Apoptosis and Necrosis In Vitro

Another common method for measuring apoptosis is the DNA labeling technique, terminal deoxytransferase-mediated dUTP nick-end labeling (TUNEL). In this method, labeling of DNA strand breaks at the 3 end is achieved by using the enzyme, terminal deoxynucleotidyl transferase (TdT) and either a radioactive or a fluorescently-Iabeled deoxynucleotide. This method provides a very sensitive measurement of DNA strands breaks. However, as DNA strand 

An alternative strategy for measuring apoptosis is the use of antibodies directed toward the enzymes involved in apoptosis. As caspase-3 activation occurs in both extrinsic and intrinsic pathways, the use of caspase-3 immunohisto-chemical staining is a common method used in measuring apoptosis. Because caspase-3 is not activated in necrosis, the immunohistochemical measurement of activated caspase-3 provides a sensitive method for measuring apoptosis. 

Measurement of Apoptosis and Necrosis In Vivo Using Functionai imaging Techniques 

A number of clinical research studies using Tc-labeled annexin V complexes have been reported over the past 5 years (Table 13.2) (20,22-30). Both Tc-I annexin V and c-BTAP annexin V have been used to measure cell death occurring in myocardial infarction. For example, Thimister et al. (26) 

99mTc-BTAP annexin V Acute myocardial infarction Thimister et al. (26) 

---

Sweet, L.I., D.R. Passino-Reader, P.G. Meier and G.M. Omann. Fish thymocyte viability, apoptosis and necrosis in-vitro effects of organochlorine contaminants. Fish Shellfish Immunol. 8 77—90, 1998. 

DEPAP, but not aniline or other 3-(A-phenylamino)- 1,2-propanediol derivatives, induced both apoptosis and necrosis in human peripheral blood lymphocytes in vitro in a time- and concentration-dependent fashion. In short-term cell cultures, possibly representative of the toxic oil syndrome acute phase, DNA degradation occurred rapidly. Apoptosis preceded membrane damage. In longer-term cultures, cytotoxicity was characterized by necrosis. As the cells die, abnormal forms of autoantigens are released, activating autoreactive lymphocytes, which could ultimately initiate autoimmune disease (Gallardo et al., 1997 Lahoz et al., 1997). 

Benzodiazepin-2-one Y demonstrates anti-ischemic activity in vitro protecting neuronal cell from apoptosis and necrosis, with the (V)-isomer more active than the (iJ)-isomer <2007BML1326>. 

The mechanisms of cisplatin-induced nephrotoxicity have not been fully elucidated. Like several nephrotoxic heavy metals (for example mercury), cisplatin can accumulate in the kidney, where it can interact with sulfhydryl compounds, resulting in increased membrane fragility and depletion of intracellular glutathione. There is some evidence that cisplatin can induce apoptosis and necrosis of kidney cells dose-dependently. In vitro studies have suggested that the constitutive expression of antiapoptotic proteins (for example bcl-X) might be inversely correlated with the sensitivity of renal tubular cells (146,195-197). 

There is an overwhelming wealth of studies on either hepatocyte necrosis or on non-hepatocyte apoptosis. However, information on specific mechanisms of apoptosis in primary hepatocytes in vitro or in experimental animals in vivo is relatively sparse. Unlike the situation in many leukemic/tumor cell lines that mostly die by apoptosis, triggering of cell death in hepatocytes by a variety of stimuli often results in necrosis (Rosser and Gores 1995). This does not necessarily imply a different death program in hepatocytes, since apoptosis and necrosis are neither mechanisms of cell death nor do they allow conclusions on the mechanism having led to cell death. In fact, similar mechanisms seem to operate in hepatocytes as in many other well characterized cell types, although the final outcome may be apoptosis or necrosis. 

The nature of tubular injury in acute renal failure (ARF) includes reversible sublethal injury (swelling, loss of apical brush border) and lethal injury (necrosis and apoptosis) [1, 2]. Proximal tubular cell death due to ischemic ARF in vivo and hypoxia in vitro results predominantly in necrosis, hence the term acute tubular necrosis or ATM. Apoptotic cell death in ischemic renal injury has been inconsistently demonstrated [3]. When apoptosis has been demonstrated in early ischemic ARF, it is present in the distal tubule [4, 5]. Apoptosis in proximal tubules may play a role in tubular regeneration and was demonstrated to occur at 3 days after ischemic injury in regenerating FT [6]. Thus, the nature of nephrotoxic injury, whether it is tubular dysfunction necrosis or apoptosis is also an important consideration. 

The ultramicrostructural alterations of the thyroid caused by overdosed iodine included a decrease of microvilli, dilation of rough endoplasmic reticulum, vacuolation of mitochondria, an increase in peroxidates and secondary lyso-somes, an increase in myelin-sheath structures in cytoplasm, and apoptosis and necrosis of thyroid epithelial cells. In contrast to Wistar rats, iodine excess could also cause infiltration of lymphocytes and proliferation of fibrous tissue in NOD. H-2 mice, which showed a positive correlation with the iodine dose. Similar results were found in other susceptible animals (Li et ai, 1993 Bagchi et at, 1995). When human thyroid cells were cultured with excessive iodine in vitro, Many et al. (1992) observed the same phenomenon under the electron microscope, such as dilated mitochondria, rough endoplasmic reticulum, increased secondary lysosomes, apoptosis and necrosis of epithelial cells, and a positive correlation between the ratio of apoptosis and iodine. 

Apoptosis and necrosis were detected using the Cell Death Detection EL1SA kit, Roche (Version 11.0), a photometric enzyme-immunoassay for the qualitative and quantitative in vitro determination of cytoplasmic histone associated DNA fragments after induced cell death. Assay is based on a quantitative sandwich enzyme immunoassay principle using mouse monoclonal antibodies directed against DNA and histones that allows specific determination of mono- and oligonucleosomes in the cytoplasmatic fraction of the cell. 

Cd " is a well known necrosis inducer, especially at higher concentrations and/or in sensitive cell lines. In vivo necrosis by Cd " has been documented in the kidney, heart, liver, and testis. A wider variety of cell types have been employed for in vitro testing and have similarly shown both apoptosis and necrosis simultaneously or necrosis alone [359]. Using pharmacological inhibitors, Yang et al. could demonstrate a role for Ca ", calpains, mitochondrial membrane potential, ROS formation and NF-kB in necrosis of CHO cells. The authors suggested that cytosolic Ca " overload might be important in the execution phase of necrotic cell death while sustained depletion of Ca " stores in ER leads to apoptosis [511]. 

Unnecessary animal testing can be avoided by relying on in vitro studies as the first stage acute toxicity and cytocompatibility test for injectable materials (Pearce et al., 2007). Many researchers have utilized in vitro studies to screen the cellular response to CNTs (Raja et al., 2007) and to investigate the various CNT-mammalian cell interactions such as oxidative stress (Manna et al., 2005 Shvedova et al., 2003, 2007), antiproliferative effects (Cui et al., 2005 Garibaldi et al., 2006), decreased cell adhesion (Cui et al., 2005), apoptosis (Bottini et al., 2006 Cui et al., 2005 Jia et al., 2005), and necrosis (Jia et al., 2005). A summary of these in vitro studies are presented in Table 12.1. 

As is implied by its name, the first TNF-a-dependent mechanism described was the induction of tumor necrosis in vivo through its role in tumor vasculature. However the mechanisms of the in vitro toxicity of TNF-a to tumor cells imply apoptosis rather than necrosis [97], Tumor necrosis in SCID (severe combined immuno-deficiency) mice treated with LPS does not lead to the rejection of tumors [98], Furthermore, necrosis and tumor regression must be dissociated since anti-IFN-y antibodies inhibit LPS-induced regression of Meth A sarcoma in mice, but not the necrotic hemorrhage attributed to TNF-a. It is now accepted that the antitumoral effect of TNF-a is indirect and dependent on acquired immune response. Matsumoto et al. [99] reported that, while TNF-a itself has no effect on hepatoma KDH-8 tumor cells in vitro, the antitumoral effect of the lipid A ONO-4007 against KDH-8 tumors in vivo is inhibited by anti-TNF-a antibodies in WKAH rat, showing an indirect effect of TNF-a. 

Activation of calpain is usually associated with the progression of a necrotic type of cell death (Wang, 2000). However, neuronal necrosis and apoptosis occur in parallel after ischemic injury in vitro and in vivo (Charriaut-Marlangue et al., 1996). Retinal ischemia causes precocious necrosis of neurons in the ganglion cell layer (GCL) and INL, whereas apoptosis appears as the delayed component of neuronal death associated with transient retinal ischemia (Joo et al., 1999). 

---