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DATE DATE DUE DATE DUE . ‘ WWW” EHESIS LIBRARY This is to certify that the thesis entitled AN IN VITRO NEPHROTOXICITY SCREENING SYSTEM FOR PLATINUM COORDINATION COMPLEXES: A CYTOCHEMICAL APPROACH, presented by Mark Andrew Batzer has been accepted towards fulfillment of the requirements for Master of Science Zoology degree in L .. . \ x ’ :D-l“ LEOCQWZ wk.) Major professor S. K. Aggamal Date O-7639 MSU is an Affirmative Action/Equal Opportuniry Institution 1932.331 AN IN VITRO NEPHROTOXICITY SCREENING SYSTEM FOR PLATINUM WORDINATION COMPIEXES: A CYTOCHEMICAL APPROACH. By Mark Andrew Bauer A THESIS submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1985 Dedicated to my parents who constantly supported my work. .-.. ACKNOWIEEGMNTS I wish to thank my major professor Dr. S.K. Aggarwal for his advice and constant support of my work. His wisdom although not always apparent was a great help. and is greatly appreciated. Appreciation is due also to Dr. RN. Band and Dr. RR. Brubaker, who served as guidance committee members and advisors during my years at Michigan State University. I would also like to thank the following people for their contributions to the project. Dr. Lippincott at Northwestern University for the use of his scanning microdonsitometer. Mr. J. Zablotny for his graphics. M. McPeek and R. Creed for their statistical help. TABLE OF CONTENTS Page LIST OI" TABLES ...................................................................................................................... V LIST OI" FIGURES ..................................................................................................................... VI INTRODUCTION ........................................................................................................................ 1 MATERIALS ANDMETHODS ......................................... 3 Cytochemical Studies ............................................................................................... 5 Biochemical Studies ................................................................................................. 7 RESULTS .................................................................................................................................. 9 DISCUSSION ............................................................................................................................. 49 REFERENCES .......... ' .................................................................................................................. 54 APPENDIX DATA AND STATISTICS ...................................................................................... 52 iv LIST OF TABLES Table Page 1 Platinum coordination complexes screened ....................................................... 4 2 Rat. weight gain/loss ............................................................................................... 37 3 111m nephrotoxicity................... ....................................................................... 46 A1 1111119. alkaline phosphatase data ....................................................................... 62 A2 1pm alkaline phosphatase statistics ............................................................... 62 A3 Maid phosphatase data ............................................................................... 64 A4 Mm acid phosphatase statistics ....................................................................... 64 A5 111.1119. urine volume data ..................................................................................... 66 A6 1pm urine volume statistics ............................................................................. 66 A7 Rat weight gain/loss data ....................................................................................... 68 A8 Rat weight gain/loss statistics .............................................................................. 68 A9 Mm alkaline phosphatase data ...................................................................... 70 A10 Walkfline phosphatase statistics .............................................................. 70 All 13113:; tubule viability data ................................................................................ 72 A12 11m tubule viability statistics ........................................................................ 72 A13 111.2110. scanning densitometry data ................................................................... 74 A14 Mscanning densitometry statistics ........................................................... 74 A15 121m scanning densitometry data. .................................................................. 76 A16 131m scanning densitometry statistics .......................................................... 76 LIST 01'" FIGURES Figure Page 1 Light micrographs (alkaline phosphatase) ....................................................... 11 2 1.111119. transport enzyme ranking .................................................................. ‘ 13 3 111119 scanning densitometry traces ................................................................ 15 4 1pm relative reaction product density .......................................................... 17 5 mm transport enzyme ranking ..................................................................... l9 6 101129. scanning densitometry traces ............................................................... 21 7 11mm relative reaction product density ......................................................... Z3 8 Wistar urine alkaline phosphatase ...................................................................... 26 9 Wistar urine acid phosphatase .............................................................................. 28 10 Long Evans urine alkaline phosphatase ............................................................. 30 11 Long Evans urine acid phosphatase .................................................................... 32 12 Wistar urine volume ............................................................................................... 34 13 Long Evans urine volume ...................................................................................... 36 14 mm culture media alkaline phosphatase ..................................................... 39 15 11mm culture media alkaline phosphatase ..................................................... 41 16 111111129. tubule viability ......................................................................................... 43 17 m tubule viability ......................................................................................... 43 18 Light micrographs (thiol groups) ....................................................................... 48 vi ABSTRACT AN IN VITRO NEPIIROTOXICITY SCREENING SYSTEM FOR PLATINUM COORDINATION COMPLEXES: A CYTOCHEMICAL APPROACH. By Mark Andrew Batzer Currently many animals and labor hours are required to screen drugs (platinum coordination complexes). To decrease the time required for screening an 111111.129. screen system is proposed and tested. Isolated rat kidney tubules were cultured for 8 hours with various drugs. Aliquots were taken at 0, 1.2, 3. 4. 5. 6 and 8 hours. frozen and analyzed for the amount of Nat/Kt ATPase. Ca2+ A'I'Pase. alkaline phosphatase. and acid phosphatase. Culture media was also analyzed biochemically for the amount of alkaline phosphatase present. The amount of alkaline phosphatase in the culture media increased by varying amounts. over time. in a drug dependent manner and equated with cytochemical analysis of tubular enzymes. Lam studies comparing urine analysis to analysis of kidney cross-sections yielded similar results. but over a longer period of time. in a strain dependant manner. This leads to the following relation: 1 hour Wis equivalent to 1 day Q vivo. Since these enzymes are responsible for transport in the kidney, their presence/absence is probably responsible for nephrotoxicity. INTRODUCTION Heavy metal platinum coordination complexes are attracting considerable attention as potential chemotherapeutic agents against a number of tumors (53). Of these cis-dichlorodiammineplatinum II ( cisplatin. DDP ) is currently used in the treatment of ovarian and testicular cancers (49). The drug is. however. not without certain toxic side effects in the kidney ( proteinuria. morphological damage ) (8. 11. 24. 57. 66). intestine ( diarrhea and anorexia ) (S7). and lymphatic system (spleenic atrophy ) (4. 57). Nephrotoxicity is the most important side effect. as it is the limiting factor in the chemotherapeutic uses of the drug (24. 25. 29. 66. 67). Currently efforts are underway to synthesize now. less toxic compounds which retain their chemotherapeutic potential (14.29. 54). Toxic effects of cisplatin have been extensively studied (4. 5. 6. 7. 18. 24. 25. 31. 35. 5'7. 66. 67). although the mechanism of action of the drug is not clearly understood (50. 53). Cisplatin has been shown to inhibit DNA replication in mammalian cells (47). as well as in prokaryotes (12. 13. 38. 42). It's mechanism of action seems to be through both inter- and intra-strand crosslinking of the DNA (46. 48). Protein synthesis and RNA synthesis may also be inhibited by the drug (26). Cisplatin has also been shown to inhibit mammalian cytokinesis (1. 3). and cause adecrease in surface associated transport enzymes (6. 11. 68). and cause embryotoxicity (31). Cisplatin also has drastic effects on metamorphosis in anurans (41 ). The exact mechanism of cisplatin‘s action with regard to its toxicities or tumor regression is not completely understood. Second-generation analogs of cisplatin am currently being synthesized and tested (29. 49). As new compounds are developed the question of the mechanism of action resurfaces. The new compounds may act in a manner similar to CDDP. or they may have their own. unique modes of action. Since the chemotherapeutic value of the platinum complexes is of primary concern. the mechanism of action is of equal importance. As new platinum analogs are synthesized there is an increasing need for a fast. efficient system to evaluate the chemotherapeutic potential and nephrotoxicity of these drugs. Currently many laboratory animals and man hours are required to test each drug. This process takes a long period of time and tends to hinder the chemotherapeutic application of these new analogs. In this study an inzjtm screen system is proposed and tested. To test the nephrotoxicity isolated kidney tubules are cultured the duration of each experiment. Since the main functions of the kidney are transport related (23. 65). membrane transport enzymes ( Cazt- activated ATPase. Na‘ /K*-activated ATPase. Alkaline Phosphame. and 5' Nucleotidase ) were demonstrated and quantitated cytochemically. then used as indicators of normal and impaired renal mnction (ll. 64). Similar studies were repeated in rats and results were compared to those in mm studies for uniformity. Attempts were also made to delineate the mechanisms of action of various platinum coordination complexes. MATERIALS AND METHODS I II' SI I‘ Inbred male Swiss Wistar and Long Evans (Hooded) rats ( Charles River lab. ). weighing 150-300 grams. were injected ( intra peritoneally ) i.p. with cis- diammine-l.1-cyclobutane dicarboxylate platinum II ( CBDCA. JM 8 ) 50 mg/kg. or cis-dichloro diammine platinum II ( DDP. Cisplatin )5.0 mg/kg ( Johnson Matthey Research Laboratories ) injection vehicles are shown inTable l. The day of the injection was taken as day 0. Sampling intervals were 0. 3. 5. 10 and 20 days post injection. Four animals were killed by cervical dislocation at each sampling interval. The kidneys from the left half were removed and mounted on cryostubs ( I. E. C.) in OCT mounting medium . frozen. and kept until use. Sections 10 nil in thickness were cut using IEC cryostat microtome for enzymatic analysis. Kidneys from the right half were removed. fixed in 100 It ethyl alcohol (-20‘ C). embedded in paraffin. Sections 5 nM in thickness were cut using a rotary microtome ( American Optical ). The sections were processed for the cytochemical localization of thiol ( -SH ) groups. Wes Three inbred Wistar rats weighing 130-300 grams were killed. and their kidneys were removed. Kidney tubules were isolated following a modified version of the procedure developed by Nagata and Rasmussen (43). The medulla of each kidney was excised. and the remaining cortices were then mechanically minced and placed in Hank's solution ( Grand Island Biological Supply Co. ). The minced “.0 Table l. Platinum coordination complexes tested. Drug Structure Abbrevlatlon Dosage Vehlcle J1 Number cl: dlcblors dlsmmlno Pl. m, Cl Clsvlalln. COW 5.0 mane .lS H Sella. --— n 2 c: o l cls dlmlno l.l-cyclebum m2 O—C . CBDCA some. 58 Glucose 0 «cu-mm. Pt >1 ><> m2 o-B mlmleolz dlmlns Oj Helmets 80 mono Off 74 “‘2 mm.»- Pl. / 1.5x Mel-C03 2x 2’“ Aux-sulfate 1.2 dmlno m2 H20 Sulfate l2 mom 5! Glucose 2O cyclonenns P! P! . NH2 H20 u: dllsoerooylsmmlns inns ms", 04 cl cm 40 rag/kg .lS n sum 9 «mam a More Pt w \ / dlclllere L2 dlmnlne 1 Cl INCH-Cl? 20 My . l5 nSsllno ~— cytldlum P! \ / ' Pt / \ NH, Cl cortices were then enzymatically digested in 100 mL of Hank's solution which also contained 40 mg collagenase ( Worthington Biochemical), 100 mg hyaluronidase ( Sigma T I. Sigma Chemical C0,). 25 mg streptomycin sulfate and. 180 mg glucose. The tubules were then washed and mechanically dispersed using a dispo pipet. The sample was then divided into 13 equal portions and incubated in minimal essential media (Grand Island Biological Supply 00.). One part of the sample served as control. while platinum coordination complexes were added to the rest at chemotherapeutic dosages as shown in Table 1. Samples were taken at l. 2, 3, 4, 5. 6 and 8 hours, and fixed in l % glutaraldehyde in .05 M cacodylate buffer ( pH 7.4 ). washed in 4.5 % sucrose-cacodylate buffer (ph 7.4), mixed with Tissue Tek II embedding media ( Lab Tek division of Miles Laboratories) and frozen until use. Sections 10 “M in thickness were cut using a CD international Harris cryostat ( International Equipment Company). and placed on dry gelatine ( 2.5 7. ) coated coverslips. placed breifly in buffered glutaraldehyde ( to firmly attach the sections to the slip ). and processed for cytochemical studies described below. Both kidney cross-sections and isolated kidney tubules sections were tested for the following phosphatases. 933W( Wham ) was detected by incubation in 0.1 M Tris- maleate buffer ( pH 7.3 ). adenosine 5' triphosphate ( ATP) 10 mM. 3 % lead nitrate. 1 mM calcium chloride. 0.2 mM magnesium chloride and distilled water (17. 22). Incubation lasted 45 minutes at 37°C. The control incubation omitted the substrate or additionally contained 15 ug/ml quercetin( an inhibitor of Ca-ATPase activity (21)) ML1W( Na‘-K*-ATPase ) was determined by incubation in media composed of Tris-maleate buffer 0.1M ( pH 7.3 ), lOmM ATP. 3 7. lead nitrate, 100 mM sodium chloride. 10 mM magnesium sulfate. 5 % sucrose and distilled water (37). Control media omitted ATP.Na. or additionally contained 0.7 mg/ml ouabain. an inhihitor of Na/K ATM activity (23. 33). Incubations were carried out for 45 minutes at 37’C W( AP ) activity was visualized by incubation in media containing 02 M Tris-maleate buffer ( pH 82 ). sodium B-glycerophosphate ( 12 7. ). l 7. lead nitrate. 0.2 mM magnesium chloride and distilled water for 45 minutes at 37°C according to the method of Hugon and Borgers (28). Control incubation contained 50 mM L—phenylalanine. an inhibitor of AP activity (15). W( 5‘ N ). For visualization of 5' N activity incubation in media consisting of 0.1 M Tris-maleate buffer ( pH 7.3 ). 1.4 mM adenosine 5'- monophosphate ( AMP ). l 7. lead nitrate. 10 mM magnesium sulfate and 5 7. sucrose was carried out at 37’C for 45 minutes according to the procedure developed by Uusitalo and Karnovsky (62). Control media omitted the substrate ( AMP ). W( Acid P ) activity was visualized by incubation in medium containing .2 M Tris-maleate buffer ( pH 5.2 ). sodium 0- glycerophosphate ( 1.2 7. ). l ‘L lead nitrate, 0.2 mM magnesium chloride and distilled water for 45 minutes at 37’C (45). Sections ( 10 MA ) of control and drug treated tissue after incubation in the appropriate incubation medium were treated with l ‘L ammonium sulfide and mounted in glycerine jelly. Slides were photographed using a Zeiss Photomicroscope II. loaded with Kodak Plus X film. The reaction product ( rp ) was quantitated on these negatives by scanning microdensitometry using a Joyce Loebl MK III C double beam recording microdensitometer( JL and Company LTD. Electron House, princesway Team Valley Gateshead -on-‘l‘yne 11 England). and by direct microscopic visualization. -.1 W Thiols were localized according to the method of Engel and Zerlotti (20). The method involves treatment of the sections with 95 7. ethanol. followed by staining with the azomercurial reagent 4-( p-dimethylamino benzene azo) phenyl mercuric acetate. and retreatment with 95 7. ethanol. A control is prepared with preincubation in N-ethyl maleimide ( 75mM ) in phosphate buffer ( pH 7.0 ) at 37'C for 2 hours (9). blocking the thiol groups. or by controlled trypsin ( protease ) digestion demonstrating that the thiol groups are protein bound. The sections were then mounted using Permount (Fisher Chemical Company). and observed with monochromatic blue ( L: 458 nM ) light. DI 'l' [II | IZ'I'I'. Tubule viability was determined by removal of control and drug treated tubules at each sampling interval. These samples were then treated with 0.2 7. trypan blue solution. and counts made of the viable kidney tubules ( viable tubules do not takeupthestain) (l9). . B' I . I E Spectrophotometric assays were performed on culture media from each sampling interval during the Mmscreening process, to determine the amount of alkaline phosphatase activity present in the media ( 34). AP activity was detected by adding 0.1 ml of culture media to a mixture of 19 mM disodium p nitrophenyl phosphate and 0.1 M carbonate bicarbonate buffer ( pH 10.0 ) which had been warmed for 10 minutes at 30’C. The mixture was then placed in a 5 ml cell with a 1 cm path length. and checked for absorbance at 400 nm using a Beckman 25 spectrophotometer. Then. using the absorbence. the number of units of alkaline phosphatase/ml of culture media was determined using Beer’s law. Rat urine was collected from 5 pairs of male Wistar and Long Evans rats at 0.1.3.5.7.10 days post injection with CBDCA or CDDP. The urine was-analyzed either immediately or frozen until use. It was spun in an IEC clinical centrifuge for 8 minutes at high speed. and dialyzed for 3 hours using Spectrapore membrane tubing ( VWR scientific Biochemistry Stores) and distilled water 4°C. The urine was then assayed for the presence of both alkaline phOSphatase and acid phosphatase (34). Acid phosphatase activity was determined by incubation in medium consisting of .l M acetate buffer pH. 4.5. .15 M substrate Na-B- glycerophosphate (Sigma 104-0 dissolved in acetate buffer). 20 7. TCA( tri-chloro acetic acid) was added after 10 minutes of incubation. Alkaline phosphatase activity was determined. as described above. for the culture media. The media was spectrophotometrically assayed and the amount of acid phosphatase and alkaline phosphatase present in the urine calculated similar to that described for culture medium analysis. with the addition of a correction for the amount of urine excreted. W All data analysis was performed on a Macintosh microcomputer using a Number Cruncher Statpak. Prior to the analysis of variance. datawas checked for equality and normality of variance using both the F max test. and Bartlett’s homogeneity test. RESULTS Cytochemical localization of alkaline phosphatase in kidney cross-sections is depicted in Figure 1A. and 5 days post CDDP treatment in Figure 13. 1211141). localization in isolated kidney tubules is shown in Figure 1C. and 5 hours post CDDP treatment in Figure 1D. The enzymatic reaction shows that alkaline phosphatase is mainly located on the lumen side of kidney tubule cells in the microvilli. A lack of reaction product is seen in both the m and MILE sections after CDDP treatment. although CBDCA does not cause a large decrease in alkaline phosphatase activity. A similar lack of reaction product is seen in negative control sections which were incubated with either L—phenylalanine or leavamisole. Sodium ATPase and calcium ATPase show a distribution similar to that of alkaline phosphatase with the addition of strong intensity on the cells of the basal lateral border of kidney tubules. Following either CDDP or CBDCA treatment both calcium and sodium ATPase are affected in the same way as alkaline phosphatase. Acid phosphatase is present in lysosomes throughout the kidney. and increases substantially in quantity after CDDP treatment. but shows only a slight increase after CBDCA treatment. The activity of 5'-nucleotidase shows a distribution similar to that of alkaline phosphatase. and is affected in a similar manner after drug treatment. The decreases correlate for both Wand Mandi”. Results of reaction densities as viewed through the light microscope are shown for mandies in Figure 2. and for Mandi” with CDDP and CBDCA in Figure 5. These results were further analyzed and quantified using a Joyce Loebl scanning microdensitometer. The results of alkaline phosphatase scans are shown for W studies in Figures 3 and 4. and for manna: in Figures 6 and 7. Enzyme levels decreased in both an analog and time dependent manner. Figure l A 10 Cross-section of a male wistar rat kidney showing alkaline phosphatase reaction product (r.p.) (arrows) in a normal animal. Original magnification X 250. Bar - 100 nM. Cross-section of male wistar rat kidney 5 days post cisplatin (5 mg/kg) treatment Showing only patches of alkaline phosphatase activity. Original Magnification X 250. Bar - 100 AM. Isolated rat kidney tubules showing alkaline phosphatase r.p. (arrows) in normal tubules. Original magnification X 950. Bar = 25AM. Isolated rat kidney tubules 5 hours post CDDP (5 mg/kg) treatment showing no alkaline phosphatase reaction product. Original magnification X 950. Bar . 25AM. 11 12 Figure 2 mainline phosphatase. Caz“ ATPase and Na*/K° ATPase reaction product density in control. CBDCA (50 mg/kg) treated and CDDP (5 mg/kg) treated male Wistar rats. Duya post injection In Vivo Transport Enzyme Activity 12 Alkaline Phosphatase (222° ATPase Na°/K° ATPase Treatment CDDP CBDCA (DDP CBDCA CDDP CBDCA o ++¢+ tn. at... t... §+e+ §++¢ 1 H t... .. +4>++ H t...» 3 :. in z N. :. H. 5 3_ it. 3 H. :. Ht 10 9 +§§¢ 4 eeee i» Mi. 1 After treatment with CDDP 5.0 mg/kg or CBDCA 50 mg/kg 2 . . . . very dense reaction: . o . dense reaction: o 0 average reaction: . poor reaction ; less than 10 7. reaction: - no reaction 1...:- 14 456393 man—o Amine 9 23¢ 23—. n u was £3839: dud—ammo A3— BSD has 3.8 m m 4838 < .2: 635.3 €338 ganqgam 258—? no 333 5.508333“. midsumg m 9.33 16 Figure 4 1111110. relative alkaline phosphatase reaction product density in male Wistar rat kidney cross-sections for control __<>._ . CDDP (5 mg/kg) +_ CBDCA (50mg/kg) _ .._ __ treatment. 1'? 20 5 O 5 528mm 20.84%. 3 >528 magma 3 INJECTION DAYS POST Figure 5 Inflmalkaline phosphatase. Ca 2’ ATPase and Na’lK’ ATPase reaction product density in control, CBDCA (50 mg/kg) treated and CDDP (5 mg/kg) treated male Wistar rats. Hours post incubation 19 in Vitro Transport Enzyme Activity 12 Alkaline Phosphatase Caz° ATPase Na‘fli‘ ATPase Treatment CDDP CBDCA CDDP CBDCA CDDP CBDCA o ++++ ++++ H” ++++ ++++ HH 1 it HM H ”H H t... 3 t H. _+_ H. 1 +4.. 5 3 H. t H. t. H. 8 - ++ - +4. - 4... 1 After treatment with cone 5.0 mg/kg or CBDCA so mg/kg‘ 2 . . . . very dense reaction; 0 . o dense reaction; 0 . average reaction: i poor reaction :iess than 10 7. reaction; - no reaction 45830.: .38 3:9: n a3? «.52. n a ES .3253: n38 Aug—Ba C .3; 9:3: m o aqua—.30.: due—ammo 3: B89 gnaw .32. _ m 4838 < he 335.3 5538 88338.3 25.3.“ .3 notau 5.308335“. mamas”; a o 9:35 Figure 7 mummiative alkaline phosphatase) reaction product density for control _<>_ CDDP (5 mg/kg + CBDCA (50mg/kg) _ _._ -treatment. 23 2 2 O 2 8 5 4. 2 O 8 6 4. 2 5:85 zoiofiw 3 C523 “.22an O INCUBATION HOURS POST Further conformation of the Mstudies was provided by analyzing rat urine enzymes for alkaline phosphatase and acid phosphatase data for male Wistar rats (Figures 8 and 9 respectively). and for male Long Evans rats (Figures 10 and 11 respectively). The urine analysis indicates an increase in alkaline phosphatase activity after injection with either CDDP or CBDCA. but that the magnitude of increase after CBDCA treatment is much less than that following CDDP treatment. Acid phosphatase levels seem to increase 6 days post injection. followed by a subsequent decrease. to normal levels. Acid phosphatase increased significantly more after CDDP treatment than after CBDCA treatment. The rat urine volumes were also monitored post injection results shown in Figure 12 for Wistar rats. and Figure 13 for Long Evans rats. A decrewe in urine volume is evident following CBDCA injection . although an increwe is found after CDDP injection. Weight gain in control. CDDP. and CBDCA treated rats is compared in Table 2. These studies indicate a weight loss after CDDP treatment. but a slight. insignificant decrease in weight following CBDCA treatment relative to control animals. 111111.11). studies were similarly confirmed using both culture media assayed for alkaline phosphatase activity (Figures 14 and 15). and tubules assayed for viability (Figures 16 and 17). 1pm studies show that the various analogs tested may be ranked from most to least toxic relative to membrane enzyme damage ( Table 3). Pictures depicting 1111110 thiol group localization are shown for control (Figure 18A). 5 days post CDDP treatment (Figure 18B) and for 5 days post CBDCA treatment (Figure 18C). The thiol groups appear to be localized on the brush and basal lateral borders of kidney tubule cells. The photnmicrographs indicate a decrease in thiol groups following CDDP treatment. but show little decrease after CBDCA treatment. similar to alkaline phosphatase. ao_o__‘- ‘K m .0 a...“ 25 Figure 8 Alkaline phosphatase activity (mU/hr/ 100 g) in male Wistar rat urine for control ,CDDP (5 mg/kg) _ . _ _ and CBDCA (50 mg/kg) + treatment. 26 l 20 my 4 30--1 oOO_\E\:E »._._>_._.o< wm._._>_._.o< wmflzznmozm Qo< INJECTION DAYS POST 29 Figure 10 Alkaline phosphatase activity (mU/hr/ 100 g) in male Long Evans rat urine for control Q. . CDDP (5 mg/kg) _ _ Q _ and CBDCA (50 mg/ kg) + treatment. 30 20 may % 4 80.235 E52 “54258:“. a 0 MEI—.334 POST INJECTION DAYS '31 Figure 11 Acid phosphatase activity (mU/hr/ 100 g) in male Long Evans rat urine for control O .CDDP (5 mg/kg) _ _ . _ and CBDCA (50 mg/kg)_‘,.__ treatment. 20 d _ o. o. 2 I 30:23:. >E>fio< mm sz: 20 I2 DAYS POST INJECTION I II III: {III {Ii ’35 Figure 13 Urine volume (ml/100 g) 8 hour sample. for male Long Evans rats control /\> . CDDP (5 mg/kg) _...___ and CBDCA (50 mg/ kg) ._A._ treatment. AK 0. O. 2 5m\aoo.\_e m230> qu: ' 3.0. 20 l2 DAYS POST INJECTION 37 Table 2 Weight gain/loss with and without drug treatment ‘2 Rat Treatment Average weight (gain/loss)/dey . Long Evans Control 6.7 t 0.9 gm/day ' coop -297 r. 1.6 gm/day CBDCA 6.2 t 2.2 gin/day Wistar Control 42 t 1.1 gm/day CDDP -2.73 t 1.14 gin/day CBDCA 3.83 z 2.08 gin/day l--6 replicates of each treatment 2~Base<1 on average weight gain/loss 5 days after injection. 38 Figure 14 1am alkaline phosphatase in units/d1 of culture media. For control _ . _ . _ , CDDP 5.0 mg/kg ..... . CBDCA 50 mg/ kg __ __ . CDDP 50 mg/ kg and CBDCA 500 mg/kg ..... treated samples. 39 I O 2 - 5 4932:: >._._>..ro< mm._._>_.ro< mm .- ..... '1‘ - v- -v s - : . 1...: -1- - hrA -; :- ~ - u '~. - .— ... L. ‘1 I Q- Q.‘ a -—- a.- A r.._, ,_p,. I ‘49. _‘ .00 K“ .60 a “ad .4 ‘ t. . L.. we... - Q h - vs (We — 2 . ~ -- - 5- in” ~ .-_ «— L. _ er s \ -vuh .5» _. . s --~ _— s- - a-»— ‘ .- A- - - .4 (SP1 6. - '- " t ' n .. ' 4 t4 \. t u -_..r p_~A—_ x.-- 4’ - -_~ J ~_ .5 h— - . ‘5 —-\ . q ‘ qa— . 0".- a \a» ‘05 rarAU-nrg \r‘ rn‘ q , 1 1 ~¢ ‘ «ca ‘ 0.. -r ~~~~~ 4—-t h_‘ -u...»i-v ‘. —~ . ~ ...... L~a b- - n ‘ - \ , .—- .‘ -""flr‘ .\ .4 5. v 6.". D _ a.» A) .- o 349‘!" H's 1’!‘ "a - n\‘—D DP- .fl'u‘ v‘ 'Hp'3L ”sq-'L- r ~r— - — —- ‘ ar- n-'1» -qu—u-ba- m-~-s— .~‘-.Jsu .‘ ~~~ my » o-h .5-.. “~" W“ v~~ 0 ".‘;¢ |:‘ I oo 7 f? f W .0. j ‘ \ L. L wk L V V 1 Treatment denotes Control. CBDCA or CD09 treated. 2 df deactes degrees or freedom Table A3 111119. urine acid phosphatase means and standard deviation. Table A4 In v11' 9 urine acid phosphatase ANOVA. Urine Acid Phosphatase Activity Data. 1 2 Rat Treatment Days post treatment 0 3 S 7 10 20 LongEvaris Control 34-. 62 _ _ _. _. — CBDCA _ 2.7 : 85 2.46 :14 2.42 :13 3.42 °_ 1.1 3.33 11.9 CDDP — 437213 562:2') 319:1: 323.4) 32112 11313.! COOUOI 2.04 z .7 _ _. _ _ — CBDCA _ 2.1 9. .2 1.58 : .'.' 2.31: 35 2.05 3. 86 1.96 : .97 CDDP ._ 4.05 i 1.6 4.6 :13 4.9 :13 2.3 : 1.1 2.1 2 .89 1Mean . Standard Deviation Shown zReported in mU/hr/lOOg 5 replicates at each point. Analyses of Variance {or Urine Acid Phosphatase Actzvztfy' ’ Day post imeczion Source of variation [312 3 5 7' IO 30.-. Treatment; 5 4 73° 1207' 305' 152 270:" Across Species Control . CBDCA vs CDDP l 1.7 92” 491" ° 13 70 ° 31 05 Across SpCC;CSC03U01VSCBDC:\ l 351 2.18 395 484 04 SpeCics reSponse ControlvsCDDP 1 355 724 09 6 37" 915‘” Error 24 1.53 353 136 S9 45 ' Mean square values are presented :3 the table with significance as noted. ’ 005 >E>001_ ” 001 :2‘0001;”° E'DOOX ‘ Treatment denotes Control. CBDCA or CDDP treated. 2 dt demise degrees of freedom 65 Table A5 In m 9 8 hour urine volume means and standard deviation. Table A6 In vivo 8 hour urine volume ANOVA. Rat Urine Volume Data. 1 2 Rat Treatment Days post treatment 0 3 5 7 10 20 Long Evans Control 1.7 g 25 _ __ _. _ __ CDDP _ 1.53 ; .74 2.37 ; 1.2 2.57; 1.3 2.26 3 1.1 1.93; .49 CBDCA _ 1.17 1.34 .83 ‘. .26 .91 ; 26 1.16 1.31 .73 ~_ .25 Wistar Control .966 1.17 _ _ __ _ _ CDDP _. 1.33: .52 2 f 1.1 1.53 1.57 1 ‘. .63 1.05; .6 CBDCA _ .95 1.3 .5712 .53 :_ .21 39 1 211+ .45 .1113 1 Mean . Standard Deviation Shown zReported in mL/lOOg/S hr 5'- replicates et each data point. Ara' . se- ot Variance to: 3 Hour sane Volume Data ’ Day post injection Source of '. ar'anon 13f"x 3 5 7 10 20 lrcunent1 5 _f0 2 32. _37’9 2.0500 115990 Across species Control . CBC-C31 vs CDDP 1 27' 10.1“ 7 07" 217’ .47 Across species Control vs CB» 1 .45 1.93 1.77 139’ .41 Species response Control vs CDDP 1 .37 1.98 3 53' 435’ ‘ 2.56”’ Error “1. .27 .34. 55 .35 .15 5 ' Mean square values are presented 1 the table with 31 *nificance as noted. ’ 0.05 >9 001; °' 0.01 ‘Eg 0 COL; ”’ P} 0.001 1 Treatment denotes Control. CBDCA or CDDP treated. Zamoraoffm 67 Table A7 Rat weight gain/loss post drug treatment means and standard deviation. Table A8 Rat weight gain/ loss post drug treatment ANOVA. Ox 0) ~ ' - ~ Weight gain/loss with and without drug treatment '2 -_o.-c..‘1-"O'_-_r' 4K Rat Treatment Average weight (gain/lossildey Long Evans Control 57 z 0.9 gin/day ' coop -297 z 1.6 gm/day CBDCA 6.2 e 22 gm/day Wistar Control 42 : 1.1 gm/day CDDP -273 e 1.14 gm/day CBDCA 3.53 : zoe gm/day 1-6 replicates of eacn treatment 2-Based on average weight gatn/toss 5 days after 1njectton. Analyses of variance for average daily weight gain ' Source of variation dfz small 5 1122’” Acres specie: control . CBNA v3 CDDP 1 530-6?“ Acres species control Y: can 1 1.03 Species response control v: CDDP 1 754 Error 54 6.35 ’ mammmpmnudinthbtnblovithminanceunoud. ' 0.05 >9) 0.01 ; ” 0.01 >23 0.001 ; m E< 0.001 'mmmmtmw.a~mopm demmmn 69 Table A9 In vitro culture media alkaline phosphatase activity means and standard deviation. Table A10 In vigo culture media alkaline phosphatase activity ANOVA and Fisher LSD. 70 in vitro culture media alkaline phosphatase activity statistics 1 2 Drug Time post incubation (hours) 0 1 2 3 4 5 6 CBDCA 50 mg/tg __ 3.0 : .7 3.0 ; .7 32 z 12 3.4 g .4 3.6 e .4 3.2 1 .3 CDDPSmg/kg _ 3.0 : 1.3 2.2.11.1 3.0 -_ 1.4 22:12 3.2 :1.1 3.6; .7 CBDCA 500 mg/Xg _ 8.5;1.8 7.3 ; .5 13.4 11.9 13.3 ; 3 14.2 _-_ 3.1 17.2 _-_3 CDDP 50 mg/ltg _ 3.4 a .7 15 a .7 18.2 :12 20; 4 Zl_-4.4 23.4 ; 4 SULFATO _ 2.3 1.3 2.7 g .4 3.5 :1 3.5; .5 5.6 ; .9 6.2 :1 MALANATO _ 1.5 -_ 5 2.0 ; .4 32 ; 1.1 2.5 ~_.s 2.3 ; .3 3.6; .7 CHIP _ 1.52;.6 1.66;.7 3.0;.9 2.3;.3 2.0; 6 3.2112 Dam-0.. _ 22 -. 12 2.11.9 3.1;1 2.5;1 32; .7 3.6;.3 comet. 1.0 :3 1.0;.5 1.12;.3 1.4;.4 1.4;.3 1.54; s 1.54;.3 DEAD 0. 3 136 . 5 _ _ _ _ _ _ 1 - 5 replicates ateach point 2 - Data reported in units/til. (culture media) Rnoue Table Source 35. Sum of P‘ean F Frtcamlzu Squares Square 3.3120 Treatments 9 1915.423 212.3247 54 39 - 000 Error 40 155.101 1 3.377528 Ad] Total ' ~49 2070 $24 Columns used in this analusts: Column 1 :Control Column 2 :CSDCA SO Column 3 ' CDDP 5 Column 4 .CSDCA $00 Column 5 :C‘DCP SO Column 5 sulfate Column 7 ‘malanato Column 3 :CHIP Column 9 .DACH—C‘. Column 10 :DEAD Fisner s teest Significant Difference Test a: .31 Columns LSD Difference Test Qesod l 2 337525 --17’.‘OOO‘. .‘CT SlCillFiCAllT l 3 s 357525 -l.536 .‘l T 3162::F1CA31T l 4 3 367525 41.548 SiGlllFlCAllT l 5 3.36752. £0218 SIENEFECANT l 6 3.36732- 6.1325 SEGPllCICANT l 7 3367-2- -3.332 SlGillF’CAtlT l 8 3.367525 -2.012 .‘lDT SIClllFECAllT 1 9 3.367525 -l.3 :‘l‘GT SICi‘llFZCAflT l 10 3.367525 55?: .‘l 1T SiCNlFZCANT 71 Table All In vitro tubule viability means and standard deviation. Table A12 1g vitgg tubule viability ANOVA and Fisher LSD. ln vitro tubule viability statistics 72 Drug Time post incubation (hours) 0 1 2 3 Columns (1‘ h) (J! (a -4 0. li) (L) Columns used :n 0115 analusis: Column, 1 :control Column 4 LCSDCA ECO Column 7 :rnalanoto LSD ‘(35 (It ‘Ch '(1\ ‘(II at 0‘ U0 (JI 0\ UI .w 3. I: L; L- I; \1‘-l‘l‘—J‘I‘J‘J\J (nth r; l\) I) I) r) to la) r) 01 UI U1 U] (H U] U] U] U! ‘1 ‘-| ‘l “1 n 1 *1 ‘ml \J 8‘ t- 1; (Jo Colm. n Column Column ' J) I.) i»: iJ- I) is u) '—~ (II (n to ,- ll) ‘1) Column 3 Column 6 Column 9 f‘erence Test - _. —- 3:137:71” nrtrtm )-}.],}, CDDP 5.3 .suifsto :DACH-Cl CDDCA‘JO rag/kg __ 98:13 96:10 92:13 87:13 87:10 81:10 79:10 CDDPSmalkg _ 93:13 88:14 81:11 80: 13 76:18 73:13 69:13 CBDCASOO nag/kg __ 100 :0 933-12 83:12 81:11 80~0 76:20 73:10 CDDPSO ins/kg _ 98:20 88:14 70:0 63-20 65~0 63:13 ‘3:18 SU‘JATO _ 92:11 85:17 81 10 77- 19 7‘2: 20 10° 16 59_~ 21 SWANATO _ 92 :17 87: 15 33 :15 81 :12 78:11 75 :13 73 z 15 El? _ 92:20 90:16 82:13 82:11 79:10 76:10 74:10 DnCH-Cl _ 94 -_ 18 92 : 13 84 : 23 78; 20 77 :22 72 : 20 68 :18 CONTROL 100 :0 100 :0 98:08 92 10 92:11 90:10 90:14 88:13 1-5 replications at each data point 2- Data reported in percent viability Rnoua Table Source 9.? Sum of Fem .-‘ ”2501193; Squares Srmore :stfo Treatment: a 5314 4 789 3 53.7 0 CCC Error 36 534,4 1.: 34:.“ Ad; foul u 5343 3 73 Table A13 In vivg scanning densitometry data. Table A14 In vivo scanning densitometry statistics. In vivo scanning WWW 43131 Treatment 13673 9033 injection 0 3 3 Control 133 : 2.x - — mp __ too 3 L4 3.4 : 2 m _ _ 123:1.9 113:2.1 lam: Statue Motionm 10 replicates at each point. Analyses of Variance tor in vivo scanning densitometry ‘ gay post injection Source of variation D12 tratmentl 2 34.73 "0 73.56". Control 8 CBDCA vs CDDP 1 59.8". 129.05" Control v's CBDCA 1 9.67 18.03 ' Error 27 2.32 42 "’ Mean square values are presented in the table with significance as acted. ' 0.05 Q 0101.- " 0.01 m; 0.001; e" at 0.001 1 treatment denote: control. CBDCA or CDDP treated. 2 or donate: degree: or freedom '75 Table A15 1;; vigo scanning densitometry data. Table A16 Mscanning densitometry statistics. to “an tanning 46‘0”me keen-oat . anaemia-um f o t 3 5 Control 75’! "' - $7.1 13:1.1 243303 CDDP - CEDC'A - ’ ‘ 1:... Std-rd 3"“an 10 replicates at each point. Analyses of mace {or in mm scanning «assumes-7 ‘ . Source or vacation 0:2 3°“: 9°“ 3‘43?“ 1 3 5 awe 2 note” mtm seam Cmmem '3 C313? 1 - 14.4.0" 3739’” 70.72”' 1:332:31 ‘B C3283 1 15.07” 931’ 21.02” Error 18 1.07 0.97 1.13 ’ :teen square values are presented in this table 11th significance as noted. 0 005 as 0.01; or 0.01 >1» acct; m g< com l treatment scoote- control 0300, or :30? treated. 2 at denotes centres of freeman: .w.) HICHIGQN STQTE ..: J..r.. t .. X I ‘ k trgs‘sfifsr . Jim: . ..~.u.‘ » - - .. , . ._ ‘51.»! 1. La... .- ..:z..:.t..$:‘~ , ”w“ “‘ ‘ .2. ).u'u. ._-..... it. 2.21.... ~ . _,_ .. 6 , .-.. r.) . J! .1 7:er ,. .7. . . ..‘r. ' ‘ r.a:...r"~ L .4. W... _..._>. . t ‘53.. t .o.-.- .-r u.-- .1 x- . ...\ . Lt, .\ 1i utt~y..,... .1}. x \ Jr; W ;,. .-'.. ~.« . mm, ... p. \ . y“ . W..,“ ‘ ., . “.‘-'v . 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