ENZYME WNQCHEMESTRY A533 'E'Hfi P'AI‘HQLQGY Q}? EKFEMEKéQNFAL MFM'FE TOXEGGSW {N THE GQENEA PEG That: he “to anm d M. 5. MECBEGAN STATE UNWEESETY Mba Uzoukwu 1968 THESIS ABSTRACT ENZYME HISTOCHEMISTRY AND THE PATHOLOGY OF EXPERIMENTAL NITRITE TOXICOSIS IN THE GUINEA PIG by Mba Uzoukwu Three groups of experiments were designed to study tissue enzymatic changes in hypoxia due to nitrite toxicosis. In the first experiment guinea pigs were given nitrite in drinking water. In the second, nitrite solution was injected subcutaneously. The third experiment, designed to provide comparative information on enzymatic alterations, consisted of making the animals hypoxia by confinement in a chamber with reduced par- tial pressure of oxygen (p02). Weight loss, or impairment of weight gain, was observed in the groups given nitrite in drinking water. Generally, the animals on the higher level (1%) lost more weight and were less active than the controls or those on 0.5% nitrite solution. Hemoglobin levels and packed cell volumes fell in guinea pigs treated with either nitrite or low p02. The fall was greater in the latter group of animals. Grossly, the livers of animals given 1% nitrite drinking water appeared mottled. Histopathologic changes in all test groups consisted mainly of marked hyperemia of liver, spleen and kidneys and edema of the lungs. Degeneration of hepatic cells and renal tubular epithelium was observed in these tissues collected from animals given 0.5% or 1% nitrite in drinking water. Mba Uzoukwu Histochemically, succinic dehydrogenase activity was decreased in some tissues of test animals. But this decrease was not consistently marked in most cases to enable valid conclusions to be drawn. Lactic dehydrogenase activities were more consistently enough depressed, es- pecially in the heart, to warrant_a suggestion that lactic dehydrogenase was specifically affected by the hypoxic state. It was not determined which of the 5 isoenzymes was most severely affected. Similar changes were observed in the guinea pigs in the low oxygen chamber, but no histo- pathologic changes developed. It is therefore suggested that alteration in tissue enzymes may be the earliest indication of deve10ping tissue hypoxia, including hypoxia that results from nitrite toxicosis. ENZYME HISTOCHEMISTRY AND THE PATHOLOGY OF EXPERIMENTAL NITRITE TOXICOSIS IN THE GUINEA PIG By Mba-Uzoukwu A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1968 ACKNOWLEDGEMENTS The author is profoundly grateful to Dr. S. D. Sleight for his sus- tained interest and guidance throughout this research. The helpful suggestions and encouragement of Dr. R. F. Langham, Dr. G. L. Waxler, and Dr. C. C. Morrill, Chairman of the Department of Pathology, are very gratefully acknowledged. I would like also to express sincere thanks to Miss Patricia Lamb, who performed the histochemical tests, and Miss Carolyn Bailey, who helped to carry out the hematologic determinations. Finally I would like to record my indebtedness to the University fia’rm of ' and the United States Agency for International Development for making it possible for me to come to Michigan State University, East Lansing. ii INTRODUCTION. REVIEW OF LITERATURE. MATERIALS AND METHODS . RESULTS . DISCUSSION. SUMMARY AND CONCLUSIONS . REFERENCES. VITA. TABLE OF CONTENTS iii Page 11 . 31 . 35 37 41 Table LIST OF TABLES Average daily fluid consumption . . . . . . Weights of group representative guinea pigs Group average hematologic values. . . . . . Summation of enzymatic activities . . . . . Average hemoglobin (Hb) and methemoglobin (MHb) trations and packed cell volumes (PCV) in nitrite- injected guinea pigs. . . . . . . . . . . . concen- Hemoglobin concentrations and packed cell volumes of individual guinea pigs on oxygen-nitrogen mixture . . . iv Page 12 13 15 20 21 26 Figure 10 11 12 13 14 15 16 17 18 LIST OF FIGURES Page Kidney. Vacuolar degeneration of epithelium (1% NaNO2 in drinking water). . . . . . . . . . . . . . . . . . 16 Liver. Vacuolar degeneration (lZ NaNO2 in drinking water)° . . . . . . . . . . . . . . . . . . . . . . . . . . l6 Lung. Edema (e) and atelectasis (a) (1% NaNO2 in drinking water) . . . . . . . . . . . . . . . . . . . . . . 17 Heart. SDH reaction (control) . . . . . . . . . . . . . . . 18 Heart. SDH reaction (12 NaN02 in drinking water). . . . . . 18 Lung. Congestion (arrow) and hemorrhage (1% NaN02 in drinking water). . . . . . . . . . . . . . . . . . . . . 22 Heart. LDH reaction (control). . . . . . . . . . . . . . . 23 Heart. LDH negative reaction (1% NaN02 injected) . . . . . 23 Kidney. SDH reaction (control) . . . . . . . . . . . . . . 25 Kidney. SDH reaction (12 NaNO2 injected) . . . . . . o . . 25 Liver. LDH reaction (control). . . . . . . . . . . . . . . 27 Liver. LDH reaction (oxygen-nitrogen mixture). . . . . . . 27 Kidney. LDH reaction (control) . . . . . . . . . . . . . . 28 Kidney. LDH reaction - clumping (oxygen-nitrogen mixture). 28 Heart. Mitochondrial staining (control). . . . . . . . . . 29 Heart. Mitochondrial staining (1% NaNOz in drinking water) 29 Liver. Mitochondrial staining (control). . . . . . . . . . 30 Liver. Mitochondrial staining (1% NaN02 in drinking water) 30 INTRODUCTION The toxicity of ingested nitrate in man and animals is ascribed to its conversion product, nitrite, which, upon absorption, induces patho— logic changes. The pathogenesis of the lesions is not clearly understood. However, the most dramatic of the observed gross changes is the color change due to the oxidation of hemoglobin to methemoglobin. This oxi- dation leads to a pr0portionate reduction in the oxygen-carrying capacity of the erythrocytes and hence to reduced oxygenation of tissues. This conceivably could be expected to decrease, or even stop, all processes that require oxygen in the cells, especially the oxidative enzyme acti— vities. The resulting anoxia could, in addition, lead to organic and structural changes that may further interfere with cellular activities. The objectives of this investigation were to identify what gross or histologic changes, if any, occur in tissues following the administration of sublethal and lethal doses of sodium nitrite to guinea pigs. It was hoped also that these changes might be correlated with any observed altera— tions in the activities of succinic and lactic dehydrogenases. Comparing the changes induced by nitrite with those caused by acutely produced hypoxia was planned with the object of establishing the mode of action of nitrite on tissues. The results of such an investigation should provide valuable basic information on the pathogenesis of tissue changes in hypoxia generally, and nitrite toxicosis particularly. They should also indicate fruitful areas for further enzyme histochemical investigation in the guinea pig or other species under the same or identical conditions. REVIEW OF LITERATURE Pathogenesis of Nitrite Toxicosis There are 2 modes of action of nitrite on tissues. The first is oxi- dation of the ferrous component of hemoglobin to the ferric state, result- ing in the formation of methemoglobin, with a consequent loss of the affinity of hemoglobin for oxygen (Briggs, 1964). This oxidation also makes the residual oxyhemoglobin less capable of dissociation (Clark 25 31,, 1943). The second mode of action of nitrites is a relaxation of smooth muscle of blood vessels. This leads to hypotension and consequent hypostatic congestion (Hueper and Landsberg, 1940; Holtenius, 1957; Beck— man, 1958; Goodman and Gilman, 1964). The net effect of these 2 actions is the inducement of a hypoxic state in tissues and organs which Simon §£_§1, (1958) regarded as the trigger mechanism for the pathologic changes associated with nitrate and nitrite toxicosis. Smith (1961) suggested that there was an alteration of protein enzymes in hepatic vitamin A depletion of rats. In 1960 O'Dell §£_§1, reported similar alteration of enzymes in vitamin E deficiency of rats. Winter and Hokanson (1964) attributed these alterations to anoxia due to nitrite. The Pathology of Nitrite Poisoning In addition to the methemoglobinization of erythrocytes by nitrites, Makgill and Mavrogordato (1897) inferred from their experiments that nitrites also cause death by direct poisoning of body organs. Subse- quently, lesions attributed to nitrate or nitrite poisoning have included 3 vascular congestion with multiple small hemorrhages, focal hyaline myo- cardial degeneration, thickening and hyalinization of the media of coro- nary vessels leading to compression of their lumens, and endocarditis (Guberlet, 1922; Hueper and Landsberg, 1940; Whitehead, 1953; Case £5,313, 1957; Simon gt a1., 1958; Smith g£;§1,, 1959; Glauser, 1966). In the cow, Smith g£_§l, (1959) observed hyalinization of pulmonary alveolar walls and interstitial and alveolar thickening due to edema, fibrin, connective tissue and infiltration of eosinOphils. Hueper and Landsberg (1940) reported no changes in the lung parenchyma of the rat. Gastritis, with or without enteritis, was described by Hueper and Landsberg (1940), Whitehead (1953), and Case (1957). Associated with gastritis was vacuolar degeneration and necrosis of gastric epithelium in all animals investigated, including man. Hemorrhagic nephritis was described by Case (1957). Hyaline degenera- tion of renal tubules with intratubular brownish material (without affinity for Prussian blue) was reported by Hueper and Landsberg (1940). These workers also described thickened and hyalinized arteries and arterioles, lymphocytic infiltration and fibrotic or atrOphic glomeruli. However, in the aborted fetus, congestion and increased cellularity of the glomeruli with vascular distention were observed by Smith g£_§l, (1958). Abortion is an important feature of nitrate poisoning in the cow (Thorp, 1938; Muhrer g£_al,, 1961; Crawford g£_§1,, 1966), in the sow (Case, 1954; Smith.g£_§l,, 1959; Tollett EEH2l°: 1960; Case, 1963), and in rats (Case, 1957). Simon ££_§l, (1958) reported edema of the fetal membranes of the cow with characteristic intercotyledonary circumscribed, necrotic, and calcified pox—like lesions. In the rat, sublethal amounts impaired reproduction (Case, 1957) as they did in the guinea pig (Atallah, 4 1966). In the latter report, atrOphy of the epithelia of the seminal vesicles and epididymides was noted. Hueper and Landsberg (1940) observed splenic hyperemia with extra- cellular and intracellular brown pigment that reacted positively with Prussian blue stain. Frank necrosis of the pulp with subcapsular edema was described by Simon.§£_al, (1958). In the brain, edema and congestion or hemorrhages, increased cellu- larity of meningeal membranes, focal or diffuse lymphocytic infiltration and degeneration of ganglion cells of experimental rats were described by Hueper and Landsberg (1940). In swine, Smith g5 31. (1959) reported only multiple hemorrhages and degeneration of blood vessels of the brain. Effect of Hypoxia on Cellular Enzymes Cellular enzymes are believed to be the primary target of toxic agents. The mitochondria which contain oxidative enzymes are remarkably sensitive to any alteration in their environment (Pearse, quoted by Novikoff, 1961). The glycolytic process in erythrocytes requires the presence of reduced phosphOpyridine nucleotides, nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH). These nucleotides are also needed for normal reduction of methemoglobin to hemoglobin in the erythrocytes (Bodansky, 1951). Sodium nitrite pro- duces methemoglobin but does not suppress g1ucose-6—phosphate dehydrogenase (G-6—PDH) which catalyzes the oxidation of glucose-6-phosphate in the presence of oxidized nicotinamide adenine dinucleotide phosphate (NADP) to form 6-phosphog1ucono-d—1actone. The byproduct of this reaction is NADPH. The absence of G-6-PDH has been reported in man. The deficiency results in cessation of production of NADPH with consequent accumulation 5 of methemoglobin in the erythrocytes and reduced oxygen-carrying capacity of the cell (Jandl, 1963; Tegeris, 1966). Wachstein £5 31, (1957) studied the early effects of hypoxia induced by ligation of the renal artery in the rat. They concluded that altera- tions in the enzymes are the earliest indication of cellular change in ischemia. In similar observations in the rat liver under conditions of hypoxia, Postupaev and Maidanova (1966) ascribed cellular enzyme altera— tions to the hypoxic state. Ziegler's (1967) investigation led to the conclusion that there is a marked inhibition of respiratory enzyme capacity to transport electrons in severe hypoxic states. Albaum and Chinn (1953) observed decreased cytochrome oxidase activity in the liver in conditions of hypoxia. Enzyme Histochemistry A number of important enzyme systems, including the oxidative and respiratory systems, are concentrated in cellular mitochondria (White g5 31,, 1964). and these are known to be highly sensitive to any alterations in their environment (Pearse, 1960). Advantage has been taken of the interaction of the various mitochondrial enzymes with appropriate sub- strates in the presence of some dyes, not only to visualize the mito- chondria in normal tissues, but also to check the effects of environmental alteration on their enzyme content. Thus tetrazolium salts have been used extensively in light and electron microscopic studies on mitochondrial dehydrogenase and diphosphopyridine nucleotide diaphorases (Foraker 25_ .51., 1953; Nachlas g£_al,, 1957; Pearse, 1960; Chason and Pearse, 1961; Novikoff g£_al,, 1961; Sedar g£_§1,, 1962; Wachstein £5 31., 1962; Hess and Staubli, 1963). Wachstein E; El- (1960) also used the lead technique 6 in unfixed cryostat sections to demonstrate mitochondrial ATPase activity, and the modified Gomori technique for acid phosphatase to delineate lyso- somal activity. Zimmermann and Pearse (1959), while attempting to demon- strate specific sorbitol dehydrogenase, obtained a reaction which they called the "nothing dehydrogenase reaction". Schafer §£_a1, (1967) found that mitochondrial NADH shifted to the oxidized state if respiration ceased because of exhaustion of oxygen. This finding has been confirmed by von Korff (1967), who showed that succinate oxidation was essential for the continuous generation of NADH, but was inhibited by anaerobiosis. The investigation was therefore designed to obtain information on the effects of nitrite toxicosis on 2 of the mitochondrial enzymes inti- mately associated with synthesis of NADH. MATERIALS AND METHODS A total of 51 guinea pigs of the smooth, short—haired variety were used in 3 investigations. The animals were kept in groups of 2, 3, or 4 and housed in Open-top sheet metal cages measuring 2.5 x 1.5 x 1.0 feet with wood shavings as bedding. They were fed guinea pig pellets* §g_1ibitum. The first group consisted of 1 series of 8 treated and 4 control guinea pigs, and a repeat series of 6 treated and 2 controls. Their ages ranged from 1 to 12 months, and they were randomly selected for treatment or use as controls. In each series the treated guinea pigs were divided into lots of equal numbers and given either a 1% or a 0.5% sodium nitrite solution in drinking water. Consumption of nitrite was measured, as was water consumption. The solutions were made by dilution from a stock solution of 60% sodium nitrite stored in a dark bottle and refrigerated at 5 C. The weights of the guinea pigs were checked at the beginning and then biweekly until death or sacrifice. Laboratory investigation included initial, biweekly, and terminal determination of hemoglobin and methemoglobin levels, and the packed cell volume. Hemoglobin was estimated by the standard cyanmethemoglobin method, and the methemoglobin was determined by the method of Evelyn and Malloy *Rockland Laboratory Animal Diets; Tekland, Inc., Monmouth, Ill. 8 (Hawk, Oser and Summerson, 1954). The capillary tube method was used for evaluation of the packed cell volume. For all determinations except the terminal bleeding, it was found most convenient to make a small cut into the dorsal metacarpal vein as it turns medially over the edge of the metacarpus. Bleeding usually stOpped after a few seconds. This method was found more convenient than incising between the toes (Atallah, 1966). The terminal blood samples were obtained under ether anesthesia by cardiac puncture. Heparinized syringes and needles were used. The second experimental group consisted of an initial trial with 8 guinea pigs divided randomly into 4 subgroups of 2 each. After initial weight checks and blood sampling these were given a single subcutaneous injection of 1% sodium nitrite solution at the rates of 70, 60, and 30 mg./kg., respectively. The fourth subgroup was left for untreated con- trols. Blood samples were taken hourly for 5 hours or until death, if this occurred earlier, for determination of hemoglobin, methemoglobin and the packed cell volume. In a subsequent experiment with 6 guinea pigs, 4 were given the 1% nitrite injections subcutaneously at the rate of 60 mg./kg., supplemented every hour by injection of 10 to 20 mg. (l to 2 m1.) of the same solution. Two were used as untreated controls. Blood samples were taken every hour until death or for a maximum of 5 hours. In a repeat experiment utilizing 7 treated and 2 control guinea pigs, treatment consisted of a single subcutaneous injection of 60 mg./kg. of nitrite given as a 1% solution. Blood was collected once every 1 or 2 hours until death or for a maximum of 5 hours. Finally, to check the direct effect of anoxia, 8 guinea pigs were divided randomly into 2 groups of 5 and 3. The latter served as controls. The 5 were individually put in a dessicating jar of about 3-liter capacity. 9 The partial pressure of oxygen in the jar was reduced by slowly letting in nitrogen at a pressure of about 75 cm. Hg (approximately 1 atmosphere). The jar was sealed* to ensure no air leakage. As soon as the pressure of 75 cm. Hg was attained in the jar (usually in about 2 minutes) the nitrogen was cut off. The pressure tended to remain steady until the animal was taken out in_extremis for collection of blood, usually about 2 hours from the start. At necropsy, portions of the heart, lung, liver and kidney were fixed in 10% buffered formalin and prepared for sectioning and routine staining by the hematoxylin—eosin method. Portions of the same organs, about 1 x 1 x 0.5 cm., were collected and quickly frozen on brass specimen holders using dry ice and acetone. These specimens were kept in the cryostat (—15 C.) until used, usually within the hour, for enzyme histo- chemistry. For investigation of succinic and lactic dehydrogenases, frozen tis- sues were cut at 10 u and picked up on coverslips. The incubating media were made up using apprOpriate substrates, succinate and lactate, in which 0.025% monotetrazolium salt (MTT)** solution was incorporated according to the method of Chason and Pearse (1961). Diphosphopyridine nucleotide (DPN),*** succinate and lactate controls were included. Dupli- cate sections were incubated for mitochondrial staining using Novikoff's (1961) method. All sections were incubated for 30 minutes at 37 C. *Lubriseal, Scientific Products, Evanston, Ill. **MTT, Sigma Chemical Co., St. Louis, Mo. ***DPN, Sigma Chemical Co., St. Louis, Mo. 10 Where necessary, differential staining for fat and glycogen was undertaken using Oil Red 0 and Best's carmine, respectively. RESULTS Amongst the guinea pigs that were given drinking water containing nitrite the reluctance to drink during the first few days was apparently proportional to the nitrite content of the water. This differential fluid consumption persisted throughout the experimental period as indicated in the calculated over-all average consumption rates (Table 1). Generally the animals given 1% nitrite solution consumed less feed and progressively lost weight and condition. Several had watery ocular discharges and rough coats toward the end of the observation period. On the other hand, guinea pigs given 0.5% nitrite solution had no charac- teristic pattern of weight changes and appeared healthier and more alert than the 1% nitrite group. The control animals remained in good health and consistently gained weight. The weights of representative animals in the 3 categories that were on the experiment for 4 or more weeks are shown (Table 2). Clinical signs seen in both the injected guinea pigs and those main- tained on the oxygen—nitrogen mixture under pressure were similar. There was a gradual acceleration of respiration which was always preceded by a period of excitement. Later the animal became hyperpneic and the coat rough. Death ensued soon after this stage if the animal was not sacrificed first. Calculated as group averages for the different times, a lowering of hemoglobin and packed cell volume values was observed in all groups, but in no case were these values outside the normal range for the guinea pig 11 12 Table 1. Average daily fluid consumption Average con- sumption per Duration of NaN02 equivalent No. of Treatment day per guinea experiment consumed per day guinea pigs (% NaNOZ) pig (ml.) (days) per guinea pig (mg.) 4 1 18.6 37 186 4 0.5 30.0 50 150 4 0.0 46.4 50 0 13 Table 2. Weights of group representative guinea pigs* Accesion Age at Weight (Gm.) No. of start Treatment (weeks) guinea pig Sex (weeks) (% NaN02) 0 2 4 7 9 10 072029 M 10 1.0 504 440 340 072232 M 10 1.0 535 490 350 072263 F 8 1.0 450 390 375 072494 F 8 1.0 425 375 390 425 074308 F 9 1.0 550 545 520 490 072495 M 10 0.5 504 525 665 625 072846 F 8 0.5 370 315 --- 455 485 072233 F 8 0.5 450 345 320 072234 M 10 0.0 509 520 —-- 630 072496 F 10 0.0 509 465 -—- 655 680 072847 M 10 0.0 519 525 --— 640 680 705 *Data for animals treated for 4 or more weeks l4 (hemoglobin, ll—l6.5 Gm./100 ml. blood; PCV 37-47%). The methemoglobin values were, for the most part, higher in the 1% nitrite group than in the 0.5% group. These values are reproduced (Table 3). The gross lesions in the 1% nitrite group consisted of brown dis— coloration of tissues, grayish-white mottling of the liver, marked con- gestion of the uterus, and slight enlargement of the spleen and adrenals as determined by visual comparison. No gross lesions were observed in the heart or kidneys; however, on microscopic examination, they were hyperemic. Vacuolar degeneration of renal tubular epithelium (Figure l) with necrosis and desquamation was noted in a number of the guinea pigs. Less commonly observed was dilation of the tubules. Centrilobular vacuo- lar degeneration of hepatic cells (Figure 2), associated with lymphocytic infiltration, was present in parts of the livers of these animals. Sec- tions of the livers and kidneys stained negatively for fat and glycogen. Microsc0pica11y, the lungs were edematous, congested or hemorrhagic and atelectatic (Figure 3). Enzymatically, succinic dehydrogenase activity in the heart was only slightly diminished (Figures 4 and 5), but the lactic dehydrogenase activity was much less than in the control animals, the degree of deple- tion appearing to increase directly with the length of treatment. Succinic dehydrogenase activity in the liver, lung and kidney was subject to indi— vidual variability. Similarly, variable results were obtained for lactic dehydrogenase in these 3 organs. As with the 1% nitrite group, but to a lesser extent, tissues in the 0.5% sodium nitrite—treated guinea pigs were brownish. HistOpatho- logically, tissue sections from these animals were not distinguishably different from the normal controls in those cases in which treatment 15 Table 3. Group average hematologic values* Treatment Time Hemoglobin Methemoglobin Packed Cell (% NaNOz) (weeks) (Gm./100 ml.) (Gm./100 m1.) Volume (%) 1 0 14.0 0.2 45.6 2 15.2 2.1 43.6 4 12.6 1.7 39.9 6 12.9 0.4 39.0 0.5 0 14.5 0.1 46.1 2 15.7 1.3 39.0 4 14.6 1.1 44.0 6 12.8 0.6 40.7 0.0 0 14.6 0.1 46.6 2 16.3 0.3 40.8 4 14.2 0.2 45.0 6 13.5 0.2 42.5 *Normal values for the guinea pig (Reid, 1958): Hb 11-16.5 (14.4) PCV 37-47 (42) 16 Figure l. Kidney. Vacuolar degeneration of epi- thelium (1% NaNO2 in drinking water). H & E stain. x 600. '5' . u.‘ Figure 2. Liver. Vacuolar degeneration (1% NaNO2 in drinking water). H & E stain. x 150. l7 Figure 3. Lung. Edema (e) and atelectasis (a) (1% NaNO2 in drinking water). H & E stain. x 150. l8 Figure 4. Heart. SDH reaction (control). MTT stain. x 600. Figure 5. Heart. SDH reaction (1% NaNO water). MTT stain. x 150. 2 in drinking 19 lasted less than 4 weeks. But in 2 guinea pigs given 0.5% nitrite for more than 4 weeks the hepatic cells had a foamy appearance and sinusoidal dilation was observed. The kidneys were hyperemic and the epithelium of the proximal convoluted tubules appeared vesicular. The lungs were also hyperemic in some animals. Special staining of sections of liver and kidney for fat and glycogen revealed none. The succinic and lactic dehy- drogenase reactions varied with the individual animals but were in most cases less than the reactions observed in normal tissues (Table 4). The hemoglobin levels amongst the guinea pigs injected with 1% nitrite solution remained within the normal range until late in the obser- vation period, about 4 hours after injection in some cases. The high degree of methemoglobinization observed early in the mono-injected guinea pigs gradually decreased with time. With multiple injections, the hemo- globin and methemoglobin values tended to remain high but the hemoglobin level fell significantly after about 4 hours following the initial injec- tion. The packed cell volume, after remaining constant like the hemoglobin, fell precipitously in the fourth hour to levels far below normal (Table 5). At autopsy, evidence of pulmonary edema was observed grossly in some guinea pigs. However, edema of the lungs was observed microscOpically in all animals injected, and this was associated with pulmonary hyperemia or hemorrhage (Figure 6). Most of the organs were hyperemic and the renal tubular epithelium had a vesicular appearance. Histochemically, both subgroups in the injection experiment manifested some decrease in succinic and lactic dehydrogenase activities. The decrease in LDH was constant and marked in the heart (Figures 7 and 8). The extent of decrease in both enzymes in the liver varied from one animal to another. There was only a slight decrease in the lung and kidney. However, there 20 Table 4. Summation of enzymatic activities* Treatment 1% NaNO2 0.5% NaN02 NaNO2 N2/02 Enzyme Tissue Control oral oral injection mixture SDH Heart 4 2.4 3.2 2.3 3.5 Lung 4 2.2 3.0 2.7 3.0 Liver 4 2.6 3.2 2.7 3.2 Kidney 4 2.4 3.0 2.5 2.7 LDH Heart 4 1.4 2.5 1.4 2.0 Lung 4 2.2 3.0 2.7 2.5 Liver 4 2.4 3.2 2.8 3.0 Kidney 4 2.8 2.6 2.5 2.7 Mito— Heart 1.0 2.0 ——- 2.5 3.0 chon— drial Lung 1.0 4.0 --- 2.0 2.0 reac— diminishing order of positivity from 4 to 1. *Figures represent average scores for animals in each group in a 21 Table 5. Average hemoglobin (Hb) and methemoglobin (MHb) concentrations and packed cell volumes (PCV) in nitrite-injected guinea pigs Mono-injected Multi-injected Time Hb. MHb. PCV Hb. MHb. PCV (min.) (Gm./100 m1.) (%)' (Gm./100 m1.) (%) 0 13.7 0.61 46.9 13.8 0.39 44.2 60 12.1 7.4 36.5 12.4 6.4 38.2 120 12.5 4.5 --- 12.7 6.3 39.0 160 11.4 1.8 --— 180 13.0 4.6 39.0 240 9.5 4.5 34.5 Figure 6. Lung. Congestion (arrow) and hemorrhage (1% NaN02 injected). H & E stain. x 150. 23 Figure 7. Heart. LDH reaction (control). MTT stain. x 600. Figure 8. Heart. LDH negative reaction (1% NaNO2 injected). MTT stain. x 600. 24 was a demonstrable localization of both enzymes, especially SDH, in the kidney (Figures 9 and 10). Results of the oxygen-nitrogen dilution experiment indicated that, following a relatively steady state, the hemoglobin concentrations and packed cell volumes fell after about 2 hours. The final values were generally lower than in the nitrite experiments (Table 6). No signifi- cant gross or histopathologic lesions were seen. Enzymatically, succinic dehydrogenase reaction in the heart, lung and liver remained strong, but was generally weaker in the kidney. Lactic dehydrogenase reaction in the liver was strong (Figures 11 and 12). The decrease in reactivity of this enzyme was most marked in the heart, with less decrease in the lung and kidney. Localization of the enzyme in the latter tissue was even more marked than in the nitrite injection experiments (Figures 13 and 14). Sections obtained from tissues of animals in all groups and stained specifically for mitochondria reacted positively. The reaction was more marked in all tissues of treated animals than in equivalent tissues of control guinea pigs (Figures 15, 16, 17, and 18). These results are included in the table of comparative enzymatic activities between the treated guinea pigs and the controls (Table 4). 25 Figure 9. Kidney. SDH reaction (control). MTT stain. x 600. . ' .\ ‘5' J. Figure 10. Kidney. SDH reaction (1% NaN02 injected). MTT stain. x 600. 26 Table 6. Hemoglobin concentrations and packed cell volumes of indi- vidual guinea pigs on oxygen-nitrogen mixture* Accession No. Hemoglobin Packed Cell Volume of guinea pig (Gm./100 m1.) (%) (Min.) 0 60 120 0 60 120 074434 14.6 --- 12.5 47.5 --- 42.0 074435 13.3 13.9 10.0 42.0 43.0 34.5 075982 ' 14.4 14.6 11.2 50.5 51.0 44.5 075983 12.7 --~ 8.9 40.0 --- 20.0** 075984 14.6 --- 12.5 47.5 --- 42.0 *The values for methemoglobin were essentially zero and are therefore not reproduced. **Slightly hemolyzed 27 Figure 11. Liver. LDH reaction (control). MTT stain. x 600. Figure 12. Liver. LDH reaction (oxygen-nitrogen mixture). MIT stain. x 600. 28 Figure 13. ‘Kidney. LDH reaction (control). MTT stain. x 600. Figure 14. Kidney. LDH reaction - clumping (oxygen— nitrogen mixture). MIT stain. x 600. 29 Figure 15. Heart. Mitochondrial staining (control). NBT stain. x 600. Figure 16. Heart. Mitochondrial staining (1% NaNO in drinking water). NBT stain. x 150. 2 30 Figure 17. Liver. Mitochondrial staining (control). NBT stain. x 600. Figure 18. Liver. Mitochondrial staining (1% NaNO in drinking water). NBT stain. x 600. 2 DISCUSSION The results of enzymatic reactions with succinate and lactate would appear to indicate that lactic dehydrogenase (LDH) was the more important component under these experimental conditions, as LDH was more consistently decreased. The depletion of heart and liver LDH agreed with the findings of Papadoupoulos g£_§l, (1967) on the effect of exercise on tissue LDH in the rat. It was not ascertained in the present experiment, however, which of the 4 isoenzymes of the heart was particularly affected. If, as Papa- doupoulos £5 31, (1967) found in the rat, Isoenzyme 5 occurs in the liver of the guinea pig, then this might be the liver LDH component depleted in this experiment. Since hypoxia was a common factor in both reports, similar depletion of cellular enzymes would be supportive evidence that this is the mode of action of nitrite on tissues. The histochemically observed decreases in succinic and lactic dehy- drogenases, with localization, probably represent early signs of cellular involvement and are likely related to the focal degenerative changes that were particularly noticeable in the renal epithelium. The supposedly positive staining of the mitochondria is the "nothing dehydrogenase" reaction described by Zimmermann and Pearse (1959). It represents the non-enzymatic production of formazan with some tetrazolium salts in systems containing NAD or NADP in the absence of substrates. The reaction was more marked in tissue sections of heart, lung and liver prepared from treated guinea pigs than in the controls. Schafer'ggngl. (1967) had reported a shift in mitochondrial NADH to a more oxidized state 31 32 (NAD) when respiration ceased because of exhaustion of oxygen. Such a shift could result in stronger activation of the non-enzymatic formazan production by mitochondria. It could, therefore, provide an explanation for the increased activation observed in tissues from animals either treated with sodium nitrite or maintained in low oxygen concentration environment. It would also support the postulate that nitrite toxicosis resulted, at least in part, from hypoxia. Nitrite in the 2 concentrations used produced some degree of inhi- bition of drinking in the guinea pigs. Those given 1% nitrite solution drank much less than the 0.5% nitrite group. However, the total average daily intake of nitrite was relatively high in the latter, 150 mg. as compared with 180 mg. by the 1% nitrite group. There was a marked and constant depression of weight response in the 1% nitrite group, whereas the response in the 0.5% subgroup varied with the individual. This indicated, as suggested by Atallah (1966), that there might be a critical quantity of nitrite that must be consumed to produce a predictable weight loss. Food consumption was most obviously depressed in the 1% nitrite group. Possibly this was part of the reason for the weight loss, but the nitrite ions might have affected the tissues directly or indirectly also. It is relevant to this that Ziegler (1967) reported that rats exposed to severe hypoxia with.§g_libitum feeding lost about.25% of body weight in 10 days. With restricted food intake there was up to 40% loss in 7 days. The "suffocated" guinea pigs survived a deficient oxygen mixture at a pressure of 75 cm. Hg for about 2 hours. In Altland's unpublished data (quoted by Reid, 1958), after exposure of dogs, rabbits, and guinea pigs to a barometric pressure of 17.8 cm. Hg at simulated altitude of 35,000 33 feet for 7 hours, 40, 100, and 52% of the dogs, rabbits and guinea pigs, respectively, died. These data tend to support the view that the guinea pig is relatively more resistant to hypoxia than some other species (Worden and Lane-Fetter, 1957). At both treatment levels, 0.5 and 1%, the hemoglobin and packed cell volume were depressed but there was no evidence of proportionality. Contrary to observations in other species (Winter and Hokanson, 1964) the relatively high level of methemoglobinization in animals given 1% nitrite did not lead to a rise in the hemoglobin value. In fact, there was a slight drop, which was in support of Atallah's (1966) findings. The absence of gross pathologic and histologic lesions in those animals that were on the lower treatment regimen (0.5% NaN02) for 28 days or less would appear to indicate that sodium nitrite at this level required a minimum period of time to exert a cumulative toxic effect on organs. The changes attributable to the more concentrated solution appeared much earlier. Their early develOpment was probably due to a more effective dilatory action on the vasculature causing hyperemia, slowing of the circulation, and decrease in the blood pressure with con- sequent hypoxia and cellular degeneration. Papadoupoulos g£_§l, (1967) observed fine droplets of fat in myo- cardial fibers and hepatic cells around the centrilobular zones in rats immediately after a 4-hour exercise. These histologic changes were not observed in equivalent tissues of guinea pigs given nitrite. Changes interpreted as hydrOpic were observed in the liver. The edema observed grossly and histologically in animals of the 1% nitrite injection group might have developed because of an immediate and more direct effect of the nitrite ions on the vasculature. This probably 34 caused hyperemia and stasis of blood. Neither in these injected animals nor in the group kept in the oxygen—nitrogen mixture was fatty change demonstrated. SUMMARY AND CONCLUSIONS 1. Sodium nitrite caused a loss of weight or impaired weight gain either by depression of appetite, induction of organic changes in the tissues, or by an undetermined mechanism. The effect on weight appeared to be quantitatively related to the amount of nitrite consumed. 2. In spite of the relatively high levels of methemoglobin, the hemoglobin and packed cell volumes remained within their normal ranges in nitrite-treated animals for the greater part of the experimental period. 3. Grossly and histologically, the liver and kidney were more severely affected than other viscera, congestion and vacuolar degenera- tion being the most frequent lesions. Pulmonary edema was also evident. 4. The greatest decrease in the enzymes occurred in LDH activity, especially in the heart. However, in the injection experiment, both SDH and LDH activities in the heart, lung, liver and kidney were appreciably decreased. 5. Succinic dehydrogenase (SDH) reactivity varied so much indi- vidually that it could not be regarded as a dependable model system for this particular study. 6. Lactic dehydrogenase (LDH) reactivity was decreased similarly in nitrite toxicosis, and with direct hypoxic state. This decrease may reflect early changes in the cells. Further investigation along this line could reveal which of the 5 LDH's is/are most involved. 35 36 7. The distinct non-enzymatic formazan production by mitochondria observed in both nitrite toxicosis and direct hypoxia should be further investigated to determine its true significance. REFERENCES Atallah, O. A.: Experimental Nitrate, Nitrite and Hydroxylamine Toxi- cosis in the Guinea Pig. Ph.D. Thesis, Michigan State University, 1966. Albaum, H. G., and Chinn, H. 1.: Brain metabolism during acclimatiza- tion to high altitude. Am. J. Physiol., 174, (1953): 141-145. Beckman, H.: Drugs, Their Nature, Action and Use. W. B. 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F.: Effect of long-term feeding of nitrate, nitrite or hydroxylamine on pregnant dairy heifers. Am. J. Vet. Res., 25, (1964): 353—361. 40 Worden, A. N., and Lane-Patter, W.: The UFAW Handbook on the Care and Management of Laboratory Animals, 2nd ed., University Federation for Animal Welfare, London, 1957. Ziegler, F. D.: Respiratory and phosphorylative responses to hypoxia and food restriction. Am. J. Physiol., 212, (1967): 197-202.. Zimmermann, H., and Pearse, A. G. E.: Limitations in the histochemical demonstration of pyridine nucleotide-linked dehydrogenases ("nothing dehydrogenases"). J. Histochem. and Cytochem., 7, (1959): 271-275. VITA The author was born at Umuahia, in what is now Biafra, on March 26, 1931. He received his primary education at the Methodist Central School, Umuahia. His secondary education was received at the Methodist College, Uzuakoli, from which he graduated in 1950. He entered a period of tech- nical training in the Federal Veterinary School, Vom, from 1951 to 1953, and the Nigerian College of Technology, Ibadan, from 1954 to 1956. Pro- fessional education was received in the Veterinary School of Glasgow University, Scotland, from 1957 to 1962, from which he graduated with the degree of B.V.M.S. and the M.R.C.V.S. For the following 2 years the author was engaged in research and teaching at the Federal Department of Veterinary Research, Vom. Late in 1964 he transferred to the Veterinary Department of the University of 01V”: if. it"; Q ' ~84e¥reg where he continued research and teaching until he came to Michigan State University, College of Veterinary Medicine, Department of Pathology, in the summer of 1966, to pursue studies leading to a Master of Science degree. He has published a scientific paper, "A Comparative Study of Vaccine Immunogenicity and Newcastle Disease Virus Strain Pathogenicity in Eastern Nigeria,‘ and co—authored a paper, "Serological Survey of Diseases of Cattle, Sheep and Coats in the Eastern Provinces of Nigeria." 41 ”'TITt'ITxMjM:MIEIEMTLEI'IQHI 9 WIT/"5