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I1} 1 1'11“ 1 3: 1m; 5;!" 111 -.’ II ""‘I.. .I‘ :I 3” ‘11' 5 IIIII “U [:I 1:41! i‘E'i 1'5111'1; j '31' I"; 1;: 1111::11; 1 11' 11 ”1111111:13113'i:1'1;i;;1i§!f "1""31' 31.2%.! if H . . '.r'§§'fi'&111}'1'11111 1"1""""'1 .' 1111.11 1.1;" ‘111114111111111.11111 1111111121. :1 “-1.1. ; IJI :2. . ‘ '1..§1'§ '1‘ .5; hp “11.111: 11: I11" 1 11111 1 1111111 11111 11111111 1 ~ 1 I: 111 11 111' ‘11 11111111111111. "'111'-'.1111-1'1-'1'1'1 1111""'11"" 1 . '11 . ""1 I II I .1 'I'1" I11! EIIII 11:1"?! 1112;“ ‘I ‘1' 9;! I ‘9' ‘ ‘ik’ 11' 1.11111 11111 v———‘———o-—. H...- fl: n...“ W 1.:an 3364.335: state glimm'emty . .. K r HESlS. This is to certify that the dissertation entitled Biochemical Mechanisms of Acetaminophen Nephrotoxicity presented by John F. Newton, Jr. has been accepted towards fulfillment of the requirements for Environmental Toxicology/ ”Pharmacology /’ ‘A/. 7‘44- 3 j o professor Ph ° D ' degree in Date April 19, 1983 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES .m—L. RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. BIOCHEMICAL MECHANISMS OF ACETAMINOPHEN NEPHROTOXICITY By John F. Newton, Jr. A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 1983 o\ fly it} I\' i? ABSTRACT BIOCHEMICAL MECHANISMS OF ACETAMINOPHEN NEPHROTOXICITY BY John F. Newton, Jr. Metabolism of acetaminophen (APAP) to a reactive arylating inter- mediate has been postulated to be a requisite step in the pathogenesis of APAP-induced renal cortical necrosis. At least two different mechanisms for the generation of reactive intermediates from APAP within the renal cortex have been suggested: (l) Direct cytochrome P- 450-dependent activation; or (2) deacetylation prior to the renal metabolism of the deacetylated product, p-aminophenol (PAP), to an electrophilic intermediate. The purpose of this investigation was to test the hypothesis that PAP formation and subsequent metabolic acti- vation are requisite steps in the pathogenesis of APAP-induced renal cortical necrosis. Urinary PAP excretion, measured as an index of PAP formation, in- creased with increasing doses of APAP. In addition, APAP was metabo- lized to PAP in isolated kidneys and in renal cortical homogenates. A reactive arylating intermediate was detected subsequent to PAP formation from APAP in renal cortical 10,000 x g supernatants. PAP administration to F344 rats resulted in renal lesions qualitatively identical to those observed following APAP administration. However, John F. Newton, Jr. PAP was 5-10 times more nephrotoxic than APAP as determined by renal functional and histopathological evaluations. The relevance of PAP formation to APAP-induced renal cortical necrosis was established with three different types of experimental approaches: (1) Strain differences in susceptibility to APAP-induced inephrotoxicity were related to the formation of arylating intermedi- ates jfl_vjvg_by a deacetylase—dependent mechanism; (2) pretreatment of F344 rats with the deacetylase inhibitor, bis-(p-nitrophenyl)phos- phate, reduced the nephrotoxicity of acetaminophen and the formation of PAP; and (3) administration of specifically labelled [14CJ-APAP to F344 rats indicated that a large portion of arylating intermediates within the kidney originated from PAP. These investigations support the hypothesis that deacetylation is an obligatory event in the pathogenesis of APAP-induced renal necrosis. ACKNOWLEDGEMENTS The following pages are a culmination of the unwavering support my mother and father have provided me over the past twenty-six years. Without their constant guidance, love and comfort it is my belief that this thesis would not have been possible. Therefore, this thesis is dedicated to my mother and father for they have been my most important advisors, my greatest fans and my financial support throughout my seemingly endless years of education. Words alone cannot express how deeply grateful I am for all my parents have done for me. I also wish to express my appreciation to my brother and sisters Reed, Christine and Mary Ellen and to my Aunt, Shirley Hespelt. I would like to express appreciation to the Monsanto Foundation for the financial support of my graduate training. I would like to thank Drs. Robert Roth, Emmett Braselton, Philip Natanabe and Theodore Brody for constructive criticism of the dissertation research. I am deeply grateful to Drs. Gilbert Mudge and Mark Gemborys for their intellectual guidance throughout the project. I would like to thank the many collaborators involved in this research, Drs. Jay Bernstein, Ken Yoshimoto, Steve Kuo, and Glenn Rush and Dale Pasino, Diane Hoefle and Marc Bailie. I am deeply indebted to my mentor, Dr. Jerry B. Hook, for the financial and moral support and intellectual guidance throughout my undergraduate and graduate studies. ii I n‘: of chick like to Erickson excellen A v deep fri their st Diane, v throng": I would like to thank Diane Hummel for the excellent translation of chicken scrawl in the preparation of this thesis. I would also like to thank Bruce Hook, Carmel Clarke, Julie Baughman-Howe, David Erickson, Julie Eldredge, Debra Finucan, and Gay DeShone for their excellent technical assistance. A very special thanks goes to Diane and the Short Boys for the deep friendship over the many years. These people never wavered in their staunch support of me. For this I am sincerely grateful. To Diane, who often bore the brunt of many of the difficult times throughout the past three years, I am especially thankful. iii LIST OF I LIST OF F LIST OF I li‘iTROD‘JC’. A. 8. C. HETHGJS-. TABLE OF CONTENTS Page LIST OF TABLES --------------------------------------------------- vii LIST OF FIGURES -------------------------------------------------- ix LIST OF ABBREVIATIONS -------------------------------------------- xii INTRODUCTION ----------------------------------------------------- l A. Role of APAP in Analgesic Nephropathy ------------------ 3 B. APAP-Induced Acute Renal Failure ----------------------- 7 C. Role of Metabolism in APAP-Induced Renal Damage -------- l0 1. Cytochrome P-450 activation of APAP --------------- l2 2. Prostaglandin endoperoxide synthetase activation of APAP ------------------------------------------- 19 3. Deacetylase-dependent activation of APAP ---------- 22 0. Purpose ------------------------------------------------ 27 E. Objectives --------------------------------------------- 28 METHODS ---------------------------------------------------------- 30 A. Specific Methods --------------------------------------- 30 l. Animals ------------------------------------------- 30 2. Toxicity tests ------------------------------------ 30 3. Histopathology ------------------------------------ 3l 4. Determination of renal and hepatic non-protein sulfhydryl (NPSH) content ------------------------- 32 5. Isolated perfused kidney (IPK) -------------------- 32 6. Quantitation of urinary metabolites of APAP and PAP from intact animals --------------------------- 34 7. Quantitation of urinary metabolites of APAP from IPKs ---------------------------------------------- 36 8. Quantitation of APAP and metabolites in liver, kidney and plasma --------------------------------- 37 9. Preparation of renal cortical and hepatic subcel- lular fractions ----------------------------------- 38 l0. In vitro microsomal mixed function oxidase acti- VTtTE§::' ------------------------------------------ 39 ll. In_vitro APAP deacetylation ----------------------- 39 iv TABLE OF C RESULTS-.. TABLE OF CONTENTS (Continued) A. l2. Cytochrome P-450-dependent binding of APAP to microsomal protein Ln vitro ----------------------- l3. Binding of PAP to microsomal protein in vitro ----- l4. Deacetylase- dependent binding of APAP Ln vitro---- 15. Determination of covalent binding of APAP* Ln vivo- 16. Radiolabelled substrate preparation --------------- 17. Statistics ---------------------------------------- Individual Experiments --------------------------------- l. APAP-induced GSH depletion and metabolism in the IPK ----------------------------------------------- 2. APAP and PAP nephrotoxicity and metabolism in the F344 rat ------------------------------------------ 3. Comparison of APAP nephrotoxicity and metabolism in F344 and 50 rats ------------------------------- 4. Comparison of PAP nephrotoxicity and metabolism in F344 and SD rats ------------------------------- 5. In vitro activation of APAP ----------------------- 6. Effect of bis-(p-nitrophenyl)phosphate (BNPP) on APAP and PAP nephrotoxicity and metabolism -------- 7. lg_vivo covalent binding of APAP ------------------ RESULTS-----------------------e ---------------------------------- APAP-Induced Glutathione Depletion and Metabolism in the IPK ------------------------------------------------ l. APAP- induced glutathione depletion ---------------- 2. Role of APAP metabolism in glutathione depletion-- 3. Metabolism of APAP by the IPK --------------------- PAP-Induced Nephrotoxicity in the F344 Rat ------------- l. PAP excretion after APAP administration ----------- 2. Effect of PAP on renal and hepatic function in the F344 rat ------------------------------------------ 3 Nephrotoxicity and hepatotoxicity of aminophenol isomers ------------------------------------------- 4. Effect of inducers of renal mixed function oxi- dases on PAP nephrotoxicity ----------------------- Strain Differences in APAP Nephrotoxicity and Metabo- lism --------------------------------------------------- l. APAP-induced histopathological and functional al- terations in kidneys of SD and F344 rats ---------- 2. Renal and hepatic mixed fugition oxidase activity- 3. Covalent binding of [ring- CJ-APAP in renal and hepatic microsomes ................................ 44 44 46 46 47 47 48 49 49 49 49 56 56 56 61 66 66 66 66 71 82 vnn '- ' '\ I. 1.1.— TABLE OF CONTENTS (continued) 4. 5. 6. Page Urinary metabolites of APAP in SD and F344 rats--- 82 In vitro deacetylation of APAP in renal and hepa- tTc—TOTOOO x g supernatants ----------------------- 861 Metabolism of APAP by the IPK --------------------- 86 0. Strain Differences in PAP Nephrotoxicity and Metabolism 90 l. PAP—induced histopathological and functional a1- terations in kidneys of SD and F344 rats ---------- 90 2. Urinary metabolites of PAP ------------------------ 96 3. Renal and hepatic NPSH concentrations ------------- 102 4. ln_vitro covalent binding of [ring-‘4CJ-PAP ------- 102 E. Renal Metabolic Activation of APAP lg_Vitro ------------ 102 l. Cytochrgme P-450-dependent covalent binding of [ring- C]-APAP ----------------------------------- 102 2. Covalent binding of [ring-14C]-PAP in vitro ------- 107 3. Deacetylation of APAP in renal and hEpatic sub- cellular fractions -------------------------------- 111 4 Covalent binding of [ring-14C]-APAP to renal and hepatic subcellular fractions --------------------- 111 5. Covalent binding of [acetyl- 4C]- and [ring-14C]- APAP ---------------------------------------------- 121 F. Effect of BNPP on APAP and PAP Nephrotoxicity and Meta- bolism ------------------------------------------------- 121 1. In vitro deacetylation and covalent binding of APAP----------------------— ----------------------- 121 2. APAP- and PAP-induced nephrotoxicity -------------- 125 3. Disposition of APAP and metabolites --------------- 131 4. Urinary metabolites of APAP and PAP --------------- 136 G. Covalent Binding of APAP and PAP ln_Vivo --------------- 146 DISCUSSION ------------------------------------------------------- 157 CONCLUSION ------------------------------------------------------- 186 BIBLIOGRAPHY ----------------------------------------------------- 187 vi Taale Table 10 11 12 T3 14 LIST OF TABLES Page PAP excretion, blood urea nitrogen and serum glutamic pyruvic transaminase activity following APAP admini- stration to F344 rats ---------------------------------- 59 APAP metabolism by the IPK ----------------------------- 60 Toxicity of aminophenols ------------------------------- 69 Effect of inducers of renal mixed function oxidases on PAP nephrotoxicity ------------------------------------- 70 Comparison of renal lesions produced in male F344 and SD rats by APAP ---------------------------------------- 75 Strain differences in renal and hepatic cytochrome P-450 content and ethoxycoumarin metabolism ------------ 81 Strain differences in covalent binding of [ring-14C]- APAP in renal and hepatic microsomes ------------------- 83 Effect of dose on urinary metabolites of APAP in F344 and SD rats -------------------------------------------- 84 Strain differences in APAP metabolism by the IPK ------- 89 Comparison of renal lesions produced in male F344 and SD rats by PAP ----------------------------------------- 95 Effect of dose on urinary metabolites of PAP in F344 and SD rats -------------------------------------------- 101 Strain differences in non-protein sulfhydryl content of liver and renal cortex --------------------------------- 103 Covalent binding of [ring-14CJ-APAP in renal and hepa- tic microsomes ----------------------------------------- 106 Covalent binding of [ring-14C]-PAP in renal and hepatic microsomes --------------------------------------------- 110 vii 22 23 24 25 26 27 28 LIST OF TABLES (continued) Table 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Page Subcellular distribution of APAP deacetylation in liver and renal cortex --------------------------------------- 114 Covalent binding of [ring-14C]-APAP to renal subcellu- lar fractions jn_vitro --------------------------------- 115 Covalent binding of [ring-14CJ-APAP to hepatic subcel- lular fractions in_vitro ------------------------------- 119 Covalent binding of [ring-14C]-APAP to renal 10,000 x g supernatants jn_vitro ---------------------------------- 120 Strain differences in the covalent binding of [ring- 14CJ-APAP to renal and hepatic 10, 000 x g supernatants- 122 Covalent binding of specifically labelled [14C]-APAP to renal and hepatic microsomes jg_vitro ------------------ 123 Covalent binding of specifically labelled [‘4CJ-APAP to renal and hepatic 10,000 x g supernatants jn_vitro ----- 124 Effect of BNPP on APAP and PAP nephrotoxicity in F344 rats --------------------------------------------------- 130 Concentrations of specifically labelled [14C]-APAP in renal cortical tissue jg_vivo -------------------------- 150 Concentrations of specifically labelled [14C]-APAP in hepatic tissue jg_vivo --------------------------------- 151 Covalent binding of specifically labelled [14C]-APAP to renal cortical protein jg_vivo ------------------------- 152 Covalent binding of specifically labelled [‘4CJ-APAP to hepatic protein in VlVO -------------------------------- 153 Distribution and 1fiovalent binding in renal cortical tissue of [ring-1 C] PAP Ln va0 ----------------------- 154 Distribution and covalent binding in hepatic tissue of [ring- 4C]- PAP Ln va0 ------------------------------ 156 viii Figure Figure 10 11 12 13 14 15 LIST OF FIGURES Page Cytochrome P-450 activation of APAP -------------------- 13 Cytochrome P-450-dependent formation of APBQI ---------- 15 Prostaglandin endoperoxide synthetase activation of APAP --------------------------------------------------- 21 Deacetylase-dependent activation of APAP --------------- 26 APAP-induced depletion of GSH concentration in the IPK- 50 Effect of P88 on APAP-induced depletion of GSH con- centration in the IPK ---------------------------------- 54 Effect of PIP BUT on APAP-induced depletion of GSH concentration in the IPK ------------------------------- 54 Excretion of the major metabolites of APAP by the IPK-- 57 Effect of PAP on BUN in F344 rats ---------------------- 62 Accumulation of PAH and TEA by renal cortical slices from F344 rats 24 hr after PAP (s.c.) administration--- 64 Effect of PAP on SGPT activity in F344 rats ------------ 67 Strain differences in APAP-induced renal necrosis ------ 72 Effect of dose on APAP-induced renal cortical necrosis in F344 rats ------------------------------------------- 75 Effect of APAP on BUN in F344 and SD rats -------------- 77 Accumulation of PAH by renal cortical slices from F344 and SD rats 24 hr after APAP (i.p.) administration ----- 79 ix LIST OF FIGURES (continued) Figure 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Page Deacetylation of APAP by renal and hepatic 10,000 x g superantants from F344 and SD rats --------------------- 87 Strain differences in PAP-induced renal necrosis ------- 91 Effect of dose on PAP-induced renal necrosis in F344 rats --------------------------------------------------- 93 Effect of PAP on BUN in F344 and SD rats --------------- 97 Accumulation of PAH by renal cortical slices from F344 and SD rats 24 hr after PAP (s.c.) administration ------ 99 Effect of substrate concentration on [ring-14CJ-PAP binding to renal microsomal protein jg_vitro ----------- 104 Effect of incubation duration (A) and protein concen- tration (B) on covalent binding of [ring-14C]-APAP to renal microsomes --------------------------------------- 108 Effect of incubation duration (A) and protein concen- tration (B) on covalent binding of [ring-14CJ-PAP to renal microsomes --------------------------------------- 112 Effect of incubation duration (A) and protein concen- tration (B) on covalent binding of [ring- C]-APAP to renal 10,000 x g supernatants -------------------------- 117 Effect of BNPP on APAP deacetylation by renal cortical 10,000 x g supernatants from F344 rats ----------------- 126 Effect of BNPP on covalent binding of [ring-14CJ-APAP to renal cortical 10,000 x g supernatants from F344 . rats --------------------------------------------------- 128 Effect of BNPP on plasma concentrations of APAP and its metabolites -------------------------------------------- 132 Effect of BNPP on renal cortical concentrations of APAP and metabolites ---------------------------------------- 134 Effect of BNPP on hepatic concentrations of APAP and metabolites -------------------------------------------- 137 LIST OF Figure 30 32 33 34 LIST OF FIGURES (continued) Figure 30 31 32 33 34 Page Effect of BNPP on urinary metabolites of APAP ---------- 139 Effect of BNPP on the absolute excretion of PAP -------- 142 Effect of BNPP on urinary metabolites of PAP ----------- 144 Time course of [ring-14C]-APAP covalent binding to renal cortical, hepatic and muscle protein ------------- 147 Renal metabolic activation of APAP --------------------- 159 xi Fin A Q R. H ~\v Van K my 815 CV PPS D1: Phl «16 a .0 U1. Uh. 94H APAP APAP-CONJ APAP-CYS APAP-GLUC APAP-GSH APAP-MAP APAP-NAC APAP-SCH3 APAP-SO3 APAP-SOCH3 APBQI BNPP BPAP BUN F344 GSH GSSG HEX IPK MFOs N-OH-APAP NPSH PAH LIST OF ABBREVIATIONS 4-hydroxyacetanilide; (acetaminophen) sum of APAP-GLUC and APAP-SO3 3-(5'-acetamido-2-'-hydroxyphenyl)-2-aminopropanoic acid; (3-cysteinylacetaminophen) 4'-acetamidophenyl-B-D-glucopyranosideuronic acid; (acetaminophen glucuronide) S-(5'-acetamido-2'-h droxyphenyl)glutathione; (3-gluta- thionylacetaminophen) sum of APAP-CYS, APAP-GSH, APAP-NAC, APAP-SCH3 and APAP-SOCH3 2-acetamido-3-(5'-acetamido-2'-hydroxyphenylthio)pro- panoic acid; (acetaminophen-B-mercapturate) 4-hydroxy-3-methylthioacetanilide; (3-methylthioaceta- aminophen) 4'-acetamidopheny1 sulfate; (acetaminophen sulfate) methyl 2-hydroxy-5-acetamidophenyl sulfoxide; (acetamino- phen-3-methylsu1foxide N-acetyl-p-benzoquinoneimine bis-(p-nitrophenyl)-phosphate 4-hydroxybutyranilide; (butyryl-p-aminophenol) blood urea nitrogen Fischer 344 reduced glutathione oxidized glutathione cycloheximide isolated perfused kidney mixed function oxidases N,4-dihydroxyacetanilide; (N-hydroxy-acetaminophen) non-protein sulfhydryl p-aminohippurate xii LIST 1 PA? PAP-:1; FES F352 roHZ PIP BL SD SEPT TEA LIST OF ABBREVIATIONS (continued) PAP PAP-NAC PBB PES PGG2 PGH2 PIP BUT SD SGPT TEA 3MC 4-hydroxyaniline; (p-aminophenol) 2-acetamido-3-(5'-amino-2'-hydroxyphenylthio)propanoic acid; (p-aminophenol-3-mercapturate) polybrominated biphenyls prostaglandin endoperoxide synthetase prostaglandin G2 prostaglandin H2 piperonyl butoxide Sprague-Dawley serum glutamic pyruvic transaminase tetraethylammonium 3-methylcholanthrene xiii fir u! INTRODUCTION N-acetyl-p-aminophenol (APAP), commonly known as acetaminophen or paracetamol, is an antipyretic analgesic that today is present in a large number of pharmaceutical preparations. In 1978, APAP sales accounted for approximately 29% and 75% of the analgesic market in the United States and Great Britain, respectively (Rumach, 1978). APAP was first introduced into therapy as an analgesic by von Mering in 1893, but gained popularity only since 1949 (Flower g§_al,, 1980). Several reasons have been suggested for the increase in popularity: 1) APAP has antipyretic analgesic properties similar to aspirin yet does not cause gastrointestinal bleeding (Czapels, 1976); 2) APAP is the major active metabolite of the more toxic coal tar analgesics phenacetin and acetanilid (Smith and Williams, 1949a); and 3) APAP has been available in the United States without prescription since 1955 (Flower gt_al,, 1980). Even though APAP has proven to be safe at therapeutic doses, large acute overdoses or long-term abuse of moderate doses often results in toxicity. There are four clinical manifestations of APAP intoxication (Duggin and Mohandas, 1982): 1) hepatic centrilobular necrosis following an acute overdose (Proudfoot and Wright, 1970; Prescott g§_al,, 1971; Williams and Davis, 1977); 2) renal cortical necr01 overd1 activl peuth and 4: by re! Chroni (Dugg‘ nepnrc 2 necrosis, often associated with hepatic damage, following an acute overdose (Boyer and Rouff, 1971; Kleinman g; al,, 1980); 3) an active hepatitis-like syndrome associated with chronic high thera- peutic intake in susceptible individuals (Bonkowsky e§_al,, 1978); and 4) the chronic renal disease, analgesic nephropathy, characterized by renal papillary necrosis and interstitial nephritis following chronic abuse of combination analgesic preparations containing APAP (Duggin, 1980; Stylges and Iuliucci, 1981; Murray, 1982). Analgesic nephropathy is the major clinical problem resulting from APAP abuse (Duggin, 1980); however, APAP overdoses resulting in acute hepatic and renal failure have increased at an alarming rate over the past two decades (Meredith e£_al,, 1981). Extensive literature exists on the biochemical mechanism of APAP- induced hepatic necrosis (for review see Hinson, 1980). Basically, the essential features that have emerged from investigations into the biochemical mechanism of APAP hepatotoxicity are as follows: A small fraction of APAP is metabolized by a cytochrome P-450 dependent mecha- nism to an arylating intermediate which combines with intracellular glutathione (GSH). If cellular stores of GSH are reduced, the reac- tive intermediate then arylates cellular macromolecules. Consequent- ly, normal cellular functions are impaired and cellular necrosis results. Several investigators have demonstrated a direct correlation between hepatic necrosis and the loss of hepatic GSH, covalent binding of APAP to hepatic macromolecules jn_gj!g_or activation of APAP 3Q gjgrg_using inducers and inhibitors of cytochrome P-450 and species differences in the constitutive forms of cytochrome P-450 responsible 3 for activating APAP (Mitchell et_al,, 1973a,b; Jollow e; al,, 1973; Potter g§_a1,, 1973; Davis g3_al,, 1974). Although extensive clinical literature exists on the role of APAP in analgesic nephropathy, few investigations have dealt with the biochemical mechanisms of APAP renal cortical and papillary necrosis. However, it has been emphasized repeatedly that the renal lesion resulting from acute overdose of APAP is markedly different than that resulting from chronic abuse. Therefore, the biochemical mechanisms of the nephropathies may be radically different, depending upon the time course of drug administration. A. Role of APAP in Analgesic Nephropathy Analgesic nephropathy is an important medical problem worldwide; a large proportion of chronic renal failure and end-stage renal disease is thought to be due to analgesic nephropathy (Murray, 1982; Kincaid-Smith, 1978). Approximately 20% of patients in the Australian dialysis and transplant program suffered from analgesic-induced renal disease (Duggin, 1980). Comparable figures reported from other countries include: South Africa 20%, Switzerland 15%, United Kingdom 10%, Canada 6%, and Western Europe 2-3%. Studies in the United States suggest that between 5 and 20% of patients with chronic renal failure and end-stage renal disease abused analgesics (Murray, 1982). Analgesic nephropathy was first recognized as a disease entity in 1953 by Spuhler and Zollinger. These investigators described inter- stitial nephritis and papillary necrosis in individuals that abused. the analgesic preparation SaridonR (Saridon is composed of 150 mg of isopropyl antipyrine, 250 mg of phenacetin and 250 mg of caffeine). 4 Ninety percent of patients suffering from analgesic nephropathy have abnormal intravenous pyelograms, markedly shrunken kidneys and non- specific calyceal abnormalities. Classic pyelonephritic scars have also been reported (Murray, 1982). Patients often exhibit reduced inulin or creatinine clearances, and increases in plasma creatinine indicative of decreased glomerular filtration rates (Bengtsson, 1975; Burry and Dieppe, 1976; Schreiner, 1978). Since almost 50% of renal function must be lost before plasma creatinine is elevated above normal ranges (Duggin, 1980), extensive papillary disease with con- siderable scarring is often present before a change in creatinine clearance is evident (Bengtsson, 1975). The inability to excrete a concentrated urine, which may occur prior to histological lesions, is consistently associated with analgesic nephropathy (Angervall and Bengtsson, 1968; Abel, 1971; Dubach g§_al,, 1975; Murray, 1982). In addition to renal disease, analgesic nephropathy patients often demonstrate peptic ulceration, psychiatric instability, anemia, hyper- tension and myocardial infarction. Furthermore, there is increasing evidence to support a causal relationship between analgesic nephro- pathy (and perhaps heavy analgesic use without overt nephropathy) and renal pelvic carcinoma (Kincaid-Smith, 1978). The conclusion that there is a causal relationship between anal- gesic abuse and the subsequent development of renal disease appears irrefutable. Four different types of studies provided support for this association (Murray, 1982). Several studies indicated that both chronic renal disease and papillary necrosis developed more often in individuals that abuse analgesics than those that do not (Larsen and Cont. 5 Mdller, 1959; Grimlund, 1963; Dubach g§_al,, 1975). Other investi- gations suggested that a history of analgesic abuse was more prevalent in patients who had papillary necrosis than those who did not have the disease (Olafsson e§_al,, 1966; Bengtsson, 1962). Several investi- gators have demonstrated a correlation between the extent of analgesic abuse and the incidence or severity of renal disease (Lindeneg e3_al,, 1959; Gault g£_al,, 1971). The incidence of analgesic nephropathy per country paralleled the per capita consumption of phenacetin in that country (Murray and Goldberg, 1976). In fact, the incidence of renal disease in Sweden has decreased since the availability of phenacetin- containing analgesics was restricted (Nordenfalt, 1972). Lastly, experiments performed in certain animal models suggested a relation- ship between renal disease and analgesic consumption (Kincaid-Smith, 1978). The precise drug responsible for analgesic nephropathy is un- known. Phenacetin and its metabolite, APAP, as well as aspirin and its metabolite, salicylate, all produce renal papillary necrosis in man and experimental animals (Duggin, 1980; Murray, 1982; Kincaid- Smith, 1978). While aspirin consistently resulted in papillary necrosis in experimental animals, the doses required often exceeded the lethal dose (Duggin, 1980). However, analgesic nephropathy has been reported in individuals that consumed only aspirin (Murray, 1982). On the other hand, a far larger number of clinical cases indicated that phenacetin alone results in analgesic nephropathy. Yet in animal studies, even large doses of phenacetin or APAP produced papillary necrosis only inconsistently. However, combination 6 analgesics containing APAP or phenacetin resulted in a striking incidence of renal disease, possibly suggesting synergistic toxicity (Duggin, 1980). Therefore, it appears that the presence of phenace- tin, and possibly its metabolite APAP, in combination or alone, is essential for the development of most cases of analgesic nephropathy. Several lines of evidence support the possibility that the role of phenacetin in analgesic nephropathy may be mediated, not by the parent drug itself, but by the major metabolite, APAP. At moderate oral doses, the first-pass metabolic conversion of phenacetin in the liver is so effective that phenacetin concentrations in the peripheral circulation are extremely low (Raaflaub and Dubach, 1975). Localiza- tion of the lesion to the renal papilla in analgesic nephropathy has been suggested to be a consequence of a high intracellular drug concentration in the papilla. Several elegant studies by Duggin and Nudge have demonstrated that APAP, but not phenacetin, was concen- trated intracellularly in the renal papilla and that intracellular papillary APAP concentration was related to urine flow. This is con- sistent with aggravation of analgesic nephropathy during oliguria or dehydration. Therefore, it appears that APAP is the primary agent present in renal cells following phenacetin administration. Thus, it is APAP and not phenacetin that undoubtedly plays a role in analgesic nephropathy subsequent to phenacetin or combination analgesic abuse. However, the exact biochemical mechanism of APAP-induced pathophysio- logy in the renal papilla is not known. 7 B. APAP-Induced Acute Renal Failure As many as ten percent of all documented APAP overdose cases develop acute renal failure (Clark g§_al,, 1973; McJunkin g§_al,, 1976). However, the appearance of renal failure is variable and may occur at different times after APAP ingestion. Human APAP intoxica- tion can be roughly divided into three clinical outcomes. Fulminant liver damage often occurs without detectable renal insufficiency (Prescott g£_al,, 1971; Clark g; 31,, 1973). Renal failure coupled with fulminant hepatic damage may result (Boyer and Rouff, 1971; Jeffrey and Lafferty, 1981). However, the onset and duration of renal failure may be different than that of hepatic failure. Several cases of acute renal failure in the absence of fulminant hepatic damage have also been reported (Cobden g3_al,, 1982; Curry g}_al,, 1981; Kleinman e§_al,, 1980; Prescott g§_al,, 1971). Such differences in the de- velopment of renal and hepatic toxicity after APAP suggest that the cellular mechanisms within each organ may not be the same. Acute renal failure following APAP overdose is characterized by an increase in serum creatinine and blood urea nitrogen concentrations and a decrease in creatinine clearance (Jeffrey and Lafferty, 1981; Cobden 35 al,, 1982). Progressive oliguria, often despite a fluid challenge, with a marked rise in the fractional excretion of sodium is indicative of the onset of renal failure in APAP-overdose victims (Jeffrey and Lafferty, 1981). Prognosis of APAP—overdose victims is usually good, as most renal function tests return to normal after several days of high urine output which, characteristically, imme- diately follows the period of progressive oliguria (Kleinman 25 al., 8 1980; Jeffrey and Lafferty, 1981; Curry e§_al,, 1981). The renal histopathologic lesion after APAP overdose was restricted to the cortex; extensive focal coagulation necrosis of the proximal tubule was primary although some distal tubule lesions have been reported (Kleinman e£_al,, 1980; Cobden e£_§l,, 1982). Ultrastructural damage, similar to that found with other forms of toxic nephropathy, includes loss of luminal brush borders, mitochondrial disarray, sloughing of cells and disruption of the tubular basement membrane (Kleinman, g£_ al,, 1980). Several mechanisms for acute renal failure following APAP over- dose have been proposed. Hypotension is a plausible mechanism of acute renal failure in patients that demonstrate extensive hepatic damage and/or coma (Boyer and Rouff, 1971). However, hypotension was not detected in many patients that developed acute renal failure following APAP consumption (Jeffrey and Lafferty, 1981; Kleinman g: 31,, 1980; Gabriel g§_gl,, 1982). Wilkinson and coworkers have suggested that renal failure in APAP overdose victims was related to the release of an endotoxin that cannot be cleared by the diseased liver (Wilkinson 23.31,, 1977). Support for their hypothesis included evidence that the occurrence of renal failure was similar in APAP overdose patients with fulminant liver failure and in patients with liver failure from other causes. However, the numerous reports of renal failure without apparent hepatic damage following APAP-overdose argue against the hypothesis set forth by Wilkinson and coworkers (Prescott g§_al,, 1971; Kleinman g£_al,, 1980; Curry g§_al,, 1981; Cobden g§_al,, 1982). In fact, acute renal failure after therapeutic 9 doses of APAP has been reported (Gabriel e1_a1,, 1982). A more likely explanation of acute renal failure is a direct nephrotoxic action of APAP and/or a metabolite. Strong evidence for this mechanism is provided by investigations of APAP-induced renal cortical necrosis in the rat (Mitchell e1_a1,, 1977; McMurtry g1_g1,, 1978). If APAP nephrotoxicity is due to a direct action of APAP and/or metabolites, then differences in renal and hepatic toxicity suggest that the bio- chemical mechanism of renal and hepatic drug-induced injury is differ- ent. Until recently, elucidation of the exact biochemical mechanism of APAP-induced nephrotoxicity was hampered by the lack of an appropriate animal model. High doses of APAP did not produce histopathological changes in kidneys of Sprague-Dawley (SD) rats or mice (Mitchell g1_ 31,, 1973). In Wistar rats, APAP at a dose of 3710 mg/kg resulted in generalized proximal tubular dilation, but not necrosis (Arnold g3 21,, 1973). Renal damage was not evident in hooded rats that were administered APAP (Calder g; 31,, 1971). Hart and coworkers (1982) demonstrated APAP-induced renal proximal tubular necrosis in female 3- methylcholanthrene pretreated, but not naive, SD rats following an oral dose of 2250 mg/kg APAP. Administration of APAP to New Zealand White female rabbits resulted in some renal biochemical, but not histopathological, abberations (Hennis £1.21,, 1981). However, Mitchell and coworkers demonstrated that administration to male Fischer 344 (F344) rats of a single sublethal dose of APAP consis- tently resulted in acute necrosis of the proximal convoluted tubules of the inner renal cortex (Mitchell g§_a1,, 1977; McMurtry g1_a1,, 10 1978). The APAP-induced renal lesion in the F344 rat was similar to that found in APAP overdose victims (McMurtry e1_a1,, 1978; Kleinman ggha1,, 1980). The identification of an appropriate animal model for acute APAP-induced renal necrosis was an important initial step in the elucidation of the biochemical mechanism of this disease. C. Role of Metabolism in APAP-Induced Renal Damage A myriad of mechanisms have been proposed for the renal lesions produced by APAP (Shelley, 1978). Recently, APAP metabolism to reac- tive intermediates in papillary tissue was suggested to be the initial obligatory biochemical event in the pathogenesis of papillary necrosis (Mudge g1_a1,, 1978; Duggin, 1980). For certain xenobiotics, corre- lations between the binding of metabolites to kidney and renal necro- genesis have been reported (Ilett e1_a1,, 1973; Reid, 1973). The correlation between papillary binding of APAP and papillary necro- genesis is not as extensive. However, covalent binding of APAP to papilla is many times the binding in non-target tissues (Mudge g1_g1,, 1978; Mudge, 1982) and was increased by dehydration, a known aggra- vating factor in the etiology of analgesic nephropathy (Mudge e;_a1,, 1978; Duggin, 1980). Extensive experimental evidence supports the involvement of metabolic activation in APAP-induced renal cortical necrosis. Aryla- tion of renal protein and depletion of renal cortical GSH produced by APAP appears to be much greater in F344 rats than $0 rats which are sensitive and insensitive to APAP-induced nephrotoxicity, respectively (McMurtry g1_g1,, 1978; Mudge eg_a1,, 1978). Following a dose of 750 11 mg/kg (i.p.) APAP to SD rats, Nudge and coworkers (1978) reported a 20 percent decrease in renal cortical GSH and covalent binding of ap- proximately 0.1 nmol APAP/mg renal cortical protein. In contrast, Mc- Murtry and coworkers (1978) reported a 50 percent decrease in renal cortical GSH and covalent binding of approximately 0.75 nmol APAP/mg renal cortical protein following a dose of 750 mg/kg (s.c.) APAP to F344 rats. In addition, agents such as cobaltous chloride and pipero- nyl butoxide, that reduced APAP-induced cortical necrosis also reduced covalent binding to renal cortical protein (Mitchell g1_g1,, 1977; McMurtry g§_g1,, 1978). Finally, autoradiographic analysis of kidneys 3H-APAP administration indicated that most of from F344 rats following the radioactivity was associated with necrotic areas in the kidney and liver (Mitchell e;_a1,, 1977). The instability of reactive intermediates generally precludes passage across cellular membranes suggesting that reactive intermedi- ates are formed in close proximity to the site of macromolecular arylation (Nelson, 1982). The presence of covalently bound APAP metabolites within the kidney, therefore, suggests that APAP activa- tion occurs directly in the kidney. There is additional evidence which is consistent with the conclusion that the reactive intermediate of APAP which arylates renal macromolecules is formed jn_§11u_and not in the liver. 3-Methy1cholanthrene (3MC) enhanced APAP-induced hepatic necrosis but had no effect on APAP-induced renal necrosis (McMurtry g1_a1,, 1978). In addition, covalent binding of APAP to renal macromolecules was not altered by total hepatectomy (Breen g3 .11., 1982). Lastly, Emslie and coworkers (1981a) have demonstrated covalent binding of APAP in the isolated perfused kidney (IPK). 12 Therefore, it appears that arylation of renal macromolecules could be the initial biochemical event in APAP-induced renal cortical and papillary necrosis, although the evidence is much more extensive in APAP-induced renal cortical necrosis. Furthermore, the metabolism of APAP to a reactive intermediate probably occurs within the kidney. At least three different biochemical mechanisms have been proposed and/or demonstrated for the generation of a reactive intermediate from APAP. Localization of these enzyme systems to specific areas of the kidney, however, may preclude their involvement in one or both types of APAP-induced renal disease. 1. Cytochrome P-450 Activation of APAP Many investigators have suggested that the biochemical mechanism of APAP activation within the kidney is similar to the hepatic mechanism of APAP activation demonstrated by Brodie and co- workers (Mitchell g1_g1,, 1977; McMurtry g§_a1,, 1978). That is, APAP could be metabolized by a cytochrome P-450 dependent reaction to a chemically reactive arylating agent which could bind covalently to cellular macromolecules resulting in pathological changes (Figure l; McMurtry g}_g1,, 1978). However, the restricted localization of cytochrome P-450 to the renal cortex dictates that this mechanism of metabolic activation, if applicable, is involved only in acute necro- sis, not analgesic nephropathy. The structural and immunological similarities between renal and hepatic microsomal P-450 suggest a similar mechanism of APAP activation. The actual reactive intermediate that arylates tissue protein is generally accepted to be N-acetyl-p-benzoquinoneimine 13 Ikdwo AJWU a :0 3330205; I - nxulwi. J/o 3330:0542 axonwiz mIOCIUOhKv T 1012 no usoczooio T 35033: no zowpu< omvum 058533 .F 95m: l4 (APBQI) (Miner and Kissinger, 1979; Hinson, 1980; Gemborys g1.g1,, 1980). However, the exact sequence of oxidation or oxygenation reactions that result in APBQI is not known. Originally, a P-450 dependent N-hydroxylation of APAP was thought to be the initial step in the formation of APBQI. The resulting N-hydroxy acetaminophen (N- OH-APAP) could then dehydrate to APBQI. However, subsequent 1n_yj§§9_ experiments demonstrated that N-OH-APAP was not formed by a P-450 dependent reaction (Hinson g1_a1,, 1979; Nelson g§_a1,, 1980). Furthermore, N-OH-APAP was not a urinary metabolite of APAP even though N-OH-APAP is surprisingly stable in biological fluids (Gemborys and Mudge, 1981; Gemborys g1_a1,, 1980). At least two possibilities can be envisioned whereby APBQI is formed directly from APAP without an intermediate N-hydroxylation (Figure 2). P-450 acting as a peroxidase rather than an oxygenase may oxidize APAP. P—450 metabolizes a variety of compounds by peroxida- tive as well as oxygenative processes (Hrycay and O'Brien, 1972; Nordblom g1_a1,, 1976). Furthermore, APAP is metabolized to an arylating intermediate by horseradish peroxidase (Nelson §1_a1,, 1981). The exact mechanism of the peroxidative metabolism of APAP by P-450 has not been defined. However, a ferryl oxyradical complex, because of its structural similarities to peroxidase Compound I, can be envisioned to initiate radical abstraction resulting in‘a radical cage complex of ferric cytochrome-hydroxyl radical and an APAP radical (Figure 2). This radical, be it a resonance-stabilized semiquinone or nitrenium radical, could easily be oxidized by a rapid second electron transfer to produce APBQI and a hydrated ferric P-450 complex (Nelson 15 O .Homaa Lo aopoastoe \. pcmucmamv- tome A. 38533 .N 95m: fol ml. +8 10 «201.02 zolw12. 210 201.92: 2019.22 m \%o 9. 1W1 a201w\2z . l6 .1E._1., 1981). Many of the characteristics of the arylating inter- mediate formed by horseradish peroxidase and P-450 are similar. However, an APAP-radical can be detected in horseradish peroxidase but not microsomal incubations (Nelson 31 31,, 1981). Alternatively, APBQI may result from P-450 reactions without the intermediate formation of radicals (Figure 2). This reaction- would be initiated by the reaction of APAP with the perferryl form of P-450 resulting in a ferric oxyamide complex. This complex could decompose at the N-O bond or at the O-Fe bond. In the case of APAP, the N-O bond is more likely to break with oxygen retaining a pair of electrons due to the resonance stabilizing influence of the phenolic group on the incipient nitrenium ion. Rapid ionization of the phe- nolic group would result in formation of APBQI (Hinson g1_§1,, 1980). Such a mechanism is an attractive explanation of the preferential N- hydroxylation of phenacetin but not APAP. Phenacetin, lacking the ionizable phenolic group, would not be resonance stabilized to the same degree as APAP, resulting in cleavage at the O-Fe bond with subsequent formation of N-hydroxy metabolites (Hinson g1_a1,, 1979). Several different types of experimental approaches have provided support for P-450 dependent renal metabolic activation of APAP and its toxicological relevance. Localization of APAP-induced renal lesions to the same discrete area of the kidney that contains the highest P-450 concentration supports the concept of P-450 depen- dent metabolic activation (Mitchell e1_a1,, 1977). Furthermore, McMurtry and coworkers (1978) demonstrated a correlation between loss of renal GSH, covalent binding of APAP metabolites to renal tissue and l7 APAP-induced renal cortical necrosis, using cytochrome P-450 inducers and inhibitors. Isolated renal proximal tubular cells and perfused kidneys metabolized APAP to its mercapturic acid (APAP-NAC), pre- sumably via the intermediate formation of a glutathione adduct of APAP (APAP-GSH) resulting from direct conjugation with a reactive inter- mediate of APAP (Jones g1_a1,, 1979; Ross e£_g1,, 1980; Emslie g1_a1,, 1981a). The metabolism of APAP to APAP-NAC was induced by the cyto- chrome P-450 inducer, 3MC (Jones §§_g1,, 1979; Emslie g1_g1,, 1981b). In addition, McMurtry and coworkers (1978) demonstrated NADPH-depen- dent covalent binding of APAP in renal microsomes that was inhibited by carbon monoxide, nitrogen, boiling and 0°C. Furthermore, a corre- lation between renal P-450 content and susceptibility to APAP-induced nephrotoxicity has been suggested (Mitchell e1_§1,, 1977). While it is apparent from 1n_!1§§9_studies that P-450 depen- dent metabolic activation of APAP does occur within the kidney, the toxicological relevance of this mechanism of activation to APAP- induced renal necrosis is unclear. Several points should be taken into consideration when evaluating the above mentioned studies. First, experiments using inducers or inhibitors of P-450 do not necessarily provide an indication of metabolic activation in the constitutive (uninduced) state. Furthermore, alternative pathways of metabolism may also be induced by xenobiotic treatment. For example, 3MC pretreatment increases the formation of APAP-NAC in isolated cells and kidneys yet has no effect on jn_!1§gg_covalent binding of APAP or APAP nephrotoxicity (Jones g1_a1,, 1979; Emslie §1_a1,, 1981b; Mc- Murtry e§_a1,, 1978). Therefore, the apparent 3MC-dependent increase 18 in P-450 activation of APAP may actually be due to the increased metabolism of the reactive intermediate (i.e., conjugation with GSH). In addition, inducers or inhibitors may alter the distribution of APAP into the kidney. For example, cobaltous chloride, a P-450 inhibitor, reduced APAP-induced nephrotoxicity which was interpreted to result from cobaltous chloride's inhibitory effect on renal P-450 (McMurtry _1__1,, 1978). However, further validation of this interpretation is required following the recent demonstration that cobaltous chloride markedly reduced delivery to the kidney 1n_!119_of the structurally similar aromatic amine, p-aminophenol (Calder g§_§1,, 1979). Extreme care, therefore, must be used in evaluating the toxicological rele- vance of renal P-450 activation from experiments utilizing inducers or inhibitors of mixed function oxidases. Experiments in non-induced animals suggest that, in addition to P-450 activation, there may be another mechanism of APAP metabolic 3”- activation. This hypothesis is supported by covalent binding of APAP to tissue macromolecules jg_ngg, After a nephrotoxic dose of APAP, binding to liver and kidney was roughly equal in the two organs even though P-450 on a nmol/mg protein basis was 10 times higher in liver than in kidney (McMurtry §1_g1,, 1978). 1n_!13§9_kinetic para- meters of P-450 dependent covalent binding of APAP also indicated that 1 vivo arylation of hepatic macromolecules should be much greater than arylation of renal macromolecules (McMurtry g§_g1,, 1978). Nevertheless, jn_vitro studies indicated that P—450 activation of APAP occurred within renal cortical cells. However, the quantitative 19 contribution of this type of metabolic activation to APAP-induced renal cortical necrosis is unknown. 2. Prostaglandin Endoperoxide Synthetase Activation of APAP Recently, APAP was demonstrated to be metabolized to an arylating.metabolite 13_vitro via an arachidonic acid-dependent pathway by prostaglandin endoperoxide synthetase (PES) (Moldeus and Rahimtula, 1980; Mohandas e1_a1,, 1981; Boyd and Eling, 1981). Pros- taglandin endoperoxide synthetase (PES) is a hemoprotein involved in the biosynthesis of eicosanoids (Samuelsson g3_a1,, 1975; Nugteren and Hazelhof, 1973; Hamberg and Samuelsson, 1974). This enzyme catalyzes the oxygenation of polyunsaturated fatty acids (primarily arachidonic acid) to the hydroxyendoperoxide PGHZ. PES is found in highest con- centration in seminal vesicle, platelets and kidney medulla (Christ and Van Dorp, 1972; Marcus, 1972; Samuelsson g§_g1,, 1978). Marnett g§_g1, (1975) observed that during the conversion of arachidonic acid to PGHZ, some structurally unrelated chemicals can be cooxygenated. For example, luminol, diphenylisobenzofuran, oxyphenylbutazone, benzidine and benzo(a)pyrene have been shown to be cooxygenated by this mechanism (Marnett g1_a1,, 1975; Zenser 23.2132 1979; Marnett, 1981; Rapp gal” 1980). ‘ In the kidney, PES exhibits a papillary to cortical gradient (highest in the papilla) which is in sharp contrast to renal mixed— function oxidases (Zenser g1_a1,, 1979). Cyclooxygenase has been localized to the renal vascular endothelial cells, collecting tubules and medullary interstitial cells by immunohistochemical techniques and was not detected in cells of proximal tubules, thick ascending limbs 20 of Henle's loop or distal tubules (Smith and Bell, 1978). Arachidonic acid-dependent jn_!11§9_covalent binding of APAP was greatest in the papilla and least in the cortex whereas NADPH-dependent binding was greatest in the cortex and undectectable in the papilla. Microsomes from the outer medulla were capable of activating APAP by either a PES- or NADPH-dependent pathway (Mohandas g1_g1,, 1981a). PES-dependent covalent binding of APAP to rabbit medullary inicrosomes was reduced by inhibitors of PES (aspirin, indomethacin, and ethoxyquin) and antioxidants (butylated hydroxyanisole and ascor- bic acid) (Mohandas e1;a_l,, 1981a; Boyd and Eling, 1981; Moldeus _ej; _a_1_. , 1982).. GSH also reduced PES-dependent covalent binding of APAP, some of which could be accounted for by the generation of a GSH conjugate (Moldeus g1_a1,, 1982). However, a larger portion of the reduction in covalent binding could be accounted for by the oxidation of reduced GSH. Linolenic acid hydroperoxide can act as a substrate for the hydroperoxidase component of PES. However, linolenic acid hydroperoxide-dependent covalent binding of APAP was not dependent on oxygen or inhibited by indomethacin (Moldeus g1_a1,, 1982). Thus, the peroxidase component of PES, which catalyzes the reduction of PGG2 to PGHZ, appeared to be responsible for the metabolic activation of APAP. The exact mechanism of PES-dependent APAP activation remains speculative. The reaction probably involves a one electron oxidation reaction which could result in hydrogen abstraction with formation of the phenoxy radical of APAP (Figure 3) (Moldeus e1_§1,, 1982). The inhibitory effect of antioxidants and the rapid oxidation of GSH support the hypothesis that a radical of APAP is formed. The APAP 21 Zhg Ado a. cowum>wpum mmmumcpczm muwxogmaoucm cavemmeumoca IO 10 383020232 28 0 333020293 0 A a 030.051: 0 I IOIU' I o 201 012 l a L Too .2 1 383020292 . 2 .49.... >159 a a 2 «cosh/t wgiggz n201.9122 22.925282. 2 m mmxood>u/ o u H van! 0.2092052 2«ou I. . n_ kidney cortex > kidney medulla. Deacetylase activity appears to reside primarily in the soluble fraction, since it is unaffected by removal of the microsomal or mitochondrial fractions (Mudge, 1982). PAP is a potent, selective nephrotoxicant that, like APAP, damages the latter third of the proximal tubule (Green g1_g1,, 1969; Calder g1_a1,, 1971; Cottrell g§_g1,, 1976). Unlike APAP, PAP did not result in hepatic histopathologic changes (Green g1_§1,, 1969). Similar biochemical sequelae have also been noted following APAP and PAP. Both compounds reduced renal GSH concentrations and arylated renal macromolecules; however, whether assessing biochemical or histopathological lesions, PAP appeared to have much greater nephro- toxic potential than APAP (Calder g§_g1,, 1971, 1975). This conclu- sion is tenuous as PAP nephrotoxicity has never been determined in an animal susceptible to APAP-induced nephrotoxicity such as the F344 rat. Certainly, the strain differences in APAP-induced nephrotoxicity suggest caution in the extrapolation of the nephrotoxic potential of PAP between strains. Nevertheless, an alternative biochemical mecha- nism of APAP-induced nephrotoxicity in the F344 rat can be envisioned: deacetylation of APAP to PAP which would then produce renal damage 25 subsequent to its own metabolic activation (Figure 4). Such a mecha- nism would be consistent with APAP-induced depletion of GSH and arylation of renal proteins reported by McMurtry and coworkers (1978), since PAP has been reported to produce similar effects in other strains of rats (Crowe e1_31,, 1979). PAP-induced nephrotoxicity, like APAP hepatotoxicity, is believed to be dependent upon metabolism to an arylating agent (Calder .EI;El:: 1979). Several biological oxidation systems such as cerulo- plasmin and cytochrome c oxidase can oxidize PAP (Frieden and Hsieh, 1976; Borei and Bjorklund, 1953). However, the relevance of these systems to intrarenal generation of reactive intermediates from PAP is unknown. Another enzyme system responsible for the oxidation of PAP to an arylating intermediate is localized to renal microsomes. The activity of this enzyme system was much greater than that found in hepatic microsomes, which correlated with the arylation of renal and hepatic macromolecules by PAP jn_gigg_(Calder g;_a1,, 1979; Crowe g1_ g1,, 1979). The actual enzyme system present within renal microsomes responsible for PAP oxidation is unknown. However, it required molecular oxygen and was inhibited by ascorbate, NADPH and GSH, sug- gesting that a radical intermediate was formed during PAP oxidation (Calder g1_a1,, 1979) (Figure 4). Furthermore, PAP oxidation does not appear to be mediated by P-450 as several inhibitors of P-450, such as piperonyl butoxide, cobaltous chloride and SKF-525A, did not affect in_ 311§Q_renal microsomal covalent binding of PAP. In addition to the undetermined enzymatic oxidation of PAP in renal microsomes, Andersson and coworkers (1982) have recently demonstrated PES-dependent 26 - 1 Demenuss Ike v1. ramsremse Acdyl CoA h 0' o _J moments GSH GSH MACROMOLECULE MACROMOLECULE CELL DEATH Figure 4. Deacetylase-dependent activation of APAP. 27 activation of PAP in ram seminal vesicle microsomes as well as rabbit renal medullary microsomes. Furthermore, the arylating intermediate resulting from PES-dependent activation of PAP, unlike that resulting from APAP, was of sufficient stability to induce DNA strand breaks in human fibroblasts (Andersson §£.§l:a 1982). Therefore, PAP can be formed in many tissues within the body. Most important to APAP-induced renal disease, however, is the formation of PAP by subcellular fractions of renal cortex and medulla. Furthermore, at least two systems of PAP metabolic activation can take palace in the kidney; one is localized in the cortex and requires only rnolecular oxygen and another is localized in the renal medulla and requires oxygen and arachidonic acid. This suggests that deacetyla- trion could play a significant role in the eventual renal metabolic akrtivation of APAP and, by implication, acute and chronic APAP-induced renal disease. 0. Purpose The primary purpose of this investigation was to test the hypo- thesis that metabolic activation is involved in the pathogenesisof chemically-induced renal injury. The mechanism of APAP-induced acute renal cortical necrosis was the focus because the drug induces a "ePr‘oducible renal lesion in a specific animal model. l\lthough there is extensive evidence that suggests APAP may be metabolically activated by mixed-function oxidases (MFOs), there is now! £a\lidence suggesting alternative means of APAP activation. In an organ such as the kidney, that contains variable amounts of MFOs in a hatePogeneous mixture of cell types, such alternative mechanisms of 28 metabolic activation may play an important role in the pathogenesis of chemically-induced renal injury. The study of non-MFO metabolic activation would be important not only when concerned with renal cortex but also within areas of the urinary tract where MFOs are not detectable such as the renal papilla and perhaps the urinary bladder. At least two mechanisms of metabolic activation could be en- visioned to generate reactive intermediates from APAP within the renal cortex, following an acute sublethal dose. These include the P-450- dependent (Figure l) and deacetylase-dependent activation (Figure 4) of APAP. It was therefore essential to document the presence of each pathway of metabolic activation within the renal cortex. Furthermore, the toxicological relevance of each mechanism was evaluated using strain differences in APAP-induced nephrotoxicity as well as specific inhibitors of deacetylation. Finally, attempts were made to quanti- tate the generation of reactive intermediates jn_xjxg_by each pathway and their relationship to the pathogenesis of APAP-induced cortical necrosis. Elucidation of the mechanism of renal cortical APAP metabolic activation and its relationship to toxicity may provide a framework from which to study the mechanism of pathogenesis of the more clini- cally relevant analgesic nephropathy. E. Objectives The specific objectives of this investigation were: 1) To document APAP- and PAP-induced nephrotoxicity in the F344 rat. 2) 3) 4) 6) 7) 8) 29 To quantify deacetylation of APAP 1n_11!9_and 13_!1159_in subcellular fractions and intact kidneys. To compare APAP nephrotoxicity and metabolism in SD and F344 rats. In contrast to F344 rats, APAP-induced renal lesions have not been reported in SD rats. To compare PAP nephrotoxicity and metabolism in SD and F344 rats. To determine the biochemical mechanisms of APAP metabolic activation in the renal cortex jn_!11§9_by a group of experi- ments using isolated cell fragments and specifically labelled APAP fortified with various cofactors. To quantify the effect of the deacetylase inhibitor, bis-(p- nitrophenyl)phosphate (BNPP), on the nephrotoxicity and metabolism of APAP and PAP in the F344 rat. To identify the exact compound, PAP or APAP, that arylates renal and hepatic macromolecules jn_ijg_in experiments that compare the covalent binding of [acetyl-14C1-APAP and [ring- 14c1-APAP. To determine the relationship of arylation to APAP-induced cortical necrosis in F344 and SD rats. METHODS A. Specific Methods 1. Animals Male F344 (200-250 g) and SD (200-250 g) were purchased from Harlan Industries, Inc. of Indianapolis, IN and Haslett, MI, respec- tively. Animals were housed in sanitary, ventilated animal rooms with controlled temperature (24-25°C) and humidity (40-50%) and regular light cycles (7 a.m.-7 p.m.). Animals were allowed free access to food (Wayne Lab-Blox, Allied Mills, Inc., Chicago, IL) and water until use. 2. Toxicity tests Rats were killed by cervical dislocation and decapitated 24 hr after APAP administration and 24 or 48 hr after PAP administration; blood was collected and the kidneys were excised and placed in ice- cold saline. The blood was allowed to clot for 60 min at room tem- perature, centrifuged and the serum was collected for measurement of serum glutamic pyruvic transaminase (SGPT) activity (Reitman and Frankel, 1957) with a commercial reagent kit (Sigma Chemical Co., St. Louis, M0); the activity was expressed as Sigma-Frankel (SF) units/ml. 4 One SF unit of SGPT forms 4.82x10' ummol glutamate/min in phosphate buffer (pH 7.5) at 25°C. Blood urea nitrogen (BUN) was determined 30 31 spectrophotometrically (Kaplan, 1965) with a commercial reagent kit (Sigma Chemical Co.). Thin renal cortical slices were prepared and incubated in 4.0 ml of phosphate-buffered medium (Cross and Taggart, 1950) which contained 7.4x1o'5M PAH and 1.0x1o'5M [‘4C]TEA (specific activity 2.0 Ci/mole). Incubations were carried out in a Dubnoff metabolic shaker at 25°C under a gas phase of 100% 02 for 90 min. After incubation, the slices were removed, weighed and homogenized in 10 ml of 3% trichloroacetic acid. Two m1 of incubation medium was treated similarly. After centrifugation, the supernatant was assayed for PAH and [14C]TEA concentrations. PAH was determined by the method of Smith §1_a1, (1945). One ml of slice or medium supernatant was added to 10 ml of ACS counting cocktail (Amersham, Arlington Heights, IL). Accumulation of PAH and TEA in renal cortical slices was expressed as a slice-to- medium (S/M) concentration ratio, where S represents mg of PAH or TEA per gram of tissue and M represents mg of PAH or TEA per m1 of medium. 3. Histopathology Specimens of kidney were cut longitudinally through the hilum and fixed in Lillie's "85" solution (sodium acetate 1.25%, mercuric chloride 6%, formalin 10%). Blocks were dehydrated in graded alcohols, cleared in xylene, and embedded in paraffin by standard procedures. Sections were stained with hematoxylin and eosin and with periodic acid-schiff reagent. Sections were coded and examined with- out other information. Sections were evaluated for necrosis, tubular protein precipitates and casts, and tubular protein resorption drop- lets. Histopathologic changes were graded on an arbitrary scale of 32 negative, mild, moderate and severe (0-3+), and the area of cortex involved by tubular necrosis was estimated visually. 4. Determination of renal and hepatic non-protein sulfhydryl (NPSH1_content In order to avoid diurnal changes in tissue NPSH content, animals were killed before 12 noon. NPSH content was measured accord- ing to the method of Ellman (1959) with a few modifications. Kidneys and liver were quickly excised and immediately homogenized in 20 volumes of ice-cold 6% trichloroacetic acid (TCA) and centrifuged at 10,000 x g for 20 min. After an adequate dilution with 6% TCA, 0.5 ml of the diluted supernatant was added to 2 ml of 0.3 m Na2P04 solution, pH 8.2. A solution (0.5 m1) of 0.04%, 5,5'-dithiobis-(2—nitrobenzoic acid) in 10% sodium citrate was then added, and the absorbance at 412 nm measured immediately after mixing. 5. Isolated perfused kidney_(IPK) The recirculating perfusion apparatus, perfusion medium and operative technique has been described in detail elsewhere (Newton and Hook, 1981). Bovine serum albumin (Fraction V) was obtained from either Sigma Chemical Company (St. Louis, MO) or Miles Biochemicals (Elkhart, IN) and used in the perfusates at concentrations of 6% and 7%, respectively. After perfusion was initiated, a 15-minute period of equilibration was allowed, followed by nine lO-min periods of urine collection. When APAP perfusate concentrations greater than 2.0 mM were used, APAP was dissolved in the perfusate (total volume, 225 ml) prior to perfusion. At lower concentrations, APAP was added to the perfusate after the first period of urine collection. When experiments 33 were designed to quantify urinary excretion of PAP, urine was col- lected into 500 pl of 10 mM N-acetylcysteine/SO mM sodium acetate (pH 5.0). At APAP perfusate concentrations greater than 2.0 mM three consecutive urine samples taken between 30 and 60 min after equili- bration were combined for analysis. At APAP perfusate concentrations less than 2.0 mM urine samples, urine samples from the last eight collection periods were combined. In certain cases, at the termination of the perfusion renal non-protein sulfhydryl content was determined. Both the perfused kidney and the contralateral nonperfused kidney (placed in ice-cold saline after the initiation of perfusion) were dissected into corti- cal, medullary and papillary (white medulla and papilla) sections, homogenized in 6% trichloroacetic acid and centrifuged to collect the denatured protein. Nonprotein sulfhydryl concentrations were esti- mated in the supernatant fraction by the method of Ellman (1959). Greater than 90% of nonprotein sulfhydryl in the kidney of the naive F344 rat has been determined to be glutathione (GSH) (W.M. Kluwe, personal communication). Percentage of depletion of GSH in the IPK was determined by the following formula: {1-([GSH] perfused kidney/[GSH] nonperfused kidney)} x 100. In most cases, APAP did not affect renal function in the IPK. However, the highest concentration of APAP (10 mM) resulted in increased urine flow and reduced sodium reabsorption compared to IPKs perfused without APAP. 34 6. Quantitation of urinary metabolites of APAP and PAP from intact animals After APAP or PAP dosing, rats were placed individually in stainless steel metabolism cages with free access to food and water. Urine collection bottles contained 10 m1 of 10 mM N-acetylcysteine/SO mM sodium acetate (pH 4.5) with a crystal of thymol. In most cases, 23 hr after dosing animals were injected i.p. with furosemide (20 mg/kg) to facilitate uniform voidings and urine collections. Twenty- four hr after APAP dosing, the cages were rinsed with warm degassed water and the washings were combined with urine and diluted prior to analysis. . APAP, several thioether metabolites (3-glutathiony1 acetamino— phen, APAP-GSH; 3-cysteinyl acetaminophen, APAP-CYS; acetaminophen-3- mercapturate, APAP-NAC; 3-methylthioacetaminophen, APAP-SCH3; 3- methylsulfoxideacetaminophen, APAP-SOCH3) and the phenolic conjugates of each (glucuronide and sulfate) were analyzed by HPLC following dilution and clarification by filtration (pore size, 0.2 pm). Amounts of phenolic conjugates were calculated from the differences of non- conjugated metabolites before and after enzymatic hydrolysis. Enzy- matic hydrolysis was carried out in the following manner: 5.0 m1 of buffer (10 mM N-acetylcysteine/SOO mM sodium acetate/20 mM barium chloride; pH 5.0) was added to 1.0 ml of diluted urine followed by a drop of chloroform and 100 pl of B-glucuronidase (Type H-2, Sigma Chemical Co., St. Louis, MO); incubation was carried out for 18 hr at 37°C. 35 Unconjugated PAP excreted following APAP administration was quantified following conversion to 4-hydroxybutyranilide (BPAP) by the method of Gemborys and Mudge (1981) with the following modifications: 2.0 ml of l M NazHPO4 (pH 7.0) was added to 3.0 ml of urine followed by 100 pl of n-butyric anhydride. 4.0 ml of l M Na HP04 (pH 7.0) was 2 added to 1.0 m1 of urine when unconjugated PAP was determined follow- ing PAP administration. After continuous agitation for 1 hr, the solution was passed through a Sep Pak (Waters Associates, Milford, MA) and eluted with 4.0 ml of methanol. The amount of BPAP was then determined by HPLC. This procedure was slightly modified to determine the total amount of PAP after enzymatic hydrolysis by adding 2.0 ml of l M NazHPO4 (pH 7.0) to 5.6 m1 of hydrolysate. Hydrolysis also pro- duced a small amount of BPAP proportional to the APAP concentration in the hydrolysate. This was not seen in samples of urine incubated without added enzyme and may be attributed to deacetylation of APAP catalyzed by contaminants in the B-glucuronidase. Experimental assays of PAP were corrected according to the concentration of APAP in the hydrolysate. APAP and metabolites were quantified on an HPLC system consisting of a model M6000-A solvent delivery system, a model 440 UV detector set at 254 nm, a model U6K injector, a Data ModuleTM , and a model RCM-100 radial compression module (Waters Associates, Inc., Milford, MA). Dedicated 5 u Radial PakTM reverse phase cartridges (8 mm x 10 cm) were used for all analyses. BPAP was quantified using a solvent system consisting of 15.5% acetonitrile/10.0 mM NaH2P04 (pH 7.0) at a flow rate of 2.5 m1/min. APAP, APAP-NAG, APAP-CYS, 36 APAP-GSH, APAP-SOCH3, and APAP-SCH3 in the urine from intact animals, were quantified using a solvent system consisting of 6.5% dioxane/1% acetic acid at a flow rate of 2.5 ml/min. Solvents were clarified by filtration (pore size, 0.2 pm) prior to analysis. Metabolites were quantified by extrapolation from peak area calibration curves of unmanipulated synthesized standards (Gemborys and Mudge, 1981). Recovery experiments with PAP revealed 60-70 percent recovery from unhydrolyzed samples and 55-65 percent recovery from hydrolyzed samples over concentration ranges encountered in these studies. Therefore, quantified BPAP was corrected for these expected reco- veries. APAP and its acetylated metabolites were not corrected for recovery. The urinary excretory pattern has been described in terms of percent of recovered dose, defined as the amount of a compound factored by the sum of the parent drug plus total metabolites, all on a molar basis. 7. Quantitation of urinary metabolites of APAP from IPKs The four major metabolites of APAP, APAP-$03, APAP—GLUC, APAP-NAC and PAP were quantified by HPLC. At APAP perfusate concen- trations less than 2.0 mM the following methodology was used. Before HPLC analysis urine samples from the last eight collection periods were combined, dissolved in eight volumes of methanol and concentrated under nitrogen. Urine samples were first chromatographed on a Waters radial compression module with a 10 u radial pak A reverse phase cartridge (8 mm x 10 cm) with methanol-water-acetic acid (l3:86:1, v/v/v) solvent system at a flow rate of 3.0 ml/min. The fractions of eluent corresponding to the elution volumes of synthesized standards 37 (Gemborys and Mudge, 1981) were collected, concentrated and injected on separate solvent systems which were optimized for the separated metabolites. All three of the metabolites were quantified at a flow rate of 3.0 m1/min using an acetonitrile/water solvent system con- taining 5 mM tetrabutylammonium phosphate. The ratio of acetoni- trile/water (v/v) was varied with the metabolite fraction chromato- graphed (APAP-SO 17.5:82.5; APAP-NAC, 15:85; APAP-GLUC, 12.5:87.5). 3. At APAP perfusate concentrations greater than 2.0 mM, meta- bolites were quantified directly, without concentration, on a 5 u radial pak A reverse phase cartridge (8 mm x 10 cm) using a solvent system consisting of 12% dioxane/5.0 mM tetrabutylammonium phosphate at a flow rate of 2.5 ml/min. Unconjugated PAP was determined in the urine of the IPK by methods described above. In all cases quantification of metabolites was by extrapolation from peak area calibration curves of unmanipulated synthesized standards. Furthermore, all samples and solvents were clarified by filtration (pore size, 0.2 uM) prior to analysis. 8. Quantitation of APAP and metabolites in liver,,kidney and plasma Rats were killed by cervical dislocation and decapitation 0.5, 1, 2, 3 or 5 hr after APAP (900 mg/kg) administration. Blood was collected in heparinized tubes and the liver and kidneys excised and weighed. Samples (1 g) of kidney cortex were rinsed with ice-cold saline and homogenized (Potter-Elvehjem homogenizer with a teflon pestle) in 4 volumes of water. Aliquots (0.25 ml) of tissue homo- genates or plasma were combined with 2.5 m1 of methanol and centri- fuged at 3,000 x g for 10 min. The supernatant was removed and the 38 pellet extracted twice with 1.0 ml methanol. All supernatants were combined and dried under M2 at 40°C. Extracts were reconstituted in 1.0 ml 10 mM N-acetylcysteine/SO mM sodium acetate buffer (pH 4.5). Enzymatic hydrolysis of APAP-CONJ was carried out as described pre- viously except that only 0.5 m1 of sample was hydrolyzed. APAP, APAP- NAC, APAP-CYS, APAP-GSH and APAP-SCH3 in plasma and tissue extracts were quantified on a dedicated 5 u Radial PakTM reverse phase car- tridge (8 mm x 10 cm) using a solvent system consisting of 6.5% dioxane/1% acetic acid/4 mM hexane sulfonic acid/1 mM octane sulfonic acid at a flow rate of 2.5 ml/min. All samples and solvents were clarified by filtration (pore size, 0.2 um) prior to analysis. Metabolites were quantified by extrapolation from peak area calibra- tion curves of unmanipulated synthesized standards (Gemborys and Mudge, 1981). Recovery experiments with plasma, and renal cortical and hepatic homogenates spiked with APAP and synthetic metabolites (APAP-CONJ, APAP-CYS, APAP-GSH and APAPNAC) revealed greater than 85 percent recovery over concentration ranges encountered in these studies. APAP and its acetylated metabolites were not corrected for recovery. 9. Preparation of renal cortical and hepatic subcellular fFactions Subcellular fractions were prepared from livers and kidneys of rats killed by cervical dislocation. Livers and kidneys were quickly excised and placed in ice-cold 1.15% KCl. Kidneys were bisected and papillary and white medullary tissue discarded. After being weighed, tissues were minced and homogenized (Potter-Elvehjem 39 homogenizer with a teflon pestle) in 3 volumes of 0.1 M sodium phos- phate buffer (pH 7.4). The homogenate was centrifuged at 10,000 x g for 20 min and, when appropriate, the postmitochondrial supernatant was centrifuged at 105,000 x g for 60 min. The resulting microsomal pellet was resuspended in 0.1 M sodium phosphate buffer (pH 7.4) to a final concentration of 15-25 mg protein per m1. Protein concentra- tions of all subcellular fractions were determined by the method of Lowry e_t_a1_. (1951). 10. In vitro microsomal mixed function oxidase activities Reaction mixtures contained microsomes (1-2 mg/ml for kidney and 0.25-0.50 mg/ml for liver) and 1 ml of 0.1 M sodium phosphate buffer (pH 7.8) containing 4.5 uM glucose-6-phosphate, 0.3 uM NADP, 0.3 uM NADH, 0.1 uM NADPH, 163 uM MgCl2 and 1 unit of glucose-6- phosphate dehydrogenase. After 3 minutes preincubation, the reaction was initiated by addition of substrate. The deethylation of ethoxy- coumarin, measured by the method of Atio (1978), was linear with respect to time and protein concentration. Cytochrome P-450 (P-450) concentrations were determined from the dithionite reduced CO differ- ence spectra (Omura and Sato, 1964). All spectral measurements were made on a Beckman dual beam spectrophotometer (Model No. UV 5260). Microsomal protein was determined by the method of Lowry g1_a1, (1951). 11. In vitro APAP deacetylation Liver and kidney cortex was homogenized as above with 3 volumes of ice-cold sodium phosphate buffer (0.067 M; pH 7.0 when the pH of the final incubation had to be adjusted; 0.1 M, pH 7.4 in all 40 other situations) and the homogenate centrifuged for 20 min at 10,000 x g at 4°C. The assay system contained 0.5 ml of the tissue super- natant and 0.7 ml of sodium phosphate buffer (0.2 M, at the desired pH when the pH of the final incubation had to be adjusted; 0.1 M, pH 7.4 in all other situations) in which APAP (final concentration, 10 mM) was dissolved. In certain cases, microsomal or cystolic suspensions were substituted (12 mg protein/ml) for 10,000 x g supernatants. Incubation was at 37°C, usually for 30 min. Deacetylation within renal and hepatic homogenates was determined to be linear for approximately 1 hr at 10 mM APAP. Reactions were terminated by the addition of 0.6 ml 10% TCA followed by centrifugation for 10 min at 3,000 x 9. One ml of supernatant was used for PAP analysis by the alkaline phenol method with minor modifications (Frings and Saloom, 1979). Enzyme activities were expressed as micromoles of PAP generated per hour per gram, wet weight of initial tissue or nmoles of PAP generated per mg protein per hour. 12. Cytochrome P-450-dependent binding of APAP to microsomal protein in vitro Reactions contained microsomes (2.5 mg/ml, unless indicated) and 2.0 ml of 0.1 M sodium phosphate buffer (pH 7.4) containing an NADPH regenerating system (Potter g1_a1,, 1974). After 2 minutes preincubation, the reaction was initiated by the addition of 4.0 umole of [ring-14C]-APAP or [acetyl-14C]-APAP (500 dpm/nmole). The reaction was terminated, usually 10 min later, by addition of 1.0 ml 10% tri- chloroacetic acid (TCA) and the tubes were centrifuged at 3,000 x g for 10 min. The resulting precipitate was washed twice with 3 ml 10% 41 TCA followed by repeated washings with aqueous 80% methanol and cen— trifugation until radioactivity could no longer be extracted from the pellet. Protein pellets were then dissolved in 1.0 m1 1 N NaOH and analyzed for radioactivity and protein content (Lowry g1_p1,, 1951). 13. Binding of PAP to microsomal protein in vitro Reactions contained microsomes (4.0 mg/ml, unless indicated) and 1.2 ml 0.1 M sodium phosphate buffer (pH 7.4). After 2 minutes preincubation, the reaction was initiated by the addition of 1.2 pmole [ring-14CJ-PAP (500 dpm/nmole). The reaction was terminated, usually 10 min later, by addition of 0.6 ml 10% trichloroacetic acid (TCA) and tubes were centrifuged at 3,000 x g for 10 min. Covalent binding was determined as indicated above. 14. Deacetylase-dependent binding of APAP in vitro Reactions contained tissue (10-14 mg/ml, unless indicated) and 1.2 ml 0.1 M sodium phosphate buffer (pH 7.4), when indicated, reaction mixtures also contained a NADPH regenerating system (Potter g1.p1,, 1974). After 2 minutes preincubation, the reaction was ini- tiated by the addition of 2.4 umoles [ring-‘4CJ-APAP or [acety1-‘4c1- APAP (500 dpm/nmole). The reaction was terminated, usually 120 min later, by addition of 0.6 ml 10% TCA and tubes were centrifuged at 3,000 x g for 10 min. Covalent binding was determined as indicated above. 15. Determination of covalent binding of APAP in vivo Rats were killed by C02 asphyxiation and decapitated; blood was collected in heparinized tubes and kidneys, liver and muscle (gastronemius) were quickly excised and placed in ice-cold saline. 42 Kidneys were bissected and papillary and white medullary tissue dis- carded. Approximately 1 g of the remaining renal tissue was homo- genized in 4 volumes of water; liver was treated similarly. Muscle was minced with a Polytron prior to homogenization. For distribution studies, 250 pl of tissue homogenate or whole blood were added to scintillation vials containing 1 ml of Soluene 350TM and allowed to stand overnight. Following decoloriza- tion, 10 ml of ACS was added to each vial for analysis of radioacti- vity. The amount of covalently bound APAP was determined in the following manner. Three m1 of 10% TCA was added to 1 ml of tissue homogenate in a 15 ml screw cap tube and the tubes were centrifuged at 3,000 x g for 10 min. The resulting precipitate was washed twice with 3 ml of 10% TCA followed by repeated washings with aqueous 80% metha- nol and centrifugation until radioactivity could no longer be ex- tracted from the pellet. Protein pellets were then dissolved in 1.0 ml of 1 N NaOH and analyzed for radioactivity and protein content (Lowry g1_g1,, 1951). 16. Radiolabelled substrate preparation [Ring-14C]-APAP and [acetyl—14CJ-APAP were purified prior to use by HPLC on a pBondapakTM C18 prep column (6.8 mm x 30 cm) using 21% methanol/0.15% phosphoric acid as a solvent system at a flow rate of 4.5 ml/min. The HPLC system consisted of a model M6000-A solvent delivery system, a model 440 UV detector set at 254 nm, a model U6K injector, and a Data ModuleTM (Waters Associates, Inc., Milford, MA). Approximately 1 mg of [ring-14C]-APAP (182 pCi/mg) and 3.5 mg of [acetyl-14C]-APAP (53 uCi/mg) were purified in individual runs at a recovery APAP, sy ceuticai New Engl and [ace systems. mm x 10 2.5 ml/r 1m x 30 rate of for puri with the 43 recovery of approximately 73% and 81%, respectively. [Ring-‘40]- APAP, synthesized by Tracer labs, was the kind gift of McNeil Pharma- ceuticals (Fort Washington, PA). [Acetyl-14C]-APAP was purchased from New England Nuclear (Boston, MA). The radiochemica1 purity of HPLC purified [ring-‘4CJ-APAP and [acetyl-14CJ-APAP was verified by HPLC on two different solvent systems. One system utilized a 5 u C18 radial compression column (5 mm x 10 cm) eluted with 4.5% dioxane/l% acetic acid at a flow rate of 2.5 ml/min. Another system utilized a uBondapakTM phenyl column (3.9 mm x 30 cm) eluted with 13% methanol/0.15% phosphoric acid at a flow rate of 1.8 ml/min. Approximately one million counts were injected for purity analysis. In both systems, 99.95% of radioactivity eluted with the uv absorbing APAP peak for both [ring-14c1-APAP and [acetyl- 14CJ-APAP. 14 [Ring- C]-PAP, in its hydrochloride form, was purchased from Pathfinder Laboratories (St. Louis, MO). The radiochemica1 purity of [ring-14C]-PAP was verified by HPLC with a uBondapakTM phenyl column (3.9 mm x 30 cm) eluted with 4% acetonitrile/lO mm NaP04, pH 7.0 at a flow rate of 1.5 ml/min. Approximately one million counts were injected for purity analysis. Radiochemical purity was determined to be >99.0%. 17. Statistics Data were analyzed by analysis of variance (completely randomized design) and treatment means were compared using Tukey's test or the least significant difference test (Steel and Torrie, 1960). The 0.05 level of probability was used as the criterion of significance in all instances. 911 DE! 3x' 6111 till St1 1111] pm Po‘ di: He he pr. vi AP. to ad; 44 8. Individual Experiments l. APAP-induced GSH depletion and metabolism in the IPK First, experiments were designed to quantify the change in glutathione content in three discrete areas of the kidney after perfusion for 80 min with various concentrations of APAP ranging from 3x10'8 to 3x10'5M. In addition, renal function (sodium reabsorption and inulin clearance) was also evaluated. The second series of experiments were designed to determine the role of drug metabolism in GSH depletion. Piperonyl butoxide (PIP BUT), used as an inhibitor of renal drug metabolism, was admini- stered as a single i.p. dose (1000 mg/kg, dissolved in peanut oil) 90 min before initiation of perfusion. Kidneys from naive and PIP BUT 5M APAP. pretreated animals were perfused with either 0 or 3x10- Polybrominated biphenyls (PBB, Firemaster BP-6), used as inducer of renal drug metabolism, was administered daily by gavage (90 mg/kg, dissolved in peanut oil) for 2 days; 24 hr after the last dose kidneys were perfused with either 0 or 3x10-8M APAP. In the third series of experiments, the excretion of APAP metabolites was quantified in IPKs from naive F344 rats or F344 rats pretreated with P88 in a manner identical to that indicated pre- viously. In these experiments all IPKs were perfused with 3x10'5M APAP for 80 min. 2. APAP and PAP nephrotoxicity and metabolism in the F344 rat , In the first series of experiments, various doses of APAP (0 to 900 mg/kg, i.p.) as a warmed (40°C) suspension (35 mg/ml) were administered to male F344 rats. Animals were placed in metabolism 45 cages for collection of urine for 24 hr and then killed. Nephrotoxi- city was evaluated by quantification of changes in BUN; hepatotoxicity was evaluated by quantification of changes in SGPT. Urinary excretion of PAP was also determined in these animals as well as in IPKs from naive F344 rats perfused with 2.5-10 mM APAP. In the next series of experiments, various doses of PAP (0- 200 mg/kg, s.c.) in its hydrochloride form as an aqueous solution (100 mg/ml) were administered to male F344 rats under light ether anesthe- sia. Rats were killed 24 or 48 hr after PAP administration and nephrotoxicity and hepatotoxicity was evaluated by quantification of changes in BUN, PAH and TEA accumulation by cortical slices and SGPT. In an attempt to determine the relationship of aminophenol structure to nephrotoxicity, several isomeric forms of PAP were evaluated for their nephrotoxic and hepatotoxic potential. Male F344 rats received 1.37 nmol/kg (s.c.) of PAP, o-aminophenol or m-amino- phenol dissolved in 0.5 mM HCl in isotonic saline (20 mg/ml). Nephro- toxicity was determined 24 hr later by quantification of changes in KW/BW ratio, BUN and PAH accumulation; hepatotoxicity was determined by quantification of changes in SGPT. In the next series of experiments, the effect of inducers of renal MFOs on PAP-induced nephrotoxicity was evaluated. To induce MFOs a group of male F344 rats were treated daily with a gavage of P88 (Firemaster BP-6, 90 mg/kg for 2 days) or B-naphthoflavone (BNF, 100 mg/kg for 4 days) dissolved in corn oil and were then administered PAP (0, 100 or 200 mg/kg, s.c.) 24 hr after the last dose of inducer. Twenty-four hr after PAP administration, BUN and PAH accumulation by renal cortical slices were determined. 46 3. Comparison of APAP nephrotoxicity_and metabolism in F344 and SD rats Various doses of APAP (0 to 900 mg/kg, i.p.) as a warmed (40°C) suspension (35 mg/ml) were administered to weight-matched F344 and SD rats. Animals were placed in metabolism cages for collection of urine for 24 hr and then killed. Nephrotoxicity was evaluated by quantification of changes in histopathology, BUN and PAH accumulation by cortical slices. Urinary excretion of APAP and metabolites was also determined in these animals as well as in IPKs from naive F344 and SD rat kidneys perfused with 5 or 10 mM APAP. In a separate series of experiments, strain differences in 1p_111§p_renal and hepatic APAP deacetylation, mixed function oxidase activities and NADPH-dependent APAP activation were determined. 4. Comparison of PAP nephrotoxicity_and metabolism in F344 and SD rats Various doses of PAP (0 to 400 mg/kg, s.c.) in its hydro- chloride form as an aqueous solution (100 mg/ml) were administered to weight-matched F344 and SD rats. Animals were placed in metabolism cages for collection of urine for 24 hr and then killed. Nephrotoxi- city was evaluated by quantification of changes in histopathology, BUN and PAH accumulation by renal cortical slices. In addition, urinary excretion of PAP and metabolites was determined in these animals. In a separate group of experiments, strain differences in renal and hepatic NPSH content and renal PAP activation 1p_gi§pp_were deter- mined. 47 5. In vitro activation of APAP In the first series of experiments, cytochrome P-450 depen- dent covalent binding of [ring-14C]-APAP to renal and hepatic micro- somes from F344 rats was determined at optimal protein concentrations and incubation durations. In addition, the covalent binding of [ring- 14C]-PAP to renal and hepatic microsomes from F344 rats was also determined at optimal protein concentrations and incubation durations. In the second series of experiments, 1p_xj§pp_incubation conditions were optimized in an attempt to identify a deacetylase- dependent mechanism of APAP activation. Therefore, the subcellular localization of APAP deacetylation and deacetylase-dependent APAP activation was determined. Specifically, labelled [14C]-APAP and specific inhibitors of APAP deacetylation and PAP activation were employed to substantiate deacetylase-dependent APAP activation. 6. Effect of Bis-(p-nitrophenyl)phosphate (BNPP) on APAP and PAP nephrotoxicity_and metabolism In the first series of experiments, rats were divided into 10 groups of 6 rats each. Five groups received BNPP pretreatment (100 mg/kg, i.p.); the remaining 5 groups were pretreated with water 30 min prior to the administration of APAP or PAP. Two groups (pretreated with water or BNPP) served as controls while the other groups received APAP (750 or 900 mg/kg, i.p.) or PAP (150 or 300 mg/kg, i.p.). Animals were placed in metabolism cages for collection of urine for 24 hr and then killed. Nephrotoxicity was evaluated by quantification of changes in histopathology, BUN and TEA and PAH accumulation by renal cortical slices. Urinary excretion of APAP or PAP and metabolites was 48 also determined. In addition, the effect of BNPP on renal APAP de- acetylation and deacetylase-dependent covalent binding 1p_!11§p_was determined. For tissue distribution studies, 2 groups of 30 rats each were used. One group received BNPP pretreatment (100 mg/kg, i.p.) while the other received water 30 min prior to APAP administration; all animals received 900 mg/kg APAP. Animals were killed at various times following APAP administration (0.5, 1, 2, 3, 5 hr) and tissue concentrations of APAP and metabolites determined. 7. In vivo covalent binding of APAP In the first series of experiments, the time course of [ring-14CJ-APAP arylation of renal cortical, hepatic and muscle pro- tein was determined. Male F344 rats received a single i.p. dose of [ring-14CJ-APAP (900 mg/kg, approximately 50 dpm/nmole) between 6 a.m. and 8 a.m. Animals were killed 3, 6 or 9 hr later and arylation of protein and tissue distribution of radioactivity determined. In a separate series of experiments, the covalent binding of [acety1-14CJ-APAP and [ring-14CJ-APAP to renal cortical, hepatic, and muscle protein was determined in male F344 and SD rats. Animals were pretreated with either water or cycloheximide (1 mg/kg, i.p.) 60 min prior to and 3.0 hr after APAP administration. Animals received a single i.p. dose of either [ring-14CJ-APAP or [acetyl-14CJ-APAP (900 mg/kg, approximately 50 dpm/nmole) between 6 and 8 a.m. Animals were killed 6 hr following APAP administration and arylation of protein and tissue distribution of radioactivity determined. RESULTS A. APAP-Induced Glutathione Depletion and Metabolism in the IPK l. APAP-induced_glutathione depletion Perfusion alone significantly depleted glutathione (GSH) concentrations in the cortex, medulla and papilla. However, APAP further reduced the GSH content in all areas of the perfused kidney 8 to 3x10'5M. The highest concen- over a concentration range of 3x10- tration of APAP used produced significant depletion of GSH concentra- tions in all three areas of the kidney (Figure 5). No alterations in function (glomerular filtration rate, urine flow, sodium reabsorption) were evident at any concentrations of APAP used (data not shown). 2. Role of APAP metabolism in glutathione depletion Treatment of rats with PBB did not alter the GSH concentra- tions in kidneys perfused without APAP. However, PBB enhanced the ability of 3x10'8M APAP to deplete cortical and medullary GSH con- tents. In contrast, PBB failed to result in a reduction in GSH con- tent in the papilla of IPKs perfused with 3x10-8M APAP (Figure 6). PIP BUT treatment alone did not significantly alter GSH concentrations in perfused kidneys. Perfusion with 3x10'5M APAP significantly lowered GSH concentrations in all three areas of the kidney. 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However, the proportion of PAP in ES[) rats was elevated (in comparison to the lowest dose of PAP) only at 1F'163 highest dose of PAP. 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This linear relationship between nonenzymatic binding and the specific activity of PAP suggests that the material that bound nonenzymatically was not PAP itself but some contaminant of the radioactive PAP. However, some of the non- enzymatic binding measured after 25 min of incubation occurred during 108 .meopxpe :oppoomo Lmo mcoppocpEmemo pomempwpo mmesp pmomp po mo .z.m.m.H mcome moo ompcmmmeomc opoo .mcpocpo oppoex~cmicoc ooo _opop :mmzpmo mocmomeepo mop Eocw ompopoopoo mo: ocpocpo pcmocmomouzoo opp: omcpeempmo mo; morocpo p:m_o>oo popop .o97% is cytosolic protein) in a 10,000 x g supernatant. Therefore, the expected de- acetylation rates for renal and hepatic 10,000 x g supernatants were similar. However, the observed deacetylation rate in renal 10,000 x g supernatant was only 69% of the expected rate, whereas the expected and observed deacetylation rates in hepatic 10,000 x g supernatants were comparable (Table 15). 4. Covalent binding of [ringrl4C]-APAP to renal and hepatic subcellular’fractions Under incubation conditions (protein and duration) that were optimized to demonstrate deacetylase-dependent covalent binding, enzymatic binding of [ring-14C]-APAP to protein was evident in renal microsomes. This enzymatic binding was inhibited by the addition of an NADPH regenerating system (Table 16). 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To timed 0010... zno .. exeéiz n5.0.2: H L = = .o e ImO j 0 o. I \H $333: one... 9101...... 2n... n l Ian-(z a ee macarooie 9 emfioeeee ea 3.3595 19;. 160 degraded within the IPK to PAP—NAC. PAP-NAC could then be reacety- lated back to APAP-NAC. However, several lines of evidence fail to support this possibility. First, although synthesized standards were not available, PAP-NAG, chromatographically, would not be expected to be dramatically different from APAP-NAG. However, all HPLC peaks within the chromatographic region of APAP-NAC corresponded to peaks in urine of IPKs perfused without APAP. Second, addition of 35S-GSH to renal incubations jg_vjtrg_reduced arylation of protein by PAP but did not result in the appearance of peaks chromatographically different 35S-GSH or 35 than from S-GSSG. While these experiments are prelimi- nary in nature, they indicate that a GSH conjugate of PAP may not be formed. Therefore, it appears that most of the APAP-NAC excreted by the IPK could have resulted from a P-450-dependent mechanism of activa- tion. This observation was corroborated by the demonstration of NADPH-dependent covalent binding of APAP to renal microsomes ig_vitrg, Furthermore, the formation of APAP-NAC by the IPK was consistent with reports of other investigators who quantified APAP-NAC production in isolated cells and isolated kidneys (Jones et_§l,, 1979; Ross gt_§1,. l980; Emslie gt al,, 1981a,b). The formation of APAP-NAG may account for a small part of the APAP-induced depletion of GSH in all three sections of the kidney. Several investigators have reported that compounds that induce and inhibit hepatic drug-metabolizing enzymes influence APAP-induced depletion of hepatic GSH concentration (Mitchell gt_al,, l973b; Mudge et_al,, 1978). This was presumably due to alterations in the l61 generation of the reactive intermediate which can conjugate with and deplete hepatic GSH. Our studies may indicate that some GSH-depleting metabolites were formed via cytochrome P-450-dependent pathways. In the present study, PIP BUT, an inhibitor of mixed-function oxidases (Mitchell gt_al,, l973a), reduced APAP-induced GSH depletion in cortex and medulla while PBB, a potent inducer of renal mixed-function oxi- dases (Kluwe and Hook, l98l), enhanced APAP-induced GSH depletion in cortex and medulla. In addition, the enhanced GSH depletion produced by PBB was accompanied by accelerated excretion of APAP-NAG, the major sulfide-containing APAP metabolite excreted by the IPK (Ross gt_gl,, l980; Emslie gt_al,, l98la,b). This was presumably the result of an enhanced formation of a reactive intermediate of APAP which could conjugate with and deplete GSH. Treatment with 3MC, another potent inducer of renal mixed-function oxidases, also enhanced the formation of sulfide-containing metabolites by isolated renal cells (Jones gt 21,, l979). Experimental protocols utilizing inducers of mixed-function oxidases should, however, be interpreted with caution as alternative pathways of metabolism may also be induced. Indeed, PBB by inducing GSH transferases may facilitate the conjugation of GSH with reactive intermediates of APAP resulting in enhanced excretion of APAP-NAG and depletion of medullary and cortical GSH. Conversely, PIP BUT, by interfering with GSH transferases, may result in less depletion of medullary and cortical GSH. While GSH can combine non-enzymatically with many electrophilic xenobiotics, the rate of conjugation of the electrophilic intermediate formed by P-450 metabolism with GSH is l62 markedly enhanced by the presence of cytosolic GSH transferases (Rollins and Buckpitt, l979). Recently, Emslie and coworkers (1981b) demonstrated that 3MC pretreatment resulted in a marked increase in the excretion of APAP-NAC in IPKs perfused with APAP. However, the covalent binding of APAP to the IPK was not enhanced. These findings were attributed to induction of GSH transferases by 3MC (Emslie gt_ al,, l98lb). Induction of renal GSH transferases and not P-450 by such compounds as PBB and 3MC may explain the apparent discrepancy between APAP activation and nephrotoxicity. Pretreatment with both compounds increased APAP-NAG excretion in isolated renal cells (3MC) and IPKs (3MC + PBB) (Jones et_al,, l979; Emslie gt_al,, 1981b). However, neither pretreatment regimen enhanced APAP-induced nephro- toxicity jg_vjvg_in the F344 rat (McMurtry g§_al,, l978; J.F. Newton and J.B. Hook, unpublished). Furthermore, 3MC pretreatment did not increase NADPH-dependent covalent binding in renal microsomes from F344 rats (McMurtry gt_al,, l978). These observations suggest that the effects of PBB and PIP BUT on APAP-induced GSH depletion and APAP- NAC excretion in the IPK may not merely be due to modulation of P-450- dependent APAP activation. Two lines of evidence suggest that the GSH-depleting metabolite produced in the papilla may not be formed via cytochrome P-450. The papilla of the rat kidney (inner medulla and papilla) does not contain detectable amounts of cytochrome P-450 (K.S. Hilliker, W.M. Kluwe and J.B. Hook, unpublished observations). In addition, the consistent lack of effect of modulators of cytochrome P-450 activity on GSH depletion in the papilla in the present study also suggests that the l63 GSH-depleting metabolite is not formed via a cytochrome P-450-mediated process in the papilla. Recently, Moldeus and Rahimtula (l980) demon- strated that an intermediate of APAP capable of conjugating with GSH was formed via a prostaglandin synthetase-dependent reaction. The renal inner medulla contains considerable prostaglandin synthetase activity (Zenser et_al,, 1979). Therefore, in our experiments, APAP may have been activated by prostaglandin synthetase in the papilla of the IPK. Recently, Boyd and Eling (l981) demonstrated arachidonic acid-dependent metabolism of APAP to an arylating metabolite by rabbit renal medulla. Alternatively, it has been demonstrated that APAP can be deacetylated to PAP in cortex and medulla (Carpenter and Mudge, l98l). The activation of PAP to a reactive intermediate capable of depleting GSH appears to occur via a cytochrome P-450-independent reaction (Calder §t_al,, l979). Therefore, in our experiments APAP may have reduced the GSH concentration in the papilla of the IPK subsequent to deacetylation and metabolic activation. In fact, both mechanisms of activation may be involved in the generation of a reactive intermediate capable of reducing papillary GSH. Recently, Andersson and coworkers (l982) demonstrated that PAP could be meta- bolized to a reactive intermediate by a PES-dependent mechanism. The reduction in GSH concentrations by APAP in the IPK is con- sistent with the effect produced jg_vjvg, McMurtry gt_§l, (1978) demonstrated that APAP could reduce the "whole kidney" GSH concentra- tion, whereas Mudge gt_al, (1978) separated APAP-induced GSH depletion into cortical and papillary components. In addition, McMurtry g§_al, (l978) demonstrated a correlation between GSH depletion and renal 164 cortical necrosis. Our studies with the IPK suggest the APAP meta- bolite responsible for GSH depletion and possibly cortical necrosis could be formed directly within the kidney. However, the depletion of GSH concentrations in the IPK cannot be accounted for entirely by the production of APAP-NAC. In fact, the formation of APAP-NAG can only account for a small fraction (