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Tissue and cellular disposition of paraquat in mice
TOXICOLOGY
AND
APPLIED
56, 127-140(1980)
PHARMACOLOGY
Tissue and Cellular WILLIAM Department
Disposition
J. WADDELL
of Pharmacology
Received
of Paraquat
AND CAROLYN
MARLOWE
and Toxicology, School of Medicine, Louisville, Kentucky 40292
March
25. 1980: accepted
June
in Mice1-2
Unh’ersity
of Louisville.
9. 1980
Tissue and Cellular Disposition of Paraquat in Mice. WADDELL, W. J., AND MARLOWE, C. Toxicol. Appl. Pharmacol. 56, 127- 140. Male C57B116J mice were injected intravenously with [methyl-‘*C]paraquat dichloride and frozen at 1,3,9, and 24 hr for whole-body autoradiography or with [merhyL3H]paraquat dichloride and the lungs were removed at 3.24, and 48 hr for cellular autoradiography. The methods do not allow thawing or exposure of the tissues to solvents; this prevents translocation or removal of radioactivity. The wholebody autoradiographs revealed localization of radioactivity at all time intervals in melanin, lung, choroid plexus, muscle, and excretory pathways such as proximal tubules of kidney, urine, liver, gallbladder, and intestinal contents. The radioactivity in lung was much higher in certain discrete. unidentified areas at all time intervals except at 1 hr. The concentration was high in myocardium at 1 and 3 hr. Cellular resolution autoradiography revealed that the radioactivity within the lung was confined almost entirely to cells having the distribution of alveolar type II cells at the three time intervals studied. The radioactivity in these cells was easily washed away indicating that an active transport process was probably involved instead of binding to a cellular constituent. The localization suggested that choline might be antidotal to paraquat toxicity. However, there was not a significant increase in survival of mice given 100 mg/kg choline chloride simultaneously to or following treatment with 50 mg/kg paraquat chloride. (1980).
Several mechanisms of the toxic action of the herbicide paraquat in animals and humans have been proposed. Manktelow (1967) provided evidence that paraquat interfered with the production of lung surfactant, Gage (1968) demonstrated that free radicals produced from paraquat increased respiration from liver mitochondrial fragments and Bus et al. (1974) suggested that paraquat toxicity may be mediated through the formation of the superoxide ion with subsequent lipid peroxidation. An early review discussed these possible I Presented at the annual meeting of the American for Pharmacology and Experimental Therapeutics in Portland. Ore.. August 22, 1979. ’ Supported by the University of Kentucky Tobacco and Health Research Institute Project No. KTRB-057, NIH Contract 263-78-M-8374, and Pharmacon Research Foundation. Inc. Society
mechanisms including the peroxidation of lipids which was suggested by Gage in 1968 as unpublished observations (Conning et al., 1969). Disagreement persists concerning the possible involvement of lipid peroxidation since vitamin E deficiency potentiates paraquat toxicity (Block, 1979) but lipid peroxidation was inhibited by paraquat in vitro (Steffen and Netter, 1979). In any event, therapy based on the proposed damage from peroxidation of lung lipids either has not been successful (Shu eC al., 1979) or has limited applicability in the clinical situation (Wasserman and Block, 1978). The preponderant damage is to the lungs although the kidneys and myocardium also are frequently involved (see review- by Haley, 1979, for references). Although 127
0041-008X/80/130127-14$02.00/O Copyright Q 1980 by Academic Press. Inc. All nghta of reproduction in any form resenrd.
128
WADDELL
AND MARLOWE
several studies have been reported on the localization of paraquat in tissues, including some using autoradiography (Litchfield et al., 1973; Larssoner al., 1978), none of these provided sufficient resolution in the tissues in which toxic effects are seen to provide clues to the mechanism of action. The present study was undertaken to identify the cells in which the compound accumulates, in vivo, in the hope that this would help clarify the mechanism of toxic action of this chemical. METHODS Whole-body autoradiography. Male, CS7Bl/6J mice were obtained from Jackson Laboratories, Bar Harbor, Maine. Food and tap water were provided ad libitum. [methyl-‘*C]Paraquat dichloride, Batch No. IS. having a specific activity of 30 mCi/mmol, was purchased from Amersham/Searle. Radiochemical purity was ascertained on cellulose thin-layer chromatographic plates developed in benzene:amyl alcohol:methyl alcohol:1 N HCl (1.3:4:8:8). A single peak with an R, of 0.74 was observed for the radioactive and standard paraquat solutions. Mice weighing 30 to 36 g were injected intravenously with 2.0 to 2.1 mg/kg [“Clparaquat dichloride dissolved in 0.9% NaCl and sacrificed 1, 3, 9. and 24 hr later. An additional mouse, weighing 41 g, was injected iv with 1.1 m&kg [“Clparaquat dichloride and sacrificed 3 hr later. All mice were sacrificed at the designated times by anesthetizing with ether and then freezing the mouse in a hexane/dry ice bath. Twentymicrometer-thick sections were taken onto Scotch tape at -22°C and the dried sections were placed on Xray film to produce autoradiographs. These wholebody autoradiographic procedures do not allow thawing or exposure of the tissues to solvents; this prevents translocation or removal of radioactivity. Details of this technique have been previously published (Waddell and Marlowe, 1977). High-resolution autoradiography. Male, C57BU6J mice w’eighing 26 to 32 g were obtained from Jackson Laboratories. Food and tap water were provided ad libitum. [methyl-3H]Paraquat dichloride was synthesized by AmershamSearle with a specific activity of 3.0 C/ mmol. A single radioactive peak, which corresponds to nonradioactive paraquat, was observed when the [3H]paraquat was spotted on silica gel thin-layer chromatographic plates and developed in N-butyl alcohol:5 N HCI (2:23). The R, of each compound was 0.50 in this system.
Nine mice were injected intravenously with 288 to 338 &kg L3H]paraquat dichloride dissolved in saline and triplicate mice were sacrificed by cervical dislocation at 3, 24, or 48 hr later. The lungs were quickly removed, blotted on Kimwipes, and trimmed to yield pieces of lung approximately 3-5 mm3. These small pieces of lung were attached to small microtome stages using minced, nonradioactive liver as the embedding material and then quickly frozen in liquid nitrogen. Duplicate samples were prepared from each mouse except four samples were prepared from one of the mice sacrificed 3 hr after receiving the paraquat. Four-micrometer-thick sections from the duplicate samples were taken at -3s”C in a Harris cryostat using an IEC rotary microtome. After drying at -3s”C the sections were attached to slides coated with Kodak NTB3 emulsion without the use of any solvents. These techniques were modified from procedures published by Stumpf (1976). The remaining two samples from the 3-hr mouse were also sectioned as described above but, instead of drying, the freshly cut sections were attached immediately to the emulsion-coated slides at room temperature. This thawmount technique does allow thawing of the section; thawing may result in translocation of the radioactivity (Stumpf, 1976). After photographic development thin sections were stained in either methyl green/ pyronine or celestine blue/Van Gieson’s (Stumpf, 1976; Kennedy and Little, 1974). Acute toxicity determinations. Adult male C57BV6J mice (Jackson Laboratories) weighing 20-32 g and Swiss Webster mice (Laboratory Supply, Indianapolis, Ind.) were provided food and tap water ad libitum. Sixteen C57BY6J mice were given a single ip injection of 50 mg/kg paraquat dichloride$ 1, 7, and 24 hr later, eight of the mice received SC 100 mgkg of an isotonic solution of choline chloride (Sigma Chemical) and the remaining eight received SC an equal volume of 0.9% NaCl. The Swiss Webster mice were administered the choline or saline and paraquat simultaneously; four mice received an ip injection of 50 mg/ kg paraquat dichloride combined with 100 mg/kg of an isotonic solution of choline chloride and three mice received the paraquat salt dissolved in an equal volume of 0.9% NaCl. All mice were observed to record the time of death.
RESULTS The whole-body autoradiographs revealed that at 1 hr there was high accumulation of radioactivity in myocardium, ’ The paraquat was generously supplied by Dr. Wyman H. Dorough, University of Kentucky.
BRAIN
SALIVARY
GLAND
BLOOD
FIG. I, A print of a whole-body autoradiograph White areas correspond to radioactivity.
MUSCLE
MELANIN
RIBS
MYOCARDIUM
LIVER
ADRENAL
INTESTINE
KIDNEY
URINE
of a 20-pm section from a male CS7BV6J mouse frozen 1 hr after an iv injection of [mPrhy/-“Clparaquat.
LUNG
PLEXUS
MUSCLE
LIVER
section
DIAPHRAGM
of a 20-@rn
LUNG
MYOCARDIUM
autoradiograph to radioactivity.
BLOOD
FIG. 2. A print of a whole-body paraquat. White areas correspond
MELANIN
CHOROID
from
a male C57B1/6J
INTESTINAL
STOMACH
mouse
frozen
CONTENTS
KIDNEY
3 hr after
an iv injection
GU
of [methyl-‘4C]-
TRACT
0
L
DISPOSITION
LUNG
HEART
OF PARAQUAT
DIAPHRAGM
LIVER
FIG. 3. A detail of an autoradiograph of a 20-pm section from a male C57BV6J mouse an iv injection of [“Clparaquat. Intense radioactivity can he seen in the diaphragm discrete spots in the lung. White areas correspond to radioactivity.
liver, intestinal contents, skeletal muscle, melanin, ribs, urine, lungs, kidney, and choroid plexus. Most of these sites can be seen in Fig. 1; the other tissues were present in other sections of the mouse. By 3 hr after administration the amount of radioactivity in the myocardium, ribs and to some extent in the liver had decreased sharply. The relative concentration in the choroid plexus, lung, and convoluted segment of the proximal tubule in the kidney had increased. The accessory sex glands in the male mice showed relatively high concentrations at this time interval (Fig. 2). Enlargements of autoradiographs from other sections of this mouse at 3 hr are shown in Figs. 3 and 4. These show the intense radioactivity in the convoluted segments of proximal tubules in the kidney and in certain discrete areas of the lung. These spots in the lung are more intense than any of the surrounding tissues, but their anatomical location within the lung could not be determined. The high concentration in the diaphragm was most notable only at 3 hr.
frozen 3 hr after and in certxin.
At 9 hr after receiving the compound. melanin, muscle, liver, intestinal contents, and urine continue to show appreciable activity, but the relative activity in lung and kidney continues to increase (Fig. 5). The discrete localizations in lung are even more prominent than they were at 3 hr and the choroid plexus is well labeled. After 24 hr, the localizations that remain have decreased to a few areas: (I) the contents of the gastrointestinal tract and urine: (2) melanin and skeletal muscle: (3) choroid plexus, lung, and kidney (Fig. 6). The activity in lung and kidney are even more clearly confined to discrete areas: these could not be identified in lung but in kidney were identified as the convoluted segment of proximal tubule. At each of the time intervals. there was considerable variability among different skeletal muscle groups. The high-resolution autoradiographs of the lung done by the dry-mount technique at each of the three time intervals revealed localization predominately in certain discrete cells. The histological pattern of
WADDELL
132
PROXIMAL
AND MARLOWE
TUBULES
OF
KIDNEY
FIG. 4. A detail of an autoradiograph of a 20-pm section from the male C57BY6J mouse frozen 3 hr after [“Clparaquat. Note the accumulation of radioactivity in the convoluted segments of the proximal tubules in the kidney. White areas correspond to radioactivity.
the distribution of this cell type appeared to correspond most closely with that of the alveolar type II. The cells were in the corners of the alveoli and occasionally along the alveolar wall (Fig. 7). When the tissue section for high-resolution autoradiography was allowed to thaw as it was applied to the warm emulsion, the radioactivity was completely translocated from the cells to the interior of the alveoli and periphery of the section (Fig. 8). No significant protection from choline on the acute toxicity of paraquat in mice was observed in the two studies. When the mice were exposed to the paraquat 1 hr before the first of three injections of choline or saline all mice in the saline group were dead after 4 days and only one of the mice in the choline group survived after 4 days. One mouse from each of the choline and saline groups survived when the paraquat was combined with the choline or saline for injection. Most of the mice died within the first 72 hr following paraquat exposure; however, the occurrence of death in the saline-treated group
was delayed in general about 24 hr in comparison to the choline-treated mice. DISCUSSION The tissue distribution of paraquat was studied by whole-body autoradiography earlier using sections of mice that were 100 pm thick (Litchfield et al., 1973). Those studies identified the major organs which accumulate the compound but the thick sections did not allow sufficient resolution to discern the cells of accumulation in lung and kidney; these are the primary organs where the toxic effects of the chemical are seen. Later autoradiographic studies with 20-,um sections examined the nature of the binding of paraquat to melanin and intervertebral cartilage (Larsson er al., 1978); however, neither of these sites is involved in an adverse effect from the chemical. Whole-organ analyses in the rat revealed high and persistent accumulation in lung, muscle, and kidney (Sharp&al., 1972).
PLEXUS
MUSCLE
IN
SALIVARY
BLOOD
FIG. 5. A print of a whole-body autoradiograph White areas correspond to radioactivity.
MELANIN
CHOROID
of a 20.pm
GLAND
HEART
section
from
RIBS
LUNG
a male CS7BV6J
LIVER
STOMACH
mouse
frozen
CONTENTS
9 hr after an iv injection
INTESTINAL
KIDNEY
of [methyl-“Clparaquat.
134
WADDELL
AND
MARLOWE
DISPOSITION
OF
13s
PARAQUAT
FIG. 7. Dry-mount autoradiographs of lungs from male C57B116J activity in the alveolar type II cells 3 hr following an iv injection celestine blue/Van Gieson‘s. (A) 88x. (B) 350x.
mice showing accumulation of [,?ic~rh~/-:‘H]paraquat.
Stained
of radiowith
136
WADDELL
AND
MARLOWE
FIG. 8. Thaw-mount autoradiographs of lungs from male C57BV6J mice showing translocation of radioactivity from the type II cells to the interior of the alveoli and periphery of the tissue. The lungs were removed from mice 3 hr after receiving iv [3H]paraquat. Stained with methyl green/pyronine. (A) 88 x, (B) 350x.
DISPOSITION
The present report was undertaken to elucidate the cells in the tissues which accumulate paraquat. Since the lung is the primary organ injured and the kidney is also to a lesser extent (see Haley, 1979, for references), attention was focused on the kinetics of accumulation in these organs. The studies reported here revealed that radioactivity was confined to what is interpreted to be essentially one cell type in the lung. The histological distribution of the cell type which accumulated the radioactivity was most probably the alveolar type II. Other possibilities, e.g., alveolar type I, macrophages, capillary endothelium, etc., were excluded because of their different distribution patterns (McNary and ElBermani, 1970; Mayo, 1974; Sorokin, 1977). The low concentration in blood eliminates the possibility that the radioactivity was localized in capillaries. Furthermore, it appears that it persists in this cell for several days. In addition, the accumulation in that cell is very easily displaced since simple thawing of the section translocated the radioactivity. Consequently, it is unlikely that the radioactivity is strongly bound to cellular constituents; it certainly is not covalently incorporated into molecules within the cell. A logical conclusion is that paraquat is actively transported into the alveolar type II cells by a mechanism that exists for a similar molecule used in its normal metabolic activities. Additional evidence for active transport of paraquat by specific cells is the persistent accumulation of radioactivity in the choroid plexus and in the convoluted portion of the proximal tubule: each of these cell layers is known to actively transport cations with structures similar to that of paraquat. This accumulation in the choroid plexus may have clinical significance since Grant et al. (1980) reported cerebral damage in patients who died following paraquat ingestion. High-resolution autoradiography of the brain and kidney were not necessary since the choroid plexus is easily identified
OF PARAQUAT
137
in 20-pm sections and the distribution in the convoluted segment of the proximal tubule of the kidney is distinctive and readily recognized (Longley, 1969). Radioactivity seen in the autoradiographs probably represents unchanged paraquat since there is no evidence for metabolism of paraquat which has been administered intravenously (Daniel and Gage, 1966). Localization of paraquat in tissues such as liver, gallbladder, intestinal contents. and urine obviously merely reveals the routes of elimination of the compound. The accumulation in melanin and intervertebral cartilage has been well studied previously and is probably due to an ionic interaction (Larsson et al., 1978). The wide variability in the accumulation in skeletal muscle may be due to the state of contraction or relaxation of these individual muscle groups at the time of freezing of the animal: the same pattern is seen with j”Ca (Appelgren rt 1961; Ericsson and Hammarstrom. al., 1964). Most of the speculation on the mechanism of toxic action of paraquat centers on possible lipid peroxidation mediated by the compound (see Haley, 1979, for references): however, there is much experimental evidence which causes serious doubt that this explains its action in viva (Shu ef (11.. 1979: Steffen and Netter, 1979). The experiments reported here suggest another mechanism which has not heretofore been considered, to our knowledge. The cellular accumulation of paraquat which is most persistent is in three cell types, i.e., alveolar type II, convoluted segment of proximal tubule, and choroid plexus, which appear to concentrate it by an active transport mechanism. The concentration in that cell in the kidney can readily e Yplain the effect in blocking the function of the proximal tubule causing its degeneration in poisoning by the chemical (Vaziri t~f ui., 1979). Rose et al. (1974) reported that the accumulation of paraquat into lung slices, in bxitro, was an energy-dependent process.
138
WADDELL
AND MARLOWE
Also, Lock et al. (1976) demonstrated that a variety of compounds could inhibit the accumulation of paraquat in rat lung slices. We should like to suggest that the accumulation in the alveolar type II cell might explain its toxic effect on the lung by the following mechanism. One function of the alveolar type II cell is the synthesis of pulmonary surfactant (see review of Batenburg and Van Golde, 1979, for references); the primary composition of this surfactant is phosphatidyl choline (King and Clements, 1972; King, 1974; Smith and Kikkawa, 1979). Choline is a quartenary ammonium compound just as is paraquat. [3H]Choline has been shown to accumulate selectively in alveolar type II cells (Chevalier and Collet, 1972). If the alveolar type II cell accumulates paraquat instead of choline, the synthesis of phosphatidyl choline may be blocked. Blocking the synthesis of the lung’s primary surfactant would allow damage to the alveoli by inhaled oxidants. This sequence of events is consistent with many observations in paraquat poisoning including loss of pulmonary surfactant (Manktelow, 1967), decreased production of phosphatidyl choline by lung after treatment with paraquat (Etherton and Gresham, 1979), enhancement of the toxicity by increased oxygen tension (Fisher et af., 1973), protection from paraquat toxicity by vitamin E (Block, 1979), and delayed onset of toxic effects. Furthermore, since the alveolar type 11 cell is not permanently damaged by the presence of paraquat and in fact may be proliferating in a compensatory manner (Dearden et al., 1978; Popenoe and Loosli, 1978; Seidenfeld et al., 1978; Newhouse et al., 1978) a possible antidotal therapy was suggested. We reasoned that treatment with choline might displace paraquat from these cells and allow them to produce pulmonary surfactant in their normal manner. The low toxicity of choline made it even more attractive as a possible antidote (Greengard, 1975). However, choline was not protec-
tive when given simultaneously to or following treatment with paraquat in the doses and schedules used in our experiments. Since renal clearance of paraquat involves active tubular secretion (Ecker et al., 1975a,b), and the kinetics of elimination of paraquat are influenced by administration of saline (Drew and Gram, 1979), it may be that choline competes with the elimination of paraquat by the kidney; this would then explain the failure of choline to protect the lung. It is interesting to note that although chlorpromazine inhibited the uptake of paraquat by rat lung slices, the toxicity of paraquat was potentiated by chlorpromazine treatment (Siddik et al., 1979). These authors found a reduction in urinary excretion of paraquat from chlorpromazine treatment. Further experiments are needed regarding the mechanism of accumulation of paraquat in alveolar type II cells, the effect of the compound on that cell’s metabolic functions, and on the mechanism of renal elimination of paraquat. ACKNOWLEDGMENT The authors wish to thank Professors James B. Longley and Frederick K. Hilton, Department of Anatomy, University of Louisville School of Medicine, and Professor Sergei P. Sorokin, Department of Physiology, Harvard University, for their kind assistance in the identification of the histological sites of accumulation of radioactivity. The patient assistance of Dr. Walter E. Stumpf, Department of Anatomy, University of North Carolina School of Medicine, in instructing the authors on the use of the high-resolution technique is gratefully acknowledged. We also wish to thank Dr. Harriet M. Maling. National Heart, Lung, and Blood Institute, NIH, for helpful discussions and supplying the [3H]paraquat.
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L.-E.,
ERICSSON,
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AND
ULLBERG,
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(1961). A comparison of the distribution of radioactive fluorine and calcium by use of double-isotope autoradiography. Actn Ph.vsiol. Scud. 53, 339-347. BATENBURG,
J. J., AND
VAN
GOLDE,
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1131)
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LOCK, E. A.. SMIIH. L. L.. A&D Rosr;. M. S. (1Y7h). Inhibition of paraquat accumulation in rat lung \lrce\ by a component of rat plasma and a variety of tJrug\ and endogenous amines. Bictc~lwrtr. Phcirrtrtrc.ct’. 25, 1769- 1772. LONCL~\ . J. B. (1969). Histochemistry of the kidney In 771~ i\‘iclnev (C. Rouiller and A. F. Muller. cd\.,. Vol. I. pp. 157-260. Academic Press. New York. MANK~~LOW, B. W. (1967). The loss of pulmrmary surfactant in paraquat poisoning: A model for rhc study of the respiratory distress syndrome. Rrit .I tsp. Ptrrhl. 48. 366-369. MAYO. M. L. ( 1974). Differentiation of type 11 alvec&tl cells-A new use for the Cason Stain .Sitr/~: 7’eclinol. 49. 407-408. McNAR>, W. F.. JR.. AND EL-BEKMANI. A.-W. (1970). Differentiating type I and type II alveolar cell\ in rat lung by OsO,-NaI staining. Sltri,i Tc~,~/m~t/ 45, Zl5-?I’). NFWHOUS~, M.. McEvoy. D., .&No ROS~YI-HAI I). (197X). Percutaneous paraquat absorption. ,1 “c/r Ikrmtrtol. 114, 1516-1519. PoPEhot. D.. AND Loosi I. C. G. (1978). The morphological effects of a single exposure of pardqual on the mouse lung. Prr~c.. W’ccr. Phcrrtntrc~~ti. .Y(H 21. 151-153. Rest. M. S.. SM~IH, L. L.. AND W~AIT. I. (1~741. Evidence for energy-dependent accumulation
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AND MARLOWE
(1972). Correlation of paraquat toxicity with tissue concentrations and weight loss of the rat. Toxicol. Appl. Pharmacol. 22, 241-25 1. SHU, H., TALCOTT, R:E., RICE, S. A., AND WEI, E. T. (1979). Lipid peroxidation and paraquat toxicity. Biochem. Pharmacol. 28, 327-331. SIDDIK,~. H.,DREw, R.,ANDGRAM, T. E. (1979). The effect of chlorpromazine on the uptake and efflux of paraquat in rat lung slices. Toxicol. Appl. Phurmacol. 50, 443-450. SMITH, F. B., AND KIKKAWA, Y. (1979). The type II epithelial cells of the lung. V. Synthesis of phosphatidyl glycerol in isolated type II cells and pulmonary alveolar macrophages. Lab. Invest. 40, 172177. SOROKIN, S. P. (1977). The respiratory system. In Histology (L. Weiss and R. 0. Creep, eds.). 4th ed., pp. 765-830. McGraw-Hill, New York.
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STUMPF, W. E. (1976). Techniques for the autoradiography of diffusible compounds. In Methods in Cell Biology (D. M. Prescott, ed.), Vol. 13, pp. 171-193. Academic Press, New York. VAZIRI. N. D.. NESS, R. L., FAIRSHTER, R. D., SMITH. W. R.. AND ROSEN, S. M. (1979). Nephrotoxicity of paraquat in man. Arch. Intern. Med. 139, 172- 174. WADDELL, W. J.. AND MARLOWE, C. (1977). Autoradiography. In Drug Fare and Metabolism: Methods and Techniques (E. R. Garrett and J. L. Hirtz. eds.), Vol. I, pp. l-26. Dekker. New York. WASSERMAN, B., AND BLOCK, E. R. (1978). Prevention of acute paraquat toxicity in rats by superoxide dismutase. Atfiution Space Environ. Med. 49, 805-809.
AND
APPLIED
56, 127-140(1980)
PHARMACOLOGY
Tissue and Cellular WILLIAM Department
Disposition
J. WADDELL
of Pharmacology
Received
of Paraquat
AND CAROLYN
MARLOWE
and Toxicology, School of Medicine, Louisville, Kentucky 40292
March
25. 1980: accepted
June
in Mice1-2
Unh’ersity
of Louisville.
9. 1980
Tissue and Cellular Disposition of Paraquat in Mice. WADDELL, W. J., AND MARLOWE, C. Toxicol. Appl. Pharmacol. 56, 127- 140. Male C57B116J mice were injected intravenously with [methyl-‘*C]paraquat dichloride and frozen at 1,3,9, and 24 hr for whole-body autoradiography or with [merhyL3H]paraquat dichloride and the lungs were removed at 3.24, and 48 hr for cellular autoradiography. The methods do not allow thawing or exposure of the tissues to solvents; this prevents translocation or removal of radioactivity. The wholebody autoradiographs revealed localization of radioactivity at all time intervals in melanin, lung, choroid plexus, muscle, and excretory pathways such as proximal tubules of kidney, urine, liver, gallbladder, and intestinal contents. The radioactivity in lung was much higher in certain discrete. unidentified areas at all time intervals except at 1 hr. The concentration was high in myocardium at 1 and 3 hr. Cellular resolution autoradiography revealed that the radioactivity within the lung was confined almost entirely to cells having the distribution of alveolar type II cells at the three time intervals studied. The radioactivity in these cells was easily washed away indicating that an active transport process was probably involved instead of binding to a cellular constituent. The localization suggested that choline might be antidotal to paraquat toxicity. However, there was not a significant increase in survival of mice given 100 mg/kg choline chloride simultaneously to or following treatment with 50 mg/kg paraquat chloride. (1980).
Several mechanisms of the toxic action of the herbicide paraquat in animals and humans have been proposed. Manktelow (1967) provided evidence that paraquat interfered with the production of lung surfactant, Gage (1968) demonstrated that free radicals produced from paraquat increased respiration from liver mitochondrial fragments and Bus et al. (1974) suggested that paraquat toxicity may be mediated through the formation of the superoxide ion with subsequent lipid peroxidation. An early review discussed these possible I Presented at the annual meeting of the American for Pharmacology and Experimental Therapeutics in Portland. Ore.. August 22, 1979. ’ Supported by the University of Kentucky Tobacco and Health Research Institute Project No. KTRB-057, NIH Contract 263-78-M-8374, and Pharmacon Research Foundation. Inc. Society
mechanisms including the peroxidation of lipids which was suggested by Gage in 1968 as unpublished observations (Conning et al., 1969). Disagreement persists concerning the possible involvement of lipid peroxidation since vitamin E deficiency potentiates paraquat toxicity (Block, 1979) but lipid peroxidation was inhibited by paraquat in vitro (Steffen and Netter, 1979). In any event, therapy based on the proposed damage from peroxidation of lung lipids either has not been successful (Shu eC al., 1979) or has limited applicability in the clinical situation (Wasserman and Block, 1978). The preponderant damage is to the lungs although the kidneys and myocardium also are frequently involved (see review- by Haley, 1979, for references). Although 127
0041-008X/80/130127-14$02.00/O Copyright Q 1980 by Academic Press. Inc. All nghta of reproduction in any form resenrd.
128
WADDELL
AND MARLOWE
several studies have been reported on the localization of paraquat in tissues, including some using autoradiography (Litchfield et al., 1973; Larssoner al., 1978), none of these provided sufficient resolution in the tissues in which toxic effects are seen to provide clues to the mechanism of action. The present study was undertaken to identify the cells in which the compound accumulates, in vivo, in the hope that this would help clarify the mechanism of toxic action of this chemical. METHODS Whole-body autoradiography. Male, CS7Bl/6J mice were obtained from Jackson Laboratories, Bar Harbor, Maine. Food and tap water were provided ad libitum. [methyl-‘*C]Paraquat dichloride, Batch No. IS. having a specific activity of 30 mCi/mmol, was purchased from Amersham/Searle. Radiochemical purity was ascertained on cellulose thin-layer chromatographic plates developed in benzene:amyl alcohol:methyl alcohol:1 N HCl (1.3:4:8:8). A single peak with an R, of 0.74 was observed for the radioactive and standard paraquat solutions. Mice weighing 30 to 36 g were injected intravenously with 2.0 to 2.1 mg/kg [“Clparaquat dichloride dissolved in 0.9% NaCl and sacrificed 1, 3, 9. and 24 hr later. An additional mouse, weighing 41 g, was injected iv with 1.1 m&kg [“Clparaquat dichloride and sacrificed 3 hr later. All mice were sacrificed at the designated times by anesthetizing with ether and then freezing the mouse in a hexane/dry ice bath. Twentymicrometer-thick sections were taken onto Scotch tape at -22°C and the dried sections were placed on Xray film to produce autoradiographs. These wholebody autoradiographic procedures do not allow thawing or exposure of the tissues to solvents; this prevents translocation or removal of radioactivity. Details of this technique have been previously published (Waddell and Marlowe, 1977). High-resolution autoradiography. Male, C57BU6J mice w’eighing 26 to 32 g were obtained from Jackson Laboratories. Food and tap water were provided ad libitum. [methyl-3H]Paraquat dichloride was synthesized by AmershamSearle with a specific activity of 3.0 C/ mmol. A single radioactive peak, which corresponds to nonradioactive paraquat, was observed when the [3H]paraquat was spotted on silica gel thin-layer chromatographic plates and developed in N-butyl alcohol:5 N HCI (2:23). The R, of each compound was 0.50 in this system.
Nine mice were injected intravenously with 288 to 338 &kg L3H]paraquat dichloride dissolved in saline and triplicate mice were sacrificed by cervical dislocation at 3, 24, or 48 hr later. The lungs were quickly removed, blotted on Kimwipes, and trimmed to yield pieces of lung approximately 3-5 mm3. These small pieces of lung were attached to small microtome stages using minced, nonradioactive liver as the embedding material and then quickly frozen in liquid nitrogen. Duplicate samples were prepared from each mouse except four samples were prepared from one of the mice sacrificed 3 hr after receiving the paraquat. Four-micrometer-thick sections from the duplicate samples were taken at -3s”C in a Harris cryostat using an IEC rotary microtome. After drying at -3s”C the sections were attached to slides coated with Kodak NTB3 emulsion without the use of any solvents. These techniques were modified from procedures published by Stumpf (1976). The remaining two samples from the 3-hr mouse were also sectioned as described above but, instead of drying, the freshly cut sections were attached immediately to the emulsion-coated slides at room temperature. This thawmount technique does allow thawing of the section; thawing may result in translocation of the radioactivity (Stumpf, 1976). After photographic development thin sections were stained in either methyl green/ pyronine or celestine blue/Van Gieson’s (Stumpf, 1976; Kennedy and Little, 1974). Acute toxicity determinations. Adult male C57BV6J mice (Jackson Laboratories) weighing 20-32 g and Swiss Webster mice (Laboratory Supply, Indianapolis, Ind.) were provided food and tap water ad libitum. Sixteen C57BY6J mice were given a single ip injection of 50 mg/kg paraquat dichloride$ 1, 7, and 24 hr later, eight of the mice received SC 100 mgkg of an isotonic solution of choline chloride (Sigma Chemical) and the remaining eight received SC an equal volume of 0.9% NaCl. The Swiss Webster mice were administered the choline or saline and paraquat simultaneously; four mice received an ip injection of 50 mg/ kg paraquat dichloride combined with 100 mg/kg of an isotonic solution of choline chloride and three mice received the paraquat salt dissolved in an equal volume of 0.9% NaCl. All mice were observed to record the time of death.
RESULTS The whole-body autoradiographs revealed that at 1 hr there was high accumulation of radioactivity in myocardium, ’ The paraquat was generously supplied by Dr. Wyman H. Dorough, University of Kentucky.
BRAIN
SALIVARY
GLAND
BLOOD
FIG. I, A print of a whole-body autoradiograph White areas correspond to radioactivity.
MUSCLE
MELANIN
RIBS
MYOCARDIUM
LIVER
ADRENAL
INTESTINE
KIDNEY
URINE
of a 20-pm section from a male CS7BV6J mouse frozen 1 hr after an iv injection of [mPrhy/-“Clparaquat.
LUNG
PLEXUS
MUSCLE
LIVER
section
DIAPHRAGM
of a 20-@rn
LUNG
MYOCARDIUM
autoradiograph to radioactivity.
BLOOD
FIG. 2. A print of a whole-body paraquat. White areas correspond
MELANIN
CHOROID
from
a male C57B1/6J
INTESTINAL
STOMACH
mouse
frozen
CONTENTS
KIDNEY
3 hr after
an iv injection
GU
of [methyl-‘4C]-
TRACT
0
L
DISPOSITION
LUNG
HEART
OF PARAQUAT
DIAPHRAGM
LIVER
FIG. 3. A detail of an autoradiograph of a 20-pm section from a male C57BV6J mouse an iv injection of [“Clparaquat. Intense radioactivity can he seen in the diaphragm discrete spots in the lung. White areas correspond to radioactivity.
liver, intestinal contents, skeletal muscle, melanin, ribs, urine, lungs, kidney, and choroid plexus. Most of these sites can be seen in Fig. 1; the other tissues were present in other sections of the mouse. By 3 hr after administration the amount of radioactivity in the myocardium, ribs and to some extent in the liver had decreased sharply. The relative concentration in the choroid plexus, lung, and convoluted segment of the proximal tubule in the kidney had increased. The accessory sex glands in the male mice showed relatively high concentrations at this time interval (Fig. 2). Enlargements of autoradiographs from other sections of this mouse at 3 hr are shown in Figs. 3 and 4. These show the intense radioactivity in the convoluted segments of proximal tubules in the kidney and in certain discrete areas of the lung. These spots in the lung are more intense than any of the surrounding tissues, but their anatomical location within the lung could not be determined. The high concentration in the diaphragm was most notable only at 3 hr.
frozen 3 hr after and in certxin.
At 9 hr after receiving the compound. melanin, muscle, liver, intestinal contents, and urine continue to show appreciable activity, but the relative activity in lung and kidney continues to increase (Fig. 5). The discrete localizations in lung are even more prominent than they were at 3 hr and the choroid plexus is well labeled. After 24 hr, the localizations that remain have decreased to a few areas: (I) the contents of the gastrointestinal tract and urine: (2) melanin and skeletal muscle: (3) choroid plexus, lung, and kidney (Fig. 6). The activity in lung and kidney are even more clearly confined to discrete areas: these could not be identified in lung but in kidney were identified as the convoluted segment of proximal tubule. At each of the time intervals. there was considerable variability among different skeletal muscle groups. The high-resolution autoradiographs of the lung done by the dry-mount technique at each of the three time intervals revealed localization predominately in certain discrete cells. The histological pattern of
WADDELL
132
PROXIMAL
AND MARLOWE
TUBULES
OF
KIDNEY
FIG. 4. A detail of an autoradiograph of a 20-pm section from the male C57BY6J mouse frozen 3 hr after [“Clparaquat. Note the accumulation of radioactivity in the convoluted segments of the proximal tubules in the kidney. White areas correspond to radioactivity.
the distribution of this cell type appeared to correspond most closely with that of the alveolar type II. The cells were in the corners of the alveoli and occasionally along the alveolar wall (Fig. 7). When the tissue section for high-resolution autoradiography was allowed to thaw as it was applied to the warm emulsion, the radioactivity was completely translocated from the cells to the interior of the alveoli and periphery of the section (Fig. 8). No significant protection from choline on the acute toxicity of paraquat in mice was observed in the two studies. When the mice were exposed to the paraquat 1 hr before the first of three injections of choline or saline all mice in the saline group were dead after 4 days and only one of the mice in the choline group survived after 4 days. One mouse from each of the choline and saline groups survived when the paraquat was combined with the choline or saline for injection. Most of the mice died within the first 72 hr following paraquat exposure; however, the occurrence of death in the saline-treated group
was delayed in general about 24 hr in comparison to the choline-treated mice. DISCUSSION The tissue distribution of paraquat was studied by whole-body autoradiography earlier using sections of mice that were 100 pm thick (Litchfield et al., 1973). Those studies identified the major organs which accumulate the compound but the thick sections did not allow sufficient resolution to discern the cells of accumulation in lung and kidney; these are the primary organs where the toxic effects of the chemical are seen. Later autoradiographic studies with 20-,um sections examined the nature of the binding of paraquat to melanin and intervertebral cartilage (Larsson er al., 1978); however, neither of these sites is involved in an adverse effect from the chemical. Whole-organ analyses in the rat revealed high and persistent accumulation in lung, muscle, and kidney (Sharp&al., 1972).
PLEXUS
MUSCLE
IN
SALIVARY
BLOOD
FIG. 5. A print of a whole-body autoradiograph White areas correspond to radioactivity.
MELANIN
CHOROID
of a 20.pm
GLAND
HEART
section
from
RIBS
LUNG
a male CS7BV6J
LIVER
STOMACH
mouse
frozen
CONTENTS
9 hr after an iv injection
INTESTINAL
KIDNEY
of [methyl-“Clparaquat.
134
WADDELL
AND
MARLOWE
DISPOSITION
OF
13s
PARAQUAT
FIG. 7. Dry-mount autoradiographs of lungs from male C57B116J activity in the alveolar type II cells 3 hr following an iv injection celestine blue/Van Gieson‘s. (A) 88x. (B) 350x.
mice showing accumulation of [,?ic~rh~/-:‘H]paraquat.
Stained
of radiowith
136
WADDELL
AND
MARLOWE
FIG. 8. Thaw-mount autoradiographs of lungs from male C57BV6J mice showing translocation of radioactivity from the type II cells to the interior of the alveoli and periphery of the tissue. The lungs were removed from mice 3 hr after receiving iv [3H]paraquat. Stained with methyl green/pyronine. (A) 88 x, (B) 350x.
DISPOSITION
The present report was undertaken to elucidate the cells in the tissues which accumulate paraquat. Since the lung is the primary organ injured and the kidney is also to a lesser extent (see Haley, 1979, for references), attention was focused on the kinetics of accumulation in these organs. The studies reported here revealed that radioactivity was confined to what is interpreted to be essentially one cell type in the lung. The histological distribution of the cell type which accumulated the radioactivity was most probably the alveolar type II. Other possibilities, e.g., alveolar type I, macrophages, capillary endothelium, etc., were excluded because of their different distribution patterns (McNary and ElBermani, 1970; Mayo, 1974; Sorokin, 1977). The low concentration in blood eliminates the possibility that the radioactivity was localized in capillaries. Furthermore, it appears that it persists in this cell for several days. In addition, the accumulation in that cell is very easily displaced since simple thawing of the section translocated the radioactivity. Consequently, it is unlikely that the radioactivity is strongly bound to cellular constituents; it certainly is not covalently incorporated into molecules within the cell. A logical conclusion is that paraquat is actively transported into the alveolar type II cells by a mechanism that exists for a similar molecule used in its normal metabolic activities. Additional evidence for active transport of paraquat by specific cells is the persistent accumulation of radioactivity in the choroid plexus and in the convoluted portion of the proximal tubule: each of these cell layers is known to actively transport cations with structures similar to that of paraquat. This accumulation in the choroid plexus may have clinical significance since Grant et al. (1980) reported cerebral damage in patients who died following paraquat ingestion. High-resolution autoradiography of the brain and kidney were not necessary since the choroid plexus is easily identified
OF PARAQUAT
137
in 20-pm sections and the distribution in the convoluted segment of the proximal tubule of the kidney is distinctive and readily recognized (Longley, 1969). Radioactivity seen in the autoradiographs probably represents unchanged paraquat since there is no evidence for metabolism of paraquat which has been administered intravenously (Daniel and Gage, 1966). Localization of paraquat in tissues such as liver, gallbladder, intestinal contents. and urine obviously merely reveals the routes of elimination of the compound. The accumulation in melanin and intervertebral cartilage has been well studied previously and is probably due to an ionic interaction (Larsson et al., 1978). The wide variability in the accumulation in skeletal muscle may be due to the state of contraction or relaxation of these individual muscle groups at the time of freezing of the animal: the same pattern is seen with j”Ca (Appelgren rt 1961; Ericsson and Hammarstrom. al., 1964). Most of the speculation on the mechanism of toxic action of paraquat centers on possible lipid peroxidation mediated by the compound (see Haley, 1979, for references): however, there is much experimental evidence which causes serious doubt that this explains its action in viva (Shu ef (11.. 1979: Steffen and Netter, 1979). The experiments reported here suggest another mechanism which has not heretofore been considered, to our knowledge. The cellular accumulation of paraquat which is most persistent is in three cell types, i.e., alveolar type II, convoluted segment of proximal tubule, and choroid plexus, which appear to concentrate it by an active transport mechanism. The concentration in that cell in the kidney can readily e Yplain the effect in blocking the function of the proximal tubule causing its degeneration in poisoning by the chemical (Vaziri t~f ui., 1979). Rose et al. (1974) reported that the accumulation of paraquat into lung slices, in bxitro, was an energy-dependent process.
138
WADDELL
AND MARLOWE
Also, Lock et al. (1976) demonstrated that a variety of compounds could inhibit the accumulation of paraquat in rat lung slices. We should like to suggest that the accumulation in the alveolar type II cell might explain its toxic effect on the lung by the following mechanism. One function of the alveolar type II cell is the synthesis of pulmonary surfactant (see review of Batenburg and Van Golde, 1979, for references); the primary composition of this surfactant is phosphatidyl choline (King and Clements, 1972; King, 1974; Smith and Kikkawa, 1979). Choline is a quartenary ammonium compound just as is paraquat. [3H]Choline has been shown to accumulate selectively in alveolar type II cells (Chevalier and Collet, 1972). If the alveolar type II cell accumulates paraquat instead of choline, the synthesis of phosphatidyl choline may be blocked. Blocking the synthesis of the lung’s primary surfactant would allow damage to the alveoli by inhaled oxidants. This sequence of events is consistent with many observations in paraquat poisoning including loss of pulmonary surfactant (Manktelow, 1967), decreased production of phosphatidyl choline by lung after treatment with paraquat (Etherton and Gresham, 1979), enhancement of the toxicity by increased oxygen tension (Fisher et af., 1973), protection from paraquat toxicity by vitamin E (Block, 1979), and delayed onset of toxic effects. Furthermore, since the alveolar type 11 cell is not permanently damaged by the presence of paraquat and in fact may be proliferating in a compensatory manner (Dearden et al., 1978; Popenoe and Loosli, 1978; Seidenfeld et al., 1978; Newhouse et al., 1978) a possible antidotal therapy was suggested. We reasoned that treatment with choline might displace paraquat from these cells and allow them to produce pulmonary surfactant in their normal manner. The low toxicity of choline made it even more attractive as a possible antidote (Greengard, 1975). However, choline was not protec-
tive when given simultaneously to or following treatment with paraquat in the doses and schedules used in our experiments. Since renal clearance of paraquat involves active tubular secretion (Ecker et al., 1975a,b), and the kinetics of elimination of paraquat are influenced by administration of saline (Drew and Gram, 1979), it may be that choline competes with the elimination of paraquat by the kidney; this would then explain the failure of choline to protect the lung. It is interesting to note that although chlorpromazine inhibited the uptake of paraquat by rat lung slices, the toxicity of paraquat was potentiated by chlorpromazine treatment (Siddik et al., 1979). These authors found a reduction in urinary excretion of paraquat from chlorpromazine treatment. Further experiments are needed regarding the mechanism of accumulation of paraquat in alveolar type II cells, the effect of the compound on that cell’s metabolic functions, and on the mechanism of renal elimination of paraquat. ACKNOWLEDGMENT The authors wish to thank Professors James B. Longley and Frederick K. Hilton, Department of Anatomy, University of Louisville School of Medicine, and Professor Sergei P. Sorokin, Department of Physiology, Harvard University, for their kind assistance in the identification of the histological sites of accumulation of radioactivity. The patient assistance of Dr. Walter E. Stumpf, Department of Anatomy, University of North Carolina School of Medicine, in instructing the authors on the use of the high-resolution technique is gratefully acknowledged. We also wish to thank Dr. Harriet M. Maling. National Heart, Lung, and Blood Institute, NIH, for helpful discussions and supplying the [3H]paraquat.
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STUMPF, W. E. (1976). Techniques for the autoradiography of diffusible compounds. In Methods in Cell Biology (D. M. Prescott, ed.), Vol. 13, pp. 171-193. Academic Press, New York. VAZIRI. N. D.. NESS, R. L., FAIRSHTER, R. D., SMITH. W. R.. AND ROSEN, S. M. (1979). Nephrotoxicity of paraquat in man. Arch. Intern. Med. 139, 172- 174. WADDELL, W. J.. AND MARLOWE, C. (1977). Autoradiography. In Drug Fare and Metabolism: Methods and Techniques (E. R. Garrett and J. L. Hirtz. eds.), Vol. I, pp. l-26. Dekker. New York. WASSERMAN, B., AND BLOCK, E. R. (1978). Prevention of acute paraquat toxicity in rats by superoxide dismutase. Atfiution Space Environ. Med. 49, 805-809.