The release of DNA into the plasma of mice following hepatic cell death by apoptosis and necrosis

The goal of these investigations was to measure levels of DNA in the plasma of mice following administration of hepatotoxic agents to induce apoptotic or necrotic cell death and determine any differences in the release of this marker depending upon death pathway. For this purpose, the effects of varying doses of anti-Fas, acetaminophen (APAP) or carbon tetrachloride (CCl₄) were assessed in normal mice. Plasma DNA was measured fluorometrically by the dye PicoGreen while lactate dehydrogenase (LDH) and caspase 3, other molecules released with cell injury or death, were measured by enzymatic assays. Histology was used to assess the occurrence of apoptosis or necrosis. Results of these experiments indicate that increased blood DNA levels occurred with all three agents and were highest with anti-Fas and CCl₄; caspase 3 levels were much higher with anti-Fas than the other agents. Histological examination confirmed the predominance of apoptotic death with anti-Fas and necrotic death with APAP and CCl₄. These results indicate that increased blood DNA is common in hepatotoxic injury and is a feature of both apoptotic and necrotic death.     

Interdigital cell death can occur through a necrotic and caspase-independent pathway.

Programmed cell death in animals is usually associated with apoptotic morphology and requires caspase activation. Necrosis and caspase-independent cell death have been reported, but mostly in experimental conditions that lead some to question their existence it in vivo. Loss of interdigital cells in the mouse embryo, a paradigm of cell death during development [1], is known to include an apoptotic [2] and caspase-dependent [3] [4] mechanism. Here, we report that, when caspase activity was inhibited using drugs or when apoptosis was prevented genetically (using Hammertoe mutant mice, or mice homozygous for a mutation in the gene encoding APAF-1, a caspase-activating adaptor protein), interdigital cell death still occurred. This cell death was negative for the terminal-deoxynucleotidyl-mediated dUTP nick end-labelling (TUNEL) assay and there was no overall cell condensation. At the electron microscopy level, peculiar 'mottled' chromatin alterations and marked mitochondrial and membrane lesions, suggestive of classical necrotic cell death, were observed with no detectable phagocytosis and no local inflammatory response. Thus, in this developmental context, although caspase activity confers cell death with an apoptotic morphotype, in the absence of caspase activity an underlying mechanism independent of known caspases can also confer cell death, but with a necrotic morphotype. This cell death can go undetected when using apoptosis-specific methodology, and cannot be blocked by agents that act on caspases.     

A novel proanthocyanidin IH636 grape seed extract increases in vivo Bcl-XL expression and prevents acetaminophen-induced programmed and unprogrammed cell death in mouse liver.

Several molecular events in the apoptotic or necrotic death of hepatocytes induced by acetaminophen (AAP) now appear to be well defined. Recent studies also indicate that select expression of bcl-Xl is possibly modified during AAP-induced liver injury. The purpose of this study was several-fold: (i) to examine the hepatoprotective ability of short-term (3-day) and long-term (7-day) exposures of a grape seed proanthocyanidin extract (GSPE) on AAP-induced liver injury and animal lethality; (ii) to monitor effects of GSPE on one of the prime targets of AAP, i.e., hepatocellular genomic DNA and associated apoptotic and necrotic death; and (iii) to unravel changes in the pattern of expression of an antiapoptotic gene, bcl-Xl in the liver. In order to investigate these events, male ICR mice (30-40 g) were administered nontoxic doses of GSPE (3 or 7 days, 100 mg/kg, po), followed by hepatotoxic doses of AAP (400 and 500 mg/kg, ip), and sacrificed 24 h later. Serum was analyzed for alanine aminotransferase activity (ALT) and the liver for histopathological diagnosis of apoptosis/necrosis. The ability of AAP to promote apoptotic DNA fragmentation and its counteraction by GSPE in the liver was also evaluated quantitatively (by a sedimentation assay) and qualitatively (by agarose gel electrophoresis). Portions of livers were also subjected to Western blot analysis (27,000g fraction of liver homogenates) to examine the pattern of expression of cell death inhibitory gene bcl-Xl. Results indicate that 7-day GSPE preexposure induced dramatic protection and markedly decreased liver injury and animal lethality culminated by AAP, when compared to a short-term 3-day exposure. Abrogation of toxicity was also mirrored in DNA fragmentation. Histopathological evaluation of liver sections showed remarkable counteraction of AAP-toxicity by this novel GSPE and substantial inhibition of both apoptotic and necrotic liver cell death. Agarose gel electrophoresis revealed that 7-day GSPE preexposure prior to AAP administration completely blocked Ca(2+)/Mg(2+)-Ca(2+)/Mg(2+)-dependent-endonuclease-mediated ladder-like fragmentation of genomic DNA and significantly altered the bcl-Xl expression. The most dramatic changes observed in this study were: (i) substantial increase in the expression of bcl-Xl in the liver by 7-day GSPE exposure alone; (ii) significant modification bcl-Xl expression by AAP alone; and (iii) dramatic inhibition of AAP-induced modification of bcl-Xl (phosphorylation?) expression by GSPE. In summary, these observations demonstrate that GSPE preexposure may significantly attenuate AAP-induced hepatic DNA damage, apoptotic and necrotic cell death of liver cells, and, most remarkably, antagonize the influence of AAP-induced changes in bcl-Xl expression in vivo.     

Methylmercury and H(2)O(2) provoke lysosomal damage in human astrocytoma D384 cells followed by apoptosis

Methylmercury (MeHg) is a neurotoxic agent acting via diverse mechanisms, including oxidative stress. MeHg also induces astrocytic dysfunction, which can contribute to neuronal damage. The cellular effects of MeHg were investigated in human astrocytoma D384 cells, with special reference to the induction of oxidative-stress-related events. Lysosomal rupture was detected after short MeHg-exposure (1 μM, 1 h) in cells maintaining plasma membrane integrity. Disruption of lysosomes was also observed after hydrogen peroxide (H₂O₂) exposure (100 μM, 1 h), supporting the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress. The lysosomal alterations induced by MeHg and H₂O₂ preceded a decrease of the mitochondrial potential. At later time points, both toxic agents caused the appearance of cells with apoptotic morphology, chromatin condensation, and regular DNA fragmentation. However, MeHg and H₂O₂ stimulated divergent pathways, with caspases being activated only by H₂O₂. The caspase inhibitor z-VAD-fmk did not prevent DNA fragmentation induced by H₂O₂, suggesting that the formation of high-molecular-weight DNA fragments was caspase independent with both MeHg and H₂O₂. The data point to the possibility that lysosomal hydrolytic enzymes act as executor factors in D384 cell death induced by oxidative stress.     

Acetaminophen induced acute liver failure via oxidative stress and JNK activation: Protective role of taurine by the suppression of cytochrome P450 2E1

Abstract

The present study was carried out to investigate whether taurine plays any beneficial role in acetaminophen (APAP)-induced acute hepatotoxicity. APAP exposure increased the plasma levels of ALT, ALP, LDH, TNF-α and NO production. Moreover, APAP treatment reduced the glutathione level and antioxidant enzyme activities, increased lipid peroxidation and caused hepatic DNA fragmentation which ultimately leads to cellular necrosis. Also, incubation of hepatocytes with APAP reduced cell viability, enhanced ROS generation and increased CYP2E1 activity. APAP overdose caused injury in the hepatic tissue and hepatocytes via the upregulation of CYP2E1 and JNK. Taurine treatment was effective in counteracting APAP-induced hepatic damages, oxidative stress and cellular necrosis. Results indicate that APAP overdose caused hepatic injury due to its metabolism to hepatotoxic NAPQI (N-acetyl-p-benzoquinone imine), usually catalysed by CYP2E1, and via the direct activation of JNK-dependent cell death pathway. Taurine possesses prophylactic as well as therapeutic potentials against APAP-induced hepatic injury.     

Oxidative stress induces the expression of Fas and Fas ligand and apoptosis in murine intestinal epithelial cells

Intestinal epithelial cell function is compromised by local immune and inflammatory responses. In this study, we examined the possibility that intestinal epithelial cell injury occurs in the presence of activated inflammatory cells, such as neutrophils and macrophages, via production of reactive oxygen species (ROS). Following exposure to 50–150 μM H₂O₂, levels of mRNA and protein for Fas and, to a lesser degree, Fas-L were increased and intestinal epithelial cells underwent apoptosis. Treatment of H₂O₂-exposed cells with agonistic anti-Fas antibody, but not isotype control antibody, significantly enhanced apoptosis. Apoptosis was associated with the activation of caspase 8, while Z-IETD, an inhibitor of caspase 8, blocked apoptosis of H₂O₂-exposed intestinal epithelial cells. Thus, ROS induced Fas and Fas-L expression in association with intestinal epithelial cell apoptosis. These data support the hypothesis that, following exposure to oxidative stress, enterocytes are primed for cell death via Fas-mediated pathways.     

S-allyl cysteine prevents CCl(4)-induced acute liver injury in rats

Aged garlic extract (AGE) possesses multiple biological activities. We evaluated the protective effect of S-allyl cysteine (SAC), one of the organosulfur compounds of AGE, against carbon tetrachloride (CCl₄)-induced acute liver injury in rats. SAC was administrated intraperitoneally (50-200 mg/kg). SAC significantly suppressed the increases of plasma ALT and LDH levels. SAC also attenuated histological liver damage. CCl₄ administration induced lipid peroxidation accompanied by increases in the plasma malondialdehyde and hepatic 4-hydroxy-2-nonenal levels, and SAC dose-dependently attenuated these increases. The hepatic total level of hydroxyoctadecadienoic acid (HODE), a new oxidative stress biomarker, was closely correlated with the amount of liver damage. These results suggest that SAC decreased CCl₄-induced liver injury by attenuation of oxidative stress, and may be a better therapeutic tool for chronic liver disease.     

柔木丹改善小鼠肝纤维化的TGF-β1/Smad4信号通路机制*

目的:探讨柔木丹(RMD)对改善CCl₄诱导的小鼠肝纤维化的TGF-β1/果蝇抗生物皮肤生长因子蛋白家族4号因子(Smad4)信号通路机制。方法:雄性BALB/c小鼠随机分为空白对照组、模型组、RMD治疗组(n=11)。腹腔注射CCl₄诱导小鼠肝纤维化模型,模型及RMD治疗组小鼠腹腔注射20 % CCl₄(CCl₄∶橄榄油=1∶4),注射量为2.5 ml/kg,空白对照组以同样方法注射等量橄榄油,每周2次;第2周起调整模型及RMD治疗组小鼠CCl₄腹腔注射量为5 ml/kg(空白对照组注射等量橄榄油),每周2次。成模后,RMD治疗组小鼠使用RMD灌胃给药(6.2 g/(kg·d);空白对照组、模型组使用等量的水灌胃),模型及RMD治疗组小鼠继续腹腔注射20 % CCl₄,注射量为1.5 ml/kg(空白对照组注射等量橄榄油),每周1次,持续3 周。采取各组小鼠血清样本检测谷丙转氨酶(ALT)、谷草转氨酶(AST)活性;采取各组小鼠肝组织样本使用HE、Masson、原位杂交、免疫组织化学染色、Western blot、Q-PCR等方法进行检测。结果:与正常组相比,CCl₄造模5 周后,模型组小鼠肝脏纤维化病理特征明显。与模型组相比,RMD治疗3 周,治疗组小鼠肝组织病理学改变减轻,小鼠的肝脏指数(P＜0.01)、血清中的ALT(P＜ 0.01)、AST(P＜0.01)活性、肝组织中羟脯氨酸的含量(P＜0.05)均降低;Ⅰ型胶原(Collagen Ⅰ,P＜0.01)、Ⅲ型胶原(Collagen Ⅲ,P＜0.01)表达减少,胶原沉积减少;肝组织中TGF-β1(P＜0.05)和α-SMA(P＜0.05)表达均降低;肝组织中Smad4阳性表达区域缩小、表达强度降低。结论:RMD通过抑制TGF-β1/Smad4通路信号转导,减少胶原沉积,进而发挥抗小鼠肝纤维化的作用。     

A possible intermediate step during apoptotic execution

Many proteases are known to be involved in apoptosis. Among them, interleukin-1beta converting enzyme (ICE) and its family proteases, which are called caspases, play critical roles in the execution stage of apoptosis. We previously reported that a proteasome-inhibitor, benzyloxycarbonyl Leu-Leu-leucinal (ZLLLal), induced apoptosis in MOLT-4 cells. In the present study, in order to analyze the detailed mechanism of ZLLLal-induced apoptosis, we examined the effect of a caspase-inhibitor, acetyl(Ac)-Tyr-Val-Ala-Asp-chloromethyl ketone (AcYVADcmk), on ZLLLal-induced apoptosis in the cells. Agarose gel electrophoresis revealed that low concentrations of AcYVADcmk efficiently suppressed apoptotic DNA fragmentation. However, the cells presented morphology different from normal, apoptotic or necrotic cells, although DNA fragmentation was suppressed. The same examination was performed on the cells with anti-Fas antibody-induced apoptosis, and the same results were obtained. Some cells with a similar morphology were found even without the caspase-inhibitor in the early stage of anti-Fas antibody-induced physiological apoptosis. In addition, apoptotic cascade was reactivated by washing out the caspase inhibitor from the DNA degradation-suppressed cells. Therefore, this newly found morphological feature shows the presence of a step prior to caspase activation in the cells, and this is the first report presenting the pre-caspase-activated step in the apoptotic cascade.     

NF-kappaB protects macrophages from lipopolysaccharide-induced cell death: the role of caspase 8 and receptor-interacting protein

Macrophages play a pivotal role in the pathogenesis of a variety of diseases. These studies were performed to characterize the mechanisms by which Toll-like receptor 4 (TLR4)-mediated NF-kappaB activation promotes resistance to cell death in macrophages. When NF-kappaB activation was inhibited by a super-repressor, IkappaBalpha, the TLR4 ligand lipopolysaccharide induced the activation of caspase 8, the loss of mitochondrial transmembrane potential (DeltaPsim), and apoptotic cell death in macrophages. The inhibition of caspase 8 activation suppressed DNA fragmentation but failed to protect macrophages against the loss of DeltaPsim and resulted in necrotic cell death. In contrast, the reduction of receptor-interacting protein 1 suppressed the loss of DeltaPsim and inhibited apoptotic cell death. Further, when caspase 8 activation was suppressed, the knock down of receptor-interacting protein inhibited the loss of DeltaPsim and necrotic cell death. These observations demonstrate that following TLR4 ligation by lipopolysaccharide, NF-kappaB is a critical determinant of macrophage life or death, whereas caspase 8 determines the pathway employed.     

Caspase inhibition in apoptotic T cells triggers necrotic cell death depending on the cell type and the proapoptotic stimulus

CD95 (Fas/Apo-1) triggers apoptotic cell death via a caspase-dependent pathway. Inhibition of caspase activation blocks proapoptotic signaling and thus, prevents execution of apoptosis. Besides induction of apoptotic cell death, CD95 has been reported to trigger necrotic cell death in susceptible cells. In this study, we investigated the interplay between apoptotic and necrotic cell death signaling in T cells. Using the agonistic CD95 antibody, 7C11, we found that caspase inhibition mediated by the pancaspase inhibitor, zVAD-fmk, prevented CD95-triggered cell death in Jurkat T cells but not in A3.01 T cells, although typical hallmarks of apoptosis, such as DNA fragmentation or caspase activation were blocked. Moreover, the caspase-independent cell death in A3.01 cells exhibited typical signs of necrosis as detected by a rapid loss of cell membrane integrity and could be prevented by treatment with the radical scavenger butylated hydroxyanisole (BHA). Similar to CD95-induced cell death, apoptosis triggered by the DNA topoisomerase inhibitors, camptothecin or etoposide was shifted to necrosis when capsase activation was inhibited. In contrast to this, ZVAD was fully protective when apoptosis was triggered by the serpase inhibitor, Nalpha-tosyl-phenyl-chloromethyl ketone (TPCK). TPCK was not protective when administered to anti-CD95/ZVAD-treated A3.01 cells, indicating that TPCK does not possess anti-necrotic activity but fails to activate the necrotic death pathway. Our findings show (a) that caspase inhibition does not always protect apoptotic T cells from dying but merely activates a caspase-independent mode of cell death that results in necrosis and (b) that the caspase-inhibitor-induced shift from apoptotic to necrotic cell death is dependent on the cell type and the proapoptotic stimulus.     

Phorbol ester (12-O-tetradecanoylphorbol 13-acetate) prevents ornithine decarboxylase inhibition and apoptosis in L1210 leukemic cells exposed to TGF-β1

Previous studies have shown that growth suppression and apoptosis of leukemic cells exposed to TGF-β₁ is associated with the inhibition of ornithine decarboxylase (ODC) — the key enzyme of polyamine pathway. The aim of the present study was to evaluate the influence of 12-O-tetradecanoylphorbol 13-acetate (TPA) — a potent ODC inducer on antiproliferative and apoptotic effects of TGF-β₁ in L1210 leukemic cells. Cells were incubated in 2%FCS/RPMI1640 medium, supplemented with TGF-β₁ (2 ng/ml), TPA (100 ng/ml) or -difluoromethyl-ornithine (DFMO) (5 mM). Cell proliferation, apoptosis and necrosis were evaluated using [methyl-³H] thymidine, electron microscopy, electrophoresis of DNA and trypan blue exclusion. Expression and activity of ODC were determinated by RT-PCR and measurement of ¹⁴CO₂ release from L-1-¹⁴C ornithine, respectively. TGF-β₁ inhibited proliferation and induced apoptotic and necrotic cell death in L1210 leukemic cells. The above effects were associated with the inhibition of ODC expression and activity, measured 2 and 4 hr after TGF-β₁ administration, respectively. The presence of DFMO, an irreversible inhibitor of ODC, led to apoptotic fragmentation of DNA, similar to that observed in TGF-β₁-treated cultures. Administration of TPA simultaneously with TGF-β₁ significantly reduced antiproliferative, apoptotic and necrotic effects of TGF-β₁, and prevented its inhibitory action on ODC expression and activity. It is concluded that: down-regulation of ODC expression may be one of the early events associated with TGF-β₁-evoked suppression of growth and apoptosis; ODC is involved in the mechanism of protective action of TPA on TGF-β₁-related growth inhibition of L1210 leukemic cells.     

虫草素对四氯化碳所致小鼠急性肝损伤的保护作用

本文探究蛹虫草活性成分虫草素对四氯化碳（CCl₄）造成的小鼠急性肝损伤的保护作用及其分子机制。首先建立四氯化碳致小鼠急性肝损伤的动物模型,通过检测血清生化指标、肝功指标的变化及HE染色观察组织切片病理的病变情况,评价虫草素的保肝效果,进一步通过Western blot检测虫草素能否通过激活Nrf-2/Keap1信号通路及其下游抗氧化因子（HO-1、NQO-1）的表达来提高机体抗氧化损伤能力以及抑制炎症因子（TNFα、TNFβ、IL-6、IL-10）的表达。对比模型组结果显示,虫草素能极显著降低（P<0.01）小鼠血清中ALT、AST及肝脏中MDA水平,并能极显著提高肝脏中SOD水平（P<0.01）;HE染色结果显示虫草素能有效降低改善受损肝组织中的炎细胞浸润及纤维组织增生;Western blot结果表明虫草素能够通过激活Nrf-2信号通路,促进下游抗氧化因子及抗炎因子的表达,从而降低炎症反应。虫草素对CCl₄致小鼠急性肝损伤具有一定的保护作用,其机制与Nrf-2信号通路相关,实验结果为后续蛹虫草及虫草素的开发应用奠定基础。     

Death receptor-induced apoptotic and necrotic cell death: differential role of caspases and mitochondria

In L929sAhFas cells, tumor necrosis factor (TNF) leads to necrotic cell death, whereas agonistic anti-Fas antibodies elicit apoptotic cell death. Apoptosis, but not necrosis, is correlated with a rapid externalization of phosphatidylserine and the appearance of a hypoploid population. During necrosis no cytosolic and organelle-associated active caspase-3 and -7 fragments are detectable. The necrotic process does not involve proteolytic generation of truncated Bid; moreover, no mitochondrial release of cytochrome c is observed. Bcl-2 overexpression slows down the onset of necrotic cell death. In the case of apoptosis, active caspases are released to the culture supernatant, coinciding with the release of lactate dehydrogenase. Following necrosis, mainly unprocessed forms of caspases are released. Both TNF-induced necrosis and necrosis induced by anti-Fas in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone are prevented by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone and the oxygen radical scavenger butylated hydroxyanisole, while Fas-induced apoptosis is not affected.     

Endonuclease G: a mitochondrial protein released in apoptosis and involved in caspase-independent DNA degradation.

A hallmark of apoptosis is the fragmentation of nuclear DNA. Although this activity involves the caspase-3-dependent DNAse CAD (caspase-activated DNAse), evidence exists that DNA fragmentation can occur independently of caspase activity. Here we report on the ability of truncated Bid (tBid) to induce the release of a DNAse activity from mitochondria. This DNAse activity was identified by mass spectrometry as endonuclease G, an abundant 30 kDa protein released from mitochondria under apoptotic conditions. No tBid-induced endonuclease G release could be observed in mitochondria from Bcl-2-transgenic mice. The in vivo occurrence of endonuclease G release from mitochondria during apoptosis was confirmed in the liver from mice injected with agonistic anti-Fas antibody and is completely prevented in Bcl-2 transgenic mice. These data indicate that endonuclease G may be involved in CAD-independent DNA fragmentation during cell death pathways in which truncated Bid is generated.     

Lipid Peroxidation and Biogenic Aldehydes: From the Identification of 4-Hydroxynonenal to Further Achievements in Biopathology

The formation, reactivity and toxicity of aldehydes originating from lipid peroxidation of cellular membranes are reviewed. Very reactive aldehydes, namely 4-hydroxyalkenals, were first shown to be formed in autoxidizing chemical systems. It was subsequently shown that 4-hydroxyalkenals are formed in biological conditions, i.e. during lipid peroxidation of liver microsomes incubated in the NADPH-Fe systems. Our studies carried out in collaboration with Hermann Esterbauer which led to the identification of 4-hydroxynonenal (4-HNE) are reported. 4-HNE was the most cytotoxic aldehyde and was then assumed as a model molecule of oxidative stress. Many other aldehydes (alkanals, alk-2-enals and dicarbonyl compounds) were then identified in peroxidizing liver microsomes or hepatocytes. The in vivo formation of aldehydes in liver of animals intoxicated with agents that promote lipid peroxidation was shown in further studies. In a first study, evidence was forwarded for aldehydes (very likely alkenals) bound to liver micro-somal proteins of CCl₄ or BrCCl₃-intoxicated rats. In a second study, 4-HNE and a number of other aldehydes (alkanals and alkenals) were identified in the free (non-protein bound) form in liver extracts from bromoben-zene or ally-1 alcohol-poisoned mice. The detection of free 4-HNE in the liver of CCl₄ or BrCCl₃-poisoned animals was obtained with the use of an electrochemical detector, which greatly increased the sensitivity of the HPLC method. Furthermore, membrane phospho-lipids bearing carbonyl groups were demonstrated in both in vitro (incubation of microsomes with NADPH-Fe) and in vivo (CCl₄ or BrCCl₃ intoxication) conditions. Finally, the results concerned with the histochemical detection of lipid peroxidation are reported. The methods used were based on the detection of lipid peroxidation-derived carbonyls. Very good results were obtained with the use of fluorescent reagents for carbonyls, in particular with 3-hydroxy-2-naphtoic acid hydrazide (NAH) and analysis with confocal scanning fluorescence microscopy with image video analysis. The significance of formation of toxic aldehydes in biological membranes is discussed.     

MRI study of the inhibitory effect of new spin traps on in vivo CCl4-induced hepatotoxicity in rats

Acute CCl₄ hepatotoxicity is thought to occur as a result of free generated from the metabolism of CCl₄ in the liver. With the use of MRI it is possible to detect in vivo a CCl₄-induced edematous region surrounding the major branch of the hepatic portal vein in the right lobe. Inhibition of the CCl₄-induced response has been obtained by pretreatment with the spin trap, PBN, 30 min prior to CCl₄ exposure. The inhibitory effect of two new traps, M₃PO or methyl-DMPO, and PhM₂PO or phenyl-DMPO, on in vivo CCl₄-induced acute hepatotoxicity was investigated. Both PhM₂PO and M₃PO were found to inhibit the CCl₄-induced response at lower concentrations (0.35 M/kg body weight) than PBN (0.70 M/kg body weight). However, both M₃PO and PhM₂PO were also found to induce and edematous response at the same concentrations used for the PBN studies (0.70 M/kg body weight). PhM₂PO, at a concentration of 0.35 M/kg body weight, was 93% as efficient as PBN, at a concentration of 0.70 M/kg body weight; whereas M₃PO, at a concentration of 0.35 M/kg, was 89% as efficient as PBN at 0.70 M/kg body weight. Electron micrographs were obtained from small liver sections taken in proximity to the major branch of the hepatic portal veins of all treatment groups. The electron microscopy investigations support the MRI findings.     

CpG DNA-mediated induction of acute liver injury in D-galactosamine-sensitized mice: the mitochondrial apoptotic pathway-dependent death of hepatocytes

Unmethylated CpG motifs present in bacterial DNA (CpG DNA) induce innate inflammatory responses, including rapid induction of proinflammatory cytokines. Although innate inflammatory responses induced by CpG DNA and other pathogen-associated molecular patterns are essential for the eradication of infectious microorganisms, excessive activation of innate immunity is detrimental to the host. In this study, we demonstrate that CpG DNA, but not control non-CpG DNA, induces a fulminant liver failure with subsequent shock-mediated death by promoting massive apoptotic death of hepatocytes in D-galactosamine (D-GalN)-sensitized mice. Inhibition of mitochondrial membrane permeability transition pore opening or caspase 9 activity in vivo protects D-GalN-sensitized mice from the CpG DNA-mediated liver injury and death. CpG DNA enhanced production of proinflammatory cytokines in D-GalN-sensitized mice via a TLR9/MyD88-dependent pathway. In addition, CpG DNA failed to induce massive hepatocyte apoptosis and subsequent fulminant liver failure and death in D-GalN-sensitized mice that lack TLR9, MyD88, tumor necrosis factor (TNF)-alpha, or TNF receptor I but not interleukin-6 or -12p40. Taken together, our results provide direct evidence that CpG DNA induces a severe acute liver injury and shock-mediated death through the mitochondrial apoptotic pathway-dependent death of hepatocytes caused by an enhanced production of TNF-alpha through a TLR9/MyD88 signaling pathway in D-GalN-sensitized mice.     

Photodynamic activity of a new sensitizer derived from porphyrin-C60 dyad and its biological consequences in a human carcinoma cell line

The photokilling activity of a porphyrin-C₆₀ (P-C₆₀) dyad was evaluated on a Hep-2 human larynx-carcinoma cell line. This study represents the first evaluation of a dyad, with high capacity to form a photoinduced charge-separated state, to act as agent to inactivate cells by photodynamic therapy (PDT). Cell treatment was carried out with 1 μM P-C₆₀ incorporated into liposomal vesicles. No dark cytotoxicity was observed using 1 μM P-C₆₀ concentration and during long incubation time (24 h). The uptake of sensitizer into Hep-2 was studied at different times of incubation. Under these conditions, a value of 1.5 nmol/10⁶ cells was found after 4 h of incubation showing practically no change even after 24 h. The cell survival after irradiation of the cells with visible light was dependent upon light exposure level. A high photocytotoxic effect was observed for P-C₆₀, which inactivated 80% of the cells after 54 J/cm² of irradiation. Moreover, the dyad kept a high photoactivity even under argon atmosphere. Thus, depending on the microenviroment where the sensitizer is localized, this compound could produce a biological photodamage through either a ¹O₂-mediated photoreaction process or a free radical mechanism under low oxygen concentration.

The mechanism of cell death was analyzed by Hoechst-33258, toluidine blue staining, TUNEL and DNA fragmentation. Cell cultures treated for 24 h with P-C₆₀ and irradiated with a dose of 54 J/cm² showed a great amount of apoptotic cells (58%). Moreover, changes in cell morphology were analyzed using fluorescence microscopy with Hoechst-33258 under low oxygen concentration. Under this anaerobic condition, necrotic cellular death predominated on apoptotic pathway. There were more apoptotic cells under air irradiation condition than under argon irradiation condition. To determine the apoptotic pathway, caspase-3 activation was studied by caspase-3 activity detection kits. The last results showed that P-C₆₀ induced apoptosis by caspase-3-dependent pathway. These results indicated that molecular dyad, which can form a photoinduced charge-separated state, is a promising model for phototherapeutic agents and they have potential application in cell inactivation by PDT.