Chlorozocin
`A Diabetogenic Analogue of Streptozocin
`with Dissimilar Mechanisms of Action
`on Pancreatic Beta Cells
`
`BROOKE T. MOSSMAN, GLENN L. WILSON, AND JOHN E. CRAIGHEAD
`
`SUMMARY
`Chlorozotocin (chlorozocin, CLZ), the 2-chloroethyl an-
`alogue of streptozocin (STZ), was evaluated in three
`species of rodents. The drug is currently being used in
`phase II chemotherapeutic trials in man, and appears to
`be effective in the treatment of certain tumors. In our
`studies, hyperglycemia was induced in hamsters as
`early as 2 days after a single intraperitoneal (i.p.) injec-
`tion of 30-60 mg/kg and was most striking at 4 days.
`Greater concentrations of CLZ (>50 mg/kg) were re-
`quired to produce hyperglycemia in CD-1 mice. Degran-
`ulation and necrosis of beta cells developed in ham-
`sters and mice, whereas alpha and acinar cells of the
`pancreas revealed no morphologic changes. Hypergly-
`cemia was not induced in rats at any concentration
`tested; however, animals showed abnormal carbohy-
`drate tolerance after administration of 100 mg/kg CLZ
`(LD50 dosage).
`The nature of damage by CLZ to beta cells was in-
`vestigated both in vivo and in vitro. Pretreatment of
`hamsters with nicotinamide (500 mg/kg, i.p.) failed to
`alter the extent of CLZ-induced beta cell injury and as-
`sociated hyperglycemia, but decreased the amount pf
`beta cell necrosis and hyperglycemia in animals receiv-
`ing STZ. The nonmetabolizable sugar, 3-O-methylglu-
`cose (3-0-MG), and 3-aminobenzamide, an inhibitor of
`the nuclear enzyme, polyADPribose synthetase, pre-
`vented STZ-associated damage to beta cells in islet cell
`cultures, but only 3-0-MG reduced CLZ-induced toxic-
`ity. Thus, in comparison to STZ, CLZ appears to be a
`diabetogenic agent with different species specificity
`and alternative mechanisms of cytotoxicity. The glu-
`cose moiety of both drugs appears critical in the induc-
`tion of beta cell damage. DIABETES 1985; 34:602-10.
`
`From the Department of Pathology, University of Vermont College of Medicine,
`Burlington. Vermont; and the Department of Anatomy, University of South Al-
`abama, Mobile, Alabama (G.L.W.).
`Address reprint requests to B. T. Mossman, Ph.D., Department of Pathology,
`University of Vermont, Medical Alumni Building. Burlington, Vermont 05405-
`0068.
`Received for publication 4 July 1984 and in revised form 16 November 1984
`
`Streptozocin (STZ), a nitrosourea linked to position
`
`C2 of D-glucose, exhibits selective toxicity for beta
`cells and is diabetogenic in laboratory animals (re-
`viewed in ref. 1). It is unclear why STZ shows a
`specificity for the beta cell, although the presence of the
`glucose moiety is thought to be important. Several experi-
`mental observations support this statement. For example,
`STZ-induced diabetogenicity is ameliorated when 3-O-meth-
`ylglucose (3-0-MG) or 2-deoxyglucose, nonmetabolized an-
`alogues of glucose, are administered to rodents.23 Large
`amounts of glucose also prevent hyperglycemia and necrosis
`of beta cells after injection into rats of either a- or p-anomers
`of STZ.3 When injected into rodents at equivalent concentra-
`tions, STZ is found in insular tissue at 3.8-fold higher amounts
`than methylnitrosourea (MNU), the non-glucose-containing
`form of STZ.4 These data suggest that the structural glucose
`renders N-nitroso compounds with a unique specificity for
`beta cells.
`This study was undertaken to determine whether another
`glucose-containing N-nitroso compound, chlorozocin (CLZ),
`a drug used in human cancer chemotherapy,5 was diabe-
`togenic in rodents (Figure 1). Moreover, we were interested
`in determining possible mechanisms of action of CLZ. Data
`presented here indicate that the interaction of CLZ with the
`DNA of the beta cell differs markedly from that observed with
`the classical diabetogenic agent, STZ.
`
`MATERIALS AND METHODS
`Experimental protocol to evaluate the diabetogenicity of
`CLZ. Male golden Syrian hamsters (Bioresearch Institute,
`Cambridge, Massachusetts), CD-1 mice, and Sprague Daw-
`ley rats (both from Canadian Breeding Laboratories, St. Con-
`stant, Quebec) were used at 6-8 wk of age. Rodents (N =
`7-12 per treatment group) having free access to food and
`water were weighed before i.p. injection of CLZ (NSC
`#178248, the gift of Dr. Robert R. Engle, Developmental Ther-
`
`602
`
`DIABETES, VOL. 34, JUNE 1985
`
`NOVARTIS EXHIBIT 2064
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`
`

`

`T. MOSSMAN, G. L. WILSON AND J. E. CRAIGHEAD
`ChUOH
`
`H
`
`OH \ OH
`
`O NO
`
`H
`
`NHC-N-CH2CH,CI
`
`Chlorozotocin (CLZ)
`
`CH2OH
`
`Histopathology. Randomized groups of rodents (N = 4-6)
`were killed on the second, fourth, and seventh days of the
`experiments. Other animals were killed either when moribund
`or after 4 wk. The liver, heart, kidneys, lung, gallbladder, and
`pancreas were fixed in Bouin's solution and 5 |xm histologic
`sections were stained with hematoxylin and eosin (H and E).
`The pancreatic tissue was also examined by the peroxidase-
`antiperoxidase technique to evaluate insulin and glucagon
`in islet cells.7
`Islet cell cultures. Islets were isolated from the pancreata
`of neonatal Sprague-Dawley rats and prepared for culture
`as described previously.8 After an initial 16-h incubation in
`60-mm culture dishes to remove adherent fibroblastoid cells,
`suspensions of islet cells were plated into 16-mm culture
`wells (24 wells/plate). Twenty-four hours after plating, the
`monolayers were treated for 48 h with serum-free, cysteine-
`free medium to reduce contamination by fibroblastoid cells.9
`Thereafter, islet cells were maintained using Medium 199
`
`O H \ O H
`
`O NO
`NHC-N-CH3
`
`Streptozotocin (SZ)
`
`400.
`
`300.
`
`200.
`
`100.
`
`0
`
`A. 2 days
`
`//
`•
`0 20 30 40 50 60
`
`600.
`
`B. 4 days
`
`FIGURE 1. Structural formulas of chlorozocin (CLZ) and streptozocin
`(STZ).
`
`500 _
`
`400.
`
`300.
`
`200_
`
`100-
`
`0-
`
`! :
`
`4- •'•
`
`•
`
`'
`
`:
`
`.
`
`—1—II—1
`1
`20 30
`0
`
`1
`40
`
`1
`50
`
`1
`60
`
`500-
`
`C.
`
`7 days
`
`(50)
`
`400.
`
`300.
`
`200.
`
`100.
`
`(0)
`
`(0)
`
`:
`(0) 7.
`
`(66)
`
`(90)
`
`E oV
`
`)o
`
`o O o_
`
`o
`CD
`
`0
`
`—1—th
`0 20 30 40 50 60
`Chlorozotocin(mg/kg body weight)
`
`FIGURE 2. Nonfasting blood glucose concentrations in control and
`CLZ-treated hamsters bled at 2 (A), 4 (B), and 7 (C) days after injection.
`Numbers in parentheses in C represent the mortality (i.e., percentage
`of animals in each group) at 7 days.
`
`apeutics Program, Division of Cancer Treatment of the Na-
`tional Cancer Institute; 20-100 mg/kg) dissolved in a final
`volume of 0.1 ml dimethyl sulfoxide (DMSO). Control animals
`received 0.1 ml DMSO alone. In comparative studies, ham-
`sters were administered nicotinamide (Sigma Chemical Co.,
`St. Louis, Missouri; 500 mg/kg)6 i.p. in 0.8% NaCI, pH 7.4,
`10 min before injection of either CLZ (40 mg/kg) or STZ
`(obtained from Dr. W. E. Dulin, Upjohn Research Laborato-
`ries, Kalamazoo, Michigan; 100 mg/kg, dissolved in 0.1 M
`citrate buffer, pH 4.4). Solutions for injection into animals were
`monitored spectrophotometrically at 232 nm to determine the
`stability of CLZ and STZ in DMSO and citrate buffer, re-
`spectively.
`All animals were bled at 2, 4, and 7 days after injection.
`In hamsters and mice, nonfasting blood glucose concentra-
`tions were determined on samples of whole blood from the
`orbital sinus. Because nonfasting blood glucose concentra-
`tions were not elevated in rats after injection of CLZ (20-100
`mg/kg), glucose tolerance tests also were performed on an-
`imals of this species. Rats were fasted overnight (12 h) at 4
`days after injection of CLZ and a blood sample obtained
`before i.p. injection in double-distilled H2O (0.1 ml) of 2 mg/
`g glucose. Blood glucose then was determined at 30, 60,
`and 120 min.
`
`DIABETES, VOL. 34, JUNE 1985
`
`603
`
`NOVARTIS EXHIBIT 2064
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`
`

`

`changed at 48-h intervals thereafter, and the spent medium
`saved for determination of immunoreactive insulin (IRI).
`Radioimmunoassay for insulin. Insulin release during each
`48-h period was determined by a back-titration method10 us-
`ing 125l-labeled porcine insulin (Cambridge Medical Diag-
`nostics, Inc., Billerica, Massachusetts) diluted with cold por-
`cine insulin (kindly provided by Dr. Ronald Chance, Eli Lilly
`and Company, Indianapolis, Indiana) and guinea pig anti-
`bovine insulin serum (Linco Research, Inc., Eureka, Mis-
`souri).
`
`RESULTS
`Blood glucose. Hamsters became hyperglycemic (defined
`as 150 mg glucose/dl) 2 days after injection of CLZ at con-
`centrations ranging from 30 to 60 mg/kg (Figure 2). The
`diabetogenic effect in the hamster was dosage dependent
`and was most pronounced 4 days after injection. Some an-
`imals failed to exhibit elevated concentrations of glucose in
`the blood even when higher dosages were administered.
`Death of some hyperglycemic animals was observed be-
`tween 4 and 7 days (Figure 2C). All of the hamsters exhibiting
`elevated blood glucose concentrations after the administra-
`tion of 50 and 60 mg/kg CLZ were dead at 7 days.
`Hyperglycemia was observed in only a few mice after in-
`jection of CLZ at doses > 50 mg/kg (Figure 3). All diabetic
`animals died between day 4, the time when hyperglycemia
`was most prominent, and day 7. In contrast, blood glucose
`concentrations in rats were normal at 2, 4, and 7 days after
`administration of CLZ (20-100 mg/kg, data not shown). The
`relative insensitivity of the rat to the drug was confirmed by
`glucose tolerance tests on day 4. Abnormal carbohydrate
`tolerance was observed only after injection of 100 mg/kg
`CLZ, the approximate LD50 dosage in this species (Figure 4).
`
`«
`
`A
`
`^S
`
`CLZ
`100 mg/kg
`
`CLZ 80mg/kg
`CLZ 60mg/kg
`
`CONTROL
`
`200
`
`-
`
`150
`
`-
`
`100
`
`50
`
`-/
`
`10
`
`I*
`
`oo
`CO
`
`1
`
`1
`
`1
`
`60
`30
`0
`120
`Minutes After Injection of 2mg/gm Glucose
`
`1
`
`FIGURE 4. Carbohydrate tolerance in control and CLZ-treated rats. Four
`days after administration of CLZ, fasting rats were injected i.p. with 2
`mg/g glucose. Blood glucose concentrations were determined at time
`0 and 30, 60, and 120 min after injection of glucose.
`
`CHLOROZOCIN: A DIABETOGENIC ANALOGUE OF STREPTOZOCIN
`
`A.
`
`2 days
`
`500-
`
`400-
`
`300-
`
`200.
`
`100.
`
`*
`
`—i—if-
`0 20 30 40 50 60
`
`100
`
`B. 4 days
`
`••"
`
`-T-
`
`f*
`
`4.
`
`*.'
`
`1— 1
`—1—11—t
`1
`1
`20 30 40 50 60
`
`-ih
`
`1—"
`100
`
`C. 7
`
`days
`
`(10
`
`•:•
`
`::
`
`'
`
`( ~ }
`
`(7..0>
`
`'f
`
`(70)
`
`(70)
`
`0-
`
`500.
`
`400.
`
`300.
`
`200.
`
`100.
`
`o
`
`500.
`
`400-
`
`300-
`
`200-
`
`100-
`
`o 0
`
`)
`•<•
`
`ose o
`
`•oo
`&
`
`0-
`
`1
`
`1
`
`1
`
`1
`
`11
`
`1
`
`ii- 1
`—1
`100
`20 30 40 50 60
`0
`Chlorozotocin (mg/kg body weight)
`
`FIGURE 3. Nonfasting blood glucose concentrations in control and
`CLZ-treated mice. All animals were bled at 2 (A), 4 (B), and 7 (C) days
`after injection. Numbers in parentheses in C represent the mortality
`(i.e., percentage of animals in each group) at 7 days.
`
`(Flow Laboratories, McLean, Virginia) supplemented with
`10% fetal bovine serum, glucose (300 mM/L), and genta-
`mycin (1 mM/L). Fresh medium was added at 48-h intervals,
`and the spent medium saved for determination of immuno-
`reactive insulin (IRI) concentration by radioimmunoassay.8
`On the eighth day of culture, STZ and CLZ, alone and with
`3-0-MG or 3-aminobenzarnide (3-AB) were added to the
`monoiayers. In brief, a 1 mM concentration of STZ or CLZ
`(Kodak Company, Rochester, New York) was prepared in
`Hanks' balanced salt solution (HBSS) alone or containing the
`nonmetabolizable glucose analogue, 3-0-MG (Sigma), or the
`inhibitor of polyADPribose synthetase, 3-AB (Sigma). The
`STZ was initially dissolved in citrate buffer (pH 4.5) before
`dilution in HBSS, whereas CLZ was dissolved in DMSO. The
`culture medium was removed and the monoiayers washed
`two times with HBSS before exposure to drugs and their
`proposed inhibitors of action for 1 h. Control cultures received
`either 0.1% DMSO or citrate, or the drugs or the inhibitors
`alone. After 1 h, the test chemicals were removed, and the
`cultures replenished with fresh culture medium. Medium was
`
`604
`
`DIABETES, VOL. 34, JUNE 1985
`
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`
`

`

`acinar tissue was mildly congested and edematous. Mor-
`phologic changes were not found in alpha cells, and there
`was no evidence of degranulation.
`STZ (100 mg/kg) induced severe necrosis and degranu-
`lation of beta cells but had no apparent effect on alpha cells
`of the hamster (Figure 7, A-C). Beta cell degranulation was
`prominent in diabetic hamsters treated with STZ, whereas
`degranulation was less striking in CLZ-injected animals. Nic-
`otinamide prevented the necrosis of beta cells caused by
`STZ, although some degranulation still was evident (Figure
`7, D-F). In contrast, nicotinamide did not ameliorate CLZ-
`induced injury (Figure 7, G-l).
`Subtle cytologic changes were evident in the renal tubules
`of hamsters receiving 30 mg/kg of CLZ and frank epithelial
`necrosis was observed after injection of 50 mg/kg. Many of
`these animals had elevated concentrations of blood urea
`nitrogen, but creatinine was not increased. Focal myocardial
`necrosis and exudative interstitial pulmonary inflammation
`were'seen in animals receiving the higher dosages. Heart,
`lung, and kidney of mice and rats appeared to be more
`resistant to the cytotoxic effects of CLZ, but necrotizing renal
`tubular changes were observed in rats receiving 100 mg/kg
`CLZ. Although the cause of death in these rodents is un-
`certain, the lesions of renal tubules were doubtlessly signif-
`icant and are consistent with the findings of previous toxicity
`studies.11 In this regard, the nephrotoxicity of STZ12 and other
`nitrosoureas13 is well documented in humans. These drugs
`are eliminated by renal excretion.
`Effects of 3-0-MG and 3-AB on STZ- and CLZ-induced
`damage to cultures of beta cells. To determine whether the
`mechanism of cytotoxicity of CLZ is similar to that of the
`known beta cell toxin, STZ, monolayer cultures prepared from
`the islet cells of neonatal rats were exposed to the two drugs
`in combination with agents (3-0-MG and 3-AB) that are
`known to inhibit the effects of STZ on beta cells. The toxicity
`of CLZ could be blocked in part by the nonmetabolizable
`sugar 3-0-MG; however, greater concentrations (100 and
`150 mM) were required than were necessary to ameliorate
`the effects of STZ (Figure 8, A and B).
`The inhibitor of polyADPribose synthetase, 3-AB, pre-
`vented STZ-induced beta cell damage (Figure 9A) but was
`ineffective in reducing CLZ-induced toxicity (Figure 9B).
`
`TABLE 1
`Insular changes in animals receiving various "dosages of CLZ*
`
`CLZ
`(mg/kg)
`
`Days
`after
`inoculation
`
`Insular
`necrosis
`
`Beta cell
`degranulation
`
`±0
`
`00
`
`2 +
`2-3 +
`0-2 +
`
`2-4 +
`1 +
`0
`3 +
`2-3 +
`0-3 +
`
`00
`
`3
`3-4
`
`747477
`
`Mice
`
`Hamsters
`
`Rats
`
`100
`50
`25
`50
`40
`30
`50
`30
`
`Insular necrosis and degranulation of beta cells was graded using
`a 4-point ordinal scale in comparison with tissues from rodents re-
`ceiving 0.1 ml DMSO. Most severe alterations are scored 4 + ,
`whereas 0 indicates no change. A ± indicates equivalent positive
`and negative changes.
`
`B. T MOSSMAN. G L WILSON AND J E. CRAIGHEAD
`
`A
`
`A A4
`
`?
`
`A A
`
`
`
`* tAi
`
`J*
`
`0
`
`0 0
`
`0
`
`A. 7 dtyt
`
`*
`
`#
`
`500.
`
`400.
`
`300.
`
`200.
`
`100.
`
`0
`
`•D
`
`5C
`
`D 500-|
`
`SZ
`(100n>Q/kg)
`
`MC
`(soomg/kg) CLZ
`+
`(40mg/kg)
`(100mg/Kg)
`
`MC
`(5oo«ng/kg) Control
`+_
`(DMSO)
`(40mg/kg)
`
`FIGURE 5. Blood glucose concentrations at 2 (A), 4 (B), and 7 (C) days
`in control, STZ-, and CLZ-treated hamsters after pre-administration of
`nicotinamide. Numbers in parentheses in C represent the mortality (i.e.,
`percentage of animals in each group) at 7 days. No deaths occurred in
`control hamsters or in those administered nicotinamide and STZ.
`
`To determine whether preadministration of pyridine nu-
`cleotides5 prevents CLZ-induced diabetes, hamsters were
`administered nicotinamide (500 mg/kg) i.p. 10 min before
`injection of either CLZ (40 mg/kg) or STZ (100 mg/kg), the
`positive control for these experiments. Nicotinamide amelio-
`rated SZ-, but not CLZ-induced hyperglycemia at 2, 4, and
`7 days (Figure 5).
`Histopathology. Table 1 summarizes observations on the
`pancreata of animals receiving various dosages of CLZ. The
`extent of insular necrosis and degranulation of beta cells is
`expressed on an ordinal scale in which 0 indicates no alter-
`ation and 4+ denotes most severe changes. Rats failed to
`develop lesions of the islets, whereas insular changes were
`consistently observed in mice and hamsters injected with
`>50 mg/kg CLZ (Figure 6). The beta cells of these animals
`were often pyknotic, and the granules appeared to be
`clumped in the cytoplasm. Occasional cells were obviously
`necrotic and exhibited varying degrees of degranulation. In-
`flammation of the pancreata was not observed, although the
`
`DIABETES, VOL. 34, JUNE 1985
`
`605
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`

`

`CHLOROZOCIN: A DIABETOGENIC ANALOGUE OF STREPTOZOCIN
`
`* * • « •«
`
`v,v<* >-•/ •*;.
`
`«*.%r.-.V2*
`
`• «S ^'.is*-'
`
`Ar.J
`
`.«P^.
`
`FIGURE 6. Insular alterations in hamsters injected with CLZ (50 mg/kg, A-C, x 148) at 4 days postinjection. After administration of 30 mg/kg CLZ
`(D-F, x84), beta cells were affected less severely. Control hamsters receiving DMSO (G-l, x42) show no insular changes. In rats receiving 100
`mg/kg CLZ, islets appear distorted and filled with vacuolated cells (J-L, X140). Hematoxylin and eosin (A, D, G, and J). The peroxidase-anti-
`peroxidase technique for insulin (B, E, H, and K), and glucagon (C, F, I, and L) illustrates the affinity of CLZ for the beta cell.
`
`606
`
`DIABETES. VOL. 34. JUNE 1985
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`

`T MOSSMAN. G L WILSON AND J. E. CRAIGHEAD
`
`FIGURE 7. Morphologic damage to hamster beta cells by STZ (100 mg/kg) (A-C, x 140) is ameliorated after injection of nicotinamide (500 mg/kg),
`although degranulation of beta cells is still observed (D-F, x 148). In contrast, nicotinamide (500 mg/kg) does not prevent CLZ (40 mg/kg)-in-
`duced injury (G-l, x 100). All photographs were taken at 7 days after administration of drugs. Hematoxylin and eosin (A, D, and G), peroxidase-
`antiperoxidase technique for insulin (B, E, and H), and peroxidase-antiperoxidase technique for glucagon (C, F, and I).
`
`DIABETES, VOL. 34, JUNE 1985
`
`607
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`

`A.
`
`48 hr
`
`B.
`
`• c
`E3 50mM 3-0-MG
`• SZ 1mM
`1! SZ + 50 mM 3-0-MG
`
`96 hr
`
`144 hr
`
`• c
`E33 150mM 3-0-MG
`•I CLZ 1mM
`• CLZ + 100mM 3-0-MG
`^ CLZ -I- 150mM 3-0-MG
`
`u ju
`* <
`
`3 00
`
`250
`
`UJ Qj 200
`
`UJ Z
`
`150
`
`"" 100
`U. </>
`oz
`
`50
`
`0
`
`48 hr
`
`96 hr
`
`144 hr
`
`FIGURE 8. Effect of 3-O-methylglucose (3-0-MG) on (A) STZ- and (B)
`CLZ-induced damage to beta cells in vitro. Immunologically reactive in-
`sulin (IRI) release by beta cells in culture (N = 6/group) was docu-
`mented at intervals (48, 96, and 144 h) after addition of STZ and CLZ
`for 1 h in HBSS alone and containing 50, 100, and 150 mM 3-0-MG.
`Culture medium was collected from individual wells before addition of
`chemicals (pretreatment controls) and at 48, 96, and 144 h thereafter.
`Results are expressed as a percentage (mean ± SEM) of the IRI re-
`leased in each culture dish during the 48-h period (pretreatment con-
`trol) immediately preceding exposure to the chemicals.
`
`DISCUSSION
`Importance of glucose in tropism of CLZ and STZ for beta
`cells. To determine why STZ has a specificity for beta cells,
`a variety of agents has been administered to rodents in at-
`tempts to block STZ-induced diabetes.1261415 Because non-
`metabolizable analogues of glucose alleviate diabetes
`caused by STZ,23 the "popular" school of thought suggests
`that the glucose moiety accounts for the beta cell tropism of
`the drug. We addressed this question using a structural an-
`alogue of STZ. Like STZ, CLZ contains glucose, a component
`of the compound that apparently renders it tropic for the beta
`cell. The simultaneous addition of 3-0-MG with STZ or CLZ
`to cultures prevents drug-associated toxicity, suggesting a
`critical role of the glucose moiety in interaction with beta-cells.
`Experiments conducted both in vivo and in vitro support this
`hypothesis. For example, the non-sugar-containing structural
`analogues of STZ and CLZ, i.e., methylnitrosourea (MNU)
`and 1,3-bis(2-chloroethyl)-1-nitrosourea
`(BCNU), respec-
`tively, are not diabetogenic when administered to golden
`Syrian hamsters (Mossman, Jean, Muir, Orfeo, and Wilson,
`manuscript in preparation). STZ and CLZ also prove to be
`selectively toxic to cultured beta cells, whereas MNU and
`BCNU are cytotoxic to beta cells and pancreatic fibroblasts
`at equimolar concentrations. Cytotoxicity in these cultures
`
`was determined both by phase microscopy and by quanti-
`tating release of insulin into the culture medium.816
`Several phenomena could explain a tropism of glucose-
`containing N-nitroso compounds for the beta cell. Assuming
`that the glucose moiety of CLZ and STZ binds to receptors
`on the plasma membrane of the beta cell, beta cells might
`possess an increased number of glucose receptors in com-
`parison with other cell types. Alternatively, glucose transport
`in beta cells might be facilitated via a Na"-dependent sym-
`port system.17 In contrast, uptake of glucose-containing N-
`nitroso compounds by "nontarget" cells might occur by pas-
`sive transport. For example, studies by Lam and colleagues
`show that uptake of CLZ by human lymphoblasts in vitro is
`a passive process.18
`Mechanisms of cytotoxicity at the molecular level. On the
`basis of their experimental results using STZ and alloxan,
`Yamamoto and colleagues propose a common mechanism
`for chemically induced diabetes.1921 After addition of dia-
`betogenic drugs to islets in vitro, single-strand breakage of
`DNA occurs either by direct interaction of chemicals with
`DNA or via reactions with oxygen-free radicals elicited by the
`compounds. The activation of polyADPribose synthetase, a
`nuclear enzyme using NAD as a substrate, then ensues. As
`a result, a significant reduction in insular concentrations of
`NAD is observed, a change that can be prevented by inhib-
`
`A.
`
`B.
`
`<
`
`-I
`
`UJ UJ
`
`UJ Z
`
`300
`
`250
`
`200
`
`150
`
`100
`
`50
`
`0
`
`UJ 300
`< 250
`
`200
`
`150
`
`100
`
`ujz
`
`U_ 0)
`°S
`
`50
`o
`
`• c
`M 100mM 3AB
`^ S Z 1 mM
`• SZ + 10mM 3AB
`• SZ + 50mM 3AB
`Eg SZ + 100mM 3AB
`
`1
`
`48 hr
`
`96 hr
`
`144 hr
`
`DC
`E3 100mM 3AB
`E3 CLZ 1mM
`• CLZ + 10mM 3AB
`m CLZ + 50mM 3AB
`Eg CLZ + 100mM3AB
`
`1
`
`1
`
`48 hr
`
`96 hr
`
`144 hr
`
`FIGURE 9. Effect of 3-aminobenzamide (3-AB) on (A) STZ- and (B) CLZ-
`induced damage to beta cells in vitro. The results are expressed as de-
`scribed in the legend to Figure 8.
`
`608
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`B T. MOSSMAN. G L
`
`itors of polyADPribose synthetase, including nicotinamide
`and methylxanthines. These substances also prevent the de-
`velopment of STZ- and alloxan-induced diabetes in rats. Ya-
`mamoto's hypothesis is attractive, although a direct role of
`polyADPribose in beta cell injury and repair remains to be
`elucidated. Alternatively, our inability to show a protective
`effect of nicotinamide on CLZ-induced beta cell damage8
`and diabetes suggests that all diabetogenic chemicals do
`not act by this mechanism. The negative results obtained in
`vitro with use of 3-AB, another inhibitor of polyADPribose
`synthetase, supports our findings with nicotinamide. In recent
`work, we have shown that 3-AB also is a potent scavenger
`of the hydroxyl radical (OH").22 Thus, CLZ-induced injury to
`beta cells does not appear to be mediated by oxygen-free
`radicals, although some experimental studies suggest that
`active oxygen species are intrinsic to STZ-2324 and alloxan-
`associated diabetes.2526
`Species specificity of CLZ and STZ. In our studies, the
`susceptibility of three species of rodents to diabetogenic
`amounts of CLZ differed. Curiously, the order of susceptibility
`to CLZ was altered markedly from that reported for STZ.
`Hamsters were most susceptible to CLZ, followed by mice
`and rats. In contrast, the opposite pattern was observed with
`use of STZ. It is generally reported in the literature that 100-
`200 mg/kg STZ is required to produce overt diabetes in the
`hamster,2728 180-200 mg/kg is necessary for the mouse,2930
`and only 70-90 mg/kg is required for the rat.3132 Therefore,
`both hamsters and mice appear to be more sensitive to the
`diabetogenic effects of CLZ, while rats are exquisitely sen-
`sitive to STZ. Although rats injected with the drug are rela-
`tively resistant, a marked tropism of CLZ for rat beta cells in
`comparison with pancreatic fibroblasts can be demonstrated
`in vitro.8
`The studies described here were initiated to document the
`diabetogenicity of CLZ and compare its mode of action to
`that of STZ. The intriguing observations that CLZ-induced
`diabetes is not ameliorated by nicotinamide, that marked
`interspecies variation occurs in the response of rodents to
`CLZ in comparison with STZ, and the fact that, in comparison
`to STZ, CLZ does not appear to act through stimulation of
`polyADPribose synthetase or production of oxygen free rad-
`icals, suggest that CLZ and STZ may exert their effects on the
`beta cells via unrelated molecular mechanisms. Evidence
`presented here also supports the hypothesis that the pres-
`ence of glucose imparts a specificity of N-nitroso compounds
`for the beta cell of the pancreas. Although the intracellular
`stability of STZ and CLZ has not been studied in the beta
`cell, the spontaneous decomposition of STZ and CLZ under
`physiologic conditions (pH 7.4) is similar.33 Thus, it is im-
`probable that the contrasting biologic responses reported
`above are the result of differential breakdown of drugs.
`
`ACKNOWLEDGMENTS
`These studies were completed with the excellent technical
`assistance of Stephanie Bellomo, Joan Carrassi, Christopher
`Evans, Nancy Green, Nancy Patton, Perry Heffelfinger, and
`Julie Ruggeri.
`This study was supported by grant NIH PHS R23-25821
`from the NIAMDD, by grants NIH PHS RO1 ES03113 and
`
`ES03066 from NIEHS, and by grants from the Kroc Foun-
`dation and the American Diabetes Association.
`
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