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I University w.— This is to certify that the thesis entitled Dietary Alteration of Paraquat Toxicity presented by William Davidson Evers has been accepted towards fulfillment of the requirements for OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: —_____________- Place in book return to remove charge from circulation records Wé DIETARY ALTERATION OF PARAQUAT TOXICITY By William Davidson Evers A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1980 ABSTRACT DIETARY ALTERATION OF PARAQUAT TOXICITY by William Davidson Evers The diet is an important variable in toxicological research. Several reports have indicated that feeding experimental animals a nutrient—adequate diet, for which the exact nutrient composition is not available (closed formula diet), can result in a different re- sponse to a xenobiotic than feeding a diet formulated from purified ingredients in specifically stated proportions (purified diet). Preliminary experimental results suggested that in mice the herbicide, paraquat, was more toxic when administered in conjunction with a purified diet rather than a closed formula diet. Paraquat is a water soluble, highly ionized herbicide used as a defoliant on broadleaf weeds and grasses. The lung is the main organ to manifest a toxic response, with the kidney also being affected. The mechanism of action is thought to involve free radical formation by a cyclic oxidation-reduction of paraquat. The objective of the research described in this dissertation was to determine if paraquat was, in fact, more toxic to mice fed a puri- fied diet rather than a closed formula diet. In addition, some of the dietary factor(s) responsible for the altered sensitivity and the metabolic alterations involved were experimentally defined. William Davidson Evers lower ET Male ICR.mice fed a purified diet had a lower LDSO’ 50, shorter survival time and lower 7-day percent survival after i.p. in— jection of paraquat than mice fed a cereal-based closed formula diet. The increased sensitivity to paraquat in mice fed the purified diet was evident as soon as 3 days after feeding the purified diet and as long as 84 days after feeding. Neither the age when the diet was started nor the sex of the ICR mice had any affect on the dietary alteration in paraquat toxicity. Some difference in strains of mice was noted. Alteration of the amount and type of lipid in the purified diet did not affect the increased sensitivity to paraquat in mice fed the purified diet. Substitution of egg white solids for casein as the protein source in the purified diet resulted in an increased survival time and ETSO' This suggested a protective effect against paraquat toxicity by the egg white protein in the purified diet. It was hypothesized that an alteration in a paraquat—induced free-radical lipid peroxidation mechanism in mice fed the purified diet would explain the increased sensitivity to paraquat. Supplemen— tation of the purified diet with vitamin E and selenium, both natural inhibitors of free radical formation, or with the antioxidant, butylated hydroxytoluene (BHT), did not decrease the sensitivity to paraquat of mice fed the purified diet. Exposure of mice fed the purified diet to carbon tetrachloride or a 100% oxygen atmosphere, both of which may cause free radical formation, did not result in an increased toxicity of these compounds when compared to the same exposure of mice fed the closed formula diet. It was concluded that the altered response to William Davidson Evers paraquat observed in mice fed the purified diet did not result from a difference in lipid peroxide formation. Mice fed the purified diet accumulated more paraquat in the liver, kidney and plasma, 3 to 12 hours after injection, than did mice fed the closed formula diet. Lung concentrations of paraquat were not changed by diet. This suggested that the feeding of the purified diet resulted in a more rapid absorption from the peritoneum and/or slower excretion of paraquat by the kidneys. Despite elevated plasma paraquat concentration, urinary excretion of paraquat over the 48 hour period was unchanged in mice fed the puri— fied diet rather than the closed formula diet. This unaltered excre— tion of paraquat plus an increase in plasma urea nitrogen 72 hours after paraquat injection in mice fed the purified diet suggested a possible impairment of renal function. However, other indicators of nephrotoxicity, urine volume and the in yit£9_accumulation of organic ions by renal cortical tissue slices were not changed after paraquat injection in mice fed the purified diet rather than the closed formula diet. The results of this study lead to the following conclusions: Mice fed a purified diet are more sensitive to paraquat than mice fed a cereal—based closed formula diet; the protein source of the purified diet is a factor in the altered toxicity; and there is a change in the tissue concentration of paraquat in mice fed the purified diet possibly resulting from or producing a greater nephrotoxic response to paraquat. The importance of diet in toxicological research is further emphasized by the results of these experiments. DEDICATION for James T. and Mary Jane Evers ii ACKNOWLEDGEMENTS The support of Joanne Evers is the main reason that I was able to complete this research. My sons, Michael and Christopher, also gave me the drive to finish my work. My advisor, Dr. Jenny T. Bond and the members of my dissertation committee, Dr. M.R. Bennink, Dr. J.B. Hook, Dr. G.A. Leveille and Dr. R.A. Roth, Jr. gave me advice, questions, support and encouragement without which I could not have continued. The technical assistance of Dr. W.E. Braselton, Jr., Dr. D.E. Ullrey, Greg Miller, Gary McLeod, Janice Spotts, Eileen Foulkes, Sue Short and Diane Hummel is gratefully acknowledged. To the graduate students and/or friends with whom I communicated; Robin Goldstein, Steve Fretwell, Ellen and Steve Rolig, Dr. Byron Noordewier, Penny Ross, Jeff Osborn, Sue Ford, Ken Wallace, Dr. Kevin McCormack, Ian McLean, Dr. Mike Armstrong and Dr. Will Forsythe; I say thank you for helping me through the rough times and thank you for enjoying with me the fun times. TABLE OF CONTENTS ACKNOWLEDGEMENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION Nutrition Methodology in Toxicological Studies Closed Formula vs. Purified Diets Paraquat METHODS Animals Diets Food Intakes Survival Time and 7—Day Percent Survival Median Lethal Dose (LD50) Median Effective Time (ETSO) Length of Feeding Time Dietary Alterations Age Sex and Strain Other Toxicants Nonprotein Sulfhydryl Group Compounds (NPSC) Glucose-G—phosphatase Activity Tissue Distribution of Paraquat Renal Accumulation of Organic Ions Hematocrit, Plasma Urea Nitrogen and Plasma Osmolality——--—— Statistical Analyses RESULTS Growth and Food and Water Consumption Median Lethal Dose (LD50) Median Effective Time (ET50) Survival Time and 7—Day Percent Survival iv Page iii vi vii 16 16 16 23 23 24 24 25 25 26 26 27 27 28 29 3O 31 32 33 33 33 33 38 TABLE OF CONTENTS (continued) Page RESULTS (continued) Initial Age of Mice 38 Dietary Alterations 38 Sex and Strain of Mice 45 Nonprotein Sulfhydryl Group Compounds (NPSC) ———————————————— 61 Glucose—6—phosphatase Activity 64 Tissue Distribution of Paraquat 64 Renal Organic Ion Accumulation 71 Urine Volume and pH 71 Hematocrit, Plasma Urea Nitrogen and Plasma Osmolality —————— 74 Food Intake—Diet vs. Paraquat Interactiou 76 DISCUSSION 78 CONCLUSIONS 96 BIBLIOGRAPHY 99 Table 10 ll 12 l3 14 LIST OF TABLES Composition of the Cereal—based Closed Formula Diet-——— Composition of the Purified Diet Composition of the Commercial Purified Diet Composition of the Cereal—based Open Formula Diet —————— Vitamin Composition of Diets Mineral Composition of Diets Effect of Diet on Food and Water Intake of Male ICR Micc Effect of Diet on 7-Day Dose—Response to Paraquat ------ Effect of Diet and Paraquat Injection on the Concentra— tion of Nonprotein Sulfhydryl Group Compounds (NPSC)-~— Effect of Diet and Carbon Tetrachloride (CC14) on the Concentration of Nonprotein Sulfhydryl Group Compounds (NPSC) and G1ucose-6—phosphatase Activity in Liver and Kidney Effect of Diet and Paraquat on Renal Organic Ion Accumulation Effect of Diet and Paraquat on Urine Volume and pH ————— Effect of Diet-Paraquat Interaction on Hematocrit, Plasma Urea Nitrogen and Plasma Osmolality Effect of Diet and Paraquat on Food Intake vi Page l7 18 19 20 21 22 36 37 62 63 72 73 75 77 Figure 10 ll 12 13 14 LIST OF FIGURES Effect of diet on body weights of male ICR mice ———————— Effect of length duration of feeding on paraquat toxi- city Age and acclimation effect on dietary alteration of Page 34 39 41 paraquat toxicity Effect of type of diet on paraquat toxicity Effect of dietary protein alteration on paraquat toxi— city Effect of dietary alteration on paraquat toxicity ------ Effect of vitamin E and selenium on the dietary altera— tion of paraquat toxicity Effect of sex or strain on dietary alteration of para— quat toxicity Effect of strain on dietary alteration of paraquat toxicity Effect of diet on oxygen toxicity Effect of diet on carbon tetrachloride toxicity ———————— Dietary effect on the concentration of paraquat in kidney and lung Dietary effect on the concentration of paraquat in liver and heart Dietary effect on the concentration of paraquat in plasma and urine vii 43 46 48 50 52 55 57 59 65 67 79 INTRODUCTION A study involving alteration of lipid membranes by dietary mani— pulation and the possible effects on the toxicity of the herbicide, paraquat, indicated a possible difference in the lethal dose of para— quat in mice fed a commercial cereal—based diet compared to mice fed a purified diet formulated in the laboratory. The study reported here grew out of this observation and an interest in understanding how diet can affect the results of toxicological studies in general. Nutrition Methodology in Toxicological Studies Many conclusions concerning the mechanisms of action and the toxicity of compounds come from research data using laboratory animals. Major decisions on the use of food additives, artifical sweeteners, birth control agents, industrial solvents, fire retardants and other chemicals have been based primarily on results obtained from studies on animals. The increased synthesis of new compounds has led to the rapid development of toxicology as a separate area of study. Toxicologists use data gathered from animal studies to predict the hazard of a chemical and its impact on the human population (Laroche, 1965; Boyd, 1968; Casarett, 1975). The present sensitivity of instruments has made it possible to measure much lower concentrations of chemicals. This has given rise to debate within the scientific community and among the public as to how decisions concerning the safety of a particular 2 substance are to be made. The problem revolves around the growing awareness that no chemical is absolutely safe, but rather that each ”substance has a certain hazard or risk associated with it. While more and more research is being done at the organ and cellu— lar level, it is necessary to remain cognizant of the whole environment that interacts with the system under study. The whole animal and the environment around the animal could affect the reactions that take place at the subcellular level, even if these reactions are studied later in a system that is isolated from the original organism (Cook, 1971). Many components of isolated systems (i.e., perfused organs, purified membranes, isolated enzymes) have been carefully defined as to blood flow, temperature, lighting, substrates and necessary cofactors (Cross and Taggart, 1950; Vegt, 1976; Browne_etmal., 1978; Eagle eg El): 1978). There are also data available concerning some environmen— tal factors such as animal housing, bedding, room lighting, tempera— ture, humidity, and noise level, all of which may have influenced the animal prior to the isolation of the system under study (Laroche, 1965; Geber gt a1., 1966; Yamauchi et 31., 1967; Boyd, 1968; Port and Kalten— bach, 1969; Plant_gtna1., 1970; Baer, 1971; Vesell et_al,, 1976). In relation to toxicological studies the importance of diet composition has received little attention. Much data concerning the feeding of diets and the alteration of organ composition or drug metabolizing enzymes has been published (Norred and Wade, 1972; Cooper and Feuer, 1973; Nash and Bender, 1976; waynforth, 1977; Chadwick et al., 1978). Many vitamins and minerals are now known to interact with various foreign compounds (Galdhar and Pawar, 1976; Makar and Tephly, 1977; Chakrabarty gt_al,, 1978; Hollinger_gt_a1., 1978; Sugawara and Sugawara, 3 1978; Menzel, 1979; Rising, 1979). However, in these types of studies the dietary regimen has been purposefully altered to produce a result. Evidence has been published over the last 25 years that uninten- tional differences in diet composition may be affecting the results of scientific studies. One study was reported as early as 1954 (Brown 35 31,, 1954), and several reviews on environmental factors (including diet composition) which might affect experiments have been published (Laroche, 1965; Boyd, 1968; Greenfield and Briggs, 1971; Newberne, 1975). ‘More recent reports have shown that when animals are fed a cereal—based diet versus a diet which is formulated from more purified ingredients, differences in toxicity are observed for lead (Mylroie_gt a1}, 1978), 5—fluorouracil (Bounous §£_§1,, l978a,b), pentobarbital (Bounous_g£nal., l978a), vanadium (Hafez and Kratzer, 1976), lindane (Chadwick et_a1,, 1978), phenacetin (Pantuck 23 31., 1975), and alcohol (Tottmar gt 31., 1978). Currently, there are several commercially prepared cereal—based diets for laboratory animals. The use of these formulations for toxi- cological studies can be faulted on several counts. Often these diets are formulated on a least-cost basis. The proximate composition of protein, fat, moisture and ash will remain the same as will the con- centrations of many vitamins and minerals. However, the ingredients will vary depending upon cost and availability, and this could alter the concentrations of trace minerals and other minerals as well as vitamins, amino acids, type of fiber, lipid and carbohydrate composi— tion (Pollak, 1965; Greenfield and Briggs, 1971; Walker, 1975; Wayn— forth, 1977). In addition, there may be present in the diets trace 4 amounts of non—nutritional xenobiotics such as heavy metals, pesticides and preservatives (ILAR Committee, 1976; Fox and Boylen, 1978; ILAR Committee, 1978; Coleman and Tardiff, 1979). If the commercial cereal— based diets are used as a control against more purified diets to which specific chemicals have been added, it would be difficult to single out those specific chemicals as being the cause of any observed differences in the animals compared to the control animals. If the commercial cereal—based diets have compounds added directly to them as a part of an experiment (rather than adding the compounds to a purified diet) it is even more difficult to show that an effect observed in the animal was due to the added compound and not due to the interaction of this compound with some unknown dietary constituent in the cereal-based diet (Greenfield and Briggs, 1971; Walker, 1975). Many journal articles do not completely describe the methodology used in the care and feeding of animals. As a result other researchers are unable to exactly duplicate the experiments and to control variables that occur from the housing and feeding of the animals. Dietary regi— mens in particular are often inadequately described (Greenfield and Briggs, 1971). The adequacy of nutrients (when comparing 2 diets or when using one diet as a control for another diet to which a toxicant has been added) is sometimes overlooked. Toxicological studies must be so designed that the effects observed can be attributed to the compound in question. Unless adequate care is taken to insure that the nutrients in the diet are sufficient for the needs of the animal, the subsequent results can be questioned as to their relationship to the toxicant being studied. 5 In recent years there have been studies and recommendations offered to standardize the reporting of diet composition (Greenfield and Briggs, 1971; Walker, 1975; Corbin, 1976; AIN Ad Hoc Committee, 1977; ILAR Committee, 1978). The American Institute of Nutrition (AIN) Ad Hoc Committee on Standards for Nutritional Studies (1977) published a uniform terminology for the reporting of diets. They defined three types of diets. A "cereal—based, unrefined or non—purified" diet would describe diets presently referred to as "stock diet”, "commercial pellets", "pelleted diet" or "laboratory chow". These diets are formu- lations composed predominantly of unrefined plant and animal materials possibly with added vitamins and minerals. The diets would be further defined as "open—formula" if the precise percentage composition of each ingredient is available or "closed—formula” if the manufacturer does not disclose the exact composition by type and amount of each ingre— dient. The second type of diet would be called a "purified" diet and would replace such terms as "synthetic", "semisynthetic” or "semipuri— fied". This diet would be composed primarily of highly refined pro— teins (such as casein or ovalbumin), carbohydrates (starch, sucrose, glucose) and fats (corn oil, lard) with added mineral and vitamin mixtures. The third diet is a "chemically defined" diet. It would be composed of pure amino acids, mono- or disaccharides, purified fatty acids or triglycerides and highly purified vitamins and minerals. The committee described a cereal based open-formula diet (NIH—7) and a purified diet (AIN—76) which were adequate for reproduction, lactation, growth and maintenance of both rats and mice. They recommended that these diets be used to standardize the types of diets used in many 6 animal studies. The adequacy of the AIN—76 diet has been questioned in a recent report by Yew 23.91' (1979). They concluded that further supplementation of the AIN-76 diet with vitamins and minerals improved reproductive performance and reduced the toxicity of aminopyrine and sodium nitrite in offspring. The Committee on Laboratory Animal Diets of the Institute of Laboratory Animal Resources (National Research Council - National Academy of Science) published their recommendations on the use of diets in laboratory animal experimentation (ILAR Committee, 1978). While recommending the same two diets as the AIN Committee on Standards for Nutritional Studies, this committee also reported on the possible contamination of feeds by industrial contaminants or by the use of unclean diet—mixing apparatus. This contamination might be a factor in toxicological studies where commercial cereal—based closed formula diets have been used. Closed Formula vs. Purified Diets - As previously mentioned, several studies have been published where an altered toxic response to various compounds was observed when animals were fed a purified diet rather than a closed formula diet. Some of these researchers (Hafez and Kratzer, 1976; Bounous et_a1,, l978a,b; Mylroie et_a1,, 1978) attempted to further delineate the possible differences in the diets by altering the amounts of protein, fat or carbohydrate. However, no attempts were made to compare the toxicity between compounds to deter- mine if the results were the same for more than one xenobiotic. Since the reports were from different laboratories, the same purified and closed formula diets were not always used. Therefore it is difficult 7 to find specific differences between the diets just by comparing the literature. By comparing three studies that did have several common features the difficulty of finding specific dietary differences can be shown (Pantuck_gtua1., 1975; Chadwick et 31., 1978; Mylroie 25 31°, 1978). In all three reports Purina Lab Chow was the cereal-based closed formula diet used and a diet called Normal Protein Test (NPT) Diet was fed as the purified diet. However, the NPT diet used by Mylroie gt_a1, (1978) was from a different supplier and contained 59% starch while the other NPT diets contained 56.8% sucrose. It has been reported by Boyd (1968) that high sucrose diets caused an increased sensitivity to oral benzyl—penicillin and caffeine when compared to starch diets. This difference in type of carbohydrate makes compari- sons more difficult. Mylroie ggflal. (1978) did report that substitu— tion of sucrose for starch did not alter the effect of the purified diet on lead toxicity. The NPT diet does not contain fiber. Mylroie g£_§1, (1978) recognized this and added fiber (cellulose) into the diet, although the percent of fiber added was not given. Addition of cellulose did not alter the results. In the study by Chadwick gt_§1. (1978) the metabolism of lindane was increased by the addition of 10% pectin fiber to the NPT diet, but not by agar or cellulose fiber. Feeding Purina Lab Chow caused even more significant increases in lindane metabolism. This further alteration of lindane metabolism indicated that other factors besides the source of fiber must also have been present in the cereal-based diet (Purina Lab Chow). Pantuck 35 31, (1975) reported a three—fold increase in the metabolism of phena- cetin when rats were fed the closed formula diet compared to rats fed 8 the NPT diet. Pantuck gt 9;- (1975) did not attempt any modification of the NPT diet to study possible dietary effects. Therefore, two of the three studies showed increased metabolism of two different xenobiotics and one study showed a decreased toxicity of another foreign compound in animals fed a closed formula diet when compared to animals fed a purified diet. This would indicate some protective factor in the closed formula diet. Chadwick_gt_a1. (1978) and Mylroie gt a1. (1978) saw no change in response when cellulose was added to the purified diet, but Chadwick_gtfla1. (1978) did see a change when another fiber, pectin, was added. Mylroie gt_al, (1978) and Pantuck_gtnal. (1975) might have altered their results by the addition of 10% pectin to the purified diet. To further complicate comparisons, Chadwick_gtfla1. (1978) used weanling female Sprague—Dawley rats and fed them for 28 days; Pantuck_§§”a1. (1975) used 170 g male Long—Evans rats and fed them for 7 days; and Mylroie §£_a1, (1978) used 60—80 g male Holtzman rats and fed them for 35 days. These age, sex and strain differences may well have added further variables. It is apparent that comparing these three studies becomes quite difficult when diet formu— lation as well as other variables are considered. Another example of research involving differences between purified and closed formula diets is the work of B.H. Ershoff (Ershoff, 1954, 1957, 1972, 1974, 1977a,b; Ershoff and Thurston, 1974). He has shown that types of fiber and carbohydrate can alter the toxicity of gluco- ascorbic acid, sodium cyclamate, chlorazanil hydrochloride, amaranth and tartrazine when these chemicals are mixed into the diet. Ershoff's 9 results with amaranth and tartrazine have recently been supported by others (Takeda and Kiriyama, 1979; Tsujita_g£_a1., 1979). Alterations in the amounts of many nutrients have been tested to determine the factor(s) which appear to be involved. So far, only some of the water holding properties of fiber in the gastrointestinal tract have been suggested as a possible explanation (Ershoff, 1977a,b; Takeda and Kiriyama, 1979; Tsujita gt a1., 1979). Fractionation of the fiber has only been superficially carried out, and further separation would seem to be a good approach to finding a specific component that might protect against the toxicity of the various compounds. A problem with Ershoff's design is that the chemicals are mixed into the diet and an equal amount of the carbohydrate source is removed. Since the chemi— cals and fibers are added at relatively high percentages (2—20% by weight) of the diet, and several chemicals plus fiber may be added at the same time, there is an overall reduction in caloric density. One of the major responses Ershoff used to assess the effects of the xeno- biotics is weight gain. It could be argued that some of the weight loss observed after feeding the chemical—containing diets was due to a decreased caloric density of the food. Ershoff also uses nutrients such as ascorbic acid, para-aminobenzoic acid and inositol which are not considered essential for rats and mice (Committee on Animal Nutri— tion, 1972). These nutrients then add other unknown variables. The type of research discussed demonstrates the need for good nutritional methodology to systematically define the differences in response to toxicants by animals and to ascertain the component(s) in a diet which might cause the noted differences. 10 Paraguat Paraquat or 1,l'-dimethy1—4,4'-bipyridylium dichloride is an herbicide used for broadleaf weeds and grasses. Two reviews (Smith and Heath, 1976; Haley, 1979) and a book (Autor, 1977) deal extensively with the toxicological aspects of paraquat in plants and animals, including humans. Paraquat has a molecular weight of 257.2 as the dichloride (186.2 as the divalent cation); is very soluble in water but quite insoluble in organic solvents; and is highly ionized over a wide range of pH (Haley, 1979). Paraquat poisoning in humans has resulted in at least 500 deaths world-wide since 1964 (Rebello and Mason, 1978; Fairshter g£_§1,, 1979). Ingestion is usually intentional (Carson and Carson, 1976; Smith and Heath, 1976; Farr, 1977; Spector 25.2i39 1978) although accidental ingestion has also been reported (Smith and Heath, 1976; Fairshter gt_§1,, 1979). .Most cases have occurred in Great Britain and Malayasia where much of the bipyridyl-based herbicides are manufactured (Smith and Heath, 1976). Four cases of human paraquat poisoning in the United States have been reported in the recent literature (Spector g; g1,, 1978; Cravey, 1979; Dabir-Vaziri ggna1., 1979; Fairshter gt 31., 1979). The concentrated liquid form of paraquat (GramoxoneR — 20% paraquat) is usually the preparation which is ingested, with few reports of ingestion of granules (WeedolR, 2.5% paraquat) and no re— ports of inhalation as being the cause of poisoning (Smith and Heath, 1976; Dabir-Vaziri g£_§1,, 1979). From studies of workers at plants which produce bipyridyl-based herbicides researchers have concluded that there were no long-term effects from inhalation of or skin ll exp05ure to paraquat (Swan, 1969; Hearn and Keir, 1971; Staiff g£_al., 1975; Howard, 1979). Paraquat is poorly absorbed from the gastrointestinal tract in all animals studied with greater than 80% of an oral dose appearing in the feces (Daniel and Gage, 1966; Conning §E_a1., 1969; Murray and Gibson, 1974). The paraquat that is absorbed is rapidly excreted by the kid— neys in its original form (Murray and Gibson, 1974; Smith and Heath, 1976; Haley, 1979). Hughes_gt.a1. (1973) reported that biliary excre— tion in rat, guinea pig and rabbit is minimal. Paraquat is actively taken up by the lung (Rose and Smith, 1977; Charles £2.2l3, 1978; Drew 25.2l3’ 1979; Siddik gt a1., 1979). The kidney also contains high concentrations of paraquat soon after administration (Sharp gt_al,, 1972; Murray and Gibson, 1974; Rose and Smith, 1977). The toxic re— sponse to paraquat appears to be biphasic. In the early stage (usually within 1—5 days) several organs, including lung, kidney and G.I. tract, may be affected (Smith and Heath, 1976; Farr, 1977; Lock and Ishmael, 1979). The second stage of toxicity (1—3 weeks after exposure) results mostly from pulmonary interstitial fibrosis (Smith and Heath, 1976). Since the lung is the organ most affected by paraquat, numerous reports describe the pathology and histology of the lungs after para- quat administration to the mouse (Brooks, 1971; Popenoe and Loosli, 1978; Etherton and Gresham, 1979; Popenoe, 1979), rat (Autor, 1974; Greenberg gt_§1,, 1978; Thompson and Patrick, 1978), rabbit (Seidenfeld g£_g1., 1978), dog (Kelly gg‘a1., 1978) and man (Smith and Heath, 1976; Farr, 1977; Cravey, 1979; Haley, 1979). In the early stage of paraquat toxicity there is destruction of alveolar epithelium and some intra— alveolar hemorrhage and edema (Smith and Heath, 1976; Greenberg §t_al., 12 1978; Etherton and Gresham, 1979; Haley, 1979). In the later stage a proliferation of fibrotic tissue and consolidation of alveolar paren— chyma takes place (Smith and Heath, 1976; Etherton and Gresham, 1979). On a biochemical level increased collagen prolyl hydroxylase activity in the lung has been reported in rats after exposure to para— quat. The increased activity of this enzyme has been reported to correlate with the histological evidence of increased fibrotic tissue (Greenberg 35 a1., 1978; Kelly gt El‘: 1978; Thompson and Patrick, 1978). However, there is contradictory evidence as to whether there is an actual increase in the concentration of both hydroxyproline and collagen in the lung after exposure to paraquat (Greenberg gt_al., 1978; Kuttan EE”§1., 1979). Protein synthesis has been reported to be increased within 3 days after paraquat treatment but in_yi£rg_studies by Kuttan gt_§1, (1979) indicate that incubation of lung slices with paraquat (10-3M) inhibits protein synthesis. Kuttan 35 a1. (1979) also reported that prolyl hydroxylase activity and collagen synthesis were decreased by paraquat (lO-IM). Fletcher and Wyatt (1970) reported that neither total lung phos- pholipid nor incorporation of palmitic acid into dipalmitoyl phospha— tidylcholine (Fletcher and Wyatt, 1972) which is the major component of lung surfactant, is altered by oral administration of paraquat to female mice. In a single experiment with an_n of l, Etherton and Gresham (1979) reported that male mice given an intraperitoneal injec— tion of paraquat showed a reduced amount of lung surfactant. The nephrotoxic response to paraquat has also been studied. Lock and Ishmael (1979) reported that toxic effects of paraquat on the rat 13 kidney included a marked diuresis, albuminuria, glucosuria and an increased plasma urea concentration 6-24 hours after oral or subcuta— neous administration. In,y}£rg_incubation studies of renal cortical slices with paraquat in the medium suggest that paraquat is secreted by the organic base transport system in mice (Ecker §E_§13, 1975a) and rats (Lock and Ishmael, 1979). From inlvivg_studies Lock (1979) and Ecker gt g1. (1975b) reported that there was a decreased excretion of organic ions and a decreased plasma volume after paraquat administra- tion. Lock (1979) also reported a decreased glomerular filtration rate in rats. He hypothesized that the paraquat-induced decrease in plasma volume altered the renal hemodynamics, which in turn caused the reduc— tion in renal excretory function. Ecker EE.§£° (1975b) did not observe a reduction in glomerular filtration rate in mice and hypothesized a direct effect of paraquat on kidney secretory function. One hypothesis to explain the mechanism of paraquat toxicity in the lung is that some form of free radical (superoxide, hydroxyl, or lipid hydroperoxide) reaction involving lipid peroxidation causes the cell damage that is observed (Bus gt El-9 1974, 1976). Under aerobic conditions paraquat undergoes cyclic reduction-oxidation. NADPH is the source of electrons which are transferred by NADPH-cytochrome c reduc- tase to the paraquat molecule. The paraquat is reoxidized when the electron is transferred to molecular oxygen to form the superoxide radical. The superoxide radical may then be converted to hydrogen peroxide by the zinc-requiring superoxide dismutase enzyme and then to water by catalase. In this manner the free radical propagation would be terminated. The superoxide radical may also nonenzymatically l4 dismutate to singlet oxygen which then can react with unsaturated fatty acids in cell membranes to produce lipid hydroperoxides. The hydroperoxides can be metabolized to lipid alcohols by the selenium— dependent glutathione peroxidase enzyme and then be excreted without harm to the animal. However, in the presence of trace amounts of transition metal ions, particularly iron, the lipid hydroperoxides may spontaneously decompose to lipid free radicals which can then initiate and propagate the lipid peroxidation-free radical chain reaction. This cyclical reaction leads to damage of the cell membrane. Vitamin E is an endogenous antioxidant and, along with the sulfydryl compound, glutathione, is thought to be one of the main terminators of this free radical chain reaction. Various factors in the free radical forming system such as superoxide dismutase (Autor, 1974; Wasserman and Block, 1978), vitamin E (Block, 1979) and zinc (Hollinger gt_gl,, 1977, 1978, 1979) have been studied as to their effect on paraquat toxicity. No consistent protective effects have been noted by the use of any of these factors. Other researchers have questioned the significance of lipid peroxidation as a factor in paraquat toxicity (Shu SE 31., 1979; Steffen and Netter, 1979; Talcott g£_a1,, 1979). Shu gt_a1, (1979) reported that prevention of paraquat-stimulated lipid peroxidation by pretreatment with an antioxidant (N,N'—diphenyl—p-phenylene diamine) did not decrease paraquat toxicity in rats. They also reported that after a toxic dose of paraquat, no evidence of lipid peroxidation, as measured by conjugated diene accumulation, could be found in mouse lung. Montgomery and Niewoehner (1979) and Steffen and Netter (1979) 15 reported an inhibition of mouse lung microsomal lipid peroxidation ig 31339, as measured by malondialdehyde formation, when paraquat (1 mM) was added to the incubation medium. Clinical treatment of paraquat poisoning has been largely un- successful since no antidote to paraquat has been discovered. Mini- mizing absorption from the G.I. tract, forced diuresis soon after poisoning, peritoneal dialysis, hemodialysis, charcoal hemoperfusion, superoxide dismutase, D-propranolol and even lung transplantation have all been used as part of therapy for ingestion of paraquat (Smith and Heath, 1976; Farr, 1977; Spector gt a1., 1978; Fairshter 35 31., 1979). Based on the initial experience that paraquat toxicity may be altered by feeding mice a purified diet rather than a closed formula diet, and the realization that diet formulation is an important compo— nent of toxicology research, the objectives of this study were to determine 1) if there was a difference in the lethality of paraquat between mice fed the closed formula or the purified diet; 2) what factor(s) in the diet(s) could be the causative agent(s) of this difference and 3) what changes are occurring in the mouse that cause the difference in response to an injection of paraquat. METHODS Animals Unless noted, male ICR mice (Spartan Research, Haslett, M1 or Harlan Industries, Inc., Indianapolis, IN), 28 days of age, were used in all experiments. Four to six mice were housed in plastic boxes with corn—cob bedding. Water and food were available ad_libitum, lights were on 11 hours per day, and room temperature was maintained at l9-23°. 21222 A pelleted cereal-based closed formula diet, Wayne Lab Blox, was purchased from Allied Mills, Inc., Chicago, IL. The ingredients and proximate composition are given in Table 1. A purified diet, prepared in the laboratory, was formulated to meet the nutritional needs of mice as recommended by the Committee on Animal Nutrition of the National Research Council (1972). The composition of this diet is given in Table 2. Unless noted, this diet (Table 2) is the one re— ferred to as the purified diet. The composition of the commerial purified diet formulated by Luecke §£_al, (1968) is shown in Table 3. The composition of a cereal-based open formula diet (Campbell §E_§13, 1966) used is given in Table 4. Tables 5 and 6 list the vitamin and mineral compositions, respectively, of the closed formula (Table l), 16 17 TABLE 1 Composition of the Cereal—based Closed Formula Dietl Ingredients: Corn and wheat flakes, ground yellow corn, soybean meal, fish meal, wheat middlings, dried whey, brewers dried yeast, soybean oil, animal liver meal, cane molasses, vitamin A palmitate, D- activated animal sterol, vitamin E supplement, menadione sodium bi— sulfite (source of vitamin K activity), riboflavin supplement, niacin, calcium pantothenate, choline chloride, thiamine, ground limestone, dicalcium phosphate, salt, manganous oxide, copper oxide, iron carbo— nate, ethylenediamine dihydriodide, cobalt carbonate and zinc oxide. Proximate Composition: Percent of Diet Nutrient Protein 24.5 Carbohydrate 61.9 Fat 4.0 Fiber 3.6 Minerals 4.3 Vitamins 1.0 Choline Cl 0.2 d,14Methionine 0.5 lWayne Lab Blox, Allied Mills, Inc., Chicago, IL. 18 TABLE 2 Composition of the Purified Diet Nutrient Ingredient Percent of Diet Protein Caseinl 20.0 Carbohydrate Dextrosel 67.5 Fat Corn oill 3.0 Fiber Cellulosel 4.0 Minerals Bernhart—Tomarelli 4.0 salt mix2 Vitamins AIN 76 vitamin mix3 1.0 Choline C12 0.2 d,1-Methionine4 0.3 lU.S. Biochemi 2Teklad Test D 3 cals, Cleveland, OH. iets, Madison, WI. AIN Ad Hoc Committee (1977). 4Sigma Chemicals, St. Louis, MO. 19 TABLE 3 Composition of the Commercial Purified Dietl Nutrient Ingredient Percent of Diet Protein Egg white solids 20.0 Carbohydrate Dextrose 63.6 Fat Corn oil 10.0 Fiber Cellulose 3.0 Minerals 2.2 Vitamins 1.0 Choline C1 0.2 lZinc control diet, Teklad Test Diets, Madison, WI. 20 TABLE 4 Composition of the Cereal—based Open Formula Dietl Ingredient Percent of Diet Ground corn 60.7 Soybean meal 28.0 Alfalfa meal 2.0 Fish meal 2.5 Dried whey 2.5 Limestone 1.6 Dicalcium phosphate 1.8 Iodized salt 0.5 Mineral mix 0.1 Vitamin mix 0.1 lCampbell gt a1. (1966). 21 TABLE 5 Vitamin Composition of Diets 1 . NAS-NRC Vitamin Purified Closed comfrflal Requirements Formula Purified Mouse mg/kg diet Thiamine HCl 6 14 10 3.2 Riboflavin 6 6.5 6 4.4 Pyridoxine HCl 7 8.1 4 l Niacin 30 60 25 11 Calcium pantothenate 16 l8 16 9.4 Folic acid 2 1.6 0.5 required Biotin 0.2 0.15 4 required g/kg diet Cyanocobalamin 10 30(B12) 20 5.6 Vitamin K 50 28002 330 603 (menadione) IU/kg diet Vitamin A 4,000 15,000 10,000 556 Vitamin D 1,000 4,410 1,250 167 Vitamin E 50 50 110 32 lDaily requirements as established by the National Research Council of the National Academy of Science (Committee on Animal Nutrition, 1972). 2As menadione sodium bisulfite. 3 . . . . . . No quantitative requirement available for the mouse, but Vitamin K is required. Value given is for the rat. 22 TABLE 6 Mineral Composition of Diets Mineral . . Closed Commercial NASINRCI (mg/kg diet) Purified Formula Purified Rquirement ouse Calcium 9,000 12,000 4,700 6,700 Phosphorus 7,456 9,900 3,400 5,600 C/P ratio l.207:1 1.212:l l.910:1 l.196:1 Sodium 800 3,900 2,200 2,205 Potassium 2,675 9,600 4,800 2,200 Magnesium 603 2,200 330 4002 Manganese 46 210 3 22 Iron 37 340 150 352 Copper 6.5 20.0 3.0 5.02 Zinc 17 60 50 57 Iodine 0.20 1.50 20.00 0.15 Selenium —- —— -— 0.043 Chloride 743 5,000 3,400 3,404 Sulfur 500 —— 400 -- Cobalt —— 0.7 0.4 —— lDaily requirements as established by the National Research Council of the National Academy of Science (Committee on Animal Nutrition, 1972). 2 . . . . No quantitative requirement available for the mouse, but the mineral is required. Value given is for the rat. Requirement not given, value is for the rat. 23 purified (Table 2) and commercial purified (Table 3) diets. The vitamin and mineral requirements as established by the Committee on Animal Nutrition of the National Research Council (1972) are also listed. Food Intakes Mice were fed either the closed formula or purified diet for 6 days and then transferred, 2 to a cage, to stainless steel metabolism cages. After a 3—day acclimation period, daily food and water con— sumption were measured for 4 consecutive days. Food was kept in glass food cups (Unifab, Kalamazoo, MI) with a stainless steel ring inserted to prevent food spillage. Water bottles with curved, stainless steel tubes, were weighed daily to determine water consumption. Survival Time and 7—Day Percent Survival Mice, usually lS/diet, were fed a diet for specified periods of time, and then an intraperitoneal (i.p.) injection of paraquat was administered. Animals were checked and dead mice removed at 12 hour intervals for the first 3 days after injection and every 24 hours from 3 to 7 days after injection. Paraquat dichloride (methyl viologen) was purchased from Sigma Chemical Co., St. Louis, MO. The doses given are for paraquat base which is 72.4% of the dichloride compound. For any given dose, a stock solution was made such that a total volume of 6-8 ml/kg body weight was injected. Thus, a 25—30 g mouse received 0.15 to 0.24 ml of solution. Solutions were made with 0.9% NaCl and brought to a pH of 7.0-7.5 with NaOH. Control animals received 6—8 ml/kg body weight of 0.9% NaCl. Time of injection was between 10:00 a.m. and 12:00 noon. 24 A similar procedure was followed when carbon tetrachloride (CC14) was administered. A stock solution in corn oil was made to give a total volume for i.p. injection of 8 m1/kg. To measure oxygen toxicity mice were fed either a purified or a closed formula diet for 14 days and then placed, 5/box, in an enclosed plastic chamber with gloved inserts (Glove Bag, model no. X—37—37, Instruments for Research and Industry, Cheltenham, PA). An atmosphere of 100% oxygen was maintained throughout an 8-day period. The volume of oxygen in the chamber was approximately 14 liters and oxygen flow averaged 3 liters per minute. Animals were checked several times each day and dead animals removed. Median Lethal Dose (LDSO) Mice were fed the closed formula or purified diet for 14 days. One of 6 doses of paraquat (17, 21, 26, 33, 42 and 52 mg/kg) was then administered i.p. The method of Litchfield and Wilcoxon (1949) was used to calculate the 7-day LD50 and to determine if there was a statistically significant difference in LD5 between mice fed the 0 cereal—based closed formula diet and mice fed the purified diet. Median Effective Time (ETSO) Mice were fed the closed formula or purified diet for 14 days. Paraquat (52 mg/kg) was then administered i.p. Mortality was followed for 7 days, and the ET50 was calculated and compared by the method of Litchfield (1949). 25 Length of Feeding Time Mice were fed either a closed formula or a purified diet for l, 2, 3, 14, 28 or 84 days and then survival time and 7-day percent survival were measured after paraquat injection (26—30 mg/kg). Body weights were measured weekly over the 84—day study. In some experi- ments, mice were switched from the closed formula diet to the purified diet before injection with paraquat. Dietary Alterations Another cereal-based closed formula diet (Purina Lab Chow, Ralston—Purina Co., St. Louis, MO), a commercially prepared purified diet (Table 3), a cereal-based open formula diet (Table 4), the regular cereal—based closed formula diet or the purified diet were fed to mice for 28 days. Survival time and 7—day percent survival were then measured after injection of paraquat (26 mg/kg). Survival time and 7—day percent survival after paraquat injection were also measured in mice fed the purified diet with one of the following modifications: 1) The 3% corn oil was replaced by either 15% corn oil (U.S. Biochemicals, Cleveland, OH), 15% safflower oil (U.S. Bio- chemicals, Cleveland, OH) or 15% lard (Food Stores, Michigan State University, E.Lansing, MI). Dextrose was removed from the diet so that caloric density (kcal/g diet) and protein, minerals and vitamins per 100 kcal of diet remained essen- tially the same. Diets were fed for 56 days before injec- tion of paraquat (26 mg/kg). 26 2) The 20% casein was replaced by 20% spray-dried egg white (Teklad Test Diets, Madison, WI). Diets were fed for 14 days prior to injection of paraquat (30 mg/kg). 3) Addition of 500 IU vitamin E, as a-tocopherol succinate (Sigma Chemical Co., St. Louis, MO), per kg diet and 0.45 ppm selenium, as sodium selenite (Delta Chemical Works, Inc., New York, NY). Diets were fed for 21 days before paraquat injection (30 mg/kg). 4) Addition of the antioxidant butylated hydroxytoluene (Sigma Chemical Co., St. Louis, M0) at 0.02% or 0.2% of the diet. Diets were fed for 14 days prior to paraquat injection (21 Ins/kg). Agg Twenty—eight—day—old mice were fed the closed formula diet until 56 days of age and then half of the animals were switched to the purified diet for 10 days. Other mice were fed 1 of the 2 diets for 84 days as described above. Survival time and 7-day percent survival, after paraquat injection, were compared between dietary groups. Sex and Strain Female ICR mice (Spartan Research, Haslett, MI), 28 days of age at the start, were fed either the closed formula or the purified diet for 7 days. Male B6C3F1 mice (Harlan Industries, Inc., Indianapolis, IN), 28 days of age at the start, were fed the closed formula or purified diet 27 for 14 days. Male CS7BL/6J mice (kindly supplied by Dr. Dale Romsos, Department of Food Science and Human Nutrition, Michigan State Univer— sity) 21-45 days of age at the start, were fed the closed formula or purified diet for 14 days. Survival time and 7-day percent survival, after paraquat injection, were then compared between dietary groups. Other Toxicants Mice were fed the closed formula or the purified diet for 14 days and then received an i.p. injection of 1.8 or 2.0 m1 CClA/kg or were exposed to an atmosphere of 100% oxygen as described under the section Survival Time and 7-Day Percent Survival. Nonprotein Sulfhydryl Group Compounds (NPSC) Mice were fed a closed formula or a purified diet for 14 days. Three hours after an i.p. injection of paraquat (42 mg/kg) or 0.9% NaCl mice were killed and lung and liver tissue were assayed for NPSC. Four and 24 hours after an i.p. injection of €014 (2.5 ml/kg for 4 hours and 1.75 ml/kg for 24 hours) or corn oil mice were killed and kidney and liver tissue were assayed for NPSC. After 84 days on the diets, mice were given an i.p. injection of CCl4 (1.75 ml/kg) or corn oil, killed 24 hours later, and kidney and liver tissue were assayed for NPSC. CCl4 was dissolved in corn oil (20—25% C014) and injected at a total volume of 8 ml/kg. The assay for NPSC is based on the 1:1 reaction of 5,5'-dithio— bis—Z—nitrobenzoic acid (DNTB) with sulfhydryl groups. The nitro- mercaptobenzoic acid anion has an intense yellow color and the absorbance 28 of this color is maximum at 412 nm. With various modifications, total, protein—bound and nonprotein sulfhydryl groups can be measured with this method (Sedlak and Lindsay, 1968). Tissue samples from liver, lung or kidney were homogenized in 20 times their volume of 6% trichloroacetic acid (TCA) for 5 seconds with a Polytron homogenizer. After centrifugation, 0.5 m1 aliquots of clear supernatant (0.2 ml aliquot + 0.3 ml 6% TCA for liver samples) were assayed for NPSC by adding 2 m1 of 0.3 M Na HPO and 0.5 m1 of 0.04% DNTB in 10% sodium 2 4 citrate, mixing, and measuring the absorbance at 412 nm. G1ucose—6—phosphatase Activity The same experiments and procedures as described for determining tissue NPSC concentration, after CCl injection, were followed up to 4 homogenization of tissue samples. Then the method of Harper (1965) was used to determine glucose—6—phosphatase (G6Pase) activity. G6Pase catalyzes the reaction: Glucose—6-phosphate (G6P) + H 0 + glucose + 2 phosphate. The rate of the reaction is measured by the increase of inorganic phosphate with time. Tissue is homogenized in citrate buffer (0.1 M; pH 6.5) in the ratio of 250 mg tissue to 9.75 ml buffer. After filtering through cheesecloth, the homogenate is heated at 37° for 5 minutes. Then 0.1 m1 of the homogenate is mixed with either 0.1 ml of 0.08 M G6P (experimental tube) or with 0.1 m1 citrate buffer (tissue blank). A second blank of 0.1 m1 citrate buffer + 0.1 ml of G6P (reagent blank) is also run. After exactly 15 minutes at 37°, 2 m1 of a 10% TCA solution is added. After centrifugation the supernatant is used for the colorimetric determination of phosphate by the method of Fiske and Subbarow (1925). After addition of 5 m1 of a 29 2x10_3M ammonium molybdate solution to 1 m1 supernatant, 1 m1 of a reducing agent, 4.2x10-2M l—amino-2-naphthol—4—sulphonic acid and 0.56 M 303—2, is added and the absorbance at 660 nm is read at the same time for each tube between 15 and 60 minutes after addition of the reducing agent. The reagent blank is used to zero the instrument. A standard is also run and the G6Pase activity is calculated as follows: (absorbance of experimental tube) — (absorbance of tissue blank) (absorbance of standard) X pmoles phosphate in the standard tube (0.5 umoles in this assay) X 2.2 (total volume of enzymatic reaction mixture) x 1000 mg/g % 2.5 mg tissue in the enzymatic reaction mixture % 15 minute period of the enzymatic reaction This will give a value in umoles phosphate/min/g tissue. Tissue Distribution of Paraquat Male ICR mice were fed either a purified diet or a closed formula diet for 14 days, then an i.p. injection of 14C—paraquat (33 mg/kg, Amersham-Searle, Arlington Heights, IL) was administered. The speci— fic activity of the l4C—paraquat was 1.2 uCi/mg paraquat. An average mouse of 25 g received approximately 1 uCi. Several animals on both diets were injected with nonradioactive paraquat and killed at differ— ent time periods. The tissues from these animals were used to obtain sample radioactive background values. At 1, 3, 6, 12, 24 and 48 hours after injection, mice were lightly anesthetized with ether and venous blood was obtained from the orbital sinus. Animals were then decapitated, the liver, lung, kidney and heart were rapidly removed, rinsed with 0.9% NaCl and kept on ice. 30 Duplicate tissue samples of 80—100 mg or 0.1 m1 plasma were put into polyethylene vials with 1 m1 of a tissue solubilizer (Soluene—lOO; Packard Instrument Co., Downers Grove, IL). After tightly capping, the vials were slowly shaken overnight to facilitate the solubilizing process. After solubilization was complete, 4 drops of glacial acetic acid were added to each vial to reduce chemiluminescence. Fifteen m1 of a scintillation cocktail [5 g 2,5—diphenyloxazole, 0.5 g p-bis(0— methylstyryl benzene), 1000 m1 toluene, as described for Soluene—lOO solubilizer; Packard Instrument Co., Downers Grove, IL] was added to each vial and the vials were stored in the dark for at least 24 hours. Vials were then counted in a refrigerated liquid scintillation counter (Beckman LS—230, Beckman Instruments, Inc., Fullerton, CA) for 20 minutes or to a preset error of 0.2%. The efficiency of the counter was determined by use of an internal standard. For assaying of l4C-paraquat in urine, mice were placed, 2/cage, in stainless steel metabolism cages 4 days prior to injection. After injection of paraquat, urine was collected under toluene. At 1, 3, 6, 12, 24 and 48 hours, urine was removed and the volume and pH were recorded. Duplicate 0.1 m1 samples were assayed for paraquat as described above for tissue and plasma samples. Renal Accumulation of Organic Ions Mice were given an i.p. injection of paraquat (42 mg/kg) or 0.9% NaCl. Three hours later mice were killed and kidney transport of organic ions was measured using an in_vitgg slice incubation system based on the method of Cross and Taggart (1950). Thin slices of renal cortex were prepared freehand and stored in cold saline until 31 incubation. Organic ion transport capacity was quantified as the ability of cortical slices to accumulate a representative anion, p— aminohippuric acid (PAH; Sigma Chemical Co., St. Louis, MO), and cation, tetraethylammonium (TEA; New England Nuclear, Boston, MA). Tissue was incubated in 2.7 ml of phosphate—buffered medium (Cross and Taggart, 1950) which contained 7.4x10'5M PAH and 1.0x10‘5M l4c-TEA (specific activity, 3 mCi/umole). Incubation was carried out in a Dubnoff metabolic shaker at 25° under 100% oxygen for 90 minutes. A 90 minute incubation was chosen because accumulation has reached a maximum and a steady state has been achieved by this time. After incubation, slices were quickly removed from the medium, blotted, and weighed. Tissue or a 2 ml aliquot of medium was added to 3 ml 10% trichloroacetic acid. The tissue was homogenized, and tissue and medium were brought to a final volume of 10 ml with distilled water. After centrifugation, PAH in tissue and medium supernatants was deter— mined by the method of Smith g£_a1. (1945). Samples of both super— natants were counted in modified Bray's solution (6 g of 2,5—dipheny1— oxazole and 100 g of napthalene per liter of dioxane) in a liquid scintillation spectrometer (Beckman LS—230, Beckman Instruments, Inc., Fullerton, CA) to determine TEA concentration. Efficiency of the scintillation counter was determined by use of an internal standard. Hematocrit, Plasma Urea Nitrogen and Plasma Osmolality Mice were fed either the purified or closed formula diet for 2 weeks and then given an i.p. injection of paraquat (30 mg/kg). Blood was collected by decapitation at 0, 8, 24 or 72 hours after injection. 32 Hematocrit was measured by collecting blood in heparinized micro— capillary tubes, centrifuging and reading the percent of packed red blood cells. Urea nitrogen was measured by the methods of Fawcett and Scott (1960) and Chaney and Marback (1962) as described in Sigma Technical Bulletin No. 640 (Sigma Chemical Co., St. Louis, MO). Plasma osmolality was measured using a vapor pressure osmometer (Model 51003, Wescor, Inc., Logan, UT). Statistical Analyses Differences in survival time were compared by the Mann-Whitney U— test for 2 samples, ranked observations, not paired (Sokal and Rohlf, 1969). Differences in 7—day percent survival were compared by the Chi square 2x2 contingency test, Model II (Sokal and Rohlf, 1969). Renal ion accumulation, NPSC concentration, G6Pase activity, hematocrit, urea nitrogen, urine volume, urine pH, food and water intake were all analyzed with analysis of variance (Sokal and Rohlf, 1969). Signifi— cant differences were further analyzed by the Student—Newman—Keuls procedure (Sokal and Rohlf, 1969). Accumulation of l4C—paraquat and body weights were compared between dietary groups using Student's t- test (Sokal and Rohlf, 1969) or a modified t—test for unequal vari— ances (Gill, 1978). The 0.05 level of probability was used as the criterion of significance. RESULTS Growth and Food and Water Consumption The growth of male ICR mice fed the closed formula or purified diet for up to 84 days is shown in Figure 1. The body weights of the mice did not differ between dietary groups over this time span. Food and water consumption are shown in Table 7. Neither average food intake nor water consumption was statistically different between the 2 groups. Median Lethal Dose (LDSO) The effect of paraquat dose on survival of mice fed either the closed formula or purified diet is given in Table 8. The LD50 was 24 mg paraquat/kg (95% confidence limits = 20—30 mg/kg) for mice fed the purified diet and 38 mg paraquat/kg (95% confidence limits = 32—46 mg/kg) for mice fed the closed formula diet. This represents a po- tency ratio of 1.6 and the difference is significant at p<0.05. Median Effective Time (ETSO) The ETSO’ after an injection of paraquat (52 mg/kg), was 2.8 days (95% confidence limits = 1.9 to 4.1 days) for mice fed the closed formula diet and 0.72 days (95% confidence limits = 0.28 to 1.36 days) for mice fed the purified diet. The ET was significantly shorter 50 for mice fed the purified diet. 33 34 Figure 1. Effect of diet on body weights of male ICR mice. Mice were 28 days old at the start of the experiment. Diets were fed for 12 weeks. Body weights were recorded on a weekly basis. Each dietary group contained 20 mice. 35 Closed Formula Diet D—El Purified Diet H .5 O 33’ . f” I 0 330 / _§~ I m ll 0 4 8 12 Weeks on Diet FIGURE 1 36 TABLE 7 Effect of Diet on Food and Water Intake of Male ICR Mice Diet N Food Consumption Water Consumption (g/day/mouse) (ml/mouse/day) Closed formula 5 5.2io.2l 5.7:0.4 Purified 5 4.8i0.2 4.3i1.1 1Values are an average over 4 days of 5 groups/diet with 2 mice/ group. Mice were fed diets for 10 days prior to the start of the experiment. 37 TABLE 8 Effect of Diet on 7—Day Dose-Response to Paraquat Diet Dose (mg paraquat/kg body wt) 17 21 26 33 42 52 Closed formula 0/121 3/12 4/12 9/12 9/12 Purified 1/6 7/12 9/12 12/12 5/6 1Values are number of mice dead after 7 days over total number in— jected. Mice were fed the diets for 14 days before i.p. administra- tion of paraquat. 38 Survival Time and 7—Day Percent Survival After injection of paraquat (26 mg/kg) mice fed the purified diet for periods of time ranging from 3 to 84 days had significantly shorter survival times and lower 7-day percent survivals when compared to mice fed the closed formula diet (Figure 2). This difference in mortality was not observed after feeding the purified diet for only 1 or 2 days. Allowing the mice to become acclimated to the animal quarters by maintaining them on the closed formula diet for 4 days after arrival and then starting them on the purified diet did not affect the difference in mortality that was observed in the previous experiments (Figure 3, top). Initial Age of Mice The mice fed the purified diet from 56 to 66 days of age (after 28 days on the closed formula diet) still had a significantly shorter survival time and lower percent survival after paraquat treatment when compared to mice fed the closed formula diet continuously from 28 to 66 days of age (Figure 3, bottom). Dietary Alterations Results from the feeding of l of 6 diets are shown in Figure 4. A powdered form instead of a pelleted form of the closed formula diet did not affect the survival time or percent survival. Use of a pelleted cereal—based closed formula diet from another commercial source (Purina Lab Chow, Ralston Purina Co., St. Louis, MO) did not result in a different response from that of the first closed formula diet (Wayne Lab Blox). The feeding of a cereal—based open formula 39 Figure 2. Effect of duration of feeding on paraquat toxicity. £92; Twenty—eight—day-old mice were fed the closed formula or purified diet for 1, 2 or 3 days and then given an i.p. injection of paraquat (26—33 mg/kg). Each dietary group contained 15 mice. *7-day percent survival and survival time are significantly different from mice fed the closed formula diet for the same length of time, p<0.05. Bottom: Twenty—eight-day-old mice were fed the closed formula or purified diet for 14, 28 or 84 days and then given an i.p. injection of paraquat (26 mg/kg). Each dietary group contained 15 mice. *7-day percent survival and survival time are significantly different from mice fed the closed formula diet for the same length of time, p<0.05. 100— Percent Survival Survival Percent 4O Closed Formula Diet D-Cl Purified Diet H I - I I I 1 day I I I I 2 days I I I I 3 days 50" 1 day 2 days d ‘3 days 0 I I I I ‘ fi 100 I I I I I I I I 14 days I I I I 28 days 50. I I 84 clays * . .14 days 28 days '84 da 5 o . .y . . . 3 5 7 Day after Iniection FIGURE 2 41 Figure 3. Age and acclimation effect on dietary alteration of paraquat toxicity. Top; Twenty—eight—day-old mice were fed either the closed formula diet for 7 days or the closed formula diet for 4 days and then switched to the purified diet for 3 days. Then an i.p. injection of paraquat (26 mg/kg) was administered. Each dietary group contained 15 mice. *7—day percent survival and survival time are significantly different from mice fed the closed formula diet, p<0.05. Bottom: Twenty—eight- day-old mice were fed either the closed formula diet for 38 days or fed the closed formula diet for 28 days and then switched to the purified diet for 10 days. Then an i.p. injection of paraquat (26 mg/kg) was administered. Each dietary group contained 15 mice. *7—day percent survival and survival time are significantly differ— ent from mice fed the closed formula diet, p<0.05. 100- Percent Survival lOO Survival Percent 50- 42 Closed Formula Diet El—L'] Purified Diet H 50- I I I I I I Closed Formula Diet El-Cl Purified Diet H 3 5 7 Day after Iniection FIGURE 3 43 Figure 4. Effect of type of diet on paraquat toxicity. Twenty—eight-day-old mice were fed 1 of 6 diets for 28 days and then given an i.p. injection of paraquat (26 mg/kg). Each dietary group contained 15 mice. *7-day percent survival and survival time are significantly different from mice fed the other 5 diets, p<0.05. lOO Survival Percent 50-ll O‘—-_'l——F I I I I 44 Pelleted Closed Formula Diet D—U Purified Diet H Commercial Purified Diet A—A Purina Lab Chow H Powdered Closed Formula Diet O—O Open FormulaDiet B—lj . ‘ [-2 V V l 3 5 7 Day after Iniection FIGURE 4 45 diet (Table 4) or a commercially prepared purified diet (Table 3) also resulted in a response to paraquat injection that was similar to the response observed in mice fed the closed formula diet and signifi- cantly different from the one observed in mice fed the purified diet. Alteration of the source of protein in the purified diet, substi- tuting egg white for casein, resulted in a significant increase in survival time (Figure 5). The median effective time after paraquat (30 mg/kg) in mice fed this modified purified diet was also increased when compared to mice fed the purified diet (4.3 days vs. 2.1 days). Seven—day percent survival was not significantly different. Mice fed the commercial purified diet which also uses egg white protein showed a response to paraquat which is very similar to the modified purified diet (ET50 = 3.1 days). Increasing the lipid content of the purified diet did not result in a change in survival time or 7—day percent survival (Figure 6, top). This was true whether the lipid was high in unsaturated fat (corn oil), polyunsaturated fat (safflower oil) or saturated fat (lard). Supplementation of the purified diet with BHT (Figure 6, bottom) or vitamin E and selenium (Figure 7) did not protect against the increased toxic response to paraquat. Sex and Strain of Mice Female ICR mice did not differ from male ICR mice in response to an i.p. injection of paraquat after being fed a closed formula or a purified diet (Figure 8, top). The mice fed the purified diet had a 46 .mo.ova .mnoae m Hosuo msu wow oofia Eoum uaouowwwv kaudmoflmwawwm ma mafia Hm>fl>usme .oofiE ma coaflmuaoo asouw kumuoflw :omm .wa\wa omV umaamumm mo noeuoomafl .m.w am ao>flw ouoB ooHE oSu ouomon m%ww «a How mom oHoB muoflp oSH .aflommo NON How vouSuHumnam mwflaom ouflne wwo NON SuHB poem moflmwusm osu no ”pomp mommauaa awflonoaaoo Ho UQHMflHDQ .maaauom vomoao a wow oHoB mafia uHo hmwluzwfiolmuaoBH .mufloflxou unavmuma ao GOHumsouHm aflououa manuoflw mo uoomwm .m ouawflm 47 m mMDme actuate. .530 >00 m m _ rll_l|_l_ll._l_l_lul o 4 4 d m II II. M” w 4 room I I I w. A .. P matron. T 0.2.50". memo—U pacts.— 2_.._>> mam MIMI. 122.5.— _o.u._oEEou oo— 48 Figure 6. Effect of dietary alteration on paraquat toxicity. Top; Twenty—eight—day-old mice were fed 1 of 4 lipid—modified purified diets for 56 days and then were given an i.p. injection of paraquat (26 mg/kg). Each dietary group contained 6 mice. Bottom: Twenty—eight—day-old mice were fed a closed formula, a purified or a purified diet with 0.02% or 0.20% butylated hydroxytoluene (BHT) added. After 14 days of feeding mice were given an i.p. injection of paraquat (21 mg/kg). Each dietary group contained 15 mice. *Survival time and 7—day percent survival significantly different from mice fed the closed formula diet, p<0.05. lOO 49 Purified Diet (3% Corn oil) H 15% Corn oil A-A 15% Safflower oil 0'0 15% Lard D-El '6 .2 t a m 50- E o 3 o m - O I I T I I I I 100 II I I I I I I I I .. Closed Formula Diet D-El T: "‘ Purified Diet H .5 ‘ .0275 BHT Diet 0-0 5 .20% BHT Diet A—A 3,550. LA A -- A r .\ E: ‘I\ II II II. 3 n O- .- A¥ A A‘ u 7.: IA 0 I I I l I I I l 3 5 7 Day after Iniection FIGURE 6 50 .mo.ova .uoww maaauom vomoao onu wow mowEISOHm uaoHoMMHw kauamowmfiamwm Hm>fl>uam uaoouom hamln can dawn Hm>fi>name .oofia calm woafimuaoo anonw mumuofiw zoom .wa\wa omv unsawumm mo aowuoomafl .m.H aw monoumfiafiawm aosu can mhmw Hm How Aamm m¢.ov eaflaoamm was Auoflw wx\DH oomv m afiamufi> SuHB wouaoaoaamam uoflw onMHuaa onu Ho mommauam .maaauom womoao asp Honuwo mom who? mafia wH0I%mwlu£wHoI%uao3H .huHoHMou umavmmmm mo GOHumHouHm humuoflw ofiu do adHaoHom mam m aflawufl> mo moommm .m ouawwm 51 m mMDUHm «.0237: 5:0 >00 n m m — I, 5' I, On I In Berna om+me> I 3:. oozesa e Dln hwm—u U_DE._O* 1¢mO_U IDAgAmS waned oo— 52 Figure 8. Effect of sex or strain on dietary alteration of paraquat toxicity. Tgp; Twenty—eight—day—old female ICR mice were fed the closed formula or purified diet for 7 days and then administered an i.p. injection of paraquat (26 mg/kg). Each dietary group contained 12 mice. *Survival time and 7—day percent survival significantly differ— ent from mice fed the closed formula diet, p<0.05. Bottom: Twenty- eight—day-old male B6C3F1 mice were fed a closed formula or purified diet for 14 days and then administered an i.p. injection of paraquat (26 mg/kg). Each dietary group contained 15 mice. *Survival time and 7—day percent survival significantly different from mice fed the closed formula diet, p<0.05. 53 100 I Female ICR Mice I I I I I I I I E I Closed Formula Diet EJ-Cl 'E Purified Diet o—o «1:: 50- E 3 E l 0 j I I I I I lOO- I I I I I I I 3’ Male 86C3Fl Mice E D ”50- E 3 3 D- .. * O 3 5 7 Day after Iniection FIGURE 8 54 significantly shorter survival time and lower 7-day percent survi— val. The B6C3Fl male mice responded to the diet—paraquat treatment in a manner similar to the ICR.mice (Figure 8, bottom). Survival time and percent survival were both less in the B6C3F1 mice fed the puri— fied diet compared to mice fed the closed formula diet. At a paraquat dose of 26 mg/kg, male C57BL/6J mice (Figure 9) on both diets had a 7- day percent survival and survival time that appeared to be less than the ICR (Figure 2) or B6C3Fl (Figure 8, bottom) mice fed the closed formula diet after the same dose of paraquat. When the paraquat dose was lowered to 23 or 21 mg/kg, the survival time and percent survival increased. However, unlike the male ICR or B6C3F1 mice, no signifi— cant dietary difference in survival time or 7—day percent survival was noted after any of the 3 doses of paraquat (Figure 9). Exposure of mice to a 100% oxygen atmosphere for 7 days resulted in the survival patterns shown in Figure 10. No deaths were observed for 4 days and then survival decreased rapidly in mice fed either diet. There was no significant difference in survival time and 7-day percent survival waa'greater in mice fed the purified diet. Figure 11 gives the results of an i.p. injection of carbon tetra— chloride (0014) in mice fed either a closed formula or a purified diet. As can be seen, the survival time and 7-day percent survival show a large decrease with a very small change in dose. However, there was no dietary effect on the toxicity of CCl4 at either dose. 55 50mm .owow wx\wa om mam Hm osu now own mo wkmw ma ou om vowamu uaoEfiHmaNo as“ mo waflaaflwon msu um mafia osu mo own ao>flw aonu mam mhwv «a mom uofiw voMMdem n so masfiyom womoao .kufloflNOu umavmumm mo coaumhouam mum .ooHE mHIHH voaHMuaoo maomw mnwuoww ow wx\ma mm man how mzmw mmufim EOMM osH .umsvmuma mo aowuoohafi .m.H an a one some none 83mg 3o: uoflv no ammuum mo moomwm .m oHDMHm 56 m HMDUHm cozooE. BIO >00 n m m _ mfg: 0N _ p . . . . o mime om - u I I I d a mass mm m. e - _U mo.\mE mm I ..Om S -- nu w A I I ml mx\mE a I I I I ...I. (7 fl 9.3.: E I I I I I I 09 IRE vetted Uluaflo 23:20”— memo—U 8:2 2:33 232 57 Figure 10. Effect of diet on oxygen toxicity. Twenty-eight—day—old mice were fed a closed formula or a purified diet for 14 days prior to exposure to an atmosphere of 100% oxygen. Each dietary group contained 25 mice. *7-day percent survival significantly different from mice fed closed formula diet, p<0.05. 58 100% OXYGEN Closed Formula Diet D—El Purified Diet H loo ‘ I. ‘- Survival I 50 '. Percent O 3 4 5 6 7 8 Days of 02 Exposure FIGURE 10 59 .omow\ouwa ma woafimuaoo macaw mnmuofiw Loam .owfluoanowuuou aopumo mo coauomhafl .a.H an monoumwaaaum amau man made ea How uoflw woflmwusa a no maaauom womoao a wow mums mafia maelkmwlusmfiolmuaose .muHoHNou ovfiuoasomuuou cognac do mafia mo moommm .HH madman 60 HH mMDon 23.00?— 330 >00 m m 00:5 ON I'III'III, SSE 3 - ole 85 some: . 1.); DID .20 0.3—:0”. mono—U emote—$00.3... coat0u iueaied Ion IDAgAJnS oo— 61 Nonprotein Sulfhydryl Group Compounds (NPSC) Paraguat — Concentrations of NPSC in lung and liver 3 hours after an i.p. injection of paraquat (42 mg/kg) are given in Table 9. No differences between dietary groups were observed in control animals (0 mg/kg). The only statistically significant difference noted was between control mice fed the purified diet and injected mice fed the closed formula diet. Carbon tetrachloride - Kidney and liver NPSC concentrations did not differ between control (0 mg/kg) mice fed a closed formula diet or a purified diet for 14 days (Table 10). Four hours after an i.p. injection of CCl4 (2.5 ml/kg) kidney NPSC concentration was signifi— cantly decreased by 30—40%. This decrease was seen in mice of both dietary groups and there was no significant difference between groups in the decrease. No differences either from control or between dietary groups were observed in liver NPSC concentrations 4 hours after CCl4 injection. Twenty-four hours after a smaller dose of CCl4 (1.75 ml/kg), kidney NPSC was unchanged from control and no differences between dietary groups were noted. Liver NPSC concentration was significantly depressed (83% decrease from control for both dietary groups) 24 hours after CCl4 administration, and the decrease was similar for both dietary groups. After feeding the diets for 84 days, a similar response to CCl4 was observed in mice 24 hours after injec- tion of 1.75 ml/kg. No effect of €014 or diet was observed in kidney NPSC, while liver NPSC was significantly lower (98%) in mice fed either diet. 62 TABLE 9 Effect of Diet and Paraquat Injection on the Concentration of Nonprotein Sulfhydryl Group Compounds (NPSC) Para uat Dose NPSC Diet (mq/k ) (mg/g wet wt) g g Lung Liver bl Closed formula 0 0.54:0.08 1.99:0.173’ 42 o.53ro.03 1.82:0.l4a Purified o 0.65i0.10 2.60:0.24b 42 0.51:0.10 2.31:0.17a’b Mice were fed diets for 7 days prior to paraquat admini- stration. Control animals (0 mg/kg) were given 0.9% NaCl. Mice were killed 3 hours after injection. n=4 mice/dose/ dietary group. Values without the same superscript are sig— nificantly different, p<0.05. 63 .omoe\noae\ouea mun .uonoH meson am no a eoHHHx eon sane mo aceuoomaw am ao>fiw aonu mam mama am no «a How uoflw pawmmuaa no massaom vomoao m wow ouo3 woes .mo.ova .uaouommflw mauamoflmwawflm ohm umfihomuoaam oemm mfiu anomuwz moaam> .wafiwoow mo haw a 00m mafia m aflnuflz .03 no: m\afia\mommoaou oumnmmona moaofi: mm ao>flw ma kufi>fiuom ammumnmwosalolomooaao N H mH.HHN.m pm.NHw.m nNo.OHmo.o mN.OH©H.H mm.H oq.HHo.NN mm.HHm.wH «MH.OHMH.N No.0me.H 0 am woflwfiusm sN mm.HHw.m nw.oao.HH nqo.ommo.o w0.0HHN.H mN.H MADEHOM m.H.oam.mN mm.HHH.mH wmm.oumH.N ma.oan.H 0 am womoao «N n¢.NHm.mH m.HHm.w Loa.oawq.o ma.OHHH.H mN.H om.HHm.om N.Hnm.oa mom.omom.N mo.oamm.a o «a poflmwuam qN no.HHm.HH o.MHN.OH awa.omnq.o dH.OHOH.H mm.a maaauom mH.HHH.ON ¢.HHo.oa mmm.oamw.N No.0me.H 0 ma momoau «N ow.NHq.qN N.NH©.OH Ha.ommm.a nmo.ommm.o om.N mq.NHw.mN q.HHN.mH mN.OHHm.N mqa.oaam.a 0 ma onMHuam q 9H.Haq.qa m.HHm.OH HH.OHON.H ma.ouow.o om.N «Haemom o.om.mnm.mm H.0no.oa Ha.onom.a oma.o neN.a o as eouoau s N Ho>flg moawfim Ho>flq koavflm wa\wfiv moan ao AGOHuoomaH hufi>fluo¢ A03 umB m\wav a mafia momma muaosv m omom H00 mzmm omaumn monaloiomoosao ommz oaHH hoawflm 0am Ho>HA 0H hufl>fiuo¢ annumsmmonmlolomoosaw 00m Aommzv mwaaoaaoo aaouo aksvmnmaam aflououaaoz mo GOHumHuaooaoo onu do Aqaoov owfiuoanomuuoH aonumo wan poem we moowmm OH mqm “mm H kmv\omsoa\woom mo w owmuo>m mm do>Hw mosam>m .GOHuoonqfl ou Howum whom «H How madam man now whoa oofiz .mmso N map How wowmuo>m mos wmabmcoo woom ofiH .wQSU woo“ N paw ooflE m wufidflmuaoo Non m mucomoumou o:am>.m.o£HH sum.osm.m ourm.suo.e ouem.auo.e sm.osm.a ee.oue.H ss.ouN.H uuoN.oue.e N eueueuse masauom ouse.oua.e ouse.ous.m oueo.sse.m ouse.osm.m usN.ouN.N oa.oum.s NossNo.ouo.e Hm eouoao e m e m N H o u uses GOHuoomaH Houm¢ Amvhmo oxmuaH boom so umswmumm paw uoflm mo uoomwm «a mamme DISCUSSION The results from this research clearly demonstrate that the letha- lity of paraquat in mice is increased by the feeding of the purified diet (Table 2) as compared to the closed formula diet (Table 1). The LD50 dose for mice fed the purified diet was only 60% of the dose needed to kill 50% of the mice fed the closed formula diet. At a para- quat dose of 52 mg/kg, there was a 75% reduction in the median effec- tive time to death for mice fed the purified diet compared to mice fed the closed formula diet. Survival time and 7—day percent survival were also significantly less (Figure 2). A 7-day period was chosen to follow lethality since much of the comparative literature has used 4- 14 day time periods (Wasserman and Block, 1978; Hollinger §£_§1,, 1977; Autor, 1974) and preliminary experiments had shown that 7 days adequately covered the time period in which all deaths occurred. Given that there was a difference in response to paraquat between dietary groups, the next objective was to determine the dietary fac— tors involved. Several possible methodological reasons for the effects had been eliminated. Paraquat dichloride in saline (0.9% NaCl) was found to have a pH of 1.5-2.5 depending upon the concentra- tion. The paraquat dichloride—saline solution used in these experi- ments was adjusted to a pH of 7.0—7.4 using 1.0 N or 0.1 N NaOH. Drew and Gram (1979) have reported that unadjusted paraquat dichloride-saline 78 79 solution had a pH of 6.1-6.2. The reason for this difference is not known. When the paraquat dichloride was dissolved in a phosphate buffer (pH 7.4) instead of the NaOH—adjusted saline, the dietary alteration of paraquat toxicity was not changed (results not shown). Using water instead of saline as a carrier has been reported to alter paraquat toxicity (Drew and Gram, 1979). This was not tested during the present study. There was also a possibility that the difference in toxicity was due to a particular batch of closed formula diet or to the particular supplier of the diet. However, several batches of Wayne Lab Blox were used and, as can be seen in Figure 4, a closed formula diet from another supplier (Ralston—Purina Co., St. Louis, MO) produced the same results as the closed formula diet that was normally fed to the mice. Although the purified diet was in a powdered form and the closed formula diet was pelleted, this difference was not a factor since feeding the closed formula diet in powdered form resulted in the same response as feeding the pelleted form (Figure 4). Acclimation to the animal quarters was also ruled out as a fac— tor. Putting the mice on the closed formula diet for 4 or 28 days and then switching them to the purified diet resulted in the same in— creased sensitivity to paraquat as observed in mice which were started on the purified diet on the day of arrival from the supplier (Figure 3). The first question to be answered was whether or not the puri— fied diet was adequate in all nutrients. The diet was similar to the one described by the AIN Ad Hoc Committee (1977). However, incorrect formulation of the vitamin mix or lack of incorporation into the diet of some necessary macronutrient could result in a nutrient deficiency 80 that might account for the altered toxic response to paraquat. This is unlikely since the diet was prepared many different times and the altered response to paraquat was still observed. In addition, growth of the mice fed the purified diet was the same as mice fed the closed formula diet (Figure l). The purified diet was also made up using an AIN 76 vitamin mix (AIN Ad Hoc Committee, 1977) purchased from Teklad Test Diets (Madison, WI) instead of the AIN 76 vitamin mix formulated in the lab. No difference in response to paraquat between mice fed the purified diets with either vitamin mix (commercial vs. lab formu- lated vitamin mix) was observed (results not shown). The time course of the altered response, as shown at the top of Figure 2, also argues against an inadequate formulation. The dietary effect on paraquat toxicity is observed as early as 3 days after feeding of the purified diet. A nutrient deficiency (other than water or total food deprivation) would not be expected to cause such an increased sensitivity within such a short period of time. Contamination of the diets with drugs, heavy metals, pesticides, food additives or other xenobiotics also was possible. Several re- ports have indicated that closed formula diets in particular may contain detectable levels of such compounds (Coleman and Tardiff, 1979; Babish 35 al., 1978; Fox and Boylen, 1978; Fuhremann et 31., 1978; Tottmar gt 31., 1977; ILAR.Committee, 1976). Purified diets may also contain unintentional additives (Fox and Boylen, 1978). Analysis of the closed formula and purified diets using gas liquid chromato— graphy with an electron capture detector showed less than 10 ppb of 81 polychlorinated or polybrominated biphenyls or chlorinated pesticidesl. Other possible contaminants were not measured but it would seem that the differences observed in paraquat toxicity would have been reversed if the closed formula diet were contaminated with low levels of some toxic compound. Several available diets were tested to determine whether a simi— lar alteration in paraquat toxicity occurred. The response to para— quat after feeding other types of diets could determine the possible ingredients that are involved. For instance, if feeding a purified diet with a different composition resulted in another response to paraquat (compared to the original purified diet) then possible dietary factors to be examined would be far easier to evaluate because composition of both diets would be precisely stated. By modifying the original purified diet, one constituent or nutrient at a time, and then comparing the lethality of paraquat, factors which cause the altered response might be isolated. The results shown in Figure 4 demonstrate that the altered re— sponse to paraquat by mice fed the purified diet was not a generalized phenomena. Not only was there no alteration by feeding a different closed formula diet (Purina Lab Chow) or the powdered form of the first closed formula diet (Wayne Lab Blox), but the feeding of an open formula diet (Table 4) or a commercially prepared purified diet (Table 3) also did not result in a different response to paraquat administra- tion. lAnalysis done by Dr. W.E. Braselton, Jr., Dept. of Pharmacology and Toxicology, Michigan State University, E.Lansing, MI. After extraction 1 mg equivalent diet was injected onto 1% OV-l at 245° for polybrominated biphenyls and 1% OV-l7 at 210° or 190° for polychlori- nated biphenyls or chlorinated pesticides, respectively. 82 The significance of these results was that now there were 2 puri- fied diets which when fed to mice resulted in different responses by the mice to paraquat. When the composition of the purified and commercial purified diets are compared, there are 2 major differences in the macronutrients used (Tables 2 and 3). The type of protein is different, casein in the purified diet and egg white in the commercial purified diet, and the amount of lipid is different, 3% corn oil in the purified diet and 10% corn oil in the commercial purified diet. Modifying the lipid fraction of the purified diet did not prevent the increased sensitivity to paraquat (Figure 6, top). It was hypo— thesized that the lipid fraction might influence paraquat toxicity by means of the suggested lipid peroxidation mechanism. If lipid mem- brane in the lung was altered by feeding high fat diets then it could be postulated that generation of free radicals by paraquat would be enhanced, thus causing more membrane damage, if the diet, and there— fore the membrane, was high in polyunsaturated lipids. On the other hand, a diet high in saturated lipids should slow down the propagation of free radicals because fewer double bonds would be available for lipid peroxidation. However, as can be seen in Figure 6, feeding a purified diet high in polyunsaturated fats (safflower oil) did not increase paraquat toxicity nor did feeding a purified diet high in saturated fat (lard) reduce the toxic response. The source of protein does appear to be a factor in the dietary alteration of paraquat toxicity. When the purified diet was modified by replacing the casein with egg white, survival time and median effective time were both significantly increased compared to mice fed 83 the original purified diet (Figure 5). The response was similar to the response observed in mice fed the commercial purified diet. This protective effect of the egg white is not understood at the present time. It is not known whether other protein sources, such as extracts from plants, animal organs or fish might also provide a protective effect. It is unlikely that there has been a change in the composi- tion of the proteins in the plasma or other organs since the proteins would not be absorbed as such from the gastrointestinal tract. Lock (1979) and Conning 25 a1. (1969) have reported unpublished observa— tions that paraquat is not bound to plasma protein. Hollinger and Giri (1978) have reported in zitrg‘protein-binding of paraquat in rat lung, although Rose and Smith (1977) give results that indicate a lack of any binding by lung tissue. It would seem then that the casein or egg white had not altered some protein—binding property of paraquat. The literature on dietary differences in response to xenobiotics suggests possible alterations in toxicity due to the type of fiber or the carbohydrate source. The purified diet used in the present experiments contained 4% cellulose. Mylroie et_al. (1978) also used cellulose as a source of fiber in their studies on lead toxicity. Chadwick gt 31. (1978) could alter the metabolism of the organochlo— ride insecticide, lindane, to a much greater degree by feeding 10% pectin than by feeding 10% cellulose. The possibility arises that by replacing the cellulose in the purified diet with either pectin or one of the plant fibers used by Ershoff (1974), the increased sensitivity to paraquat might be prevented. However, the commercial purified diet also contained cellulose (3% of the diet) and the increased sensiti— vity to paraquat was not observed in mice fed this diet. Ershoff's 84 studies always involved the addition of the chemical to the diet (Ershoff, 1954, 1957, 1972, 1974, 1976, l977a,b). It is possible that the protective effect seen with the various fibers in his studies resulted from a decreased absorption due to binding of the chemicals in the gastrointestinal tract. The present experiments involved intraperitoneal injection of paraquat and this would argue against the type of fiber as the factor which altered the toxicity of paraquat, because no interactions between paraquat and the contents of the gastrointestinal tract would be expected. Even if the type of fiber did cause paraquat to be transferred from the blood to the intestines, the fact that both the purified and the commercial purified diets contained the same type and amount of fiber suggests that a fiber— induced excretion of paraquat into gastrointestinal tract was not the cause of the altered sensitivity to paraquat toxicity. Ershoff (1977b) was also able to decrease the toxicity of sodium cyclamate in the diet by switching from.sucrose or glucose to corn— starch as the dietary source of carbohydrate. As was mentioned in the Introduction, Boyd (1968) reported that high sucrose diets caused an increased sensitivity to oral benzylpenicillin and caffeine. This suggests that the use of glucose in the purified diet could be the factor which caused the altered toxicity of paraquat. However, Mylroie 35 a1, (1978) did not see any difference in the increased susceptibility to lead toxicity when they switched from starch to sucrose in their purified diet. In addition, the commercial purified diet used in the present study had glucose as the carbohydrate source, yet animals fed this diet responded to paraquat as did animals fed the 85 closed formula diet. Pritchard and Warner (1977) also reported that while calcium cyclamate was more toxic to animals fed a purified diet than animals fed a closed formula diet, this was only true when cycla— mate was added to the diet (as was done by Ershoff) and not when cyclamate was given in an i.p. injection. The third objective of this study was to determine differences in paraquat metabolism that might be responsible for the altered response to paraquat by mice. Several broad aspects were examined first. In the initial studies the diets were fed for 14 days. Mice fed the purified diet for longer periods of time might adapt to the diet, either as a result of maturational processes or because of metabolic adaptations to the factor(s) in the diet which were causing the altered response. How- ever, feeding the purified diet for as long as 84 days did not result in a change in the increased sensitivity to paraquat (Figure 2, bottom). These mice were 112 days old and could be classified as adult animals at the time of paraquat injection. Therefore no adapta- tion to the diet was occurring. The mice used in most of the experiments were started on the diets at 28 days of age. If the mice were more mature when they were initially fed the purified diet the response to paraquat might not be different from that of mice fed the closed formula diet. However, when the mice were fed the closed formula diet from 28 to 56 days of age and then fed the purified diet for 10 days, the toxic response to paraquat injection was still greater than the response observed in 86 mice fed the closed formula diet throughout the whole experiment (Figure 3, bottom). Male and female mice were treated with paraquat to determine whether the sex of the mice was affecting the dietary-induced altera- tion in paraquat toxicity. As can be seen at the top of Figure 8, the female mice fed the purified diet were more sensitive to paraquat. These mice were 28 days of age at the start of the feeding and were fed the diet for 7 days. It is possible that a longer feeding time or starting the purified diet after sexual maturity might change the increased sensitivity that is observed. Other strains of mice were compared to determine whether the ICR mouse might have some specific metabolic difference which was respon- sible for the altered sensitivity to paraquat. The B6C3Fl mouse is used in cancer studies because it has a high rate of spontaneous tumor-formation. The B6C3Fl was chosen for comparison because, like the ICR.mouse, it is used frequently in research and any altered response to paraquat due to dietary regimen would be noteworthy to investigators using this strain. After 14 days of feeding the B6C3F1 mice fed the purified diet showed the same increased sensitivity to paraquat as did the ICR.mice (Figure 8, bottom). At a comparable dose of paraquat (26 mg/kg) male C57BL/6J mice had a shorter survival time and 7-day percent survival than ICR or B6C3F1 mice (Figures 2, 8- bottom and 9), and this increased sensitivity to paraquat was not altered by feeding the purified diet instead of the closed formula diet. It should be noted, however, that at every paraquat dose tested (21, 23, or 26 mg/kg) the survival curve appeared steeper and 7-day percent survival appeared lower, although not significantly, for the 87 C57BL/6J mice fed the purified diet. While there may be a strain difference in regard to overall sensitivity to paraquat, the dietary alteration in the sensitivity to paraquat may still occur in all 3 strains although it is less apparent in the C57BL/6J strain. The original hypothesis to explain an alteration in paraquat toxi— city in the mouse was that feeding the purified diet resulted in an increase in the susceptibility to free radical generation and/or in the resulting damage to lipid membrane. Carbon tetrachloride (C014) is an hepatotoxin whose mechanism of action has been linked to forma- tion of free radicals and lipid peroxidation (Recknagel and Glende, 1973; Recknagel, 1967; Slater, 1966; Recknagel and Ghoshal, 1966). While the morphological and histological changes in the lung from exposure to 100% oxygen differ from those observed after paraquat administration (Montgomery et al,, 1979; Smith and Heath, 1976) super- oxide radicals may still be involved in the process. If the purified diet altered a free radical-lipid peroxidative process in the mice, then both C014 and 100% oxygen may be more toxic to mice fed the purified diet. The results in Figures 10 and 11 do not support this hypothesis. Neither injection of CCl4 nor exposure to 100% oxygen appeared to alter the survival of mice fed the purified diet rather than the closed formula diet. If anything, 7-day percent survival after exposure to 100% oxygen was higher in mice fed the purified diet. When nonprotein sulfhydryl compounds (NPSC) were measured in liver and kidney after CCl4 and in liver and lung after paraquat, no dietary differences were observed (Tables 7 and 8). Since NPSC are depleted during free radical formation (Bus gt al., 1976; Younes and 88 Siegers, 1980) an increased formation of free radicals in mice fed the purified diet should have resulted in greater depletion of the NPSC. G6Pase activity was measured as an indicator of microsomal damage resulting from lipid peroxidation in kidney and liver after treatment of mice with CCl4 (Recknagel and Ghoshal, 1966). While CCl4 admini— stration did result in a decrease in G6Pase activity, the type of diet fed to the mice did not affect the decreased activity. The lack of dietary alteration of liver microsomal damage after C014, as evidenced by similar changes between dietary groups in G6Pase activity, would also argue against a dietary change in susceptibility to free radical lipid peroxidative damage. An antioxidant in the diet should also hinder the propagation of free radicals by scavenging and thus terminating the free radical reaction. This assumes that the antioxidant is absorbed and taken up into the tissues. Feeding 0.02% or 0.20% butylated hydroxytoluene (BHT) in the purified diet did not decrease the toxic response to paraquat in these experiments (Figure 6, bottom). Selenium and vitamin E are both involved in the lipid peroxida— tion system and selenium.(Bus et al., 1976) and vitamin E (Block, 1979; Bus gt_al,, 1976) deficient diets have been reported to increase the toxicity of paraquat. Selenium was not added to the mineral mix used in the present experiments. However, analysis of the purified and closed formula diets gave selenium concentrations of 0.04 ppm and 0.40 ppm, respectivelyz. The National Research Council requirement 2Analysis done by Dr. D.E. Ullrey, Dept. of Animal Husbandry, Mich. State Univ., E.Lansing, MI. After sample digestion and oxidation, selenium was complexed with 2,3—diaminonapthalene and dissolved in cyclohexane. Fluorescence was measured at 518 nm on an Aminco-Bowman Spectrophotofluorometer (American Instrument Co., Inc., Silver Springs, MD) with a range of 0.60 to O. 05 ppm. 89 for selenium is 0.04 ppm in rats (Committee on Animal NUtrition, 1972). Since selenium was present at this level in the purified diet, it is doubtful that a deficiency could have occurred. Supplementation of the purified diet with vitamin E (500 IU/kg) and selenium (0.45 ppm) did not prevent the increased toxic response (Figure 7). The C57BL/6J strain of mice are known to be less susceptible than other strains to the toxicity of the halogenated hydrocarbon, chloroform (Vesell_etnal., 1976). Since another halogenated hydrocarbon, carbon tetrachloride (CC14), and paraquat are both postulated to generate free radicals (Recknagel and Ghoshal, 1966; Bus gt 31., 1976), it might have been hypothesized that the C57BL/6J mouse would be more resistant to the effects of paraquat, since the mechanism of action would be similar to that of C014. The results showing an increased sensitivity to paraquat in the CS7BL/6J mice (Figure 9) do not support this hypothesis. The results given above demonstrate that the increased suscepti- bility to paraquat seen in mice fed the purified diet does not result from an increase in free radical formation and/or increased lipid peroxidation. The organ distribution of radiolabelled paraquat was measured to determine if there had been any changes in the concentration of para- quat in various organs over time. A higher organ concentration of paraquat in mice fed the purified diet compared to mice fed the closed formula diet could imply an increased toxicity to that organ and could help to explain the increase in lethality of paraquat. The results given in Figures 12—14 indicate that paraquat was concentrated to a 90 greater extent in the kidney and liver of mice fed the purified diet when compared to the same organs from mice fed the closed formula diet. The paraquat concentration in the lung did not differ between dietary groups during the 48 hour time period, even though the lung actively transports paraquat. In agreement with Sharp (1972), paraquat concentrations in the lung remained elevated while concentrations in other organs declined (Figures 12-14). Using in yitrg incubation of lung slices with 10 or 100 uM paraquat, Drew §E_§l, (1979) have shown that even after 4 hours there is virtually no efflux of paraquat from lung tissue. Rose and Smith (1977) have reported that paraquat uptake in 21E£2.by lung tissue is dependent upon the paraquat concentration of the incubation medium. If efflux of paraquat from the lung was minimal and the plasma concentration was higher, then the lungs of mice fed the purified diet would be expected to have higher concentra- tions of paraquat. This was not observed in the present experiments. Rose and Smith (1977) reported that the lung uptake of paraquat obeys saturation kinetics. If there was saturation of the paraquat trans- port system in the lungs of mice fed the purified diet then there could be higher concentrations of paraquat in the plasma, compared to concentrations in the plasma of mice fed the closed formula diet, without having higher concentrations of paraquat in the lungs. For mice fed the purified diet the average concentration of paraquat in the plasma from 3 to 12 hours after paraquat injection was 0.5 ug/ml or approximately 3xlO_6M. Rose and Smith (1977) did not find satura- O 0 O O O -5 tion of in_v1tro or $2 Vivo lung transport of paraquat at 10 M. 91 Therefore, the transport of paraquat into the lungs of mice fed the purified diet would not appear to have been saturated. Alternatively, Lock 3; a1, (1976) reported that several endoge- nous amines inhibited the uptake of paraquat into lung slices. Among these amines was the amino acid, lysine, which inhibited paraquat uptake by 35% at a concentration of 0.1 mM. Tryptophan, tyrosine and histidine at 1 mM did not affect paraquat uptake. If the purified diet had higher concentrations of lysine, and if feeding this diet resulted in higher blood lysine than in mice fed a closed formula diet, then this might explain the lack of increase in lung paraquat concentration. The lysine content of egg white and casein has been reported as 739 mg/100 g and 1077 mg/100 g of food, respectively (Food Policy and Food Science Service, 1970). Wayne Lab Blox (the closed formula diet used in the present research) contains 1640 mg lysine/100 g of food (circular from Specialty Feeds Dept., Allied Mills, Inc., Chicago, IL). The largest possible difference in lysine concentration was between the closed formula and egg white protein diets, and yet both the closed formula dietary group and the egg white protein purified dietary group were less sensitive to paraquat than the casein protein purified dietary group. Therefore, it does not appear that the lysine content of the diet was the factor in the protein which caused the difference in paraquat toxicity. Lock (1979) and Ecker et_al. (1975b) reported a decrease in plasma volume after paraquat treatment of rats and mice. A decreased plasma volume might result in higher plasma concentrations of paraquat as was observed in mice fed the purified diet (Figure 14). If the 92 purified diet alone, or by the interaction with paraquat, caused a further reduction in plasma volume, then the organs would be exposed to higher concentrations of paraquat which could result in increased toxicity. Therefore, hematocrit was measured in mice fed either the purified or closed formula diet. The purified diet alone did not affect hematocrit (Table 13) but within 8 hours after injection of paraquat, hematocrit rose in mice fed the purified diet and remained elevated for at least 72 hours. This elevated hematocrit could indi- cate a lower plasma volume which might partially explain the higher plasma paraquat concentrations that were observed up to 48 hours after injection (Figure 14). The higher plasma concentrations of paraquat could lead to higher organ concentrations and possibly increased toxicity. However, using decreased plasma volume to explain increased plasma and tissue concentrations of paraquat does not seem feasible in this research since the percentage increase in hematocrit was only 20% by 72 hours after injection (Table 13) while the plasma concen- tration of paraquat from 3 to 48 hours after injection was from 200 to 1000% higher in mice fed the purified diet compared to mice fed the closed formula diet. Even though plasma concentrations of paraquat were higher in mice fed the purified diet (Figure 14), excretion of paraquat into the urine was the same (Figure 14). Since urine volume did not differ between dietary groups in these experiments (Table 12) the actual quantity of paraquat excreted in the urine was also the same. Of the total amount of paraquat excreted in the urine over the 48 hour period, approximately 85% was excreted during the first 3 hours and 93 approximately 95% within 12 hours after injection. Approximately 17— 20% of the average total dose of 900 pg paraquat-base per mouse was excreted in the urine during the 48 hour period studied. Therefore, a large percentage of the dose either remained in tissues or had not been absorbed from the peritoneum in the first 48 hours after injec— tion. Murray and Gibson (1974) reported that 14% of an oral dose of paraquat was excreted in the urine 32 hours after administration, while Sharp gt_al, (1972) reported only a 3% excretion in the urine 24 hours after an oral dose of paraquat. On the other hand, Daniel and Gage (1966) reported 90-100% recovery of a subcutaneous dose in urine after 48 hours. Since practically all of the absorbed paraquat is excreted by the kidneys (Sharp gt al,, 1972; Murray and Gibson, 1974), several para- meters were examined in this organ to determine whether the purified diet had altered the renal handling of paraquat. The pH of the urine was significantly lower in mice fed the purified diet (Table 12). A urine pH which is more acidic might increase the excretion of com- pounds which are more basic since they would become more ionized in an acidic urine and less apt to diffuse back across the renal tubules (Foulkes, 1977). Since paraquat is already highly ionized before reaching the urine, it should not become more or less ionized in the urine. Because of the highly ionized form in which paraquat is usually present, very little passive tubular reabsorption would take place in the nephrons of the kidney. Therefore, a lower urine pH would not result in an increased concentration of paraquat in the plasma due to increased reabsorption from renal tubules. In mice, paraquat has been shown to be transported by the renal organic cation 94 system (Ecker 25 31., 1975a). In the present study, injection of paraquat caused a reduction in the ability of mice fed the closed formula diet to accumulate the prototype cation, tetraethylammonium (TEA), in renal cortical slices while mice fed the purified diet did not show an altered accumulation of TEA after paraquat injection (Table 11). The renal organic anion transport system appeared to be slightly stimulated by paraquat injection in mice fed the purified diet. These results do not demonstrate any major nephrotoxic effect on organic ion transport 3 hours after an acute dose of paraquat. A 3 hour time period had been chosen to correlate changes in transport with changes in the concentration of NPSC (Table 9). Since the ele— vated plasma and kidney paraquat was only first observed after 3 hours, a difference in ion accumulation might have occurred at 6 or 12 hours after injection as the exposure to higher plasma paraquat con- tinued. Blood urea nitrogen concentration is used as one indicator of kidney damage, primarily in relation to glomerular function (Berndt, 1976). There was a dietary difference in plasma urea nitrogen after injection of paraquat (Table 13). Plasma urea nitrogen appeared to increase as early as 8 hours after injection of paraquat in mice fed the purified diet although the difference was not significant until 72 hours after injection. It is not likely that the dietary difference in concentration of plasma urea resulted from increased protein catabolism due to a starvation type of situation (Cahill and Owen, 1970). While food intake did significantly decrease during this time period, the decrease was similar in both dietary groups (Table 14). 95 As was mentioned, urine concentration of paraquat was the same in both dietary groups but plasma concentration of paraquat was higher in mice fed the purified diet (Figure 14). This would indicate that paraquat was not being filtered at the same rate in mice fed the purified diet. Along with the elevated plasma urea nitrogen in mice fed the purified diet, the apparent decrease in glomerular filtration of paraquat would suggest some alteration in kidney function. Deter- mination of renal blood flow by measurement of para-aminohippurate clearance and determination of glomerular filtration rate by measure— ment of inulin clearance in paraquat—treated animals would be impor- tant in defining the possible alteration in renal function that is occurring. From these results it is not clear whether the elevated concentration of paraquat in the kidney that was observed from 3 to 12 hours after injection caused a more nephrotoxic response in mice fed the purified diet, was a result of increased nephrotoxicity, or resulted from other factors such as a greater absorption from the peritoneum or possibly less excretion into the gastrointestinal tract 0 CONCLUSIONS The results of this research and the research reviewed in the literature further substantiate the obvious need for a careful consi- deration of the nutritional methodology that is used in toxicological (and other) research. If dietary factors are not stringently con— trolled and kept consistent across treated groups, conclusions drawn concerning the actions of xenobiotics must be questioned as to their validity. Several conclusions may be drawn from the results presented in this study. First, based on LD survival time and 7—day 50’ ETSO’ percent survival, mice fed the purified diet were more susceptible to the toxic effects of the herbicide, paraquat, than were mice fed a cereal-based closed formula diet. This difference in susceptibility was apparent within 3 days after feeding the purified diet and re- mained after feeding the diet for up to 84 days. Neither age nor sex of the mice altered this dietary difference, while there may have been some strain differences. Second, one factor in the diet that was responsible for the altered paraquat toxicity appeared to be the type of protein used. Changing from casein to egg white as the protein source resulted in a less toxic response as measured by survival time and ETSO' It is not known whether other purified protein sources would also help prevent the increased mortality observed with a para- quat injection when casein is used as the protein ingredient. While 96 .. ‘r—r . . . . i ,. 11%.!![1‘1 97 the amino acid composition of the proteins may be the actual factor involved, an interaction of lysine with lung uptake of paraquat does not appear to have been altered by changing the protein source. The type and amount of lipid in the diet was not a factor in the increased toxic response observed in mice fed the purified diet. Based on a comparison of the composition of the purified diet and the commercial purified diet, neither the type of carbohydrate nor the type of fiber were responsible for the effects observed. It is possible that the type and amount of lipid, carbohydrate and fiber might be factors through an interaction with the type and amount of protein, but no conclusions can be drawn from this study. Third, it can be concluded that feeding the purified diet causes the organ concentration of paraquat to change. While lung paraquat concentrations remained the same, plasma and kidney concentrations of paraquat in mice fed the purified diet rather than the closed formula diet were elevated from 3 to 12 hours after paraquat injection. It is not understood why, given the higher plasma paraquat concentration, the lungs of mice fed the purified did not accumulate more paraquat. Based on a greater plasma urea nitrogen concentration at least by 72 hours, possibly as early as 8 hours, after paraquat injection, mice fed the purified diet may develop greater and earlier nephrotoxicity as a result of greater exposure of the kidney to paraquat. However, renal transport of organic ions as an indicator of kidney damage was not significantly affected by a dietary effect on paraquat toxicity. 98 A dietary alteration in a possible paraquat-induced free radical formation and/or lipid peroxidation was not responsible for the dietary effects that were observed. 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