L‘\ ‘ ' «an war n ; ,. “Th;9':'HHIII-‘IIIII’MeIz’... TERATOGENIC EFFECT OF 2.s£c- Bum-4, 6- DINITROPHENOL (DINOSEB) m we FETAL RAT Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY AFAF IZZEL‘DIN ABUELGASIM. 1977 . TERATOGENIC EFFECT OF 2—SEC—BUTYL-4,6—DINITROPHENOL (DINOSEB) IN THE FETAL RAT BY Afaf Izzeldin Abuelgasim Two-sec-butyl-4,6-dinitrophenol (dinoseb) was administered daily to 32 pregnant Sprague-Dawley rats on days 10 to 12 of gestation by the intraperitoneal route; seven females were held as controls. Fetal organogenesis occurs during this period. The rats were divided into groups and dosed as follows: (1) control, (2) 6.3 mg/kg, (3) 8.mg/kg, (4) 9 mg/kg, (5) 11.2 mg/kg, (6) 12.5 mg/kg, (7) 15.8 mg/kg, (8) 17.7 mg/kg. Doses of 11.2 mg/kg to 17.7 mg/kg were lethal to the dams. Doses of 8 mg/kg and 9 mg/kg resulted in a significant fetal body weight reduction at days 20 and 21 of gestation but not at one day of age. The gross lesions consisted of hydroureter, dilation of the urinary bladder and dilation of the renal pelvis. Microscopically, lesions were hydropic degeneration of the epithelial lining of the ureter, edema in the lamina propria of the bladder and dilation of the renal tubules. Vacuolation of hepatic cells was also seen. A dose of 6.3 mg/kg did not cause significant lesions. TERATOGENIC EFFECT OF 2-SEC-BUTYL-4,6-DINITROPHENOL (DINOSEB) IN THE FETAL RAT BY Afaf Izzeldin Abuelgasim A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1977 Dedicated to my parents, my husband Abdu and my son Ayemen ii ACKNOWLEDGEMENTS The author very sincerely wishes to express her gratitude to Dr. V. L. Sanger, her major advisor, for encouragement, patient guidance in her graduate study program and valuable suggestions and assistance in the preparation of her thesis. Also to Dr. J. B. Hook and Dr. S. D. Sleight for their willingness to serve on her guidance committee and to give useful comments and suggestions. The author also appreciated the cooperation of Mr. K. McComick. The author also expresses her gratitude to the Government of the Democratic Republic of Sudan for the financial support she received during her stay in the United States. The writer wishes to acknowledge her gratitude to her husband, who wholeheartedly encouraged her in her study. Because of the above-mentioned, this thesis was made possible. iii TABLE OF CONTENTS Page INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . 2 Kidney Development . . . . . . . . . . . . . . . . . . . 2 Abnormal Kidney Development. . . . . . . . . . . . . . . 5 Hydronephrosis . . . . . . . . . . . . . . . . . . . . . 5 The Effect of Chemicals on the Kidney. . . . . . . . . . 9 Dinoseb. . . . . . . . . . . . . . . . . . . . . . . . . 11 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . 14 Animals 0 O O O O O O O O O O O O O O O O O O O O O 0 O O 14 Preparation of Dinoseb . . . . . . . . . . . . . . . . . 14 Treament. O O O O O O O O O O O O O O O O O O O O O O O 14 Methods of Examination . . . . . . . . . . . . . . . . . 16 Statistics 0 O O O O O O O O O O O O O O I O O O O O O O 17 “SULTS O O O O O O O O O O O O O O O O O O O O O O O O O O O O 18 General Observations . . . . . . . . . . . . . . . . . . 18 Effect of Dinoseb on Fetal Growth. . . . . . . . . . . . 18 Pamo logic Findings 0 O O O O O O O O O O O O O O O O O O 2 2 DISCUSSION. 0 O O O O O O O O O O O O O O O I O O O O O O O O O 40 SUMMARY 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O 4 3 LIST OF REF'E ENCES O O O O O O O O C O O O O O O O O O O O O O . 4 5 iv Table LIST OF TABLES Page The number of dams treated and the dose level of dinoseb in each group. . . . . . . . . . . . . . . . . . 15 Number of fetuses, resorption rate and mean fetal size from control rats . . . . . . . . . . . . . . . . . 19 Number of fetuses, resorption rate and mean fetal size from dams treated with 6.3 mg/kg dinoseb (2-sec—buty1-4,6-dinitrophenol). . . . . . . . . . . . . 20 Number of fetuses, resorption rate and mean fetal size from dams treated with 8.0 mg/kg dinoseb (2-sec—butyl-4,6-dinitrophenol). . . . . . . . . . . . . 21 Number of fetuses, resorption rate and mean fetal size from dams treated with 9.0 mg/kg dinoseb (2-sec-butyl-4,6-dinitrophenol). . . . . . . . . . . . . 23 Mean number of fetuses and fetal size from treated and control dams . . . . . . . . . . . . . . . . . . . . 24 Percentage of fetuses with gross lesions from dams treated with 8.0 mg/kg dinoseb (2-sec-butyl-4,6- dinitrophenol) . . . . . . . . . . . . . . . . . . . . . 25 Percentage of fetuses with gross lesions from dams treated with 9.0 mg/kg dinoseb (2-sec—buty1-4,6- dinitrophenol) . . . . . . . . . . . . . . . . . . . . . 26 Figure 10 ll 12 13 14 15 16 17 LIST OF FIGURES Chemical structure of dinoseb. . . . . . . . . . . . . Dissected urinary system of a 20-day fetal rat from a dam that was dosed with 8 mg/kg of dinoseb . . . . . Dissected urinary system of a 20-day fetal rat from control dam. . . . . . . . . . . . . . . . . . . . . . Kidney from one—day-old rat from a dam that was given 9 mg/kg of dinoseb . . . . . . . . . . . . . . . Kidney from one-day-old rat from a dam that was dosed with 9 mg/kg dinoseb . . . . . . . . . . . . . . . . . Kidney from one-day-old rat from a control dam . . . . Kidney from one-day-old rat from a control dam . . . . Cross section of the urinary bladder of a 21-day fetal rat from a dam treated with 8 mg/kg of dinoseb . Urinary bladder from the animal seen in Figure 8 . . . Urinary bladder from a 21-day fetal rat from a control dam. O O O O 0 O O O O O O O O O O O O O O O 0 Higher magnification of the normal structure of the urinary bladder wall from the animal seen in Figure 10 Cross section of a ureter from a one-day-old rat from a dam treated with 9 mg/kg of dinoseb . . . . . . Ureter from the same animal seen in Figure 12. . . . . Cross section of a ureter from a control animal the same age as the animal seen in Figure 12 . . . . . . . Cross section of a ureter from the same animal shown in Figure 14, in which normal structures of the ureter can be seen. . . . . . . . . . . . . . . . . . . . . . Liver from a 20-day fetal rat from a control animal. . Higher magnification of the normal liver seen in Figure 16. O O O O O O I O O O O O O O O O O O O O O 0 vi Page 11 27 28 3O 3O 31 31 33 33 34 34 35 35 36 36 37 37 Figure 18 19 Page Liver from a 20-day fetal rat from a dam treated with 8 mg/kg of dinoseb . . . . . . . . . . . . . . . . . . . 38 Higher magnification of the liver from the same animal seen in Figure 18 . . . . . . . . . . . . . . . 38 vii INTRODUCTION Teratology is the study of congenital abnormalities produced by interference of the normal developmental processes. It has been described in many animal species. Kidney anomalies have been fre- quently encountered in teratology. The development of the mammalian kidney begins early in fetal life. It is vulnerable to injury at various stages. Among the anomalies which have been recognized in kidneys are unilateral or bilateral renal agenesis, renal hyp0p1asia, dysplasia, polycystic kidneys, and most frequently, hydronephrosis. Various perinatal manipulations may have an effect on develop- mental processes. These may include nutritional factors as well as the effects of various chemicals. Hypervitaminosis A during pregnancy in man (Bernhardt, 1974) and rats (Baba and Tsuruhar, 1959) induced renal anomalies in new- borns. On the other hand, pteroylglutamic acid deficiency in rats (Monie et al., 1957) resulted in malrenal development. The treatment of plants with herbicides such as dinoseb may result in accidental or incidental ingestion of these chemicals, which may lead to malformations in newborn animals. However, very few studies related to the toxicity and teratogenicity of dinoseb have been done. The objective of this research was to study the capability of dinoseb to induce teratogenicity in fetal rats. 1 LITERATURE REVIEW Kidney Development The mammalian kidney arises from the mesoderm. During its embryonal development there are three sets of excretory organs: the pronephros, mesonephros and metanephros. The first two are transi- tory while the last is the future functional kidney. The pronephros is the first and simplest excretory organ. It consists of a pair of pronephric tubules arising as buds off the nephrotome plate. Their ends fuse together to form the pronephric duct, which persists as the mesonephric duct (Arey, 1974). The pro- nephros is characterized by the presence of external glomeruli (Jordan and Kindred, 1942). The mesonephros is a compound tubular organ that opens into the mesonephric duct (Wolffian duct). The tubules arise from a nephro- genic cord which is unsegmented compared to the segmented nephrotome (Jordan and Kindred, 1942). The lateral end of the tubules grows to contact the mesonephric duct. The medial end of each tubule expands and becomes invaginated by the blood capillaries to form the glomerular (Bowman's) capsule within which is a glomerulus (Jordan and Kindred, 1942; Arey, 1974). The two together form the mesonephric corpuscle. The tubules are differentiated into secretory and collecting portions. Two types of mesonephri have been described by Bremer (1916). One type of mesonephros which is found in the cat, sheep and pig 2 3 functions until the functioning kidney develops; the other type of mesonephros is nonfunctional. It is seen in the guinea pig, rabbit, rat and in man and degenerates before the functioning kidney is developed. The mesonephric duct is incorporated into the genital duct of males. The metanephric kidney arises far caudal in the body (Arey, 1974). It originates from the ureteric bud and metanephrogenic mass. The ureteric bud arises from the mesonephric duct and its distal end dilates into the renal pelvis, which bifurcates into major calyces. The branching of the major calyces forms the minor calyces into which Open the papillary ducts (Cotchin and Roe, 1967). The collecting tubules grow out of the minor calyces. These make up a large part of the medulla and project into the cortex as medullary rays (Cotchin and Roe, 1967). The proximal end of the ureteric bud differentiates into the ureter. The metanephric mass is composed of two layers. The internal layer differentiates into secretory tubules and the external layer becomes the interstitial connective tissue and the capsule of the kidney. Cell clusters in the secretory tubules unite with the collecting tubules at one end. The thinner walled blind end of the secretory tubule becomes Bowman's capsule where the glomerulus is found. The secretory and collecting tubules form the nephron. Nephron lengthening results in the proximal convoluted tubule, the loop of Henle and the distal convoluted tubules. The proximal con- voluted tubules make up the bulk of the cortex. They are lined by cuboidal cells with abundant granular cytoplasm. The surface towards the lumen has a brush border. From the glomerulus each proximal convoluted tubule makes a series of convolutions and passes down the 4 medullary rays to become a descending limb of Henle's 100p. The epi- thelium of the descending loop is flattened with no brush border. These tubules continue as ascending loops of Henle with a cuboidal granular epithelium. The ascending Henle's loop comes into contact with the juxtaglomerular part of the efferent vessels of its respective glomerulus after which it forms the distal convoluted tubule. The crowded cells at this point of contact form the macula densa (Cotchin and Roe, 1967). Small cuboidal cells with no brush border line the distal convoluted tubules. The diameters of the lumens of distal convoluted tubules are greater than that of the proximal convoluted tubules. The smaller collecting tubules are lined by cuboidal epithelium and the larger ones are lined by columnar epithelium. The renal pelvis and ureters have a transitional type of epithelium. The development of the renal cortex in most mammals is incom- plete at birth (Arataki, 1926). In the rat the number of cortical glomeruli doubles during the first week of extrauterine life. The first nephrons to be formed are those in the juxtamedullary area and the last are the superficial ones (Arataki, 1926; Gersh, 1937). In utero kidney function serves for the formation of amnionic fluid and assists in regulating fetal water and solute composition. The newly born rat does not regulate its renal function as effectively as the adult animal (Dlouha and Goncarevska, 1974; Falk, 1955; Heller, 1949). The single glomerular filtration rate increases during early postnatal development (Solomon and Capek, 1972). On the other hand, the reabsorption capacity of the proximal tubules is lower in weanling rats than in adults (Capek et al., 1968). Thus, younger rats have a higher capacity to filter than to reabsorb or 5 secrete. Consequently, urine flow is higher in young animals (Dlouha, 1976). The urine excreted by newborn rats tends to be diluted and of unvarying composition and volume (McCance and Wilkinson, 1947; Heller, 1949). Abnormal Kidney Development Kidney anomalies have been reported in many species. Some of these anomalies may be inherited, whereas the cause of others is unknown. Among the defects frequently reported in animals is renal agenesis or aplasia. One or both kidneys can be affected. If only one kidney is affected, there will be compensatory hypertrophy of the other kidney. The cause of renal agenesis is related to anomalous growth of the ureteric bud or when the ureteric bud fails to reach the metanephrogenic mass, or a combination of both (Campbell, 1963). Renal hypoplasia is seen in newborn or young animals in which the kidney is smaller than normal with little or no function. It results from a decreased induction of the metanephric tissues (Campbell, 1963). Abnormal development of nephric and ductal struc- tures can lead to a dysplastic kidney. The kidney can be of any size or shape. Dysplasia may be cortical or medullary, total or partial, focal or segmented (Bernstein, 1971). Failure of union between glomeruli and collecting tubules may result in polycystic kidneys (Hildebrandt, 1894). Hydronephrosis Hydronephrosis is one of the kidney defects encountered in teratology. It is the dilation of the renal pelvis and calyces with compression or atrophy of renal parenchyma due to obstruction of the outflow of urine and is often accompanied by hydroureter. It results 6 in atrOphic kidney tissue with predominant damage to the renal medulla and distal tubules (Machado and Lozzio, 1972). In the absence of infection the tissue of the renal cortex remains intact several weeks after obstruction. The dilation of the renal pelvis can subside after removal of a longstanding obstruction (Fylling, 1952; Geisinger, 1937; Walters, 1933). Irreversible disturbances of kidney function occur a few days after obstruction of the ureter. In severe complete obstruction of both sides, the animal dies from uremia without extensive gross changes in the kidney. Sudden complete obstruction on one side may cause atrophy of the corresponding kidney. The urinary obstruction can be at any level from the urethra to the renal pelvis. Hydro— nephrosis usually develops when the obstruction is partial. However, complete obstruction will lead to mild hydronephrosis because of abrupt suppression of renal glomerular filtration. Dilation of the renal pelvis and calyces compresses the renal vessels resulting in venous stasis and arterial insufficiency (Hinman and Morison, 1926). The urinary obstruction can be congenital as well as acquired. Heritable hydronephrosis has been reported in albino rats (Hain and Robertson, 1936, 1937), Brown Norway rats (Bennett et al., 1970), and Wistar rats (Lozzio et al., 1967). Similar heritable hydro- nephrosis has been reported in inbred mice (Wallace and Spickett, 1967) and in man (Campbell, 1963). Two reports of Spontaneous hydro- nephrosis have been described in rats (Sellers et al., 1960; Astarabadi and Bell, 1962). Machado and Lozzio (1972) found that 52% of 1824 rats examined had hydronephrosis with a majority having unilateral defects. The right kidney was most commonly involved and urinary obstruction was observed in some severe cases. They considered that congenital hydronephrosis is inherited as an autosomal dominant gene. Monie et a1. (1957) stated that during normal development in the rat a temporary closure of the orifice of the ureter occurs in 16 to 19 day fetuses. If the orifices are closed longer than normal, dis- tention will take place because of continuous urine formation, resulting in hydronephrosis and/or hydroureter. Woo and Hoar (1972) observed that in late gestation in rats the renal papilla slowly and steadily increases in length and the renal parenchyma rapidly increases in weight. This disparity in growth rates results in a kidney with an enlarged renal pelvis. They referred to such dilation as hydronephrosis. The condition disap- peared shortly after birth and was thus called apparent hydronephrosis. Some investigators studied the effects of ureteral obstruction on the kidneys. Thomasson et al. (1970) observed that intrauterine ureteral ligation during the last third of gestation in fetal rabbits resulted in hydronephrosis, rapid dilation of collecting tubules and Bowman's space, and thinning of the cortex. Schubert et a1. (1975) ligated the left ureter of rats and observed the eXpansion of Bowman's space and the lumens of proximal convoluted tubules which diminished within 24 hours. On the other hand, distention of the lumenscflfdistal tubules and collecting tubules persisted up to 14 days. These findings are in harmony with those of Sheehan and Davis (1959) and Thomasson et al. (1970) in the rabbit and Strong (1940) in the dog. Hydronephrosis results in both morphological and functional changes. The weight of the kidney with an obstructed ureter increased to a maximum after ligation and then decreased. Schubert et al. 8 (1975) attributed such an increase to the accumulation of fluid and to hypertrophy in the distal portions of the nephron. Swann et al. (1955) and Swann (1960) explained that an increase in hydronephrotic kidney weight occurred because of accumulation of fluid at different sites in the kidney. In contrast, Herlant (1948) and Threlfall et a1. (1966, 1967) stated that the greater weight found in the hydro- nephrotic kidney was caused by an increased synthesis of DNA, RNA and protein. The expansion of the distal portion of the nephron in a hydro- nephrotic kidney is irreversible (Schubert et al., 1975). Micro- puncture studies indicated that persistent dilation of proximal tubules will not occur in an obstructed kidney as long as the contra- lateral kidney remains intact (Yarger et al., 1972). The severity of hydronephrosis is directly related to the duration and extent of obstruction (Strong, 1940; Deming, 1951). The function of the kidney depends on the type and extent of malformation. In the dog, experimental hydronephrosis causes mild impairment of total kidney glomerular filtration rate and increased sodium excretion. Thus, there is decreased functional mass with an increased glomerular filtration rate per nephron and a decreased proximal tubular sodium reabsorption (Suki et al., 1966). In con- trast, Wilson's (1972) studies in rats showed a marked diminished total kidney glomerular filtration rate, decreased glomerular filtra- tion per nephron and increased proximal tubular sodium reabsorption. The impairment of the function in the hydronephrotic kidney causes a compensatory hypertrophy and hyperfunction in the contralateral normal kidney. The degree of compensatory hypertrophy depends upon the severity of the hydronephrosis. 9 The Effect of Chemicals on the Kidney A variety of chemical agents may cause renal abnormalities in prenatal animals. Teratogenic effects in man and rats were produced by 2,4,5—trichlor0phenoxyacetic acid (Courtney et al., 1970). Later it was discovered that the compound was contaminated with 2,3,7,8- tetrachlorodibenzo-p-dioxin. Courtney and Moore (1971) studied the effect of pure trichlorphenoxyacetic acid and tetrachlorodibenzo-p— dioxin (TCDD) on CD-l, DBA/ZJ, and C57Bl/6 strains of mice and in the CD strain of rats. Trichlorophenoxyacetic acid given subcu— taneously to mice and orally to rats at doses of 50 to 150 ug/kg and 10 to 80 ug/kg body weight, respectively, on days 6 to 15 of gesta- tion produced little malformation. On the other hand, TCDD adminis— tered subcutaneously at a dose of 3 ug/kg body weight on days 6 to 15 of pregnancy had a teratogenic effect in all three strains of mice. It produced cleft palate and kidney anomalies. The C57Bl/6 mice were found to be the most sensitive and almost 100% of the fetuses had kidney anomalies. Administration of TCDD to rats at the rate of 3 ug/kg of body weight on days 6 to 15 of gestation had only a slight effect on kidney development. However, a dose of 2 ug/kg of body weight on days 9 and 10 or days 13 and 14 of gestation produced an effect of 1% and 34%, respectively, on kidneys but no cleft palate lesions were observed. The anomalies were described as unilocular cystinephrotic kidneys and hydronephrosis. In additional studies with TCDD, 3 ug/kg body weight administered orally to mice on gesta- tion days 10 through 13 produced a 55% incidence of cleft palate and a 95% incidence of kidney anomalies. By decreasing the dose to l ug/kg body weight, the incidence of cleft palate was 2%, while the kidney anomalies persisted at a high level. A single dose of l ug/kg 10 administered on day 10 resulted in considerable kidney anomalies but had no effect on the palate. In a series of cross fostering and reciprocal cross fostering eXperiments there were prenatal and post- natal effects associated with TCDD treatment (Moore et al., 1973). Pups from TCDD treated mothers who nursed untreated mothers had no kidney lesions. However, pups born of untreated mothers and which nursed TCDD treated mothers had hydronephrotic kidneys. Therefore, exposure of females to TCDD during the nursing period was a major factor in the develOpment of renal hydronephrosis. No signs of renal obstruction were observed. It was suggested that pups were exposed to TCDD through the milk. It was observed that in unilateral hydro- nephrosis the right kidney was almost always the one affected. This was attributed to anatomical variation. Exposure of rats to methylsalicylate during 10 and 11 days of gestation resulted in an apparent hydronephrosis in their offspring. Intraperitoneal injection of 0.1 ml methylsalicylate on days 10 and 11 of gestation resulted in less weight gain, fewer and smaller offspring with more resorbed and malformed young. The growth of the kidney was retarded but comparatively the growth of the renal papilla was even more severely retarded. Thus, the incidence of renal abnormalities near term was increased (Woo and Hoar, 1972). Nearly 10% of the kidneys from treated rats had gross dilation of the renal pelvis and reduction of renal parenchyma at weaning, which was thought to be a permanent hydronephrosis (Woo and Hoar, 1972). At birth the renal papillae were shorter in most kidneys of treated animals compared to control kidneys. In addition, there was a higher frequency of kidneys with absent papillae in the treated group than in the controls. Thus, methylsalicylate reduced renal growth with ll advanced fetal and postnatal age. The renal papilla in both treated and control animals grew steadily from short to full length. The fetal kidney weight doubled from day 19 to day 20 and increased over 60% from day 20 to day 21. The rapid growth of the renal parenchyma coupled with constant but slower increase in renal papillary length may result in a transitory formation of a large pelvis (Woo and Hoar, 1972). Dinoseb The chemical structure of dinoseb is illustrated in Figure 1. . . . . . a . It is the active constituent of a premergence herbiCide and is OH H I CH3 N02 c __c -——CH3 I H CH3 NO2 Figure 1. Chemical structure of dinoseb. produced in the form of ethanol and isopropanol amine salts of dinoseb. It is used widely to control seedling weeds. In rabbits and rats, dinoseb is metabolized and carboxylic acid is formed by oxidation of the terminal carbon of the secondary side chain (Ernst and Bar, 1964). In vitro, dinoseb in ruminal fluid was converted to aDow Chemical Company, Midland, MI. 12 6-amino-derivative with successive reduction to diamino-compounds. In vivo, diamino-compounds were not found in the blood of cows (Forslie and Karlog, 1970). In mice metabolites of dinoseb were found in liver and kidney but not in blood (Gibson and Rao, 1973). Dinoseb can cross the placenta, although a barrier was present since the level of dinoseb in the embryo never exceeded 2.5% of the maternal plasma levels (Gibson, 1972). Dinoseb was found to be teratogenic to mice (Gibson, 1973; Gibson and Rao, 1973). Intraperitoneal injection of 17.7 mg/kg on days 10 to 12 or 14 to 16 of gestation was toxic to the dams, but in dams which survived teratogenic effects were found only in fetuses from dams which were treated at the earlier period of gestation. A 30 to 40% incidence of hydronephrosis was produced at a dose level of 12.5, 15.8 and 17.7 mg/kg on days 10 to 12 of gestation. How- ever, 5 mg/kg given intraperitoneally on days 8 to 16 or 14 to 16 of gestation produced no effect. On the other hand, a dose of 17.7 mg/kg subcutaneously on days 14 to 16 of pregnancy produced 10.6% cleft palates. A dose of 20 to 32 mg/kg given orally during early or late organogenesis had no significant teratogenic effect. Thus, the prenatal toxicity depends on both dose level and route of administration. It was concluded that 2—sec-butyl-4,6-dinitrophenol had a low potentiality to induce teratogenicity in mice. Environmental factors can play a role in the teratogenicity as well as the embryotoxicity of dinoseb. By increasing the environmental temperature, the toxic effect in adult mice was increased and the prenatal effect was also enhanced (Preache and Gibson, 1975a). Food 13 deprivation for 24 hours increased the embryotoxicity of the herbi- cide while 48 hours of deprivation had no effect. Pretreatment with 2—diethy1aminoethyl-2-2-diphenylvalerate (SKF-525A) increased the teratogenicity of dinoseb while treatment with phenobarbitone protected against its teratogenicity (Preache and Gibson, 1975b). In general, exposure of pregnant mice to dinoseb caused fetal anomalies under certain conditions. This is also true for certain other chemicals which have been studied. The effects of dinoseb on fetal rats has not been studied. MATERIALS AND METHODS Animals Pregnant Sprague-Dawley ratsa ranging from 295 to 310 grams body weight were placed singly in stainless steel wire cages with wood chip bedding. The rats were received at 7 days of gestation and were held for two days before treatment. Feedb and water were given ad libitum. Preparation of Dinoseb A freshly prepared aqueous solution of dinoseb (Lot no. 7200206, 1966)c was mixed in a concentration so that 2 ml/kg of body weight provided the appropriate dose. The herbicide was dissolved in 0.1 N NaOH and the pH was adjusted to 7.4 by using 0.1 N HCl. The solution of each concentration was made up to 50 ml with double distilled water. Treatment The 39 pregnant rats were weighed and then were divided into eight groups. Group I (7 animals) was kept as controls. The herbicide was given to each group at the dose level indicated in Table l. A aSpartan Research Animals, Inc., Haslett, MI. bSource: Wayne Lab Blox, Allied Mills, Inc., Chicago, IL 60606. CDow Chemical Company, Midland, MI. 14 15 Table l. The number of dams treated and the dose level of dinoseb in each group Group No. of dams Dose level of dinoseb no. treated mg/kg I 7 control II 4 6.3 III 8 8.0 IV 7 9.0 V 3 11.2 VI 3 12.5 VII 4 15.2 VIII 3 17.7 16 daily dose of dinoseb was administered intraperitoneally during early organogenesis (day 10 through day 12 of gestation). The dose level .of dinoseb ranged from 6.3 mg/kg body weight in Group II to 17.7 mg/kg body weight in Group VIII. The dosage levels were chosen so that maternal toxicity and fetal teratogenicity would be manifested in one or more groups. The seven control rats were given an equivalent amount of 0.1 N NaCl at a rate of 2 mg/kg body weight. The dams were observed during the gestation period for any clinical changes. Methods of Examination The fetuses were examined on either day 20 or day 21 of gestation or at one day of age. The dams were killed by a blow on the head, aafter which the gravid uterus was removed. The number and position (Df live or resorbed fetuses was recorded. Fetuses were removed by ssevering the umbilical cords with a cautery knife to prevent loss of Ifetal blood, then dried and sexed. The fetal crown-rump length was Ineasured to 0.1 centimeter using a vernier caliper. Fetal weight ‘vas recorded in grams, and each fetus was examined for cleft palate éand external skeletal anomalies. Fetuses were killed by strangulation and placed on a necropsy kxDard. The skin was then reflected from the abdomen and thorax. “Hie internal organs were examined under a dissecting microscope for Scxft tissue anomalies. Kidney, bladder, ureter, liver, lung, heart, mlliscles and skin were fixed in either 10% buffered formalin or Carnoy' s fluid. Tissues were paraffin embedded and cut 6 microns thick. Sec- tiCnis were stained with hematoxylin and eosin, periodic acid-Schiff l7 (PAS) and Gomori's trichorome. Frozen sections were stained by oil red 0. Techniques used were essentially those suggested by the Armed Forces Institute of Pathology (Luna, 1968). Statistics Statistical analysis of measured parameters was made by analysis of variance. Group difference was detected by the t-test (Steel and Tarrie, 1960). The level of significance was chosen as p<0.05. RESULTS General Observations Adult rats dosed with dinoseb at a rate of 17.7 mg/kg, 15.2 mg/kg and 12.5 mg/kg body weight died after receiving the first dose. Two of the adult rats given 11.2 mg/kg died after the first injection and the third one died after having received the last of three doses. Neither clinical signs nor lesions were demonstrated. The remainder of the adult rats were in good condition with normal behavior and appetite throughout gestation. Effect of Dinoseb on Fetal Growth Group I. The number and average size of control fetuses at different ages are given in Table 2. Group II. Four pregnant rats were injected with dinoseb at a rate of 6.3 mg/kg body weight. The fetuses were examined on day 20 of gestation. The mean weight and crown-rump length are given in Table 3. The size or resorption rate of fetuses from dams treated with this level were not affected. Group III. Animals in this group were examined at different ages, namely, 20 and 21 days of gestation and one day of postnatal life. The parameters measured are given in Table 4 for the three periods in time. At days 20 and 21 of gestation the weights of treated fetuses were significantly reduced to 4.175 gm and 5.445 gm, 18 19 Table 2. Number of fetuses, resorption rate and mean fetal size from control rats Fetal body Fetal crown Dam Number of Number weight (gm) rump length (cm) number fetuses resorbed Mean SD Mean SD 20 days gestation 1 9 0 4.510 .t 0.013 3.75 i 0.05 2 10 0 4.39 i 0.11 3.44 _t 0.04 3 14 o 4.579 + 0.05 3.778 + 0.05 21 days gestation 4 l4 0 5.923 :_0.012 3.76 :_0.09 5 12 0 5.716 :_0.09 4.04 :_0.07 1 day old 6 16 0 6.451 :_0.11 3.95 :_0.05 7 17 0 6.660 :_0.02 4.15 :_0.05 20 Table 3. Number of fetuses, resorption rate and mean fetal size from dams treated with 6.3 mg/kg dinoseb (2-sec-buty1-4,6- dinitrophenol) Fetal body Fetal crown Dam Number of Number weight (gm) rump length (cm) number fetuses resorbed Mean SD Mean SD 1 10 0 4.567 :_0.05 3.65 :_0.05 2 8 1 4.089 i_0.15 3.53 :_0.06 3 15 0 4.148 :_0.06 3.59 i_0.04 4 14 0 3.89 :_0.09 3.46 + 0.03 21 Table 4. Number of fetuses, resorption rate and mean fetal size from dams treated with 8.0 mg/kg dinoseb (2-sec-butyl-4,6- dinitrophenol) Fetal body Fetal crown Dam Number of Number weight (9m) rump length (cm) number fetuses resorbed Mean SD Mean SD 20 days gestation 1 14 0 4.665 :_0.09 3.34 :_0.22 2 10 1 4.440 :_0.06 3.49 :_0.04 3 12 0 4.229 :_0.06 3.38 :_0.04 21 days gestation 4 l4 0 5.151 :_0.17 3.87 :_0.05 5 10 1 5.741 :_0.07 3.85 :_0.03 1 day old 6 10 0 6.259 i_0.ll 3.97 :_0.04 7 8 0 6.088 + 0.11 4.07 :_0.03 8 16 O 5.936 + 0.91 3.911 + 0.03 22 respectively, compared to 4.513 gm and 5.815 gm of their corresponding controls. The reduction in weight at one day of age was not signifi- cant. The resorption rate and the reduced crown-rump length compared to controls were not statistically significant. Group IV. The fetuses in this group had lower values in all parameters measured than fetuses in other treated groups (Table 5). The weights of the fetuses at days 20 and 21 of gestation were reduced to 3.485 gm and 4.225 gm, respectively. At one day of age the reduction in weight was no longer significant. There was no significant change in the values of crown-rump length and fetal resorption rate. The mean values of the parameters measured in Groups I, II, III and IV at different ages are summarized in Table 6. Pathologic Findings Gross Lesions. No pathological changes were found in fetuses from control rats. Fetuses from dams treated with dinoseb at a rate of 6.3 mg/kg did not have any skeletal or soft tissue anomalies. Fetuses from dams which received dinoseb at a rate of 8 mg/kg and 9 mg/kg, respectively, had a high incidence of dilated bladders and hydroureters at 20 and 21 days of gestation and at one day of age. The percentage incidence of these changes at different ages is given in Tables 7 and 8. The right ureters were markedly dilated on the proximal ends (Figure 2) as compared to the control (Figure 3). The incidence of hydroureters reached 100% at one day of age in fetuses from mothers which received 9 mg/kg dinoseb. At day 21 of gestation there was an increase in the kidney size (hydronephrosis) 23 Table 5. Number of fetuses, resorption rate and mean fetal size from dams treated with 9.0 mg/kg dinoseb (2-sec-butyl-4,6- dinitrophenol) Fetal body Fetal crown Dam Number of Number weight (gm) rump length (cm) number fetuses resorbed Mean SD Mean SD 20 days gestation 1 12 1 3.325 i_0.05 3.31 :_0.15 2 11 1 3.650 : 0.15 3.40 :_0.10 21 days gestation 3 8 3 4.280 1 0.17 3.56 :_0.3 4 12 0 4.176 :_0.08 3.33 :_O.l4 1 day old 5 12 0 6.410 :_0.11 4.05 :_0.04 6 2 0 6.463 :_0.10 3.83 + 0.10 7 14 0 5.740 i 0.10 3.71 i 0.05 24 Table 6. Mean number of fetuses and fetal size from treated and control dams Dose level Mean num- Fetal body Fetal crown in dams ber of weight (gm) rump length (cm) (mg/kg) fetuses Mean SD Mean SD 20 days gestation 0 11 4.513 t. 0.06 3.65 _+_; 0.10 6.3 11 4.098 i 0.07 3.42 1 0.06 8.0 12 4.372 1 0.12 3.43 i 0.07 9.0 11 3.485 .t 0.15 3.40 + 0.10 21 days gestation 0 13 5.815 1 0.10 3.90 i 0.14 8.0 12 5.445 1 0.29 3.86 i 0.01 9.0 10 4.225 1 0.06 3.44 t. 0.11 1 day old 0 15 6.559 1: 0.11 3.95 1 0.02 8.0 11 6.427 .t 0.09 3.98 .t 0.04 9.0 9 6.204 1 0.23 3.86 i 0.09 25 Table 7. Percentage of fetuses with gross lesions from dams treated with 8.0 mg/kg dinoseb (2-sec-butyl-4,6-dinitrophenol) Dam Percentage Percentage Percentage number hydroureter dilated bladder hydronephrosis 20 days gestation 1 71.4 21 - 0 2 53 33 0 3 85 4O 0 21 days gestation 4 71 64 14 5 94 71 18 1 day old 6 60 50 0 7 63 63 O 26 Table 8. Percentage of fetuses with gross lesions from dams treated with 9.0 mg/kg dinoseb (2-sec-buty1-4,6-dinitrophenol) Dam Percentage Percentage Percentage number hydroureter dilated bladder hydronephrosis 20 dayspgestation l 91 64 0 2 83 58 O 21 days gestation 3 85 54 16 4 88 75 18 1 day old 5 100 58 O 6 100 100 0 7 100 57 O 27 — ‘ —_ Figure 2. Dissected urinary system of a 20—day fetal rat from a dam that was dosed with 8 mg/kg of dinoseb. Notice the size of the right ureter (arrow) compared to the left ureter. 28 Figure 3. Dissected urinary system of a 20-day fetal rat from control dam. 29 in fetuses from dams which had been dosed with 8 mg/kg and 9 mg/kg (Tables 7 and 8). The kidneys were Opened for examination. On cross section the renal pelvis was dilated in fetuses of all ages from Groups III and IV. There were no cleft palates or other skeletal anomalies in fetuses from animals treated at different dose levels. Likewise, other tissues such as liver, lung, heart, muscle and skin had no gross pathological changes. Microscopic Lesions. The histopathological changes at days 20 and 21 of gestation and one day of age will be discussed together unless otherwise specified. The microscopic findings in each organ will be discussed separately. K'dn y. The cross section of the fetal kidneys from dams dosed with dinoseb at a rate of 8 mg/kg and 9 mg/kg had an enlarged and dilated renal pelvis (Figure 4) as compared to the controls (Figure 6). There were many distended tubules seen, mostly at the corticomedullary junction (Figure 5), compared to kidneys from con- trol animals (Figure 7). These included proximal and distal convo- luted tubules and Henle's loops. The distal tubules were highly dilated resulting in flattening of the epithelial lining (Figure 5). Mesenchymal tissue was seen in kidneys of fetuses from both control and treated dams. The glomeruli were highly cellular in both. Gomori's trichrome stain showed absence of collagen fibers on kidneys from both groups. The changes at days 20 and 21 of gestation are similar to those seen at one day of age (Figures 4 and 5). Urinagy bladder. The transitional epithelium was thinner in fetuses from animals treated with dinoseb at a rate of 8 mg/kg and 3O Figure 4. Kidney from one-day-old rat from a dam that was given 9 mg/kg of dinoseb. Enlarged renal pelvis and tubules. H & E stain X40. Figure 5. Kidney from one-day-old rat from a dam that was dosed with 9 mg/kg dinoseb. The distal tubules are dilated to an extent that the epithelium is flattened. Henle's loop and proximal tubules are also dilated. There is a remnant of mesenchymal tissue. H & E stain X250. 31 Figure 6. Kidney from one-day-old rat from a con- trol dam. Notice the size of the renal pelvis and tubules. H & E stain X40. Figure 7. Kidney from one-day-old rat from a con- trol dam. Normal structure of the kidney. H & E stain X250. 32 9 mg/kg body weight (Figure 8) when compared to controls (Figure 10). There was some vacuolation in the epithelial cells. The lamina propria in control animals had a fairly dense layer of fibrous tissue containing lymphatic vessels (Figure 11). In the treated animals there were large empty spaces in the lamina prOpria (Figure 10) which extended to the muscle layer and apparently led to atrophy of muscle fibers (Figure 9). These large spaces were suggestive of edema. Hemorrhage was also present in the lamina propria. The lesions at 20 days of gestation and one day of age were similar to those seen in Figures 8 and 10. Ureters. The main lesion in fetuses from animals given dinoseb at a rate of 8 mg/kg and 9 mg/kg body weight was the appearance of vacuoles in the cytoplasm of transitional epithelial cells (Figures 12 and 13). The vacuoles varied in size. The material accumulated in the cytoplasm and displaced the nuclei peripherally. In some cells the nucleus was not visible. This material in the vacuoles was not a lipid or glycogen as indicated by the negative reaction to oil red O and PAS stains, respectively. These vacuoles might have been caused by hydropic degeneration. The changes at days 20 and 21 of gestation are similar to those shown in Figures 12 and 13. The ureters from control fetuses were unaffected (Figures 14 and 15). giygg. The liver of control fetuses had some vacuolation of hepatic cells (Figures 16 and 17) caused by the high content of glycogen in the developing animals. The liver was active in hemato- poiesis and contained many megakaryocytes. The liver of fetuses from animals dosed with dinoseb at a rate of 6.3 mg/kg, 8 mg/kg and 9 mg/kg body weight was severely vacuolated at days 20 and 21 of gestation (Figures 18 and 19). The nuclei were not visible in some vacuolated 33 Figure 8. Cross section of the urinary bladder of a 21-day fetal rat from a dam treated with 8 mg/kg of dinoseb. The transitional epithelial lining of the bladder is thinning; edema is also present. H & E stain X64. Figure 9. Urinary bladder from the animal seen in Figure 8. Edema in the lamina propria extended to the muscle resulting in atrophy of the muscle. H & E stain X400. 34 Figure 10. Urinary bladder from a 21-day fetal rat from a control dam. Normal structures of the uri— nary bladder wall are shown. H & E stain X64. Figure 11. Higher magnification of the normal structure of the urinary bladder wall from the animal seen in Figure 10. H & E stain X400. 35 Figure 12. Cross section of a ureter from a one- day-old rat from a dam treated with 9 mg/kg of dinoseb. There is vacuolation in the transitional epithelium. H & E stain X100. Figure 13. Ureter from the same animal seen in Figure 12. Hydropic degeneration of the epithelial cells can be seen. Notice the nuclei are pushed to the periphery. Some cells seem to have lost their nuclei. H & E stain X250. 36 Figure 14. Cross section of a ureter from a con— trol animal the same age as the animal seen in Figure 12. H & E stain X100. Figure 15. Cross section of a ureter from the same animal shown in Figure 14, in which normal struc- tures of the ureter can be seen. H & E stain X250. 37 Figure 16. Liver from a 20-day fetal rat from a control animal. Notice the presence of vacuolation in normal liver cells. H & E stain X64. Figure 17. Higher magnification of the normal liver seen in Figure 16. H & E stain X400. 38 Figure 18. Liver from a 20-day fetal rat from a dam treated with 8 mg/kg of dinoseb. Notice the vacuo- lation in the liver cells. H & E stain X64. Figure 19. Higher magnification of the liver from the same animal seen in Figure 18. Notice the size of the vacuoles. In some cells the nuclei appear to be pressed aside and in some nuclei cannot be seen. H & E stain X400. 39 cells or were pressed to one side if they were present. Hematopoiesis was less apparent than in the controls and there were fewer megakaryocytes. There were no significant pathological changes in the lung, heart, muscle and skin. DISCUSSION Dinoseb, a dinitrophenol derivative, has been used as a herbi- cide for some plants. Although animals and man can contact this chemical by ingestion of treated plants, no report of accidental toxicity of dinoseb in man and animals was found. In pregnant Swiss Webster mice intraperitoneal administration of dinoseb at a rate of 15.8 mg/kg body weight on days 10 to 12 of gestation caused no maternal toxicity, while a dose of 17.7 mg/kg given at the same period of time killed one mouse out of 14 (Gibson, 1973). In the present study in Sprague-Dawley rats, doses of dinoseb given intra- peritoneally at a rate of 11.2 mg/kg, 12.5 mg/kg, 15.8 mg/kg and 17.7 mg/kg of body weight on days 10 through 12 of gestation killed all the dams dosed. These results indicate that Sprague-Dawley rats may be more sensitive to dinoseb than Swiss webster mice when both are treated intraperitoneally. However, pregnancy may play a role. Negherbon (1959) found that the lethal dose of dinoseb in adult rats was 37 mg/kg body weight when it was given orally. This dose is more than three times the amount of dinoseb which killed rats when it was given intraperitoneally. Gibson (1973) reported that dinoseb was absorbed more completely when given intraperitoneally than when given orally. It may also be that the chemical was altered in some manner in the digestive tract which may have reduced its toxicity. Teratogenicity of dinoseb had not been demonstrated in rats but it had been studied in mice (Gibson, 1973; Gibson and Rao, 1973). A 40 41 dose of 17.7 mg/kg administered intraperitoneally to pregnant mice caused teratogenic lesions in fetuses. In the present studies a similar dose was found to be lethal to pregnant rats but doses of only 8 mg/kg and 9 mg/kg caused teratogenic lesions in their offspring. The weight of fetuses from dams that received 8 mg/kg and 9 mg/kg of dinoseb were significantly reduced at days 20 and 21 of gestation. This may be due to the toxic effect of dinoseb on the growing cells. It is not known why the weight reduction did not carry over to the first day of age. Furthermore, in spite of the fetal weight reduction there was no corresponding reduction in crown- rump length or the resorption rate at the same ages. The reason for the hydroureters, dilated bladders and hydro- nephrosis is not clear because there was no evidence of obstruction either grossly or microscopically. The explanation given by Monie et a1. (1954) and Woo and Hoar (1972) may not apply to the lesions seen in the ureters. Even though rats may suffer from severe hydro- nephrosis resulting in renal damage, there was no observable systemic effect on the animal (Lozzio et al., 1967). In contrast, a similar condition in man has a grave prognosis (Campbell, 1963). The microscopic lesions, consisting of hydropic degeneration of the transitional epithelial cells of the ureters, edema in the lamina propria of the urinary bladder, and dilation of tubules, were reversible once the cause was removed. The presence of vacuoles in the livers of fetuses from treated mothers may be a result of an effort by the liver to metabolize the chemical. In adult rats dinoseb was metabolized and carboxylic acid was formed (Earnest, 1964). Gibson and Rao (1973) reported the presence of unchanged dinoseb and an unidentified metabolite of 42 dinoseb in the liver and kidney of mice, but neither was found in blood. Fetal anomalies have been induced by some chemicals when the dams were treated at day 10 through day 12 of gestation. Rats exposed to methyl salicylate during early organogenesis had off- spring with apparent hydronephrosis (Woo and Hoar, 1972). Likewise, TCDD was reported to induce cleft palate and kidney anomalies in rats and mice (Courtney and Moore, 1971; Moore et al., 1973). In the present study, dinoseb did not seem to be markedly teratogenic to fetal rats. This may be related either to the nature of the chemical itself or to the strain of rat used. It can be concluded from this study that dinoseb caused some renal teratogenesis. However, whether these changes were transitory or permanent needs further investigation. SUMMARY Thirty-nine pregnant Sprague-Dawley rats were used in this investigation. Two-sec-buty1-4,6-dinitrophenol (dinoseb) was administered daily during early organogenesis on days 10 through 12 of gestation by the intraperitoneal route. For treatments with dinoseb the animals were divided into the following groups: (1) control, (2) 6.3 mg/kg, (3) 8.0 mg/kg, (4) 9.0 mg/kg, (5) 11.2 mg/kg, (6) 12.5 mg/kg, (7) 15.8 mg/kg, and (8) 17.7 mg/kg of body weight. The dose levels of 11.2 mg/kg to 17.7 mg/kg were found to be lethal to the mothers. The fetuses were measured for growth rate, and examined for soft tissue and skeletal anomalies. Doses of 8.0 mg/kg and 9.0 mg/kg of body weight resulted in a significant weight reduction at days 20 and 21 of gestation but not at one day of age. The dose of 6.3 mg/kg had no effect on growth rate. Gross lesions caused by dose levels of 8 mg/kg and 9 mg/kg included hydroureter, dilated bladder and a dilated renal pelvis at days 20 and 21 of gestation and one day of age. At day 21 of gestation the incidence of hydronephrosis was 16% and 17% at the dose of 8 mg/kg and 9 mg/kg, respectively. Microscopic lesions included hydropic degeneration of the epithelial lining of the ureter, edema in the lamina propria of the bladder, dilation of the proximal and distal convoluted tubules, Henle's loOps and collecting tubules, and vacuolation of the liver cells. 43 44 There were no gross lesions at a dose of 6.3 mg/kg but micro- scopically vacuolation of the hepatocytes was seen. It is concluded that dinoseb has a low potential for terato- genic effects. LI ST OF REFERENCES LIST OF REFERENCES Arataki, G. M. 1926. The postnatal growth of the kidney with special reference to the number and size of the glomeruli (albino rat). Am. J. Anat., 36:399-436. Arey, L. B. 1974. Developmental Anatomy. A Text Book and Labora- tory Manual of Embryology, 7th ed. W. B. Saunders Company, Philadelphia and London. Astarabadi, T. and Bell, E. T. 1962. Spontaneous hydronephrosis in albino rats. Nature, 195:392-393. Baba, T. and Tsuruhara, T. 1959. The relationship between the environment and the development of fetuses. XIII. Malforma- tions caused by excess administration of vitamin A. 5. The genesis of the malformation of the urinary organs. Trans. Soc. Path. Jap.,48:1227-1228. Bennett, C. J., DeBruin, R. W. and Kort, W. J. 1970. Heritable hydronephrosis in a mutant strain of Brown Norway rats. Lab. Anim. Care, 20:489-493. Bernhardt, I. B. and Dorsey, J. D. 1974. Hypervitaminosis A and congenital renal anomalies in a human infant. Obstet. Gynec., 43:750-755. Bernstein, J. 1971. The morphogenesis of renal parenchyma mal- development (renal dysplasia). Pediatr. Clin. North Am., 18:395-407. Bremer, J. J. 1916. The interrelations of the mesonephros, kidney and placenta in different classes of animals. Am. J. Anat., 19:179-209. Campbell, M. F. 1963. Urology, 2nd ed., Vol. 2. Saunders, Phila- delphia. Capek, K., Dlouha, H., Hernandez, J. and Popp, M. 1968. Regulation of proximal tubular reabsorption in early postnatal period of infant rats. Micropuncture study. Proc. 24th Int. Congr. Physiol. Sci., Washington, 8:72-75. Cotchin, E. and Roe, F. J. 1967. Pathology of Laboratory Rats and Mice. Blackwell Scientific Publication, Oxford and Edinburgh. 45 46 Courtney, K. D., Gaylor, P. W., Hogan, M. D., Falk, H. C., Bates, R. R. and Mitchell, I. 1970. Teratogenic evaluation of 2, 4,5-trichlorophenoxyacetic acid. Science, 168:864-866. Courtney, K. D. and Moore, J. A. 1971. Teratology studies with 2, 4,S-trichlorophenoxyacetic acid and 2,3,7,8-tetrachlorodibenzo- p-dioxin. Toxicol. Appl. Pharmacol., 20:396-403. Deming, C. L. 1951. The effects of intrarenal hydronephrosis on the components of the renal cortex. J. Urol., 65:748-753. Dlouha, H. and Goncarevska, 0. A. 1974. The development of the single nephron structure in young rats. Physiol. Bohemoslov., 23:138-141. Dlouha, H. 1976. A micropuncture study of the development of renal function in the young rat. Biol. Neonate, 29:117-128. Ernst, W. U. and Bar, F. 1964. Die Unwandlung des 2,4-dinitro-6- sec-butylphenols und seiner Ester imtierischen Organismus. Arzneimittel Forsch.,l4:81-84. Falk, G. 1955. Maturation of renal function in infant rat. Am. J. Physiol.. 181:157-170. Froslie, A. and Karlog, O. 1970. Ruminal metabolism of DNOC and DNBP. Acta Vet. Scand., 11:114-132. Fylling, R. 1952. Restitution of rat kidney after temporary ureteral ligature. Acta Pathol. Microbiol. Scand. Supp. 93:224-229. Geisinger, J. F. 1937. The recuperative power of the kidney: A report of three cases. J. Urol., 37:639-650. Gersh, L. 1937. The correlation of structure and function in the deve10ping mesonephros and metanephros. Contrib. Embryol. , 153:35-39. Gibson, J. E. 1972. Placental transfer, embryotoxicity and terato- genicity of the herbicide 2-sec-butyl—4,6-dinitrophenol in mice. 5th Int. Congr. on Pharmacology, San Francisco. Gibson, J.H. 1973. Teratology studies in mice with 2-sec-buty1- 4,6-dinitrophenol (dinoseb). Food Cosmet. Toxicol., 11:31-42. Gibson, J. E. and Rao, K. S. 1973. Disposition of 2-sec-butyl-4,6- dinitrophenol (dinoseb) in pregnant mice. Food Cosmet. Toxicol., 11:45-52. Hain, A. M. and Robertson, E. M. 1936. Congenital urogenital anomalies in rats including unilateral renal agenesia. J. Anat., 70:566-576. 47 Hain, A. M. and Robertson, E. M. 1937. Congenital urogenital anomalies ixlrats including unilateral renal agenesia. Further data in support of their inheritance. J. Anat., 72:83—100. Heller, H. 1949. Effects of dehydration on adult and newborn rats. J. Physiol., 108:303-314. Herlant, M. 1948. Experimental hydronephrosis studied by the colchi- cine method. Nature (Lond.), 162:251-252. Hildebrandt, A. 1894. Weiterer beitrag zur pathologischen anatomic der nierengeschwalste. Arch. fur Klin. Chir., 48:343-371. Hinman, F. and Morison, D. M. 1926. Experimental hydronephrosis. Surg. Gynec. Obst., 42:209-214. Jordan, H. E., and Kindred, J. E. 1942. Text Book of Embryology, 4th ed., D. Appleton-Century Company Incorporated, New York. Lozzio, B. 8., Chernoff, A. L., Machado, E. A. and Lozzio, C. B. 1967. Hereditary renal disease in a mutant strain of rats. Science, 156:1742-1744. Luna, L. G. (ed.). 1968. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed., McGraw- Hill Book Company, New York. Machado, E. A. and Lozzio, B. B. 1972. Congenital renal structural alterations in a mutant strain of rats. Invest. Urol., 10: 78- 83 o McCance, R. A. and Wilkinson. 1947. The response of adult and suckling rats to the administration of water and hypertonic solutions of urea and salt. J. Physiol., 106:256-263. Monie, I. w., Nelson, M. M. and Evans, H. M. 1954. Abnormalities of the urinary system of rat embryos resulting from maternal ptyeroyglutamic acid deficiency. Anat. Rec., 120:119—136. Monie, I. w., Nelson, M. M. and Evans, H. M. 1957. Abnormalities of the urinary system of rat embryos resulting from transitory deficiency of pteroyglutamic acid during gestation. Anat. Rec., 127:711-723. Moore, J. A., Gupta, B. N., Zinkl, J. G. and Vos, J. G. 1973. Post- natal effects of maternal exposure to 2,3,7,8-tetrachloro- dibenzo-p-dioxin (TCDD). Environ. Health PerSpect., 5:81-85. Negherbon, W. O. 1959. Handbook of Toxicology, Vol. III. Insecti- cides. W. B. Saunders, London. Preache, M. M. and Gibson, J. E. 1975(a). The effect of food depri- vation, phenobarbital and SKF-SZSA on teratogenicity induced by 2-sec—butyl-4,6-dinitrophenol (dinoseb) and on diSposition of 14C dinitrophenol in mice. J. Toxicol. Environ. Health, 15:109-118. 48 Preache, M. M. and Gibson, J. E. 1975(b). Effects in mice of high and low environmental temperature on the maternal and fetal toxicity of 2-sec-butyl-4,6-dinitrophenol (dinoseb) and on disposition of (14c)-dinoseb. Teratology, 12:147-156. Schubert, G. E., Staudhacmer, K. R. and Kneissler, U. 1975. Tubular dimensions and juxta-glomerular granulation index in rat kidney after unilateral obstruction of the ureter. Urol. Res., 3: 115-122. Sellers, A. L., Rosenfelds, S. and Friedman, N. B. 1960. Spontaneous hydronephrosis in the rat. Proc. Soc. Exp. Biol. Med., 104: 512-515. Sheehan, H. L. and Davis, J. C. 1959. Experimental hydronephrosis. Arch. Pathol., 68:185—225. Solomon, 8. and Capek, K. 1972. Regulation of superficial single nephron. Glomerular filtration rates in infant rat. Proc. Soc. Exp. Biol. Med., 139:325-329. Steel, R. G. D. and Torrie, H. H. 1960. Principles and Procedures of Statistics. McGraw-Hill Book Company, Inc., New York. Strong, K. C. 1940. Plastic studies in abnormal renal architecture. Parenchymal alterations in experimental hydronephrosis. Arch. Pathol., 29:77-119. Suki, w., Eknoyan, G., Rector, E. C. and Seldin, D. W. 1966. Pat- terns of nephron perfusion in acute and chronic hydronephrosis. J. Clin. Invest., 45:122-131. Swann, H. G., Feist, F. W. and Lowe, H. J. 1955. Fluid draining from functionally distended kidney. Proc. Soc. Exp. Biol. Med., 88:218-221. Swann, H. G. 1960. The functional distension of the kidney. Tex. Rep. Biol. Med., 18:566-595. Thomasson, B. H., Esterly, J. R. and Ravitch, M. M. 1970. Morphologic changes in the fetal rabbit kidney after intrauterine ureteral ligation. Invest. Urol., 8:261-271. Threlfall, G., Taylor, D. M., and Buck, A. 1966. The effect of folic acid on growth and deoxyribonucleic acid synthesis in the rat kidney. Lab. Invest., 15:1477-1485. Threlfall, G., Taylor, D. M., and Buck, A. T. 1967. Studies of the changes in growth and DNA synthesis in the rat kidney during experimental induced renal hypertrophy. Am. J. Pathol., 50: 1-13. Wallace, M. E. and Spickett, S. G. 1967. Hydronephrosis in mouse, rat and man. J. Med. Genet., 4:73-82. 49 Walters, W. 1933. Restoration of renal function following removal of obstructing lesions. Z. Urol. Chir., 36:264-275. Wilson, D. R. 1972. Micropuncture Study of chronic obstructive nephropathy before and after release of obstruction. Kidney Int., 2:119-130. Woo, D. C. and Hoar, R. M. 1972. Apparent hydronephrosis as a normal aspect of renal development in late gestation of rats. The effect of methyl salicylate. Teratology, 6:191-196. Yarger, W. E., Aynedjian, H. S. and Bank, N. 1972. A micropuncture study of postobstructive diuresis in the rat. J. Clin. Invest., 51:625-637. "71111111711 if! Mill [i @iflfq’ffliflflflaflfljfioiiimflfl