Apoptotic Lesions Research Articles

Spatiotemporal evolution of apoptotic neurodegeneration following traumatic injury to the developing rat brain

Closed head injury to the developing rat brain causes an acute excitotoxic lesion and axonal disruption at the impact site followed by a delayed pattern of apoptotic damage at various distant sites. Using an electromagnetic impact device to deliver a precisely controlled degree of mechanical deformation to the P7 infant rat skull, we studied the distribution of distant apoptotic lesions and the sequence and time course with which these lesions evolve following relatively mild closed head injury. The first major wave of apoptotic neurodegeneration occurred at 8 h postimpact in the retrosplenial cortex and pre- and parasubiculum. The next major wave occurred in the 16- to 24-h interval and was localized to the anterior thalamic nuclei. A third wave was detected at 36 to 48 h in the mammillary nuclei. We propose that the first and second waves were triggered by injury to a specific fiber tract, the corpus callosum/cingulum bundle that conveys reciprocal connections between the anterior thalamic nuclei and retrosplenial/pre- and parasubicular neurons. This fiber tract passes through a zone of maximum mechanical strain, as measured by tagged MRI. The third wave affecting mammillary neurons occurred because the principal synaptic targets of these neurons are the anterior thalamic neurons that were destroyed in the second wave of degeneration. Prevention of these apoptotic waves of brain damage is a realistic goal in view of the long delay between the impact event and onset of apoptotic degeneration.

Ribosome-inactivating proteins: progress and problems.

Ribosome-inactivating proteins (RIPs), mostly from plants, are enzymes which depurinate rRNA, thus inhibiting protein synthesis. They also depurinate other polynucleotide substrates. The biological activity of RIPs is not completely clarified, and sometimes independent of the inhibition of protein synthesis. There are differences in the cytotoxicity of RIPs and, consequently, in their toxicity to animals. Some RIPs are potent toxins, the best known being ricin, a potential biological weapon. New toxins have recently been identified. RIPs cause apoptotic and necrotic lesions, and induce production of cytokines causing inflammation. RIPs are potentially useful in agriculture and medicine because (i) they have antiviral activity and (ii) they are used for the preparation of conjugates with antibodies ('immunotoxins') or other carriers, rendering them specifically toxic to the cell target of the carrier, which may be helpful in therapy. The distribution, mechanism of action and role in nature of RIPs are not completely understood, and we can expect several future developments in their practical application.

Imaging apoptosis in vivo using 124I-annexin V and PET

Abnormal regulation of apoptosis is an important pathogenic mechanism in many diseases including cancer. Techniques to assess apoptosis in living organisms are limited and, in the case of solid organs, restricted to histological examination of biopsy samples. We investigated the use of 124I-annexin V, which binds to phosphatidylserine (PS) on the surface of apoptotic cells, as a potential positron emission tomography (PET) radioligand for the noninvasive measurement of apoptosis in vivo. Annexin V and a similar-sized protein, ovalbumin, were directly labelled with 124I. We report the validation of 124I-annexin V in vitro and in an animal model of liver apoptosis that has not previously been used to test iodinated annexin V. Also, for the first time, we report metabolite analysis of 124I-annexin V and the correlation of 124I-annexin V uptake with apoptotic density (AD). Sixfold more 124I-annexin V was associated with Jurkat cells after apoptosis induction, indicating that PS binding by annexin V was preserved after iodination. 124I-ovalbumin did not demonstrate increased uptake in apoptotic cells. In normal BDF-1 mice, the radioligand was rapidly cleared, but some in vivo dehalogenation resulted in the accumulation of activity in the thyroid and stomach content. PET images demonstrated uptake of 124I-annexin V but not 124I-ovalbumin in apoptotic liver lesions. In vivo 124I-annexin V uptake, derived from PET images, correlated with histologically derived AD ( r=.86, P<.01). These results demonstrate that 124I-annexin V is localised to anti-Fas-induced apoptosis, in contrast to 124I-ovalbumin, which did not show preferential uptake in the apoptotic liver.

Functional comparison of annexin V analogues labeled indirectly and directly with iodine-124

We are interested in imaging cell death in vivo using annexin V radiolabeled with 124I. In this study, [ 124I]4IB-annexin V and [ 124I]4IB-ovalbumin were made using [ 124I] N-hydroxysuccinimidyl-4-iodobenzoate prepared by iododestannylation of N-hydroxysuccinimidyl-4-(tributylstannyl)benzoate. [ 124I]4IB-annexin V binds to phosphatidylserine-coated microtiter plates and apoptotic Jurkat cells and accumulates in hepatic apoptotic lesions in mice treated with anti-Fas antibody, while [ 124I]4IB-ovalbumin does not. In comparison with 124I-annexin V, [ 124I]4IB-annexin V has a higher rate of binding to phosphatidylserine in vitro, a higher kidney and urine uptake, a lower thyroid and stomach content uptake, greater plasma stability and a lower rate of plasma clearance. Binding of radioactivity to apoptotic cells relative to normal cells in vitro and in vivo appears to be lower for [ 124I]4IB-annexin V than for 124I-annexin V.

Reply

Our study1 referred to by Professor Pender describes a very early stage in the formation of new multiple sclerosis (MS) lesions characterized by the presence of broad areas of palely staining (but still largely intact) myelin sheaths, containing apoptotic oligodendrocytes and activated microglia, but few or no myelin phagocytes or lymphocytes. The absence of T lymphocytes in the immediate vicinity of apoptotic oligodendrocytes is especially significant, because such changes have not been reported in any form of the animal model, experimental allergic encephalomyelitis (EAE). As noted by ourselves and Dr Trapp,2 this raises the possibility of important differences in the pathogenesis of these two diseases. Professor Pender suggests that steroid administration shortly before death may have cleared the central nervous system (CNS) of T cells; this possibility can be ruled out first by the fact that not all seven patients (in whom apoptotic lesions were identified) received such treatment, and second by the presence of numerous lymphocytes in zones of active phagocytosis adjacent to or contiguous with areas exhibiting prominent oligodendrocyte apoptosis in each of the 10 apoptotic lesions described. In the patient with the 17-hour symptomatic lesion, we described an absence of lymphocytes in zones of oligodendrocyte apoptosis, but relatively numerous CD4, CD8, and especially CD45RO+ T cells together with myelin phagocytes elsewhere in the lesion.1 Also as noted in our study, the findings were consistent in each of the nine other apoptotic lesions described. Our findings argue strongly against direct T-cell–mediated destruction of oligodendrocytes in early MS lesions. Rather, they suggest that dissolution of myelin by macrophages is largely or entirely scavenging activity directed against dead myelin sheaths, that is, plasma membrane of apoptotic oligodendrocytes. As noted by Professor Pender, however, our results do not rule out antibody or other humoral factors as effectors of oligodendrocyte apoptosis in early MS lesions. John W. Prineas MB, BS*, Michael H. Barnett MB, BS*, * Department of Medicine, The University of Sydney, Sydney, Australia.

Plasma membrane alterations during apoptosis: role in corpse clearance.

Apoptosis is a program of cellular self-destruction culminating in the clearance of cell corpses by neighboring macrophages. Studies in recent years have served to characterize a number of structural and molecular plasma membrane alterations that act in concert to mediate efficient engulfment of cell corpses. Hence, "eat me" signals, including the anionic phospholipid phosphatidylserine (PS) and its oxidized counterpart, PS-OX, as well as the PS-binding protein, annexin I, are exposed on the surface of effete cells and function to mediate engulfment by neighboring phagocytic cells. Plasma membrane blebbing (zeiosis), a common feature of the apoptotic program, provides a structural context for the exposition of recognition signals insofar as PS molecules aggregate on the surface of these membrane protrusions. Apoptotic cells also secrete chemotactic factors ("seek me" signals), such as the phospholipid lysophosphatidylcholine, that recruit phagocytes to the site of the apoptotic lesion. Taken together, these events serve to mediate the disposal of effete cells prior to their necrotic disintegration, thus preventing the inflammation and tissue scarring that would otherwise ensue.

Loss of ATM sensitizes against O 6-methylguanine triggered apoptosis, SCEs and chromosomal aberrations

A critical pre-cytotoxic and -apoptotic DNA lesion induced by methylating carcinogens and chemotherapeutic drugs is O 6-methylguanine (O 6MeG). The mechanism by which O 6MeG causes cell death via apoptosis is only partially understood. The current model ascribes a role to DNA replication and mismatch repair, which converts O 6MeG into a critical distal lesion (presumably a DNA double-strand break) that is finally responsible for genotoxicity and apoptosis. Here we analysed whether the PI3-like kinase ATM is involved in this process. ATM is a major player in recognizing and signaling DNA breaks, but most reports are limited to ionizing radiation. Comparing mouse ATM knockout fibroblasts (ATM−/−) with the corresponding wild-type (ATM+/+) we show that ATM−/− cells are hypersensitive to the cytotoxic and apoptosis-inducing effect of N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). Inhibition of O 6-methylguanine-DNA methyltransferase (MGMT) activity by O 6-benzylguanine enhanced cell killing whereas the increase of MGMT activity by transfection with an expression vector provoked MNNG resistance. This was more pronounced in ATM−/− than in ATM+/+ cells, suggesting that O 6MeG is responsible, at least in part, for increased MNNG sensitivity of ATM−/− cells. Cytogenetic studies showed that MNNG-induced sister-chromatid exchange frequencies were the same in ATM−/− and ATM+/+ cells in the first mitoses following treatment, but higher in ATM−/− cells than in the wild-type in the second post-treatment mitoses, when MGMT was depleted. Also, a significant higher frequency of MNNG-induced chromosomal aberrations was observed in ATM−/− than in ATM+/+ cells when analysed at a late recovery time, which is consistent with O 6MeG being the inducing lesion. In summary, we conclude that ATM is not only involved in resistance to ionizing radiation but also to methylating agents, playing a role in the repair of secondary DNA damage generated from O 6MeG lesions. The data also show that ATM is not required for activating the apoptotic pathway in response to O 6MeG since ATM−/− cells are able to undergo apoptosis with high frequency.

Platelet storage under in vitro condition is associated with calcium-dependent apoptosis-like lesions and novel reorganization in platelet cytoskeleton

Platelets are cleared from circulation after a life span of 8–10 days. The molecular mechanisms underlying platelet senescence remain poorly characterized. Here we report that, progressive functional impairment in the platelets incubated in vitro in a plasma-free isotonic medium for up to 24 h at 37 °C is associated with release of cytochrome c from platelet mitochondria and cleavage of procaspase-9, but without evidence of caspase-3 activation. Concomitantly, there was proteolysis of survival proteins like focal adhesion kinase, Src, gelsolin, and specific cytoskeleton-associated peptides, in a manner regulated by extracellular calcium and calpain activity. Cytoskeleton played a critical role as evidenced from the association of these proteins and their degradation products, as well as procaspase-3 and the actin regulatory small GTPase, CDC42Hs, with the cytoskeleton of the stored platelets. The cytoskeletal enrichment with specific proteins was not associated with increase in the content of F-actin and was cytochalasin-resistant, thus signifying a novel mechanism of interaction of the translocating proteins with the pre-existing cytoskeleton. There was progressive exposure of phosphatidylserine on the outer leaflet of platelet membrane and specific electron microscopic changes suggestive of apoptotic lesions. Based on these observations we discuss the caspase-independent but calpain-mediated signaling events in the stored platelets resembling the features of apoptosis in the nucleated cells.

Coxsackievirus B3 Induces Apoptosis in the Early Phase of Murine Myocarditis: A Comparative Analysis of Cardiovirulent and Noncardiovirulent Strains

Objective: To investigate the relationship between enteroviral infection and myocardial tissue apoptosis during the development of viral myocarditis in a murine model. Methods: C3H/HeJ mice were inoculated with two strains of coxsackievirus B3, specifically CVB3 (cardiovirulent Nancy strain) and CVB3/0 (noncardiovirulent strain). Mice were sacrificed at 4, 7 and 10 days postinfection (p.i.). Hearts were removed, and plaque assays and RT-PCR were performed to detect the presence of viruses. Myocardial tissue sections were additionally evaluated by hematoxylin and eosin staining for inflammation, VP1 and Bax immunohistochemical staining for detection of virus and Bax expression, and TUNEL and Apostain for localization of apoptosis. Results: CVB3 replicated to significantly higher titers than CVB3/0 at all time points. Histopathological analyses revealed significant inflammatory changes at all time points in CVB3-infected mice, in contrast to minimal changes in CVB3/0-infected mice. TUNEL and Apostain assays of myocardial tissues from mice infected with CVB3 disclosed maximum apoptotic lesions at 4 days p.i. and to a lesser extent at 7 and 10 days p.i. Moreover, CVB3-infected myocardial tissues displayed significantly enhanced Bax expression at 4 days p.i., and lesions overlapped with VP1-stained areas. Conclusions: These data indicate that (1) the cardiovirulent strain CVB3 induces more severe inflammation and apoptosis than the noncardiovirulent CVB3/0 strain, (2) viral replication is localized in inflammatory and apoptotic lesions in myocardial tissues, (3) apoptotic changes are observed in the early stages of myocarditis and (4) Bax may be associated with the apoptosis process in CVB3-induced myocarditis.

DNA repair in resistance to alkylating anticancer drugs.

The major critical target of alkylating antineoplastic drugs belonging to the group of methylating and chloroethylating agents is DNA. DNA alkylation lesions can be repaired by the action of alkyltransferase (MGMT) and base excision repair enzymes. The major cell killing and apoptotic alkylation lesions are O6-methylguanine (O6MeG) and O6-chloroethylguanine. O6MeG causes mispairing with thymine which is erroneously processed by mismatch repair (MMR), leading to secondary lesions that potently trigger the mitochondrial apoptotic pathway. Apoptosis induced by O6MeG is a late cellular response that requires cell proliferation to occur. Data are available indicating that DNA double-strand breaks are actively involved as the ultimate trigger of apoptosis. O6MeG and O6-chloroethylguanine are repaired by the specific action of MGMT thus counteracting the killing effects of the lesions. The expression of MGMT is highly variable and is often increased in tumors compared to normal tissue. Determination of MGMT activity in various tumors showed low expression in brain, pancreas and skin and high expression in testicle, breast, colorectal, lung and ovarian tumors. Distribution profiles of MGMT revealed non-random distribution indicating the existence of subpopulations exhibiting low and high activity. Since MGMT is one of the most important factors determining drug resistance to alkylation, strategies have been developed to inhibit MGMT in tumors with the aid of MGMT inhibitors and overexpression of MGMT in healthy, non-target tissue (e.g. blood stem cells) by transferring a mutated form of MGMT inaccessible to inhibition. Targeting MGMT inhibitors to tumors may further enhance the antineoplastic efficiency of alkylating agents. The role of base excision repair, Fos and p53 in drug resistance to alkylation is also discussed.

Endothelial Cell Apoptotic Genes Associated with the Pathogenesis of Thrombotic Microangiopathies: An Application of Oligonucleotide Genechip Technology

Idiopathic thrombotic thrombocytopenic purpura (TTP) is a disease characterized by the apoptotic injury of all microvascular endothelial cells (MVEC) except those of pulmonary origin. It notably also spares EC of large vessel origin. It is fatal unless treated with plasma exchange. The EC lineage restriction of the apoptotic lesions in vivo is reproduced in vitro following exposure of primary human MVEC derived from various tissues to TTP plasma. Oligonucleotide genechip technology was used to identify genes that may contribute to the resistance of lung MVEC to apoptosis induced by TTP plasma and to explore the intrinsic genotypic heterogeneity between MVEC of TTP-sensitive (skin) versus resistant (lung) lineage. Exposure of cells to TTP or normal plasma yielded 157 genes that were differentially expressed in primary human lung MVEC. A global change in expression of pro- and anti-apoptotic genes was seen, including increases in caspase 1, Fas, and Bcl-xl, already shown by experimental means to be involved in TTP pathogenesis. Additional differences suggest the importance of pathways related to the death receptor ligand TRAIL, as well as a role for disruption of EC–extracellular matrix interactions in the initiation of apoptosis. Maintenance of specific prosurvival signals at baseline may be a feature of lung MVEC resistance in TTP as suggested by higher expression than skin EC of the TRAIL antagonist, osteoprotegerin, and the vascular endothelial growth factors, VEGF/VPF and VEGF-C, and their receptors, VEGFR-2 (KDR) and VEGFR-3 (Flt4).

Apoptotic and necrotic brain lesions in a fatal case of carbon monoxide poisoning

A 41-year-old man was accidentally exposed to carbon monoxide (CO) gas and found in a state of cardiopulmonary arrest while he took bath. After admission, he was resuscitated and underwent artificial ventilation in a comatose state and died about 19 h later. Computed tomography (CT) examination disclosed bilateral low density area in the basal ganglia and the thalamus, a well-known finding in the CO intoxication. Necropsy, histological examination, DNA ladder assay gave the first line of evidence for the presence of apoptosis as well as necrosis in the human case of CO intoxication. TdT-mediated dUTP-biotin nick-end labeling (TUNEL) positive apoptotic cells were more predominant in the CA2 area than in CA1 area. There is general co-relation between the ratio of TUNEL-positive cells and the DNA laddering on the agarose gel. Basal ganglia and thalamus, which showed bilateral low density area in CT, were revealed to be severe edema. The two types of cell death occurred in the cortex, basal ganglia, hippocampus, thalamus, and cerebellum. Hypoxia caused by CO–hemoglobin formation alone cannot explain the phenomena.

BER, MGMT, and MMR in defense against alkylation-induced genotoxicity and apoptosis

Methylating carcinogens and cytostatic drugs induce different methylation products in DNA. In cells not expressing the repair protein MGMT or expressing it at a low level, O6-methylguanine is the major genotoxic, recombinogenic, and apoptotic lesion. Genotoxicity and apoptosis triggered by O6-methylguanine require mismatch repair (MMR). In cells expressing O6-methylguanine-DNA methyl transferase (MGMT) at a high level or for agents producing low amounts of O6-methylguanine, N-alkylations become the major genotoxic lesions. N-Alkylations are repaired by base excision repair (BER). In mammalian cells, naturally occurring mutants of BER have not been detected, which points to the importance of BER for viability. In order to ascertain the role of BER in cellular defense, BER was modulated either by transfection or mutational inactivation. It has been shown that overexpression of N-methylpurine-DNA glycosylase (MPG) does not protect, but rather sensitizes cells to SN2 agents. This has been interpreted in terms of an imbalance in BER. Regarding abasic site endonuclease (APE), transient but not stable overexpression of the enzyme was achieved upon transfection in CHO cells, which indicates that unphysiologic APE levels are not tolerated by the cell. Besides the repair function, APE (alias Ref-1) exerts redox capability by which the activity of various transcription factors is modulated. Therefore, it is possible that stable overexpression of mammalian APE impairs transcriptional regulation of genes, whereas transient overexpression may exert some protective effect. DNA polymerase beta (Pol beta) transfection was ineffective in conferring resistance to methylmethane sulfonate (MMS). On the other hand, Pol beta-deficient cells proved to be highly sensitive to methylation-induced chromosomal aberrations and reproductive cell death. The dramatic hypersensitivity in the killing response is largely due to induction of apoptosis. Obviously, nonrepaired BER intermediates are clastogenic and act as a strong trigger of the apoptotic pathway. The elements of this pathway are currently under investigation.

Influence of liver hepatitis C virus RNA and hepatitis C virus genotype on FAS-mediated apoptosis after liver transplantation for hepatitis C.

Recurrent hepatitis C virus (HCV) infection after liver transplantation is characterized by a high level of intrahepatic HCV replication and more severe liver damage in case of genotype 1b infection. We investigated the involvement of apoptosis in recurrent HCV liver disease, and its possible links with histological findings, HCV genotype, liver HCV RNA level, and liver Fas mRNA level. We studied 61 liver graft biopsy specimens from 25 patients transplanted for HCV-related cirrhosis. DNA fragmentation was determined semi-quantitatively by in situ end labeling. HCV RNA and liver Fas mRNA were determined in parallel by quantitative polymerase chain reaction, with ribosomal 28S RNA as internal standard. Apoptotic lesions were predominantly portal (nonhepatocytic) or lobular (hepatocytic). Both were correlated with serum aminotransferase levels. The degree of portal apoptosis correlated with acute rejection (P<0.001), although lobular apoptosis was associated with lobular hepatitis (P<0.02), and HCV genotype 1b (P=0.04). In multivariate analysis, liver Fas mRNA level independently correlated with HCV-related chronic active hepatitis (P=0.04), age (P=0.01), and liver HCV RNA level (P=0.0007). After liver transplantation, 1) apoptosis is involved in HCV-related liver damage; 2) HCV type 1b may induce more severe apoptotic lesions than other genotypes; and 3) Fas transcription may be up-regulated by intrahepatic HCV replication.

Molecular mechanism of monocyte predominant infiltration in chronic inflammation: mediation by a novel monocyte chemotactic factor, S19 ribosomal protein dimer.

A novel monocyte chemotactic factor, a cross-linked homodimer of S19 ribosomal protein (RP S19) was initially isolated from a rheumatoid arthritis synovial lesion. The RP S19 dimer causes the monocyte specific chemotaxis in vitro and the monocyte predominant infiltration in vivo, via its agonistic and antagonistic effects on the C5a receptors of monocytes and polymorphonuclear leukocytes, respectively. The agonistic effect is attributed to the similarity of regional structures between RP S19 and C5a, the complement C5-derived leukocyte chemotactic factor, although overall homology of the amino acid sequence between these molecules is only 4%. The antagonistic effect depends upon the C-terminal portion of RP S19. The RP S19 dimer is produced and released by apoptotic cells, and this dimer recruits monocytes from the circulation to the apoptotic lesion. The infiltrated monocytes/macrophages engulf the apoptotic cells, translocate to regional lymph nodes via lymphatics and present the antigenic information of the apoptotic cells to the T cell repertoire. In this manner, the apoptotic cell clearance system connects to the acquired immune system. The innate and acquired immune mechanisms, mediated by the RP S19 dimer, participate in the pathology of inveterate chronic inflammation such as rheumatoid arthritis.

Behavioral alterations associated with apoptosis and down-regulation of presenilin 1 in the brains of p53-deficient mice.

Presenilin 1 (PS1) expression is repressed by the p53 tumor suppressor. As shown herein, wild-type PS1 is an effective antiapoptotic molecule capable of significantly inhibiting p53-dependent and p53-independent cell death. We analyzed, at the functional and molecular levels, the brains of p53 knockout mice. Surprisingly, we found that lack of p53 expression induces apoptotic brain lesions, accompanied by learning deficiency and behavioral alterations. p53-deficient mice show an unexpected overexpression of p21(waf1) with subsequent down-regulation of PS1 in their brains. This process is progressive and age-dependent. These data indicate that the p53 pathway, besides affecting tumor suppression, may play a major role in regulating neurobehavioral function and cell survival in the brain.

Apoptotic cellular damage in mice after T-2 toxin-induced acute toxicosis.

By histopathologic, electron microscopic, and immunochemical observation, the mechanism of cellular death was investigated in thymus, spleen, and liver of mice given intraperitoneally sublethal doses of T-2 toxin, a trichothecene mycotoxin. In the thymus and spleen of mice given 5.0 mg/kg body weight of T-2 toxin and killed 12 hours later, a massive cellular destruction characterized by chromatin condensation was evident, and electron microscopy analysis revealed the presence of apoptotic bodies. In the liver of mice given 2.5 mg/kg of T-2 toxin and killed 2 hours later, the induction of apoptotic cellular lesions was observed by electron microscopy, and Kupffer cells phagocytosed the apoptotic bodies. Such lesions were not observed in the mice killed 12 hours after receiving the toxin. In situ nick translation analysis (Tunel method) revealed DNA fragmentation in thymus, spleen, and liver shortly after administration of T-2 toxin. As previously observed in vitro, these findings indicated that T-2 toxin is a potent inducer of apoptotic cell death in thymus, spleen, and liver in vivo; especially in liver, apoptosis is induced rapidly as compared with the other tissues observed, and Kupffer cells play an important role for clearance of apoptosis.

Dismantling in cell death: molecular mechanisms and relationship to caspase activation.

The notion of a cell death programme was introduced in view of the reproducibility of its occurrence in time and space (e.g. in the developing embryo) and of its genetic determination. Programmed cell death can be schematically subdivided into three steps: a signalling phase, an execution phase and a dismantling phase. This review focuses on the latter. Apoptosis is the most studied form of dismantling of animal cells. The molecular pathways leading to certain apoptotic lesions appear to be dependent on the proteolytic activity of caspases. Death itself can, however, be caspase-independent. Also, non-apoptotic forms of cell death exist, even in animal cells; their molecular bases are still unknown. The relationship between cell death, apoptosis and caspases is discussed.

Metallothionein (MT)-Null Mice Are Sensitive to Cisplatin-Induced Hepatotoxicity

cis-Diamminedichloroplatinum (cisplatin) is an important anticancer drug used to treat solid tumors. The nephrotoxicity of cisplatin is recognized as the most important dose-limiting factor, but high doses of cisplatin also produce hepatotoxicity. However, little is known about cisplatin-induced liver injury and the role of metallothionein, a cysteine-rich, metal-binding protein, in modulating its hepatotoxicity. This study was designed to examine cisplatin hepatotoxicity in control and metallothionein-I/II knock-out (MT-null) mice. Animals were given a single injection of cisplatin (50–200 μmol/kg ip), and liver injury was evaluated 3–16 h later. Cisplatin produced dose- and time-dependent liver injury, as evidenced by increased serum activity of alanine aminotransferase (ALT), as well as by histopathology. Apoptosis, rather than necrosis, predominates in cisplatin-induced liver injury, as indicated by increased numbers of apoptotic cells (hematoxylin and eosin staining),in situapoptotic DNA detection, and DNA fragmentation on agarose gel electrophoresis. MT-null mice were more sensitive than controls to cisplatin-induced hepatotoxicity. Cisplatin (200 μmol/kg) was lethal to 12% of control mice, but 60% of MT-null mice died within 16 h. At the dose of 150 μmol/kg, serum ALT activities were increased 2-fold in control mice compared to 6.5-fold in MT-null mice. Apoptotic lesions were more pronounced in MT-null than in control mice. MT-null mice were also more susceptible than controls to cisplatin-induced nephrotoxicity, as evidenced by having higher blood urea nitrogen concentrations. Furthermore, cultured MT-null hepatocytes were more sensitive than control cells to the cytotoxicity of cisplatin (50–200 μM), as indicated by lactate dehydrogenase leakage into the medium. These results demonstrate that (1) high doses of cisplatin produce hepatotoxicity, with apoptosis as the major lesion, and (2) MT protects against cisplatin-induced liver injury.

Apoptotic cellular damage in mice after T‐2 toxin‐induced acute toxicosis

AbstractBy histopathologic, electron microscopic, and immunochemical observation, the mechanism of cellular death was investigated in thymus, spleen, and liver of mice given intraperitoneally sublethal doses of T‐2 toxin, a trichothecene mycotoxin. In the thymus and spleen of mice given 5.0 mg/kg body weight of T‐2 toxin and killed 12 hours later, a massive cellular destruction characterized by chromatin condensation was evident, and electron microscopy analysis revealed the presence of apoptotic bodies. In the liver of mice given 2.5 mg/kg of T‐2 toxin and killed 2 hours later, the induction of apoptotic cellular lesions was observed by electron microscopy, and Kupffer cells phagocytosed the apoptotic bodies. Such lesions were not observed in the mice killed 12 hours after receiving the toxin. In situ nick translation analysis (Tunel method) revealed DNA fragmentation in thymus, spleen, and liver shortly after administration of T‐2 toxin. As previously observed in vitro, these findings indicated that T‐2 toxin is a potent inducer of apoptotic cell death in thymus, spleen, and liver in vivo; especially in liver, apoptosis is induced rapidly as compared with the other tissues observed, and Kupffer cells play an important role for clearance of apoptosis. Nat. Toxins 5:141–145, 1997. © 1997 Wiley‐Liss, Inc.