally with protocatechuic acid and tannic acid in the dose of 50mg/kg every 3 days for 2 weeks. MC was administered at the 12th day of phenolics treatment. The activities of EROD (CYP1A1), MROD (CYP1A2), PROD (CYP2B), PNPH (CYP2E1), GST, UDPGT, NQO1 were measured in the liver and kidney. Protocatechuic acid treatment minimally reduced the MC-induced EROD and MROD, but the observed differences were statistically significant. This compound was also a weak inhibitor of hepatic PNPH. Moreover, Western blot analysis with CYP1A1/1A2- and CYP2E1-specific antibodies showed the same effect in the levels of hepatic CYP1A1/1A2 and CYP2E1. Minimal decrease of renal constitutive (by 23%) and more significant reduction of induced form (by 66%) of PNPH was found as result of treatment with protocatechuic acid. Tannic acid alone had no effect on cytochrome P450 enzymes while in combination with MC this polyphenol minimally enhanced the MC induction of MROD and in greater extent PNPH in liver. The treatment with protocatechuic acid alone enhanced slightly the activities of all three phase II enzymes in liver. The pretreatment with this phenolic of the MC-induced rats however significantly increased the activities of hepatic GST and NQO1 in comparison with MC-treated group. In kidney MC-induced activity of NQO1 was reduced (about 43%) to the control level by tannic acid pretreatment. The results of our present study indicate that in rat the prolonged treatment with protocatechuic acid affects differently the activities of CYP and phase II enzyme when compared to tannic acid. Moreover, the effect of this polyphenols significantly depends on the method of treatment.     

Preventive effect of neutropenia on carbon tetrachloride-induced hepatotoxicity in rats

The preventive effect of neutropenia on carbon tetrachloride (CCl4)-induced hepatotoxicity was examined in rats. In rats treated once with CCl4 (1 ml kg(-1), i.p.), the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indices of liver cell damage, and the hepatic activity of myeloperoxidase (MPO), an index of tissue neutrophil infiltration, increased at 6 h after the intoxication and further increased at 24 h. The liver of CCl4 -treated rats showed an increase in the concentration of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, and decreases in superoxide dismutase (SOD) activity and reduced glutathione (GSH) concentration at 6 h after the intoxication followed by a further increase in TBARS concentration and further decreases in SOD activity and GSH concentration at 24 h with increased xanthine oxidase (XO) activity at 24 h. Neutropenic treatment with anti-rat neutrophil antiserum (2 ml kg(-1), i.p.) at 0.5 h after CCl4 intoxication attenuated the increases in serum ALT and AST activities and hepatic MPO activity and TBARS concentration and the decreases in hepatic SOD activity and GSH concentration found at 6 and 24 h after CCl4 intoxication and the increase in hepatic XO activity found at 24 h after the intoxication. This neutropenia reduced the necrotic and degenerative changes with inflammatory cell infiltration in the liver cell of CCl4 -treated rats. These results indicate that neutropenia prevents CCl4 -induced hepatotoxicity in rats by attenuating the disruption of hepatic reactive oxygen species metabolism mediated by neutrophils accumulating in the liver tissue.     

Role of osteopontin in hepatic neutrophil infiltration during alcoholic steatohepatitis

Alcoholic liver disease (ALD) is a major complication of heavy alcohol (EtOH) drinking and is characterized by three progressive stages of pathology: steatosis, steatohepatitis, and fibrosis/cirrhosis. Alcoholic steatosis (AS) is the initial stage of ALD and consists of fat accumulation in the liver accompanied by minimal liver injury. AS is known to render the hepatocytes increasingly sensitive to toxicants such as bacterial endotoxin (LPS). Alcoholic steatohepatitis (ASH), the second and rate-limiting step in the progression of ALD, is characterized by hepatic fat accumulation, neutrophil infiltration, and neutrophil-mediated parenchymal injury. However, the pathogenesis of ASH is poorly defined. It has been theorized that the pathogenesis of ASH involves interaction of increased circulating levels of LPS with hepatocytes being rendered highly sensitive to LPS due to heavy EtOH consumption. We hypothesize that osteopontin (OPN), a matricellular protein (MCP), plays an important role in the hepatic neutrophil recruitment due to its enhanced expression during the early phase of ALD (AS and ASH). To study the role of OPN in the pathogenesis of ASH, we induced AS in male Sprague-Dawley rats by feeding EtOH-containing Lieber-DeCarli liquid diet for 6 weeks. AS rats experienced extensive fat accumulation and minimal liver injury. Moderate induction in OPN was observed in AS group. ASH was induced by feeding male Sprague-Dawley rats EtOH-containing Lieber-DeCarli liquid diet for 6 weeks followed by LPS injection. The ASH rats had substantial neutrophil infiltration, coagulative oncotic necrosis, and developed higher liver injury. Significant increases in the hepatic and circulating levels of OPN was observed in the ASH rats. Higher levels of the active, thrombin-cleaved form of OPN in the liver in ASH group correlated remarkably with hepatic neutrophil infiltration. Finally, correlative studies between OPN and hepatic neutrophil infiltration was corroborated in a simple rat peritoneal model where enhanced peritoneal fluid neutrophil infiltration was noted in rats injected OPN intraperitoneally. Taken together these data indicate that OPN expression induced during ASH may play a significant role in the pathogenesis of ASH by stimulating neutrophil transmigration.     

Mechanisms of hepatotoxicity.

This review addresses recent advances in specific mechanisms of hepatotoxicity. Because of its unique metabolism and relationship to the gastrointestinal tract, the liver is an important target of the toxicity of drugs, xenobiotics, and oxidative stress. In cholestatic disease, endogenously generated bile acids produce hepatocellular apoptosis by stimulating Fas translocation from the cytoplasm to the plasma membrane where self-aggregation occurs to trigger apoptosis. Kupffer cell activation and neutrophil infiltration extend toxic injury. Kupffer cells release reactive oxygen species (ROS), cytokines, and chemokines, which induce neutrophil extravasation and activation. The liver expresses many cytochrome P450 isoforms, including ethanol-induced CYP2E1. CYP2E1 generates ROS, activates many toxicologically important substrates, and may be the central pathway by which ethanol causes oxidative stress. In acetaminophen toxicity, nitric oxide (NO) scavenges superoxide to produce peroxynitrite, which then causes protein nitration and tissue injury. In inducible nitric oxide synthase (iNOS) knockout mice, nitration is prevented, but unscavenged superoxide production then causes toxic lipid peroxidation to occur instead. Microvesicular steatosis, nonalcoholic steatohepatitis (NASH), and cytolytic hepatitis involve mitochondrial dysfunction, including impairment of mitochondrial fatty acid beta-oxidation, inhibition of mitochondrial respiration, and damage to mitochondrial DNA. Induction of the mitochondrial permeability transition (MPT) is another mechanism causing mitochondrial failure, which can lead to necrosis from ATP depletion or caspase-dependent apoptosis if ATP depletion does not occur fully. Because of such diverse mechanisms, hepatotoxicity remains a major reason for drug withdrawal from pharmaceutical development and clinical use.     

Cadmium-induced acute hepatic injury is exacerbated in human interleukin-8 transgenic mice

It is reported repeatedly that severe hepatocellular necrosis along with infiltration of neutrophils occurs after acute cadmium exposure. Neutrophils, which migrate by the gradient of chemoattractants such as interleukin-8, are believed to play an important role in inflammation at the damaged sites. To investigate whether neutrophils aggravate or repair the liver injury induced by cadmium, we checked the hepatotoxic effects of cadmium on human interleukin-8 transgenic mice (hIL-8Tg), which overexpressed IL-8 and displayed an inability of neutrophil migration resulting from both the lack of chemotactic gradient and the downregulation of l-selectin on the surface of neutrophils. A significantly lower survival rate was observed in hIL-8Tg compared with wild-type mice after subcutaneous administration of cadmium. Evident liver injury characterized by abrupt increases in plasma GOT and GPT levels was found in hIL-8Tg at 18 h after cadmium administration. Histological examinations, including H & E staining and esterase staining, revealed the infiltration of numerous neutrophils into the damaged liver tissues in wild-type mice, and the inhibition of the neutrophil migration into the liver as well as enhanced hepatocellular necrosis in hIL-8Tg. Peripheral white blood cell and polymorphonuclear cell counts increased and reached their peaks at 12 h after cadmium administration in wild-type mice, whereas the increase in blood leukocyte counts was delayed in hIL-8Tg. There was no significant difference in the amounts of cadmium accumulated in liver and kidneys between wild-type mice and hIL-8Tg. In conclusion, an acute cadmium hepatotoxic effect was exacerbated in hIL-8Tg resulting from inhibited neutrophil migration, suggesting that migrated neutrophils can prevent aggravation of liver injury by acute cadmium administration.     

Rhein protects against acetaminophen-induced hepatic and renal toxicity

This study investigated the possible protective effects and mechanism of rhein on Acetaminophen (APAP)-induced hepatotoxicity and nephrotoxicity in rats. Treatment of rats with APAP resulted in severe liver and kidney injuries, as demonstrated by drastic elevation of serum glutamate-pyruvate transaminase (GPT), glutamate-oxaloacetic transaminase (GOT), total bilirubin (TBIL), creatinine (CREA), urea nitrogen (UREA) levels and typical histopathological changes including necrosis, phlogocyte infiltration and fatty degeneration in liver, tubules epithelium swelling and severe vacuolar degeneration in kidney. APAP caused oxidative stress, as evidenced by increased reactive oxygen species (ROS) production, nitric oxide (NO) and malondiadehyde (MDA) levels, together with depleted glutathione (GSH) concentration in the liver and kidney of rats. However, rhein can attenuate APAP-induced hepatotoxicity and nephrotoxicity in a dose-dependent manner. Our results showed that GPT, GOT, UREA and CREA levels and ROS production were reduced dramatically, NO, MDA, GSH contents were restored remarkedly by rhein administration, as compared to the APAP alone treated rats. Moreover, the histopathological damage of liver and kidney were also significantly ameliorated by rhein treatment. These findings suggested that the protective effects of rhein against APAP-induced liver and kidney injuries might result from the amelioration of APAP-induced oxidative stress.     

Protective effect of caffeic acid phenethyl ester on tert-butyl hydroperoxide-induced oxidative hepatotoxicity and DNA damage

Increased oxidative stress and associated high levels of free radical generation have been described to occur during the pathogeneses of various diseases in animal models. In the present work, we investigated the protective effects of the phenethyl ester of caffeic acid (CAPE), an active component of honeybee propolis, on tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity in a cultured HepG2 cell line and in rat liver. CAPE was found to significantly reduce t-BHP-induced oxidative injury in HepG2 cells, as determined by cell cytotoxicity, and lipid peroxidation and reactive oxygen species (ROS) levels in a dose-dependent manner. Furthermore, CAPE protected HepG2 cells against t-BHP-induced oxidative DNA damage, as determined by the Comet assay. Consistently, CAPE reduced hydroxyl radical-induced 2-deoxy-d-ribose degradation by ferric ion-nitrilotriacetic acid and H2O2, and also removed the superoxide anion generated by a xanthine/xanthine oxidase system. Our in vivo study showed that pretreatment with CAPE prior to the administration of t-BHP significantly and dose-dependently prevented increases in the serum levels of hepatic enzyme markers (alanine aminotransferase and aspartate aminotransferase) and reduced lipid peroxidation in rat liver. Moreover, histopathological evaluation of livers consistently revealed that CAPE reduced liver lesion induction by t-BHP. Taken together, these results suggest that the protective effects of CAPE against t-BHP-induced hepatotoxicity may, at least in part, be due to its ability to scavenge ROS and protect DNA from oxidative stress-induced damage.     

Sulforaphane prevents microcystin-LR-induced oxidative damage and apoptosis in BALB/c mice

Microcystins (MCs), the products of blooming algae Microcystis, are waterborne environmental toxins that have been implicated in the development of liver cancer, necrosis, and even fatal intrahepatic bleeding. Alternative protective approaches in addition to complete removal of MCs in drinking water are urgently needed. In our previous work, we found that sulforaphane (SFN) protects against microcystin-LR (MC-LR)-induced cytotoxicity by activating the NF-E2-related factor 2 (Nrf2)-mediated defensive response in human hepatoma (HepG2) and NIH 3T3 cells. The purpose of this study was to investigate and confirm efficacy the SFN-induced multi-mechanistic defense system against MC-induced hepatotoxicity in an animal model. We report that SFN protected against MC-LR-induced liver damage and animal death at a nontoxic and physiologically relevant dose in BALB/c mice. The protection by SFN included activities of anti-cytochrome P450 induction, anti-oxidation, anti-inflammation, and anti-apoptosis. Our results suggest that SFN may protect mice against MC-induced hepatotoxicity. This raises the possibility of a similar protective effect in human populations, particularly in developing countries where freshwaters are polluted by blooming algae.     

Hesperetin attenuated acetaminophen-induced hepatotoxicity by inhibiting hepatocyte necrosis and apoptosis,oxidative stress and inflammatory response via upregulation of heme oxygenase-1 expression

Acetaminophen (APAP) is a common antipyretic and analgesic drug, but its overdose can induce acute liver failure with lack of effective therapies. Hesperetin, a dihydrogen flavonoid compound, has been revealed to exert multiple pharmacological activities. Here, we explored the protective effects and mechanism of hesperetin on APAP-induced hepatotoxicity. The results showed that pretreatment with hesperetin dose-dependently attenuated APAP-induced acute liver injury in mice, as measured by alleviated serum enzymes activities, hepatic pathological damage and apoptosis. Moreover, hesperetin mitigated APAP-induced oxidative stress and inflammatory response in mice by inhibiting oxidative molecules but increasing antioxidative molecules production, reducing inflammatory cells infiltration and proinflammatory cytokines production, blocking Toll-like receptor (TLR)-4 signal activation. In vitro experiment indicated that hesperetin dose-dependently inhibited APAP-primed cytotoxicity, apoptosis, and reactive oxygen species (ROS) in murine AML12 hepatocytes. Notably, hesperetin up-regulated expression of heme oxygenase-1 (HO-1) mRNA and protein in the liver of mice and AML12 cells exposed to APAP. Furthermore, knockdown of HO-1 by adenovirus-mediated HO-1 siRNA reverted these beneficial effects of hesperetin on APAP-induced hepatocytotoxicity as well as ROS and inflammatory response in vivo and in vitro. These findings demonstrated that hesperetin exerted a protective prophylaxis on APAP-induced acute liver injury by inhibiting hepatocyte necrosis and apoptosis, oxidative stress and inflammatory response via up-regulating HO-1 expression.     

Osteopontin is an initial mediator of inflammation and liver injury during obstructive cholestasis after bile duct ligation in mice

Osteopontin (OPN) is a chemotactic factor which can be cleaved to the pro-inflammatory form by matrix metalloproteinases (MMPs). To test the hypothesis that OPN can modulate inflammatory liver injury during cholestasis, wild-type (WT) C57BL/6 and OPN knockout (OPN-KO) mice underwent bile duct ligation (BDL). OPN-KO mice showed significant reduction in liver injury (plasma ALT and necrosis) and neutrophil recruitment compared with WT animals at 24 h but not 72 h after BDL. In WT mice, a 4-fold increase in hepatic MMP-3 mRNA and elevated MMP activities and cleaved OPN levels were observed in bile. WT mice subjected to BDL in the presence of the MMP inhibitor BB-94 showed reduced liver injury, less neutrophil extravasation and diminished levels of cleaved OPN in bile. Thus, during obstructive cholestasis, OPN released from biliary epithelial cells could be cleaved by MMPs in bile. When the biliary system leaks, cleaved OPN enters the parenchyma and attracts neutrophils. In the absence of OPN, other chemoattractants, e.g. chemokines, mediate a delayed inflammatory response and injury. Taken together, our data suggest that OPN is the pro-inflammatory mediator that initiates the early neutrophil-mediated injury phase during obstructive cholestasis in mice.     

In vivo protective effect of protocatechuic acid on tert-butyl hydroperoxide-induced rat hepatotoxicity.

Increasing evidence regarding free-radical generating agents and the inflammatory process suggests that accumulation of reactive oxygen species (ROS) can involve hepatotoxicity. Previously, we found that protocatechuic acid (PCA), a polyphenolic compound from Hibiscus sabdariffa L. possessing free radical-scavenging capacity, protected against oxidative damage induced by tert-butylhydroperoxide (t-BHP) in rat primary hepatocytes. In this study, first PCA was evaluated by its capacity of inhibiting xanthine oxidase (XO) and lipoxygenase (LO) activity in vitro, then it was used to induce hepatotoxicity to assess the antioxidant and anti-inflammatory bioactivity of PCA in vivo. Our investigation showed that pretreatment with PCA (50-100 mg/kg) by gavage for 5 days before a single dose of t-BHP (ip; 0.2 mmol/kg ) significantly lowered serum levels of the hepatic enzyme markers lactate dehydrogenase (LDH) and alanine (ALT) and aspartate (AST) aminotransferase, and reduced oxidative stress of the liver by evaluating malondialdehyde (MDA) and glutathione (GSH). Histopathological evaluation of the rat livers revealed that PCA reduced the incidence of liver lesions, including hepatocyte swelling, leukocyte infiltration, and necrosis induced by t-BHP. In addition, PCA inhibited t-BHP-induced tyrosine phosphorylation, an implication of the activation of a stress signal pathway, in the liver. These results indicate that PCA protects against t-BHP-induced hepatotoxicity by its antioxidant and anti-inflammatory characteristics accompanied by blocking of stress signal transduction.     

Cyclopamine attenuates acute warm ischemia reperfusion injury in cholestatic rat liver: hope for marginal livers

Cholestasis is a significant risk factor for immediate hepatic failure due to ischemia reperfusion (I/R) injury in patients undergoing liver surgery or transplantation. We recently demonstrated that inhibition of Hedgehog (Hh) signaling with cyclopamine (CYA) before I/R prevents liver injury. In this study we hypothesized that Hh signaling may modulate I/R injury in cholestatic rat liver. Cholestasis was induced by bile duct ligation (BDL). Seven days after BDL, rats were exposed to either CYA or vehicle for 7 days daily before being subjected to 30 min of ischemia and 4 h of reperfusion. Expression of Hh ligands (Sonic Hedgehog, Patched-1 and Glioblastoma-1), assessment of liver injury, neutrophil infiltration, cytokines, lipid peroxidation, cell proliferation and apoptosis were determined. Significant upregulation of Hh ligands was seen in vehicle treated BDL rats. I/R injury superimposed on these animals resulted in markedly elevated serum alanine transaminase (ALT), aspartate transaminase (AST), total bilirubin accompanied with increased neutrophil recruitment and lipid peroxidation. Preconditioning with CYA reduced the histological damage and serum liver injury markers. CYA also reduced neutrophil infiltration, proinflammatory cytokines such as TNF-α and IL-1β expression of α-smooth muscle actin and type 1 collagen resulting in reduced fibrosis. Furthermore CYA treated animals showed reduced cholangiocyte proliferation, and apoptosis. Hepatoprotection by CYA was conferred by reduced activation of protein kinase B (Akt) and extracellular signal regulated kinase (ERK). Endogenous Hh signaling in cholestasis exacerbates inflammatory injury during liver I/R. Blockade of Hh pathway represents a clinically relevant novel approach to limit I/R injury in cholestatic marginal liver.     

Differential hepatotoxicity induced by cadmium in Fischer 344 and Sprague-Dawley rats.

A number of reports document that Fischer 344 (F344) rats are more susceptible to chemically induced liver injury than Sprague-Dawley (SD) rats. Cadmium (CdCl2), a hepatotoxicant that does not require bioactivation, was used to better define the biological events that are responsible for the differences in liver injury between F344 and SD rats. CdCl2 (3 mg/kg) produced hepatotoxicity in both rat strains, but the hepatic injury was 18-fold greater in F344 rats as assessed by plasma alanine aminotransferase (ALT) activity. This difference in toxicity was not observed when isolated hepatocytes were incubated with CdCl2 in vitro, indicating that other cell types contribute to Cd-induced hepatotoxicity in vivo. Indeed, the sieve plates of hepatic endothelial cells (EC) in F344 rats were damaged to a greater degree than EC in SD rats. Additionally, Kupffer cell (KC) inhibition reduced hepatotoxicity in both strains, suggesting that this cell type is involved in the progression of CdCl2-induced hepatotoxicity. Moreover, enhanced synthesis of heat shock protein 72 occurred earlier in the SD rat. Maximal levels of hepatic metallothionein (MT), a protein associated with cadmium tolerance, were greater in SD rats. These protective factors may limit CdCl2-induced hepatocellular injury in SD compared with F344 rats by reducing KC activation and the subsequent inflammatory response that allows for the progression of hepatic injury.     

Effects of phenobarbital and 3-methylcholanthrene on the in vivo distribution,metabolism and covalent binding of 4-ipomeanol in the rat; implications for target organ toxicity

The effects of phenobarbital (PB) and 3-methylcholanthrene (MC) on the distribution, metabolism and covalent binding of 4-ipomeanol were examined in the rat. An analysis of tissue extracts by high-pressure liquid chromatography (HPLC) showed that both treatments markedly decreased the concentrations of unmetabolized 4-ipomeanol at all times examined. PB treatment increased the urinary excretion of nonbound 4-ipomeanol metabolites, while MC treatment did not alter their excretion. Analysis of urine by HPLC indicated that the increased concentration of urinary metabolites found in the phenobarbital-treated rats was attributable primarily to an increased excretion of ipomeanol-4-glucuronide. These data indicate that the decreased pulmonary covalent binding and lethality of 4-ipomeanol in the rat after MC and PB were caused by alterations in the tissue distribution of the parent compound. Pulmonary concentrations of unmetabolized 4-ipomeanol were decreased by MC through an increased metabolism of 4-ipomeanol in the liver, primarily to toxic products that bind covalently in that tissue and lead to hepatotoxicity. PB produced a similar decrease in unmetabolized 4-ipomeanol concentrations in lung but by an enhanced in vivo metabolism and clearance of 4-ipomeanol, primarily through a “nontoxic” pathway, glucuronidation, and did not lead to hepatotoxicity.     

Microcystin congener- and concentration-dependent induction of murine neuron apoptosis and neurite degeneration

Cyanobacterial microcystins (MCs) represent a toxin group with > 100 variants, requiring active uptake into cells via organic anion-transporting polypeptides, in order to irreversibly inhibit serine/threonine-specific protein phosphatases. MCs are a human health hazard with repeated occurrences of severe poisonings. In the well-known human MC intoxication in Caruaru, Brazil (1996), patients developed signs of acute neurotoxicity, e.g., deafness, tinnitus, and intermittent blindness, as well as subsequent hepatotoxicity. The latter data, in conjunction with some animal studies, suggest that MCs are potent neurotoxins. However, there is little data to date demonstrating MC neuron-specific toxicity. MC exposure-induced cytotoxicity, caspase activity, chromatin condensation, and microtubule-associated Tau protein hyperphosphorylation (epitopes serine199/202 and serine396) were determined. Neurite degeneration was analyzed with confocal microscopy and neurite length determined using image analysis. MC-induced apoptosis was significantly increased by MC-LF and MC-LW, however, only at high concentrations (≥ 3μM), whereas significant neurite degeneration was already observed at 0.5μM MC-LF. Moreover, sustained hyperphosphorylation of Tau was observed with all MC congeners. The concentration- and congener-dependent mechanisms observed suggest that low concentrations of MC-LF and MC-LW can induce subtle neurodegenerative effects, reminiscent of Alzheimer's disease type human tauopathies, and thus should be taken more seriously with regard to potential human health effects than the apical cytotoxicity (apoptosis or necrosis) demonstrated at high MC concentrations.