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THE EFFECTS OF CISPLAIIN ON CARBOHYDRATE METABOLISM by Robin Sheryl Goldetein A DISSERIAIION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1982 ABSTRACT A THE EFFECTS or CTSPLATTN 0N CARBOHYDRATE METABOLISM by Robin Sheryl Goldstein This study was designed to characterize the effects of cisplatin (cis-DDP) and other divalent platinum compounds on carbohydrate meta- bolism and to elucidate the underlying biochemical and endocrine mecha- nisms. Glucose metabolism was evaluated in male F-344 rats treated with equimolar platinum doses of cis-DDP, trans-DDP or ammonium tetrachloro- platinate. A group of pair-fed controls was also studied to correct for the metabolic effects of reduced food intake. Administration of cis-DDP, but not trans—DDP or tetrachloroplatinate, resulted in fasting and nonfasting hyperglycemia. Impaired glucose utilization contributes in part to cis-DDP hyperglycemia, as indicated by a marked and persis- tent hyperglycemia following an exogenous glucose load in treated animals. Glucose intolerance was apparent 2 and 4, but not 7 and 14, days following cis-DDP (S'mg/kg) treaUment, indicating that it is a transient phenomenon. cis-DDP glucose intolerance was accompanied by a relatively deficient plasma insulin response. Neither trans-DDP nor tetrachloroplatinate impaired glucose tolerance at any time examined, suggesting that cis-DDB glucose intolerance is unique to the geometry Robin Sheryl Goldstein of the complex and is related to properties other than the presence of a divalent platinum atom. Mannitol pretreatment reduced cis-DDP nephrotoxicity and glucose intolerance, suggesting that impaired renal function by cis-DDP contri- butes in part to the observed glucose intolerance. Administration of other nephrotoxicants, however, did not uniformly impair glucose intol- erance. cis-DDP treated animals did not exhibit a blunted hypoglycemic re- sponse to exogenous insulin. Rather, decreased glucose utilization following cis-DDP treatment was associated with impaired insulin secre- tion, an effect not related to starvation, adrenaldmediated stress, hypo- kalemia or hypocalcemia. No histopathological damage of cis-DDP treated pancreata was evident. cis-DDP treatment resulted in hyperglucagonemia, an effect not associated with increased hepatic or renal gluconeogenic enzyme activi- ties. A.component in addition to the 3500 MN form of plasma immunoreac- tive glucagon was significantly elevated by cis-DDP treatment. cis-DDP hyperglucagonemia was reduced by ameliorating cis-DDP nephrotoxicity via mannitol pretreatment. Other nephrotoxicants also increased plasma glucagon. These results suggest that cis-DDP hyperglucagonemia is pro- bably related to decreased renal degradation of glucagon. In summary, the results of this study indicate that cis-DDP in- duces hyperglycemia in association with impaired glucose utilization; the latter is mediated by decreased insulin secretion. Robin Sheryl Goldstein of the complex and is related to properties other than the presence of a divalent platinum atom. Mannitol pretreatment reduced cis-DDP nephrotoxicity and glucose intolerance, suggesting that impaired renal function by cis-DDP contri- butes in part to the observed glucose intolerance. Administration of other nephrotoxicants, however, did not uniformly impair glucose intol- erance. cis-DDP treated animals did not exhibit a blunted hypoglycemic re- sponse to exogenous insulin. Rather, decreased glucose utilization following cis-DDP treatment was associated with impaired insulin secre- tion, an effect not related to starvation, adrenaldmediated stress, hypo- kalemia or hypocalcemia. No histopathological damage of cis-DDP treated pancreata was evident. cis-DDP treatment resulted in hyperglucagonemia, an effect not associated with increased hepatic or renal gluconeogenic enzyme activi- ties. A.component in addition to the 3500 MW form of plasma immunoreac- tive glucagon was significantly elevated by cis-DDP treatment. cis-DDP‘ hyperglucagonemia was reduced by ameliorating cis-DDP nephrotoxicity via mannitol pretreatment. Other nephrotoxicants also increased plasma glucagon. These results suggest that cis-DDP hyperglucagonemia is pro- bably related to decreased renal degradation of glucagon. In summary, the results of this study indicate that cis-DDP in- duces hyperglycemia in association with impaired glucose utilization; the latter is mediated by decreased insulin secretion. To my mother for teaching me the meaning of perseverance To my father for teaching me the values of an education To my brother and sister for their unconditional love and support and to my husband for making it all worthwhile ii ACKNOWLEDGEMENTS I would like to extend my warm appreciation to Drs. Jenny Bond, Gilbert Mayor and Jerry Book for their advice, criticisms, unselfish support, and above all, for their contributions to my professional and intellectual development. I would also like to express my gratitude to Drs. Robert Rosenbaum, Barnett Rosenberg and Dale Romsos for their advice and helpful discussions in preparation of this dissertation. I am indebted to Dr. Ronald Gingerich for performing the radio- immunoassays and to Adina Klein, Keith Rhinehart, Georgejeian Maderas and Beth Robinson for their excellent technical assistance. I am also especially grateful to Diane Hummel for her expert assistance in typing this dissertation. ‘ I would like to offer special thanks to all of the members of Dr. Hook's lab with whom I have had the pleasure to work. Finally, I would like to thank my friends, Sue Ford, Bill Evers and Byron Reorde- ‘wier for making my graduate career a pleasurable and memorable one. 111 TABLE OF CONTENTS Page DEDICATION ii ACKNOWLEDGEMENTS iii LIST OF TABLES vii LIST OF FIGURES viii INTRODUCTION 1 Cisplatin 2 History and Development 2 Chemistry and Mbde of Action 3 Pharmacokinetics: Plasma Clearance and Protein Binding 13 Pharmacokinetics: Excretion 16 Pharmacokinetics: Tissue Distribution 18 Pharmacokinetics: Biotransformation 20 Toxic Effects of cis-DDP: Kidney 21 Modulation of Kidney Toxicity 27 Other Toxic Effects 28 Pharmacology and Toxicology of Other Platinum Compounds 30 Endocrine Regulation of Glucose Homeostasis 30 General Aspects of Biological Actions of Insulin and Glucagon 30 Effects of Heavy Metals on Carbohydrate Metabolism 33 Rationale e 36 MATERIALS AND METHODS 37 General 37 Animals and Diet 37 Platinum Treatment 37 Glucose Tolerance Tests 38 Insulin and Glucagon Determination 38 Specific Protocols 40 Effect of Platinum on Serum.Glucose in Nonfasting Ani- mals 40 Effect of cis—DDP on Serum Glucose in Fasting Animals-- 40 Effect of cis-DDP on Glucose Tolerance 41 iv TABLE OF CONTENTS (continued) Page Effect of Platinum on Glucose Tolerance: Time Course and Dose Response 41 Effect of Platinum on Selected Organ Weights and Serum Amylase Activity 41 Histopathological Examination of Selected Tissue from cis-DDP Treated Animals 42 Role of Adrenal Glands in cis-DDP Glucose Intolerance-- 43 Role of the Kidney in cis-DDP Glucose Intolerance ----- 43 Effiect of Platinum on In Vivo Renal Function-- 43 Effect of Platinum on Renal Organic Ion Transport- 44 Effect of cis-DDP on Renal Clearance of Inulin and PAR 45 Effect of Mannitol Pretreatment on cis-DDP Glucose Intolerance 46 Effect of Selected Nephrotoxicants on Glucose In- tolerance 47 Biochemical Correlates of cis-DDP Glucose Intolerance-- 48 Effects of cis-DDP on Serum Sodium, Potassium, Calcium and Phosphorus 48 Gluconeogenic Enzyme Activity 48 Endocrine Correlates of cis—DDP Glucose Intolerance--- 50 Plasma and Pancreatic IRI and IRG Concentrations-- 50 Components of Plasma IRC— 51 Glucagon Resistance . 52 Insulin Resistance ' 52 Statistical Analyses 52 RESULTS 53 Food Intake 53 Body weight 53 Effect of Platinum on Serum.Glucose in Nonfasting Animals--- 59 Effect of cis-DDP on Serum Glucose in Fasting Animals--—--- 59 Effect of cis-DDP on Glucose Tolerance 65 Effect of Platinum on Glucose Tolerance: Time Course and Dose Response 70 Effect of cis-DDP on Plasma IRI and IRC 77 Organ‘Weights 96 Role of Adrenal Glands in cis-DDP Glucose Intolerance----- 96 TABLE OF CONTENTS (continued) Page Role of the Kidneys in cis-DDP Glucose Intolerance lOl Renal Function Following Platinum Treatment 101 Urine Volume and Fluid Intake 101 Urine Glucose ‘ 107 Urine Osmolality 107 Sodium and Potassium~ ll6 Blood Urea Nitrogen 127 Organic Ion Accumulation 127 Renal Clearances of Inulin and PAH 134 Histopathological Effects of cis—DDP on the Kidney 134 Effect of Mannitol Pretreatment on cis-DDP Glucose Intolerance 140 Effect of Other Nephrotoxicants on Glucose Tolerance- ------ 140 Effect of cis-DDP on Pancreatic Structure and Function---- 149 Biochemical Correlates of cis-DDP Glucose Intolerance ------ 149 Serum Sodium, Potassium, Calcium and Phosphorus------ 149 Fasting Plasma Glucose and Hepatic and Renal Gluconeo- genic Enzyme Activity 149 Endocrine Correlates of cis-DDP Glucose Intolerance-- -- 156 IRI and IRG Concentrations in Plasma and Pancreas-—--- 156 Components of Plasma IRG 156 Glucagon Sensitivity 162 Insulin Sensitivity 162 DISCUSSION 169 SUMMARY 192 BIBLIOGRAPHY 196 vi Table 10 11 12 13 LIST OF TABLES Page Effect of Platinum Treatment on Daily Food Intake (g food/24 hours) 56 Effect of Platinum Treatment on Percent of Initial Body Weight 60 Effect of cis-DDP on 2 Plasma Immunoreactive Insulin (IRI) Response to Glucose— 95 Effect of Platinum Treatment on Adrenal Weight/Body Weight (AW/BW), Kidney Weight/Body Weight (KW/BW) and Liver Weight/Body Weight (LW/BW) 97 Effect of Bilateral Adrenalectomy (Adx) on Plasma Immunoreactive Insulin (IRI) and Glucagon (IRG) in cis- DDP Treated Rats 102 Effect of cis—DDP (5 mg/kg) on Renal Clearance of ' Inulin (cinulin) and PAH (CPAH) 135 Effect of Mannitol Pretreatment on Blood Urea Nitrogen (BUN) and Plasma Immunoreactive Glucagon (IRG) of cis- DDP Treated Rats 140 Effect of Selected Nephrotoxicants on Renal Function and Glucose Tolerance 146 Effect of cis—DDP on Serum Sodium, Potassium, Calcium, and Phosphorus 156 Selected Biochemical and Endocrine Measurements of Glucose Metabolism Following cis-DDP Treatment-------- 155 Components of Plasma Immunorsactive Glucagon (IRG) Following cis-DDP Treatment 161 Effect of Selected Nephrotoxicants on Plasma Immuno- reactive Glucagon (IRG) 163 Effect of cis-DDP on Glycemic Response to Exogenous Glucagon 164 vii Figure 10 11 12 LIST OF FIGURES Page cis-Dichlorodiammineplatinum (II) 4 Aquation of cis-DDP 5 Proposed differences in binding of cis-DDP (top) and trans-DDP (bottom) to guanine 10 Effect of cis—DDP on daily food intake 54 Effect of cis-DDP on body weight and Effect of reduced ‘food intake (pair-feeding) on body weight 57 Effect of cis-DDP (top), trans-DDP (center) and ammo- nium tetrachloroplatinate (bottom) on serum glucose concentration in nonfasting animals 61 Effect of cis—DDP on serum glucose concentration in fasting animals 63 Effect of cis-DDP on plasma glucose concentration in the fasting state (0) and 15, 30, 60, and 120 minutes following a glucose load (2 g/kg, i.p.) 66 Effect of cis-DDP on plasma immunoreactive (IRI) in the fasting state (0) and 15, 30, 60, and 120 minutes following a glucose load (2 g/kg, i.p.) Effect of cis-DDP on plasma immunoreactive glucagon (IRG) in the fasting state (0) and 15, 30, 60, and 120 minutes following a glucose load (2 g/kg, i.p.) Effect of cis—DDP (left) and trans-DDP (right) on glucose tolerance two days following DDP administration Effect of cis-DDP (left) and trans-DDP (right) on glu- cose tolerance four days following DDP administration—- viii 67 71 73 75 LIST OF FIGURES (continued) Figure 13 14 15 16 17 18 19 20 21 22 23 24 Effect of cis-DDP (left) and trans-DDP (right) on glu- cose tolerance seven days following DDP administration- Effect of cis-DDP (left) and trans-DDP (right) on glu- cose tolerance fourteen days following DDP administra- tion Effect of ammonium tetrachloroplatinate on glucose to- lerance two (left) and four (right) days following treatment Effect of ammonium tetrachloroplatinate on glucose to- lerance seven (left) and fourteen (right) days followb ing treatment Effect of cis-DDP on plasma immunoreactive insulin (IRI) (left) and glucagon (IRG) (right) two days fol- lowing treatment Effect of cis-DDP on plasma immunoreactive insulin (IRI) (left) and glucagon (IRG) (right) four days fol- lowing drug treatment Effect of cis-DDP on plasma immunoreactive insulin (IRI) (left) and glucagon (IRG) (right) seven days fol- lowing drug treatment Effect of cis-DDP on plasma immunoreactive insulin (IRI) (left) and glucagon (IRG) (right) fourteen days following drug treatment Effect of bilateral adrenalectomy (Adx) on cis-DDP in- duced glucose intolerance Effect of cis-DDP on daily water intake (left) and urine volume (right) Effect of trans-DDP (left) and ammonium.tetrachloro- platinate (right) on daily water intake and urine vol- ume Effect of cis-DDP on concentration (left) and daily excretion (right) of urinary glucose Page 78 80 82 84 86 88 90 92 99 103 105 108 LIST OF FIGURES Figure 25 26 27 28 29 30 31 32 33 34 35 36 37 Effect of trans-DDP on concentration (left) and daily excretion (right) of urinary glucose Effect of ammonium tetrachloroplatinate on concentra- tion (left) and daily excretion (right) of urinary glucose Effect of cis-DDP on urine osmolality (left) and daily urine osmolar excretion (right) Effect of trans-DDP (left) and ammonium tetrachloro- platinate (right) on urine osmolality and daily urine osmolar excretion Effect of cis-DDP on concentration (left) and daily excretion (right) of urinary sodium= Effect of trans-DDP (left) and ammonium tetrachloro- platinate (right) on concentration and daily excretion of urinary sodium Effect of cis-DDP on concentration (left) and daily excretion (right) of urinary potassium— Effect of trans-DDP (left) and ammonium tetrachloro- platinate (right) on concentration and daily excretion of urinary potassiumr Effect of cis-DDP (top), trans-DDP (center) and ammo- nium tetrachloroplatinate (bottom) on blood urea nitro- gen (BUN) concentrations PAH and TEA accumulation (slice/medium) (S/M) by renal cortical slices four days following cis-DDP treatment- Effect of addition of cis-DDP to incubation medium on PAH and TEA accumulation (S/M ratio) by renal cortical slices Renal tissue four days following cis-DDP treatment---- Renal tissue four days following cis-DDP treatment-—- Page 110 112 114 117 119 121 123 125 128 130 132 136 138 LIST OF FIGURES (continued) Figure 38 39 40 41 42 43 44 45 46 Page Effect of mannitol pretreatment on cis-DDP induced glucose intolerance 142 Effect of mannitol pretreatment on plasma immunoreac- tive insulin (IRI) and glucagon (IRG) concentrations of cis-DDP treated animals 144 Effect of selected nephrotoxicants on glucose tolerance 147 Pancreatic tissue from rats treated with cis-DDP- ----- 150 Serum amylase activity four days following cis-DDP treatment 152 Pancreatic concentrations of immunoreactive insulin (IRI) four days following cis-DDP treatment 157 Pancreatic concentrations of immunoreactive glucagon (IRG) four days following cis-DDP treatment 159 Area under the glucose curve following exogenous gluca- gon (1 mg/kg, ip) administration in cis-DDP treated animals 165 Effect of cis-DDP on glycemic response to exogenous insulin (0.28 IU/kg, ip) 167 xi INTRODUCTION Cisplatin (cis-dichlorodiammineplatinum) is the first of a group of platinum coordination complexes to be introduced as a cancer chemo- therapeutic agent. This agent, both alone or in combination chemo- therapy, has been demonstrated to be effective in the treatment of testicular, ovarian, bladder, prostate, lung, and head and neck cancers. However, clinical use of cisplatin is often limited by its toxic side effects, including renal toxicity, gastrointestinal disturbances, myelosuppression and ototoxicity (Madias and Harrington, 1978; Von Hoff 3.5 _a_]_._. , 1979). In addition, administration of cisplatin has also been reported to induce hyperglycemia in laboratory rats (Kociba and Sleight, 1971). Several divalent metal ions are known to induce hyperglycemia by affect- ing pancreatic islet function (Ghafghazi and Mennear, 1975; Horak and Sunderman, 1975a,b); therefore, a divalent platinum compound such as cis-DDP may similarly impair carbohydrate metabolism. Although an extensive number of studies have characterized specific organ toxicities of cisplatin, very little is known about its effects on intermediary metabolism. The primary objective of the research reported herein is to elucidate the effects of cisplatin and other divalent platinum compounds on carbohydrate metabolism. Implicit in this objective ‘ 2 is an assessment of the biochemical and endocrine correlates of platinum toxicity. CISPLATIN History and Development The biological effects of platinum complexes were first noted in Escherichia coli (E. coli) exposed to an electrical field generated across two platinum electrodes (Rosenberg 35 51,, 1965). VOltage applied across these electrodes resulted in a changed appearance of E. coli from its normal rod shape to an elongated filament form (Rosenberg 535 g” 1965). Further investigations revealed that one or more long- 1ived chemical species were produced by the electrical current and induced bacterial filamentation by inhibiting cellular division but not cellular growth (Rosenberg 23 51., 1965). These chemical by-products were later identified as cis-dichlorodiammdneplatinum (II) (cis-DDP) and cis-tetrachlorodiammineplatinum (IV). Further studies established that neutral platinum complexes, such as cis-DDP, not only specifically inhibit cell division without affecting growth rate but derepress latent viral genomic information in lysogenic bacteria (Reslova, 1972), an effect commonly observed with other anti-tumor agents. On this basis, Rosenberg postulated that neutral platinum comp plexes, by virtue of their action on bacteria cells, may similarly inhibit cell division in rapidly growing cancer cells. This postulate was confirmed by the successful tumor regression of solid sarcoma 180 in mice by cis-DDP treatment (Rosenberg £5 31,, 1969). Since then, the broad spectrum anti-tumor activity of cis-DDP has been well 2 is an assessment of the biochemical and endocrine correlates of platinum toxicity. CISPLATIN History and Development The biological effects of platinum.complexes were first noted in Escherichia coli (E. coli) exposed to an electrical field generated across two platinum electrodes (Rosenberg gtflgl,, 1965). Voltage applied across these electrodes resulted in a changed appearance of E. coli from its normal rod shape to an elongated filament form (Rosenberg ‘25“313, 1965). Further investigations revealed that one or more long- 1ived chemical species were produced by the electrical current and induced bacterial filamentation by inhibiting cellular division but not cellular growth (Rosenberg 35 51,, 1965). These chemical by-products were later identified as cis—dichlorodiammineplatinum (II) (cis-DDP) and cis-tetrachlorodiammineplatinum (IV). Further studies established that neutral platinum complexes, such as cis—DDP, not only specifically inhibit cell division without affecting growth rate but derepress latent viral genomic information in lysogenic bacteria (Reslova, 1972), an effect commonly observed with other anti-tumor agents. On this basis, Rosenberg postulated that neutral platinum come plexes, by virtue of their action on bacteria cells, may similarly inhibit cell division in rapidly growing cancer cells. This postulate was confirmed by the successful tumor regression of solid sarcoma 180 in mice by cis-DDP treatment (Rosenberg 35 31,, 1969). Since then, the broad spectrum anti-tumor activity of cis-DDP has been well 3 established as indicated by its effectiveness against transplantable, chemical and viral induced tumors, slow as well as rapidly growing tumors, and disseminated as well as solid tumors (Rosenberg, 1978a). Currently, cis-DDP, either alone or in combination chemotherapy, is primarily used in the management of testicular and ovarian cancers, although its use has also been extended to treating cancers of the head and neck, bladder, prostate and lung. Chemistry and Mode of Action cis-DDP is a square planar coordination complex containing a central platinum atom surrounded by two chloride atoms and two ammonia moieties (MW - 300.1) (Figure 1). It has a melting point of approximately 270°C and is soluble in aqueous vehicles at maximum concentrations of 1 mg/ml. cis-DDP appears to be highly reactive in in ziggg_systemsn Several reports clearly demonstrate that aqueous solutions of cis-DDP degrade via nucleophilic displacement of the chloride_ligands by water molecules (Greene E£“2£:: 1979; Lee and Martin,'l976), yielding various combina- tions of equated and/or hydroxylated species of the parent drug (Figure 2). The predominating species appear to be determined by pH; the dihydroxy species are formed at a pH greater than 7.37 while the diaquo species are formed at a pH less than 5.51. Ighzigrg, the lability of the chloride ligands appears to be re- lated to the chloride concentration of the solvent such that addition of chloride to aqueous solutions of cis-DDP results in an increased proportion of intact cis—DDP molecules (Greene g£_§1,, 1979). Converse— ly, preparation of cis-DDP solutions in water with no added chloride c: / NH3 \P / \c. "”3 . cis -DD P. Figure l . cis-Dichlorodiamineplatinum (II) . .manlmuo mo coauo=v< .N enough a £96 .923... / o Jouscmnzz... flu «$122.... . “Archers... ”fizgousmfze... / + of / «.o«:.«.n:z....§ of «... 6 increases the aquated/hydroxylated complexes (Greene ggba13, 1979). Furthermore, the reversibility of this reaction has been indicated by the reformation and stabilization of cis-DQP following addition of 0.9% NaCl to an already degraded sample of cis-DDP (Greene §£_§l,, 1979). Extrapolating these E 32533 data to the in v_i_\2_ disposition of cis-DDP has led to the hypothesis that cis-DDP exists as an un- charged neutral complex in extracellular fluid (ECF) since the chlor- ide concentration of ECF (=112 mM) is sufficiently high to stabilize the complex and prevent hydrolysis. However, the markedly lower intra- cellular concentration of chloride (=4 mM) facilitates the displace- ment of chloride by water molecules, yielding a positively charged aquated/hydroxylated platinum complex (Rosenberg, 1978). It is the equated/hydroxylated intracellular complex which is believed to be active in inhibiting DNA synthesis. The screening of cis-DDP and other platinum analogues for anti- tumor activity has provided valuable information regarding structure- activity relationships. One striking feature of these studies is that the geometry of these complexes is clearly a determinant of anti-tumor activity, as indicated by the marked anti-tumor activity of the cis, but not the trans, configuration of DDP (Leonard 35 51., 1971). Furthermore, it has been suggested by Cleare and Hoeschele (1973) that complexes exhibiting anti-tumor activity share definite chemical properties: (1) they are electrically neutral (although the ultimate active form may be charged following ligand exchange ig;xi!g), (2) they exchange only some of their ligands rapidly, (3) the complexes are 7 either square planar or octahedral, (4) they contain a pair of cis- monodentate (or one bidentate) leaving groups (corresponding trans isomers are generally inactive), (5) the leaving groups are spaced approximately 3.3 A apart, and (6) ligands trans to the leaving groups 'appear to be strongly bound and inert. The lability of the cis leaving groups is believed to be important in determining anti-tumor activity and is consistent with the pbstulate that the anti-tumor effects of cis-DDP are mediated by a modified form of cis-DDP. The equated/hy- droxylated forms of cis-DDP are electrophilic and may react at nucleo- philic sites of DNA. That the intact parent drug does not react with DNA is indirectly suggested by the reduced affinity of cis-DDP for DNA when chloride concentrations are increased in 33552 (Horacek and Drobnick, 1971), an effect presumably related to decreased formation of the aquated/hydroxylated platinum species. Several lines of evidence indicate that the primary intracellular target of cis-DDP (or metabolite) is DNA. In bacteria, cis-DDP inhi- bits cell division (Rosenberg g£_§1,, 1965), inactivates transforming DNA (Munchausen, 1974) and DNA containing bacteriophage (Shooter 25 21,, 1972), and reduces viability of DNA repair—deficient mutants (Beck and Brubaker, 1973). In addition, cis-DDP has been reported to be mutagenic (Beck and Brubaker, 1975), as indicated by its.ability to ' induce base-pair substitutions (Mbnti-Bragadin'ggual., 1975) and frame shift mutations (Andersen, 1979), In eukaryotic cells, cis-DDP selec- tively inhibits DNA synthesis in cultured human embryonic AV cells 3 (Harder and Rosenberg, 1970), Erlich ascites tumor cells (Howle and 8 Gale, 1970) and Chinese hamster cells (Van den Berg and Roberts, 1976). More platinum molecules are bound per molecule of DNA than per mole- cule of RNA or protein in HeLa cells treated with cis-DDP (Pascoe and Roberts, 1974), further indicating that DNA is a likely target for ' cis-DDP. In addition, cis-DDP also appears to be mutagenic in eukary- otic cells (Zwelling‘g£_§1,, 1979). The binding of cis-DDP (or metabolite) to DNA.may occur in several possible ways, including interstrand crosslinking (crosslinking of two bases on opposite DNA strands), mono- and bi-functional chelation to bases, and/or crosslinking between RNA and protein. Several studies have suggested that interstrand crosslinking or crosslinking to protein does not play a critical role in cis-DDP cytotoxicity (Pascoe and Roberts, 1974; Shooter gghal., 1972).. Rather, it has been proposed that intrastrand crosslinking represents a likely mechanism of plati- nuvaNA.interaction. Indirect in gi!g_evidence supporting this concept has been provided by Roos (1977) who measured the binding of an inter- calating agent (9-aminoacridine) (9AA) to DNA, with or without platinum pretreatment, as a means of assessing DNAvplatinum interaction. Roos (1977) postulated that if cis-DDP (or metabolite) is avidly cross- linking two bases 3.4 A apart on a single strand, then this intrabase separation will be preserved and prevent intercalation by 9AA. Con- versely, interstrand crosslinking by cis-DDP (or metabolite) could still allow 9AA intercalation. Binding of 9AA to DNA, which increases base separation from 3.4 to 6-7 A, is indeed decreased by prior reac- tion of DNA with active platinum compounds, indicating the likelihood of intrastrand crosslinking to DNA as a primary mode of interaction 9 by active platinum complexes. Unfortunately, the platinum complex under investigation was dichloro(ethylenediammine)platinum (II) and not cis-DDP. Although this compound and cis-DDP have similar anti- tumor activities it would have been of interest to examine the effects of cis-DDP on 9AA binding to DNA. It is generally believed that active platinum complexes react pri- marily with purine bases, rather than sugar-phosphate moieties of DNA. Furthermore, it has been suggested that DNA intrastrand crosslinking may involve adjacent guanine bases and may represent an important lesion induced by cis-DDP (or metabolite) (Kelman 22.2l3: 1977). In this re- gard, several investigators have sought to explain the stereospecific anti-tumor activity of cis-DDP complexes by invoking a possible reac- tion site of DNA specific to the cis configuration. Guanine has been postulated as a likely reaction site since a cis geometry would facili- tate formation of a closed ring bidentate chelate structure with the N7 and 06 sites of guanine. In contrast, the trans isomer may pri- marily form a monodentate ligand attachment at the N7 site of guanine; its geometry prohibiting formation of a closed ring chelate (Figure 3) (Goodgame gt“51,, 1975; Macquet and Theophanides, 1976). Chelation at the 06 site of guanine may be particularly signifi- cant to the stereospecific effects of DDP complexes on DNA integrity since other adducts (i.e., alkylated products) at this specific site have been causally linked to mutagenesis. On this basis, Rosenberg (1975; 1978b) has suggested that a "platinated" adduct, presumably at the 06 site of guanine, becomes fixed as a mutation, modifying the DNA 9 by active platinum complexes. Unfortunately, the platinum complex under investigation was dichloro(ethylenediammine)platinum (II) and not cis-DDP. Although this compound and cis-DDP have similar anti— tumor activities it would have been of interest to examine the effects of cis-DDP on 9AA binding to DNA. It is generally believed that active platinum cmmplexes react pri- marily with purine bases, rather than sugar-phosphate moieties of DNA. Furthermore, it has been suggested that DNA intrastrand crosslinking may involve adjacent guanine bases and may represent an important lesion induced by cis-DDP (or metabolite) (Kelman.g£_al,, 1977). In this re- gard, several investigators have sought to explain the stereospecific anti-tumor activity of cis-DDP complexes by invoking a possible reac- tion site of DNA specific to the cis configuration. Guanine has been postulated as a likely reaction site since a cis geometry would facili- tate formation of a closed ring bidentate chelate structure with the N7 and 06 sites of guanine. In contrast, the trans isomer may pri- marily form a monodentate ligand attachment at the N7 site of guanine; its geometry prohibiting formation of a closed ring chelate (Figure 3) (Goodgmme‘ggna1., 1975; Macquet and Theophanides, 1976). Chelation at the 06 site of guanine may be particularly signifi- cant to the stereospecific effects of DDP complexes on DNA integrity since other adducts (i.e., alkylated products) at this specific site have been causally linked to mutagenesis. On this basis, Rosenberg (1975; 1978b) has suggested that a "platinated" adduct, presumably at the 06 site of guanine, becomes fixed as a mutation, modifying the DNA 10 Figure 3. Proposed differences in binding of cis-DDP (top) and trans-DDP (bottom) to guanine. Binding of cis-DDP to guanine results in formation of a closed ring bidentate chelate structure with attachments at N7 and 06 sites of guanine. The geometry of trans-DDP prohibits formation of closed ring chelate. ll Figure 3 12 template and resulting in selective inhibition of DNA replication by cis, but not trans, DDP. To account for the selective cytotoxicity to cancer cells, Rosenberg (1978b) has further postulated that the platinated adduct may be removed and repaired only by normal tissue; cancerous tissue may lack the necessary repair mechanisms. Along these lines, recent evidence suggests the presence of repair mechanisms for cis-DDP induced DNA damage: (1) cells harvested from Xeroderma pigmen- tosum patients are more sensitive to cytotoxicity of cis-DDP than normal fetal lung cells, indicating that deficient DNA repair mechanisms enhance cis-DDP cytotoxicity, (2) platinum is removed from DNA of expo- nentially growing Chinese hamster cells at a constant rate with a t% of 28 hrs and (3) there is a positive correlation between the extent of platinum binding to DNA and cell death, indicating that cell death may be‘a consequence of unexcised‘platinum lesions of DNA (Roberts and Fraval, 1980). Another possible mechanism of the selective destruction of cancer cells has been proposed by Rosenberg (1972) as the enhanced antigeni- city hypothesis. This hypothesis proposes that cis-DDP enhances immunological response of the host to cancer cells. Cancer cells produce new antigens at the cell surface and generate a host immune reaction resulting in destruction of these cells. According to Rosen- berg (1972), these strong antigens of cancer cells are masked by weaker antigens, such as nucleic acids, and thereby escape host immune re- sponses. Inasmuch as these cell surface nucleic acids are no longer apparent following cis-DDP treatment, it has been postulated that cis- DDP may disrupt antigen masking and expose the underlying stronger l3 antigens of cancer cells to stimulate an immune response. Direct evi- dence substantiating this attractive hypothesis is lacking. Pharmacokinetics: Plasma Clearance and Protein Binding Similar patterns of plasma clearance of cis-DDP have been observed. In all species studied to date, plasma concentrations following intra— venous administration of cis-DDP decline in a biphasic manner, charac- terized by a rapid a t% and a more prolonged B t%; however, the specific plasma clearance rates differ quantitatively according to species, dose, route of drug administration and time points analyzed. For example, the estimated ck of cis-DDP in the mouse is 1.6-5 hours (Hoeschele and Van Camp, 1972). In the rat, Litterst g£_al. (1976b) initially reported the a t% as 43 minutes and the 8 t% as 2 days, and reported in a later study, the a tk and 8 t% as 9.7 minutes and 35.7 hours, respectively, using more time points for their analysis (Litterst gghal., 1979). In dogs, the initial rapid phase of plasma clearance of cis-DDP is similar to rats; however, the more prolonged B t% approximates 4-5 days in dogs (Litterst gghal,, 1976a). In humans, a biphasic mode of plasma clear- ance of platinum has also been observed, with an initial t% of 25-49 minutes and a secondary phase of 58-73 hours (DeConti 35 51,, 1973). These findings are consistent with a recent clinical study by Gormely 35 '51, (1979); however, in conducting experiments over a 21 day period, these investigators also observed a slow tertiary phase of plasma platinum clearance (Gormley g£_al,, 1979). All of these studies, how- ever, are difficult to interpret since only total platinum concentra- tions were determined and therefore may not reflect clearance of intact cis-DDP. 14 Plasma protein binding of platinum occurs within the first two hours of drug administration and may account for as much as 90% of administered dose (DeConti £5 21., 1973). Further analysis of platinum concentrations in blood have revealed its presence in at least three distinct pools: a free, protein bound and erythrocyte bound fraction (Hanaka and wolf, 1980). Inasmuch as total serum platinum concentra- tions apparently does not reflect active drug concentrations (Gormley ‘25 El): 1979), meaningful pharmacokinetic studies must discriminate between the disposition of free circulating drug from that of total drug (bound plus unbound) concentrations. Recent analytical developments utilizing centrifugal ultrafiltration and atomic absorption spectro- scopy have provided the necessary means to distinguish free filterable platinum from protein bOund species following cis-DDP administration (Bannister 3331., 1977). The kinetics of protein binding of cis-DDP are characterized by a first order reaction with a t% of 220 minutes, as indicated by the in ‘git£2_disappearance of platinum with time in ultrafiltrates of solutions containing human serum and cis-DDP (Gormley at 51,, 1979). Similar studies suggest that the rate of protein binding to platinum is too slow to be accounted for by affinity reactions (i.e., hydrogen bonding, van der waals forces, etc.) (Long and Repta, 1981). Although the plasma clearance of non-protein bound platinum appears to be biphasic in humans, the terminal half-lives for filterable pla- tinum (32-53 minutes) are considerably shorter than those for total platinum (>67 hours) (Gormley 25 El}: 1979; Himmelstein 32 a1., 1981; 15 Patton g£_al3, 1978). The nature of the protein-platinum reaction has been recently evaluated by equilibrium dialysis in which human plasma or 22 serum albumin in phosphate buffer was equilibrated with cis-DDP (Repta and Long, 1980). Theoretically, if protein-platinum reaction is reversible, then equilibrium would be established and characterized by equivalent p1atinum.concentrations on either side of the dialysis membrane. Results, however, indicate no evidence of dialysis for periods of up to 24 hours, suggesting that the rate of release of p1atinum.from the protein-platinum complex is slow and the reaction is not readily reversible (Repta and Long, 1980). Long and Repta (1981) therefore suggested that the protein-platinum reaction is pro- bably one of covalent bond formation. In most of these studies, analytical techniques are based upon the detection and quantification of the platinum atom; i.e., atomic absorption, Xpray fluorescence, neutron activation or isotopic assays. In all cases, it has been assumed that elemental or radiolabeled pla- tinum represents the parent drug, thereby precluding the possibility of biotransformation. Thus, the relatively nonspecific nature of these techniques provides little information regarding the pharmaco- kinetics specific to the parent drug or its metabolic products. Howh ever, recently Chang 55.21, (1978) reported a method capable of speci- fically isolating and quantifying cis-DDP in the presence of other platinum containing species utilizing high performance liquid chromato- graphy (HPLC) with a strong anion-exchange resin. Utilizing this technique, the pharmacokinetics of intact cis-DDP, total platinum and l6 non-protein bound platinum have been studied in patients receiving intravenous drug administration (Himmelstein £5 31., 1981). While total platinum concentrations in plasma appear to decline in a tri— phasic mode (with a terminal tk greater than 24 hours), both non-protein bound platinum and cis-DDP decline in a monophasic manner (with a ter- minal t% of 20-30 minutes). Furthermore, the ratio of cis-DDP to total non-protein bound platinum in plasma approximates 0.6—0.8 within 5 minutes of cis-DDP administration and remains in this range throughout the time course of sampling. The authors therefore suggest that cis- DDP is converted to other non-protein bound platinum forms only shortly after drug administration. Thus, measurement of total non-protein bound platinum probably provides a reasonable index of circulating cis-DDP concentrations, although overestimations are likely. Using HPLC techniques, the half-life of cis-DDP in human plasma and plasma ultrafiltrates has been estimated as 1.5 and 2.2 hours, respec- tively (Long 53 21;, 1980). The in $33 disappearance of cis-DDP from ultrafiltrates may be due to (l) association of cis-DDP with other low'molecular weight plasma components or (2) biotransformation reac- tions. In support of the former, sulfhydryl groups of amino acids and/or peptides have been indicated as likely nucleophilic sites for cis-DDP reactivity (Repta and Long, 1980). Pharmacokinetics: Excretion cis-DDP and/or its related metabolic products are excreted primarily in the urine although a small amount of biliary and fecal excretion has 17 also been detected (DeSimone £5.3l3’ 1979). Using radiolabeled plati— num, DeConti 35 31. (1973) reported a bimodal pattern of urinary pla- tinum excretion in humans, characterized by a rapid initial phase and a more prolonged and incomplete later phase. The amount of cis-DDP recovered as platinum in the urine has been estimated as 352 at 4 hours, 402 at 24 hours and 502 four days following intravenous drug administration (Lagasse ggnal., 1981). In rats,.7SZ of the administered dose is recovered as platinum in urine 30 days following drug admini- stration (Litterst £5 31,, 1979). However, these studies suffer from non-specific determination of platinum and provide little information regarding the chemical forms of cis-DDP excreted. Recently at least two platinum.species in the urine have been identified using ion exchange chromatography: a water elutable (presumably cis-DDP) and HCl elutable (presumably aquated/hydroxylated complex) fraction (LeRoy 35 31., 1980). During cis-DDP infusion to patients, a rapid urinary excretion of both of these species appears in proportions similar to the composition of ' drug infusate (LeRoy £5 31., 1980). Following drug treatment, urinary excretion of the water elutable fraction decreases while that of the acid-elutable fraction increases with time. These authors suggested that the quantitative aspects of urinary platinum excretion may depend on factors such as urinary concentration of chloride and/or pH. Recently, the renal handling of cis-DDP has been investigated in patients receiving a 24 hour drug infusion. Using plasma ultrafiltrate and atomic absorption techniques, it has been reported that free platinum clearance exceeds creatinine clearance, suggesting tubular 18 secretion of cis-DDP (or metabolite) (Jacobs gE_§l,, 1980). Although these results have been confirmed by a recent preliminary report (Sa- firstein 32 31., 1981a), more definitive information regarding the nature of the platinum species excreted in the urine and their respec- tive handling by the kidney is needed. Pharmacokinetics: Tissue Distribution Studies examining the in gigg_disposition of platinum following drug administration indicate an initial distribution to nearly all tissues, followed by a specific accumulation in kidney, liver, muscle and skin (Litterst st 31., 1979). Thus far, similar patterns of drug distribution have been documented in all species studied including mice (Hoeschele and Van Camp, 1972; Lange 33 31., 1972), rats (DeSimone st 32., 1979; Litterst it. 31.,1976b), rabbits (Lange 3511;, 1972), dogs (LeRoy £5 51., 1979; Litterst gtugl., 1976) and dogfish sharks (Litterst 35;3;,, 1979). A consistent finding of these studies is the localiza- tion and prolonged retention of platinum in the kidney (tk - 50 hours) and liver (£35 - 32 hours) (Choie 3 _a_]._., 1980). When tissue mass is taken into account the absolute amount of platinum in the rat is greatest in skin, bone, muscle and liver for up to three days following drug treatment (wolf and Manaka, 1976). In contrast, very low concen- trations of platinum are present in brain tissue (Hoeschele and Van Camp, 1972; Lange ggnal,, 1972; Litterst g£“§1., 1976b); although re- cently it has been reported that brain concentrations of platinum in— crease two-to-three fold following the first day of drug treatment in dogfish sharks (Litterst £5 31., 1979). 19 Interestingly, the distribution of cis-DDP to target organs of anti-tumor activity does not reveal any consistent correlation; that is, although high platinum concentrations are detected in ovarian and uterine tissue, very low concentrations are reported in the testes (Litterst st 31., 1976b). Comparisons of platinum tissue distribution in tumored versus non-tumored animals have also been investigated in an attempt to account for the preferential cytotoxicity of cis-DDP to can- cer cells. Although there appears to be no specific uptake of platinum by tumor tissue (TothPAllen, 1970), consistently higher platinum con- centrations have been reported in tissues of tumored, compared to non- tumored, mice 108 hours after drug administration, an effect which may be attributable to reduced drug excretion by tumored animals (Hoeschele and Van Camp, 1972). The subcellular distribution of cis-DDP in HeLa cells appears to be greatest in the nuclear fraction whereas the cytoplasm contains little or no platinum (Kahn and Sadler, 1978). Although there is a paucity of information regarding subcellular platinum.distribution following £11.32 drug administration, Choie _e£ a_1_. (1980) recently reported that specific platinum concentrations (us Pt/mg protein) in kidney nuclei and microsomes are significantly greater than those in kidney mitochondria, plasma membrane or cytosol. In contrast to the kidney, no specific subcellular accumulation of platinum is observed in hepatic tissue (Choie 25 s;,, 1980). The lack of association of platinum'with hepatic microsomes is consistent with the absence of any 20 effect of cis-DDP on the activities of hepatic microsomal drug metabo- lizing enzymes following single drug administration (Litterst 33 31., 1979. Pharmacokinetics: Biotransformation Biotransformation of cis—DDP is suggested by the in ziggg_dis- appearance of cis-DDP and concomitant appearance of other platinum compounds in plasma and plasma ultrafiltrates. Utilizing gel filtration chromatography techniques, at least seven platinum species have been identified in plasma ultrafiltrates 12 hours following addition of cis- DDP (Long and Repta, 1980). Further characterization by X-ray fluor- escence indicates the presence of both sulfur and platinum in approxi- mately equimolar concentrations. Molecular weights of the four major species are estimated as 600, 500, 465 and 440 daltons (Repta and Long, 1980). Ighvizg_studies indicate that the chromatographic profile of plati- num species in plasma ultrafiltrates and urine from a patient receiving cis-DDP are markedly different than that observed in 21552 (Repta and Long, 1980). However, a peak with an estimated molecular weight of 440 is evident in both ultrafiltrate and urine and corresponds closely to in ziggg_resu1ts. The apparently low molecular weights of these plati- num compounds coupled with the presence of sulfur suggests that cis-DDP probably reacts non-enzymatically with low molecular weight proteins, peptides and/or amino acids. To date, studies on the in zigg metabolism of cis-DDP in both extracellular and intracellular compartments are 21 limited and require further attention if the physiological disposition and molecular actions of cis-DDP are to be understood. Toxic Effects of cis-DDP: Kidney Clinical use of cis-DDP is primarily limited by its dose-related and cumulative renal toxicity, an effect which is well-documented in all species studied to date including mice (Leonard gt §l°: 1971; Schaeppi £31., 1973), rats (Choie £31., 1980; Kociba and Sleight, 1971; Safir- stein 2211., 1981b; Ward and Fauvie, 1976), dogs (Cvitkovic $511., 1977; Litterst 35 21,, 1976a; Schaeppi 35 31., 1973), monkeys (Leonard st 21,, 1971; Schaeppi 35 31., 1973) and humans (Dentino £5 31., 1978; Gonzalez-Vitale 25 31,, 1977). In laboratory animals, cis-DDP nephro- toxicity is characterized histologically by acute tubular necrosis in the corticomedullary region (Choie 55 a1., 1981; Dobyan £3 31,, 1980; Hard and Fauvie, 1976). Although there is uniform agreement that cis- DDP nephrotoxicity primarily involves degenerative changes in the proximal tubule, there are conflicting reports regarding its effects on the distal tubules, with some studies reporting either moderate to severe (Aggarwal 25 31., 1980; Choie £5 31., 1981) or an absence of (Dobyan e_t_a_1_., 1980; Lehane 3921., 1979; Safirstein _e_t_al_., 1981b) damage to distal tubules of rat kidneys. In humans, reports of cis-DDP nephropathy are limited; however, GonzalezAVitale and coworkers (1977) reported focal acute tubular necrosis, affecting primarily the distal convoluted and collecting tubules at autopsy. To date, glomerular or vascular lesions following cis—DDP treatment have not been detected. 22 The time course of cis-DDP nephropathy in laboratory animals is characterized by degenerative changes in the proximal tubule as early as 1-2 days following treatment and consists of cytoplasmic vacuoliza- tion, tubular dilatation, pyknotic nuclei and hydropic degeneration (.ChOie £31., 1980; Dobyan 3511;, 1980; Ward and Fauvie, 1976). By days 3-5 pathologic changes are the most profound and are characterized by widespread tubular necrosis of the corticomedullary region, predomi- nantly in the third segment (83) or straight portion of the proximal tubule (pars recta) (Choie 25Ha1., 1980; Dobyan gtflal., 1980; Lehane ‘ £31., 1979; Safirstein 3551;, 1981b; Ward and Fauvie, 1976). Elec- tron microscopic studies reveal several ultrastructural changes in the pars recta including: profound thinning or focal loss of brush border, cellular swelling, condensation of nuclear chromatin, cytoplasmic vacuolization, rounded mitochondria with swollen cristae, dissociation of mitochondria from basal infoldings, loss of basal infoldings and an increased number and size of pinocytotic vesicles and lysosomal bodies in the apical region bordering the lumen (Asgarwal‘g£_al,, 1980; Dobyan gghgl,, 1980). Animals surviving cis-DDP nephrotoxicity demon- strate renal tubular regeneration as indicated by enlarged nuclei and mitotic figures. However, the presence of necrotic debris in tubular lumen coupled with persistent tubular damage for six months following single administration of cis-DDP (Dobyan 35H21., 1980) suggests income plete recovery. In addition, chronic treatment with cis-DDP may result in cyst formation (Dobyan £5 21,, 1981), and interstitial fibrosis and thickening of tubular basement membranes (Choie g£_al,, 1980), causing irreversible renal damage. 23 Functional correlates of cis-DDP nephrotoxicity include transient elevations in blood urea nitrogen (BUN) and serum.creatinine concen- trations (Gonzalez-Vitale st 31., 1977; Kociba and Sleight, 1971; ward and Fauvie, 1976) and polyuria despite diminished glomerular filtration rate (GFR) (Safirstein st 31., 1981b). Micropuncture studies indicate that cis-DDP treatment compromises superficial single nephron GFR (SNGFR); however, the magnitude of its reduction is insufficient to account for the decline in whole kidney GFR, suggesting that cis-DDP profoundly affects GFR in juxtamedullary nephrons (Safirstein 25 51., 1981b). A defect in concentrating ability is also a striking feature of cis-DDP nephrotoxicity and appears to be related to a diminished corti- copapillary solute gradient, an effect associated with a failure to recycle urea (Safirstein at 31., 1981b). The mechanisms underlying cis-DDP nephrotoxicity, and in particu- lar the profound necrosis of the pars recta, remain unclear. .Choie and coworkers (1980) have postulated that the localization and severity of tubular necrosis following cis-DDP treatment is related to the regional platinum distribution within the kidney. This postulate is supported by the observed concentration gradient of platinum in the kidney with the highest concentration in the corticomedullary junction and the lowest platinum concentration in medullary tissue, an effect correlating with the localization of tubular necrosis (Choie 35 21., 1980). The mechanisms responsible for the selective accumulation of platinum in corticomedullary tissue are not well-identified. However, it is known that the pars recta is a major site of active tubular secretion, a process which initially involves active transport from 24 peritubular fluid into proximal tubules, resulting in intracellular accumulation of the transported molecule. On this basis, it has been postulated that cis-DDP (or metabolite) may be transported by a similar mechanism and may account for selective intracellular accumulation of platinum in the pars recta (Dobyan 25 21., 1980). Although cis-DDP appears to undergo renal tubular secretion (Jacobs‘ggual., 1980; Safir- stein g£_al,, 1981a), characterization of this transport mechanism and direct evidence linking it to intracellular platinum accumulation in the pars recta is lacking. An important aspect of cis-DDP nephrotoxicity which has not been adequately addressed is the chemical nature of the immediate nephro- toxicant. Inasmuch as the gross characteristics of cis-DDP nephropathy appear similar to the histopathological changes observed following administration of other heavy metals, particularly mercury, it has been presumed that the nephrotoxicity of cis-DDP is related to the toxicity of the platinum atom (Choie £5 21., 1980; Nadias and Harring- ton, 1978). Characteristics common to both cis-DDP and mercuric- chloride nephrotoxicity include: (1) acute tubular necrosis affecting primarily the pars recta (Dobyan gtflal., 1981), (2) increased size and number of renal lysosomes (Aszarwal‘ggual., 1980), and (3) depletion of protein-bound SH groups (Levi 35H31., 1980). However, several lines of evidence suggest that the differences between cis-DDP nephrotoxicity and mercuric chloride toxicity are quite distinct: (1) Time courseand development. The earliest detectable changes in the kidney appear several days following cis-DDP treatment. Although 25 GFR is compromised 3, 7, 14 and 22-30 days following a single admini— stration of cis-DDP (5 mg/kg) to rats, no differences are observed on days 1 and 2 (Safirstein 35 21., 1981). Similarly, changes in uri- nary composition and volume are not evident until several days followb ing treatment (Safirstein 25 31., 1981). In contrast, alterations in renal function following administration of even a small dose of mer- curic chloride are rapidly induced within 24 hours (Haagsma and Pound, 1979). Thus, the delayed appearance of cis-DDP nephrotoxicity suggests that its underlying mechanisms probably differ from.mercuric chloride and may be related to the time needed for biotransformation. In addition, tubular regeneration is complete 9-14 days following mercuric chloride (Haagsma and Pound, 1979), but not cis-DDP (Choie 35 31,, 1980; Dobyan 25 31., 1981), treatment, further suggesting differences in the time course of kidney damage and repair between these compounds. (2) Chelation therapy. Mercuric chloride nephrotoxicity is often reversible following treatment with sulfhydryl reacting chelating agents, i.e., cysteamine, penicillamine and N-acetylcysteine. In con- trast, cis-DDP nephrotoxicity is not reduced by metal chelators (Graziano £2 31., 1981), suggesting a dissociation between the nephro- toxic actions and molecular reactivity of cis-DDP and mercury. Furthermore, several lines of evidence suggest that cis-DDP nephro- toxicity may not be solely attributable to the platinum atom: (1) Stereospecificity of cis-DDP. Although cis-DDP and trans- DDP result in similar renal concentrations of platinum (Van Camp and 26 Hoeschele, 1972), the trans isomer does not induce renal toxicity (Leonard £2 21,, 1971; Van den Berg 25 El-: 1981), indicating that the geometry of these complexes probably plays a crucial role in producing nephrotoxicity and that the presence of the platinum moiety alone may not be sufficient for inducing nephrotoxicity. (2) Structure-activity relationships. Modification of ligands of DDP complex alters the nephrotoxicity. The following chemical proper— ties appear to be related to nephrotoxicity: (a) presence of N-H in coordinating amines, (b) absence of bulky alkyl groups in coordinating amines, and (c) chelate ring size, with smaller ring associated with increased toxicity (Broomhead 35 31., 1980). Thus, modulation of nephrotoxicity by altering the ligands of platinum complexes indicates that toxicity is not solely related to the platinum atom. Inasmuch as the immediate nephrotoxicant of cis-DDP has not been identified as of yet, the molecular mechanisms of cis-DDP nephrotoxicity remain poorly understood. Since cis-DDP (or metabolite) is known to' react with nuclear DNA, it has been suggested that its nephrotoxicity may result from a similar mechanism. The selective accumulation of platinum in renal nuclei (Choie £5 31., 1980) may be particularly signi- ficant in this regard. Other mechanisms of renal toxicity which have been suggested, but not adequately tested, include depletion of renal sulfhydryl groups (Dobyan £5 21,, 1980) inhibition of renal Na,KrATPase (Guarino ggugl,, 1979), and activation of renin-angiotensin system (Medias and Harrington, 1978). 27 Modulation of KidneLToxicity Since nephrotoxicity limits the clinical use of cis-DDP, numerous attempts have been made to reduce kidney damage and increase the thera- peutic index of this drug. Cvitkovic e_t_ 31. (1977) demonstrated that vigorous intravenous hydration before, during and immediately after cis-DDP administration reduces the incidence and severity of renal toxicity in dogs. Similarly, mannitol (10 g/hr) in conjunction with hydration (0.452 saline, 200 ml/hr) for six hours after cis-DDP admini- stration significantly improves the therapeutic index of cis-DDP (Hayes 9331;, 1977). Although these manipulations reduce cis—DDP nephrotoxi- city, as indicated by the prevented rise in BUN concentrations, drug half-life and plasma clearance and tissue distribution of platinum are unaltered (Pera 3511,, 1979; DeSimone 5311., 1979). Furthermore, the anti-tumor properties of cis-DDP are not compromised (Hayes e_t_ 31. , 1977; Pera e_t_ ;a_1_., 1979). The reduction in cis-DDP nephrotoxicity associated with mannitol pretreatment appears to be related to reduced urinary concentration of platinum, although total urinary platinum excretion is not affected (Pera and Harder, 1979). Histopathological evaluation of kidneys on days 1-4 indicate, however,,.an equivalent degree of proximal tubular necrosis in rats treated with cis-DDP alone or in combination with mannitol. Thereafter, a trend developed toward less persistence of histopathological damage in mannitol treated groups (Pera £31., 1979). Other attempts to modify cis-DDP nephrotoxicity include pretreat- ment with probenecid (Ross and Gale, 1979), thiosulfate (Howell and 28 Taetle, 1980), superoxide dismutase (McGinness gtflal., 1978), furosemide (ward 25 21,, 1977) and chelating agents (Graziano g£_§1,, 1981). Re- cently, Litterst (1981) has reported that alterations in the vehicle concentration of NaCl influence renal toxicity. Specifically, ' nephrotoxicity is increased when cis-DDP is prepared in distilled water. Conversely, cis-DDP nephrotoxicity is reduced when prepared in increas- ing concentrations of NaCl. Furthermore, kidney platinum concentra- tions and plasma protein binding of platinum are both markedly elevated following administration of drug prepared in distilled water, compared to 0.92 or 4.52 NaC1 drug solutions. These results therefore suggest that cis—DDP prepared in a water vehicle facilitates the formation of an aquated/hydroxylated platinum species, resulting in enhanced plasma pro- tein and tissue binding. Although efforts to modify cis-DDP nephro- toxicity have been somewhat successful, complete protection from nephrotoxicity will only be feasible when the mechanism of cis-DDP nephrotoxicity is more fully understood. Other Toxic Effects In addition to kidney toxicity, gastrointestinal disturbances, characterized by nausea, vomiting and diarrhea, also represent major clinical problems associated with cis-DDP therapy. Although the patho- genesis of these gastrointestinal disturbances has not been defined in humans, several reports indicate histological damage of the intestinal tract of laboratory animals. Kociba and Sleight (1971) reported edematous villi of intestinal mucosa as early as one day following cis-DDP treatment. On days 3-4 histopathological lesions are the most 29 severe, coinciding with development of fluid distension and diarrhea, and are characterized by denuded mucosal surface and a cyst-like ap- pearance of the crypts of Lieberkuhn. The intestinal epithelium of surviving rats demonstrate increased mitotic activity of cells lining the crypts, indicating regeneration. A more recent study has confirmed and extended these observations, indicating that cellular necrosis is most severe in the ileum, followed by the jejunum.and duodenum, while neither the stomach nor colon are markedly affected (Choie 351§1., 1981a). Inasmuch as tissue concentrations of platinum are comparable in all segments of the GI tract, the effect of cis-DDP on GI mucosa may be related to other factors, e.g., pH (Choie at $1., 1981a). Pancreatitis has also been observed in cis-DDP treated dogs (Schaeppi 25 $1., 1973) and there has been one report of liver toxicity in a treated patient (Cavalli 35.31., 1978). Myelosuppression, characterized by leukopenia, thrombocytopenia and anemia, is also observed following cis-DDP treatment (Kociba and Sleight, 1971 ; Von Hoff ggual., 1979). Other toxic side effects of cis-DDP include ototoxicity (tinnitus, hearing loss) (Helson st 21., 1978; Von Hoff £5 21,, 1979), allergic reactions (eczema, anaphylactic reactions) and peripheral neuropathy (Vbn Hoff £5 31., 1979). A poten- tially serious toxic effect of cis-DDP is drug induced hypomagnesemia. A phase II prospective clinical trial has indicated the development of hypomagnesemia in 23 of 44 patients receiving cis-DDP (Schilsky gt_al,, 1979), an effect which may be attributable to impaired tubular reab- sorption of magnesium, resulting in inappropriate urinary losses. 30 Hypocalcemia has also been observed in 4 of 8 patients (Hayes 25 31., 1979). Pharmacologyfiand Toxicology of Other Platinum Compounds The increasing utility of platinum as the active component of the automotive catalytic converters has stimulated interest in its toxi- city following inhalation. Moore and coworkers (l975a,c) reported that immediately following inhalation of the soluble platinum seats, 191Pt is distributed in the gastrointestinal and respiratory tract. In addi- tion, kidney and bone contain the greatest amount of radioactivity. Intravenous administration of platinum to rats results in significant platinum accumulation in kidney, liver, spleen, adrenal gland and pan- creas (Mbore 35 21., 1975a,b,c). Following a single oral dose, almost all of the 191Pt is excreted in the feces due to malabsorption; where- as, similar quantities are excreted in both urine and feces following intravenous administration. Information regarding the tOxicology of platinum salts is extremely limited. However, it has been reported that workers chronically exposed to platinum metal salts in refineries are subject to p1atinosis, a con- dition characterized by dermatitis, eczema, skin ulcerations and respi- ratory distress (LeRoy, 1975). ENDOCRINE REGULATION OF GLUCOSE HOMEOSTASIS General Aspects of Biological Actions of Insulin and Glucagon Despite differing nutritional states, blood glucose concentrations in normal humans are generally maintained within the narrow range of 31 3-7 mM. Such stringent regulation of blood glucose is probably reflec— tive of the importance of glucose as a primary respiratory fuel, par- ticularly by the central nervous system. Thus, if blood glucose con- centrations are to remain fairly constant, both influx and efflux of glucose from extracellular fluid (ECF) must be balanced. In this re- gard, the liver plays a major regulatory role by controlling glucose fluxes in response to variations in dietary intake and to variations in fuel homeostasis. For example, in response to low glucose concen- trations in portal venous blood net glucose production and released into ECF is increased. Conversely, in response to increased glucose concen- trations in portal vein, net uptake and metabolism of glucose is in— creased. In this way, the liver is able to exert tight regulation of peripheral blood glucose concentrations. The intrinsic ability of the liver to respond appropriately to glycemic stimuli is governed, in part, by the biological actions of insulin and glucagon perfusing the liver. Both insulin and glucagon are synthesized in pancreatic islets; the insulinscontaining beta cells form more than 602 of the islet cell population and are arranged in a rela- tively homogeneous central mass while the glucagon containing alpha cells are situated at the periphery of the islet and comprise approxi- mately 302 of islet cells. Somatostatin containing delta cells form the remaining 102 of islet cell population. Both insulin (MW =6000) and glucagon.(MH’=3485) are proteolytic products of larger precursor mole- cules, i.e., proinsulin (MW 39000) and proglucagon (MW =9000-18,000), respectively. 32 Proinsulin, synthesized by ribosomes associated with the rough endoplasmic reticulum of beta cells, is translocated to the Golgi apparatus by an energy dependent system where hydrolytic cleavage yields insulin and C-peptide. These products, packaged into granules, ‘migrate to the periphery of the cell, fuse with the plasma membrane and are ultimately extruded by an exocytotic process into ECF. The normal secretory products of the beta cell, therefore, include insulin, an equimolar concentration of C-peptide and a small amount (=52) of uncon- verted proinsulin. Once secreted, the initial action of insulin depends upon specific receptor binding in the plasma membrane of target tissues. It is generally believed that following binding, the insulin-receptor complex generates one or more signals and may include changes in con- 2, Mg+2) and/or nucleo- formation of plasma membrane, ion flux (Na+, Ca+ tides (ATP, cyclic AMP) (Czech, 1981). These signals or second messen- gers then interact with a variety of effector units which ultimately mediate the host of biological actions attributable to insulin. The cardinal feature of insulin action is the promotion of nutrient storage and/or utilization. With respect to carbohydrate metabolism, insulin stimulated glucose utilization is characterized by increased tissue uptake and metabolism of glucose (glycolysis) while storage of carbo— hydrates is effected by increased glycogen synthesis. In contrast, the effects of glucagon on carbohydrate metabolism are characterized by mobilizing nutrients from storage depots and are mediated by the action of cyclic AMP. The biological effects of glucagon are thus antagonistic to those of insulin and include increased glycogenolysis and gluconeo- genesis. 33 The molar ratio of insulin to glucagon (I/G) has been suggested to provide a more accurate index of net biological action than the absolute level of either hormone alone (Unger, 1971). Thus, an elevated I/G would promote glycogen storage and inhibit endogenous glucose produc- tion. Conversely, a reduced I/G would favor mobilization of stored glycogen and increase gluconeogenesis. For example, in fasting animals, the concentration of glucagon, relative to insulin, must be sufficiently high to maintain hepatic fuel production to meet the fuel needs of the organism. On the other hand, when the need for endogenous glucose production is diminished by glucose ingestion, I/G increases markedly and promotes carbohydrate storage and/or utilizatidn (Unger, 1971). In this way, the maintenance of normoglycemia is achieved through a "push- pull" system of two tightly coordinated biological antagonists, insulin and glucagon. EFFECTS OF HEAVY METALS ON CARBOHYDRATE METABOLISM Several divalent metal ions, including cadmium, nickel, cobalt, copper, zinc and mercury affect carbohydrate metabolism (Horak and Sunderman, 1975b). Of these metals, cadmium and nickel have been 4 studied the most extensively. Ghafghazi and Mennear (1973) were the first to demonstrate that administration of a single dose of cadmium (6 mg/kg, i.p.) impairs glucose tolerance in mice. Glucose intolerance is observed 1 hour, but not 24 hours, following cadmium.administration. In addition, these authors reported a reduced immunoreactive insulin response to glucose stimulation in cadmium treated animals. In 34 contrast to these acute effects, repeated injections of cadmium (4 mg/kg/day x 14 days) does not impair glucose tolerance (Ghafghazi and Mennear, 1973). The authors attributed the absence of glucose intoler- ance to the possible induction of metallothionein synthesis, which presumably sequesters the cadmium ion. To more clearly define. the role of the pancreas in cadmium induced glucose intolerance, Ghafghazi and Mennear (1975) utilized the isolated perfused rat pancreas to demon- strate a direct inhibitory effect of cadmium on pancreatic insulin secretory activity. The inhibition of insulin secretion by cadmium appears to be nonspecific, immediate in onset and is not reversed by washout of the pancreas with perfusion medium. However, perfusion of cadmium treated pancreata with both glucose and theophylline results in a partial restoration of normal insulin secretion. The partial restora- tion of insulin secretion in cadmium treated pancreata by theophylline may be due to the effects of theophylline on intracellular calcium flux and/or on pancreatic islet cyclic AMP (cAMP) metabolism (Ghafghazi and Mennear, 1975). . Administration of cachnium chloride (1 mg/kg/day x 21 days) has also .been reported to markedly elevate hepatic cAMP levels (Merali _e_t_ a_1., 1975). Chronic treatment with cadmium chloride (0.25 mg/kg/day x 45 days) markedly elevates the activities of key gluconeogenic enzymes (Merali _e_t_ 31;, 1975). Single administration of cadmium (60 mg/kg) does not however affect gluconeogenic enzyme activity one hour follow- ing treatment, although elevated blood glucose and hepatic cAMP levels are evident at this time (Singhal 3331., 1976). Since alterations in 35 cAMP precede elevations in gluconeogenic enzyme activity, it has been proposed that the primary lesion mediating cadmium induced hypergly- cemia involves altered cAMP metabolism (Singhal ethal,, 1976). The effects of cadmium.an adrenal catecholamine metabolism have also been investigated. Daily intraperitoneal injections of cadmium chloride (1 mg/kg/day x 45 days) significantly augments adrenal weight and adrenal norepinephrine and epinephrine concentrations (Rastogi and Singhal, 1975). Inasmuch as catecholamines may inhibit insulin secre- tion, the reduced insulin secretory response following cadmium treatment may be secondary to the effects of catecholamines. To further examine the influence of the adrenal glands on cadmium induced hyperglycemia, Ghafghazi and Mennear (1973) measured blood glucose levels in adrenal- ectomized mice treated with a single dose of cadmium. Since cadmium induced hyperglycemia was abolished in adrenalectomized mice, the authors concluded that the hyperglycemic response to cadmium may be adrenal- mediated. Nickel (11) also induces a transient elevation in plasma glucose (Horak and Sunderman, 1975; Clary, 1975). Peak plasma glucose levels are observed 0.5 hours following intraperitoneal injection of nickel (II) as either NiCl or 111804 and is associated with elevated levels 2 of plasma glucagon in rats (Horak and Sunderman, 1975), suggesting that hyperglucagonemia may be responsible for nickel induced hyperglycemia. Plasma concentrations of glucagon and glucose returned to control values within 2-4 hours following nickel administration. Horak and Sunderman (1975) further observed that the hyperglycemic response to 36 nickel was suppressed, but not completely abolished, by adrenalectomy. To date, the exact mechanism(s) of nickel induced hyperglycemia re- main(s) poorly defined. RATIONALE Recent investigations have suggested that several divalent metal ions, particularly cadmium and nickel, markedly affect carbohydrate metabolism. Therefore, a divalent platinum compound, such as cis-DDP, may similarly alter carbohydrate metabolism. Several lines of evidence suggest that cis-DDP may affect glucose homeostasis: (l) cis-DDP treatment in rats results in random hyperglycemia (Kociba and Sleight, 1971) and (2) adrenal hyperplasia in mice (TothrAllen, 1970) and pan- creatic necrosis in dogs (Schaeppi gt 31., 1973) have been observed following cis-DDP treatment. This study was therefore designed to characterize the effects of cis-DDP and other divalent p1atinum.comr pounds on carbohydrate metabolism and to elucidate the underlying bio- chemical and endocrine mechanisms. MATERIALS AND METHODS GENERAL Animals and Diet Adult male Fischer-344 (F-344) rats, weighing 175-200 g, were pur- chased from either Charles River (Boston, MA) or Harlan Industries (Indianapolis, IN). All animals were housed in sanitary, ventilated animal rooms with controlled humidity, temperature, and 12-12 light-dark cycle for the duration of the experiments. A minimum of four days prior to experimental manipulation was allowed for acclimation to animal quarters. Unless indicated otherwise, all animals were offered a closed formula laboratory chow (Wayne Lab Blox, Allied Mills, Inc., Chicago, IL) and distilled water ad-libitum. P1atinum.Treatment The platinum compounds tested were cis-DDP and trans-DDP (kindly provided by Dr. Barnett Rosenberg, Department of Biophysics, Michigan State University, E. Lansing, MI) and ammonium tetrachloroplatinate [(NHA)2Pt014] (Aldrich Chemical Company, Milwaukee, WI). All agents, prepared in 0.92 NaC1, were administered intravenously as a single bolus injection. Rats were lightly anesthetized with ether and received p1a- tinum or a saline vehicle via a jugular vein. The relative insolubility of the DDP complexes in concentrations exceeding 1 mg/ml necessitated a volume injection of 16 m1/kg. Therefore, all treatment groups, 37 38 including controls receiving saline, were administered this volume un- less indicated otherwise. Twenty-four hours prior to treatment was designated as day 0. Glucose Tolerance Tests Animals were fasted 4-5 hours prior to glucose tolerance tests. Glucose tolerance was evaluated by serially sampling blood (approxi- mately 400 pl) before and 15, 30, 60 and 120 minutes following a glucose load (2 g/kg, i.p., 8 ml/kg). Venous blood was sampled by orbital sinus puncture in unanesthetized animals and was collected in test tubes con- taining EDTA (14 mg/ml blood) as an anticoagulant and 12 NaF to inhibit glycolysis. Following centrifugation at 2000 x g for 10 minutes, the plasma fraction was separated, frozen at 0°, and later analyzed for glucose using glucose oxidase, peroxidase and o—dianisidine as reagents (Sigma Chemical Company, St. Louis, MO). Insulin and Glucagon Determination For measurement of plasma immunoreactive insulin (IRI) and glucagon (IRG), blood was collected in test tubes containing EDTA (14 mg/ml blood) as an anticoagulant, and Traysylol (aprotinin) (FBA Pharmaceu- tical, New York, NY) (1000 kallikrein inactivator units/ml blood) to inhibit the proteolytic degradation of glucagon. Both IRI and IRG assays were conducted under the supervision of Dr. Ronald Gingerich (Department of Pediatrics, washington University, St. Louis, MO) using a double antibody system. 39 IRI was assayed according to the method of Morgan and Lazarow (1963) and is based on a two step reaction involving: (1) Incubating sample, 1251-insulin (300-350 uCi/ug), and anti- insulin plasma obtained from.guinea pigs, in a 0.05 M sodium, potassium phosphate buffer containing 0.025 M EDTA and 12 bovine serum.albumin. Incubations were conducted overnight at 4°C, allowing adequate time for equilibration and formation of a soluble insulin-antibody complex. (2) Following overnight incubation, anti-guinea pig plasma, ob- tained from rabbits, was added to samples and were incubated at 4° for 2.5 hours. Addition of anti-guinea pig plasma results in precipitation of the once soluble insulin-antibody complex. Samples were then cen- trifuged and the precipitate quantified for 1251 activity utilizing gamma counting techniques. The percent of radioactivity in the pre- cipitate is inversely proportional to the concentration of IRI in the plasma samples. Calibration curves are based on porcine insulin stan- dards. IRI concentrations are expressed as uUnits/ml. IRG was assayed according to the method of Leichter g£_§l, (1975) and was conducted in a similar manner to the IRI assay with the follow- ing exceptions: (1) Incubation reaction included plasma sample and anti-glucagon plasma obtained from guinea pigs. Samples were incubated for three days at 4° in a 0.2 M glycine buffer containing 102 Traysylol and 12 bovine serum albumin. Following incubation, 125I-glucagon (400-445 uCi/ug) was added to samples and incubated at 4° overnight. 40 (2) Following overnight incubation, anti-guinea pig plasma ob- tained from goats, was added to samples, incubated for 2.5 hours at 4° and centrifuged. 1251 activity was determined in the precipitate using gamma counting techniques. Calibration curves are based on porcine glucagon standards. IRG concentrations are expressed as pg/ml. SPECIFIC PROTOCOLS Effect of Platinum on Serum Glucose in-NonfastingiAnimals Male F-344 rats were administered a single intravenous dose of O, 2.5, 5, 7.5 or 15 mg/kg cis-DDP or trans-DDP. Ammonium.tetrachlorop1a- tinate was administered in a similar manner in doses equimolar in p1a- tinum concentration to those used in the DDP studies, yielding final doses of 0, 3, 6, 9 or 18 mg/kg of the platinum salt. Since preliminary studies indicated a dose-related anorexia associated with cis-DDP treat- ment, a group of controls was pair-fed on a daily basis to cis-DDP treated animals at each dose studied to correct for the metabolic effects of reduced food intake. Immediately before and l, 2, 4, 7 and 14 days following platinum treatment, animals were lightly anesthetized with ether and serum obtained from orbital sinus blood was analyzed,for glu- cose as previously described. Effect of cis-DDP on Serum Glucose in Fasting Animals Male F-344 rats were administered a single bolus injection of 0, 2.5, 5, 7.5 or 10 mg/kg cis-DDP (5 m1/kg). cis-DDP (1.5 mg/ml) was prepared in 0.92 NaCl and required gentle heating prior to injection. Animals were fasted 4-5 hours prior to each blood sampling. Orbital blood was sampled in anesthetized animals every 12 hours for the first five days 41 and then daily from days 5 to 7. Serum was analyzed for glucose as previously described. Effect of—eis-DDP on Glucose Tolerénce Rats were treated with a single intravenous dose of O, 2.5 or 7.5 mg/kg cisFDDP (5 ml/kg). A.pair-fed control group, restricted to the amount of food consumed by the 7.5 mg/kg group, was also studied. Since meal-feeding is known to alter carbohydrate tolerance, an attempt was made to simulate ad-libitum consumption in pair-fed animals by offering the allotted food over several time points during the day. Two days following treatment, glucose tolerance was measured by collecting serial blood samples before and after an intraperitoneal glucose load as pre- viously described. Plasma from three identically treated rats was pooled, frozen at 0° and later assayed for glucose, IRI and IRS concen- trations. EffectLof Platinum on Glucose Tolerance: Time Course and Dose Response Glucose tolerance was evaluated 2, 4, 7 and 14 days following treatment with cis-DDP (O, 2.5 or 5 mg/kg), trans-DDP (0, 5, 7.5 or 15 .mg/kg), or ammonium tetrachloroplatinate (0, 6 or 18 mg/kg). Plasma from three identically treated rats was pooled and analyzed for glucose. In specific incidences, plasma was also analyzed for IRI and IRG. Effect of Platinum on Selected Organ Weights and Serum Amylase Activity Following completion of glucose tolerance tests on day 4, and in some specific incidences on day 14, animals treated with cis-DDP, trans- DDP or ammonium tetrachloroplatinate in above experiments were sacrificed. 42 Liver, kidneys and adrenal glands were rapidly excised and wet weight determined. On day 4, rats treated with cis—DDP were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and blood sampled from.the abdominal aorta. Serum was colorimetrically analyzed for amylase activity using a standardized extract of corn starch as substrate (Sigma Chemical Company, St. Louis, MO). Results are expressed as Somogyi Units/d1. A Somoygi unit is defined as that amount of amylase causing formation of reducing power equivalent to 1 mg glucose in 30 minutes at 37°. Histopathological Examination of Selected Tissue from cis-DDP Treated Animals Male F-344 rats were treated with 0, 2.5, 5 or 7.5 mg/kg cis-DDP. Animals were sacrificed on day 4 and samples from the pancreas and kid- ney were fixed in buffered formalin (3.72 formaldehyde in 0.3 M sodium phosphate buffer, pH 7.2). After fixation, tissues were embedded in paraffin blocks, sectioned at 5 microns, mounted on glass slides and stained with hematoxylin and eosin under the supervision of Dr. Vance Sanger (Department of Pathology, Michigan State University, E. Lansing, MI). Histopathological examination of pancreatic tissue was performed by Dr. George Padgett (Department of Pathology, Michigan State Univer- sity, E. Lansing, MI). Sections were evaluated for presence of intact islets, cellular necrosis, vacuolization and inflammatory cell infiltra- tion. Histopathological examination of kidney tissue was performed by Dr. Keizo Maita (Department of Pathology, Center for Environmental Toxicology, Michigan State University, E. Lansing, MI). Renal tissue 43 was evaluated for coagulative necrosis, vacuolization, loss of brush border and nuclear enlargement. Role of Adrenal Glands in cis-DDP Glucose Intolerance Bilateral adrenalectomies (Adx) were performed in male F-344 rats anesthetized with sodium pentobarbital (50 mg/kg, i.p.) following re- troperitoneal incisions. A control group of rats was subjected to sham operations in which the adrenal glands were inspected but not excised. All rats were allowed a 7 day recuperative period following surgery. During this period and for the duration of the experiment, adrenalecto- mized animals were allowed free access to saline (0.92) in lieu of water to help maintain sodium balance. Following the recuperative period, rats were lightly anesthetized with ether and administered a single intravenous dose of 5 mg/kg cis-DDP or saline vehicle. The experimental groups included: Adx/control, Adx/cis-DDP and Sham/cis-DDP. Glucose tolerance was evaluated 2 and 4 days following drug or vehicle admini- stration. Plasma from.three identically treated animals was pooled and assayed for glucose as previously described. Plasma IRI and IRG was assayed in the fasting state and 15 minutes following a glucose load on day 2. Role of the Kidney in cis-DDP Glucose Intolerance Effect of Platinum on In Vivo Renal Function Male F-344 rats, individually housed in stainless steel metabolism cages, were administered a single intravenous dose of 0, 2.5, 5, 7.5 or 15 mg/kg cis-DDP or trans-DDP or equimolar platinum doses of ammonium 44 tetrachloroplatinate (0, 3, 6, 9, or 18 mg/kg). A group of controls, pair-fed on a daily basis to each cis-DDP treatment group, was also studied. Twenty-four hour urine volume was determined following urine collection into glass flasks containing toluene to prevent microbial contamination and evaporation. Urine samples were collected prior to (day 0) and l, 2, 4, 7 and 14 days following platinum treatment. Urine samples were also analyzed for: osmolality by a vapor pressure osmo- meter (Wescor, Inc., Logan, UT), sodium and potassium by flame photo- metry (IL Flame Photometer, Lexington, MA) and glucose by a spectrophoto- metric assay using a hexokinase reagent (Calbiochem, La Jolla, CA). Twenty-four hour urinary osmolar, sodium, potassium.and glucose excre- tion was calculated from the observed volume and measured concentra- tions. At the end of days 0, l, 2, 4, 7, and 14, animals were lightly anesthetized with ether and approximately 1 ml blood was sampled from the orbital sinus cavity.l Blood was allowed to clot, centrifuged for 10 minutes at 2000 x g and the serum.fraction separated and analyzed for urea nitrogen using a urease-Berthelot reagent (Sigma Chemical Co., St. Louis, MO) . Effect of Platinum on Renal Organic Ion Transport Four days following cis-DDP administration (0, 1.25, 2.5 or 5 mg/kg), animals were weighed and killed by decapitation. Kidneys were quickly removed, decapsulated, weighed and placed in cold isotonic saline (0.92 NaC1). The ability of renal cortical slices to actively accumulate an organic anion, p-mminohippurate (PAH), and an organic cation, tetra- ethylammonium (TEA), was determined. Renal slices were prepared 45 freehand and approximately 100 mg tissue was incubated in phosphate buffered medium (Cross and Taggart, 1950) containing 7.4x10-5M [14CJPAH (0.02 mCi/ml) (New England Nuclear, Boston, MA) and lxlO-BM [140]TEA (0.02 mCi/ml) (New England Nuclear, Boston, MA). Incubations were performed in a Dubnoff metabolic shaker at 25°C for 90 minutes under 1002 oxygen. After incubation, slices were quickly removed, blotted, weighed and prepared for analysis of PAH and TEA concentration of both slice and media preparations by liquid scintillation spectroscopy. Data are expressed as slice (dpm/g tissue) to medium (dpm/ml media) or S/M concentration ratios. To determine the iggzi££g_effect of cis-DDP on renal organic ion transport, renal cortical slices were prepared from untreated adult male F-344 rats as described above. Slices were incubated in phosphate- buffered medium (Cross and Taggart, 1950) containing 7.4x10-5M PAH, 1x10'3n [14CJTEA (0.02 mCi/ml), and o, 300, 400, 500 or 600 ug/ml cis- DDP. Following incubation procedures as described above, tissue and media were prepared for the spectrophotometric determination of PAH according to Smith 35 31, (1945). TEA concentrations of tissue and media was determined by liquid scintillation spectroscopy. Data are expressed as SIM concentration ratios. Effect of cis-DDP on Renal Clearance of Inulin and PAH The effects of cis-DDP on the renal clearance of inulin (Cinulin) and PAH (CPAH) were determined _i_r_1_ 11:32 four days following administra- -tion of 5 mg/kg cis-DDP or a saline vehicle. A pair-fed control group was also studied. Rats were anesthetized with 50 mg/kg sodium.pento- barbital, intraperitoneally. Body temperature was maintained at 37° 46 using heat lamps. A PE50 cannula was inserted into the bladder and urine was collected into preweighed vials. The femoral vein was cannu- lated for infusion of a saline solution containing 2x10-3M [3H]inulin (0.5 uCi/ml) and 3x10-2M [IACJPAH (0.5 uCi/ml) infused at 0.018 ml/ minutes using a Harvard infusion pump. The duration of the clearance experiment approximated 3 hours and included: an equilibration period of 90 minutes from the beginning of infusion to the initiation of urine collections, two 20-minute urine collections (prevolume expansion period), one S—minute volume expansion period followed by an equili- bration period of 10 minutes and a final 20—minute urine collection (post-volume expansion period). Blood (300 pl) was sampled from the femoral artery at the midpoint of each urine collection. Volume expan- sion was accomplished by infusing a 1:4 rat plasma:saline solution at [14C]PAH and [3H]inulin in plasma and urine was simul- 42 body weight. taneously determined by dual-label counting techniques using a Packard Tri-Carb liquid scintillation counter. Effect of Mannitol Pretreatment on cis-DDP Glucose Intolerance Pretreatment of animals with mannitol was conducted according to the method described by Pera 35 El: (1979). Rats were anesthetized with pentobarbital (50 mg/kg, i.p.) and the right jugular vein cannulated with PESO tubing. A 102 mannitol solution (in 0.452 NaCl) was infused at a dose of 2.4 g/kg over a 30 minute period using a Harvard infusion pump. After 25 minutes, a bolus injection of cis—DDP (5 mg/kg) or saline vehicle was infused via the intravenous tubing. Mannitol was infused for the remaining few minutes. Control animals also received 47 mannitol and were pair-fed to the mannitol/cis-DDP group. In addition, another group of animals was subjected to similar surgical procedures with the exception that they received cis-DDP alone, without mannitol pretreatment (Sham/cis-DDP). Thus, there were three experimental *groups: Mannit01/Control, Mannitol/cis-DDP and Sham/cis-DDP. On day 4, glucose tolerance was evaluated as previously described. Plasma ob- tained from three identically treated rats was pooled and assayed for glucose, IRI and IRG. Following completion of glucose tolerance tests, rats were anesthetized with pentobarbital (50 mg/kg, i.p.) and blood sampled from the abdominal aorta. Plasma was assayed for urea nitrogen as previously described. Effect of Selected Nephrotoxicants on Glucose Tolerance In addition to cis-DDP (5 mg/kg), three agents known to impair renal function were studied for their effects on glucose tolerance: cephaloridine (1000 mg/kg, i.p.) (Eli Lilly and Co., Indianapolis, IN), gentamicin (30 mg/kg, i.p. x 2x/day x 8 days) (Schering Pharmaceutical Corp., Kenilworth, NJ)-and glycerol (10 ml/kg, i.m., 502 wfv solution). Controls received a saline vehicle. Glucose tolerance was evaluated on those days in which renal functional impairment was manifested as an elevation in blood urea nitrogen as determined by preliminary experi- ments. Thus, glucose tolerance was evaluated on: Day 1 -— glycerol Day 4 -- cis-DDP and cephaloridine Day 9 -- gentamicin 48 Injections were staggered in such a way that evaluation of glucose tolerance for all treatments was conducted on the same day. Serial plasma samples were assayed for glucose as previously described. Following the two hr glucose tolerance test, animals were anesthetized with pentobarbital (50 mg/kg, i.p.) and blood sampled from the abdomi- nal aorta. Serum samples were measured for urea nitrogen and creati- nine concentrations (Sigma Chemical Co., St. Louis, MO). Following exsanguination, kidneys were removed and wet weight determined. Biochemical Correlates of CiSfQDP Glucose Intolerance Effects of cis-DDP on Serum Sodium, Potassium, Calcium and Phos— phorus ' Male F-344 rats were treated with a single intravenous dose of 0, 2.5, 5 or 7.5 mg/kg cis-DDP. Controls pair-fed to the 7.5 mg/kg group were also studied. On day 4, serum.was obtained from abdominal aortic blood of anesthetized animals and assayed for sodium and potassium by 'flame photometry as previously described. Serum.ca1cium was quantified using a Varian AAP375 atomic absorption flame photometer. For calcium determinations, potassium (5000 ug/ml) was added to serum samples and calcium measured at 422.7 nm using a nitrous oxide acetylene flame. Serum.phosphorus was measured using a modification of the method de- scribed by Fiske and Subbarow (1925). Gluconeogenic Enzyme Activity Hepatic and renal glucose-6-phosphatase (G-6-Pase) and fructose 1,6-diphosphatase (PDPase) activities were determined in male F-344 rats four days following a single intravenous administration of 0, 2.5, 5 or 49 7.5 mg/kg cis-DDP. A group of controls pair-fed to the 7.5 mg/kg group was also studied. All animals were fasted overnight (16 hours) prior to the day of experimentation. On day 4, animals were sacrificed by decapi-V tation, blood collected in chilled test tubes for glucose determination and liver and kidneys were rapidly removed and placed in ice-cold buffers. ' For measurement of G-6-Pase activity, 250 mg tissue was weighed, minced, rinsed and homogenized in 9.75 ml of ice-cold 0.1 M citrate buffer, pH 6.5, using 4 passes of a motor driven Potter-Elvehjem.homo- genizer. The homogenates were then filtered through cheesecloth and used for the igngiggg determination of G-6-Pase activity according to the method of Harper (1965). Following five minutes of preincubation of homogenates (0.1 ml) in a Dubnoff metabolic shaker at 37°, either 0.1 m1 of 0.08 M g1ucose-6-phosphate (substrate) or citrate buffer (tissue blank) was added to samples. _After exactly 15 minutes, 2 ml of 102 TCA was added to the reaction mixture and samples placed on ice. Following centrifugation of samples (2000 x g for 10 minutes), product formation was estimated in the resulting supernatant fraction by the spectrophoto- metric determination of phosphate according to Fiske and Subbarow (1925). Protein concentration of whole homogenates was determined according to Lowry SE 21, (1951) and results expressed as umoles phosphate/hour/mg protein. FDPase activity was quantified in hepatic and renal tissue by pre- paring 52 homogenates in ice-cold 0.15 M KCl buffer, pH 7.4 using four passes of a motor driven homogenizer. The homogenates were centrifuged 50 at 100,000 x g at 0° for 30 minutes using a Beckman L8-55 ultracentri- fuge. The supernatant fraction was then assayed for FDPase activity by a modification of the method of Pogell and McGilvery (1952). Incubation media contained 0.05 M sodium.borate buffer, pH 9.5, 0.1 m1 of 0.05 M MgSOA, 0.1 ml of 0.05 M fructose 1,6—diphosphate and supernatant fluid (kidney, 0.3 m1; liver, 0.2 m1), yielding a total reaction volume of 1.0 m1. Following preincubation.of samples containing buffer, MgSO4 and supernatant for,5 minutes at 37°, substrate was added and samples were incubated for exactly 15 minutes. The enzymatic reaction was stopped by the addition of l‘ml of 102 TCA. Following centrifugation (2000 x g for 10 minutes), samples were measured for product formation by the spectro- photometric determination of phosphate (Fiske and Subbarow, 1925). Protein was determined in the 100,000 x g supernatant according to the method of Lowry g£_§l. (1951) and results expressed as umoles phos- phate/hr/mg protein. Both enzyme assays were carried out under strictly linear conditions with respect to time and protein concentration. Endocrine Correlates of cis-DDP Glucose Intolerance Plasma and Pancreatic IRI and IRG Concentrations Blood from treated animals in above experiment was collected in chilled test tubes containing EDTA and Traysylol for determinations of plasma IRI and IRG. In addition, pancreatic tissue from these animals was rapidly excised, weighed and homogenized for 10 seconds in an ice- cold 2.12 sulfuric acid/802 ethanol mixture. Pancreatic tissue from the 51 duodenal lobe (head) and the remaining tissue adjacent to the spleen and stomach (body and tail) were individually analyzed for IRI and IRG concentrations. Extractions were carried out under ice-cold conditions using a ratio of 100 mg tissue:l ml acid/ethanol solution. Samples were incubated overnight at 4°. The following day samples were centrifuged. at 2000 x g for 10 minutes at 4°C. Supernatants were appropriately di- luted with 0.05 M phosphate buffer, pH 7.6, containing 0.025 M EDTA, 0.92 NaCl, 12 bovine serum albumin and 102 Traysylol. Samples were frozen at 0° and later assayed for IRI and IRG as previously described. Components of Plasma IRG Four days following treatment with 0, 2.5, 5.or 7.5 mg/kg cis-DDP, animals were anesthetized with sodium.pentobarbital (50 mg/kg, i.p.) and blood sampled from the abdominal aorta twenty minutes following admini- stration of the anesthetic. Blood was collected in chilled test tubes containing EDTA and Traysylol. Plasma was frozen at 0° and later ana- lyzed for IRG as previously described ("total" plasma IRG). In addi- tion, plasma reactivity to Unger's 30K antibody, which utilizes an anti- porcine/bovine glucagon obtained from rabbits, was also measured and designated as "true pancreatic" plasma IRG. "Extra-pancreatic" plasma IRG was calculated as the difference between "total" and "true pan- creatic" plasma IRG. Both "total" and "true pancreatic" IRG were also measured in plasma of animals from experiments evaluating the effect of mannitol pretreat- ment and the effect of selected nephrotoxicants. 52 Glucagon Resistance Four days following drug treatment (0, 2.5, 5 or 7.5 mg/kg cis- DDP), rats were fasted 4-5 hours and were evaluated for glucagon re- sistance by measuring the glycemic response to an intraperitoneal ad- ministration of porcine glucagon (1 mg/kg) (Sigma Chemical Co., St. Louis, MO). Blood was sampled by orbital sinus puncture in unanesthe- tized animals before and 10, 20, 30 and 60 minutes following glucagon administration. Plasma was assayed for glucose as previously described. ’Insulin Resistance Four days following administration of 5 mg/kg cis-DDP or a saline vehicle, rats were fasted 4-5 hours and then evaluated for insulin re- sistance by measuring the glycemic response to an intraperitoneal ad- mnistration of porcine insulin (0.28 IU/kg) (Sigma Chemical Co., St. Louis, MO). Blood was sampled from the orbital sinus cavity in un- anesthetized animals before and 10, 15, 20, 30 and 60 minutes following insulin administration. Plasma was assayed for glucose as previously described. Statistical Analyses All data are expressed as means a SEM. All data were analyzed by analysis of variance and treatment means were compared using the Student- Newman-Keuls or the Least-Significant-Difference tests (Sokal and Rohlf, 1969). The criterion of significance was p<0.05. RESULTS Food Intake A dose-related depression in food intake was observed following treatment with cis-DDP (Figure 4). Daily food consumption averaged 16, 12, 11, 9 and 8 grams one day following treatment with 0, 2.5, 5, 7.5 or 15 mg/kg cis-DDP, respectively (Figure 4). Animals treated with 7.5 or 15 mg/kg cis—DDP did not survive the duration of the 14 day experiment. In animals treated with 5 mg/kg cis—DDP, food intake was depressed through day 4, increased modestly on day 7, and was comparable to con- trols on day 14 (Figure 4). In contrast, administration of equimolar doses of trans-DDP did not significantly alter daily food intake at any time examined (Table 1). Animals treated with 18 mg/kg tetrachloro- platinate consumed significantly less food than controls one day followb ing treatment; however, food consumption in this treatment group was not different from controls on days 2, 4, 7 and 14 (Table l). Smaller doses of tetrachloroplatinate did not significantly affect daily food intake (Table 1). Body Weight Significant weight loss was apparent 2 and 4 days following treat- ment with 5, 7.5 or 15 mg/kg cis-DDP (Figure 5). Treatment with 5 mg/kg cis-DDP also resulted in a significant reduction in body weight on day 53 S4 .Amc.ovev moo moaomoemouuoo men so maouuooo Bonn mucoummmwv unmoqmwcmam m oncogene mxmfiuoum< .uooawuomuo men «a can no coauouno one o>u>unm no: can include wx\wa ma no n.~ sows ooumouu mHmaqm< .mmouumouauuuoo o>wu on know no ZEm H some use musemoueou mead Hmofiuuo> seas uofioe seem .exeusw ooom haweo mo manlmao mo acumen .e munwwm 55 .V snow“...— A;s3 m2: 3 a e a m— I .. mK E .. m I eon—...... 9:9: mfi OIO .9539. 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I V a 0 N o (u: rz/B)a)mm aoos V N 56 TABLE 1 Effects of Platinum Treatment on Daily Food Intake (g food/24 hours) Time (days after administration) o 1 2 4 7 14 Trans-DDP (mg/kg) 0 17:28 11:2 13:1 14:1 15:1 12:1 2.5 15:1 11:2 12:1 13:1 16:1 18:2 5 16:2 13:2 13:2 16:1 16:1 16:1 7.5 16:2 11:1 12:1 14:1 14:1 14:2 15 14:1 11:2 9:1 13:1 15:1 16:1 (NH‘)2Pt014 (mg/kg) 0 15:1 12:2 15:1 15:1 15:2 18:3 3 13:1 12:1 14:2 15:1 '15:1 19:1 6 15:1 9:1 11:1 13:2 15:1 20:1 9 17:1 11:2 14:1 15:2 15:1 16:2 18 14:1 7:2 10:1 13:2 12:2 18:2 8Values are expressed as means 1 SEM of four to five deter- minations. b day (p<0.05). Significantly different from controls on corresponding 57 Figure 5. 222; Effect of cis-DDP on body weight. Data are expressed as percent of initial or pretreatment (day 0) body weight. Each point *with vertical line represents the mean i SEM of four to five determina- tions. Animals treated with 7.5 or 15 mg/kg cis-DDP did not survive the duration of the 14 day experiment. Asterisks indicate a significant difference from controls on the corresponding day (p<0.05). Bottom: Effect of reduced food intake (pair-feeding) on body weight. Animals were offered the measured amount of food consumed by their drug treated partners. Each symbol indicates the drug treatment group to which animals were pair-fed. Asterisks indicate a significant differ- ence from controls on the corresponding day (p<0.05). 2 INITIAI. BODY WEIGHT 58 H control 0-0 2.5 mg/kg cis-DDP H 5 ” A-A 7.5 ” N 15 " "0 cis-DDP 100 A 90 a so 70 12 4 l4 7 TlME(doys) Figure 5 59 7; however, by day 14, body weights of these treated animals were com- parable to those of ad—libitum fed controls (Figure 5). No effect on body weight was evident following treatment with 2.5 mg/kg cis-DDP (Figure 5). Animals pair-fed to cis-DDP treated groups (5 or 7.5 'mg/kg) similarly lost a significant amount of weight on days 4 and 7 (Figure 5). Body weights of pair-fed partners to the 5 mg/kg group were comparable to controls on days 1, 2 and 14 (Figure 5). In con- trast, neither trans-DDP nor tetrachloroplatinate significantly affected body weight at any time examined (Table 2). Effect of Platinum on Serum Glucose in Nonfasting Animals Hyperglycemia was evident one day following treatment with 7.5 or 15 mg/kg cis-DDP (210i15 and 210:3 mg/dl, respectively) (Figure 6). Hyperglycemia persisted in these animals through day 2, reaching a concentration of 541:89 mg/dl in the latter treatment group (Figure 6). Treatment with 5 mg/kg cis-DDP resulted in a significant elevation in serum.glucose only on day 2 (Figure 6). In contrast, administration of equimolar doses of trans-DDP or ammonium.tetrachloroplatinate did not significantly alter serum glucose concentration at any time examined (Figure 6). Effect of cis-DDP on Serum Glucose in Fasting Animals Animals treated with 7.5 or 10 mg/kg cis-DDP exhibited fasting hyperglycemia 0.5 days following drug treatment (Figure 7). In both of these treatment groups, hyperglycemia was apparent immediately prior to death, reaching concentrations of 527ilOl mg/dl 1.5 days following 60' TABLE 2 Effects of Platinum Treatment on Percent of Initial Body Weight Time (days after administration) 1 2 4 7 14 Trans-DDP (mg/kg) 0 99:13 97:1 97:2 97:2 100:3 2.5 99:1 96:1 97:1 100:2 105:1 5 98:1 96:1 96:1 97:2 102:2 7.5 , 98:1 95:1 96:1 98:2 101:2 15 97:1 95:1 94:1 96:2 98:1 (NH‘)2PtC14 (mg/ 532 0 99:1 97:2 95:2 97:2 102:2 3 97:1 97:1 95:1 96:2 100:3 6 98:1 95:2 96:2 97:1 101:1 9 98:1 96:1 96:1 96:2 99:2 18 95:1 93:2 91:2 93:3 99:1 aValues are expressed as means 3 SEM of four to five determi- nations. Values represent percent of body weight on day 0. 61 Figure 6. Effect of cis-DDP (top), trans-DDP (center) or ammonium tetrachloroplatinate (bottom) on serum glucose concentration in non- fasting animals. Each point with vertical line represents the mean i SEM of four to five determinations. Animals treated with 7.5 or 15 mg/kg cis-DDP did not survive the duration of the 14 day experiment. The stippled area represents the mean i 952 confidence interval of pair-fed animals. Asterisks indicate a significant difference from both ad—libitum and pair—fed controls (p<0.05). Serum Glucose(mgldl) 62 700 P H control 600 - 0-0 2.5 rug/kg cis-DDP at H 5 " 500 I H 7.5 " H 15 " aoo . 25355; pair-fed 300 I- 200 - 100 300 "' O-O control O-O fling/kg trons-DDP .. H5 " A-A 7.5 " 0'9 15 " 200 — 100 .— n 1 L I L # ' H control 30° F o-o 3mg/kglNl-l4) no, a—a 6 ' 2 - H 9 " H18 '- 200 - 100 — I L L | 4L I 0 l 2 4 7 14 Timoldoys) Figure 6 63 needs asasnbsne .Amo.oVev zoo wcfiocoemouuoo men so mHouuooo Bonn monoumwmao unmoamanmfim m sumo .ucoaauooxo moo m use mo sowumuoo emu o>w>pnm no: can manlmao wx\ma ca no m.m nufi3 commons mamafis< .moowumoaaumuoo snow mo sz H some men mucmmmueou mafia Hmofiuuo> nude uoaoe zoom .mamaficm woaummm ca oofiumuunoomoo omoonam abuse so manlmfio mo muommmm .n munwam 64 n ousmwm 7.33 05:. .4. D 5. 2: m. 5 a. 03 m m CON man an m s O Gnu m .P P .. nv—.A'rL-V Avmufiw .nHU .. mN mugs ucwoe comm .A.e.« .wx\m NV omoa omoonaw m wsfisoaaow mounowa ONH one .00 .om .mH use AoV mumum mouummm use me soaumuucmoooo omoonaw remade co manlmwo mo uuowmm .w ounwfim 67 ON— mwmgawflh 325:: 00 m— 0 * 1 OOOOO OOOOOOOOOOOOOOOO STE...- '0. 0..qu n.“ .5... dOBhZOU I *- OO— O O O o O c O c In 1' m N (IF/Bun) 3503019 “my“ 0 O 0 can con 68 Figure 9. Effect of cis-DDP on plasma immunoreactive (IRI) in the fasting state (0) and 15, 30, 60, and 120 minutes following a glucose load (2 g/kg, i.p.). Each point with vertical line represents the mean i SEM of four determinations. Plasma pooled from three identi- cally treated animals was used for one determination. Pair-fed animals were offered the measured amount of food consumed by 7.5 mg/kg group. Asterisks and daggers indicate a significant difference from ad- libitum fed and pair-fed controls, respectively, at the corresponding time point (p<0.05). (”units/ml) I20 69 H control 0...... 2.5 mg/kg 100‘ .000. 7.5 rug/kg ”.000. Pair-led 80 - 60,- 40 — 1' 20 z 0 O O O O 15 30 60 120 TIME (min) Figure 9 70 Fasting plasma IRG of the 7.5 mg/kg group averaged 777:155 pg/ml whereas ad-libitum and pair-fed controls averaged 254:17 and 178:3 pg/ml, respectively (Figure 10). Similarly, plasma IRG concentrations in the 7.5 mg/kg group were significantly elevated throughout the two hour glucose tolerance test (Figure 10). Administration of 2.5 mg/kg cis-DDP did not significantly affect plasma IRG concentrations in either the fasting or glucose stimulated state (Figure 10). Effect of Platinum on Glucose Tolerance: Time Course and Dose Response Fasting plasma glucose concentrations were not significantly ele- vated 2, 4, 7 or 14 days following administration of cis-DDP (Figures 11-14), trans-DDP (Figures 11-14) or ammonium tetrachloroplatinate (Figures 15 and 16). However, on day 2, plasma glucose was markedly elevated 15, 30, 60 and 120 minutes following a glucose load in the 5 mg/kg cis-DDP group compared to both ad-libitum and pair-fed controls (Figure 11). In contrast, equimolar or greater than equimolar doses of ~trans—DDP did not result in plasma glucose concentrations different than controls during the two hour glucose tolerance test (Figure 11). On day 4, animals treated with 5 mg/kg cis-DDP, but not those treated with equimolar or greater than equimolar doses of its trans isomer, exhibited profound hyperglycemia following a glucose load, reaching a concentration of 379i10 mg/dl at 15 minutes compared to ad- 1ibitum and pair-fed control values of 232:20 and 300:13 mg/dl, respec- tively (Figure 12). Hyperglycemia in the 5 mg/kg cis-DDP group was evident following a glucose load at every time point examined (Figure 12). Plasma glucose was significantly elevated only at 15 minutes following glucose administration in the pair-fed group (Figure 12). 71 Figure 10. Effect of cis-DDP on plasma immunoreactive glucagon (IRG) in the fasting state (0) and 15, 30, 60, and 120 minutes following a glucose load (2 g/kg, i.p.). Each point with vertical line represents the mean i SEM of four determinations. Plasma pooled from three identi- cally treated animals was used for one determination. Pair-fed animals were offered the measured amount of food consumed by 7.5 mg/kg group. Asterisks and daggers indicate a significant difference from.ad—libitum fed and pair-fed controls, respectively, at the corresponding time point (p<0.05). 72 H control M 2.5 mg/ kg Coooo 7.5 Ina/k9 ’"OPoir-fed l200 68*! 000000 ooooo A I *1 00°00 - *9 00° % A50°°° °°°° ooo 3:: oo° a a“ o. 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OON 3 >m 66.2 26.2 22.6 22.2 66.6 66.2 no.2 62.2 62.6 62.2 62.6 o2xmm a6.m 66.2 mm.m 66.2 65.2 66.2 66.m 26.6 mn.n 62.6 66.2 2 32 QNoHH moHH NoHH Non“ Non“ Nona «cHH 26H“ unnoHH NoHH moHH o2xmm 66 mm om as on as mm mm 26 2 mm mm 2 :2 ANoHH 26H“ 26H“ 26”“ 26.2 26”“ 26.6 26”“ 622oH6 2oHH . 62on6 622mm 62 N2 62 a2 62 62 62 62 22 22 62 m :6 2 22 6.2 m o 662 m m.~ o m 6 o -6266 wa\wav 6266222622V wa\wav manumnmuu 262:652 226.626 6 >62 0 mgma oo.H2m.m no.Hnm.m wH.H~m.m OH.Hnm.m 22.222.2 22.222.2 22.222.2 ~222mm 222.222. 22.222. 22.222. 22.222. 22.222. 22.222. 22.222. 2226mm 22.222. 22.222. 22.222. 22.222. 22.222. 22.222. 22.222. 222mm 2 2 2 32 0 222:262 22662222222 22.262 222:222 222-626 26666266662 2 22222 99 .Amo.ov2v 22202 2222 m222202222200 222 22 2222222 manlm2o\a22m 6022 2022222222 22202222w22 2 22202222 222ww2n .22202 2222 w222202222200 222 22 220222oo\x2¢ 2022 2022222222 22202222w22 2 22202222 222222222 .N >22 20 2222 202222202 220022» 2:02 032 222 2>2>2=2 202 222 2222222 20222oo\x2< .2022222222222 220 202 222: 223 2222 2222222 22220222222 22222 2022 222002 222222 .22022222222222 2202 we sum 2 2222 2222222222 2222 2202222> 2223 22202 £02m .A.2.2 .wx\w NV 2202 220022m 2 22222 2222 22222w2 2222022 22 2022222220200 220022m 222222 .20222222222822 220222> 20 maalm20 w223022om 2222 a22w2uv c 222 A2222v N 2222222>2 223 202222202 2200220 .22222» 2222222 22222022 202 222 w222022222 22 222202222 2223 2202222220 E22m .20222222222222 h222222v 220222> 20 2222:220 m22ma mv w222 02 20222 2222 22>22 222202222 2223 222302022222222 .20222220222 2200=2w 2202222 222:220 20 Ax2 no A.>.w .wx\wa mv mnnlmfio mewsoaaom mumo oBu wouososoo who: musoauuoexm .mamaaam ooumouu adamoauaoow manna Scum omaooe mammae mo omumfimcoo unoauumexw 50mm .musoaflumexo usom mo sum H momma mm commounxo mum mooam>n .A.e.fi .wx\w NV omoa omoous m wsfisoaaom mouoawa ma one A.ov oumum wawumom onu as ooaeamm was oooamn mofimmm omHmwm owe Hmom . Nawca omfiom manlm«o\amnm wwaaqq coficmq mmHHomm oe Hue oefima maelmao\xo< «Namnm ONHNwN mmaumme amass ANHNH Houusoo\xo< ma o Hmwowwmmmmmn ma o usoaumouh Ammusswav mmoosau Houm< mafia camousouav omoosflu Houm< mafia Asa\wav oeH mammam Aaa\m8fi:=:v HmH saunas mama woumoue manlmao ea AUMHV commonau can aHmHv cwasmsH o>fiuommuoc=aaH mammam so Axowuooemmu .maouucoo oomlufime can own anuwnualom aoum mosonommao unmofiuficwam m mama Ifioaa mumwwmo use mxmfiuoum< .ucoefiumaxm mom «a onu mo coaumuso onu o>w>usm uoc ofio manlmfio wx\ma ma no m.m cues omumouu mamsaq< .usoauwouu mono some now muosuuma oomluwoe usommueou upon some .msofiumsfienmuoo m>am ou Meow «0 2mm H some ecu musomoueou mafia Hmoauu0> cuHB non zoom .Aunwwuv oESHo> mean: one Auwmav mxmusa momma haflmo co manlmao mo uommmm .NN munwfim 104 Fenian—oat ... . 3...! .u n.“ .238 ‘ C. @- . Nu «mamas, . 3:95 So - :9 ... n. as n ma .228 ...” - - .. 3 on :53 3 H ” 2 I W- @— .... 2 2 .... . an n s>Hm on “now no saw H some ecu mucomouamu mafia Hmofiuuo> saws use zoom .oadao> mean: one axons“ nouns zafimo so Aunwauv mumsfiuoaeouoanomuuou asfiaoaao ocm Aumoav manumsmuu mo uoowmm .mN ousmam 106 (sq num) omn'oA ouyn 222 882 2322 322 :22 322 , *-||Il i *-||I|l| *-||I|l , 1 2 z %a :% a z 2» m w m m m Mn 3 a a fi 3 so 0 - N v N 3 if, > 1| Ell “IIII g E %% %% 2%%% %% we eei "mm mm mmw 1mm mmw mmn as we we we as . 222 222 2:22 222 322 882 ("I'Z/IW) equal" JMDM IIS (“i 73"”) “PA “‘90 On man o- u.— I 02.55 2515 (Jq pagan-noun 1.89M Figure 23 (""4)2P'C|4(m9lkl) trans- DDP ( ing/kg) 107 affect daily fluid intake or urine volume on days 7 and 14 (Figure 23). Smaller doses of tetrachloroplatinate did not affect daily urine volume or fluid intake at any time examined (Figure 23). Urine Glucose. Urinary glucose concentrations were markedly elevated two days following treatment with 15 mg/kg cis-DDP and four days following treatment with 5 or 7.5 mg/kg cis-DDP (Figure 24). On days 7 and 14, urinary glucose concentrations of the 5 mg/kg cis-DDP group were comparable to controls (Figure 24). Daily urinary glucose excretion was increased in the 5, 7.5 and 15 mg/kg cis-DDP groups, averaging 241:58 mg/24 hours 4 days following administration of 5 mg/kg cis-DDP (Figure 24). Urinary glucose excretion was also increased in the 5 mg/kg group on day 7, but not on day 14 (Figure 24). In contrast, administration of equimolar doses of trans-DDP did not affect urinary glucose concentration or total daily excretion at any time examined (Figure 25). Urinary glucose concentration and excretion was increased 1,“ 2 and 4 days following administration of 18 mg/kg tetrachloroplatinate (Figure 26). However, glycosuria was no longer apparent on days 7 and 14 in this treatment group (Figure 26). Smaller doses of tetrachloro- platinate did not significantly affect urinary glucose concentration or total daily excretion (Figure 26). Urine Osmolality. Administration of 5, 7.5 or 15 mg/kg cis- DDP resulted in reduced osmolality on day 2 and this effect persisted in surviving animals at all times examined during the 14 day experiment (Figure 27). Decreased urine osmolality was also apparent in the 2.5 108 .Amo.ovev moo wcnoaoemonnoo men so .mHo>nuooemon .maonusoo oow Innme ocm Eounnnalom aonw moconomwno unmonmncwnm m oumonoan mnowwmo one mxmnnOum< .mHMEHam oomlnnme mo Hm>nmusn mocoonmsoo Nmm w some men mucmmoneon mono ooammnum one .uaoannmaxo zoo on may no sonuonso men o>n>nsm no: ono mnolmno wx\wa ma no m.n nun: oouoonu mamans< .maonu ImsnSnouoo o>nm on nsow wo saw n some men musommnaon mane Hoonunm> :uHB ounce zoom .omoonaw mnmanns mo Aunwnnv conuonoxo zanmo ocm Aumoav sonumnuumocoo so manlmfio mo noowwm .em onsmfim 109 .. 8-... gem... “mm .9359 I ON 0? oo oo Oa— .ON— a?" OON can (Int/II") Momma asoomo mmnm cu onswnm 3...... : m—.Ol. can-..» 9.3... .....4. co .233 I 0009 Dean cow? (IF/5W)aso:>n1o aman 110 Figure 25. Effect of trans-DDP on concentration (left) and daily excretion (right) of urinary glucose. Each bar with vertical line represents the mean : SEM of four to five determination. 111 :13 0.9.33 manozo: 353A 2; 6 4 2 .o 4 2 6 4 2 I I I :1. 5. vi 5) Kw as 2 A nu a. 2 ,m D. M D D D m ... s m 7. .r. 5 is a ...... 0‘ swam. o I I I :4 nu :4 4 3 .I :39: V 333.0 05.5 Figure 25 112 .Amo.onv xmo wsnoaoemonnoo men so mHonucoo Bony mononommno usmunmnownm m ouoonosn mxmnnonm< .msonumsnanouoo o>fim on nnom mo 2mm n some men muaomonemn mend Hmonunm> :un3 nanoe 50mm .omoosaw unmann: mo Aunwnnv sown Ionoxm hanmo onm Aumoav conumnucooooo so ouocnumaaonoanoonuou sansoaam mo uuommm .oN onamnm 113 363 2.: — o 0 Jo... «.4sz 9.3... n .233 z 4.4 «to 0.0 0.0 mu one»; 72930:...» 2 n v . a _ o 0 fi . on 8. cc . 8 ._ 3a m . 8a m - Su M m . o8 m . can u m . “o9. 03 u m. . 2:. V 0 co m, . 0!. m . 82 m . one 1 co: - can . con. - . oom— .. 2 I .. 0 I . .. o .1... . 8: :UENAVIZvuéuEn OIO. _On.:°u I - ... ...v Loon. (IP/wamfllo own 114 .Amo.ovnv zoo wcnoaoeoonnoo men do .zHo>nuooemon .mHonucoo oowlnnoe oao oom abunnnanoo Bonm oudonommno neoonmnsmao o muoonosn mnmmwoo oso oxmnnoumo .ucoanoonu mono zoom now onosnnoe oomunnoe unoooneon anon ammo .msonu loonsnoumo m>nm on noom mo 2mm n coma o£n ousoooneon mend Hoununo> nuns non zoom .Ausmnnv .nN musmnn conuonoxo noHoamo manna zHHoo oao humoav znnaoaoaoo mean: so manlono mo uuowmm 115 n— nN o >nm on noom «0 2mm n coma osu mucommnemn mafia Hoonuno> nuHS non zoom .sonuonoxo noHoEmo moan: zanoo oao zunHoHoamo manna no Aunmnnv ouosnnoaeonoaeoonuou anacoaao oso Aumoav manlmconu mo noommm .mN onownm 118 (Jq yz/unom ) uogunxg qomso ouyn llllllllll, llllllllll lllllllll llllllllll llllllllllll lllllllllll ... m m m m m m ° - 1 1 1| 1| I r 2 O A Illllllll llllllllllll lllllllllllllll llllllllll llllllllllll lllllllllllll . g 0mm“, was >- >- k — 3 3 3 a E E 3 5 6 DO 000 O O 88§ 888 8§813§8 3§8 §8§ (“l/MOW) Mum wen Figure 28 “I rum-om) nosa'ma 01°W'O own ( N0 ONO ONO ONO NO - a-I- _— ...— t I18 I'II rt. 0 2 6 III [9 W as as *%% mm 1mm 1mm 1mm mmm nmmni can aaflzaa'amaksma' a? 3 E E 3‘ E 3 E | 1 :3 5 an as ex ea za zesé mw mm 1mm 1mm "mm mm: an no sea gee axe one ( 8)./meow )Mllfllmo Own 119 .Amo.ovev zoo manoeoeoonnoo ozu so .zHo>nuooaoon .ononusoo oowlnnoe oao oom aounnnaloo Eonm ooaonomuno usoonmnawao o onoonoan onowwoo oao oxmnnouo< .ucoannoaxo zoo ea onn mo conuonno onu o>n>nno no: ono manlono wx\wa ma no m.z nuns oouoonu maoans< .uooauoonu mono nooo now onocunoa ooulnnoe naoooneon onon some .ooonuosnsnouoo o>nm On nsow mo ZMm n some men ousooonmon osna Hoonuno> nun3 non zoom .aonooo znoannn mo Aunwnnv acnuonoxo zanoo oao Auwoav conuonuaoocoo no manlono mo nooumm .mu onownm 120 *r 5 DIN‘4 DlN 7 '2 {- {- 3 I I. . D IIIIIIIIIII Illllllllllllll I III m - - _ :23 223 223 223 233 (aura/ham) new”: +°N Nun Illlll " * o a- ‘I’ N ‘I' * I' N >- >- >- >- " a a 3 a 3 a . 3 3 .. (Ellunrlod (”bat") "ovum-“~03 +°N mun l5 DWNI4 25 -'" +”mung A D J x O E ‘1’ CI I .2 U m m m? 333 ZS IS Figure 29 DINI4 5 2J5 ti cis-DDPhng/kg) conuol 119 .Amo.ovev zoo wonoaoeoonnou may no .zHo>nuooaoon .oHonuooo oomlnnoe oco oom aounnnaloo Eonm ooconommno ncoonmnswno o ouoofioan onowwoo oao oxonnouo< .ucoannoexo zoo «H man no conuonoo wen o>n>n=o no: ono mnolono wx\wa ma no n.n nuns oouoonu mHoans< .uooauoonu mono zoom now onosnnoe oomlnnoa noooonmon onon some .oconuoonanouoo o>nm on noow mo ZMm n some man onaoooneon moan Hoonuno> nuns non zoom .aonooo znosnno mo Anzwnnv conuonoxo zanoo ooo Auuoav conuonuaoocoo no manlowo mo noomwm .mm onawnm 120 2 ° // " // 2 * 2 3 -’3' I a I a '3 ‘I' ‘l i I - _I I I El ||||||||||| lllllllllllllll * Ill lllllll! IIIIIIIIIIII - - - - m m m m m - - - - - 2 ° 3 ‘2 ° 3 3 3 3 1'3 9 3 '0 ° 3 (aura/haw) new”: :11 ~an llllll " * O ‘- * N ‘I ' I' N > >- >- >- " a a a a 3 D 90er it I ..... :o:o:o:o:o:o:o:o: 0:0...o.o.o.o.o.o.o.o .O.O.O.O:.:'.'.‘ o‘e‘o’o’o‘o’o’ :.:.:.:.o.:. ..... ooooooooooooooooooooooooooooooooooooooooooooooooo gee §§§8 22.2.8 age 32.38 (Ilbaw) nonhuman: +°N own Dle4 L5 L0 (15 DINId _L__ I1 *I luuunfl 125 ‘15 IS 5 '5 ‘ 125 IS cis-DDngllm) 1L5 cis-DDP (mg/Ito) Figure 29 121 .Amo.ovmv haw wcqvcoamwuuou mnu so maouuaou Eoum mucmumHMHv acmofimwafim m mumofivaa mxmfiuoum< .mucmaummuu asafiuman umaoafinaw ucwmmuamu mumn wwaumuuma Adamowuame .mfioaumcaEHOumv 0>Hm ou “new mo Sum I came «nu mucwmmuamu mafia Hmuaupw> sags umn comm .Esacom manna“: we coauouoxm adamv cam GOfiu + Imuuamocoo no Auswfiuv mumcfiumHQOHOHnumuumu abacoaam tam Aummav manlmcmuu mo uumwwm .om musmfim 122 (fig 73/ ham) uogsuax3+oN '“I‘n “‘°— “2Q"! “10.3 nee-q mom I. II. -'- I—It ... -II 2 M m 7% WA 7/////. iI/o mu mm" mu um mm IWW: wa aaw 3% fig“ am? ago * >3. * * 3* >3. %%% *%% *% %% 2%% awai llllllllllll Illlllllllllllllll ||||||||l llllllllli llllllllllllll |||||||||||||| n mim m m m m o O In 50 I 50 I00 50 50 00 50 I 50 00 50 DO 22m 88 ("b3”) MINIUONOD+DN w" (nan/haw) uoswuagm own ' '2 °. '2 n. q "2 n a v? n O. "a .— .— c -— - O -' —' O - '- O * I I II E 7% % 7///x. W .4 "III" "III!“ IIIIIIIII "NH" 3 fig, @35 fig, aao z z a» 4 . :3 *z% '%% %% %%: |||||||||||| llllllllllllll ||||||||||||| ||||||||||| 13 m m m M O 222 88% 883 222 (”53'”) MIDJWOW+DN ougan ) (NH )2 "UAW/lug Figure 30 tram-DDNmIng) 123 .Amo.0vav hue wawvaommmuuoo msu so >H0>fiu looammu .maouuaoo vmwluwma cam vmm ananHHIvm Baum mocmummmwv ucmuwmaawwm m mumoavafi mumwwmv cam mxmfiumum< .usmawuwmxm mac «a mnu mo coaumunu «nu m>a>u=m uo: vav manlmfio wx\wa ma no n.n nufi3 voumouu mamaaa< .ucoaummuu wand 50mm pom muocuumm wmmlufimm accumuamu mama ammo .mcoau Iwcaaumumv m>wm ou u30m «0 saw H cams mnu muammwuamu mafia Hmowuum> sags Han sown .aafimmmuoa zumaau: mo Aunwauv coaumuuxm mafimw cam Aummav coaumuuamuaou co montage mo yummmm .Hm muswam 124 m— ‘llll 3:93:54? 95 a nd .2289 __m_ _m_ .m— .2. .2." .m— N fl 3 >3 N {- I I llllllllll N— V )- 3 fl * I I I. * IIIIIIIIIII N ’////////. - - N— O Hm unamflm (“I 73/53“) film-13x3} cum .- II | I 3333..-... 95 m ad .9530 m Gm— 000 Got 2.,— can a! IIIIIIIIIIIII 5 as a? 74 m .... .... W- m om— .. 23 on: W on. H can 94 m- m— H can . >3 :3 On — on» On v I IIIIIIIIIIIIII' _m_ (l/ba‘“) MINJHOMD+3 00an 125 .Amo.ovmv haw wafivcoammuuou mnu so maouuaoo Scum moamumMMHv uamuwmaawwm m mumoacaa mxmwuoum< .ucmaummuu ascfiuwaa umaoaascm ucmmmuamu mums omcumuuma xaamuwucme .maOHumaaahmumv m>wm ou know no zmm H same may mucmmmuamu mafia Hwoauum> sufiz umn :umm .Esfimmmuoa %um¢au: no coauwuuxa mafimv can coau Imuucwuaou so Auswguv mumcuumamouoazumuumu anacoaam vam muwmav manlmamuu mo uoommm .Nm Guzman 126 (Jul yz/baw) uogcuaxg +)| ouyn 883 883 883 883 883 83 rI TI I I I I I I I II.O *- I I -l I I 2 mmnr IIIIIIIIII numn mum mumm IIIIIIIIIIIIIT ... m_ m“ m? .W'c 3 ’3' E E 3 S IIIIIIIIIIII IIIIIIIIII mmm ‘ munm nmmnn umnmm .. m m m m m m o as: 283 as: as: 222 282 (l/baW)0°!mw-auog+x cum ("I vz/baw) lam-ma} own 2' 0 2.5 5 7.5 I5 (NH 4I2 PIC|4ImgIlKgI Figure 32 Inns-DDP (mg/kg) 127 Treatment with 9 or 18 mg/kg tetrachloroplatinate resulted in de- creased urinary sodium.(Figure 30) and potassium (Figure 32) concen- trations on days 1 and 2 and this effect persisted in the latter group through days 4 and 7. Urinary potassium (Figure 32), but not sodium (Figure 30), excretion was elevated 1 day following treatment with 18 mg/kg tetrachloroplatinate. Smaller doses of tetrachloroplatinate did not affect urinary sodium (Figure 30) or potassium (Figure 32) concen- tration and excretion. Blood Urea Nitroggn, Treatment with 7.5 or 15 mg/kg cis-DDP resulted in marked elevations of BUN concentration on days 1 and 2 (Figure 33). Animals treated with 5 mg/kg cis-DDP exhibited a 3-fold increase in BUN on day 4, but not on days 7 or 14 (Figure 33). In contrast, administration of the trans isomer of DDP did not affect BUN concentrations at any time examined (Figure 33). Treatment with 18 mg/kg ammonium tetrachloroplatinate, but not smaller doses, resulted in increased BUN concentrations, an effect observed on days 1 and 2, but not on days 4, 7 or 14 (Figure 33). Organic Ion Accumulation. The ability of renal cortical slices to accumulate PAH or TEA was not significantly altered in cis-DDP treated animals when compared to pair-fed partners (Figure 34). In con- trast, addition of cis-DDP (500 or 600 ug/ml) directly into incubation medium significantly depressed organic ion accumulation by kidney slices (Figure 35). In the presence of 500 or 600 ug/ml cis-DDP, PAH SIM ratios were approximately 48 and 342 of controls, respectively. Simi- larly, the ability of kidney slices to accumulate TEA in the presence of 500 or 600 ug/ml cis-DDP was 40-50% of controls (Figure 35). 128 Figure 33. Effect of cis-DDP (top), trans—DDP (center) and ammonium tetrachloroplatinate (bottom) on blood urea nitrogen (BUN) concentra- tions. Each point with vertical line represents the mean i SEM of four to five determinations. Identical symbols indicate equimolar platinum treatment. Animals treated with 7.5 or 15 mg/kg cis-DDP did not sur- vive the duration of the 14 day experiment. The stippled area repre- sents the mean i 952 confidence interval of pair-fed controls. Asterisks and daggers indicate a significant difference from ad-libitum fed and pair-fed controls, respectively, on the corresponding day (p<0.05): auu hula) 129 I05 - H control 90 . 0-0 2.5 Ina/kg cis-DDP ’ ‘ ” H 5 " 75 *I at H 7.5 I H 15 " 60 . ”I'd.‘ I u 45 I’ I" , I 30 P A x ............. - IS - 332.5% nun-DDP £12” 7.5 " HI: ' ' i n I 1 1 ' l J H control 0-0 3 lug/kg (Madmen. H 6 " H 9 " W ' I’I Fmfifii 130 .ucoaummuu waum Sumo How muoauuma comluflmm usommumou mama ammo .mcofiumcfiauouom prom mo 2mm H cmoa osu mucmmouqou amp 20mm .uaoaumowu moalmwo wafikoHHOM whom usom moowam Hmofiuuoo Hmcou he Az\mv AEDvaa\ooHHmv aofiumasaaoom o Aunwama hvon Rev aoauaflom onwammumammua um» and m mew looms“ mo voumwmaoo uaoaauoaxo comm .mummaauonxo mic mo sum H mm vommouaxm mum moaam>o .Ano.ovmv maouuaoo mowluwma mam asuwnaalvm :uon aouw uaouomuwm mauemoumqamfimn .aoauomaaoo mean: nomo mo uafiommwa one um humane Hmuoeom on» aoum moaaemm was ad: oomV voon .maowuooadoo mean: ouaaaaiom oBu mo voumwmaoo ucoefiuoexo zoom .mucoawuomxo mic mo sum « mnmoa mm mommouaxm mum mosam>m «a.oama.o pea.oamo.~ Nq.afioa.aa oe.o«oa.m nam.oamn.a Hm.oaum.m _ mama o o I. I o o o o I o “H Hag.“ ~H.¢Hmm.~ BHN oaom o umm.o+~e.~ o~.o+n~ H aha oamn c «an o+om H o uoeuuaam maoumao Houuaoo somsuaam mannoau Houuaoo coamcmnxm mamao>lumom aowmmmmxm masao>loum Am a,“ \ )\ ‘ ' '. . _ T ‘ at} 7 i ‘ V ‘ . .‘I '7. . , ‘, ‘ k, ?W ‘7' ,. s. ,w L - .. «l. M.:‘, h ff.” :iirlb :L .1" . '8' ., Figure 36 138 Figure 37. Renal tissue four days following cis-DDP treatment. Hema- toxylin and eosin, 100x. Proximal tubular cells of the 5 and 7.5 mg/kg cis-DDP group were atrophic with pale vesicular cytoplasm. Desquamated epithelial cells and amorphous cellular debris filled the tubular lumens. Proximal tubular lumens of kidneys of animals treated with 7.5 mg/kg were markedly dilated. Administration of 2.5 mg/kg cis-DDP did not markedly affect proximal tubules. 139 Figure 37 140 Effect of Mannitol Pretreatment on cis-DDP Glucose Intolerance Administration of mannitol prior to cis-DDP treatment resulted in a marked reduction in BUN concentration (Table 7). Although mannitol pretreatment did not affect cis-DDP hyperglycemia fifteen minutes following a glucose load, a significant reduction in plasma glucose concentrations was observed at 30, 60 and 120 minutes (Figure 38). Nevertheless, plasma glucose concentrations were elevated in the Manni- tol/cis-DDP group compared to controls at 15, 30 and 60 minutes (Figure 38). Plasma IRI concentrations of cis-DDP treated animals in both the fasting and glucose stimulated state were not significant affected by mannitol pretreatment (Figure 39). Plasma IRG concentrations were sig- nificantly elevated in both ciseDDP treated groups (sham and mannitol pretreated) (Figures 39); however, plasma IRG of cis-DDP drug treated animals was significantly reduced by mannitol pretreatment at all time points examined (Figure 39). Effect of Other Nephrotoxicants on Glucose Tolerance Blood urea nitrogen and serum creatinine concentrations were sig- nificantly elevated following administration of cis-DDP, gentamicin or glycerol (Table 8). Kidney weight to body weight ratios were ele- vated following treatment with any of the tested nephrotoxicants (Table 8). Glucose tolerance, however, was only impaired following treatment with cis-DDP or glycerol, and not following treatment with cephalori- dine or gentamicin (Figure 40). Similarly, the total integrated 141 TABLE 7 Effect of Mannitol Pretreatment on Blood Urea Nitrogen (BUN) and Plasma Immunoreactive Glucagon (IRG) of cis-DDP Treated Rats Plasma IRG (pg/m1) Treatment BUN (mg/d1) Totald True Pancreatice Extra-Pancreaticf Control 30: 2a 154: 6 74: 8 80:10 Mannitol/ 48: 3b 321:33b’c 122: 3b 1991331“c cis-DDP ’ Sham/cis- 101:19C 835i56c 251143c 602i69c DDP 8‘Values are expressed as means i SEM of four determinations. Plasma pooled from 3 identically treated rats was used for one determina tion. Mannitol pretreatment (2.4 g/kg) involved intravenous infu- sion of 102 mannitol solution (in 0.452 NaCl) over a 30 minute period using a Harvard infusion pump. Following 25 minutes of mannitol infusion, cis-DDP (5 mg/kg) (mannitol/cis-DDP) or saline vehicle (controls) was intravenously infused. Mannitol was then infused for remaining 5 minutes. Sham/cis—DDP animals were sub- jected to similar surgical procedures but did not receive mannitol. bSignificantly different from Sham/cis-DDP (p<0.05). cSignificantly different from controls (p<0.05). dTotal plasma IRG was determined using an antibody which cross- reacts with all immunoreactive components of glucagon (Leichter gt a_l, , 1975). eTrue pancreatic plasma IRG was determined using Unger's 30K antibody which cross-reacts with 3500 MW form of glucagon. fExtra-pancreatic plasma IRG was calculated as the difference between total and true-pancreatic IRG. 142 .Amo.ovav uaHoa.oaHu weavcommouuoo use um mamafiam manlmHo \HouHcama aoum monoquMHm unmoHMchHm m ouoochH muowwmn .unHoe oaau waHmcoamouuoo one um mHoHu Iaoo aoum monoummem unmonchHm m oumoncH mmeuoum< .aoHumaHauouov oao Mom mom: on: mHmEHam anemone hHHmoHucomH mouse Scum moaoom memmHm .maoHumcHaumuov know no :Mm H same one mason Ioueou ocwa HmoHuuo> :uHB ucHoa comm .conmmnH Hoanama uaonuas .maoam mnnImHo vo>HmooH zone umnu noHuaooxo one :uHB mounmooouq Hmonuom HmHHaHm ou vouooflnom ouo3 mamaHom mnnImHo\amnm .mouncHE o>Hm wchHmaou ocu wow momomcH HouHcama can zamsoco>muuaw momsmaH mm3 Aaouucoov oHoHno> ocHHmm no AmmalmHo\HouHccmav wa\me my manlmfio mo cowuoofinH mSHon m .mouaafia mm umum< .coHuom ounnHa an m uo>o HOuchmE Noa mo con5wcH meoao>muumH mo>ao>cw wa\w «.mv HouHcamE :uH3 acme lumonuoum .oocmuoaoucH omoonaw moosmcH manlmHo no ucoEumouuoua HouHcama mo uoommm .mm oustm 143 mm oustm Assn—$2.5... 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ON— oe ounwfim $5525... 0mm— 0 cc OO— OON can GOV (IF/5‘“) esoamg ' nmsnld 149 glycemic response to glucose administration was elevated only in ani- mals treated with cis-DDP or glycerol (Table 8). Effect of cis-DDP on Pancreatic Structure and Function Pancreatic tissue from cis-DDP treated rats (2.5, 5 or 7.5 mg/kg) appeared similar to that of controls (Figure 41). No signs of islet degeneration, necrosis, or inflammation were apparent at any of the drug doses tested (Figure 41). cis-DDP had no effect on serum.amy1ase activity (Figure 42). Biochemical Correlates of cis-DDP Glucose Intolerance Serum Sodium, Potassium, Calcium and Phosphorus cis-DDP treatment (2.5, 5, or 7.5 mg/kg) did not affect serum sodium, potassium or calcium concentrations on day 4 (Table 9). How- ever, administration of 7.5 mg/kg cis-DDP resulted in serum phosphorus concentrations approximately two times greater than controls (Table 9). Fasting;?1asma Glucose and Hepatic and Renal Gluconeogenic Enzyme Activity ‘ Fasting plasma glucose concentrations of animals treated with 2.5 or 5 mg/kg cis-DDP were comparable to controls (Table 10). In contrast, fasting plasma glucose was markedly elevated four days following admini— stration of 7.5 mg/kg cis-DDP (Table 10). cis-DDP treatment (2.5, 5 or 7.5 mg/kg) had no effect on hepatic or renal G-6-Pase or FDPase acti- vities (Table 10) on day 4. 150 Figure 41. Pancreatic tissue from rats treated with cis-DDP. Hema- toxylin and eosin stain, x100. Lighter stained tissue represents pan- creatic islets. No gross differences in histological appearance of pancreatic tissue are detectable following treatment with 2.5, 5 or 7.5 mg/kg cis-DDP. 151 C 2.5 mg/kg 7.5 mg/kg' . ....fi...€.....a.- an... a, _- «335% m. .9... a. “83%.... was . . '-x I \ .v ..b ..Ir . A 4 w of i r y . ANNEX .* fixe- In. a, p-I } .~ id? "9!" 1*. ‘ 5 559‘ ‘ if; .. ‘1' . . “2‘21 ’ if. A‘ 5 3‘- at? I" .3 «a mamhwnnhwmw 0‘. .2 Y ,‘1 .. 5.; 'Hfi- . n wig. ‘2‘ Figure 41 152 monommo ouo3 mamefiom momlufimm .aaouw m&\wa m ago an mmaomooo doom wo unseen monommoa onu .ooaumcwahouov moo you mom: was mamawam mmumouu adamowuommw wounu aoum vmaooo sonmm .mnofiumoaauouom know no sum H some one muconouoou mafia amoauuo> 53 :5 comm .ocoaumouu mannmwo woasoaaom ammo snow >ua>auoo mamazao souom .Nc muowam 153 107......“ Na .uawam 3:95 eon-..» m Wu w > oaaamm .muH>HuHmoom human mo muHEHH macho: moo Ha\wa oooHA mums mooam> HHo moummm mums mamaHam HH< .aoHumuumHoHamm oHoHno> no manlmHo waH uBoHHow mxmm q mouoomaoo ouo3 muomsHuooxm .mooHumoHaHOuov a unmoa um mo mum H momma no commounxo mum mooam>m m qua «Hmm 5 Hum ca.OHHq.~ mH.0Hom.H . oq.HHom.m 0H.HHma.¢ amoHIHHmm voooHA oano ocHHNmH mm.OHmo.N NH.OH~m.H om.oH~m.m oo.HHmH.m m.n ooanqm oHNN 5 Hum om.oHo~.N «H.OHmm.H Hm.OHma.o em.HHHo.HH m w waH «Hum a Ham mH.oHnm.~ -.0Hns.a wo.HHoh.h w~.HHm~.HH m.~ m Hmma chc c Hmm c~.0Hoo.N mq.0qu.H we.oH~m.m mmw.0HHm.m o :3an 3353 32s: 32 033...: 32. 3:5 335 mwmwam mmwwam oMMMMMw AoHououa wa\un\oum:nmo£a moHoanv AoHououm wa\us\oum:emosa moaoanv mnaImHo ammumnomonanlo.Huomouoaum amoumnnmonmlouomooaau unmaumouh mnaImHo wnHaoHHom EmHHonmuoz onoooau mo muooaouommoz oaHuoomnm mom HmoHamsoon mouooaom OH mam<9 156 Endocrine Correlates of cis-DDP Glucose Intolerance IRI and IRG Concentrations in Plasma and Pancreas Although fasting plasma IRI concentrations of animals treated with 2.5 or 5 mg/kg cis-DDP were comparable to controls, those treated with 7.5 mg/kg cis-DDP were significantly elevated compared to ad-libitum and pair-fed controls (Table 10). Fasting hyperglucagonemia was apparent four days following treatment with either 5 or 7.5 mg/kg cis- DDP . cis-DDP treatment at doses of 2.5, 5 or 7.5 mg/kg, did not signi- ficantly affect pancreatic concentrations of IRI (Figure 43) or IRG (Figure 44) in either the head portion (duodenal lobe) or body and tail region (adjacent to spleen and stomach). Components of Plasma IRG Treatment with S or 7.5 mg/kg cis-DDP resulted in a profound in- crease in total circulating concentrations of IRG on day 4 (Table 11). When an antibody specific to the 3500 MW form of IRG ("true pancreatic" glucagon) was used, a significant elevation in plasma IRG was also observed in the 7.5 mg/kg group when compared to ad-libitum fed controls (Table 11). An elevation in "true pancreatic" plasma IRG was also apparent in the 5 mg/kg cis-DDP group when compared to pair-fed part- ners (Table 11). A.marked elevation in "extra-pancreatic" plasma IRG (total - "true" IRG) was evident in these latter two treatment groups (Table 11). Plasma IRG ("total", "true pancreatic", and "extra-pancre- atic") in cis-DDP (5 mg/kg) treated animals was significantly reduced by mannitol pretreatment (Table 7). Nevertheless, total and "extra—pancre- atic" plasma IRG of Mannitol/cis-DDP treated animals was elevated 157 .ooouw wx\we m.m one an vasomeoo voom mo unsoEm monommoa mnu mmummmo mama mHmEHom mom nuHmm .Acomaem mom :omfioum on unoomflmmv Ham» mam hoop onu mam Aaoaovoom ou unoomfimmv mmouo lama onu mo :oHuuoa mam: men :H wmmHEumuoo mos ooHumuunmoooo HmH oHumoHoamm .mnoHumoHsuouoo o>Hm on snow mo Sam H acme mnu munmmmHamu oaHH HmoHuuo> noHB awn :omm .uomaumouu mnnImHo mGHBoHHom mama snow AHmHV :HaamoH m>HuomouoaoaaH mo mnoHumuuaooaoo oHumouoomm .mq muomHm 158 mo ouome 32.13-... . 0. he... as m ...S .26 Eon I 9:: U .md .223 8 8 2 3 (“PM 5/5'0 m O In O O 159 .enouw wx\wa m.m msu an mosomcoo voow mo uaooam consumes can mmummmo mum? mHmEHcm momIHHmm .Anomaem mam nomaoum ou unmomnmmv HHmu mom anon onu mom Aaooomoom ou unmomhvmv mmmuoama may mo coHuuoo emu: mnu oH mmoHahouom mos :oHumuucooooo me oHummuoomm .mnoHumoHEuouoo o>Hm ou snow mo mam H some mSu mucmmounou ocHH HmoHuuo> nqu no; comm .uooaummuu manlmHo ononHom mane snow AomHv commooaw m>HuomouocoaaH mo mcoHumuuaooooo oHumoHoamm .qc ouame 160 as «names 33.: :3 -... ...fi... 3 n 3 3:8... :5 Fa Eon I nHm on snow mo saw H cams mam muawmounou moHH HmoHuum> nqu Hon zoom .ucoaummuu mnaImHo wnHaoHHom mama snow AcmHv commooaw m>HuomoHocsaBH mo meoHumnuaooeoo oHuoouoamm .qq ouanm 160 «a «games 3:95 can ...u .3... m N m . 2 3:8. :5 .2558 I 04;: D 0.— ad 6.? (1M 40M 6I3") 92“ 161 TABLE 11 Components of Plasma Immunoreactive Glucagon (IRG) Following cis-DDP Treatment Plasma IRG (log) cis-DDP (mg/kg) Totala True-Pancreaticb Extra-Pancreaticc o 2.32:0.04d 2.04:0.04 0.26:0.03 2.5 I 2.34:0.06 2.04:0.02 0.31:0.06 5 2.61:0.03e’f 2.10:0.01f 0.51:0.02""f 7.5 3.56:0.069"f 2.73:0.058’f 0.79:0.02‘2’f Pair—fed 2.15:0.02e 1.94:0.05e 0.21:0.03e aTotal plasma IRG was determined using an antibody which cross-reacts with all immunoreactive components of glucagon (Leichter ££¢EL-: 1975). bTrue pancreatic IRG was determined using Unger's 30K antibody which cross-reacts with 3500 MW form of glucagon. cExtra-pancreatic IRG was calculated as difference between total and true pancreatic IRG. dValues are expressed as means i SEM of five determinations. Experi— ments were conducted four days following drug or vehicle administra- tion. Pair-fed animals were offered the measured amount of food con- sumed by the 7.5 mg/kg group. eSignificantly different from ad-libitum fed controls (p<0.05). fSignificantly different from pair-fed controls (p<0.05). 162 compared to controls (Table 7). "Total", "true pancreatic" and "extra- pancreatic" plasma IRG was also elevated following treatment with gentamicin and glycerol (Table 12). Glucagon Sensitivity Ten minutes following exogenous glucagon administration, plasma glucose concentrations were similar between control and treated (2.5, 5 or 7.5 mg/kg) animals (Table 13). However, a significant elevation in plasma glucose was observed 20 and 30, but not 60, minutes following glucagon administration in the 7.5 mg/kg cis-DDP group. No differences were noted between controls and animals treated with smaller doses of the drug (Table 13). The total integrated glycemic response to exo- genous glucagon was elevated only in the 7.5 mg/kg group (Figure 45). Insulin Sensitivity Ten, fifteen and twenty minutes following exogenous insulin admini- stration, a marked elevation in plasma glucose was evident in the 5 mg/kg group compared to controls (Figure 46). However, plasma glucose concentrations of treated animals declined to similar levels as controls 30 and 60 minutes following insulin administration (Figure 46). 163 .Amo.ovmv maouuooo aoum econoMMHm maucmoHMchHmo .mooHumoHauouom «In no sum H momma no mommouexo mum moaam>v .mammam oHumouoomn mama moo Hmuou consume mucoHoMMHm ozu mm mounaaoamo mus emu mammam UHumouocmaouuxmo .oowmooaw mo BMOM 3: comm :uHs muomouImmouo noHs3 hmoAHuom Mom m.uow:= mnHmo vooHauouov mos umH mamman oHumouoome woman .Ansma ..mmqmm.umunoHoAv command» mo muconoaaoo o>Huooouoo=aaH Ham nuHa muommuImmouo noHss mmonuuom so mchs moanuouom mos mam mamoae Hmuoem A>\s Non moqafimmoa usuafisoa a so as .ma\aa oao Houmohao Anzac w x mmm\x~ usamfiamm omwaamme ummaammaa a x as .ma\ma one aHoHamuoou A>H .ma\ma no quHHmmm ooanmmm onaHHnam a monImHo . Asa .ma\wa.oooav hm HmNN w Hema on Hnnn a oaHUHHoamnamo an HNoH n HmNH va Hhmu e Houuooo ooHuoouocmm muuxm noHummHoamm onus mamuoa ucoaumoue uaoaumoua Houw< oaHa Aaa\wav omH «amuse AomHv :owmoaao o>HuomouoonaaH mammam no mucmonououseoz vouooaom mo uoomum NH mqm<8 164 TABLE 13 Effect of cis-DDP on Glycemic Response to Exogenous Glucagon Plasma Glucose (mg/d1) %;:;£:§ Time (minutes) Following Glucagon (1 mg/kg, i.p.) O 10 20 30 60 o 113: 4a 182:13 155:15 159:11 176:14 2.5 131:14 l73il7 153:17 151118 168:22 5 104:12 175:30 175110 177:14 199:25 7.5 122:15 221:68 229:57b 243:80b 254:98 Pair-fed 82: 7 122: 9 107: 2 106: 7 129:1? aValues are expressed as means 1 SEM of four determinations. Experi— ments were conducted 4 days following drug or vehicle administration. Pair-fed animals were offered the measured amount of food consumed by the 7.5 mg/kg group. bSignificantly different from pair-fed controls (p<0.05). 165 .Amo.ovav >Ho>Huoonmou .maouuooo moMIuHmn mom aoanHHImo aoum monoHoMMHo oomoHMHome m oumonoH muowwmu mom mmeuoum< .mcoHumcHaHoumm snow mo sum H coma may mason Iouaou ocHH HmoHuuo> :uHS Han scam .eoouw wx\ws m.h onu kn moabmaoo moow mo ucaoam monommoa may monommo whoa mamaHom voquHmm .ooHuouuchHamo oHoHno> no maoImHo onSoHHow whom Hoom mouoovooo ouo3 mucoaHuoaxm .ooHumuuchHEom oomoooaw mcHonHom mouooHa om mom on .om .oa .Q on cowumuu Ioooooo omoooflw mammae mucomouoou o mam .m .o .n .m muonz new I Aooo + mom + now + non + moavw I mono “maoshom ononHom onu onm: moumaaoamo mos mou< .mHmaHam mouoouu anImHo oH ooHu ImuuchHBom AQH .wx\wa HV commooaw moocowoxo waHBoHHow o>uoo mooooaw as» Home: mou< .mq ouame O m 166 *I ‘6 O In 0 ($1 (\I I-- l-- ("Iw-IP/B) asoams VWSV'ld I!) 7.5 5 2.5 - cis- DDP (mg/ kg) control Figure 45 165 .Amo.oVov >Ho>Huooomou .maouuooo ooquHoe mom aoanHHImm aoum monopoMMHo oomoHMchHm m oumoHocH muowwmm mom mmeuoum< .mcoHumcHEHouom Hoom mo sum H some onu mason Ioueou omHH HmoHuuo> :uH3 Hon Loom .ooouw wx\wa m.n one km moaomooo moom mo uaooam monommoa one monomwo mums mHmBHnm moquHmm .ooHumuumHoHamo oaoHno> no mnaImHo wcHBOHHom ammo Hoom couoomooo ouoz muaoaHuoexm .ooHumuumfioHEom commoaHm on3oHHom mouoaHa cm mom on .om .OH .O on coHumuo Ioooaoo omoooaw mammaa mucomoueou o mom .v .o .n .m ouona new I Amos + mom + oon + pom + moavw u mono "casewow wcHSoHHow one onmo moumaooamo mo3 mou< .mHmBHom mouoouu mnnImHo :H ooHu ImuumHnHBom AoH .mx\wa Av nomoooaw moocowoxo on3oHHow o>uoo omoooaw any Home: mou< .mc ouome O 0') 166 *f In N a :2 2 ("!W-lP/5) asoomo vwsm In cis- DDP (mg/ kg) Figure 45 167 .Ano.0vov unHoe oaHu waHvooemouuoo onu um mHouu Iooo aoum monoHoMMHm unmoHuHome m oumoHvoH mmeuoum< .mooHumaHauouov o>Hm «0 2mm H some osu muoomoueou ooHH HmoHuHo> :uH3 uoHoo 50mm .mamchm moumouu ou moquHmo ohms maouuoou .aofiumuuchHEom oHoHno> no wa\ma mv mnaImHo onBoHHom whom Hoom vouoomooo ouoB muaoaHuoexm .AQH .wx\DH ww.ov swaomoH moooowoxo ou uncommon oHaooxam co anImHo mo uoommm .oq ouome 168 A5575; on r) I non—um? 9:9: m I .933 0.. ON O— o O O O on ~o t N O 2 0503le unsold u! ofiuqqax ON— ov— DISCUSSION Glucose homeostasis is characterized by a balance between the opposing, but tightly coordinated, processes of glucose utilization and glucose production. Consequently, a chemical lesion or insult dis- turbing this delicate balance may result in hyperglycemia by impairing glucose utilization and/or increasing glucose production. Although regulation of these processes is primarily mediated by the "push-pull" actions of insulin and glucagon, the individual contributions of these hormones in producing hyperglycemia have been controversial. Recently, two schools of thought have emerged. The "unihormonal abnormality con- cept" proposes that hyperglycemia is the direct consequence of decreased glucose utilization associated with impaired insulin secretion and/or action. In contrast, the "bihormonal" or "double-trouble" hypothesis assigns to glucagon the role of an essential co-mediator (Unger and Orci, 1975). According to the latter postulate, hyperglycemia is not only a consequence of a relative or absolute deficiency of insulin, but in addition results from massive overproduction of glucose mediated by excessive circulating glucagon (Unger and Orci, 1975). Although'Unger's bihormonal theory remains controversial, it is generally agreed that impaired insulin metabolism plays an important role in glucose utilization and therefore may determine the severity of 169 170 hyperglycemia. Consequently, hyperglycemia may result, in part, from a lesion affecting any aspect of insulin metabolism, ranging from pan- creatic beta cell damage to impaired insulin action at target tissues. With respect to the bihormonal theory, hyperglycemia may also result in part from alpha cell hypersecretion of glucagon and/or increased sensi- tivity to glucagon action at target tissues. Several divalent metal cations influence carbohydrate metabolism by their ability to alter insulin and/or glucagon metabolism (Eaton, 1973; Ghafghazi and Mennear, 1975; Horak and Sunderman, l975a,b; It- hakissios 35 al., 1975). Therefore, it was the central hypothesis of this dissertation that a divalent platinum compound, such as cis-DDP may similarly affect glucose homeostasis. Although the paucity of data documenting the effects of divalent metals on glucose homeostasis does not support a common single mechanism, hyperglycemia appears to be a common manifestation associated with metal toxicity (Ghafghazi and Mennear, 1973; Horak and Sunderman, 1975b). Results of this study suggest that cis-DDP (S, 7.5 or 15 mg/kg) induces hyperglycemia in nonfasting animals (Figure 6). In contrast, admdnistration of equimolar doses of trans-DDP did not result in hyper- glycemia (Figure 6), indicating that this action is unique to the geo- metry of the cis—DDP complex. Furthermore, chemical properties other than the presence of a divalent platinum atom appear to contribute to cis-DDP hyperglycemia since administration of tetrachloroplatinate did not elevate blood glucose concentrations (Figure 6). Treatment with 5 mg/hg cis-DDP resulted in hyperglycemia observed only in the nonfasting state and was no longer apparent upon fasting, 171 when exogenous (dietary) carbohydrate is removed. These results there- fore suggest that decreased utilization of exogenous glucose contributes in part to cis-DDP hyperglycemia. To examine the effects of cis-DDP treatment on glucose utilization, glucose tolerance was evaluated by measuring plasma glucose following an exogenous glucose load. A glu- cose challenge in cis-DDP treated (5 mg/kg) animals resulted in a marked and persistent hyperglycemia (Figures 11 and 12), an effect consistent with impaired glucose utilization. Impaired glucose metabolism follow- ing treatment with 5 mg/kg cierDP was manifested to a modest degree on day 2 (Figure 11) and to a more profound extent on day 4 (Figure 12). However, this phenomenon appears to be transient as indicated by the negligible and complete absence of cis-DDP glucose intolerance on days 7 (Figure 13) and 14 (Figure 14), respectively. Although the transient nature of cis-DDP glucose intolerance is a pattern consistent with that of other divalent metals, its exact time course is markedly different. For example, glucose tolerance is impaired 1 hour following treatment with cadmium and returns to control values by24 hours (Ghafghazi and Mennear, 1973). In contrast, at least four days are required for com- plete expression of cis-DDP glucose intolerance. This apparent delay of cis-DDP toxicity is not unique to the development of glucose intol- erance; cis-DDP nephrotoxicity requires several days before histopatho- logical or functional damage becomes apparent (Dobyan £5 31., 1980; Safirstein st 51,, 1981b; ward and Fauvie, 1976). Thus, although the effects of cis-DDP on glucose tolerance are similar to those of other heavy metals, its delayed appearance may indicate that (l) the mecha- nisms mediating cis-DDP toxicity differ from those of other heavy metals 172 and/or (2) the biological handling of this platinum complex is uniquely different from metal salts. Both of these possibilities are consistent with the suggested biotransformation of cis-DDP to a more reactive and cytotoxic molecule (Long and Repta, 1981). Neither trans-DDP nor tetrachloroplatinate impaired glucose toler- ance at any time examined (Figures 11-16), indicating the specificity of action of the cis-DDP complex. Tissue platinum concentration peruse probably does not account for this specificity since similar tissue distribution patterns of radiolabelled platinwm have been reported fol- lowing administration of cis-DDP or trans-DDP (Hoeschele and Van Camp, 1972). Although trans-DDP or tetrachloroplatinate may affect glucose metabolism prior to day 2, mechanisms mediating these acute changes are probably different than those mediating the more delayed actions of cis—DDP. Since insulin plays a fundamental role in tissue uptake and meta- bolism.of glucose, decreased utilization of exogenous glucose following cis-DDP treatment:may be secondary to impaired insulin secretion and/or target tissue action. The deficient insulin response to a glucose stimulus 2 days following treatment with 7.5 mg/kg cis-DDP (Figure 9) is consistent with an impairment of insulin release by cis—DDP. Hows ever, plasma IRI concentrations following glucose stimulation in animals treated with 5 mg/kg cis-DDP was not significantly different from.ad- libitum fed controls on days 2 (Figure 17), 4 (Figure 18), 7 (Figure 19) or 14 (Figure 20). Thus, although marked impairment of glucose utilization was manifested 2 days following treatment with either 5 mg/kg 173 (Figure 11) or 7.5 mg/kg (Figure 8) cis-DDP, plasma insulin response was only significantly impaired in the latter group. This apparent discrepancy may be attributable to a dose-dependent impairment in insulin metabolism. Decreased glucose utilization in the 5 mg/kg cis-DDP group was evident despite an apparently normal plasma IRI response to a glucose stimulus, suggesting that cis-DDP may impair insu- lin action. Insulin resistance, by definition, exists when a known quantity of insulin produces a less than normal biological response (Olefsky, 1981); therefore, an apparently normal IRI response accom- panied by decreased glucose utilization following cis-DDP treatment may suggest the presence of insulin resistance. In uremia, impaired glucose utilization may be due to insulin re- sistance (DeFronzo, 1978; DeFronzo and Alvestrand, 1980; DeFronzo gt 5%,, 1981). Therefore, impaired glucose metabolism following cis-DDP treatment may be related to cis-DDP induced uremia. Impaired renal function in cis—DDP treated animals (5, 7.5 or 15 mg/kg) was charac- terized by polyuria (Figure 22), glycosuria (Figure 24) and elevated BUN concentrations (Figure 33). Since polyuria preceded polydipsia in treated (5 mg/kg) animals (Figure 22), increased water intake is pro- bably secondary to volume depletion and is not a cause of increased 24 hour urine volume. Although polyuria following ciséDDP treatment may be a consequence of impaired concentrating ability (Safirstein ggugl., 1981b) or profound glycosuria (Figure 24), cis-DDP (5 mg/kg) glycosuria was transient (Figure 24) whereas polyuria persisted throughout the 14 day experiment, suggesting a dissociation between these two phenomena. Interestingly, those cis-DDP treated animals which were glycosuric were also hyperglycemic (Figure 6). Inasmuch as hyperglycemia 174 appeared to precede glycosuria following cis-DDP treatment, glycosuria may not only reflect a proximal tubular lesion, but may also be a con- sequence of hyperglycemia, conceivably by increasing the filtered load of glucose beyond the transport capacity for proximal tubular reabsorp- tion. Additionally, the transient nature of cis-DDP glycosuria and hyperglycemia in surviving treated (5 mg/kg) animals further suggests an interrelationship between the two phenomena. In contrast to cis-DDP, equimolar doses of the trans isomer did not affect urine volume (Figure 23) or urinary glucose excretion (Figure 25); indicating the specificity of cis-DDP action on the kidney. Howe ever, administration of 18 mg/kg tetrachloroplatinate resulted in both polyuria (Figure 23) and glycosuria (Figure 26) on days 1, 2, and 4. Similar to the effects of cis-DDP, polyuria preceded increased fluid intake in tetrachloroplatinate treated (18 mg/kg) animals, indicating that polyuria is not a consequence of increased fluid intake. Glycos- uria accompanied polyuria at all times examined; therefore, the osmotic effect of increased urinary glucose may contribute to increased urine volume. Since tetrachloroplatinate did not result in hyperglycemia (Figure 6), glycosuria in treated (18 mg/kg) animals may primarily re- flect impaired proximal tubular reabsorption glucose. Since cis-DDP treated and pair-fed animals consumed equivalent quantities of food, urinary electrolyte excretion should be similar between the two groups unless cis-DDP selectively affects gastrointesti- nal absorption and/or renal excretion of electrolytes. Although cis-DDP (5 mg/kg) decreased urinary sodium (days 7 and 14 (Figure 29) and potas- sium.concentration (days 2, 4, 7, and 14) (Figure 31) compared to pair- fed partners, the concomitant increase in 24 hour urinary volume in 175 treated animals resulted in renal electrolyte excretion equivalent to pair-fed animals. Only animals treated with 15 mg/kg cis-DDP exhibited increased urinary potassium excretion compared to pair-fed partners (Figure 31) and presumably resulted in net negative potassium balance. Similarly, neither trans-DDP nor tetrachloroplatinate affected urinary sodium and potassium excretion (Figures 30 and 32). BUN concentrations were elevated following cis-DDP (5, 7.5 or 15 mg/kg) or tetrachloroplatinate (18 mg/kg), but not trans-DDP, treatment CFigure 33). cis-DDP induced elevation in BUN is a finding consistent with other reports (Leonard 35 21., 1971; Safirstein egual., 1981b; Schaeppi 35 31., 1973; Ward and Fauvie, 1976) and may be attributable to impaired renal clearance of urea, suggested by the decreased GFR in treated animals (Table 6). The decreased GFR may be related to the profound depression in effective renal plasma flow (CPAH) (Table 6). Although kidneys of animals treated with 5 mg/kg cis-DDP tended to exhibit a decreased capacity to accumulate PAH and TEA, no differences in these renal functions were noted compared to their pair-fed partners (Figure 34), suggesting that the observed effect was probably related to decreased food intake. In contrast, the presence of 500 or 600 ug/ml cis-DDP in incubation medium profoundly impaired the ability of renal cortical slices to accumulate both PAH and TEA (Figure 35). Insofar as PAH and TEA are secreted by independent systems, competitive inhibition for both transport carriers by cis-DDP is unlikely. Rather, cis-DDP related depression in organic ion accumulation is nonspecific and is probably related to a generalized depression in renal metabolism. 176 The apparent discrepancy between the profound depression in organic ion accumulation observed ig_vitro and the modest effects observed ig_vivo may be due, in part, to the relative differences in cis-DDP exposure in the two studies. Four days following cis-DDP treatment (5 mg/kg), kidneys of F-344 rats contain 10 pg platinum/g wet tissue (Litterst st 51,, 1976b). Interpolation of these data suggest that kidneys of drug treated animals are exposed to 30-40 times less platinum than slices incubated in medium containing 500 or 600 ug/ml cis-DDP. Taken collectively, these results indicate that administration of cis-DDP (5, 7.5 or 15 mg/kg), but not equimolar doses of trans-DDP, impairs specific renal functions. Therefore, the effects of cis-DDP on the kidney may contribute to the disturbances observed in glucose meta— bolism. Evidence supporting this postulation is based on the observa- tion that administration of cis-DDP only at doses which are frankly nephrotoxic appear to impair glucose tolerance. Furthermore, BUN con- centrations of the 5 mg/kg cis—DDP group were profoundly elevated on day 4 (Figure 33), a time at which glucose tolerance was also profound- ly impaired (Figure 12). Hemodialysis in uremic patients improves glucose tolerance and may be associated with improved tissue sensitivity to insulin (DeFronzo 25 31,, 1981). By analogy, if cis-DDP induced glucose intolerance were secondary to uremia, an amelioration in renal function should theoreti- cally improve glucose metabolism. To determine the effect of cis-DDP induced uremia on glucose tolerance, the effects of mannitol pretreatment on cis-DDP nephrotoxicity and glucose intolerance were evaluated. Since mannitol pretreatment reduces cis-DDP nephrotoxicity without altering 177 its pharmacokinetic or therapeutic properties (Pera £5 31., 1979), any effect of this manipulation on cis-DDP glucose intolerance is presumably related to alterations in kidney function. Although mannitol pretreat- ment reduced plasma glucose concentrations in drug treated animals 30, 60 and 120 minutes following a glucose load, hyperglycemia relative to controls was still apparent at 15, 30 and 60 minutes (Figure 38). The partial improvement in glucose utilization by reducing cis-DDP nephroe toxicity suggests that impaired renal function contributes, at least in part, to cis-DDP glucose intolerance. Furthermore, mannitol pretreatment improved glucose tolerance with no apparent improvement in plasma IRI response to glucose (Figure 39), suggesting that improved glucose meta- bolism may be due to enhanced tissue sensitivity to IRI. Although these results indicate that cis-DDP induced uremia may contribute to the observed glucose intolerance, administration of other agents which also induce uremia, i.e., cephaloridine, gentamicin or glycerol, did not uniformly impair glucose tolerance (Figure 40). These results therefore suggest that cis-DDP induced glucose intolerance is probably independent of uremia. To determine if cis-DDP produces insulin resistance, the hypogly- cemic actions of exogenous insulin were evaluated. Theoretically, if insulin action were impaired at the target tissue level by cis—DDP, then administration of exogenous insulin to treated animals should result in a blunted hypoglycemic response.‘ However, treated animals exhibited a maximal hypoglycemic response 30 minutes following insulin administration, an effect which was similar to controls (Figure 46). 178 Insulin resistance may be due to decreased sensitivity to insulin (i.e., a shift in the dose-response curve to the right) and/or decreased maximal response to insulin (Kahn, 1978; 1980). Since only 10% of receptor occupancy is required for maximal response to insulin (Kahn, 1978), a reduction in receptor binding of up to 90% produces decreased sensitivity with no change in maximal response. Therefore, although maximal biological response to insulin was elicited with the given dosage of insulin, administration of smaller doses of insulin to treated ani- mals may have produced a more blunted hypoglycemic response compared to controls. These results therefore do not rule out the possibility of decreased insulin sensitivity following cis-DDP treatment. Since manni- tol pretreatment improved cis-DDP glucose intolerance without improving IRI secretion, the increased metabolism of glucose may be related to enhanced tissue sensitivity to insulin. An alternative explanation for decreased glucose utilization accom- panying an apparently normal plasma IRI response in cis-DDP treated animals (5 mg/kg) may relate in part to the presence of a less biologi- cally active, although immunoreactive, beta cell secretory product. .The insulin antibody used in the routine immunoassay cross-reacts with pro- insulin (Rubenstein st 31., 1968). Proinsulin in the normal state com- prises only 52 of total plasma IRI concentration; therefore, cross- reactivity with this molecule does not usually confound interpretation of IRI data. Hewever, in renal failure, fasting proinsulin concentra- tions are elevated 7.5-fold (Baba 35 31,, 1979), a finding consistent with the primary role of the kidney in proinsulin degradation (Constan 179 §£_§l,, 1978; Izzo.§£.a1., 1978; Jaspan 35 31., 1977; Katz and Ruben- stein, 1973; Kitabchi, 1977). The renal handling of proinsulin and insulin are characterized by high extraction rates (36 and 40%, respec- tively) and low urinary clearances, suggesting that almost all of the polypeptide extracted is metabolized by the kidney (Katz and Rubenstein, 1973). Despite these similarities, renal clearances of insulin and proinsulin represent one-third and two—thirds of their total metabolic clearance, respectively; the differential between the two polypeptides is related to the significant metabolism of insulin, but not proinsulin, by the liver (Katz and Rubenstein, 1973). In renal failure, plasma proinsulin levels are also elevated follow— ing glucose stimulation and represent a significantly large percentage of total circulating IRI (Baba 25 31., 1979). On this basis, plasma IRI of cis-DDP treated animals in both the fasting and glucose-stimulated states may represent a disproportionately large amount of proinsulin, an effect secondary to diminished renal degradation associated with cis- DDP nephrotoxicity. If this were the case, then the less potent hypo- glycemic actions of this beta cell product may explain: (1) fasting hyperglycemia accompanied by elevated plasma IRI in the 7.5 mg/kg cis— DDP group (Table 10) and (2) decreased utilization of exogenous glucose despite an apparently normal plasma IRI response in animals treated with 5 mg/kg cis-DDP (Figures 11, 12, 17 and 18). Since experiments were not designed to examine the composition of plasma IRI following cis-DDP treatment, these interpretations remain speculative. In summary, these results indicate that cis-DDP (5 mg/kg) hypergly- cemia accompanied by an apparently normal plasma IRI response to glucose, 180 may either reflect decreased tissue sensitivity to insulin and/or the presence of a less biologically active, although immunoreactive, beta cell secretory product. However, it might be argued that although absolute plasma IRI concentrations were apparently similar between treated and controls, they were deficient relative to the elevated blood glucose concentrations in animals treated with 5 mg/kg cis-DDP. Thus, the absence of an exaggerated IRI response to glucose following cis-DDP treatment suggests that impaired insulin synthesis and/or secretion contributes significantly to the observed impairment in glucose utili- zation (Figure 9). Although biosynthetic rates of insulin were not' determined, pancreatic IRI concentrations were not affected by cis-DDP treatment (Figure 43), indicating that pancreatic stores of IRI are not deficient. Thus, impaired glucose utilization following exogenous glucose is probably related to impaired insulin secretion. Several factors may contribute to impaired insulin secretion following cis-DDP treatment. Starvation is known to result in glucose intolerance by impairing insulin release (Hedeskov, 1978) and therefore, reduced food intake following cis-DDP treatment (Figure 4) may contribute, in part, to impaired glucose metabolism. However, inasmuch as animals pair-fed to drug treated partners did not exhibit glucose intolerance (Figures ll-14), the decreased utilization of exogenous glucose following cis-DDP treatment appears to be independent of starvation. Furthermore, animals pair-fed to the 7.5 mg/kg group did not exhibit a blunted plasma IRI response to glucose stimulation (Figure 9), indicating that reduced food intake of drug treated animals did not significantly affect insulin 181 metabolism. Similarly, the relative response in plasma IRI from the fasting to the glucose stimulated state at 15 minutes was not signifi- cantly affected in animals pair-fed to the 5 mg/kg cis-DDP group (Table 3). An impairment in insulin secretion may also result from drug-induced stress. The stress-induced release of adrenal catecholamines and gluco- corticoids produces hyperglycemia; the former by a direct adrenergic effect on pancreatic islet function, resulting in inhibition of insulin release, and the latter by decreasing receptor binding to insulin. Consequently, impaired glucose utilization may not necessarily result from a direct insult to pancreatic islet function; but rather to changes in insulin metabolism effected by a stress related increase in catechol- amdnes and glucocorticoids. In addition to influencing pancreatic islet. functions, catecholamines may also directly activate hepatic glycogeno- lysis and gluconeogenesis or inhibit tissue uptake of glucose. These well documented effects of adrenal catecholamines and gluco- corticoids on carbohydrate metabolism coupled with the observation of adrenal medullary and cortical hypertrophy following cis-DDP treatment (Tech-Allen, 1970), suggest that cis-DDP glucose intolerance may be adrenal mediated. The adrenal weight to body weight (AN/BU) ratios were elevated 4 days following treatment with 5 mg/kg cis-DDP (Table 4), an effect which is not due to decreased body weight since these ratios were significantly different from pair—fed partners of comparable body weight (Figure 5). Furthermore, changes in relative adrenal weights (Table 4) were accompanied by changes in glucose tolerance (Figures 12 and 14) 4 and 14 days following treatment with 5 mg/kg cis-DDP. 182 Theoretically, if glucose intolerance following cis-DDP treatment were adrenal mediated, then adrenalectomy should prevent these abnorma- lities in carbohydrate metabolism by improving insulin metabolism. Bi- lateral adrenalectomy reduced plasma glucose concentrations in both the fasting state and 120 minutes following a glucose load in cis-DDP treated animals (Figure 21). This improvement in glucose metabolism by adrenalectomy appears to be related to enhanced tissue sensitivity to insulin as indicated by the absence of fasting hyperglycemia (Figure 21) and hyperinsulinemia (Table 5) in the Adx/cis-DDP group. Enhanced tissue sensitivity to insulin in adrenalectomized animals may be due to increased receptor binding affinity to insulin associated with the presumed absence of glucocorticoid activity (Kahn, 1978). Nevertheless, impaired glucose utilization following exogenous glucose administration, was apparent during the early stages of the glucose tolerance test (15, 30, and 60 minutes) in both cis-DDP groups, regardless of the presence or absence of adrenal glands (Figure 21). These results sugest that although partial improvement in cis-DDP glucose intolerance was observed, a stress related effect on catecholamine and/or glucocorticoid metabolism cannot entirely account for the abnormalities in carbohydrate ‘metabolism. Therefore, factors in addition to those related to adrenal function.must mediate cis-DDP glucose intolerance. One of the critical events in glucose stimulated insulin release is an increased flux of ionized calcium into the cytosol of pancreatic beta cells. Diminished insulin response to glucose has been reported in hypocalcemia (Bansal st 2;}: 1975; Gero 35 al., 1976), an effect which 183 may relate to the dependence of insulin secretion upon extracellular calcium. Hypocalcemia may result from cis-DDP treatment (Hayes et 31., 1979) and therefore may contribute to glucose intolerance. However, serum calcium concentrations of drug treated animals were comparable to controls (Table 9). Hypokalemia is also known to result in impaired insulin secretion (Rowe £5 91,, 1980); however, serum potassium concen- trations were also unaltered by cis-DDP treatment (Table 9). Although neither serum potassium nor calcium concentrations were affected by cis- DDP treatment, an effect on their beta cell metabolism cannot be dis- counted. Alternatively, impaired insulin secretion following cis-DDP treat- ment may be a result of a direct pancreotoxic effect of the drug. However, administration of cis-DDP at doses known to impair glucose metabolism did not result in histopathological damage to the pancreas, as indicated by the absence of islet degeneration, necrosis and inflam- mation using light microscopy (Figure 41). Although no gross histo- pathological damage to the pancreas was evident, cis-DDP may alter islet cell population and/or subcellular architecture. Further studies would be needed to explore these aspects. Serum amylase activity, which provides a gross estimate of pancreatic function, was not affected by cis-DDP treatment (Figure 42), indicating the unlikelihood of cis-DDP induced pancreatic damage. Since glucagon has been assigned the role of an essential co— mediator in diabetes (Unger and Orci, 1975), it was hypothesized that hyperglycemia following cis-DDP treatment may also be mediated in part by drug induced hyperglucagonemia. Glucagon may induce hyperglycemia 184 by increasing endogenous glucose production beyond the capacity for glucose utilization. Plasma IRG concentrations were elevated following cis-DDP treatment (5 or 7.5 mg/kg) and accompanied the observed glucose intolerance (Figures 10, 17 and 18). Furthermore, administration of 7.5 or 10 mg/kg cis-DDP induced hyperglycemia in both the fasting and nonfasting state (Figures 6 and 7), indicating the likelihood of endo- genous glucose overproduction. Endogenous glucose overproduction may be characterized by increased rates of glycogenolysis and/or gluconeogenesis. Although fasting hyper- glycemia accompanied by hyperglucagonemia, was evident following treat- ment with 7.5 mg/kg cis-DDP, increased hepatic and renal activities of the gluconeogenic enzymes, glucose-6-phosphatase and fructose-1,6-di- phosphatase, were not observed (Table 10). Several possible explana- tions may account for the absence of increased gluconeogenesis accomr panying fasting hyperglycemia following cis-DDP treanment (7.5 mg/kg). Mechanisms mediating fasting hyperglycemia may not involve an absolute increase in glucose production; rather, glucose production may be in- creased only relative to rates of glucose utilization. Since cis-DDP treatment impairs glucose utilization, normal rates of glucose produc- tion may exceed the capacity for utilization. Further studies would be needed to determine the relative rates of glucose production and utili- zation following cis-DDP. Secondly, glucagon mediated glucose produc- tion is a short-lived phenomenon, resulting in only a transient increase in glucose production (Fradkin g£_§l,, 1980). Therefore, since glucose utilization is impaired by cis-DDP, a brief burst in glucose production (prior to day 4) may result in prolonged hyperglycemia unaccompanied by 185 a sustained elevation in glucose production. Finally, an alternative explanation may be that fasting hyperglycemia in treated animals is mediated by an increased glucose production due either to a preferen- tial mobilization of glycogen stores or to increased gluconeogenesis which was for some reason not detected in the present experiments. In contrast to the 7.5 mg/kg cis—DDP group, hyperglucagonemia following treatment with 5 mg/kg cis-DDP was not associated with fasting hyperglycemia (Table 10). Although it may be argued that profound glycosuria induced by cis-DDP (Figure 24) may mask the hyperglycemic actions of glucagon, hyperglycemia was evident in these animals in the nonfasting state (Figure 6). Thus, the paradoxical appearance of hyper- glucagoenmia and normoglycemia is probably not related to urinary losses of glucose. Rather, this phenomenon may be attributed to the effects of cis-DDP on the biological activities of circulating IRG and/or to meta- bolic_adaptation to glucagon action. Plasma glucagon immunoreactivity comprises at least four different components with differing molecular~ weights, averaging 150,000, 9000-12,000, 3500 and 2000 daltons (Jaspan 35 31,, 1981).. The 3500 MW form corresponds with "true pancreatic" glucagon and is believed to be the biologically active form.of glucagon. In contrast, the nature of the higher MW forms of plasma IRG are not ‘well defined, although the 9000-12,000 MW fraction may represent a pre- cursor (proglucagon) or intermediate of glucagon biosynthesis. The marked heterogeneity of plasma IRG is further complicated by the pre- sence of glucagon like immunoreactive (GL1) components, which are prin- cipally of intestinal origin. 186 Since the antibody used to measure plasma IRG in these studies cross-reacts with all immunoreactive components of glucagon, it was hypothesized that the elevation in plasma IRG following cis-DDP treat- ment (5 or 7.5 mg/kg) may represent a form other than the 3500 MW. To test this, plasma IRG was quantified using an antibody which cross- reacts with all IRG components ("total") and one which cross-reacts with only the 3500 MW form ("true pancreatic"). Although plasma concentra- tions of "true pancreatic" IRG of the 5 mg/kg group were comparable to ad-libitum.fed controls, they were significantly greater than pair-fed controls (Table 11). "Extra-pancreatic" plasma IRG ("tota1" - "true pancreatic") of animals treated with 5 or 7.5 mg/kg cis-DDP represented a significantly large proportion of total circulating IRG (70 and 84%, respectively) compared to ad-libitum or pair-fed controls (43 and 38%, respectively). These data suggest that elevated plasma IRG following cis-DDP treatment is primarily due to an increase in a form other than the 3500 MW component. Nermoglycemia accompanied by an elevation in "extra pancreatic" plasma IRG in animals treated with 5 mg/kg cis-DDP might suggest decreased hyperglycemic activity of this IRG component. However, this suggestion remains speculative since the biological acti- vity of these IRG fractions was not determined in these studies. Despite the relatively large proportion of "extra pancreatic" plasma IRG in the 7.5 mg/kg group, "true pancreatic" plasma IRG concentration was elevated 5 to 7 fold (Table 11). The magnitude of this elevation would be expected to produce severe fasting hyperglycemia and massive increases in glucose production. However, neither of these phenomena 187 was observed, suggesting the possibility of metabolic adaptation to the biological actions of "true pancreatic" glucagon following cis-DDP treatment. The effects of hyperglucagonemia are short-lived; thus, a chronic and sustained elevation in circulating glucagon is not charac- terized by continued glucose overproduction (Fradkin £2 21., 1980; Sherwin and Felig, 1980). Adaptation to chronic hyperglucagonemia is well documented and appears to be characterized by either down regula- tion of glucagon receptors (Bhathena SE 31., 1978) and/or the develop- ment of hepatic refractoriness to the enzymatic effects of glucagon (e.g., decreased activity of adenylate cyclase, increased activity of phosphodiesterase) (Fradkin e£_al,, 1980). Regardless of the exact mechanism, it is likely that chronic hyperglucagonemia following cis-DDP treatment results in metabolic adaptation and this may account for the absence of severe fasting hyperglycemia and glucose overproduction normally elicited by an elevation of glucagon. An alternative, but less likely, explanation for hyperglucagonemia unaccompanied by severe fasting hyperglycemia following cis-DDP treat- ment, is the possibility of an impaired glycemic response to glucagon at the target tissue level. This phenomenon has been reported to con- tribute to the hyperglucagonemia and normoglycemia of cobalt chloride treated animals (Eaton, 1973). To test this possibility in cis-DDP treatment, animals were evaluated for their glycemic response to exoge- nous glucagon. A blunted response to glucagon was not observed in treated animals (Table 13); therefore, glucagon resistance at the target tissue level is unlikely following cis-DDP treatment. In fact, animals treated with 7.5 mg/kg cis-DDP demonstrated marked hyperglycemia 20 and 30 minutes following glucagon administration (Table 13). Since the 188 maximal increase in blood glucose was not different between treated and control animals, the hyperglycemia following treatment with 7.5 mg/kg cis-DDP probably reflects decreased tissue uptake and metabolism of glucose rather than continued increase in glucose production. In summary, these results indicate that the absence of severe fast- ing hyperglycemia and massive glucose overproduction in cis-DDP hyper- glucagonemia may be related in part to the biological activities of the plasma IRG components and/or to the development of metabolic adaptation to a chronic and sustained elevation in plasma IRG. It is likely that cis-DDP hyperglucagonemia is due to decreased de- gradation of IRG rather than increased secretion. The kidney represents the principal site of glucagon degradation as indicated by the relatively high extraction and low urinary clearance of this hormone (Bastl e£_§1,, 1977; Lefebvre 35 51}, 1974). Furthermore, hyperglucagonemia, indepen- dent of pancreatic IRG secretion, has been documented following acute bilateral kidney exclusion (Lefebvre and Luyckx, 1976), renal artery. clamping (Lefebvre 35 21., 1974), bilateral ureteral ligation (Bilbrey £31., 1974; Emanouel _e£_;a_]_._., 1976), 702 nephrectomies (Bastl it; 9.1., 1977) and in chronic renal failure patients in which hyperglucagonemia is reversed by renal transplantation (Bilbrey 35 $1,, 1975). In this manner, hyperglucagonemia of renal failure has been attributed to im- paired renal degradation rather than increased alpha cell secretion. A similar mechanism associated with cis-DDP nephrotoxicity may therefore mediate cis-DDP hyperglucagonemia. 189 Reduced cis-DDP nephrotoxicity by mannitol pretreatment also re— sulted in reduced plasma IRG concentrations in treated animals (Figure 39 and Table 7). Furthermore, administration of other agents which im- pair renal function, such as glycerol and gentamicin, also resulted in an elevation in plasma IRG (Table 12). These results are consistent with the postulate that cis-DDP hyperglucagonemia is primarily mediated by impaired renal degradation of glucagon, an effect secondary to cis- DDP nephrotoxicity. The mechanisms by which glucagon is handled by the kidney appear to involve glomerular filtration followed by proximal tubular reabsorption (Nahara e£_al,, 1958); the proximal tubules contain glucagon degrading enzymes located in both the brush border and cytosol (Duckworth, 1976a). Kuku egual. (1975) reported that hyperglucagonemia of chronic renal failure was largely due to a striking elevation in a 9000 MN component, comprising 571 of total circulating IRG, and a moderate elevation in . the 3500 MN fraction. The 3500 MW form appears to be handled primarily by glomerular filtration and tubular reabsorption whereas the renal handling of the 9000 MW component may involve peritubular uptake from postglomerular capillaries (Emmanouel SE 31., 1976). These results, when extrapolated to the present studies, suggest that the moderate elevation in the 3500 MW form of IRG may be related to reduced GFR of drug treated animals (Table 6). Since the 3500 MW IRG component is the only IRG peptide to be metabolized by both kidney and liver (Jaspan SE 51., 1977), hepatic metabolism of this component in cis-DDP treated animals may prevent a profound elevation in the circulation. In contrast, the kidney is the major site of catabolism 190 of the 9000 MW form of IRG. Therefore, the presumed impairment in renal degradation of IRG associated with cis-DDP nephrotoxicity may result in increasing circulating levels of this IRG component in treated animals. Although experiments were not designed to identify the immunoreactive components of plasma IRG in cistDP treatment, it seems reasonable to speculate that a large proportion of "extra pancreatic" plasma IRG in treated animals is represented by the 9000 MW form. An alternative explanation for cis-DDP hyperglucagonemia is in- creased glucagon secretion. Glucagon hypersecretion from the alpha cells of the pancreas may be secondary to catecholamine release asso- ciated with drug induced stress. If such a mechanism mediates cis-DDP hyperglucagonemia, then adrenalectomy should prevent cis-DDP hypergluca- gonemia. However, since plasma IRG concentrations were not signifi- cantly elevated 2 days following cis-DDP treatment (Table 5), it is difficult to evaluate the role of adrenal mediated stress in cis-DDP hyperglucagonemia. In summary, these results indicate that cis-DDP treatment results in profound hyperglucagonemia, an effect which is probably related to decreased renal degradation of IRG. Since hyperglucagonemia in treated animals was not associated with severe fasting hyperglycemia, the biolo- gical activity of this hormone and its contribution to cis-DDP glucose intolerance must be questioned. Several lines of evidence suggest that cis-DDP hyperglucagonemia probably does not mediate cis-DDP glucose in- tolerance: (l) hyperglucagonemia was apparent on days 2, 4, and 7 (Figures 17-19) following treatment with 5 mg/kg cis-DDP, yet glucose tolerance was impaired only on days 2 and 4 (Figures 11-14), (2) although 191 treatment with cis-DDP, gentamicin and glycerol resulted uniformly in hyperglucagonemia (Table 12), glucose tolerance was differentially affected by these agents (Figure 40), and (3) hyperglucagonemia was not associated with fasting hyperglycemia in animals treated with 5 mg/kg cis-DDP. These results therefore suggest that hyperglucagonemia per §e_does not necessarily result in impaired glucose tolerance. Al- though glucagon may contribute to cis-DDP hyperglycemia by acutely in- creasing glucose production, the abnormalities in carbohydrate metabo- lism persist for at least several days following cis-DDP treatment, indicating that other mechanisms must be invoked. It is likely that the primary lesion mediating cis-DDP hyperglycemia involves impaired glu- cose utilization associated with impaired insulin secretion. The exact mechanisms underlying the impairment in insulin secretion by cis-DDP treatment cannot be evaluated from these studies and therefore merit further investigation. SUMMARY This study was designed to characterize the effects of cis-DDP and other divalent platinum compounds on carbohydrate metabolism and to elucidate the biochemical and endocrine correlates mediating these metabolic abnormalities. Glucose metabolism was evaluated in male F-344 rats treated with equimolar platinum doses of cis-DDP, trans-DDP or ammonium tetrachloro- platinate. Since preliminary studies indicated a dose-related anorexia associated with cis-DDP treatment, a pair-fed control group was also studied to correct for the metabolic effects of reduced food intake. Administration of cis-DDP, but neither trans-DDP nor tetrachloroplati- nate, resulted in fasting (7.5 or 10 mg/kg) and nonfasting (5, 7.5 or 10 mg/kg) hyperglycemia. Impaired glucose utilization appears to con— tribute to cis-DDP hyperglycemia as indicated by the marked and persis- tent hyperglycemia following exogenous glucose administration in treated (5 or 7.5 mg/kg) animals. Impaired glucose utilization was apparent 2 and 4, but not 7 and 14, days following treatment with 5 mg/kg cis-DDP, indicating that it is a transient phenomenon. Neither trans-DDP nor tetrachloroplatinate impaired glucose tolerance at any time examined. These results suggest that glucose intolerance following cis-DDP treat- ment is unique to the gemmetry of the complex and that properties other than the presence of a divalent platinum atom must contribute to cis- DDP glucose intolerance. 192 193 The mechanisms mediating cis-DDP glucose intolerance are related to impaired insulin metabolism. Decreased utilization of exogenous glucose was observed in treated (5 mg/kg cis-DDP) animals despite an apparently normal plasma IRI response. Although amelioration of cis-DDP induced uremia was associated with an improvement in cis-DDP glucose intoler- ance, other nephrotoxicants which induced uremia did not uniformly impair glucose tolerance. These results therefore suggest that cis-DDP glucose intolerance is mediated by mechanisms other than those asso- ciated with uremia. Although improvement in glucose metabolism by reducing cis-DDP nephrotoxicity suggests that cis-DDP impairs insulin action at target tissues, treated animals did not exhibit a blunted hypoglycemic response to exogenous insulin. Although plasma IRI con- centrations following glucose stimulation were similar between control and treated animals, it is likely that the composition of plasma IRI is different following cis-DDP treatment. cis-DDP impairs renal function; therefore, renal degradation of proinsulin, which is a less biologically ‘active, although immunoreactive, beta cell secretory product, may be compromised in treated animals and therefore may constitute a large proportion of plasma IRI. Further studies would be needed to explore this possibility. Although it is likely that proinsulin is elevated following cis-DDP treatment, an exaggerated total plasma IRI response to glucose stimula- tion was not apparent, indicating that cis-DDP glucose intolerance is primarily mediated by impaired insulin release. Pancreatic concentra- tions of IRI were not significantly affected by cis-DDP treatment; therefore, pancreatic insulin depletion probably does not account for 194 impaired release. Impaired insulin release appeared to be independent of starvation, uremia and adrenal-mediated stress. Furthermore, neither hypocalcemia nor hypokalemia was evident in treated animals; therefore, glucose intolerance associated with a blunted insulin re- sponse cannot be attributed to these phenomena. A direct pancreotoxic effect of cis-DDP also seems unlikely as indicated by the normal appear- ance of the pancreas upon histological examination by light microscopy following cis-DDP treatment. Since glucagon is known to contribute to hyperglycemia by increas- ing glucose production, glucagon metabolism was evaluated following cis— DDP treatment. cis-DDP glucose intolerance was accompanied by hyper- glucagonemia. However, several lines of evidence indicate that the elevation in plasma IRG may not directly mediate cis-DDP glucose in- tolerance: (l) hyperglucagonemia following cis-DDP treatment did not increase hepatic or renal G6Pase or FDPase activities, suggesting an absence of glucagon mediated glucose production, (2) a form other than the 3500 MW form of plasma IRG constituted a large proportion of total plasma IRG in treated animals, (3) animals treated with 5 or 7.5 mg/kg cis-DDP exhibited a significant increase in the 3500 MW form of IRG; however, this was not accompanied by severe fasting hyperglycemia and (4) the biological effects of glucagon are known to be short-lived; therefore, chronic hyperglucagonemia of treated animals may not result in continued increase in glucose production. The elevation in plasma IRG following cis-DDP treatment is probably related to decreased renal degradation of this hormone. 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