PATHOGENESlS 0F ABORTION IN ACUTE NiTRiTE TOXiCOSiS lN GUiNEA P‘GS Thesis for the Degree of Ph. D. MlCHIGAN STATE UNIVERSITY D\NESHWAR PRASAD StNHA 1968 “131': W” ‘mum 0-169 WLI-W‘M‘Ph. ”may ' i . .e 1! 'fi 1:; A 1:1 :5. A“ f\ Nikki f n State I. V L1 ,1— University This is to certify that the thesis entitled Pathogenesis of Abortion in Acute Nitrite Toxicosis in Guinea Pigs presented by Dineshwar Prasad Sinha has been accepted towards fulfillment of the requirements for PhoDo degree inPathOlog! ,1}: ; ”I f4 ” /(:L -i _4/Iy{'{r’f-A U /‘ 6&4? V” fl I Date ’3'éI/7'tW/LI ”1&1 / 9.1 57 Major professor / Bl'ElNG BY AG & SUNS' nnnx nmnrnv mu, ‘ iii-‘7”; “at! g a ABSTRACT PATHOGENESIS 0F ABORTION IN ACUTE NITRITE TOXICOSIS IN GUINEA PIGS by Dineshwar P. Sinha This work was designed to study the effects of acute nitrite toxi— cosis on reproduction in guinea pigs. Sodium nitrite was used as the source of nitrite. Since formation of methemoglobin is the major factor in the patho- genesis of nitrite toxicosis, methemoglobinization of blood ingiggg_was studied and the data obtained were used to build a model for the in give. studies. The lethal dose of NaNOz for guinea pigs was determined. Methemo- globin and plasma nitrite levels after administration of NaNOz were also determined. The pathologic and embryotoxic effects of acute NaNOz toxi- cosis at various stages of gestation were studied. A postmortem examina- tion of each animal was performed and sections of various tissues were prepared for histopathologic examination. The protective effect of methylene blue on NaNOz toxicosis was evaluated. Maternal and fetal P02, PC02 and blood pH values were determined after administration of NaNOz to the dam. Methemoglobinization occurred at a faster rate and higher levels were attained in gi££g_with fetal blood than with maternal blood. However, the reduction of methemoglobin to hemoglobin was faster in fetal than in maternal blood. After parturition the differences in methemoglobinization Dineshwar P. Sinha between maternal blood and blood from the newborn were reduced considerably. Studies in yi££g_indicated that even in the case of a high degree of methemoglobinization, the enzyme system was capable of reducing met- hemoglobin to hemoglobin. Following subcutaneous administration, NaNOZ was absorbed rapidly and the plasma nitrite level reached a peak between 7.5 and 15 minutes. The highest level of methemoglobin was observed about an hour after NaN02 administration. When dams were given NaNOZ subcutaneously, maternal plasma nitrite values were higher than fetal plasma nitrite values, indicating that there may be a partial placental barrier to transport of nitrite to the fetus. In the last quarter of pregnancy guinea pigs given NaNOz, 50 mg./kg., underwent normal parturition, whereas in those given NaNOz, 60 mg./kg., fetal mortality occurred and was followed by abortion 1 to 4 days after treatment. The fetal deaths occurred when the maternal and fetal met- hemoglobin levels were at their peak. At the time of fetal death there were no significant changes in the placenta; pathologic changes developed after death of the fetuses. The necrotic changes observed in the pla- centas were therefore a consequence rather than a cause of the fetal deaths. In pregnant guinea pigs given NaNOZ, 60 mg./kg., and treated simul- taneously with methylene blue, 10 mg./kg., no fetal deaths occurred. There were lower P02 and higher PCOZ values in the fetuses of the guinea pigs treated with NaNOZ, 60 mg./kg., than in fetuses of the con- trol animals. The results strongly suggest that fetal deaths resulted from hypoxia induced by methemoglobinemia. Dineshwar P. Sinha The administration of NaNOZ, 60 mg./kg., to guinea pigs in the first half of pregnancy produced less fetal mortality than when given in the last quarter of pregnancy. When NaNOZ, 60 mg./kg., was given to guinea pigs on or about the 30th day of pregnancy, deformity of the hind leg was observed in 2 of 16 newborn guinea pigs. PATHOGENESIS OF ABORTION IN ACUTE NITRITE TOXICOSIS IN GUINEA PIGS By Dineshwar Prasad Sinha A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1968 Dedicated to Mary Lynne ii ACKNOWLEDGEMENTS Many persons have contributed significantly to the development of this work. To them I wish to express my gratitude, not only for their influence on my academic activities, but also for the part they have played in shaping my personal life during this period. The brevity of this section by no means reflects the nature of my recognition of their importance, which I believe will be lifelong. Some persons will of necessity go unnamed, but to some in particu- lar, I am bound to pay public tribute: To Dr. S. D. Sleight, my major professor, for sharing his vast experience in the field of toxicopathology and my thanks for his many personal efforts on my behalf. His unsolicited support and cooperation have provided me with an example I shall long remember. To Dr. C. C. Morrill, my admiration for his outstanding ability to offer constructive criticism in a truly professional manner. To Dr. R. F. Langham, my high regard for his unfailing interest in teaching pathology, and for the many valuable suggestions he offered when I was planning my graduate program. Since few pieces of graduate level scientific investigation have ever been accomplished without close academic supervision or without collaboration, advice and assistance of persons allied elsewhere in the field, I would be failing in my duty if I did not deem it a special privilege and an enlightening experience to have had Dr. 0. Mickelsen on my committee. iii Last in mention but certainly not in importance, I wish to sin- cerely thank Dr. V. L. Sanger and Dr. G. H. Conner for their genuinely selfless encouragement and guidance. Also, my sincere gratitude to Dr. G. L. waxler for reviewing this manuscript. Finally, I would like to thank all who helped directly or indirectly, knowingly or unknowingly. iv TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . . . . . . Nitrate and Nitrite Intoxication. . . . . Methemoglobinemia Produced by Nitrites. . Effect of Nitrite on Reproduction . . . . Hypoxia and Teratogenic Effects . . . . MATHIALS AND MTHODS O O O O C O O O O O O O O O Technics, Chemicals and Care and Management Methemoglobinization of Guinea Pig Blood Induced by NaNOZ of the Animals. (ig_vitro) . . . . . . . . . . . . . . . . . . . . . Determination of Lethal Doses of NaNO for Guinea Pigs. 2 Determination of Levels of Methemoglobin and Plasma Nitrite after Administration of NaNO2 to Guinea Pigs Pathologic and Embryotoxic Effects of Acute NaN02 Toxicosis in the Last Quarter of Pregnancy . Prevention of Acute Toxicity of NaN02 in Pregnant Guinea Pigs by Treatment with Methylene Blue. . . . Effects of NaN02 Administration on Pregnant Guinea Pigs During First 30 Days of Gestation. . . . . . . . . Determination of Maternal and Fetal Blood Po2 , PCO and Methemoglobin Values after Administrati SOdium Nitrite O I O O O O O O O 0 RESULTS 0 O O O I O O I O O O O O O O O O I O O O Methemoglobinization of Guinea Pig Blood by NaN02 (in vitro). Determination of Lethal Doses of NaNOz for Guinea Pigs. . . . 8 .1311 n of Page 10 10 ll l3 14 16 18 18 19 21 24 DISCUSSION Determination of Levels of Methemoglobin and Plasma Nitrite after Administration of NaNO2 to Guinea Pigs . Pathologic and Embryotoxic Effects of NaNOZ Toxicosis . . Prevention of Acute Toxicity of NaN02 in Pregnant Guinea Effects of NaNO During F Pigs by Treatment with Methylene Blue. . . . . it Administration on Pregnant Guinea Pigs st 30 Days of Gestation. . . . . . . . . Maternal and Fetal Blood P0 , Values after Adminisfra 0 SUMMARY AND CONCLUSIONS. REFERENCES . VITA . vi PCOZ’ pH and Methemoglobin tion Of NaN02 o o o a o o Page 29 35 46 46 . 52 6O 65 67 7O Figure 10 ll 12 l3 14 15 LIST OF FIGURES Effect of NaNO (40 mg./100 ml.) on whole blood (A), blood diluted 50% in plasma (B) and RBC's suspended in physiological saline (C). . . . . . . . . . . . . . . . Methemoglobinization of guinea pig blood induced by NaNOZ, 40 Inga/100 ml. 0 O O O O O O O O O I O O O O O O O Methemoglobinization of guinea pig blood induced by NaN02 ’ 80 mg. I100 m1. 0 O O O O O O O O O O O O O O O O O Methemoglobinemia in guinea pig induced by NaNOz, 60 mgO/kg. (subcu.) O O O O O O O O O O O O O O O I O O O O O Methemoglobinemia in the pregnant guinea pig induced by NaNOZ ’ 60 mg I /kg 0 (Schu 0 ) O O O O O O O O O I O O O O O O Fetus of NaNOz-treated (60 mg./kg.) guinea pig killed 24.5 hours after treatment. Notice subcutaneous edema and discoloration (arrow). . . . . . . . . . . . . . . . . Fetus of control guinea pig killed same time as in Figure 6 I O O O O O O O O O O O O O O O O O O O O O O O O Hepatic lipidosis in NaNOZ-treated guinea pig. . . . . . . Higher magnification of Figure 8 . . . . . . . . . . . . . Necrosis of placenta in NaNOz-treated guinea pig . . . . . Higher magnification of Figure 10. . . . . . . . . . . . . Young (1 day old) of the guinea pig (No. 7) given NaNOz, 60 mg./kg. (subcu.). Notice deformity of hind leg (arrOWS) O O I O O O O O I O O O O O O O O O O O O O O O O The mean P02 value of treated and untreated guinea pigs. . The mean PCO2 value of treated and untreated guinea pigs. The effect of NaNO2 (6O mg./kg., subcu.) on maternal and fetal methemoglobin and P02 values . . . . . . . . . . . . vii Page 23 25 27 32 39 45 45 47 47 48 48 55 56 57 58 Table 10 ll 12 13 LIST OF TABLES ;g_vitro methemoglobinization of guinea pig blood induced by NaNOZ I I I I I I I I I I I I I I I I I I I I I Ig_vitro methemoglobinization of blood from guinea pigs of different ages. Dose level of NaNOz equivalent to 40 mgI /100 m]- I Of bloOdI I I I I I I I I I I I I I I I I I Mortality rate in male guinea pigs produced by subcu— 1231180118 adminis trat ion Of NaNOZ o o o o o o o o o o o o o 0 Determination of lethal dose of NaNOZ for fetuses of sninea pigs I I I I I I I I I I I I I I I I I I I I I I I I Methemoglobinemia induced by NaNOZ in guinea pigs. . . . . Plasma nitrite-nitrogen levels after administration of NaNoz to guinea pigs I I I I I I I I I I I I I I I I I I I Methemoglobinemia in male guinea pigs produced by NaNOz and methylene blue . . . . . . . . . . . . . . . . . . . . Methemoglobinemia in pregnant guinea pigs produced by NaNO I I I I I I I I I I I I I I I I I I I 2 o o o o o o I o Methemoglobin and plasma nitrite levels in the maternal and fetal blood after administration of NaNOZ (40 mg. /kg.) to dam I I I I I I I I I I I I I I I I I I I I I I I I I I Methemoglobin and plasma nitrite levels in the maternal and fetal blood after administration of NaNOz (60 mg. /kg.) to dam I I I I I I I I I I I I I I I I I I I I I I I I Effect on body temperature of subcutaneous administra- tion of NaNO2 (50 mg./kg.) to pregnant guinea pigs . . . . Reproductive performance of guinea pigs given NaN02 or NaCl subcutaneously. . . . . . . . . . . . . . . . . . . . Methemoglobin and plasma nitrite levels in the maternal and fetal blood after administration of NaNOz (60 mg. /kg. ) to dam I I I I I I I I I I I I I I I I I I I I I I I I I I viii Page . 22 26 28 30 31 33 34 36 37 38 4O 42 43 1,-...__ ..._ Table Page 14 Fetal mortality after subcutaneous administration of NaNOz or NaCl (60 mg./kg.) to pregnant guinea pigs . . . . . 44 15 Effect of NaNOz or NaCl (60 mg./kg.) on the fetuses of guinea pigs treated with methylene blue. . . . . . . . . . . 49 16 Reproductive performance of pregnant guinea pigs treated with NaN02 and methylene blue. . . . . . . . . . . . . . . . 50 17 Effect of NaNO administered subcutaneously to guinea pigs on the 10th day of pregnancy (estimated) . . . . . . . . . . 51 18 Effect of NaN02 administered subcutaneously to guinea pigs on the 20th day of pregnancy (estimated) . . . . . . . . . . 53 19 Effect of NaNOz administered subcutaneously to guinea pigs on the 30th day of pregnancy (estimated) . . . . . . . . . . 54 20 Maternal and fetal blood pH of NaNOz-treated (subcu.) and untreated guinea pigs I I I I I I I I I I I I I I I I I I 59 ix INTRODUCTION Nitrite poisoning among animals and man has been observed for centuries and reported from time to time as different syndromes by various observers. 4 With the advancement of technology, the use of nitrite is increas- ing as a food preservative, drug, herbicide and fertilizer. This may 3 lead to various health hazards, including abortion and possible terato- genic effects. Cases of abortion have been observed with nitrite poisoning in animals. The purpose of this experiment was to elucidate the patho- genesis of abortion in acute nitrite poisoning. Sodium nitrite was used in all the experiments to eliminate variables which might be intro- duced by using different nitrite compounds. The guinea pig was selected as the test animal for reasons of economy, litter size, size of fetuses and because the animal is easy to work with. Nitrite reacts with hemoglobin and forms methemoglobin. Methemo- globin leads to hypoxia or anoxia depending upon the level of methemo— globin present in the circulating blood. Hypoxia produces teratogenic effects, so it was important to study the effects of nitrite early in gestation when teratogenic effects would be most likely. REVIEW OF LITERATURE Nitrate and Nitrite Intoxication Nitrate fertilizer is palatable, especially to cattle, and has caused poisoning. Plants containing 22 or more of nitrate (dry weight) are dangerous to feed and may cause poisoning. In nitrate poisoning the major toxicant is nitrite which has been formed by the reduction of nitrate, probably by bacterial or enzymatic action within the digestive tract of the animal (Binns, 1956). The reduction of ingested nitrate in the digestive tract occurs readily in ruminants and other herbivorous animals. This is not the case in omnivorous and carnivorous species. However, all species are susceptible to poisoning by dietary nitrites (Jones, 1965). Riggs (1945) reported that nitrate as such is relatively nontoxic. The toxicity is prOportional to the amount of nitrate reduced to nitrite, which is readily absorbed from the digestive tract. Nitrite combines with hemoglobin to form methemoglobin, which produces signs similar to cyanide poisoning. His experiment, conducted with oat hay and turkey feed moistened with distilled water, showed that approximately half of the nitrate was reduced to nitrite and was present as such after 20 hours. He concluded that this reduction to nitrite may account for the poisoning associated with the ingestion of high nitrate hays following rains. McIlwain and Schipper (1963) reported that diets containing more than 0.5% potassium nitrate are toxic. Acute signs of intoxication are 2 3 related to an interference with oxygen-carrying elements of the blood. Chronically a vitamin A deficiency may occur possibly due to digestive irritation that results in decreased conversion of carotene. Goodrich (1962) reported that sheep fed 32 sodium nitrate in rations with or without the addition of vitamin A had significantly lower hepatic vitamin A stores. Muhrer ethal, (1956) observed signs of vitamin A deficiency in cattle that had consumed nitrate. Garner EEHEL- (1958) reported in- creased depletion of vitamin A in rats fed nitrate. Flynn (1961) found that hepatic vitamin A levels were low in sheep fed nitrate (0.1% to 0.752). O'Dell (1960) mentioned that nitrite caused depletion of vitamin A, and also precipitated a vitamin E deficiency in rats on a diet nor— mally adequate in vitamin E (5 mg./100 Gm.). Further evidence of the vitamin E deficiency was shown by the improvement of appetite and muscu- lar coordination when vitamin E concentrate was fed to the rats. It has been suggested that the nitrate ion may serve as a thyroid depressant by interfering with iodine metabolism (McIlwain and Schipper, 1963). Welsch (1961) reported enlarged thyroid glands in rats consuming a diet containing 2.52 KN03. Experimental results indicated that 0.31% and 0.92% dietary nitrate, consumed by rats and sheep, respectively, can affect the normal iodine metabolism of the thyroid gland (Bloomfield §£_§l,, 1961). The function of the thyroid gland in the conversion of carotene to vitamin A is still debated. However, Johnson and Baumann (1947) showed that a functioning thyroid gland is necessary for this conversion. 4 Nitrites in the body lower the tonus of arterial muscles producing a prompt fall in blood pressure. This action is directly on the smooth muscles and is mutually antagonistic with epinephrine (Sollmann, 1957). Naider and Venkatrao (1945) reported 30 cases of fatal nitrite poisoning in man. They estimated the lethal dose for an adult to be about 2 Gm. of sodium nitrite or about 2.6 Gm. of potassium nitrite. Simon gt El° (1959) stated that a lethal dose of potassium nitrate for cattle is 25 Gm./100 lb. of body weight. Case (1957) concluded that signs of nitrate intoxication vary from abortion and decreased production to severe intoxication that soon ends in death. The severity depended on the excess (over 0.52) of nitrate in the rations. Cattle and sheep fed high quality rations could tolerate more (1.52) nitrate in the total ration, whereas cattle on poor or deficient rations died with less (0.7%). Addition of carbohydrate and vitamdn A helped to reduce the toxicity if the nitrate content of the total ration was not over 1.52. Methemoglobinemia Produced by Nitrites Nitrites, as oxidizing agents, react with hemoglobin to form met- hemoglobin; this decreases the nitrite content of the plasma. The oxi- dation continues as long as the nitrite ions are in contact with the red blood cells. When nitrite-containing plasma or serum is separated from the red blood cells, the reaction ceases (Marich, 1965). Methemoglobin, also known as hemiglobin and ferriglobin, is a deriva- tive of hemoglobin in which the ferrous porphyrin complex is converted to the ferric form which does not combine with oxygen and is therefore of no value in respiration. The presence of the reducing enzymes in the 5 blood prevents complete oxidation of hemoglobin to methemoglobin. These enzymes are known as diaphorase I and II. The cofactor for diaphorase I is diphosphopyridine nucleotide (DPN), and triphosphopyridine nucleo- tide (TPN) is the cofactor for diaphorase II. In nitrite poisoning, the nitrite ion is a sufficiently good oxidizer to overcome the normal reducing effects of the diaphorase enzymes on methemoglobin. The nitrate ion does not have this effect (Jones, 1965). Austin and Drabkin (1935) noted that 0.5 to 0.7 mole of sodium nitrite is effective in converting 1 mole of oxyhemoglobin to methemoglobin. The alteration of the shape and position of the oxygen dissociation curve in methemoglobinemia implies that the tissues are liable to anoxia, not only because of loss of the oxygen carrying capacity of the blood, but also because the residual oxyhemoglobin is less capable of dissociating and thereby releasing oxygen in the tissues (Bodansky, 1951). Methemoglobin has a characteristic light absorption at 635 mu. This absorption is abolished in the presence of cyanide, which converts methemoglobin to cyanmethemoglobin. The difference in light absorption at 635 mu. before and after adding cyanide is a measure of the methemo- globin present (Evelyn and Malloy, 1938). Winter (1962) reported that hydroxylamine was formed from nitrite in the blood and was itself capable of converting hemoglobin to methemo- globin. Treatment with nitrate or nitrite had little effect on blood ammonia levels. Abbanat and Smith (1964) induced methemoglobinemia in female mice 'by the intraperitoneal injection of sodium nitrite (75 mg./kg.). The peak.methemoglobin formation (342) was achieved in about 40 minutes. 6 Keohane and Metcalf (1960) stated that there are gradual changes in the sensitivity of erythrocytes to sodium nitrite during childhood with a rapid increase in resistance at about the time of puberty. Hemo- globin of cord blood is more susceptible to methemoglobinization by sodium nitrite solution than adult hemoglobin. Ross (1963) reported that the blood of premature infants generally has a higher methemoglobin concentration than does that of infants born at term, and that they are more susceptible to the development of met- hemoglobinemia upon exposure to aniline dye. The TPN dependent methemo- globin reductase is decreased in cord blood. Emerick gt 5;. (1965) disclosed that pigs, given an intravenous injection of 0.03 Gm. sodium nitrite (22) per kg. body weight had a lower degree of methemoglobinemia when treated at 1 week of age (32%) than the same pigs when similarly treated at approximately 3 months (69%) and 5.5 months (802) of age. Studies by Stolk and Smith (1966) revealed that the hemoglobin of man, mouse, and rabbit show equivalent sensitivity to oxidation by sodium nitrite. The greater resistance of certain species to methemoglobin producing agents is due to higher levels of methemoglobin reductase activity. It is likely that the cat metabolizes aromatic amino com— pounds to methemoglobin-forming intermediates more actively than the other species. Setchell andeilliams (1962) observed that after oral administra- tion of sodium or potassium nitrate to sheep (nitrate equivalent to 2% of the food intake) the methemoglobin conversion reached a maximum of 40 to 65% of the hemoglobin. The maximum usually occurred 4 hours after drenching and then slowly declined. Repeated drenching for 6 days had 7 no cumulative effect on the concentration of nitrate or nitrite nitrogen of the plasma or the level of methemoglobin formation. Effect of Nitrite on Reproduction Sund and Wright (1957) reported several cases of abortion in cattle due to grazing on weeds of high nitrate content. The placentas of the aborted fetuses had degeneration of the cotyledons with numerous focal areas of mineral deposits. Garner gtnal. (1958) fed 4 lots of sows, 2 sows in each lot (starting 35 days after breeding), a ration containing 0.0%, 0.5%, 1.0%, and 2.0% of potassium nitrate. Serum nitrate levels were elevated with increasing amounts of nitrate in the ration. They mentioned that the litter size was not affected, but viability and the number of strong pigs decreased with the higher levels of the nitrate. Simon g£_§l, (1958) reported that in the lowland abortion syndrome in Wisconsin the characteristic pathologic changes included perirenal hemorrhage and marked kidney degeneration in the fetus, numerous circum- scribed, calcified necrotic lesions in the intercotyledonary areas of the fetal membranes, and pleural thickening and vascular changes in the lungs of the dam and aborted fetuses. A11 pathologic changes observed, including the abortion, could be attributed to anoxia. They found that abortions were associated with grazing on weeds. Simon'ggnal, (1959) placed KN03 (100 Gm.) in the rumen of pregnant dairy cattle. The blood of 1 heifer had 44.2% methemoglobin at the time of abortion. Another heifer had 65.5% methemoglobin in the blood and 2 days later aborted. 0n the day of abortion the methemoglobin value was 8.622. However, Winter (1964) stated that heifers given a balanced ration can maintain normal pregnancies despite prolonged ingestion of nitrate 8 or nitrite sufficient to convert 40 to 50% or more of the hemoglobin to methemoglobin. Muhrer g£_§l, (1956) concluded that a 1.0% to 2.0% concentration of potassium nitrate in the ration of the female rat interfered with normal reproduction. When female guinea pigs were given sodium nitrite in drinking water (0.5% or more), abortion, absorption or mummification of fetuses occurred (Sleight and Atallah, 1968). _Hypoxia and Teratogenic Effects Since nitrite poisoning leads to hypoxia or anoxia, it was important to consider the effect of hypoxia on fetuses. Oxygen deprivation, if complete, causes immediate death of the fetuses and, if partial, produces detrimental changes, the degree of which depends on the duration and severity of the lack of oxygen (Potter and Adair, 1949). A study on the effects of moderate hypoxia on chick embryos has shown that many of the induced malformations are caused by extensive edema followed by the formation of clear‘blisters and hematomas. The malformations have been attributed to a disturbance in fluid balance (Grabowski, 1961a). Grabowski (1961b) exposed 2000 three-day-old chick embryos to an environment containing from 0 to 21% oxygen, at normal pressure for periods of 3 to 24 hours. Abnormal development was initially obtained after exposures of 6 to 12 hours at an oxygen level of 13%, whereas the greatest maldeveloPment (in up to 40% of the embryos) occurred at about 52 oxygen. There was no maldevelopment at 2% oxygen because of high.death rate. v A'Ifl 9 A considerable variety of anomalies were detected in chick embryos (18 hours to 9 days of age) treated for 6 hours with less than 60% of normal oxygen concentrations. These abnormalities ranged from very slight defects to those making the embryo a complete monstrosity. A progressively increasing sensitivity to the teratogenic effect of hypoxia with advancing age was noted (Grabowski and Paar, 1958). MATERIALS AND METHODS Technics, Chemicals and Care and Management of the Animals Bleeding. To obtain large quantities ( > 2 ml.) of blood, cardiac punctures were performed on anesthetized animals. The puncture site was swabbed with 70% alcohol. A 1.5 inch, 20—gauge needle was inserted lateral to the sternum and aimed toward the point of the heart beat. When smaller amounts ( < 2 m1.) of blood were required, the bleeding was done by incising the paw. Whenever this procedure was employed, the feet were washed and swabbed with 70% alcohol. Hemostasis was accomplished with tannic acid and a bandage was applied. Anticoagulant. Heparin* was used as the anticoagulant (approximately 1 drOp from a 25-gauge needle for 5 m1. of blood). Determination of Methemoglobin (MetHb.). Methemoglobin values were determined photometrically using the Evelyn and Malloy (1938) procedure. As soon as possible the blood was mixed in M/60 phosphate buffer** in order to prevent reduction of the methemoglobin (Sleight and Sinha, 1968). * Heparin (Ammonium Salt) - Biological Research, Inc., St. Louis, Mo. ** A M/15 stock phosphate buffer (pH 6.6) was prepared by dis- solving 9.0 Gm. of NazHP04°12H20 and 5.7 Gm. of anhydrous KH2P04 in water and diluting to 1 liter. Three volumes of water were added to 1 volume of the stock buffer to prepare M/60 buffer. 10 11 Determination of Plasma Nitrite. The blood was centrifuged soon after collection and the plasma was removed. The procedure of Diven 35 31, (1962) was used. The readings were taken on a Coleman Junior Spec— trophotometer.* Sodium Nitrite. For all experiments a fresh solution of sodium nitrite (NaN02)** was prepared and used within an hour. r» Care and Management of the Guinea Pigs. They were housed in 30 x *** 18 x 12 inch steel boxes. Pelleted Rockland guinea pig diet and water were provided 2g.libitum. H ‘JXJHJ‘ '(2 Methemoglobinization of Guinea PiggBlood Induced by NaMQz (in vitro) Determgnation of Effect of Different Concentrations of NaNOz. Blood samples were collected from 4-month-old male guinea pigs by cardiac punc- ture. Each guinea pig was used only once. Test tubes (15 x 125 mm.) were placed in a 37 C. water bath, and 5 ml. of the heparinized blood was added to each tube. Sodium nitrite solution (1%) was added to the blood so that the tubes contained the equivalent of 5 mg., 10 mg., 30 mg., 40 mg., 80 mg., and 100 mg. per 100 ml. of blood, respectively. During the test, the tubes were slowly rotated in the water bath in order to keep the red blood cells in suspension. Hemoglobin, methemoglobin and packed cell volume (PCV) were determined at specific intervals for each sample. * Coleman model 6D Junior SpectrOphotometer — Coleman Instru- ments, Inc., 42 Madison Street, Maywood, Ill. ** J. T. Baker Chemical Co., Phillipsburg, N.J. *** Teklad, Inc., Monmouth, Ill. 12 Determination of Effect of Dilution and Plasma Removal on in vitro Methemoglobinization of Blood bnyaNQg. A heparinized sample of blood was collected from a guinea pig by cardiac puncture. The specimen was then divided into 3 parts as follows: 5 ml. of whole blood were trans- ferred to the first test tube; for the second tube 5 ml. of blood were centrifuged, the plasma removed and the cells washed in physiological saline (0.85%) and centrifuged again; the supernatant was discarded, the cells resuspended in physiological saline and the volume brought to 5 ml.; 2.5 ml. of whole blood were added to 2.5 m1. of plasma from the same animal for the third tube. Two-tenths milliliter of 1% NaN02 solution was added to all 3 test tubes so that each contained the equivalent of 40 mg./100 m1. This ex- periment was repeated. Determiningglnfluence of Age on Methemoglobinization. Blood was obtained from guinea pigs of the following ages: 1, 3, and 11 days, 3 and 9 weeks, and 4.5 months. Sodium nitrite solution was added to each tube so that the blood contained the equivalent of 40 mg./100 ml. of NaNOZ. Similar studies were also made on fetal and maternal blood samples. Reduction of High Levels of Methemoglobin to Hemoglobin in vitro. Sodium nitrite solution (1%) was added to 5 ml. of heparinized guinea pig blood so that it contained the equivalent of 80 mg./100 ml. The tube was placed in a water bath at 37 C. Hemoglobin and methemoglobin levels were determined on the sample at 38 and 68 minutes after addition of the NaNOz. Next the blood was centrifuged, plasma removed and the cells were washed 3 times with physiological saline to remove l3 extraerythrocytic nitrite. Following the last wash the supernatant was discarded and the cells were resuspended in the guinea pig's plasma; the volume was brought to 5 ml. The tube was placed back into the 37 C. water bath and the hemoglobin, methemoglobin and PCV values were de— termined at various intervals. Determination of Lethal Doses of NaN09 for Guinea Pigs, P‘ Males. A freshly prepared 2% NaN02 solution was given subcutaneously as follows: 100 mg./kg. to 10 guinea pigs, 90 mg./kg. to 10 guinea pigs, 80 mg./kg. to 10 guinea pigs, 70 mg./kg. to 10 guinea pigs and 60 mg./kg. A man body weight to 10 guinea pigs. The LD50 was estimated from the mortality data by using logarithms of the doses plotted against the percentage of mortality on a particular scale (DuBois and Ceiling, 1959). Pregnant Females. Sodium nitrite was given subcutaneosuly to 36 pregnant guinea pigs at the rate of 60 mg./kg. body weight. Four other pregnant guinea pigs were given a subcutaneous injection of NaNOz at the rate of 70 mg./kg. body weight. Fetuses. Three pregnant guinea pigs in the last quarter of gesta— tion were used for this study. The guinea pigs were anesthetized by giving pentobarbital sodium* (28 mg./kg.) and laparotomies were performed. The number of fetuses was counted and the approximate weight of each fetus was estimated. Varying doses of NaN02 were administered subcu- taneously to the fetuses. At least 1 fetus in each litter was left untreated. The incision was closed and an hour after treatment the * Pentobarbital Sodium Solution - Haver-Lockhart Laboratories, Kansas City, Mo. l4 fetuses were examined. At that time the fetuses were weighed and the exact amount of NaN02 given on a body weight basis was calculated. Determination of Levels of Methemoglobin and Plasma Nitrite After Administration of N3N09 to Guinea Pigg Different Doses of NaNOz to Male Guinea Pigs. A specific amount of NaNOz (10, 20, 30, 40, 60, 70 or 80 mg./kg. body weight) was injected subcutaneously. At timed intervals blood was drawn; hemoglobin and methemoglobin values were determined. Plasma nitrite was also determined in guinea pigs given 40, 60 or 80 mg./kg. of NaNOzo Evaluation of Protective Effect of Methylene Blue on NaNOZ Toxicosis. Methylene blue (1%) was administered intraperitoneally to a guinea pig at the rate of 40 mg./kg. body weight. Two guinea pigs received 60 mg./ kg. body weight of NaNOz subcutaneously and l of these guinea pigs was also given 40 mg./kg. of methylene blue (12) intraperitoneally. By incising the paw, blood samples were taken from both guinea pigs before injection and l, 2 and 3 hours after administration of the chemicals. Hemoglobin and methemoglobin levels were determined on each sample. The same experiment was done by using 100 mg./kg. body weight of NaNOZ and 10 mg./kg. body weight of methylene blue. This time methemoglobin and hemoglobin were determined before and 75 minutes after administration of the chemicals. Determination of Methemoglobin and Plasma Nitrite in Maternal and Fetal Blood During Last Quarter of Pregnancy. Sodium nitrite (40 mg. or 60 mg./kg.) was administered subcutaneously to pregnant guinea pigs. Blood samples were taken at intervals of 10, 20, 40 and 60 minutes after 15 injection for the determination of hemoglobin and methemoglobin. Blood was obtained by incising the paw and each toe was used only once. Four pregnant guinea pigs were given NaN02 subcutaneously at the rate of 40 mg./kg. body weight and a hysterotomy was performed on 1 each at 20, 40, 60 and 80 minutes after NaNOz administration. Ether was used as the anesthetic. Fetal blood was collected by cutting the umbilical cord and maternal blood was obtained by incising the paw. Levels of hemoglobin, methemoglobin and plasma nitrite were determined for each blood sample. Five pregnant guinea pigs were each given a subcutaneous injection of NaNOz at the rate of 60 mg./kg. body weight. A similar method as described above was used to obtain maternal and fetal blood at intervals of 20, 60, 100 and 140 minutes after injection. Hemoglobin, methemo- globin and plasma nitrite were determined on maternal and fetal blood. For controls, similar procedures were performed on normal untreated preg- nant guinea pigs. To follow maternal and fetal blood values in the same animal a technic was devised to take repeated samples of maternal and fetal blood at intervals. Pregnant guinea pigs were each given a subcutaneous in- jection of NaNOz (6O mg./kg.) and after 15 minutes an anesthetic, pento- barbital sodium (28 mg./kg.), was given. Laparotomy was then performed. Hemorrhages were controlled by ligation and clamping of the blood ves- sels. An incision was made in the uterine wall, 1 fetus was taken out, and the incision was closed. Fetal blood was collected by cutting the umbilical cord. At the same time maternal blood was collected by incising the paw. Manipulation was kept to a minimum in order to avoid any physi- cal interference with blood circulation. Gauze soaked in physiological l6 saline was placed over the exposed tissues to avoid dehydration. In a similar way, maternal and fetal blood samples were taken at 25, 40 and 60 minutes after administration of the NaNOz. Pathologic and Embryotoxic Effects of Acute NaNOZ‘Toxicosis in the Last (Quarter of Pregnancy Experiment 1. Six pregnant guinea pigs were divided into 3 groups of 2 each, and each group was placed in a separate box. Two groups were given subcutaneous injections of NaN02 (2%) at the rate of 50 mg./kg. body weight. One group, used as controls, was each given a subcutaneous injection of 2% NaCl at the rate of 50 mg./kg. body weight. The rectal temperature was recorded before and 0.5, l, 2, and 4 hours after administra- tion of the NaNOz. Reproductive performance was recorded. Experiment 2. Two groups of 2 pregnant guinea pigs each were used. One group was each given a subcutaneous injection of NaNOz at the rate of 50 mg./kg. body weight. The other group served as controls, and each was given a subcutaneous injection of NaCl (2%) at the rate of 50 mg./kg. body weight. An experimental and control animal each were killed at 12 and 24 hours, respectively, after administration of the chemicals. Blood samples were collected before injection of the chemicals and just before killing. Determinations for hemoglobin and methemoglobin were done on each blood sample. At the time of necropsy samples of adrenal, brain, 'heart, intestine, kidney, liver, lung, pancreas, placenta, spleen and 'uterus were fixed in 10% buffered formalin or in Zenker's fluid. Paraf- fin sections were made and stained with hematoxylin and eosin. Formalin- fixed sections of liver were also stained with Oil Red 0 stain. l7 Experiment 3. Six guinea pigs were divided at random into 3 groups of 2 each. Two groups were given NaN02 (2%) subcutaneously at the rate of 60 mg./kg. body weight. One group received NaCl (2%) subcutaneously at the rate of 60 mg./kg. body weight. Reproductive performances were recorded. Experiment 4. Twenty pregnant guinea pigs were divided at random into 10 groups of 2 each and each group was housed in a separate box. Hemoglobin, methemoglobin, total and differential leukocyte counts, PCV, and plasma nitrite were determined on all the guinea pigs before treatment. In 5 groups NaNOz solution (2%) was injected subcutaneously at the rate of 60 mg./kg. body weight. In the other 5 groups NaCl solu— tion (2%) was injected subcutaneosuly at the rate of 60 mg./kg. body weight to serve as controls. One experimental and one control guinea pig each were killed at the following intervals after administration of the NaN02 or NaCl: 0.25, 1.5, 3, 6, 12, 18, 24, 24.5, 48 and 56 hours. The guinea pigs were given a general anesthetic (ether) before killing. Hysterotomies were performed and the fetuses removed. Fetal and maternal blood samples were collected. The fetuses were examined to see if they were dead or abnormal. From the maternal blood, hemoglobin, total and differential leukocyte counts, PCV's, and methemoglobin and plasma nitrite levels were determined. From the fetal blood, hemoglobin, met- hemoglobin and plasma nitrite levels were determined. Blood samples were stained with 1% new methylene blue and examined for the presence of Heinz bodies. Tissues from adrenal, brain, heart, intestine, kidney, liver, lung, pancreas, placenta, spleen and uterus were fixed in 10% buffered formalin or in Zenker's fluid. Paraffin sections were made and stained 18 with hematoxylin and eosin. Formalin-fixed sections of liver were also stained with Oil Red 0 stain. Prevention of Acute Toxicity of NaNOz in Pregpant Guinea Pigs by Treatment with Methylene Blue Group 1. In 6 pregnant guinea pigs (last quarter of pregnancy) methylene blue was administered intraperitoneally at the rate of 10 mg./ kg. body weight, and in 3 of these animals NaN02 was given subcutaneously at the rate of 60 mg./kg. body weight. One nitrite-treated animal and 1 given only methylene blue were anesthetized with ether and hysterotomies were performed at the following intervals, after administration of chemi- cals: 3, 24 and 100 hours. Group 2. Six pregnant guinea pigs of the same age and approximately in the last quarter of pregnancy were selected for this experiment. In 2 guinea pigs methylene blue was injected intraperitoneally at the rate of 10 mg./kg. and NaNOz at the rate of 60 mg./kg. body weight. Two guinea pigs were given only methylene blue intraperitoneally at the rate of 10 mg./kg. body weight. The 2 other guinea pigs were given only NaN02 subcutaneously at the rate of 60 mg./kg. body weight. Reproductive per- formances were recorded. Effects of NaNOo Administration on Pregpant Guinea Pigs During First 30 Days of Gestation Group 1. Six pregnant guinea pigs at about 10 days of gestation were used. Four guinea pigs were given NaNOZ subcutaneously at the rate of 60 mg./kg. body weight. Two guinea pigs were given NaCl subcutaneously 19 at the rate of 60 mg./kg. body weight and served as controls. One nitrite-injected animal was killed on the 7th day and l on the 14th day after administration of the nitrite. Two nitrite-injected animals and 2 controls were allowed to complete gestation. Group 2. Nine pregnant guinea pigs at about the 20th day of gesta- tion were selected. Six animals were given NaNOz subcutaneously at the p“: rate of 60 mg./kg. body weight. Three guinea pigs were given NaCl sub- cutaneously at the rate of 60 mg./kg. body weight and served as controls. One experimental animal was killed at each of the following intervals after the start of the experiment: 2nd, 3rd, 8th and 11th day. One control animal was killed on the 2nd and another on the 8th day of the experiment. Two experimental animals and 1 control were kept under ob- servation and allowed to proceed to term. Group 3. Nine pregnant guinea pigs at approximately the 30th day of gestation were used. Six were given NaN02 subcutaneously at the rate of 60 mg./kg. body weight. The 3 controls were given NaCl (2%) subcu- taneously at the rate of 60 mg./kg. body weight. One of the sodium nitrite-injected animals was killed on the 2nd, another on the 7th, and a third on the 14th day of the experiment. One of the controls was killed on the 7th day. Two experimental and 2 control animals were not killed but were kept under observation. Determination of Maternal and Fetal Blood P02, PCO2,pH and Methempglobin Values after Administration of Sodium Nitrite For this experiment 9 pregnant guinea pigs in the last week of ges- tation were used. Three were injected subcutaneously with NaNOz at the 20 rate of 60 mg./kg. body weight. Blood samples were then collected at 30, 60 and 90 minutes after the injection. Maternal blood was drawn from uterine blood vessels and the fetal blood from umbilical blood vessels. Three of the pregnant guinea pigs were given NaNOz subcutaneously at the rate of 45 mg./kg. body weight and maternal and fetal blood sam- ples were collected at 30, 60 and 90 minutes after administration. 5mm Maternal and fetal blood samples were also drawn from 3 untreated ‘ guinea pigs, to determine normal values. Immediately after the blood was obtained a Radiometer* was used to _ determine the P02, PCO2 and pH. * * Radiometer A/S - 72, Emdrupvej, COpenhagennv, Denmark. RESULTS Methemoglobinization of Guinea Pig Blood by NaNOZ (in vitro) Effect of Different Concentrations of NaNOa. The methemoglobin level in blood containing NaNOz (5 mg./lOO ml.) reached a peak of 12.5% after 30 minutes and returned to less than 1% by the end of 2.5 hours. With increasing concentrations of NaNOZ there was a tendency towards prolongation of the time before attaining the methemoglobin peak. Also the time for reduction of methemoglobin was prolonged and incomplete at the higher levels of NaN02 (Table 1). There was no appreciable varia- tion in the PCV values at the various intervals for each blood sample. Effect of Dilution and Plasma Removal on in vitro Methemoglobiniza- tion of Blood by NaNQy. The whole blood, after addition of NaNOz, had a methemoglobin peak of 86.4% at 1.5 hours, which was gradually reduced to 11.8% at 5 hours. Methemoglobin levels in the samples of RBC's suspended in physio- logical saline were progressively elevated with time and little if any reduction had taken place by 5 hours (Figure 1). Blood diluted 50% with plasma also reached a methemoglobin peak, this time 89%, after 1.5 hours. By the 5th hour the methemoglobin level was 53%. Effect of Age of the Animal on Methemoglobinization. Fetal blood txnderwent methemoglobinization at a faster rate and higher levels were 21 22 m.m~ n.mo 0.00 0.00 0.00 0.00 m.~m 0.50 n.00 n.Hm 0.0m 0.mm 0.0 00H 0.0 <.H0 «.00 o.mw m.mn o.qm 5.0m 0.0m m.ne H.e¢ w.mm 0.0m 0.0 00 0.0 m.H m.< III m.me III n.0m 0.00 III III e.nn 5.00 «.0 0e III 0 m.H ~.0 III ~.q «.0H m.mN III m.am N.NN III 0.0 on III 0 0 e.~ ~.H 0.0 n.e H.HH n.mH ~.ma 5.0 ¢.H 0.0 0H III 0 0 0.0 m.0 ~.H 0.H c.¢ n.m m.~H H.¢ 0.0 0.0 m .mun .mun .mug .mun .mus .mp: .mp5 .Hs .dHa .sHa .swa .sfia .sfia A.HS 00H\.wav 2 m s m m . N N m A H 2 on 3 m 0 uses mam>umuaH um ANV Hm>ma nanofiwoaonumz Nozmz he pooswsfl vooan wHe mmsfisw mo coaumnwswnoawoamnuma ouufi>.mm_ .H magma METHEHOGLOBIN % 23 Figure 1. blood (A), blood diluted 50% in plasma (B), and RBC's suspended in physiological saline (C). ._____——-C C / \o I .-“\ . 1 x , \ /\- ‘3/ \. \‘ \‘ ,)\ A’ \ \ \ § \. \ \s B i \ \‘ \. \lt ‘ \* ' I U I fi 2 3 4 5 HOURS Effect of NaNO (40 mg./100 ml.) on whole 24 attained than with maternal blood. Also, the reduction of methemoglobin to hemoglobin was quicker than in maternal blood (Figure 2). These dif- ferences were reduced considerably after parturition. Three days follow- ing birth only a minimal difference was noted. Only slight differences in the rate of formation or reduction of methemoglobin could be detected in the blood from guinea pigs 9 weeks of age or older (Table 2). Reductipn of High Levels of Methemoglobin to Hemoglobin (in vitro). After the addition of NaNOz to the blood, methemoglobin levels of 89.6% and 96.5% were obtained at 38 and 68 minutes, respectively. washing the cells in physiological saline took 30 minutes, after which the cells were resuspended in the guinea pig's plasma. The methemoglobin level at this time was 80.4%. Three hours and 15 minutes later the methemoglobin level was zero (Figure 3). Determination of Lethal Doses of NaNOo for Guinea Pigg Mglgg. No deaths occurred when NaNOz was given at the rate of 60 mg./kg. to 10 guinea pigs. Deaths did occur, however, when NaN02 was given at the rate of 70 mg./kg. or more. In guinea pigs given NaNOz, 100 mg./kg., the mortality was 100% (Table 3). The estimated LD50 was 79 mg./kg. Signs of intoxication observed were hyperpnea, drowsiness, sprawl- ing of hind legs while walking, cyanosis, lowering of body temperature, unsteady gait, unconsciousness, and gasping. These signs were followed by death or recovery. Although the majority of deaths occurred approxi- mately an hour after administration of the NaNOZ, the range was from 36 to 70 minutes. One guinea pig gradually recovered after being comatose for an hour. 25 100‘} 404 -—-----K unusuostosm x -J ”-1 a. . a C Figure 2. Methemoglobinization of guinea pig blood induced by NaNOz, 40 mg./100 ml. 26 ”Iain”. =5. IIAD . 0.0 ~.n ~.~« H.m0 III «.00 N.n« 0.0« III 0.0m ~.mN 0.0m III 0.0 maumww m.H 0.« 0.NH 0.~« III 0.00 0.00 0.00 0.50 N.m0 0.00 0.00 m.0 0.0 mxmma 0 0.0 0.0 0.H 0.00 III 0.05 n.Hm 0.«0 0.0m 0.0m 0.0m 0.0m m.H 0.0 axomz m 0.0 H.« 0.0a m.m« n.00 0.Nn 0.Hm 0.00 III ~.«m 0.00 0.m« III m.H mmmv Ha n.« «.0 w.« III III 0.H0 III 0.00 III 0.00 m.~m 0.00 «.mm ~.0 mmmv m III 0.0 0.0 0.0 0.0 0.0a m.«m m.mm 0.0m m.0« «.00 III 0.«« 0.0 0pc H .mun .mun .mp3 .mun .mu: .mus .mun .u£ .awa .swa .nwa .afia .aHa .sfia mw¢ 0 0 « n 0.N N m.H H 0« cm 0H 0H 0 0 mHm>umuaH um ARV Ho>mu nanoawdamnumz 802 so 4a 82.? 3 3 333,33 Nozmz mo Ho>ma mmon .mmwm usouommaw mo mwam mosfisw aouw pooan mo sowumNHsfinonoamnuma ouufi>.mw .N manma 27 In fi—oaeroaa uAsIIIIIc s °---'DURING WASHING \ \\'—-AFTER WASHING mmmootouu x Figure 3. Methemoglobinization of guinea pig blood induced by NaNOz, 80 mg./100 ml. 28 Table 3. Mortality rate in male guinea pigs produced by subcu- taneous administration of NaNOZ Dose No. Mortality Rate (mg./kg.) Injected (%) 100 10 100 90 10 90 80 10 60 70 10 10 60 10 0 29 Pregnant Females. The mortality rate was 100% when 70 mg./kg. of NaNOz was given to 4 pregnant guinea pigs; however, the survival rate was 100% when 60 mg./kg. of NaNOz was given to 36 pregnant females. Fetuses. The data are given (Table 4). Fetuses died which were given NaNO2 at levels of 73 mg./kg. or higher. Determination of Levels of Methempglobin and Plasma Nitrite after Administration of NaNOz to Guinea Pigs Different Doses of NaNO2 to Males. The degree of methemoglobinemia produced by different doses of NaNOz is given (Table 5). With a dose of NaN02 of 60 mg./kg., the highest level of methemoglobin was observed about an hour after subcutaneous administration. The methemoglobin was then gradually reduced to hemoglobin with only 2% methemoglobin present at 5 hours and 15 minutes after injection (Figure 4). The plasma nitrite level reached a peak 7.5 to 15 minutes after sub- cutaneous administration of the NaNOz (Table 6). Evaluation of Protective Effect of Methylene Blue on NaNOZ Toxicosis. Methylene blue, 40 mg./kg., alone produced a small amount (2.3%) of methemoglobin. The methemoglobin level was only 3.8% when 60 mg./kg. of NaN02 and 40 mg./kg. of methylene blue were given simultaneously (Table 7). In the guinea pig given NaNOz at the rate of 100 mg./kg. and methy- lene blue, 10 mg./kg., the methemoglobin level in the blood was 7.5% at 75 minutes after treatment. There was no methemoglobin in the blood sample taken before administration of the NaNOz. 30 Table 4. Determination of lethal dose of NaNOz for fetuses of guinea pigs Length of NaN02 Body Dam Gestation Fetus (mg./kg.) Wt. No. (days) No. subcu. (Gm.) Remarks 1 55 1—1 23 88 Live 1-2 61 82 Live 2 55 2-1 0 70 Live 2-2 73 82 Dead 2-3 109 73 Dead 2-4 141 71 Dead 3 60 3-1 92 54 Dead 3-2 71 57 Live 3-3 0 59 Live 3-4 0 53 Live 31 Table 5. Methemoglobinemia induced by NaNOZ in guinea pigs* Dose MetHb. Levels (%) at Intervals (mg-lkg-) subcu. 30 min. 60 min. 90 min. 120 min. 10 2 3 l 1 20 12 8 5 3 30 32 25 16 4 40 49 58 46 32 6O 40 67 66 63 70 55 75 72 31 80 70 84** -—- --— * Results given for l representative animal for each dose. ** Died after blood sample was drawn. 32 100- “F 60- METHEMOGLOBIN X 0 O l u: 0 HOURS Figure 4. Methemoglobinemia in guinea pig induced by NaNOz, 60 mg. /kg. (subcutaneously). 33 III III III n«.~ III 00.~ III 0 00 0 III III III III 8 .3 no . 3 3 . N o 8 e III III III 00.H III om.~ III 0 00 m 0.0 0.0 0.0 0H.H III III III 0 00 N III III III 00.0 III mm.H III 0 0« H 83 om 8 on m . «N 3 m . a 0 ea: .mav .oz A.sHav mHm>umuaH um A.Ha 00H\nmav mwwduqu muHuqu mamMHm mmon me mmsHso mem mmaHsm ou Noamz mo GOHumuumHaHavm msomsmusonsm Houmm mHo>oH :monuHs muHuuHa mammHm .0 «Hams 34 Table 7. Methemoglobinemia in male guinea pigs produced by NaNOz and methylene blue Guinea Methemoglobin Levels (%) Pig at Intervals (Hgs.) No. Treatment 0 1 2 3 2 Methylene blue, 0 0.9 2.3 0.0 40 mg./kg. 3 NaNOZ, 60 mgo/kgo, O 308 203 008 and methylene blue, 40 mg./kg. 35 Determination of Methemoglobin and Plasma Nitrite in Maternal and Fetal Blood. The degree of methemoglobinemia produced by NaNOz (40 or 60 mg./kg.) in pregnant guinea pigs is shown (Table 8). The methemoglobin and plasma nitrite values after administration of NaN02 (40 mg./kg.) in pregnant guinea pigs are given (Table 9). The fetal blood samples con- tained small amounts of methemoglobin and plasma nitrite. The guinea pigs given NaNOZ at the rate of 60 mg./kg. had higher levels of methemo— globin than those receiving 40 mg./kg. of NaN02 (Table 10). In anesthe- tized dams in which repeated samples were taken, the highest methemoglobin level in maternal blood was 51.5%, while the highest in fetal blood was 10.6% (Figure 5). All the fetuses were alive. Pathologic and Embryotoxic Effects of NaNOzToxicosis Experiment 1. The body temperature of the guinea pigs was lowered approximately 1 C. by 1 hour after administration of the NaNOz (50 mg./kg.); the body temperature returned to normal within the next 3 hours (Table 11). All guinea pigs, those given NaNOz as well as the controls, gave birth to normal litters. Experiment 2. Methemoglobin or plasma nitrite was not detected in the blood before, 12 or 24 hours after administration of the chemicals, either in the experimental or control animals. All the fetuses were alive. No visible gross lesions were detected during postmortem examination. On microscOpic examination, centrolobular hepatic lipidosis was observed in the experimental animal killed 24 hours after treatment. The remain- ing organs appeared normal. 36 Table 8. Methemoglobinemia in pregnant guinea pigs produced by NaN02 X- Dose of NaN02 MetHb. (%) at Intervals (subcu.) 0 min. 10 min. 20 min. 40 min. 1 hr. 40 mg./kg. 0 6.5 30.4 38.9 11.8 60 mg./kg. 0 2.0 30.1 60.0 68.8 * One representative guinea pig in late pregnancy was selected for each dose. 37 .000Hn Haumm :Hmuno ou vauo GH vmauomumm was haououmumzn 0am vaHumnumosm mas awn « No.o 3.3 no.0 N.m~ om so.o m.o o3.o m.mm 00 00.0 o.~ m3.o o.m os so.o o.o m3.o o.e om 00.0 o.o oo.o 0.0 o “.3a coa\.wav saw any “.3a OOH\.wsV saw Axe A.aaav .00032 «030032 mammflm .nmuoz Iouusz «usuusz mammflm .pmumz Nozmz mo nosu vOOHm Hmuom voon Hangman: ImuumHsHav¢ umumm mngsmm mo «EHH same ou A.mx\.ma osv Nozmz mo sOHumuumHsHsvm Hound 000H0 Hmumm 0cm Hmsumuma mAu sH mHm>oH ouHuuHs mammHm 0am sHAOHwoamsumz. .0 mHan 38 .UQMU NH”? WNmSUMh fl 00.0 0.« 00.0 0.0 s0«H 0 III III 00.0 «.0H «00H « no.0 0.0N 00.0 0.«0 «00 0 III 0.5 0H.0 «.m« 0m N 00.0 0.0 00.0 0.0 0 H 03 8:03 8» 8 T? 8:90 8» 00 033 .oz Iouqu ouHuqu mammHm .Amuoz Iouqu ouHuqu mammHm .nmumz Nozmz mo sOHu me voon Huumm voon Hmsumumz ImuumHsHa0< umumm mwsHsc mngamm mo «EH9 amp ou A.wx\.wa 00v ~02mz mo GOHumuumHsHavm umuum woOHn Hmuwm 00m Hmcumuma we» oH mHm>mH muHuuHs mammHa 0am anOHwoamnuoz .0H oHan 39 6‘9 ‘ o- METHEMOGLOBIN n 0 1 l 0 ‘ so so MINUTES Figure 5. Methemoglobinemia in the pregnant guinea pig induced by NaNOZ, 60 mg./kg. (subcutaneously). 40 0.00 0.00 0.00 0.00 0.00 III 00 0 «.00 0.50 «.00 0.00 H.00 III 00 0 0.00 «.00 «.00 0.00 0.00 III 00 « 0.00 0.00 0.00 0.00 0.00 III 00 0 0.00 0.00 0.00 0.00 0.00 00 III N H.0« 0.00 0.00 0.00 0.00 00 III H « N H 0.0 unmaummua A.wx\.wsv A.wx\.wav .oz Adwunv me>uousH um sawsumoue uwum< ouomom Homz Nozmz me H.00 musumuoaawaxwvom moast me0 mmsst usmsmmum ou A.wx\.wa 000 Nozmz mo sOHumuumHsHavm msomsMusonsm mo musumummamu 0000 so uoomwm .HH mHnma 41 Experiment 3. The reproductive performance is tabulated (Table 12). Experiment 4. Neither methemoglobin nor plasma nitrite was found in any of the blood samples from the control animals. In the experimental animals methemoglobin was present in both ma— ternal and fetal blood up to 6 hours after administration of NaNOz. After this no significant amount of methemoglobin could be detected (Table 13). However, no plasma nitrite was found in either the maternal or fetal blood samples taken at 3 hours after administration of NaNOz. There was a slight absolute leukocytosis in the guinea pigs given NaNOz; no significant changes appeared in the leukocyte counts of the guinea pigs given NaCl. No Heinz bodies were demonstrated in the blood stained with new methylene blue either in the experimental or control animals. All the fetuses were alive in both the experimental and control animals at 0.25 and 1.5 hours after treatment. Fetuses of the treated guinea pigs, examined at 3 hours and onward following administration of NaNOz, had a mortality rate of 72%. No fetal deaths occurred in the control animals (Table 14). Gross Lesions. The blood, mucous membranes and viscera had a dark brown appearance in the experimental animals killed at 1.25, 1.5 and 3 hours after administration of NaNOz. Placentas of the experimental animals killed 18 hours after treatment and onward were pale in contrast to the bright red placentas of the control animals. Placentas from ex— perimental animals killed at 24 hours and onward were friable. Fetuses of the nitrite-treated animals killed at 24, 24.5, 48 and 56 hours after treatment were dead and edematous (Figures 6 and 7). 42 Table 12. Reproductive performance of guinea pigs given NaNO2 or NaCl subcutaneously Time of Treatment Abortion or Condition of Fetuses (60 mg./kg.) Parturition Live Dead Total Remarks NaNOz < 24 hrs. 1 3 4 Premature NaNOZ < 24 hrs. 0 2 2 Premature NaNOZ 4 days 0 5 5 Premature NaNOz --- - - - Not pregnant NaCl 6 days 3 0 3 Normal birth NaCl --- - - - Died* * Three days after treatment, she died due to pneumonia. 43 Table 13. Methemoglobin and plasma nitrite levels in the maternal and fetal blood after administration of NaNO2 (60 mg./kg.) to dam Time of Sampling Maternal Blood after Administra- Plasma N02-N tion of NaN02 (hrs.) MetHb.(%) (mg./100 ml.) Fetal Blood Plasma NOZ-N MetHb.(%) (mg./100 m1.) 0.25 27.0 0.19 1.1 0.02 1.50 23.3 0.07 9.2 0.02 3.00 18.6 0.00 8.8 0.00 6.00 0.2 0.00 2.3 0.00 12.00 0.0 0.00 0.0 0.00 18.00 0.0 0.00 0.0 0.00 24.00 0.0 0.00 0.0 0.00 24.50 0.0 0.00 0.0 0.00 48.00 0.0 0.00 0.0 0.00 56.00 0.0 0.00 0.0 0.00 44 0N 0 0 0N 0 0 00.00 0H NN 0 0 00 0 0 00.0« 0 00 0 0 N0 « 0 00.«N 0 00 o 0 00 H H 00.«N N 00 0 « «N 0 0 00.0H 0 0H 0 « 0 0 0 00.NH 0 00 0 « 00H 0 0 00.0 « «0 0 N 00 « 0 00.0 0 00H 0 N 00 0 N 00.H N N0 0 N 00 0 0 0N.0 H H.300 amen 0>HH A.ewv 0mon o>HH A.mnsv .oz .u3 owmuu>< .u3 owmum>< usoaummua Hmumm mHm AHomzv mmmsuuh ANozmzv mumsumh sOHumsHamxm mo mEHH mmsHsU me0 mosst usmswoum ou A.wx\.wa 00v Homz Ho Nozmz mo GOHumuumHsHavm msoosmusonsm umumm muHHMuuoa Hmumm .«H MHan 45 Figure 6. Fetus of NaNOZ-treated (60 mg./kg.) guinea pig killed 24.5 hours after treatment. Notice subcutaneous edema and discoloration (arrow). Figure 7. Fetus of control guinea pig killed same time as in Figure 6. 46 Microscopic Lesions Liver. In experimental animals a mild degree of hepatic lipi- dosis was confirmed by Oil Red 0 stain. Lipidosis was more prominent in the centrolobular areas (Figures 8 and 9). Placenta. Placentas of experimental animals killed at 0.25, 1.5 and 3 hours were hyperemic. Coagulative necrosis was observed in the placentas of experimental animals killed at 18 hours and onward (Figures 10 and 11). The extent of the lesions progressed with time. Prevention of Acute Toxicity of NaNOa in Pregnant Guinea Pigs by Treat- ment with Methylene Blue Group 1. The fetuses were apparently protected by the administra- tion of the methylene blue to the dams at the time of nitrite administra- tion (Table 15). Group 2. The guinea pigs given NaNOZ aborted 3 to 4 days after treatment, while those given NaNOZ and methylene blue or methylene blue alone farrowed normally (Table 16). Effects of NaNO., Administration on Pregpant Guinea Pigs During First 30 Days of Gestation Group 1. Among the nitrite—treated guinea pigs only 2 fetuses died. Results for this group are given (Table 17). Group 2. The guinea pig killed 8 days after NaN02 administration had 3 dead fetuses. The fetuses were all alive in the experimental as well as the control animals killed on the 2nd, 3rd and 11th days. The 47 Hepatic lipidosis in NaNOz-treated guinea Figure 8. x 75. H & E stain. pig- Higher magnification of a portion of Figure x 750. Figure 9 H & E stain. 8. 48 Figure 10. Necrosis of the placenta in NaNOz- treated guinea pig. H & E stain. x 75. Figure 11. Higher magnification.of a portion of Figure 10, to show karyorrhexis (A) and pyknosis (B). H & E stain. x 750. 49 .usmaummuu umuwm musos «N aHnuHB 0HHmauos vmaonumm « 0NN 0 0 0 00H Homz 0H 00 SN s o a 83 Nozmz 3 3 onN 0 0 0 ««N Humz 0H 00 can 0 o s 3 Nozsz 3 8 00N 0 H 0 0 Humz 0H 00 03 o 3 m m Nozé 3 S H.800 Hsuoa 0mon m>HH A.wunv usuaumous Hound Homz H.wx\.wav Ammmvv uanos HmuoH sOHumsHawxm mo msHH so msHm soHumummo momsumm Nozmz mamthumz mo mmMum man msmHhauwa :qu vmummuu mem mmsst mm mmmsumm meu so A.wx\.wa 00v Homz no Nozmz mo uommmm .0H mHnme 50 Table 16. Reproductive performance of pregnant guinea pigs treated with NaNO2 and methylene blue Time* of Methylene blue NaNOz Abortion or Condition of Fetuses mg./kg. (I.P.) mg./kg. (subcu.) Parturition Live Dead 10 60 9 days 4 0 10 60 9 days 4 0 10 60 4 days 3 0 --- 60 3 days 0 3 ___ 60 4 days 0 2 10 --- 15 days 3 l * Post-treatment. 51 Table 17. Effect of NaNO administered subcutaneously to guinea pigs on the 10th day of pregnancy (estimated) Guinea Exam. of Pig NaNOZ NaCl Fetuses after Condition of Fetuses No. (mg./kg.) (mg./kg.) Treatment Live Dead 1 60 --- 7 days 6 0 2 60 --- 14 days 4 2 3 60 --- at term 4 0 4 --- 60 at term 3 0 5 --- 60 at term 4 0 52 2 experimental and 1 control animals which were kept under observation farrowed without any apparent complications (Table 18). Group 3. The fetuses were alive in the nitrite—injected animals killed on the 2nd, 7th and 14th days, as well as the control animal killed on the 7th day of the experiment. Two experimental and 1 control animal, which were not killed, farrowed normally. One of the control animals was not pregnant (Table 19). Guinea Pigs 5 and 7, which were given NaNOz, each had 1 young with deformity of the hind leg (Figure 12). Maternal and Fetal Blood P02‘_§COZ, pH and Methempglobin Values after ’Administration of NaN92 In guinea pigs given NaNOz, 60 mg./kg., there was a corresponding reduction in the maternal and fetal blood P02 values (Figure 13) and an elevation in P302 values (Figure 14) with the increases of methemoglobin levels in the maternal blood (Figure 15). The reduction of P02 values in the maternal and fetal blood of guinea pigs given NaNOz, 45 mg./kg., was less than those given NaNOz, 60 mg./kg. There was a drop in the fetal blood pH after administration of NaNO 60 mg./kg., to the dam (Table 20). 29 53 Table 18. Effect of NaNO administered subcutaneously to guinea pigs on the 20th day of pregnancy (estimated) Guinea Exam. of Pig NaNOz NaCl Fetuses after Condition of Fetuses No. (mg./kg.) (mg./kg.) Treatment Live Dead 1 60 --— 2 days 3 0 2 --- 60 2 days 5 0 3 60 --- 3 days 2 0 4 --- 60 8 days 5 0 5 60 --- 8 days 0 3 6 60 --- 11 days 5 0 7 --- 60 at term 5 0 8 60 --- at term 4 0 9 60 --- at term 3 0 54 Table 19. Effect of NaNO2 administered subcutaneously to guinea pigs on the 30th day of pregnancy (estimated) Guinea Exam. of Pig NaNOz NaCl Fetuses after Condition of Fetuses No. (mg./kg.) (mg./kg.) Treatment Live Dead 1 60 --- 2 days 4 0 2 60 --- 7 days 5 0 3 --- 60 7 days 4 0 4 60 --- 14 days 2 0 5 60 --- at term 2* l 6 --- 60 at term 3 0 7 60 --- at term 3* 0 8 --- 60 at term 2 0 * One young in the litter had a deformity of the hind leg. 55 Figure 12. Young (1 day old) of the guinea pig (No.7 ) given NaNOz, 60 mg./kg. (subcutaneously). Notice deformity of hind leg (arrows). 56 g) '01 .MATERNAL 70. — FETAL .0. I CONTROL sonxun NITRlTE sonxun NITRITE 45 mec. 60 mm. Figure 13. The mean P02 value of treated and untreated guinea pigs. 57 la] q S“ k M) F E T A L (Hfilll (“I I'm mum .‘E (l({(({t ‘ w W " W 8“ 00 4.2;; W 0. ‘ 2:435“; } , N 'Il({\\(ll w ,y, w ) ((t ‘I ‘ 00 “f 0:09 ’5 383‘ -I 61%)? 51;: “31 / NW 0* gt ‘ ‘ ‘ (I rm . ("‘3‘ 0005)) 0:0 $31.9: "Mu. ‘ fig-“f . I 551001 W“ " $55))“ A) 0%? ; WW ‘ WW W W‘HH‘II- ‘M‘N‘f‘ Hull“ 3") CONTROL SODIUMNITRITESODIUMNITRITE 45 f-‘G./k6. 60 .‘IG./K6. Figure 14. The mean PCO2 value of treated and untreated guinea pigs. 58 mm b m .—. Maternal Po, m4 .o—cp. Maternal MetHb. . a) H Fetal Pot F._._. Fetal MetHb. 70‘ P m N 60. ”,0.“ . 60 f - ,.- ‘~~-‘ 2 3L 0. p m _‘ .2“ ’ ‘9 I O S: ‘0‘ I Into LU ’ O :r: I \ O"- / u; I I m< I , Z] I ’.’.l. ............. . 10‘ I, ’ I ’. m I .’ \ o ’ a- ‘ -.-.’ o ' j v ' o N H) H I I U T E 5 Figure 15. The effect of NaN02 (60 mg./kg., subcu- taneously) on maternal and fetal methemoglobin and P02 values. 59 Table 20. Maternal and fetal blood pH of NaNOz—treated (subcu.) and untreated guinea pigs Guinea -- Sampling Time J‘Fi Pig NaN02 After Admin. of Blood pp; No. (mg./kg.) NaNOz (min.) Maternal Fetal l 0 --- 7.44 7.18 2 0 --- 7.19 7.25 3 0 --- 7.44 7.20 4 45 30 7.22 7.03 5 45 60 7.42 7.01 6 45 90 7.37 7.27 7 60 30 7.42 6.88 8 60 60 7.26 6.80 9 60 90 7.37 6.93 DISCUSSION Since the formation of methemoglobin is the major factor in the pathogenesis of nitrite toxicosis, it was of prime importance to first study the methemoglobinization of blood ipgyippg. The data obtained were then used to build a model for the _i_r_:_ 1:552 studies. As reported, 0.5 to 0.7 mole of NaN02 converts 1 mole of oxyhemo- globin to methemoglobin (Austin and Drabkin, 1935). The red blood cell absorbs the nitrite ion which causes the oxidation of hemoglobin to methemoglobin, consequently lowering the nitrite content of the plasma. The reducing enzymes present in the normal RBC's simultaneously reduce the methemoglobin formed back to hemoglobin. However, the oxidation of hemoglobin continues as long as the nitrite ions are in contact with the RBC's. Thus, higher doses of NaNOZ increased not only the level of methemoglobin but also the period of reduction of methemoglobin to hemoglobin. There was no significant variation in the rate of methemoglobin formation in the 3 different concentrations of red blood cells (Figure 1). However, it was apparent that the reduction of methemoglobin to hemoglobin in blood diluted 50% with plasma was slower than in the whole blood. This may be explained by the fact that there was twice as much nitrite per red blood cell in the diluted blood sample as in the whole blood. The information gained here indicates that in an anemic animal, a dose of nitrite could produce a higher degree of methemoglobinemia than in a healthy animal. 60 61 The enzyme, methemoglobin reductase, utilizes reduced DPN and TPN which are derived from glycolysis and the phosphogluconate oxidative pathway (White, Handler and Smith, 1959). Thus, the absence of reduction of methemoglobin in the sample of RBC's suspended in physiological saline solution was probably due to the exhaustion of DPNH and TPHN, which sug- gests that plasma plays a role in the reduction of methemoglobin to hemoglobin. Lower amounts of methemoglobin reductase in fetal blood (Ross, 1963) is not adequate to explain the faster and higher level of methemoglobin formation and quicker reduction in comparison to maternal blood as observed ipuyippp_by the author. If the difference were simply due to a decreased amount of the reductase, there would have been a slower reduc- tion ofmethemoglobin rather than faster, as was observed and shown in Figure 2. It has been reported that when 80 to 90% of the hemoglobin is con- verted to methemoglobin, death occurs as a result of anoxia (Bodansky, 1951). However, it was not clear whether the anoxia was preceded by destruction of the intraerythrocytic enzymic system. In order to clari- fy this, a high level (96%) of methemoglobin was produced ipuyippp, after which the cells were thoroughly washed so that extraerythrocytic nitrite would not be in contact with the RBC's. The reduction of methemoglobin to hemoglobin did not take any longer in the blood containing a high level of methemoglobin than it did in the sample with only a moderate amount (Table 1). This observation supports the view that even in the case of a high degree of methemoglobinemia, the enzymic system remains intact. 62 Following subcutaneous administration, NaNOz was absorbed so rapidly that the plasma nitrite level reached a peak between 7.5 and 15 minutes. As the nitrite ions came in contact with the erythrocytes, the nitrite content of the plasma was gradually reduced to zero. The highest level of methemoglobin was observed about an hour after treatment, which corresponded with the results of the ippgippp_studies. Plasma nitrite was detected in the fetuses. This proves that the nitrite ion can pass through the guinea pig placenta. Maternal plasma nitrite values, however, were higher than fetal plasma nitrite values, thus indicating that there may be a partial placental barrier to the transport of nitrite to the fetuses. It is possible that some of the nitrite ions form a complex with the plasma protein, which cannot pass through the placenta. Pregnant guinea pigs given NaNOz, 50 mg./kg., had a normal parturi- tion, and there was no detectable effect on the fetuses. When pregnant guinea pigs were given NaNOZ, 60 mg./kg., abortions with fetal mortality occurred 1 to 4 days later. It is important to emphasize that there was a sharp demarcation between the doses of NaNOz which produced abor- tion and those which had no effect upon reproduction. As a general rule, the fetuses died between 1.5 and 3.5 hours after the dam was given NaNOZ, 60 mg./kg., subcutaneously. By correlating the information gained from Figures 4 and 5, it is apparent that the fetal deaths occurred when maternal and fetal methemoglobin levels were at their peak, but not during the plasma nitrite peak which was attained between 7.5 and 15 minutes after administration of the NaNOZ (Table 6)o This indicates that nitrite as such was not lethal to the fetuses, but rather that its ability to form methemoglobin, with the resulting hypoxia 63 or anoxia, was responsible for the fetal deaths. At the time of the fetal deaths there were no significant changes in the placentas. Upon death of the fetuses, the fetal blood circulation was stopped, thus interrupting the normal mechanism of blood flow in the placenta. It is believed that the pathologic changes seen in the placentas, there- fore, developed after the death of the fetuses. If this be true, the necrotic changes observed in the placentas were a consequence, rather than a cause, of the fetal deaths. Methylene blue seems to be a very good protective agent against nitrite poisoning, since only a small amount of methemoglobin was detected in nitrite—treated animals receiving it. In the body, methy- lene blue is reduced to leuco-methylene blue and is readily available for the reduction of methemoglobin to hemoglobin (Jones, 1965), the effect being the reduction of the methemoglobin level in the blood. In pregnant guinea pigs given NaNOz (60 mg./kg.) and treated with methylene blue (10 mg./kg.) no fetal deaths occurred. The findings strongly suggest that fetal deaths were produced due to methemoglobinemia which led to hypoxia. In order to confirm thatr fetuses were dying because of the hypoxia, the maternal and fetal P02 and PCO2 of control and nitrite-treated animals were determined. The data obtained definitely indicated that there was a lower P02 along with a higher PCO2 value in the fetuses of guinea pigs treated with NaN02 (60 mg./kg.) in comparison to the control animals. Fetal Po2 values from dams treated with NaNOz at the rate of 45 mg./kg. were not appreciably lowered. It seems that there is a critical level of maternal methemo- globin up to which fetal P02 is not affected, but beyond this point fetal P0 is reduced and the end result is a hypoxic condition in fetuses. 2 64 Severe degrees of hypoxia obviously may lead to death. In this study when NaNOZ, 60 mg./kg., was given to guinea pigs on the 30th day of pregnancy, a deformity of the hind leg was observed among 13% of the young. The author's finding is similar to that of Grabowski (1961a), in which teratogenesis was observed when chick embryos were exposed to hypoxia. . n_ The administration of NaNOZ, 60 mg./kg., to guinea pigs in the first F‘ half of pregnancy produced less fetal mortality than when given in the last half of pregnancy. This could be explained by the fact that in the latter stages of pregnancy the fetuses comprise a greater percentage of the body weight of the pregnant guinea pig than earlier in pregnancy. Thus, even though the same dose of NaNOZ was given on a body weight basis (mg./kg.) there would be less nitrite per red blood cell in early preg- nancy than in late pregnancy. However, there may be some other factors responsible for the difference; but further investigation is needed in order to clarify this. In the guinea pig, sodium nitrite, depending upon the dose, produced a mild to severe degree of methemoglobinemia but did not produce Heinz bodies. This observation is comparable to work done in pigs (Sinha and Sleight, 1968). SUMMARY AND CONCLUSIONS This work was designed to study the effects of acute nitrite toxicosis on reproduction in guinea pigs. Basic to the characteriza- tion of these effects was a series of experiments on the extent of methemoglobin formation caused by NaNOz ip 1E2. and ip m. The pathologic and embryotoxic effects of acute NaNOz toxicosis at various stages of gestation were studied. Maternal and fetal P02 and P002 values were determined after administration of NaNOz to the dam. From the experimental results, the following observations and conclusions can be made: 1. When pregnant guinea pigs were given NaNOz, 60 mg./kg., abortion with fetal mortality occurred. 2. The fetal deaths occurred when maternal and fetal methemoglobin levels were at their peak but not during the plasma nitrite peak. Met- hemoglobinemia led to hypoxia which caused the fetal deaths. 3. Following subcutaneous administration of NaNOz, 60 mg./kg., the plasma nitrite level reached a peak between 7.5 and 15 minutes. The highest level of methemoglobin was observed about an hour after treatment. 4. There were lower P02 and higher Pco2 values in the fetuses of the guinea pigs treated with NaNOz, 60 mg./kg., than in fetuses of the control animals. 5. The pathologic changes seen in the placentas were a consequence, rather than a cause, of the fetal deaths. 65 66 6. In the pregnant guinea pigs given NaNOZ, 60 mg./kg., and treated with methylene blue, 10 mg./kg., no fetal deaths occurred. 7. When NaNOZ, 60 mg./kg., was given to guinea pigs on the 30th day of pregnancy, a deformity of the hind leg was observed among 13% of the young. 8. When dams were given NaNOZ subcutaneously, maternal plasma nitrite values were higher than fetal plasma nitrite values, which indi- cates that there may be a partial placental barrier to transport of nitrite to the fetuses. 9. Even with a high.degree (96%) of methemoglobinization, the enzyme system was capable of reducing methemoglobin to hemoglobin. ~iw: 10. 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J., 49, (1957): 278-279. Welsch, C., Bloomfield, R. A., Garner, G. B., and Muhrer, M. B.: Effect of dietary nitrate on thyroid and adrenal gland weight. J. Anim. Sci., 20, (1961): 981. Wendel, W. B.: The control of methemoglobinemia with methylene blue. J. Clin. Invest., 18, (1939): 179-185. White, A., Handler, P., and Smith, E. L.: Principles of Biochemistry, 3rd ed. McGraw-Hill Book Co., New York, 1964. Winter, A. J.: Studies on nitrate metabolism in cattle. Am. J. Vet. Res., 23, (1962): 500-505. Winter, A. J., and Hokanson, J. E.: Effects of long-term feeding of nitrate, nitrite, or hydroxylamine on pregnant dairy heifers. Am. J. Vet. Res., 25, (1964): 353-361. VITA Dineshwar Prasad Sinha was born in Patna, India, on May 1, 1940. He graduated from Patna Collegiate High School, Patna, in 1955. From 1955 to 1957 he attended L. S. College, Bihar University, and received the Intermediate in Science diploma which was the pre- requisite for veterinary school. In 1957 he entered Bihar Veterinary College, Patna University, and graduated in 1961 with the Bachelor of Veterinary Science and Animal Husbandry degree. The author received honors in Animal Husbandry Part I. In the fall of 1962 he came to Michigan State University as a graduate student and received his M.S. in Veterinary Surgery and Medicine in March, 1964. In April, 1964, he joined the Department of Pathology as a Ph.D. candidate. The author is a member of the American Veterinary Medical Associ- ation, the Indian Veterinary Association, and Phi Zeta. 70 ml] M” BHI Hill“ Y" I" u "I Ill-l " u u H H H 7 9 3 9 4 7 1 3 0 3 9 2 4| 3