Ibuprofen administration attenuates serum TNF-alpha levels, hepatic glutathione depletion, hepatic apoptosis and mouse mortality after Fas stimulation

Fas stimulation recruits neutrophils and activates macrophages that secrete tumor necrosis factor-α (TNF-α), which aggravates Fas-mediated liver injury. To determine whether nonsteroidal anti-inflammatory drugs modify these processes, we challenged 24-hour-fasted mice with the agonistic Jo2 anti-Fas antibody (4 µg/mouse), and treated the animals 1 h later with saline or ibuprofen (250 mg/kg), a dual cyclooxygenase (COX)-1 and COX-2 inhibitor. Ibuprofen attenuated the Jo2-mediated recruitment/activation of myeloperoxidase-secreting neutrophils/macrophages in the liver, and attenuated the surge in serum TNF-α. Ibuprofen also minimized hepatic glutathione depletion, Bid truncation, caspase activation, outer mitochondrial membrane rupture, hepatocyte apoptosis and the increase in serum alanine aminotransferase (ALT) activity 5 h after Jo2 administration, to finally decrease mouse mortality at later times. The concomitant administration of pentoxifylline (decreasing TNF-α secretion) and infliximab (trapping TNF-α) likewise attenuated the Jo2-mediated increase in TNF-α, the decrease in hepatic glutathione, and the increase in serum ALT activity 5 h after Jo2 administration. The concomitant administration of the COX-1 inhibitor, SC-560 (10 mg/kg) and the COX-2 inhibitor, celecoxib (40 mg/kg) 1 h after Jo2 administration, also decreased liver injury 5 h after Jo2 administration. In contrast, SC-560 (10 mg/kg) or celecoxib (40 or 160 mg/kg) given alone had no significant protective effects. In conclusion, secondary TNF-α secretion plays an important role in Jo2-mediated glutathione depletion and liver injury. The combined inhibition of COX-1 and COX-2 by ibuprofen attenuates TNF-α secretion, glutathione depletion, mitochondrial alterations, hepatic apoptosis and mortality in Jo2-treated fasted mice.     

Inhibition of acetaminophen hepatotoxicity by chlorpromazine in fed and fasted mice

Acetaminophen hepatotoxicity has been shown previously to be potentiated by fasting, and the mechanism of hepatotoxicity has been correlated with depletion of reduced glutathione and the resulting elevation of cytosolic calcium. Chlorpromazine inhibited the hepatotoxicity of acetaminophen in a dose-dependent manner in fed and fasted mice. A 6 mg/kg dose of chlorpromazine prevented the acetaminophen-promoted increase in SGPT levels and prevented hepatic necrosis. Chlorpromazine did not prevent the depletion of reduced glutathione by acetaminophen in fed or fasted mice, although it did decrease the extent of reduced glutathione depletion caused by acetaminophen in fed mice from 80% depletion to 67% depletion. We propose that chlorpromazine causes a negative sensitivity modulation to calcium in hepatocytes, as evidenced by chlorpromazine preventing the acetaminophen-stimulated rise in phosphorylase a activity. We also propose that fasting potentiates acetaminophen hepatotoxicity by causing a positive sensitivity modulation to calcium in hepatocytes via the actions of glucagon.     

Redox control of hepatic cell death

In necrotic liver failure like upon acetaminophen overdose, loss of the major intracellular thiol antioxidant glutathione was shown to be causal for hepatic dysfunction. In sharp contrast, fulminant apoptotic liver destruction upon overstimulation of the death receptors TNFR1 and CD95 was not associated with reduced hepatic glutathione levels. In view of the importance of the role of reactive oxygen intermediates versus antioxidants for apoptosis, we investigated the effect of phorone-induced enzymatic GSH depletion on the sensitivity of the liver towards CD95- or TNFR1-mediated hepatotoxicity. Our findings demonstrate in vivo that receptor-mediated hepatic apoptosis is disabled when glutathione is depleted, i.e. that an intact glutathione status is a critical determinant for the execution of apoptosis. In vitro, we did mechanistic studies in lymphoid cell lines and found that pro-caspase-8 at the CD95 death receptor and the mitochondrial activation of pro-caspase-9 are the enzyme targets that require sufficient intracellular reduced glutathione for their activation.     

Vascular and hepatocellular peroxynitrite formation during acetaminophen toxicity: role of mitochondrial oxidant stress.

Peroxynitrite may be involved in acetaminophen-induced liver damage. However, it is unclear if peroxynitrite is generated in hepatocytes or in the vasculature. To address this question, we treated C3Heb/FeJ mice with 300 mg/kg acetaminophen and assessed nitrotyrosine protein adducts as indicator for peroxynitrite formation. Vascular nitrotyrosine staining was evident before liver injury between 0.5 and 2 h after acetaminophen treatment. However, liver injury developed parallel to hepatocellular nitrotyrosine staining between 2 and 6 h after acetaminophen. The mitochondrial content of glutathione disulfide, as indicator of reactive oxygen formation determined 6 h after acetaminophen, increased from 2.8 +/- 0.6% in controls to 23.5 +/- 5.1%. A high dose of allopurinol (100 mg/kg) strongly attenuated acetaminophen protein-adduct formation and prevented the mitochondrial oxidant stress and liver injury after acetaminophen. Lower doses of allopurinol, which are equally effective in inhibiting xanthine oxidase, were not protective and had no effect on nitrotyrosine staining and acetaminophen protein adduct formation. In vitro experiments showed that allopurinol is not a direct scavenger of peroxynitrite. We conclude that there is vascular peroxynitrite formation during the first 2 h after acetaminophen treatment. On the other hand, reactive metabolites of acetaminophen bind to intracellular proteins and cause mitochondrial dysfunction and superoxide formation. Mitochondrial superoxide reacts with nitric oxide to form peroxynitrite, which is responsible for intracellular protein nitration. The pathophysiological relevance of vascular peroxynitrite for hepatocellular peroxynitrite formation and liver injury remains to be established.     

Theanine prevents doxorubicin-induced acute hepatotoxicity by reducing intrinsic apoptotic response

Doxorubicin (DOX) is widely used as an antitumor agent with topoisomerase II inhibiting activity; however, its dosage and duration of administration have been strictly limited due to dose-related organ damage. The present study investigated whether theanine, an amino acid found in green tea leaves, could reduce DOX-induced acute hepatotoxicity and the apoptotic response in mice. Activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum, biomarkers of hepatic impairment, were markedly increased after the administration of 20 mg/kg DOX, whereas the degree of these elevations was significantly attenuated by 10 mg/kg theanine, which was consistent with histological hepatic images assessed by microscopic examination. The hepatic expression of Bax and Fas, representative intrinsic and extrinsic apoptotic molecules, respectively, was significantly increased by dosing with DOX. However, the elevation in the hepatic expression of Bax, but not Fas, was suppressed to control levels by theanine. The formation of cleaved caspase-3 protein in the group given DOX with theanine was significantly lower than that in the group treated with DOX alone. These results suggest that theanine can protect against acute hepatic damage induced by DOX, which is attributed to the suppression of intrinsic caspase-3-dependent apoptotic signaling.     

Mitochondrial signaling pathway is also involved in bisphenol A induced germ cell apoptosis in testes

Bisphenol A (BPA) is a potential endocrine disruptor and testicular toxicant. An earlier study showed that BPA-induced germ cell apoptosis through the Fas/FasL apoptotic pathway. In the present study, we aimed to investigate whether the mitochondrial pathway is also involved in the process of BPA-mediated germ cell apoptosis in testes. Male mice were administered with BPA (160 or 480 mg/kg) by gavage daily from postnatal day 35 (PND35) to PND49. Germ cell apoptosis in testes was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). As expected, the number of TUNEL+ germ cells per tubule and the percentage of tubules with TUNEL+ germ cells were significantly increased in testes of mice treated with BPA during puberty. TUNEL+ germ cells were observed mainly in stages VII–VIII seminiferous tubules in testes. An increase in the level of Fas and FasL was observed in testes of mice exposed to BPA during puberty. In addition, pubertal BPA exposure evoked the activation of caspase-8 and caspase-3 in testes. Interestingly, pubertal BPA exposure also caused the translocation of cytochrome c from mitochondria into cytosol. In addition, pubertal BPA exposure upregulated the level of Bax and active caspase-9 in testes. Taken together, these results suggest that pubertal BPA exposure induces germ cell apoptosis in testes through not only the Fas/FasL signaling pathway but also the mitochondrial apoptotic pathway.     

Acetaminophen-induced hepatic glycogen depletion and hyperglycemia in mice

Two hours following administration of a hepatotoxic dose of acetaminophen (500 mg/kg, i.p.) to mice, liver sections stained with periodic acid Schiff reagent showed centrilobular hepatic glycogen depletion. A chemical assay revealed that following acetaminophen administration (500 mg/kg) hepatic glycogen was depleted by 65% at 1 hr and 80% at 2 hr, whereas glutathione was depleted by 65% at 0.5 hr and 80% at 1.5 hr. Maximal glycogen depletion (85% at 2.5 hr correlated with maximal hyperglycemia (267 mg/100 ml at 2.5hr). At 4.0 hr following acetaminophen administration, blood glucose levels were not significantly different from saline-treated animals; however, glycogen levels were still maximally depleted. A comparison of the dose-response curves for hepatic glycogen depletion and glutathione depletion showed that acetaminophen (50–500 mg/kg at 2.5 hr) depleted both glycogen and glutathione by similar percentages at each dose. Since acetaminophen (100 mg/kg at 2.5 hr) depleted glutathione and glycogen by approximately 30%, evidence for hepatotoxicity was examined at this dose to determine the potential importance of hepatic necrosis in glycogen depletion. Twenty-four hours following administration of acetaminophen (100 mg/kg) to mice, histological evidence of hepatic necrosis was not detected and serum glutamate pyruvate transaminase (SGPT) levels were not significantly different from saline-treated mice. The potential role of glycogen depletion in altering the acetaminophen-induced hepatotoxicity was examined subsequently. When mice were fasted overnight, hepatic glutathione and glycogen were decreased by 40 and 75%, respectively, and fasted animals showed a dramatic increase in susceptibility to acetaminophen-induced hepatotoxicity as measured by increased SGPT levels. Availability of glucose in the drinking water (5%) overnight resulted in glycogen levels similar to those in fed animals, whereas hepatic glutathione levels were not significantly different from those of fasted animals. Fasted animals and animals given glucose water overnight were equally susceptible to acetaminophen-induced hepatotoxicity, as quantitated by increases in SGPT levels 24 hr after drug administration. The potential role of a reactive metabolite in glycogen depletion was investigated by treating mice with N-acetylcysteine to increase detoxification of the reactive metabolite. N-Acetylcysteine treatment of mice prevented acetaminophen-induced glycogen depletion.     

Oncotic necrosis and caspase-dependent apoptosis during galactosamine-induced liver injury in rats

The mode of cell death during galactosamine (Gal)-induced liver injury was originally thought to be oncotic necrosis but recently it was suggested to be apoptosis. Thus, the objective was to assess whether apoptosis and oncosis are sequential or independent events in the pathophysiology. In addition, the role of caspases in Gal-induced apoptotic signaling was investigated. A dose of 500 mg/kg Gal caused a time-dependent increase in plasma alanine transaminase (ALT) levels (24 h: 430 +/- 122 U/L) in female Sprague-Dawley rats. This was accompanied by processing of procaspase-3 and significant increases in hepatic and plasma caspase-3 activities. Using morphology and TUNEL staining, apoptotic and oncotic cells were quantitated. The number of apoptotic hepatocytes increased from 0.14% in controls to 5.4 +/- 1.0% 24 h after Gal treatment. In addition, the number of cells with oncotic morphology increased from 0 to 6.9% of total hepatocytes. Treatment with the pan-caspase inhibitor IDN-7314 (10 mg/kg) or pretreatment with uridine (1 g/kg), reduced all parameters of apoptosis to baseline. However, IDN-7314 administration did not affect plasma ALT activities and the number of oncotic cells at 6 h and only modestly reduced these parameters at 24 h. Uridine, on the other hand, prevented the increase of plasma ALT levels and reduced the number of apoptotic and oncotic cells by >80%. In conclusion, galactosamine-induced hepatocellular apoptosis in rats is caspase dependent. Although some of the apoptotic cells may undergo secondary necrosis, a significant number of hepatocytes die through oncotic necrosis as an independent mechanism of cell death.     

Evaluation of caspase-dependent apoptosis during fluoride-induced liver lesion in pigs

Sixteen barrows (Duroc×Landrace×Yorkshire) were randomly divided into two groups, each consisting eight pigs. The groups received the same basal diet supplemented with 0 and 400 mg/kg fluoride, respectively. Histological examinations, including in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), Haematoxylin and Eosin staining (HE) and transmission electron microscopy observation, found apoptotic hepatocytes 50 days after additional 400 mg/kg fluoride treatment. The obvious DNA ladder and the significantly increased both hepatic caspase-9 and caspase-3 activity indicated that fluoride induced caspase-dependent apoptosis in vivo. In addition, serum glutamate pyruvate transaminase (GPT) activity and hepatic lipid peroxides (LPO) concentration was significantly increased. The activity of serum glutamate oxaloacetate transaminase (GOT) showed an increased trend. The results suggest that fluoride induces apoptosis by elevating the oxidative stress-induced lipid peroxidation, causing mitochondrial dysfunction and further activating caspase-9 and caspase-3. The name of the laboratory where the work was carried out: Feed Science Institute in Zhejiang university     

Mouse strain-dependent caspase activation during acetaminophen hepatotoxicity does not result in apoptosis or modulation of inflammation

The mechanisms of acetaminophen (APAP)-mediated hepatic oncotic necrosis have been extensively characterized. However, it was recently demonstrated that fed CD-1 mice have a transient caspase activation which initiates apoptosis. To evaluate these findings in more detail, outbred (Swiss Webster, SW) and inbred (C57BL/6) mice were treated with APAP with or without pan-caspase inhibitor and compared to the apoptosis model of galactosamine (GalN)/endotoxin (ET). Fasted or fed APAP-treated C57BL/6 mice showed no evidence of caspase-3 processing or activity. Interestingly, a minor, temporary increase in caspase-3 processing and activity (150% above baseline) was observed after APAP treatment only in fed SW mice. The degree of caspase-3 activation in SW mice after APAP was minor compared to that observed in GalN/ET-treated mice (1600% above baseline). The pancaspase inhibitor attenuated caspase activation and resulted in increased APAP-induced injury (plasma ALT, necrosis scoring). The caspase inhibitor did not affect apoptosis because regardless of treatment only < 0.5% of hepatocytes showed consistent apoptotic morphology after APAP. In contrast, > 20% apoptotic cells were observed in GalN/ET-treated mice. Presence of the caspase inhibitor altered hepatic glutathione levels in SW mice, which could explain the exacerbation of injury. Additionally, the infiltration of hepatic neutrophils was not altered by the fed state of either mouse strain. Conclusion: Minor caspase-3 activation without apoptotic cell death can be observed only in fed mice of some outbred strains. These findings suggest that although the severity of APAP-induced liver injury varies between fed and fasted animals, the mechanism of cell death does not fundamentally change.     

鲨肝活性肽对对乙酰氨基酚致小鼠急性肝损伤的保护作用

目的探讨鲨肝活性肽(sHSS)对对乙酰氨基酚(AAP)致小鼠急性肝损伤的保护作用。方法用AAP(200 mg·kg^-1,ip)诱导小鼠急性肝损伤,用改良赖氏法测血清ALT和AST,通过光镜和电镜观察肝细胞显微和亚显微结构的变化,用流式细胞仪分析肝细胞凋亡,同时用RT-PCR方法分析Fas mRNA表达水平。结果sHSS 3.0和1.5 mg·kg^-1可显著降低肝损伤小鼠血清ALT和AST的水平;改善模型鼠肝组织细胞坏死及炎症反应;高剂量sHSS(3 mg·kg^-1)对肝线粒体具有保护作用,下调Fas mRNA的表达水平,并具有抗凋亡作用。结论sHSS对AAP诱导的肝损伤具有明显的保护作用,其机制可能与保护肝线粒体、抑制Fas基因表达及肝细胞凋亡有关。     

Toxicologic enhancement by a combination of drugs which deplete hepatic glutathione: Acetaminophen and doxorubicin (Adriamycin)

A number of chemicals are metabolized to reactive and toxic intermediates. Some reactive metabolites are detoxified by conjugation with glutathione (GSH). When endogenous GSH levels are low, or when excessive quantities of reactive metabolites are produced, the metabolite can bind to essential cellular macromolecules causing toxicity. Since both acetaminophen and doxorubicin (Adriamycin) deplete hepatic GSH, it was hypothesized that under certain conditions, doxorubicin might potentiate the hepatic centralobular necrosis characteristically induced by high doses or chronic administration of acetaminophen. Such an interaction could have clinical significance since acetaminophen is used in high doses as an analgesic for cancer patients being treated with doxorubicin. In male Swiss ICR mice, doxorubicin 20 mg/kg ip enhanced the toxicity of acetaminophen in doses of 250 to 500 mg/kg. Compared with acetaminophen alone, combination treatment produced a 15-fold increase in lethality, a dose-dependent, 90-fold increase in SGPT concentration, and an increase in the incidence and severity of hepatic centralobular necrosis. Dororubicin pretreatment caused both a 66% increase in the covalent binding of acetaminophen to hepatic protein at 4 hr, and a further depletion of hepatic GSH, 18% below that induced by acetaminophen alone. The timing of treatments to allow congruence of peak hepatic GSH depletion was necessary for toxicologic enhancement, suggesting a crucial protective role for GSH, although contributions from other toxicologic mechanisms cannot be excluded. Under certain clinical circumstances, this interaction could occur in cancer patients being treated with doxorubicin and acetaminophen.     

Differential protection with inhibitors of caspase-8 and caspase-3 in murine models of tumor necrosis factor and Fas receptor-mediated hepatocellular apoptosis

Excessive apoptosis has been implicated in a number of acute and chronic human diseases. The activation of caspases has been shown to be critical for the apoptotic process. The objective of this investigation was to evaluate the beneficial effects and mechanism of action of the caspase-8 inhibitor IETD-CHO and the caspase-3 inhibitor DEVD-CHO against tumor necrosis factor (TNF)-induced hepatocellular apoptosis in vivo and compare these results to effects of the same inhibitors against Fas-induced apoptosis. Treatment of C3Heb/FeJ mice with 700 mg/kg galactosamine/100 microg/kg endotoxin induced parenchymal apoptosis (indicated by caspase-3 activation and morphology) and severe liver injury (indicated by the increase in plasma alanine aminotransferase activities and histology) at 7 h. Treatment with IETD-CHO or DEVD-CHO (10 mg/kg at 3, 4.5, and 5.5 h) significantly attenuated caspase-3 activation and liver injury. Western analysis showed that DEVD-CHO had no effect while IETD-CHO substantially reduced procaspase-3 and procaspase-9 processing. On the other hand, caspase-3 activation and liver injury by the anti-Fas antibody Jo-2 was completely prevented by a single dose of DEVD-CHO and, as previously shown, by IETD-CHO at 90 min. Both inhibitors prevented procaspase-3 and procaspase-9 processing. Thus, there are fundamental differences in the efficacy of caspase inhibitors in these two models. We conclude that Fas may rely exclusively on caspase-8 activation and mitochondria to activate caspase-3, which can process more procaspase-8 and thus propagate the amplification of the apoptotic signal. TNF can activate a similar signaling pathway. However, alternative signaling mechanisms seem to exist, which can compensate if the main pathway is blocked.     

Melatonin attenuates lipopolysaccharide (LPS)-induced apoptotic liver damage in D-galactosamine-sensitized mice

D-Galactosamine (GalN) depletes UTP primarily in liver, resulting in decreased RNA synthesis in hepatocytes. When given together with a sublethal dose of lipopolysaccharide (LPS), GalN highly sensitizes animals to produce apoptotic liver injury with severe hepatic congestion, resulting in rapid death. Melatonin is a cytokine modulator, antioxidant and anti-apoptotic agent. In the present study, we investigated the effect of melatonin on LPS-induced apoptotic liver damage in GalN-sensitized mice. Female CD-1 mice were intraperitoneally (i.p.) injected with melatonin (5.0mg/kg) 30min before GalN/LPS (700mg10microg/kg, i.p.), another two doses of melatonin (2.5mg/kg, i.p.) being administered 1 and 2h after GalN/LPS. Results showed that serum alanine aminotransferase (ALT) activities were markedly increased 8h after GalN/LPS treatment, massive hemorrhage being observed in histological sections of liver from GalN/LPS-treated mice. Melatonin significantly attenuated GalN/LPS-induced elevation of serum ALT. In parallel, melatonin distinctly improved GalN/LPS-induced congestion. Additional experiment showed that melatonin significantly attenuated GalN/LPS-induced hepatic apoptosis, measured by inhibition of caspase-3 activities and attenuation of DNA laddering. Furthermore, melatonin markedly increased hepatic Se-dependent glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) activities and attenuated hepatic glutathione (GSH) depletion in GalN/LPS-treated mice. Increases in serum tumor necrosis factor alpha (TNF-alpha), which were observed in GalN/LPS-treated mice, were significantly reduced by melatonin. However, melatonin had no effect on LPS-evoked nitric oxide production in GalN-sensitized mice. Taken together, these results indicate that melatonin protected against LPS-induced liver damage in GalN-sensitized mice through its strong ROS-scavenging, antiinflammatory and antiapoptotic effects.     

Effectiveness of metyrapone in the treatment of acetaminophen toxicity in mice

Metyrapone tartrate, 400 mg/kg i.p. raised the LD₅₀ for acetaminophen from 340 mg/kg i.p. to 540 mg/kg i.p. in fasting male Swiss white mice. The minimum protective dose of metyrapone was 200 mg/kg. Metyrapone was effective in preventing death when given up to 2 h after acetaminophen administration. The LD₅₀ for metyrapone tartrate was 760 mg/kg i.p. Metyrapone decreased or prevented acetaminophen induced hepatic damage measured either by histology or plasma glutamate pyruvate transaminase activity. Metyrapone tartrate, 400 mg/kg i.p., inbibited the severe liver glutathione depletion seen with acetaminophen alone. It is proposed that metyrapone protects mice from acetaminophen induced liver toxicity and death by inhibiting the oxidation of acetaminophen to a toxic intermediate.     

Liver toxicity and apoptosis: role of TGF-beta1, cytochrome c and the apoptosome

Transforming growth factor-β₁ (TGF-β₁), is involved in controlling liver size, by inducing apoptotic cell death in hepatocytes. However the mechanism by which TGF-β₁ induces caspase activation and cell death is unknown. Apoptosis can be initiated either by receptor-mediated (e.g. Fas/CD95) or non-receptor chemically mediated (stress-induced) processes. With Fas/CD95 receptor mediated cell death, a multi-protein complex (DISC) is assembled at the plasma membrane, which activates the downstream caspases and cell death. In stress-mediated apoptosis, a cytosolic DISC equivalent, the apoptosome is formed that activates the effector caspases. We have characterised this complex in THP.1 cells, and shown that this is a cytochrome c dependent process that induces the formation of an 700 kDa apoptosome caspase processing complex. This is formed by oligomerisation of apoptotic protease-activating factor 1 (Apaf-1), and recruitment and processing of caspase-9. We have now shown that TGF-β₁-induced apoptosis also occurs via the release of cytochrome c and the subsequent oligomerisation of Apaf-1 into an 700 kDa apoptosome complex. Our studies show that, even though TGF-β₁ induction of apoptosis is a receptor-mediated event, it operates through the mitochondrial/Apaf-1 caspase activation pathway that appears to act as a common execution pathway for many diverse apoptotic stimuli.     

Role of oxidative stress and apoptosis in cadmium induced thymic atrophy and splenomegaly in mice

Cadmium immunotoxicity in rodents is primarily characterized by marked thymic damage and splenomegaly. To understand the toxicity of Cd on lymphoid cells in vivo, a single dose of Cd as CdCl2 (1.8 mg/kg, i.p.) was administered to male BALB/c mice and cytotoxicity (MTT assay), oxidative stress indicators (glutathione, reactive oxygen species) and apoptotic markers (mitochondrial membrane potential, caspase-3 activity, phosphatidylserine externalization, apoptotic DNA, intranucleosomal DNA fragmentation) were assessed in thymic and splenic single cell suspensions, at various time intervals. Lowering of body weight gain and cellularity and a loss in cell viability was seen in the Cd treated mice. The earliest significant increase in ROS at 18 h, followed by mitochondrial membrane depolarization, caspase-3 activation and GSH depletion at 24h in spleen and later at 48 h in thymus, strongly implicate the possible involvement of ROS. A pronounced inhibition of cell proliferative response at 48 h and 72 h may also be linked to Cd induced apoptosis. The morphological alterations including thymic cortical cell depletion and an increase in red pulp with diminished white pulp in spleen were observed at 48 h and beyond. The splenic cells appeared more susceptible than thymus cells to the adverse effects of Cd. The present study, therefore, demonstrates potentiation of oxidative stress followed by mitochondrial-caspase dependent apoptotic pathway. This may, in part, be responsible for causing suppression of cell proliferative response, thymic atrophy and splenomegaly.     

Effect of amlodipine,lisinopril and allopurinol on acetaminophen-induced hepatotoxicity in rats

BackgroundExposure to chemotherapeutic agents such as acetaminophen may lead to serious liver injury. Calcium deregulation, angiotensin II production and xanthine oxidase activity are suggested to play mechanistic roles in such injury.ObjectiveThis study evaluates the possible protective effects of the calcium channel blocker amlodipine, the angiotensin converting enzyme inhibitor lisinopril, and the xanthine oxidase inhibitor allopurinol against experimental acetaminophen-induced hepatotoxicity, aiming to understand its underlying hepatotoxic mechanisms.Material and methodsAnimals were allocated into a normal control group, a acetaminophen hepatotoxicity control group (receiving a single oral dose of acetaminophen; 750 mg/kg/day), and four treatment groups receive N-acetylcysteine (300 mg/kg/day; a reference standard), amlodipine (10 mg/kg/day), lisinopril (20 mg/kg/day) and allopurinol (50 mg/kg/day) orally for 14 consecutive days prior to acetaminophen administration. Evaluation of hepatotoxicity was performed by the assessment of hepatocyte integrity markers (serum transaminases), oxidative stress markers (hepatic malondialdehyde, glutathione and catalase), and inflammatory markers (hepatic myeloperoxidase and nitrate/nitrite), in addition to a histopathological study.ResultsRats pre-treated with amlodipine, lisinopril or allopurinol showed significantly lower serum transaminases, significantly lower hepatic malondialdehyde, myeloperoxidase and nitrate/nitrite, as well as significantly higher hepatic glutathione and catalase levels, compared with acetaminophen control rats. Serum transaminases were normalized in the lisinopril treatment group, while hepatic myeloperoxidase was normalized in the all treatment groups. Histopathological evaluation strongly supported the results of biochemical estimations.ConclusionAmlodipine, lisinopril or allopurinol can protect against acetaminophen-induced hepatotoxicity, showing mechanistic roles of calcium channels, angiotensin converting enzyme and xanthine oxidase enzyme in the pathogenesis of hepatotoxicity induced by acetaminophen.     

Nonylphenol induces apoptosis via mitochondria- and Fas-l-mediated pathways in the liver of adult male rat

Nonylphenol (NP) is an environmental contaminant known to possess estrogenic properties. Humans are constantly exposed to NP by contaminated water and food products. In the present study we sought to investigate whether treatment with low doses of NP induces apoptosis in the liver of adult rats. Rats were administered with NP by oral gavage at the doses of 15,150 and 1500 μg/kg body weight per day for 45 days. Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assayed. Apoptosis-related proteins namely cytochrome c, caspase-3, caspase-8, caspase-9, Fas and Fas-l, and expression of bcl-2 mRNA and bax mRNA were examined in the liver. Levels of AST and ALT were increased in the treated rats. Western blot analysis revealed elevation in the levels of cytochrome c, caspase-3, caspase-8, caspase-9, Fas and Fas-l in the liver of NP-treated rats. Decreased expression of bcl-2 mRNA (anti-apoptotic) and increased expression of bax mRNA (apoptotic) were observed in the liver of treated rats. Increased localization of caspase-3 in the hepatocytes and DNA damage were observed in the liver of treated rat. It is concluded that NP induces apoptosis in liver involving both mitochondria-dependent and Fas–Fas-l pathways and thereby, leading to hepatic damage in rats.