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STZ, derived from the soil microorganism Streptomycetes achromogenes in 1960, has been found to have a significant antimicrobial action for a wide spectrum of organisms.2,3 However, during the preclinical toxicology studies, it was found that this compound causes hyperglycemia when given by intravenous administration in rats and dogs within a few hours.4,5 Therefore, its use as an antimicrobial agent was abandoned. At the same time, its antitumor activity was demonstrated.6

27.2.1 Diabetogenic Action of STZ

Tumor studies in the murines4,5 and toxicological studies in dogs and rhesus monkeys4,7 demonstrated the temporary or permanent hyperglycemic and potent diabetogenic action of STZ.4,8 Therefore, STZ is generally used to induce both insulin-dependent and non-insulin-dependent diabetes in animal studies.

Characteristically, STZ primarily damages p-cells, leading to alterations in blood glucose and insulin levels. Hyperglycemia is observed as early as 2 hours after STZ administration with a concomitant drop in blood insulin levels. However, about 6 hours later, the opposite situation occurs: blood glucose levels drop while insulin levels begin to increase. Eventually, blood insulin levels decrease and hyperglycemia occurs. The responsiveness of p-cells to glucose, which temporarily returns to normal after an initial abolishment, is lost permanently.9 Immunohistochemical studies in STZ-treated animals revealed that the insulin-positive areas were decreased significantly and exhibited vacuoles of the remaining p-cells.10-12 In multiple low-dose model, after administration of STZ, a rapid loss of islets occurs within 3 days.13,14 There are conflicting reports about the islet size after STZ administration. Li et al.14 have found the islet area increased to an average of about 50% of the original size 28 days after low-dose STZ administration. However, Bonnevie-Nielsen et al.15 found a reduction of the islet area was down to 31% on Day 6 and a further decline to 1% of the original area at Day 14. Li et al.14 reported that after multiple low-dose STZ treatments of C57BL/Ks male mice, they observed a fraction of the islets of Langerhans disappeared and in the remaining islet tissue an expansion of a-cells occurred.

The dose range of STZ is wider than alloxan, another diabetogenic agent (see below). The toxic action of STZ on islet p-cells generally induced in two different ways, with multiple low doses or with a single high dose. When given in multiple low doses (40 mg/kg body weight/day/5 days), it induces insulitis and progresses to nearly complete destruction of p-cells associated with atrophy of the islets and diabetes.16,17 When given as a single high dose, generally between 40 and 60 mg/kg body weight in rats18 and hamsters and between 75 and 150 mg/kg body weight in other species, it rapidly destroys islet p-cells.19

In some studies, inflammation in sites, other than the islets of Langerhans, has been noticed following a multiple-dose treatment. Papaccio et al.20 observed that histologically the ductal cells in close proximity to islets were also affected by inflammatory cells that extend from the islets, whereas ducts far from islets were generally free from inflammation. These results suggest that the initial action of STZ is wider spread and is a less specific process that later undergoes restriction.

27.2.2 The Mechanisms of Streptozotocin Action

STZ is a monofunctional nitrosourea derivative and a member of alkylni-trosoureas, a group of alkylating antineoplastic drugs, which are clinically active against a broad range of tumors.21

p-cell toxicity of STZ requires its uptake into the cells.22 STZ (2-deoxy-2-(3-(methyl 3-nitrosoureido)-D-glucopyranose) consists of a 2-deoxyglu-cose moiety substituted in position C-2 with nitrosourea23 and is a D-glucopyranose derivative of N-methyl-N-nitrosourea (MNU). Although both STZ and MNU are potent alkylating agents,24 highly toxic, and carcinogenic,25,26 only STZ has selective p-cell toxicity.27 It is generally believed that this selective p-cell toxicity is related to the glucose moiety in its chemical structure. This specific structure is believed to be responsible for its affinity to the p-cell via the low affinity glucose transporter GLUT2, which is not merely a structural protein specific for the p-cell membrane but is a crucial constituent for recognition and entry of glucose as well as glucose-like molecules, such as STZ, in the plasma mem-brane.22,28,29 The observation that the RINm5f rat insulinoma cell line, which does not express this glucose transporter, resists STZ toxicity30,31 and becomes sensitive to the toxic action of this compound only after the expression of the GLUT2 glucose transporter in this cell line28 confirms this hypothesis. It is also demonstrated that in insulin-producing cells, which do not express GLUT2, the cellular uptake and the toxicity of STZ is low. Accordingly, it appears that the reduced expression of GLUT2 prevents the diabetogenic action of STZ.28,32 However, it is also observed that STZ itself restricts GLUT2 expression both in vivo and in vitro when administrated in multiple doses.33,34

The exact mechanism underlying the diabetogenic action of STZ has not been fully understood. It is generally believed that its toxic action relates to the DNA alkylating activity of its MNU moiety22,24,35; particularly at the O6 position of guanine (see also Chapter 9 and Chapter 27).36,37 STZ damages DNA comprising its fragmentation that leads to apparent depletion of nicotinamide adenine dinucleotide (NAD)+, which in turn, inhibits insulin biosynthesis and secretion, and finally causes p-cell death through adenosine triphosphate (ATP) depletion.19 The demonstration of various methylated purines in the tissues of STZ-treated rats provides further evidence for the DNA damaging action of STZ.24 Poly ADP-ribosylation, which is activated with the DNA damaging effect of STZ, has been proposed to lead to these unfavorable consequences in the p-cells.38-41 This notion has been confirmed by studies showing that the inhibition of this process with 3-aminobenzamide and nicotinamide (poly ADP-ribose inhibitors) prevents the toxicity of STZ.42,43

Nitric oxide (NO), which carries biological information, is a free radical gas44 and has been proposed as a possible mediator in the damage to the insulin-producing p-cells.45,46 It has been found that STZ generates NO in aqueous solutions.47 Kroncke et al.48 also demonstrated NO generation during cellular metabolization of STZ. Hence, it was speculated that NO contributes to STZ-induced DNA damage.47-49 It has also been shown that scavenging with NO protects against STZ-induced DNA strand breakage.48 This issue, however, is controversial.50,51 Moreover, in a recent study, it has been shown that multiple low doses and a high single dose of STZ does not stimulate NO production at islet cell levels.52 It seems that NO at least partially takes part in STZ islet toxicity. The certain role of NO, however, still is not clear.

In addition to NO, STZ generates reactive oxygen species (ROS), which also contribute to DNA fragmentation in the p-cells.53,54 STZ inhibits the Krebs cycle47 and consequently decreases oxygen consumption by mitochondria considerably.41 The result is a strong limitation of mitochondrial ATP production and the depletion of this nucleotide in p-cells.41,55 This process enhances O2- radical generation by the xanthine oxidase system of the pancreatic p-cell,56,57 stimulates hydrogen peroxide and hydroxyl radicals generation, and causes DNA fragmentation in isolated rat pancreatic islets.53,56,58 It has been found that the inhibition of the formation of these radicals restricts the p-cells cytotoxicity of STZ in vitro.58 Although there are a few reports that suggest that free radicals may not be involved in the DNA damage by STZ,19,59 most of the studies support the idea that the free radicals may play a role in the DNA toxicity action of this agent.60 NO and ROS, however, can act separately or form the highly toxic peroxynitrite (ONOO-). Recent evidence claims that peroxynitrite (and not

NO) is the potent trigger of DNA strand breakage.19 Peroxynitrite formation activates poly ADP-ribosylation and could play a role in the pathogenesis of islet cell damage in response to STZ or NO compounds.

STZ also induces cell death by apoptosis and necrosis in pancreatic islet cells.49,61 Cell death by apoptosis was observed in cultured pancreatic p-cell HIT-T15 and RINm5F after treatment with STZ.49 In addition, Saini et al.62 showed that STZ at low doses induces apoptosis and, at high doses, causes necrosis in a murine pancreatic p-cell line, INS-1. STZ also causes p-cell apoptosis by the generation of toxic radicals.59,63

One of the primary actions of the diabetogenic chemicals (STZ, alloxan) is at the plasma membrane level of p-cells.64,65 STZ modifies the molecular structure of phospholipids, particularly phosphatidylcholine, which is a major phospholipid of the outer leaflet of the plasma membrane, resulting in a decrease of membrane fluidity in p-cells.66-68 Hence, it was suggested that STZ acts directly on the plasma membrane and induces alterations of islet cell membrane properties64 and preservation of membrane integrity could protect p-cells from cytotoxic insults.

Recently, a new glycosylation pathway (other than the better-known N-linked pathway), O-linked protein glycosylation, has been described and implicated in diseases as diverse as cancer and Alzheimer's. The p-cells appear to be especially susceptible to disruption of the O-linked protein glycosylation pathway and an important link between p-cell O-linked protein glycosylation and p-cell apoptosis has been recently shown.69 It is assumed that STZ irreversibly increases the O-glycosylation and subsequent p-cell apoptosis, as claimed in some recent reports that the diabetogenic agent STZ causes p-cell toxicity by this pathway.70

In brief, STZ induces DNA damage by alkylation of specific sites on DNA and that free radicals and NO generated during STZ metabolism seems to play a role in the mechanism by STZ. Severe DNA damage by STZ also results in cell death by apoptosis or necrosis. Furthermore, some other pathways like an increase in O-linked protein glycosylation and modification of the cellular membrane by STZ are also proposed as the mechanisms that underlie the toxic action of STZ.

27.2.3 Species Differences of STZ Action

STZ causes necrosis or marked degenerative changes in the p-cells with nuclear pyknosis and cytoplasmic vacuolization and produces permanent diabetes in different species, including the rat, mouse, guinea pig, Chinese hamster, Syrian golden hamster, and monkey.8,71-73 Syrian hamsters have been shown to be more useful than rats as an animal model of human diabetes mellitus.74 Most species indicate a high acute mortality to STZ injection.75 Syrian hamsters also respond to a single dose of STZ with p-cell necrosis, but in contrast to other species, do not result in a higher percentage of permanent diabetes. p-cells regenerate and a high percentage of hamsters subsequently recover spontaneously from their diabetes.76 Multiple daily doses of STZ, however, result in permanent diabetes in a large number of hamsters. Recovery from STZ-induced diabetes has also been reported in rats.77 However, recovery required 8 to 18 months in rats and the induced diabetes was relatively mild.

We examined the regenerative properties and capacity of p-cells in STZ-treated hamsters, some of which received insulin.10 A single dose (50 mg/kg body weight) of STZ induced diabetes in all hamsters, causing degeneration and depletion of p-cells, proliferation of glucagon and soma-tostatin cells, and their derangement within the islets as observed in other species.78-80 However, 10 days after STZ treatment, degeneration stopped and pancreatic islet and ductal cells began to proliferate, which peaked 14 days post-STZ treatment (Figure 27.1A). These hamsters also recovered from their diabetes spontaneously. It was assumed that the regeneration of islet cells was triggered by hypoinsulinemia through a feedback mechanism. Confirming this mechanistic event was that insulin therapy prevented the spontaneous recovery and produced a persistent severe hyperglycemia.10 Insulin also inhibited DNA synthesis in ductal, ductular, and acinar cells in STZ-pretreated hamsters, but not in normoglycemic control hamsters treated with insulin alone.10 These results demonstrated a controversial deleterious effect of exogenous insulin in the course of STZ-induced diabetes in hamsters. In this study, the recovery and lack of recovery was associated with p-cell regeneration and lack of regeneration.

In a succeeding study, we found that p-cell regeneration in STZ-induced diabetic hamsters occurred primarily from undifferentiated cells within the islet (Figure 27.1A) and exogenous insulin inhibited this differentiation.81 Nagasao et al.82 have examined whether centroacinar and intercalated duct cells can serve as stem cells to induce recovery after the administration of STZ in rats. They found that rat pancreatic endocrine cells seemed to recover from newly generated cells derived from intercalated ductal and centroacinar cells. In a recent study, the recovery from Type 1 diabetes induced by multiple low doses of STZ in transgenic mice expressing the insulin-like growth Factor I (IGF-I) was studied (see also Chapter 13). It was found that the expression of IGF-I in p-cells restored p-cell mass and normoglycemia.83 Thus, the authors speculated that IGF-I expression in p-cells of these transgenic mice might protect them against the oxidative and apoptotic effects of STZ.

The susceptibility of human islets to STZ damage, which is not well established, is a controversial issue. In a study, it was found that human islets are remarkably resistant to the diabetogenic effect of STZ in vivo.84 Among the potential mediators of p-cell damage, cytokines and cytokine-

Glycogen Streptozotocin

Figure 27.1 Streptozotocin-induced lesions. A: An islet of a hamster treated with a single dose of STZ 1 week earlier. Degeneration of the bulk of p-cells (center) surrounded by glucagon cells (black). Four cells in necrotic area show labeling with tritiated thymidine (*). Labeling is seen in one glucagons cell (arrow). Combined immunohistochemistry and autoradiography, ABC methods, x75. B: The intact p-cells contain a large amount of glycogen (dark black). Islet of a STZ-treated hamster. x50. C: The proliferative pattern of an islet 20 weeks after STZ-treatment. Note the presence of several cells with giant nuclei or foamy cytoplasm (arrow). One cell has a large opaque cytoplasm (upper middle field). Strikingly, regeneration seems to take place in the islet periphery (see Figure 27.1D). H&E x75. D: In a STZ-treated hamster, the peripheral portion of an enlarged islet is replaced by atypical cells with foamy cytoplasm and pleomorphic nuclei. This hamster had an islet cell tumor in another part of the pancreas. H&E x75. E: Two islet cell tumors in a hamster. Tumors show different size and morphology. The smaller tumor had hemorrhagic area. Ductular formation is present in the upper pole of the small tumor. This hamster was treated with a combination of STZ and BOP. H&E x32. F: STZ-induced tumors generally show various histological architecture. They may show trabecular, medullary, or cystic patterns. The staining with antibodies against islet hormones is remarkably heterogeneous. H&E x50.

induced NO production has received special attention. It has been suggested that these agents damage rat islets in vitro, but their effects on cultured human islets are less pronounced, despite the production of similar amounts of NO.46 Eizirik and coworkers85 investigated the differences between humans and rodents and have found that human p-cells are resistant to nitroprusside (a NO donor),86 STZ, or alloxan (a generator of free radicals)87 at concentrations that decrease survival and function of rat or mouse p-cells. It is reported that human fetal islets grafted into nude mice were not destroyed by injections of STZ, in spite of the adequate uptake of the drug by the human tissue.88 It is well known that the time between the appearance of islet autoimmunity and the clinical onset of insulin-dependent diabetes mellitus (IDDM) is much longer in humans than in mice (non-obese diabetic mice) and, in particular, rats. Because the degree of resistance to various toxins suggested from the findings of Eizirik et al.85 seems to follow a similar pattern (i.e., humane mouse^rat), increased resistance to STZ injury may contribute to a longer period in humans. It appears that STZ does not produce any significant clinical diabetogenic effect in humans.25 Rabbits have also shown to be highly resistant to STZ with little metabolic or histologic evidence of p-cell damage at doses up to 300 mg/kg and with higher doses having severe systemic toxicity.89

De Vos et al.90 demonstrated interspecies differences in the expression of GLUT2 in vitro, which may explain the difference in sensitivity to toxic action of STZ. They found that human islet cells express little GLUT2 compared to GLUT1 or GLUT3 and that in the human p-cell the GLUT2 expression level is markedly lower than in rat p-cells. This low expression of GLUT2, which is a crucial factor for STZ uptake to p-cells, in the human p-cells could be responsible for the resistance to the toxic action of STZ in man. The exact mechanism of this process warrants further studies.

27.2.4 Tumorigenic Action of STZ

In addition to its antibiotic and diabetogenic properties, the genotoxic and tumorigenic actions of STZ have been demonstrated. This property, however, is not restricted to the pancreas as STZ induces tumors in other organs as well, including the liver and kidney.91,92

Pancreatic islets are also a target of the tumorigenic action of STZ. It has been found that STZ, in combination with nicotinamide, which prevents the acute toxicity of STZ, induces islet cell tumors with a high frequency after a long latency period.93 The tumors have been described as a well-differentiated type and resemble the normal islet tissue, both morphologically and functionally, as they are rich in the typical p-cells and release insulin in response to glucose, both in vivo and in culture.93

In this study, it was also suggested that there was a different sensitivity of the different islet cell types to the chemically induced transformation. Doi94 also observed functioning pancreatic islet cells tumors 407 days after STZ administration. However, Yoshino et al.95 found that STZ induces two types of islet cell tumors: one is insulin-producing and insulin-secreting, whereas the other is insulin-producing but not insulin-secreting.

The activation of poly-ADP ribosylation due to fragmented DNA is described previously in the diabetogenic action of STZ. Although it has been shown that the prevention of poly-ribosylation with poly(ADP-ribose) synthetase inhibitors leads to the maintenance of p-cell functions normally, DNA strand breaks have not been prevented at all96; therefore, residual DNA damage may continue to affect the p-cells. In line with this concept, Okamoto and Yamamoto96 demonstrated that about 1 year after the combined administration of alloxan or STZ with poly(ADP-ribose) syn-thetase inhibitors to rats, diabetes did not develop but islet p-cell tumors were found frequently. These results suggested that DNA breaks initiate two kinds of pathological states in p-cells — one is degenerative and the other is oncogenic. A human insulinoma case, after STZ therapy for metastatic gastrinoma, was observed by Bar et al.97

Yagihashi and Nagai98 demonstrated that STZ-induced tumors mostly consisted of p-cells. Over half of the tumors examined showed mixed cellularity with considerable numbers of A cells and small numbers of D or PP cells. They claimed that the multiplicity of the endocrine cells of rat islet cell tumors might be an expression of cellular dedifferentiation of tumor cells, which could redifferentiate into the whole range of components of the endocrine pancreas.

The histologic patterns of islet cell tumors in hamsters are comparable to those induced in rats.98 The induced islet cell tumors appear to be hormonally inactive, and no changes in blood glucose level were detectable in islet cell tumor-bearing hamsters. In a study in STZ-treated ham-sters,99 islet cell adenomas were primarily of a pleomorphic cell type and had pseudoinfiltrative tendencies. These neoplastic cells were found to develop from the most peripheral portion of islets, most probably from the periinsular ductules, which are believed to give rise to islet cells.78,100 If this is the case, then, ductular cells also seem to be principal targets for STZ, which argues with the concept that STZ toxicity is p-cell specific. The induction of ductal and ductular lesions by STZ treatment alone and a significantly higher incidence of ductal-ductular carcinomas by STZ plus BOP (a pancreatic ductal cell cancer-producing nitrosamine) than by BOP alone99 are further support for this possibility. In this context, Shepherd et al.101 demonstrated the potent carcinogenic action of STZ on cultured rat pancreatic duct epithelial cells.

Because STZ and BOP have been shown to exert their oncogenic effect by the methylation of DNA24,102 and large doses of BOP analogues cause islet cell necrosis,103-104 it appears that STZ and BOP act by similar mechanisms. Yet, it is unclear why the affected ductular cells differentiate toward islet cells after STZ and to ductular cells after BOP. Theoretically, the quantity of DNA alkylation induced by STZ and BOP may be a determining factor in the differentiating process. Much of N-methylurea, the carcinogenic moiety of STZ, taken up by islet cells can decompose within the islet cells and generate cytotoxic substances, whereas only a smaller portion affects ductular cells. In fact, the uptake of STZ in the exocrine pancreas and its alteration at a subcellular level have been shown.105 Not only the quantity, but also the quality of DNA alkylation may be important in the phenotypic expression of resulting tumors (STZ+BOP).

STZ has been found to be carcinogenic in rats, mice, and hamsters. In primary cultures of human and rat kidney cells, STZ induces neoplastic transformation.106 Hence, STZ may be a potential carcinogen also in humans; although this possibility has been claimed,97 it is not clear yet.

Differences seem to exist about the target cells of STZ on its acute and chronic effect. Although its acute toxicity is directed toward p-cells, its chronic action is on other cell types. Although it is believed that tumor cells derive from the regenerated or surviving p-cells, histological observations clearly pointed to the development of malignant cells in the islet periphery, where the cells initially show the presence of a large amount of glycogen (Figure 27.1B to Figure 27.1D). As in human tumors, the STZ-induced islet cell lesions show various morphological patterns even within the same animal with multiple tumors (Figure 27.1E and Figure 27.1F). Also, the immunoreactivity of the tumor cells to the antibodies against islet cell hormones shows great variations.

27.2.5 Antitumor Action of STZ

The potential antitumoral activity of STZ was demonstrated against mouse leukemia L1210.6 This finding opened clinical trials for tumor therapy with STZ. It was used in several cancer types as an antitumor agent, particularly in metastatic insulinoma. In patients with insulinoma, tumor growth was significantly decreased.107 Subsequently, the efficacy of STZ as a therapeutic agent on this rare tumor has been reported from numerous clinical studies.

Both p-cell and non-p-cell islet tumors that secrete various hormones (e.g., insulin, glucagons, gastrin) are suggested to be responsive to STZ, in both biochemical and tumor responses.25 It has also been shown that STZ have similar effects on non-functioning islet cell tumors. A comparable response rate (36%) was found, regardless of the function of the tumor, by Moertel et al.108 The antitumor activity of STZ against metastatic pancreatic islet tumors was also demonstrated in dogs.109 In humans, interestingly, diabetes mellitus was not identified, though some patients had reversible glucose tolerance.110 However, in dogs with insulinoma diabetes mellitus occurred following STZ treatment.109

In general, the overall success of STZ in the treatment of insulinoma is marginal because islet tumors are usually composed of mixed cell populations unresponsive to STZ.

Hyperinsulinemic hypoglycemic infancy, a rare genetic disorder, has two major different histologic appearances111: diffuse nesidioblastosis and a focal nodular form with a discrete regional adenomatous hyperplasia.112 Due to selective p-cell toxicity, STZ was used in the treatment of this disease. Treatment of some patients with STZ has resulted in tumor cell damages ranging between 0 and 60% (Pour; unpublished observation).

Remarkably, STZ-treatment has been found to suppress or even abolish the development of pancreatic cancer in the BOP-hamster model.113 Notably, the degree of this inhibition closely paralleled the severity of diabetes induced by STZ.114 The inhibitory effect of STZ on pancreatic ductal cell adenocarcinoma occurred only when diabetes was present.115 This observation supported the hypothesis that the origin of pancreatic ductal cell cancer resides in STZ-responsive islet cells.116 The assumption that the protective effect of STZ was related to the absence of insulin, which has a cell growth-promoting effect, could not be confirmed117 as the treatment with insulin did not overcome the inhibitory action of STZ on pancreatic carcinogenesis. Remarkably, although STZ-treated hamsters fully recovered from diabetes after 70 days, those treated with insulin remained hyperglycemic despite daily insulin treatment and showed a profound atrophy of islets.10 The results indicated that the preventive effect of STZ on pancreatic cancer induction is unrelated to insulin or the action of insulin on tumor induction and growth is local or paracrine.10,117 A single injection of STZ, at a dose that destroys only a portion of islet p-cells, inhibited pancreatic tumor induction; whereas multiple doses, leading to a complete destruction of islet cells, prevented pancreatic carcinogenesis, even when hamsters are treated with high doses of the carcinogen weekly.118

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