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Histological changes in kidney, liver and duodenum of the mouse following the acute and subacute administration of diethylenetriaminepentaacetic acid
Toxicology, 2 (1974) 153--163 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
HISTOLOGICAL CHANGES IN KIDNEY, LIVER AND DUODENUM OF THE MOUSE FOLLOWING THE ACUTE AND SUBACUTE ADMINISTRATION OF DIETHYLENETRIAMINEPENTAACETIC ACID
RAE M. MORGAN and HYLTON SMITH School of Pharmacy, Sunderland Poly technic, Sunderland, and National Radiological Protection Board, Harwell, Didcot (Great Britain) (Received October 18th, 1973) (Revision received November 22nd, 1973) (Accepted November 26th, 1973) .
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SUMMARY CaNa3 diethylenetriaminepentaacetic acid (DTPA) was found to produce transient histological changes in liver and kidney but not intestine in mice after the acute and subacute administration of doses between 6 and 30 times above the m a x i m u m recommended therapeutic dose.
INTRODUCTION
The polyaminopolycarboxylic acid, DTPA has become established as the most effective drug currently available for the removal of internally deposited actinides. Toxic side-effects have been reported in the rat following the administration of the calcium chelates of EDTA (CaNa2 EDTA) and DTPA (CaNa3DTPA). These consist of hydropic change in the kidney [1], vacuole production in hepatic parenchymal cells [2] and necrosis of the duodenal mucosa with atrophy of the villi [3]. These data, together with information on the early use of DTPA in man [4], have led to the imposition of limitations of the use of this chelate in human therapy, particularly in its use with workers accidentally contaminated with plutonium. However, the absence of suitable alternative therapy for the removal of contaminating higher actinides has made it necessary to re-evaluate the toxicity of this chelate. This communication presents evidence of reversible histological changes in mice receiving CaNa 3 DTPA either acutely or subacutely, at dose levels above the recommended therapeutic range.
Abbreviation: DTPA, diethylenetriaminepentaacetic acid. 153
MATERIALS AND METHODS CaNa3DTPA solution (250 mg/ml) suitable for intravenous use, was obtained from Geigy Pharmaceuticals Ltd., Macclesfield, Cheshire. The dose regimes have been planned to cover as many as possible of the reported clinical uses of the chelate [5], ranging from its use in the treatment of iron overload (200 mg/kg} to its use in therapy for plutonium contamination (14 mg/kg).
Acute administration of CaNa3DTPA (i) Light microscope studies. Groups of 10 adult, male C3H mice, weighing 25--30 g, were given CaNaaDTPA by intraperitoneal injection, at doses equivalent to 1.0, 2.5 and 5.0 g/kg body weight in a dose volume of 0.25 ml/25 g body weight. A group of 10 control animals received 0.9% saline. At time intervals of 1, 2, 4 and 24 h after chelate administration, test animals were sacrificed by cervical dislocation and samples of kidney, liver and d u o d e n u m removed for histological examination. The control group were sacrificed after 24 h. Tissues were fixed for 4 h in Bouin's fluid, wax embedded and sectioned at 5 p thickness. The stain used was haematoxylin and eosin, according to established procedures [6]. (ii) Electron microscope studies. Groups of adult male C3H mice, weighing 25--30 g, were given CaNaaDTPA by intraperitoneal injection at doses equivalent to 100 and 1000 mg/kg body weight, in a dose volume of 0.25 ml/25 g body weight. A control group of 3 animals received 0.9% saline in similar volumes. A 5000 mg/kg body weight group was not included in this series. The justification for this omission is that the ultrastructural changes would be expected to occur at the lower dose of 1000 mg/kg body weight, which is an enormous dose compared to that used in the therapeutic range. At time intervals of 10, 20, 30 min and 1, 2 and 24 h after chelate administration, groups of 3 test animals were sacrificed by cervical dislocation and samples of liver and kidney rapidly removed into ice-cold 3% glutaraldehyde solution in 0.2 M phosphate buffer (pH 7.4). The control group were sacrificed after 24 h. Approx. 0.5 mm cubes of tissue were cut from the samples and placed in fresh ice-cold glutaraldehyde solution and fixed at 4 ° for 24 h. After fixation, tissue samples were washed in 0.2 M phosphate buffer and placed in 1% osmic acid for 3 h at room temperature, rewashed in buffer and dehydrated through an alcohol series and finally passed through three changes of propylene oxide for 20 min. Tissue samples were then steeped in successively stronger concentrations of " E p o n " for 1-day periods (50/50, 75/25 Epon/propylene oxide} and finally cured in 100% " E p o n " for 48 h at 60 °, Sections 6--8 • 10 -a m thick were cut using an LKB Ultratome fitted with a diamond knife, and stained with uranyl acetate and lead citrate. Electron micrographs were obtained using an AEI 801 electron microscope.
Subacute administration of CaNa3DTPA Groups of 10 adult, male C3H mice, weighing 25--30 g were given CaNa3154
DTPA by intravenous injection at doses equivalent to 10, 100 and 250 mg/kg body weight in a dose volume of 50 gl. A group of 10 control animals received 0.9% saline. Chelate administration was repeated once daily for 5 days, followed by 2 treatment-free days, for a total of 30 treatment days. Throughout the period of chelate administration animals were housed communally, according to dose group, and allowed food and water ad libitum. At the end of the 30-day period of chelate administration, control and test animals were sacrificed and samples of kidney, liver and d u o d e n u m removed for histological examination as described above. RESULTS
Sections of tissue taken after acute and subacute administration of CaNa 3 DTPA were examined microscopically at 100 X magnification (L.P.) and 400 X magnification (H.P.).
Kidney The acute administration of CaNa 3DTPA at doses between 1.0 g and 5.0 g/kg body weight produced glomerular tuft swelling as the only morphologically detectable change in the kidney. At the three dose levels tested, this swelling of the glomerular t u f t was detectable at 1 and 2 h but was not detectable at 4 and 24 h after the injection of CaNa3 DTPA. Fig. 1 shows an
Fig. 1. E f f e c t s o f a c u t e a d m i n i s t r a t i o n o f 1 g/kg C a N a 3 D T P A on glomerulus o f the m o u s e kidney. N o t e occlusion o f capsular space 1 h a f t e r chelate. × 400
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Fig. 2. Section of renal cortex from kidney of untreated mouse, showing variation in tubule lumen size. X 100
H.P. photomicrograph of the typical degree of swelling of the glomerular t u f t 1 h after chelate administration. In this section the glomerular t u f t has b e c o m e distended and has completely occluded the capsular space. The lumen of the proximal and distal convoluted tubules in b o t h control and treated animals was often found to be reduced, and on occasion was found to be almost occluded. This was n o t considered to be an abnormality and was assumed to be a reflection of the metabolic activity of the kidney at the time of sacrifice. Fig. 2 shows an L.P. section of a typical control renal cortex in which these variations in the size of the tubular lumens can be seen. One feature observed in this series of experiments was evidence of a marked diuretic effect, demonstrable after the administration of 5.0 g/kg b o d y weight CaNa3 DTPA. At this dose level, the lumen of the renal tubules became distended as shown in Fig. 3. This was observed 1 h after chelate administration. There is clear evidence of well defined tubular cell nuclei and a distinct brush border, but no evidence of tubular cell damage. The subacute administration of CaNa3DTPA, at doses up to 250 mg/kg b o d y weight per day for 30 t r e a t m e n t days, produced no demonstrable histological changes in renal tissue. It would appear that, in contrast to the findings of other workers [ 1 - - 3 ] ,
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Fig. 3. E f f e c t of a c u t e a d m i n i s t r a t i o n o f 5 g/kg C a N a 3 D T P A o n t u b u l e s of m o u s e kidney. N o t e d i s t e n s i o n o f t u b u l e l u m e n 1 h a f t e r chelate. N o cellular d a m a g e has o c c u r r e d . × 4OO
no evidence of vacuolisation or hydropic change was detected in the mouse, even at doses well above the therapeutic range. Electron microscopic examination of renal tissue from animals receiving 100 mg and 1.0 g/kg b o d y weight CaNa3DTPA revealed no demonstrable cellular lesions at any of the time intervals tested after chelate administration. Fig. 4 shows an electron micrograph (× 6300) of a typical proximal convoluted tubule cell 1 h after the administration of 1.0 g/kg b o d y weight CaNas DTPA. A clearly defined cell membrane may be seen, and all subcellular organelles appear intact. Mitochondria are of normal appearance with well defined and clearly visible cristae. Liver Definite morphological changes were observed in liver sections at various time intervals after the acute administration of large doses of the chelate. Cloudy swelling was detectable only at the highest dose of the chelate used (5.0 g/kg b o d y weight). This p h e n o m e n o n was detectable at 2 h after chelate administration and persisted until 24 h. Vacuolisation, which was histologically defined as that of fatty degeneration and not hydropic degeneration, was observed at all three dose levels tested, b u t did n o t appear until 2 h after chelate administration. Fig. 5 shows 157
Fig. 4. E l e c t r o n m i c r o g r a p h of p r o x i m a l c o n v o l u t e d t u b u l e cell f r o m m o u s e k i d n e y 1 h after a d m i n i s t r a t i o n o f 1 g/kg C a N a 3 D T P A . N o t e n o r m a l cell m e m b r a n e a n d m i t o c h o n dria. × 6 3 0 0
Fig. 5. E f f e c t of acute a d m i n i s t r a t i o n o f 1 g/kg C a N a 3 D T P A o n p a r e n c h y m a l cells of m o u s e liver. N o t e vacuoles 24 h a f t e r chelate.-× 100
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Fig. 6. Electron micrograph of parenchymal cell from mouse liver, 10 rain after the administration of 1 g/kg CaNa3DTPA. Note vacuoles but no organelle damage. × 2500
an L.P. photomicrograph of a section of liver 24 h after the administration of 1.0 g/kg b o d y weight CaNa3 DTPA. Clearly defined vacuoles may be seen b u t there is no disruption of hepatic cords. The distribution and intensity of vacuolisation diminished over the 24-h period. However, an unexplained observation was that vacuolisation was still detectable 24 h after 1.0 g/kg and 5.0 g/kg b o d y weight b u t n o t after 2.5 g/kg b o d y weight of chelate. Electron microscopic studies of hepatic tissue following the administration of 1 g/kg b o d y weight CaNa3 DTPA revealed an incidence of vacuoles in hepatocytes as early as 10 min after chelate administration. Fig. 6 shows an electron micrograph (X 2500) of hepatic tissue 10 min after the administration of the chelate. Distinct vacuoles may be seen within the cell cytoplasm, some of which show a degree of osmic acid staining, characteristic of the presence of phospholipid and unsaturated fatty acids. Fig. 7 shows another electron micrograph (X 1 6 0 0 0 ) of hepatocytes 1 h after chelate administration showing the vacuoles at higher magnification. The absence of any definite membrane around the vacuoles suggests that the mechanism is not pinocytic in nature, and supports the view that they consist of fat globules. No evidence could be seen of membrane damage, pinocytic changes in the membrane or damage to organelles. After 30 days' treatment at chelate doses up to 250 mg/kg b o d y weight 159
Fig. 7. Electron micrograph of parenchymal cell from mouse liver 1 h after administration of 1 g/kg CaNa3DTPA. The vacuole possesses no demonstrable membrane, x 16000
per day, occasional vacuoles, similar to those described above, were detectable at the 100 mg/kg b o d y weight level, but n o t at other doses above and below this value. The n u m b e r of vacuoles was m uch smaller after subacute administration of chelate. Intestine Neither the acute nor the subacute administration of CaNa 3DTPA produced any histologically demonstrable changes in the d u o d e n u m of test animals at any of the dose levels or time intervals tested. No detectable evidence of toxicity, in the form of a t r o p h y of the villi and reduced nucleation of the Paneth region, was observed. DISCUSSION
Traumatic damage to a cell, following exposure to a toxic drug, can result in cell degeneration which occurs in 4 phases. Initial damage consists of cloudy swelling and this is followed progressively by vacuolar degeneration, necrosis and finally calcification of the cell remnants. Renal and hepatic cells are particularly susceptible to damage by drugs if concent rat i on of the drug occurs in these organs as a result of metabolism and excretion. 160
In the kidney, cloudy swelling is seen initially in the cells of the proximal convoluted tubule and the loop of Henle. It is caused by degeneration of mitochondria leading to a depletion of energy and osmotic disequilibrium with imbibition of extracellular water. Cloudy swelling may be accompanied by congestion of the glomeruli and the inter-tubular capillaries. A similar picture is seen in the liver. H e p a t o c y t e s become swollen and the degenerating mitochondria present the appearance of coarse granules in the cytoplasm. Hepatic sinusoids may become congested with erythrocytes in the early stages, b u t become partially occluded as the hepatocytes swell. The sequel to cloudy swelling is vacuolisation. In the kidney, vacuolar formation is thought to be due to a collection of watery fluid in the tubular cells, a so-called " h y d r o p i c change". The tubules are enlarged and the tubule cells b e c o m e distended and agranular. It is accompanied simultaneously by glomerular tuft swelling and often by venous congestion. In the liver, if mitochondrial function is depressed b e y o n d a certain degree, the cell accumulates dispersed fat which cannot be oxidised. The excess fat is deposited as globular fat. This condition is characterised in conventional histological techniques as vacuoles since the fat is dissolved o u t of the cells during histological processing and staining procedures. These primary cellular changes are usually reversible, b u t if they persist as a result of continued exposure to the toxic c o m p o u n d , they can lead to secondary irreversible changes which ultimately lead to cell necrosis. The primary changes can persist for long periods of time; for example, the fatty change in the liver may persist for several months before the cells are irreversibly damaged. Histological evidence in the mouse of transient cellular changes after acute and subacute administration of CaNa 3DTPA has been demonstrated in the kidney and liver at doses well above the therapeutic range, b u t no damage has been detected in the d u o d e n u m . This is in contrast to the observations of Foreman [4], who demonstrated hydropic degeneration in the proximal tubule cells of the rat kidney at doses as low as 110 mg/kg b o d y weight of the chelate. Catsch [1 ] has also described widespread hydropic degeneration in the rat following the administration of 1.25 g/kg b o d y weight CaNa3DTPA. It is important to recognise that hydropic change, seen as a distention of renal tubular cells with watery fluid can be caused by diuretic agents. CaNa 3DTPA has been shown to be a diuretic and thus these authors may have observed a diuretic change in the renal tubular cells which is reversible. In the experiments reported here, cloudy swelling'and vacuole formation in the kidney were n o t observed after acute chelate doses up to 5.0 g/kg b o d y weight, although swelling of the glomerular tuft was observed at 1.0 g/kg b o d y weight. This t u f t swelling may be indicative of a transient change, but other morphological evidence of cellular damage would have been expected. The fact that such involvement was not seen does not preclude the possibility that mild impairment of cellular function may have occurred which was n o t demonstrable histologically. However, the glomerular tuft 161
swelling only occurred at 2 and 4 h after chelate administration. The repeated administration of up to 250 mg/kg per day for 30 treatment days of the chelate also produced no demonstrable renal lesions, again in contrast to the observations of other workers. Any explanation of these discrepancies is speculative. Early reversible changes in the renal tissue of the rat have been reported [1,4] at doses above those used therapeutically. Lack of evidence in this series of experiments to demonstrate any pathological change in the mouse kidney may be explained as a variation in species sensitivity. However, Foreman [4] has indicated that no detectable lesions could be demonstrated in the renal tissue of the rat at chelate doses below 62.5 mg/kg body weight. The liver appeared to be more sensitive to the acute administration of CaNa3DTPA than the kidney. Definite vacuoles were observed after the acute administration of 1.0 g/kg body weight and above of CaNa 3 DTPA, and the number of these vacuoles reached a maximum between 2 and 4 h after chelate administration, and subsequently diminished over the remaining 24-h period. At electron microscope examination, vacuoles were visible at the 1.0 g/kg body weight dose level after 10 min, with an occasional vacuole after the administration of 100 mg/kg body weight of the chelate. No other histological changes were observed, and it has been noted that CaNa3 DTPA does not alter hepatic function as measured by the sulphobromophthalein retention test [7 ]. In this series of experiments, the evidence implicates a " f a t t y " vacuole rather than an " a q u e o u s " vacuole. This is substantiated by the presence of osmic acid-stainable material in the vacuoles and the absence of any membrane surrounding the vacuoles. Furthermore, no vesicles were demonstrated in the cell membrane and these would certainly be seen if pinocytosis was occurring as a major mechanism after the administration of CaNa3DTPA. The rapid appearance of globular fat inside a cell is possible, as fat metabolism is continuous within the hepatic cells, and any damage to mitochondria would rapidly result in depletion of the energy necessary to maintain an equilibrium between cellular dispersed fat and fatty acid oxidation. The partial restoration of the normal state after 24 h again emphasises the transient nature of the process after the cessation of chelate administration. There was no histologically demonstrable lesion evident in the d u o d e n u m after any of the acute or subacute dosage schedules used in this series of experiments. This is in contrast to the observations of Weber [3] who reported severe atrophy of the villi and detachment of mucosal cells at chelate doses up to 8 g/kg body weight. However our finding is not unexpected since mucosal cell damage presumably requires contact between the drug, or its metabolites, and the intestinal lumen. Metabolic studies [5] have shown that less than 0.5% of a parenterally administered dose of CaNa3DTPA reaches the intestinal lumen. There is no evidence of detectable biliary excretion of CaNa3DTPA or its metabolites or of intraceUular penetration of mucosal cells via the blood capillaries. In summary, the toxic effects resulting from the administration of 162
CaNaaDTPA have been r e p o r t e d by others in the kidney, liver and duodenum o f rats [ 1 - - 3 ] . In the course of an intensive re-evaluation of the alleged t o x i c i t y o f CaNa3DTPA, we r e p o r t one aspect of this study, namely the effect o f this chelate after acute and subacute administration on kidney, liver and intestine in the mouse. The administration of CaNa3 DTPA at dose levels between 6 and 30 times the m a x i m u m r e c o m m e n d e d therapeutic dose produced no histologically demonstrable lesion in the d u o d e n u m . Changes were detectable in kidney and liver, b u t in our opinion, t hey were transient. No lesions were observed when the chelate was administered at doses within the therapeutic range. In view of the potential value o f CaNa3DTPA as a chelating agent in h u man therapy, these findings justify a re-appraisal of the use of this chelate. It is possible that one explanation o f the discrepancy between different authors is tha t the mouse is less sensitive to CaNa 3 DTPA than the rat. This could be resolved by a study in o t h e r species. ACKNOWLEDGEMENTS This work f o r m e d part of a project for a Ph.D. thesis by R.M.M. and it was financially supported by the National Radiological Protection Board. We wish to acknowledge the assistance of Mr. D. S n o w d o n who prepared the photographs and Mrs. E. H e n r y for typing the script. We are grateful to Mr. A. Peat for preparing the sections for electron microscopy and for his helpful c o m m e n t s in interpreting the electron micrographs. REFERENCES 1 A. Catsch, Radioactive Metal Mobilisation in Medicine, Thomas, Springfield, 1964, p. 65. 2 M.D. Reuber, Toxicol. Appl. Pharmacol., 11 (1967) 321. 3 K.M. Weber, Z. Ges. Exptl. Med., 150 (1969) 354. 4 H. Foreman, Metal Binding in Medicine, Lippincott, Philadelphia, 1960, p. 82. 5 R.M. Morgan, Studies on the Metabolism and Toxicity of Diethylenetriaminepentaacetic Acid, Ph.D. Thesis, Sunderland Polytechnic (CNAA), 1973, p. 16. 6 H.M. Carleton, Histological Techniques, Oxford University Press, New York, 1967, p. 99. 7 R.M. Morgan and H. Smith, in preparation.
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HISTOLOGICAL CHANGES IN KIDNEY, LIVER AND DUODENUM OF THE MOUSE FOLLOWING THE ACUTE AND SUBACUTE ADMINISTRATION OF DIETHYLENETRIAMINEPENTAACETIC ACID
RAE M. MORGAN and HYLTON SMITH School of Pharmacy, Sunderland Poly technic, Sunderland, and National Radiological Protection Board, Harwell, Didcot (Great Britain) (Received October 18th, 1973) (Revision received November 22nd, 1973) (Accepted November 26th, 1973) .
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SUMMARY CaNa3 diethylenetriaminepentaacetic acid (DTPA) was found to produce transient histological changes in liver and kidney but not intestine in mice after the acute and subacute administration of doses between 6 and 30 times above the m a x i m u m recommended therapeutic dose.
INTRODUCTION
The polyaminopolycarboxylic acid, DTPA has become established as the most effective drug currently available for the removal of internally deposited actinides. Toxic side-effects have been reported in the rat following the administration of the calcium chelates of EDTA (CaNa2 EDTA) and DTPA (CaNa3DTPA). These consist of hydropic change in the kidney [1], vacuole production in hepatic parenchymal cells [2] and necrosis of the duodenal mucosa with atrophy of the villi [3]. These data, together with information on the early use of DTPA in man [4], have led to the imposition of limitations of the use of this chelate in human therapy, particularly in its use with workers accidentally contaminated with plutonium. However, the absence of suitable alternative therapy for the removal of contaminating higher actinides has made it necessary to re-evaluate the toxicity of this chelate. This communication presents evidence of reversible histological changes in mice receiving CaNa 3 DTPA either acutely or subacutely, at dose levels above the recommended therapeutic range.
Abbreviation: DTPA, diethylenetriaminepentaacetic acid. 153
MATERIALS AND METHODS CaNa3DTPA solution (250 mg/ml) suitable for intravenous use, was obtained from Geigy Pharmaceuticals Ltd., Macclesfield, Cheshire. The dose regimes have been planned to cover as many as possible of the reported clinical uses of the chelate [5], ranging from its use in the treatment of iron overload (200 mg/kg} to its use in therapy for plutonium contamination (14 mg/kg).
Acute administration of CaNa3DTPA (i) Light microscope studies. Groups of 10 adult, male C3H mice, weighing 25--30 g, were given CaNaaDTPA by intraperitoneal injection, at doses equivalent to 1.0, 2.5 and 5.0 g/kg body weight in a dose volume of 0.25 ml/25 g body weight. A group of 10 control animals received 0.9% saline. At time intervals of 1, 2, 4 and 24 h after chelate administration, test animals were sacrificed by cervical dislocation and samples of kidney, liver and d u o d e n u m removed for histological examination. The control group were sacrificed after 24 h. Tissues were fixed for 4 h in Bouin's fluid, wax embedded and sectioned at 5 p thickness. The stain used was haematoxylin and eosin, according to established procedures [6]. (ii) Electron microscope studies. Groups of adult male C3H mice, weighing 25--30 g, were given CaNaaDTPA by intraperitoneal injection at doses equivalent to 100 and 1000 mg/kg body weight, in a dose volume of 0.25 ml/25 g body weight. A control group of 3 animals received 0.9% saline in similar volumes. A 5000 mg/kg body weight group was not included in this series. The justification for this omission is that the ultrastructural changes would be expected to occur at the lower dose of 1000 mg/kg body weight, which is an enormous dose compared to that used in the therapeutic range. At time intervals of 10, 20, 30 min and 1, 2 and 24 h after chelate administration, groups of 3 test animals were sacrificed by cervical dislocation and samples of liver and kidney rapidly removed into ice-cold 3% glutaraldehyde solution in 0.2 M phosphate buffer (pH 7.4). The control group were sacrificed after 24 h. Approx. 0.5 mm cubes of tissue were cut from the samples and placed in fresh ice-cold glutaraldehyde solution and fixed at 4 ° for 24 h. After fixation, tissue samples were washed in 0.2 M phosphate buffer and placed in 1% osmic acid for 3 h at room temperature, rewashed in buffer and dehydrated through an alcohol series and finally passed through three changes of propylene oxide for 20 min. Tissue samples were then steeped in successively stronger concentrations of " E p o n " for 1-day periods (50/50, 75/25 Epon/propylene oxide} and finally cured in 100% " E p o n " for 48 h at 60 °, Sections 6--8 • 10 -a m thick were cut using an LKB Ultratome fitted with a diamond knife, and stained with uranyl acetate and lead citrate. Electron micrographs were obtained using an AEI 801 electron microscope.
Subacute administration of CaNa3DTPA Groups of 10 adult, male C3H mice, weighing 25--30 g were given CaNa3154
DTPA by intravenous injection at doses equivalent to 10, 100 and 250 mg/kg body weight in a dose volume of 50 gl. A group of 10 control animals received 0.9% saline. Chelate administration was repeated once daily for 5 days, followed by 2 treatment-free days, for a total of 30 treatment days. Throughout the period of chelate administration animals were housed communally, according to dose group, and allowed food and water ad libitum. At the end of the 30-day period of chelate administration, control and test animals were sacrificed and samples of kidney, liver and d u o d e n u m removed for histological examination as described above. RESULTS
Sections of tissue taken after acute and subacute administration of CaNa 3 DTPA were examined microscopically at 100 X magnification (L.P.) and 400 X magnification (H.P.).
Kidney The acute administration of CaNa 3DTPA at doses between 1.0 g and 5.0 g/kg body weight produced glomerular tuft swelling as the only morphologically detectable change in the kidney. At the three dose levels tested, this swelling of the glomerular t u f t was detectable at 1 and 2 h but was not detectable at 4 and 24 h after the injection of CaNa3 DTPA. Fig. 1 shows an
Fig. 1. E f f e c t s o f a c u t e a d m i n i s t r a t i o n o f 1 g/kg C a N a 3 D T P A on glomerulus o f the m o u s e kidney. N o t e occlusion o f capsular space 1 h a f t e r chelate. × 400
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Fig. 2. Section of renal cortex from kidney of untreated mouse, showing variation in tubule lumen size. X 100
H.P. photomicrograph of the typical degree of swelling of the glomerular t u f t 1 h after chelate administration. In this section the glomerular t u f t has b e c o m e distended and has completely occluded the capsular space. The lumen of the proximal and distal convoluted tubules in b o t h control and treated animals was often found to be reduced, and on occasion was found to be almost occluded. This was n o t considered to be an abnormality and was assumed to be a reflection of the metabolic activity of the kidney at the time of sacrifice. Fig. 2 shows an L.P. section of a typical control renal cortex in which these variations in the size of the tubular lumens can be seen. One feature observed in this series of experiments was evidence of a marked diuretic effect, demonstrable after the administration of 5.0 g/kg b o d y weight CaNa3 DTPA. At this dose level, the lumen of the renal tubules became distended as shown in Fig. 3. This was observed 1 h after chelate administration. There is clear evidence of well defined tubular cell nuclei and a distinct brush border, but no evidence of tubular cell damage. The subacute administration of CaNa3DTPA, at doses up to 250 mg/kg b o d y weight per day for 30 t r e a t m e n t days, produced no demonstrable histological changes in renal tissue. It would appear that, in contrast to the findings of other workers [ 1 - - 3 ] ,
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Fig. 3. E f f e c t of a c u t e a d m i n i s t r a t i o n o f 5 g/kg C a N a 3 D T P A o n t u b u l e s of m o u s e kidney. N o t e d i s t e n s i o n o f t u b u l e l u m e n 1 h a f t e r chelate. N o cellular d a m a g e has o c c u r r e d . × 4OO
no evidence of vacuolisation or hydropic change was detected in the mouse, even at doses well above the therapeutic range. Electron microscopic examination of renal tissue from animals receiving 100 mg and 1.0 g/kg b o d y weight CaNa3DTPA revealed no demonstrable cellular lesions at any of the time intervals tested after chelate administration. Fig. 4 shows an electron micrograph (× 6300) of a typical proximal convoluted tubule cell 1 h after the administration of 1.0 g/kg b o d y weight CaNas DTPA. A clearly defined cell membrane may be seen, and all subcellular organelles appear intact. Mitochondria are of normal appearance with well defined and clearly visible cristae. Liver Definite morphological changes were observed in liver sections at various time intervals after the acute administration of large doses of the chelate. Cloudy swelling was detectable only at the highest dose of the chelate used (5.0 g/kg b o d y weight). This p h e n o m e n o n was detectable at 2 h after chelate administration and persisted until 24 h. Vacuolisation, which was histologically defined as that of fatty degeneration and not hydropic degeneration, was observed at all three dose levels tested, b u t did n o t appear until 2 h after chelate administration. Fig. 5 shows 157
Fig. 4. E l e c t r o n m i c r o g r a p h of p r o x i m a l c o n v o l u t e d t u b u l e cell f r o m m o u s e k i d n e y 1 h after a d m i n i s t r a t i o n o f 1 g/kg C a N a 3 D T P A . N o t e n o r m a l cell m e m b r a n e a n d m i t o c h o n dria. × 6 3 0 0
Fig. 5. E f f e c t of acute a d m i n i s t r a t i o n o f 1 g/kg C a N a 3 D T P A o n p a r e n c h y m a l cells of m o u s e liver. N o t e vacuoles 24 h a f t e r chelate.-× 100
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Fig. 6. Electron micrograph of parenchymal cell from mouse liver, 10 rain after the administration of 1 g/kg CaNa3DTPA. Note vacuoles but no organelle damage. × 2500
an L.P. photomicrograph of a section of liver 24 h after the administration of 1.0 g/kg b o d y weight CaNa3 DTPA. Clearly defined vacuoles may be seen b u t there is no disruption of hepatic cords. The distribution and intensity of vacuolisation diminished over the 24-h period. However, an unexplained observation was that vacuolisation was still detectable 24 h after 1.0 g/kg and 5.0 g/kg b o d y weight b u t n o t after 2.5 g/kg b o d y weight of chelate. Electron microscopic studies of hepatic tissue following the administration of 1 g/kg b o d y weight CaNa3 DTPA revealed an incidence of vacuoles in hepatocytes as early as 10 min after chelate administration. Fig. 6 shows an electron micrograph (X 2500) of hepatic tissue 10 min after the administration of the chelate. Distinct vacuoles may be seen within the cell cytoplasm, some of which show a degree of osmic acid staining, characteristic of the presence of phospholipid and unsaturated fatty acids. Fig. 7 shows another electron micrograph (X 1 6 0 0 0 ) of hepatocytes 1 h after chelate administration showing the vacuoles at higher magnification. The absence of any definite membrane around the vacuoles suggests that the mechanism is not pinocytic in nature, and supports the view that they consist of fat globules. No evidence could be seen of membrane damage, pinocytic changes in the membrane or damage to organelles. After 30 days' treatment at chelate doses up to 250 mg/kg b o d y weight 159
Fig. 7. Electron micrograph of parenchymal cell from mouse liver 1 h after administration of 1 g/kg CaNa3DTPA. The vacuole possesses no demonstrable membrane, x 16000
per day, occasional vacuoles, similar to those described above, were detectable at the 100 mg/kg b o d y weight level, but n o t at other doses above and below this value. The n u m b e r of vacuoles was m uch smaller after subacute administration of chelate. Intestine Neither the acute nor the subacute administration of CaNa 3DTPA produced any histologically demonstrable changes in the d u o d e n u m of test animals at any of the dose levels or time intervals tested. No detectable evidence of toxicity, in the form of a t r o p h y of the villi and reduced nucleation of the Paneth region, was observed. DISCUSSION
Traumatic damage to a cell, following exposure to a toxic drug, can result in cell degeneration which occurs in 4 phases. Initial damage consists of cloudy swelling and this is followed progressively by vacuolar degeneration, necrosis and finally calcification of the cell remnants. Renal and hepatic cells are particularly susceptible to damage by drugs if concent rat i on of the drug occurs in these organs as a result of metabolism and excretion. 160
In the kidney, cloudy swelling is seen initially in the cells of the proximal convoluted tubule and the loop of Henle. It is caused by degeneration of mitochondria leading to a depletion of energy and osmotic disequilibrium with imbibition of extracellular water. Cloudy swelling may be accompanied by congestion of the glomeruli and the inter-tubular capillaries. A similar picture is seen in the liver. H e p a t o c y t e s become swollen and the degenerating mitochondria present the appearance of coarse granules in the cytoplasm. Hepatic sinusoids may become congested with erythrocytes in the early stages, b u t become partially occluded as the hepatocytes swell. The sequel to cloudy swelling is vacuolisation. In the kidney, vacuolar formation is thought to be due to a collection of watery fluid in the tubular cells, a so-called " h y d r o p i c change". The tubules are enlarged and the tubule cells b e c o m e distended and agranular. It is accompanied simultaneously by glomerular tuft swelling and often by venous congestion. In the liver, if mitochondrial function is depressed b e y o n d a certain degree, the cell accumulates dispersed fat which cannot be oxidised. The excess fat is deposited as globular fat. This condition is characterised in conventional histological techniques as vacuoles since the fat is dissolved o u t of the cells during histological processing and staining procedures. These primary cellular changes are usually reversible, b u t if they persist as a result of continued exposure to the toxic c o m p o u n d , they can lead to secondary irreversible changes which ultimately lead to cell necrosis. The primary changes can persist for long periods of time; for example, the fatty change in the liver may persist for several months before the cells are irreversibly damaged. Histological evidence in the mouse of transient cellular changes after acute and subacute administration of CaNa 3DTPA has been demonstrated in the kidney and liver at doses well above the therapeutic range, b u t no damage has been detected in the d u o d e n u m . This is in contrast to the observations of Foreman [4], who demonstrated hydropic degeneration in the proximal tubule cells of the rat kidney at doses as low as 110 mg/kg b o d y weight of the chelate. Catsch [1 ] has also described widespread hydropic degeneration in the rat following the administration of 1.25 g/kg b o d y weight CaNa3DTPA. It is important to recognise that hydropic change, seen as a distention of renal tubular cells with watery fluid can be caused by diuretic agents. CaNa 3DTPA has been shown to be a diuretic and thus these authors may have observed a diuretic change in the renal tubular cells which is reversible. In the experiments reported here, cloudy swelling'and vacuole formation in the kidney were n o t observed after acute chelate doses up to 5.0 g/kg b o d y weight, although swelling of the glomerular tuft was observed at 1.0 g/kg b o d y weight. This t u f t swelling may be indicative of a transient change, but other morphological evidence of cellular damage would have been expected. The fact that such involvement was not seen does not preclude the possibility that mild impairment of cellular function may have occurred which was n o t demonstrable histologically. However, the glomerular tuft 161
swelling only occurred at 2 and 4 h after chelate administration. The repeated administration of up to 250 mg/kg per day for 30 treatment days of the chelate also produced no demonstrable renal lesions, again in contrast to the observations of other workers. Any explanation of these discrepancies is speculative. Early reversible changes in the renal tissue of the rat have been reported [1,4] at doses above those used therapeutically. Lack of evidence in this series of experiments to demonstrate any pathological change in the mouse kidney may be explained as a variation in species sensitivity. However, Foreman [4] has indicated that no detectable lesions could be demonstrated in the renal tissue of the rat at chelate doses below 62.5 mg/kg body weight. The liver appeared to be more sensitive to the acute administration of CaNa3DTPA than the kidney. Definite vacuoles were observed after the acute administration of 1.0 g/kg body weight and above of CaNa 3 DTPA, and the number of these vacuoles reached a maximum between 2 and 4 h after chelate administration, and subsequently diminished over the remaining 24-h period. At electron microscope examination, vacuoles were visible at the 1.0 g/kg body weight dose level after 10 min, with an occasional vacuole after the administration of 100 mg/kg body weight of the chelate. No other histological changes were observed, and it has been noted that CaNa3 DTPA does not alter hepatic function as measured by the sulphobromophthalein retention test [7 ]. In this series of experiments, the evidence implicates a " f a t t y " vacuole rather than an " a q u e o u s " vacuole. This is substantiated by the presence of osmic acid-stainable material in the vacuoles and the absence of any membrane surrounding the vacuoles. Furthermore, no vesicles were demonstrated in the cell membrane and these would certainly be seen if pinocytosis was occurring as a major mechanism after the administration of CaNa3DTPA. The rapid appearance of globular fat inside a cell is possible, as fat metabolism is continuous within the hepatic cells, and any damage to mitochondria would rapidly result in depletion of the energy necessary to maintain an equilibrium between cellular dispersed fat and fatty acid oxidation. The partial restoration of the normal state after 24 h again emphasises the transient nature of the process after the cessation of chelate administration. There was no histologically demonstrable lesion evident in the d u o d e n u m after any of the acute or subacute dosage schedules used in this series of experiments. This is in contrast to the observations of Weber [3] who reported severe atrophy of the villi and detachment of mucosal cells at chelate doses up to 8 g/kg body weight. However our finding is not unexpected since mucosal cell damage presumably requires contact between the drug, or its metabolites, and the intestinal lumen. Metabolic studies [5] have shown that less than 0.5% of a parenterally administered dose of CaNa3DTPA reaches the intestinal lumen. There is no evidence of detectable biliary excretion of CaNa3DTPA or its metabolites or of intraceUular penetration of mucosal cells via the blood capillaries. In summary, the toxic effects resulting from the administration of 162
CaNaaDTPA have been r e p o r t e d by others in the kidney, liver and duodenum o f rats [ 1 - - 3 ] . In the course of an intensive re-evaluation of the alleged t o x i c i t y o f CaNa3DTPA, we r e p o r t one aspect of this study, namely the effect o f this chelate after acute and subacute administration on kidney, liver and intestine in the mouse. The administration of CaNa3 DTPA at dose levels between 6 and 30 times the m a x i m u m r e c o m m e n d e d therapeutic dose produced no histologically demonstrable lesion in the d u o d e n u m . Changes were detectable in kidney and liver, b u t in our opinion, t hey were transient. No lesions were observed when the chelate was administered at doses within the therapeutic range. In view of the potential value o f CaNa3DTPA as a chelating agent in h u man therapy, these findings justify a re-appraisal of the use of this chelate. It is possible that one explanation o f the discrepancy between different authors is tha t the mouse is less sensitive to CaNa 3 DTPA than the rat. This could be resolved by a study in o t h e r species. ACKNOWLEDGEMENTS This work f o r m e d part of a project for a Ph.D. thesis by R.M.M. and it was financially supported by the National Radiological Protection Board. We wish to acknowledge the assistance of Mr. D. S n o w d o n who prepared the photographs and Mrs. E. H e n r y for typing the script. We are grateful to Mr. A. Peat for preparing the sections for electron microscopy and for his helpful c o m m e n t s in interpreting the electron micrographs. REFERENCES 1 A. Catsch, Radioactive Metal Mobilisation in Medicine, Thomas, Springfield, 1964, p. 65. 2 M.D. Reuber, Toxicol. Appl. Pharmacol., 11 (1967) 321. 3 K.M. Weber, Z. Ges. Exptl. Med., 150 (1969) 354. 4 H. Foreman, Metal Binding in Medicine, Lippincott, Philadelphia, 1960, p. 82. 5 R.M. Morgan, Studies on the Metabolism and Toxicity of Diethylenetriaminepentaacetic Acid, Ph.D. Thesis, Sunderland Polytechnic (CNAA), 1973, p. 16. 6 H.M. Carleton, Histological Techniques, Oxford University Press, New York, 1967, p. 99. 7 R.M. Morgan and H. Smith, in preparation.
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