THE. RENAL RANDLING AND NEPHROTOXKCITY 0F PARAGUAT Thesis ‘09 H20 Degree 0‘. M. S. MICHMAN STATE UNIVERSITY James Lee Ecker 1975 THESI“. G - _- - - r , h A. ~ 3 I, ’4. ‘l.,_ ! i. .3, c ‘ I " - ,.- _ €c~ «- . ‘ ABSTRACT THE RENAL HANDLING AND NEPHROTOXICITY OF PARAQUAT By James Lee Ecker Paraquat (l,l'-dimethyl 4,4'-bypyridylium) is a broad spectrum herbicide which is highly toxic to man and other animals. The ob- jectives of this investigation were: (1) To determine the processes involved in the renal elimination of paraquat; (2) to evaluate the nephrotoxic potential of this compound; and (3) to utilize the infor- mation from these studies to gain a better understanding of the mech- anisms responsible for paraquat toxicity. Accumulation of various compounds by renal cortical slices in vitro is related to the capacity of the kidney to actively secrete these same compounds in vivo. Accumulation of paraquat by mouse re- ' nal cortical slices was determined to identify an active secretory component that might be involved in the renal elimination of para- quat. Paraquat, an organic base, was accumulated by slices. The amount accumulated was related to the concentration of paraquat in the medium and the duration of incubation. Paraquat accumulation was depressed by incubation of slices under nitrogen or by addition of metabolic inhibitors. Accumulation of a second organic base, N~methyl- nicotinamide (NMN), was depressed by a concentration of paraquat which failed to influence accumulation of the organic acid p-aminohippurate (PAH). The uptake component of NMN accumulation was inhibited by para- quat. The data suggest that paraquat is accumulated by an energy- James Lee Ecker requiring process and that this accumulation occurs via the organic base transport system. In addition, an apparently toxic effect of paraquat on cortical slice function was observed when the incubation temperature was raised from 25° to 37°C. At this temperature, lO-BM paraquat de- pressed not only NMN accumulation but PAH accumulation and slice oxy- gen consumption as well. Thus, paraquat can be toxic to slice func- tion and this effect appears to be temperature-dependent. The nephrotoxic potential of paraquat was evaluated by determin- ing renal function in mice acutely and chronically poisoned with paraquat. Renal function was estimated utilizing both in vitro and in vivo techniques. Proximal tubular function was monitored in vitro by measuring accumulation of PAH and NMN into renal cortical slices. Disappearance of phenolsulfonphthalein (PSP) and 14C-para— quat from plasma was used to monitor tubular function in intact ani- mals. Glomerular function was approximated using disappearance of iothalamate from plasma. Slices prepared from mice chronically poisoned with paraquat (50 ppm of paraquat in the drinking water) were not different from control slices in their ability to accumulate PAH or NMN. Renal function was also evaluated in mice surviving an LDSO (7 day) dose of the herbicide. Renal cortical accumulation of PAH and NMN in vitro was not markedly altered following acute paraquat poi- soning. In contrast, dramatic effects were measured in vivo. The rate of disappearance of both 14C-paraquat and PSP from plasma was James Lee Ecker significantly reduced in poisoned mice. In contrast, the rate of disappearance of iothalamate was not affected by paraquat. However, the concentration of iothalamate was higher in the plasma of treated animals, suggesting that paraquat poisoning results in a smaller vol- ume of distribution for iothalamate but fails to alter glomerular filtration. The renal excretion of paraquat apparently involves an active secretory process. In addition, paraquat appears to interfere with renal function in the proximal tubule of the kidney. Since secre- tion of paraquat from the body involves accumulation of the herbi- cide within the proximal tubule cells, it is not surprising that paraquat interferes with the function of these same cells. Further- more, it follows that, should toxic concentrations of paraquat be reached in the kidney, subsequent impairment of renal function would impede elimination of the herbicide, leading to more profound toxi- city in organs other than the kidney. THE RENAL HANDLING AND NEPHROTOXICITY OF PARAQUAT by James Lee Ecker A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pharmacology 1975 ACKNOWLEDGEMENTS I would like to express my sincere appreciation to the gradu- ate committee members: Drs. Theodore M. Brody, James E. Gibson, Jerry B. Hook and David R. Rovner for their helpful assistance in the preparation of this thesis. I would especially like to acknow- ledge Dr. James E. Gibson for his guidance, constructive criticism and encouragement throughout the course of this investigation. In addition, the efforts of Dr. Jerry B. Hook throughout my college education have been greatly appreciated. The technical assistance of Ms. Harriet Sherman, Ms. Carla Gauger, Mr. Robert Clark, Mr. Grant Moore and the typing skills of Ms. Joan Ecker are gratefully acknowledged. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION Renal Elimination of Paraquat Nephrotoxic Effects of Paraquat Renal Secretion of Organic Compounds In Vitra Slice Technique In Viva Measurements of Renal Function Purpose METHODS In Vitra Determination of Renal Function Description of the Slice Technique Effect of Paraquat on NMN and PAH Accumulation Paraquat Accumulation Effect of Inhibitors on Paraquat Accumulation Uptake of NMN Determination of Slice 0 Consumption Slice Function as an Indicator of Nephrotoxicity \IO‘UIbUJNH O n Viva Determination of Renal Function Disappearance of Paraquat from Plasma Disappearance of PSP from Plasma Disappearance of Inulin from Plasma . Disappearance of Iothalamate from Plasma H bUJNH 0 Statistical Analyses iii Page ii iii Vitra Analysis of the Renal Handling of Paraquat Paraquat Accumulation by Mouse Renal Cortical Slices Effect of Paraquat on NMN and PAH Accumulation Effect of Paraquat on NMN Uptake Effect of Paraquat on Slice Oxygen Consumption Nephrotoxicity of Paraquat in Mice 1. Slice Function as an Indicator of Nephrotoxicity 2. Effect of Nephrectomy on the Disappearance of Para- quat from Plasma 3. Effect of Paraquat Poisoning on the Disappearance of Paraquat from Plasma 4. Effect of Paraquat Poisoning on the Disappearance of PSP from Plasma 5. Effect of Paraquat Poisoning on the Disappearance of Inulin from Plasma 6. Effect of Paraquat Poisoning on the Disappearance of Iothalamate from Plasma DISCUSSION SUMMARY BIBLIOGRAPHY iv l7 l7 l7 l7 l8 18 18 18 19 20 20 20 21 22 28 58 Table LIST OF TABLES Effect of inhibitors of organic base transport on paraquat accumulation (S/M) by mouse renal corti— cal slices. Effect of paraquat on PAH and NMN accumulation (S/M) by mouse renal cortical slices. Effect of paraquat on oxygen consumption by mouse renal cortical slices. Accumulation of NMN and PAH by renal cortical slices prepared from mice chronically poisoned with paraquat. Accumulation of PAH and NMN by renal cortical slices prepared from.mice acutely poisoned with paraquat. Body and kidney weights of mice acutely poisoned with paraquat. Page 30 31 32 33 34 35 Figure 10 11 LIST OF FIGURES Page Structure of paraquat 37 Effect of medium concentration and duration of incubation on C-labeled paraquat accumulation 39 (S/M ratio) by mouse renal cortical slices in- cubated under oxygen at 25°C. Accumulation (S/M ratio) of 14C-labeled para--__5 quat at an initial medium concentration of 10 M 41 by mouse renal cortical slices incubated at 37°C under oxygen and nitrogen. Effect of paraquat on accumulation (S/M ratio) of PAH and NMN by mouse renal cortical slices 43 incubated under oxygen at 25°C for 90 minutes. Effect of lO-BM paraquat on the rate of 14C- labeled NMN uptake by mouse renal cortical 45 slices incubated under oxygen and nitrogen at 37°C. -3 14 Effect of 10 M paraquat on the rate of C- labeled NMN uptake by mouse renal cortical slices incubated under oxygen and nitrogen at 25°C. 47 Effect of bilateral nephrectomy or shameopera- tion on the disappearance of paraquat from 49 plasma 16 hr later. Effect of 30 mg/kg i.p. paraquat on the subse- quent plasma disappearance of a second paraquat 51 dose determined 2 and 24 hr later. Effect of 30 mg/kg paraquat i.p. on the disap- pearance of phenosulfonphthalein (PSP) from 53 plasma 24 hr later. Effect of 30 mg/kg paraquat i.p. on the disap- pearance of H-inulin from plasma 24 hr later. 55 Effect of Solmg/kg paraquat i.p. on the disap- pearance of I iothalamate from plasma 24 hr 57 later. vi INTRODUCTION The herbicide, paraquat (l,1'—dimethyl 4,4'-bypyridylium dichloride) has caused more than 200 poisonings in man and numerous farm animals since its introduction (Rogers, et aZ., 1973 and Kimbrough, 1974). The fatal dose in man has been estimated to be between 4 mg/kg (Editorial, 1972) and 50 mg/kg (Murray and Gibson, 1972) with the overall mortal- ity in reported poisonings to be approximately 33-50% (Editorial, 1971). The clinical symptoms following acute paraquat poisoning are simi- lar in man and animals. Initially, vomiting is observed with ulcera- tion of the mouth and pharynx observed later. Patients show signs of renal functional impairment, often including oliguria, albuminuria, in- creased blood urea nitrogen and altered serum electrolyte concentra- tions. After a latent interval, signs of respiratory failure ensue, including dyspnea and cyanosis. Death, usually attributal to respira- tory failure resulting from progressive pulmonary fibrosis, often occurs from 1 to 4 weeks later. Additional symptoms sometimes noted in labora- tory animals include anorexia, diarrhea and tachycardia. Pathological examinations have consistently demonstrated lung and kidney lesions, with occasional mention of alterations from normal in the liver, spleen, thymus and adrenal cortex. Histologically, the lungs appear hemorrhagic and edematous showing signs of increased fibroblastic activity and pro— liferation of the alveolar lining epithelium (Toner, at al., 1970). The distribution of 14C-labeled paraquat in the mouse has been studied following i.p. and p.o. administration by Bus, et al. (1975a). The highest concentrations of paraquat (14C) were obtained in the liver and kidney. These values were severalfold greater than those seen in other tissues or plasma. The concentrations of paraquat (14C) in the 2 liver and kidneys declined quickly; whereas in the lung, the concentra- tion of the herbicide apparently increased with time. Similar observa~ tions using whole body radioautography have been made by Litchfield, at al. (1973) following an intravenous injection of paraquat (140) in mice. Sharp, at al. (1972) reported that the concentration of paraquat in the kidney was greater than that observed in plasma for several days following the administration of the herbicide to rats. In laboratory animals, paraquat is poorly absorbed following oral administration (Daniel and Gage, 1966 and Murray and Gibson, 1974). When paraquat was given subcutaneously to rats in a single dose, 80 to 98% of the dose was excreted in the urine within 24 hrs. No signifi- cant biliary excretion or metabolism of the compound was noted (Daniel and Gage, 1966). Significant urine concentrations of paraquat have been detected weeks following administration in both man and animals (Beebeejaum, et al., 1971; Fisher, at al., 1971 and Murray and Gibson, 1974. . Many procedures for the treatment of acute paraquat poisoning in man have been attempted, yet no specific antidote has been found and no therapeutic procedure has been shown to be exceptionally effective. Rational therapeutic procedures have not been developed partly because of the lack of information concerning the tolerable dose, precise mech- anism of action and optimal methods for removal of the poison (Fisher, et aZ., 1971). Following paraquat ingestion, attempts have been made to reduce absorption by the use of gastric lavage, cathartics and adsor- bents. Staiff, at al. (1973) screened various materials to assess their adsorbent potential. The ion exchange resin, Amberlite CG-120 (100-200 or 200—400 mesh) was shown to be most effective, adsorbing paraquat in vitra and in viva. 0n the other hand, Browne (1971) 3 recommended Fuller's Earth over Amberlite as an adsorbent. Smith, et al. (1975) recently suggested a treatment program to prevent the adsorp- tion of paraquat into the plasma. Treatment consists of a stomach wash followed by the adsorbant bentonite and purgatives. Apparently this treatment effectively reduces paraquat lethality in rats even when treat- ment is delayed 10 hrs after paraquat administration. Forced diuresis has been shown to increase urinary excretion of paraquat in man and dog (Kerr, at aZ., 1968; Fisher, at aZ., 1971 and Ferguson, 1971). Most commonly mannitol has been used; however, the use of furosemide has also been reported (Beebeejaum, 1971). Apparently, peritoneal dialysis is ineffective in reducing the paraquat plasma concentration; however, hemodialysis may be effective (Grundies, at al., 1971). Various thera- peutic procedures have been recommended for paraquat poisonings includ- ing treatment with steroids, d-propranolol and superoxide dismutase (Ed- itorial, 1973). Recently, Maling, at al. (1975) have shown that pro- pranolol and other B-adrenergic blocking agents reduce paraquat mortal- ity in rats. Oxygen therapy is contraindicated in cases of paraquat poisonings unless significant cyanosis is present. Fisher, et a1. (1973) have shown enhanced paraquat toxicity in rats during oxygen treatment. Bus, et a2. (1974) recently presented evidence that paraquat toxicity may be a consequence of paraquat-induced lipid peroxidation. Paraquat has long been known to undergo oxidation and reduction under appropriate condi- tions. Paraquat (methyl viologen) is used as an indicator of oxidation— reduction reactions; the reduction of paraquat produces a deeply colored compound. Paraquat exists in the form of a reactive radical in the re- duced state (Figure 1). This reactive state may account for the herbi- cidal properties of the compound. It has been suggested that paraquat 4 is reduced in plants by certain enzyme systems, then reacts with oxygen, leading to the formation of free radicals and/or hydrogen peroxide (Calderbank, 1968). Free radicals formed are proposed to react with lipids producing more free radicals and subsequently resulting in des— tructive biochemical changes described as lipid peroxidation (Barber and Bernheim, 1967). The effectiveness of bypyridylium compounds as herbi- cides has been shown to depend on light and oxygen (Mees, 1960). Dodge, at al. (1970) demonstrated lipid peroxidation in plant membranes result- ing from the toxic effect of paraquat. Recently, pulse radiolysis studies have confirmed the existence of free radicals in plants follow- ing paraquat, which could account for the resulting lipid peroxidation (Farrington, et al., 1973). A similar mechanism of action might account for paraquat toxicity in mammalian systems. Free radicals have been demonstrated in broken- cell rat liver preparations with the addition of paraquat or diquat (Gage, 1968). More recently, Bus, at al. (1974) have shown that para- quat is reduced by mouse lung microsomes when incubated anaerobically with NADPH by the action of NADPH cytochrome c reductase. Reduced para- quat apparently reacts with oxygen to form superoxide anion which may non-enzymatically dismutate to singlet oxygen which initiates lipid per- oxidation. As predicted, reduced paraquat increases the in vitra per— oxidation of rat liver microsomal lipid (Bus, et aZ., 1974). Thus, the toxic effect of paraquat in animals may occur by superoxide and singlet oxygen induced lipid peroxidation. Specifically, the chain of events following paraquat poisoning resulting in toxicity might be as follows. Paraquat is first reduced in various tissues, then reoxidized by oxygen generating free radicals. These highly reactive compounds induce 5 membrane destruction via lipid peroxidation, resulting in impaired organ function and later morphological changes. Paraquat-induced lipid peroxidation has not been demonstrated in viva. However, there is indirect in viva evidence to suggest paraquat toxicity is mediated via lipid peroxidation. Vitamin E and selenium are known to protect against lipid peroxidation in viva. Mice placed on vitamin E and selenium-deficient diets were more susceptible to paraquat toxicity, in that mice on these deficient diets had a lower paraquat LD50 value compared to mice on control diets (Bus, at aZ., 1975b). Superoxide dismutase is an enzyme known to detoxify superoxide radicals and thus prevents superoxide and single oxygen induced lipid peroxida- tion. Autor (1974) has shown that superoxide dismutase administered i.v. reduced paraquat mortality in rats. Fisher, et a1. (1973) have shown enhanced paraquat toxicity in rats during oxygen treatment and Rhodes (1974) has shown that hypoxia protects against paraquat poison- ing. Thus, several lines of evidence suggest that paraquat toxicity may result from lipid peroxidation. Renal Elimination of Paraquat Elimination of paraquat from the body occurs via the kidneys with little or no biliary excretion of the compound (Daniel and Gage, 1966). Sharp, et a1. (1972) observed that the elimination of paraquat from the plasma was biphasic. Murray and Gibson (1974) likewise reported that the disappearance of paraquat from plasma in several species was charac- terized by an initial rapid component followed by a period of prolonged excretion lasting several days. Ferguson (1971) studied the renal han- dling of paraquat in anesthetized dogs following an i.v. infusion of the drug. He suggested that paraquat was filtered at the glomerulus, then 6 35 to 65% of the filtered load was passively reabsorbed in the proximal portion of the nephron. Based on the observations that the renal excre— tion of paraquat was independent of the plasma concentration and that excretion was closely related to urine flow, he concluded that an active component of paraquat secretion was unlikely. In the oxidized form, paraquat is ionic in nature with a plus 2 charge. Due to the ionic na- ture of paraquat, it seems unlikely that paraquat is passively reab- sorbed. Structurally, paraquat is similar to other bases known to be actively secreted into the urine (Peters, 1960). Thus, the renal excre- tion of paraquat may involve an active secretory component in addition to glomerular filtration. An active secretory component is consistent with the initial rapid disappearance of paraquat from plasma and the high renal concentration of paraquat compared to plasma and other tis- sues (Bus, et aZ., 1975a). Nephrotoxic Effects of.Paraquat The ingestion of paraquat by man has typically resulted in signs of renal functional impairment and histological changes in the proximal tu- bule of the kidney (Bullivant, 1966 and Beebeejaum, at al., 1971). Clin- ical symptoms of kidney damage in man following paraquat poisoning in- clude elevated plasma levels of blood urea nitrogen and creatinine, pro- teinuria, oliguria and altered serum electrolytes. Proximal renal tubu- lar necrosis was also reported in mice chronically poisoned with para- quat (Fowler and Brooks, 1971). In mice, paraquat administration in- duced proximal tubular necrosis, increased amount of smooth endoplasmic reticulum and produced a number of large lamellate cytosomes, presumably filled with a lipid material. The histological picture was similar in several respects to that seen following the administration of other 7 nephrotoxic compounds like carbon tetrachloride, phenacetin and cepha- loredine. Morfamquat, another bypyridylium herbicide, apparently also induces similar changes (Balogh and Mark, 1973). Since the primary route for paraquat elimination is via the kidneys, perhaps the nephrotoxic properties of paraquat impair renal function re- sulting in the retention of the poison. This might partially explain the biphasic nature of paraquat elimination. Sharp, et a1. (1972) show- ed a relationship between paraquat toxicity and the extended exposure to low plasma and tissue concentrations of the chemical. These observa- tions suggest that effective treatment of paraquat poisoning may require removal of the toxin from the body. The toxic properties of the com- pound may interfere with its own excretion enhancing toxicity in the lungs and other organs. Renal Secretion of Organic Compounds The elimination of a considerable number of organic compounds oc- curs via the urine by active energy-requiring processes located in the proximal tubule of the kidney, as well as by the passive process of glo— merular filtration. These transport processes have been studied exten- sively and two specific transport mechanisms have been described. The tubular excretion of compounds like PAH and PSP occurs via a transport mechanism specific for several organic acids. Several organic bases in- cluding various amines and quaternary ammonium compounds, like hexameth- onium and NMN, are excreted by a second mechanism which apparently acts independent from organic acid excretion (Peters, 1960). In Vitra Slice Technique The secretory mechanism for organic compounds can be studied util— izing both in vitra and in viva techniques. One commonly used in vitra 8 technique is the cortical slice method developed by Cross and Taggart (1950). In this technique, thin renal cortical slices are prepared free- hand and incubated in a physiological medium containing dilute concentra- tions of the drug or drugs to be studied. Accumulation of a compound against a concentration gradient is used as an indication of active transport. Results are expressed as the slice/medium.(S/M) concentra- tion ratio. Results obtained in vitra utilizing this technique corre- late well with results obtained in viva. Foulkes and Miller (1959) have shown that the maximum capacity of rabbit kidney cortex to accumulate PAH is similar to the concentrating capacity of rabbit renal cortical slices. Park, et al. (1971) studied various competitive inhibitors of PAH transport utilizing a kinetic model for studying organic acid trans— port in vitra. Compounds which inhibit PAH transport in vitra are often effective inhibitors of PAH secretion in viva. Other inhibitors of PAH transport, for example, dinitrophenol which uncouples oxidate phosphory- lation, inhibit PAH transport both in vitra and in viva. The slice technique employing mouse renal cortical slices has been used to charac- terize the renal handling of compounds similar in structure to paraquat. Holm (1970) observed that mouse renal cortical slices accumulate the or- ganic base, hexamethonium. The uptake of hexamethonium by cortical sli- ces could be blocked by sodium azide and dinitrophenol (McIssac, 1965) and by incubating slices under nitrogen (Holm, 1970), suggesting an ac- tive energy-requiring component in the uptake process. Tetraethylammon- ium and decamethonium antagonized the renal accumulation of hexamethon- ium in vitra presumably by competitive inhibition of the organic base transport process (McIssac, 1965). Due to the structural similarity be- tween paraquat and hexamethonium, it seemed likely that paraquat might 9 be accumulated by mouse renal cortical slices by the same active energy— requiring process. The in vitra slice technique can also be used to assess the nephro- toxic potential of various compounds. Watrous and Plaa (1972) injected mice and rats with halogenated hydrocarbons, then measured the ability of renal cortical slices to actively accumulate organic ions. Hirsch (1973) has performed these and other renal functional tests following the administration of various nephrotoxic agents. Possibly paraquat is nephrotoxic in the mouse and this toxicity can be demonstrated utiliz- ing the in vitra slice technique. In Viva Measurements of Renal Function Many techniques are available for determining various aspects of renal function in viva. Most of these techniques require extensive ex- perimental procedures which are typically best performed on large ex- perimental animals. For example, the classical clearance technique for determining renal blood flow and/or glomerular filtration requires can- nulation of arteries and veins, catheterization of the ureters or blad- der and the maintenance of relatively constant plasma concentrations by infusing drugs. To assess the nephrotoxic potential of paraquat in the mouse, it is important to determine renal function both in vitra and in viva. Thus, it was necessary to employ in viva techniques which could be applied to mice in order to determine the effect of paraquat poison- ing on renal function. Sakai, at al. (1969) have used a simplified clearance technique in children. The rate of glomerular filtration was estimated by determin- ing the disappearance of iothalamate (1251) from plasma following a single i.v. injection of the compound. There was good correlation 10 between this technique and the classical clearance technique. Utilizing a similar approach, Silkalns, at al. (1973) have accurately estimated renal plasma flow by determing the disappearance of PAH from plasma. It was of interest to determine if paraquat poisoning might alter renal function in the mouse as indicated by a change in the plasma disappear- ance of a compound known to be excreted via the kidney. Purpose The basic thesis underscoring this research is that a rational ther- apeutic program for paraquat poisoning is contingent on a better under- standing of the renal handling and nephrotoxicity of paraquat. The re— nal handling of paraquat may be similar to the excretion of other bases which are known to be handled by the organic base secretory system of the kidney. The nephrotoxic effects of paraquat may be related to the relatively high concentrations of paraquat found in the kidney, which subsequently might impair renal function, leading to paraquat retention and more profound toxicity in other organs in the body. METHODS In Vitra Determination of Renal Function 1. Description of the Slice Technique Female Swiss-Webster mice1 weighing 25-35g were stunned by a blow on the head and the kidneys quickly removed and placed in ice- cold saline. Thin renal cortical slices were prepared free-hand and placed in beakers containing 2.0 m1 of Ringer solution contain- ing 10 mM Na acetate and the drug(s) to be studied. Beakers were incubated in a Dubnoff-metabolic shaker at 25° or 37°C under a gas phase of 100% oxygen except for the few experiments when a gas phase of 100% nitrogen was employed. The duration of incubation and con- centration of drug(s) in the medium varied with the experiment being conducted. Following incubation, slices were removed from the med- ium, blotted on gauze and weighed. Tissue and medium were treated as outlined by Cross and Taggart (1950). Concentrations of PAHZ, NMN3 and paraquat4 were determined using 14C—labeled compounds ex- cept for the few experiments where PAH concentrations were deter— mined by the spectrophotometric method of Smith at al. (1945) fol- lowing acid hydrolysis of samples to ensure recovery of the PAH. Radioactivity was determined by placing a 0.5 m1 aliquot into a vial containing 10 ml PCSR-solubilizer and the disintegrations per 1Spartan Research Animals, Inc., Haslett, Michigan. 2New England Nuclear, Boston, Massachusetts. 3New England Nuclear, Boston, Massachusetts. 4Amersham/Searle Corporation, Arlington Heights, Illinois. 11 12 minute (dpm) in each sample were determined using a Packard model 3380 liquid scintillation spectrometer. Data concerning accumula- tion of drugs were expressed as the slice to medium (S/M) ratio, calculated by dividing the concentration of drug per g of tissue by the concentration of drug per ml of incubation medium, or, in the case of radio-labeled compounds, by dividing dpm per g of tis- sue by dpm per m1 of medium. 2. Effect of Paraquat on NMN and PAH Accumulation Accumulation of PAH and NMN by slices incubated for 90 min was 5M PAH and 6.0 x 10-4M NMN. determined in medium containing 7.4 x 10- The effect of various concentrations of paraquat on PAH and NMN ac- cumulation was determined at 25° and 37°C. 3. Paraquat Accumulation Accumulation of paraquat by renal cortical slices was deter- mined after 30, 90 and 180 minutes of incubation at 25°C. Para- quat accumulation was also determined at 37°C and compared to ac- cumulation of paraquat by slices incubated under nitrogen. 4. Effect of Inhibitors on Paraquat Accumulation Slices were incubated for 180 minutes at 25°C in the presence of 10-5M paraquat. The inhibitors cyanine dye #863, 2,4-dinitro- phenol and iodoacetic acid were present at final medium concentra- tions of 5 ug/ml, 10—4M and 10-3M respectively. 5. Uptake of NMN Uptake of NMN into slices was determined following pre—incuba- tion of slices for 30 min. 14C-Labeled NMN was added to the medium to produce final concentrations of 0.75, 1.5, 3.0 and 6.0 x 10-4M. l3 Slices were assayed for l4C-NMN following 30 min incubation in the presence of the drug. Results were expressed as pg NMN per g tis- sue per min. 6. Determination of Slice O2 Consumption Oxygen consumption of slices was measured using a Yellow Springs model 53 oxygen monitor employing a Clark-type polarographic elec- trode. Stirring rate of the sample was slowed in order to minimize tissue damage. The instrument was calibrated before each determina- tion with 4 ml of bathing medium saturated with oxygen at either 25 or 37°C. Slices were weighed and added in a volume of 2 ml to 4 m1 of saturated medium. Oxygen consumption was then measured over a 10 min period. Data were expressed as ul of O consumed per mg tis— 2 sue o 7. Slice Function as an Indicator of Nephrotoxicity Renal function was determined in vitra following acute and chronic paraquat poisoning. The ability of renal tissue to accumu— late PAH and NMN was measured in renal cortical slices prepared from various treated and control animals. In chronic toxicity studies, pregnant mice were obtained from timed pregnancies started in this laboratory. Paraquat was added to the drinking water supply of preg- nant mice at 50 ppm beginning on day 8 of gestation. Animals were placed in individual cages on day 19 and allowed to litter. Paraquat in the drinking water was continued. At weaning, the litter was sep- arated from the mother and maintained on paraquat water until the ex- perimental age (7 weeks). In acute toxicity studies, adult female mice were treated i.p. with an LD50 (7 day) dose of paraquat (30 mg/kg) l4 and renal function determined in vitra at 1, 3, 5 and 7 days following the paraquat poisoning. 1% Viva Determination of Renal Function 1. Disappearance of Paraquat from Plasma Mice were anesthetized with ether and bilaterally nephrectomized or sham-operated using the dorsal approach (Becker and Gibson, 1967). Paraquat (50 mg/kg) was injected via the tail vein 16 hr following sur- gery. The concentration of paraquat in the plasma was determined at 5, 10, 15, 20 and 30 minutes using individual mice for each time point. Blood was collected by cardiac puncture under ether anesthesia. Plasma was separated from blood by centrifugation. The concentration of para- quat in the plasma was determined colorimetrically following the addi— tion of 0.5 m1 of 2% Na dithionite in 1N NaOH to 2.5 m1 of a 1:25 water dilution of plasma. Optical density was measured at 395 nm. The addi- tion of plasma did not alter the standard curve for paraquat. In other experiments, the effect of paraquat poisoning on the abil- ity of the kidney to excrete a second paraquat dose was determined. At 2 and 24 hr following paraquat poisoning (30 mg/kg i.p.; 7 day LD50), mice were challenged with 50 mg/kg paraquat i.v. and the plasma disap- pearance of paraquat determined as described above. Control mice were treated with an equal volume of water (5 m1/kg i.p.) 24 hr prior to the i.v. injection of paraquat. 2. Disappearance of PSP from Plasma The disappearance of phenolsulfonphthalein (PSP) from plasma, 50 mg/kg i.v., was determined in a similar manner 24 hr following para— quat poisoning (LD50 dose) or a water injection. 15 3. Disappearance of Inulin from Plasma In control and paraquat-poisoned mice, the disappearance of inulin from plasma was also determined. Twenty-four hr following an LD50 dose of paraquat or a water injection, 3H-(methoxy)-inulin5 (503.6 mCi/g specific activity) was injected i.v. (25 uCi/kg) and the plasma concentration of inulin was determined at various times. Inulin concentrations were determined by dissolving 100 pl of plasma in 1 ml of Soluene 100R. Toluene—counting solution (15 ml) was added and radioactivity determined employing a Packard model 3380 liquid scintillation Spectrometer. 4. Disappearance of Iothalamate from Plasma The disappearance of 1251 iothalamate6 from plasma was deter- mined in mice 24 hr following an LD50 dose of paraquat or a water in- jection. Iothalamate (229 uCi/ml specific activity) was administered i.v. in a dose of 0.03 mg/kg and radioactivity in the plasma was de- termined 5, 10, 20, 30 and 60 minutes later. A similar experiment was conducted utilizing longer time intervals (30, 60, 90 and 180 min) and a higher dose of iothalmate (1.5 mg/kg). Radioactivity in the plasma was determined by counting 0.1 ml of a plasma in a well- type crystal scintillation counter. Statistical Analyses Statistical analyses were performed using analysis of variance followed by Student-Newman-Keuls test for the difference between means 5New England Nuclear, Boston, Massachusetts. 6Glofil-125R, Abbott Laboratories, North Chicago, Illinois. l6 (Sokal and Rohlf, 1969). Regression lines were determined by the method of least squares and slopes were compared using Student's "t" test (Steel and Torrie, 1960). The 0.05 level of probability was used as the cri- terion of significance. RESULTS In Vitra Analysis of the Renal Handling of Paraquat 1. Paraquat Accumulation by Mouse Renal Cortical Slices Slices prepared from mouse renal cortical tissue and incubated under oxygen at 25°C, accumulated 14C-labeled paraquat, achieving S/M ratios significantly greater than 1 (Fig. 2). Incubation of slices under nitrogen at 37°C produced paraquat S/M ratios not significantly different than 1 (Fig. 3). The magnitude of aerobic accumulation was related to the duration of incubation and the concentration of para- quat in the medium, the most dilute concentration of the herbicide yielding the greatest S/M ratios (Fig. 2). In addition, the magni- tude of accumulation under oxygen was related to the incubation temr perature with higher S/M ratios observed at 37° (Fig. 3) than 25°C (Fig. 2) at comparable medium concentrations. Various metabolic in- hibitors and cyanine dye #863 significantly reduced the accumulation of paraquat (Table 1). 2. Effect of Paraquat on NMN and PAH Accumulation Accumulation of l4C-NMN by cortical slices incubated at 25°C was depressed by paraquat in a dose-related fashion (Fig. 4). In contrast, accumulation of PAH was not inhibited by 10-4 or 10-3M paraquat. The inhibitory effect of paraquat was temperature-depen- dent (Table 2). At 25°C, both 10-4 and lO-BM concentrations of paraquat inhibited NMN accumulation without affecting PAH accumula- tion. However, at 37°C, 10-3M paraquat inhibited accumulation of both PAH and NMN. 17 18 3. Effect of Paraquat on NMN Uptake The effect of paraquat on the uptake component of 14C-NMN ac- cumulation by cortical slices was estimated. The initial rate of NMN uptake was determined at 25° and 37°C. The uptake of NMN was directly related to the concentration of NMN present in the medium (Fig. 5 and 6). NMN uptake under oxygen was greater at 37°C than at 25°C, whereas uptake under nitrogen was similar at the two incubation temperatures. At both 25° and 37°C, uptake of NMN under oxygen was depressed by 10-3M paraquat. Incubation of slices under nitrogen further depressed NMN uptake. The uptake of NMN under nitrogen at 37°C was not influenced by the presence of paraquat (Fig. 5), sug- gesting that the depressant effect of paraquat on NMN uptake was on the oxygen-dependent component of the uptake process. 4. Effect of Paraquat on Slice Oxygen Consumption Slice oxygen consumption at 37°C was not altered by preincuba- tion of slices at 25°C in the presence of paraquat (Table 3). How- ever, slice oxygen consumption determined at the same temperature was depressed by lO-BM paraquat when slices were preincubated at 37°C for 60-90 minutes. Nephrotoxicity of Paraquat in Mice 1. Slice Function as an Indicator of Nephrotoxicity The effect of paraquat administration on in vitra estimates of renal function was determined in two experimental protocols. In the first group, paraquat was administered chronically, beginning pre- natally and extending until 7 weeks of age. In this treatment regi- men, 50 ppm of paraquat in the drinking water had no significant l9 effect on accumulation of PAH and NMN by renal cortical slices. An interesting sidelight of this protocol was the observation that PAH accumulation was greater in renal cortical slices from male than fe- male mice (Table 1). In the second protocol, the accumulation of PAH and NMN was determined in renal cortical slices at 1, 3, 5 and 7 days following the 7-day LD50 dose of paraquat (Table 2). Mice surviving the acute paraquat poisoning weighed significantly less than control mice at 1 and 3 days but this difference was not evi- dent at 5 and 7 days following the poisoning (Table 3). One day following the LD50 dose of paraquat, PAH accumulation was signifi- cantly less than that in controls (Table 2). This difference was not maintained however, for at 3, 5 and 7 days there was no signi- ficant difference in accumulation of PAH or NMN by tissue from in— toxicated animals. 2. Effect of Nephrectomy on the Disappearance of Paraquat from Plasma The role of the kidney in elimination of paraquat from the plasma of intact mice was determined by comparing disappearance of the herbicide from the plasma in nephrectomized animals to the dis- appearance observed from plasma of sham-operated animals. Disap- pearance of paraquat from the plasma of sham-operated mice was rapid with an approximate half time of 7.0 minutes. The disappearance ap- peared to follow first-order kinetics over the time interval tested (Fig. 7). Following bilateral nephrectomy, however, mice were unable to significantly reduce the plasma concentration of the herbicide (Fig. 7). 20 3. Effect of Paraquat Poisoning on the Disappearance of Paraquat from Plasma The effect of an LD50 dose of paraquat on renal function was estimated by measuring the disappearance of the herbicide from the plasma 2 and 24 hrs following the toxic dose. Two hours following a toxic dose of paraquat, the disappearance of a radiolabeled dose of the herbicide appeared to be somewhat depressed though this ef- fect was not statistically significant. However, by 24 hrs the abil- ity of the poisoned animals to eliminate the herbicide was signifi- cantly depressed (Fig. 8). Whereas, in the control animals the con— centration of herbicide was reduced to less than 2 mg/100 ml of plasma within 30 min, in the intoxicated animals the concentration at 30 minutes was not significantly different than that observed at 5 minutes, i.e., the slope of the disappearance curve was not signi- ficantly different than zero. 4. Effect of Paraquat Poisoning on the Disappearance of PSP from Plasma Pretreatment with the LD50 dose of paraquat also significantly retarded the elimination of PSP from the plasma (Fig. 9). Thirty minutes after an injection of PSP, the plasma concentration in the treated animals was approximately 4 times greater than that in con- trols, but the estimated volume of distribution was no different (Fig. 9). 5. Effect of Paraquat Poisoning on the Disappearance of Inulin from Plasma The slope of the plasma disappearance curve for 3H-methoxyinulin was not altered by paraquat treatment (Fig. 10). However, at each 21 time measured following inulin administration, the plasma concentra- tion of inulin was significantly higher in the paraquat-treated mice. The disappearance of inulin over this 30-minute interval was very rapid (t8 = 8.5 min). 6. Effect of Paraquat Poisoning on the Disappearance of Iothala- mate from Plasma The disappearance of iothalamate from plasma was determined be- tween 5 and 60 min following administration, and in a second group of animals, between 30 and 120 min following administration. The disappearance of iothalamate appeared to be biphasic in that there was a rapid decrease in plasma concentration followed by a more pro- longed period of disappearance from the plasma (Fig. 11). This pat- tern was changed somewhat by pretreatment with an LD50 dose of para- quat in that the initial rapid fall in plasma concentration was not observed. Consequently, higher concentrations of iothalamate were observed at each time measured in the plasma of paraquat-poisoned animals. Between 30 and 120 min, the slope of the disappearance was not influenced by paraquat administration. DISCUSSION Organic bases like NMN and tetraethylammonium are actively accumulated by renal cortical tissue in viva and then excreted into the urine. This active accumulation of drug by renal cortical tissue is a process necessary for the phenomenon of tubular secretion (Peters, 1960). The in vitra accumulation of drugs by renal cortical slices correlates well with the in viva process and was chosen as a model for characterizing paraquat transport in this study (Ross, et aZ., 1959). The structural similarity between paraquat and other organic bases like hexamethonium and decamethonium suggested that the herbi- cide may be transported by the organic base transport system (McIssac, 1969 and Holm, 1970). Indeed, paraquat was accumulated by mouse re- nal cortical slices (Fig. 2 and 3). This process appears to require cellular energy in that accumulation was depressed by incubation of slices under nitrogen or in the presence of the metabolic inhibitors iodoacetic acid and dinitrophenol (Table l). Cyanine dye #863 at low doses blocks the uptake of organic bases by slices, apparently by competing for the organic base transport system (Farah, et aZ., 1959). Depression of paraquat accumulation by cyanine #863 could reflect competition between paraquat and the dye for uptake by the base transport system. In order to demonstrate the specificity of paraquat for this transport system, accumulation of PAH and NMN were determined in the presence of various concentra- tions of paraquat. As predicted from the structure of paraquat, NMN S/M ratios were selectively depressed whereas organic acid transport (PAH) was not affected (Fig. 4). 22 23 An S/M ratio does’not reflect only active uptake of a drug by a transport system, but rather reflects net intracellular accumulation and the respective rates of influx and efflux of the compound studied (Ross, et aZ., 1968). Perhaps depression of the NMN S/M ratio by para- quat resulted from more than a simple competition between the two com- pounds for a common transport system. To better characterize the in- teraction between paraquat and a second base for active transport, the effect of paraquat on the uptake component of NMN accumulation was de— termined. Paraquat depressed NMN uptake under oxygen at both incuba- tion temperatures (Fig. 5 and 6). At 37°C, uptake under oxygen was greater than that observed at 25°C, although paraquat inhibition of NMN uptake was similar at both temperatures. Uptake under nitrogen was less than that observed under oxygen and was not influenced by in- cubation temperature or by paraquat. Thus, paraquat inhibition of NMN accumulation (S/M ratio) is apparently specific for the oxygen-requir- ing component of NMN uptake. To evaluate the possibility that a portion of paraquat inhibi- tion of NMN accumulation was secondary to depression of slice metabo- lism, the effect of paraquat on total slice oxygen consumption was determined. Incubation of slices at 25°C in the presence of para- quat did not alter slice oxygen consumption, supporting the conten- tion that paraquat inhibits organic base transport by competition for a common transport mechanism (Table 3). However, at 37°C, 10-3M para- quat depressed slice oxygen consumption. Subsequent to this observa- tion, the effect of paraquat on NMN and PAH accumulation was deter— mined at 25° and 37°C (Table 2). In contrast to 25°C, at 37°C PAH accumulation and slice oxygen consumption was depressed by the herb- icide. This suggests that at 37°C paraquat may competitively inhibit 24 NMN transport and, in addition, produce a general depression in slice function. Bus, et a1. (1975) have demonstrated that paraquat can un— dergo reduction when incubated with lung microsomes in the presence of NADPH and that the reduced paraquat formed reacts with oxygen to form superoxide and singlet oxygen which initiates lipid peroxida- tion. Perhaps the general depression of slice function under certain conditions is a consequence of paraquat-induced lipid peroxidation. Alternatively, paraquat might interfere with other processes essen- tial for slice function. The in vitra evidence for active transport presented here, in addition to the rapid disappearance of the poison from plasma observed in viva (Fig. 7), suggests that paraquat is actively secreted into the urine via the organic base secretory system. The plasma disap- pearance of paraquat from plasma appears to be biphasic, with an ini- tial component, followed by a phase of prolonged elimination (Sharp, at aZ., 1972). The present results suggest active transport may be involved in the initial rapid removal of drug from plasma. However, the data fail to explain the prolonged period of paraquat excretion. Perhaps other tissues initially accumulate paraquat then slowly re- lease the compound. Or perhaps paraquat is tightly bound to a frac- tion of plasma. Furthermore, the nephrotoxic properties of paraquat may result in a diminished capacity of the kidney to excrete the poi- son. Sharp, at al. (1972) reported a correlation between paraquat concentrations in the lung and kidney and the toxicity of the drug. A high concentration of drug in these organs may result from a com- promised capacity of the kidney to excrete the poison. Apparently paraquat excretion and the nephrotoxic properties of the compound are 25 related in that paraquat poisoning altered the excretion of a second paraquat dose administered 24 but not 2 hrs later (Fig. 8). Thus, it appears that the nephrotoxic properties of the herbicide are not expressed initially when the kidney concentrations are greatest, but rather are observed later when most of the poison has been removed from the body. The renal functional impairment produced by paraquat was not limited to organic base excretion since elimination of the organic acid, PSP, was likewise affected (Fig. 9). Perhaps the nephrotoxic effects of paraquat are due to a direct effect on renal cortical tissue, impair- ing function in general. When renal function was examined in viva, only a small change in PAH transport was noted (Table 5). Similarly, renal function measured in vitra was not affected following chronic paraquat poisoning (Table 4). Apparently, the effects of paraquat on renal function are best detected in viva. The in vitra slice tech— nique estimates only one step in drug excretory processes which occur in viva and thus may lack the capacity or sensitivity to quantitate changes in function that occur in viva. For instance, the slice tech- nique measures steady-state accumulation; possibly the effect of para— quat is overcome in the steady-state and can only be measured in the dynamic state that occurs in the intact animal. Alternatively, para- quat poisoning may affect parameters other than the transport process. For example, paraquat poisoning may decrease renal blood flow and in— directly depress renal excretion of drugs. The disappearance of inulin may be used to approximate glomerular filtration rate. Therefore, the effect of paraquat on the disappear— ance of inulin from plasma was determined in order to estimate changes in renal hemodynamics subsequent to herbicide intoxication. The initial 26 rate of disappearance of inulin from the plasma was not affected by paraquat pretreatment (Fig. 10). However, at each time point measured, the concentration of inulin in the plasma was higher in the paraquat- poisoned animals, suggesting a smaller volume of distribution for inu- lin following paraquat. A diminished volume of distribution is consis- tent with the marked loss in body weight noted 24 hrs following paraquat administration (Table 6). The disappearance of inulin over the 30~min interval measured was too rapid to reflect glomerular filtration in that the plasma half life for inulin was not different than that of PSP. Thus, the possibility existed that disappearance of inulin from plasma was not an adequate estimate of glomerular filtration rate in the mouse. The disappearance of iothalamate, like inulin, may also be used to approxi- mate glomerular filtration (Sigman, at al., 1965). Iothalamate elimin- ation was biphasic with an initial rapid disappearance from plasma fol- lowed by a period of slower elimination (Fig. 11). Following paraquat, plasma concentration of iothalamate was higher at each time tested, pos- sibly reflecting a smaller volume of distribution of the drug. Paraquat poisoning failed to alter the slope of the disappearance curve for io— thalamate between 30 and 120 min, suggesting that depression of blood flow to the kidney cannot account for the diminished secretory capacity in paraquat-poisoned animals. The curves for PSP and paraquat disappearance from plasma follow- ing paraquat administration lack the change in the apparent volume of distribution that was evident with inulin and iothalamate. This dis— crepancy probably lies in the difference of binding of these agents to plasma proteins. Paraquat, like PSP, may be tightly bound to plasma proteins in contrast to iothalamate and inulin, which are not. Thus, a paraquat-induced decrease in plasma volume would lead to an apparent 27 decrease in the volume of distribution for iothalamate and inulin. How- ever, assuming that the amount of plasma protein would not be changed by paraquat, the apparent volumes of distribution of these highly protein- bound components would not be altered. Since paraquat is accumulated by renal cortical tissue, this organ is likely to be more sensitive to the toxic effects of paraquat compared to other organs which do not accumulate the drug. In addition, since the kidney, like the lung, has a high oxygen tension relative to other organs, it seems reasonable that the kidney may be predisposed to para- quat-induced lipid peroxidation. If the nephrotoxic effects of paraquat could be prevented, then the elimination of paraquat via the kidney might be facilitated, decreasing the potential toxicity to the kidney and other organs. SUMMARY Paraquat, a bypyridylium herbicide, has caused numerous poison- ing in man and farm animals since its introduction. Elimination of paraquat from the body occurs via the kidneys with little or no bil- iary excretion of the compound (Daniel and Gage, 1966). Paraquat is accumulated by mouse renal cortical slices by an active energy-requir- ing process. Accumulation of paraquat apparently occurs by the or- ganic base secretory system of the kidney. Thus, the renal excre- tion of paraquat likely involves an active secretory component in addition to glomerular filtration. Under certain conditions, paraquat has an apparently toxic ef- fect on cortical slice function. This toxic effect appears to be concentration and temperature-dependent. The nephrotoxic potential of paraquat was evaluated by deter- mining renal function both in vitra and in viva in mice acutely and chronically poisoned with paraquat. Slices prepared from.mice chronically poisoned with paraquat (50 ppm of paraquat in the drinking water) were not different from control slices in their ability to accumulate PAH or NMN. Similarly, renal cortical slices prepared from mice acutely poisoned with para- quat (LD50 - 7 day) were similar to control slices in their ability to accumulate PAH or NMN. In contrast, paraquat poisoning produced a marked effect on renal function when renal function was assessed in viva. Disappearance of PSP and paraquat from plasma was signifi- cantly reduced in paraquat-poisoned mice (LD50 - 7 day), suggesting a depression of tubular function. In contrast, the rate of disap- pearance of iothalamate was not affected by paraquat poisoning, 28 29 indicating that the depression of tubular function was not secondary to a change in glomerular filtration. The renal excretion of paraquat apparently involves an active secretory process. In addition, paraquat appears to interfere with renal function in the proximal tubule of the kidney. Since secretion of paraquat from the body involves accumulation of the herbicide within the proximal tubule cells, it is not surprising that paraquat interferes with the function of these same cells. Furthermore, it follows that should toxic concentrations of paraquat be reached in the kidney, subsequent impairment of renal function would impede elimination of the herbicide, leading to more profound toxicity in organs other than the kidney. 30 TABLE 1 Effect of inhibitors of organic base transport on paraquat accumulation (S/Mg by mouse renal cortical slices Inhibitor Paraquat S/Mb Control 2.39 Cyanine Dye #863 1.33c 2,4—Dinitrophenol 1.09C Iodoacetic Acid 0.92c aSlices were prepared and incubated at 25°C in the presence of 10-5M paraquat for 180 minutes. Cyanine dye #863, 2,4-dinitrophenol and iodoacetic agid were pgesent at final medium concentrations of 5 ug/ml, 10 M and 10 M respectively. Values represent the mean of 4 experiments. bCoefficient of variability was 9%. cSignificantly different from control (p < 0.05). 31 TABLE 2 Effect of paraquat on PAH and NMN accumulation (S/M) by mouse renal cortical slices PAH S/M Ratio NMN S/M Ratio Pazaquat 0 10-4M 10'3M (c.v.)b 0 10’4M 10’3M (c.v.)b 011C 0 25°C 7.92 8.84 8.41 (23%) 18.02 13.15 7.47 ( 8%) 37°C 7.82 6.93 3.49 (19%) 8.00 5.78 3.35 (12%) aSlices were prepared and incubated at 25° or 37°C with paraquat for 90 minutes. Values represent the mean of 4 experiments. Any values underscored by the same line were not significantly different. bCoefficient of variability. 32 TABLE 3 Effect of paraquat on oxygen consumption by mouse renal cortical slices Paraquat Concentration Preincubation 0 Determination 0 10-4M 10-3M (C.V.)b Temperature Temperature 25°C 37°C .077 .078 .086 (15%) 37°C 37°C .068 .062 .048 (13%) aSlices were prepared and incubated with paraquat for 60 to 90 min- utes, then oxygen consumption (pl/mg tissue/min) was determined for a 6 to 10 minute period. Values represent the mean of 4 ex- periments. Any values underscored by the same line were not sig- nificantly different. bCoefficient of variability. 33 TABLE 4 Accumulation of NMN and PAH by renal cortical slicesa prepared from mice chronically poisoned with paraquat Incubation Time 90 180 S/M Ratio PAH NMN PAH NMN Sex Male Female Male Female Male Female Male Female Control 11.5 5.7 20.2 18 7 28.2 12.0 29.4 28.4 10.6 10.6 10.9 11 1 15.7 10.5 13.3 12.2 Treated 12.5 6.8 19.4 16.8 23.7 12.4 37 5 27.7 11.4 11.0 10 5 12.2 10.7 11.8 11.8 11.9 aPregnant mice were given 50 ppm paraquat in drinking water beginning on day 8 of gestation. At weaning the litter was separated from the mother and maintained on paraquat water until the experimental age at 7 weeks. Values represent the mean 1 S.E. for 4 determinations. 34 TABLE 5 Accumulation of PAH and NMN by renal cortical slicgs prepared from mice acutely poisoned with paraquat S/M Ratio PAH NMN Days Following LD50 Dose 1 3 5 7 1 3 5 7 Control 9.7 9.5 7.3 7.1 14.4 16.9 13.4 12.6 10.8 11.4 11.1 11.4 10.9 12.6 12.1 10.6 Treated 6.3b 7.4 6.4 10.3 14.2 10.6 10.2 11.9 11.1 11.2 11.3 11.5 12.7 11.9 11.9 11.3 aAdult female mice were treated i.p. with an LD50 dose of para- quat (30 mg/kg). Values represent the mean 1 S.E. for 4 experi- ments. bSignificantly different from control (p < .05). 35 TABLE 6 Body and kidney weight of mice acutely poisoned with paraquat Body Weight (g) Kidney Weight (g) Days Following LD50 Dose 1 3 5 7 1 3 5 7 Control 32 31 31 30 0.39 0.39 0.40 0.39 +1 +1 11 +1 10.01 10.01 10.01 10.02 Treated 27b 26b 30 32 0.38 0.37 0.39 0.41 11 11 11 11 10.01 10.02 10.02 10.01 3Adult female mice were treated i.p. with an LD50 dose of paraquat (30 mg/kg). Values represent the mean 1 S.E. for 12 animals. bSignificantly different from control (p < .05). 36 Figure 1: Structure of paraquat in its oxidized and reduced states. PARAGUAT (oxidized) — + (reduced) Figure l 38 Figure 2: Ezfect of medium concentration and duration of incubation on C-labeled paraquat accumulation (S/M ratio) by mouse renal cortical slices incubated under oxygen at 25°C. Bars repre- sent the mean 1 S.E.M. of three experiments. 39 N musmflm V2355 DE: cog—33:. 00 on "'llllllllllllllllllllllllll 3 III .. 909090 . .0 0000 O ’6’6’6.;.'. 1-2 I c3 «036010.. US l m 103 w/s smbmod OI 40 Figure 3: Accumulation (S/M ratio) of 14CZ§abe1ed paraquat at an ini- tial medium concentration of 10 M by mouse renal cortical slices incubated at 37°C under oxygen and nitrogen. Bars represent the mean 1 S.E.M. of three experiments. 41 om— m shaman 7.22%: V «E: 5:33... 00 m N 0908 W/S sonbmod 0‘) Figure 4: 42 Effect of paraquat on accumulation (S/M ratio) of PAH and NMN by mouse renal cortical slices incubated under oxygen at 25°C for 90 minutes. Points represent the mean 1 S.E.M. of three (NMN) or seven (PAH) experiments. The S.E.M. was within the diameter of the circle for the points represent- ing PAH S/M ratios. 43 92 a shaman As: co__2.coucou 53020.. To- »...0— 9.9 1-0— .2500 h m 2 Z .0 $5 13. 0 is 222 O 1 1 fi 1 a O O O 0908 W/ S Figure 5: 44 Effect of 10-3M paraquat on the rate of 14C-labeled NMN up- take by mouse renal cortical slices incubated under oxygen and nitrogen at 37°C. The clear area of each bar repre— sents the uptake of NMN observed under a nitrogen atmos- phere. The total area represents the uptake of NMN observed under oxygen. The difference between the oxygen and nitro- gen uptake, represented by the shaded area, reflects the oxygen-requiring component of NMN uptake. Bars represent the mean 1 S.E.M. of three experiments. 45 m musmflm 192x21 812.1818 222 6.0 O.” m.— mud . w 7 o l 0— II.QN mm W N 1 n will 1 on M. 00009 H “I am. II", M an”. In]... 1 3 .w 000. ,l W ”a a m. H. z I I ( H, . H I On Ill Aisha; .2.cou % 1 3 IL I 46 Figure 6: Effect of 10-3M paraquat on the rate of 14C-labeled NMN up- take by mouse renal cortical slices incubated under oxygen (shaded area) and nitrogen (clear area) at 25°C. Bars rep- resent the mean 1 S.E.M. of four experiments. 47 o muswfim 3-9 x 3 512.1850 2:: as 6.. db M .3230 «2 3:09.05 195.. NO .3230 NO OP ON Om (II'm/B/Od) 01pm“ NWN Figure 7: 48 Effect of bilateral nephrectomy or sham-operation on the disappearance of paraquat from plasma 16 hr later. Each point represents the plasma concentration of an individual animal following the i.v. injection of 50 mg/kg paraquat. Only the slope for the sham-operated group was significant (p < .05). ”noun CONCENTRATION (mg/100 ml mum) 49 PLASMA DISAPPEARANCE OF PARAGUAT 16 hrs. FOLLOWING NEPHRECTOHY r I I I I o o Sham o O Nophnctomlzod .1 ‘ I L J l 0 5 1O 15 20 30 TI as (minutes) Figure 7 Figure 8: 50 Effect of 30 mg/kg i.p. paraquat on the subsequent plasma disappearance of a second paraquat dose determined 2 and 24 hr later. The plasma concentration of paraquat was de- termined at various times following the second injection of paraquat, 50 mg/kg i.v. Each point represents the mean 1 S.E.M. of 3 or 4 animals. Control values are shown on both figures. Only the slopes of the 2 hr and control groups were significant. Slopes of the 2 hr and control groups were not significantly different (p < .05). 51 1 “NE \m\““‘°°\ Figure 8 Figure 9: 52 Effect of 30 mg/kg paraquat i.p. on the disappearance of phenosulfonphthalein (PSP) from plasma 24 hr later. The plasma concentration of PSP was determined at various times following the i.v. injection of 50 mg/kg PSP. Each point represents the mean 1 S.E.M. of 3 animals. Slopes of the plasma disappearance curves were significantly different (p < .05). PSP CONCENTRATION (mg/100ml pmm) 53 PLASMA DISAPPEARANCE OF PSP 24 hr FOLLOWING PARAGUAT ADMINISTRATION 20 _ —COntrOl -- Treated \ ‘\ 1 l L l I m o 5 10 15 20 so 'I’IME (mInum) Figure 9 54 Figure 10: Effect of 30 mg/kg paraquat i.p. on the disappearance of 3H- inulin from plasma 24 hr later. Each point represents the mean 1 S.E.M. of 3 animals. Slopes of the plasma disappear- ance curves were not different. INuLIN CONCENTRATION (pg/100m plum) 00 55 PLASMA DISAPPEARANCE OF INULIN 24 hr FOLLOWING PARAGUAT ADMINISTRATION — Control --- Ttootod I l J 10 15 20 TIME (mlnutoo) Figure 10 56 Figure 11: Eggect of 30 mg/kg paraquat i.p. on the disappearance of I iothalamate from plasma 24 hr later. Two separate ex- periments are presented. Closed circles and squares repre- sent the disappearance of iothalamate from plasma following a 0.03 mg/kg dose i.v. The right hand ordinate corresponds to the open circles and squares and represents the disap- pearance of iothalamate from plasma following a 1.5 mg/kg dose i.v. Each point represents the mean 1 S.E.M. of 3 ani- mals. Slopes of the plasma disappearance curves between 30 and 120 min (open circles and squares) were not different. 57 ° . ConIrol "OQDOO — 40000 -+2QDOO - “1000 a 0000 -*4000 - 2000 g fl “' I .— I D F——°———" I—o—I IhFfla—H m4 twin—I 19.. 1 41 I—H l I l 1 1 l — LOOO 5 "3 mo 30 §§§§ § 8 s (“"9" WOOL/W43) "°!I°JIIIOONO3 ogomnpqgo' ON 8 00 120 TIME (mimics) Figure 11 BIBLIOGRAPHY BIBLIOGRAPHY Autor, A.P.: Reduction of paraquat toxicity by superoxide dismu- tase. Life. Sci._14, 1309-1319, 1974. Balogh, K. and Merk, F.B.: Ultrastructure of renal collecting tubules following ingestion of a bipyridinium herbicide (Morfam— quat). Experientia 22, 1101-1103, 1973. 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