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The effect of chlorpromazine on the uptake and efflux of paraquat in rat lung slices
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
50,
443-450
(1979)
The Effect of Chlorpromazine on the Uptake and Efflux of Paraquat in Rat Lung Slices’ ZAHID H. SIDDIK, Laboratory
of Toxicology,
ROGER DREW, AND THEODORE E. GRAM
National Cancer Institute, National Bethesda, Maryland 20014
Received February The Effect of Chlorpromazine SIDDIK,
Z. H., DREW,
R.,
AND
Institutes of Health,
15, 1979; accepted May 18, 1979
on the Uptake and Efflux of Paraquat in Rat Lung Slices. GRAM,
T. E. (1979).
Toxicol.
Appl.
Pharmacol.
50,
443-450.
The uptake of paraquat by rat lung slices was inhibited by chlorpromazine in a concentration- and time-dependent manner. In addition, the efflux of paraquat from lung slices was enhanced by chlorpromazine in a similar fashion. These in citro findings suggested that chlorpromazine might be useful in ciao in reducing pulmonary paraquat content and, in turn, its pneumotoxicity. However, chlorpromazine potentiated the lethal toxicity of paraquat rather than ameliorating it. This potentiation by chlorpromazine was found to correlate with a reduction in the urinary excretion of paraquat and concomitant increase in pulmonary paraquat concentrations.
There is a growing concern over the continued widespread use of paraquat since the herbicide, when ingested, is known to cause fatalities in both man and animals (Smith and Heath, 1976). While paraquat produces toxic alterations in liver, kidney, and brain, death is more commonly the result of degenerative pulmonary changes, including interstitial and intraalveolar fibrosis, terminating in pulmonary insufficiency. Despite extensive research on the pathogenesis and biochemistry of the paraquat pulmonary lesion, an “antidote” for the herbicide is still not known. Clinical treatment of human cases is usually ineffective and consists essentially of removing the unabsorbed (by gastric lavage) and absorbed (by hemodialysis and forced diuresis) paraquat as rapidly as possible, followed by attempts at suppression ’ Presented in part at the joint meeting of the American Society for Pharmacology and Experimental Therapeutics and the Society of Toxicology, University of Houston, Tex., August 13-17, 1978.
of fibroblastic proliferation with corticosteroids or immunosuppressive drugs (Smith and Heath, 1976). Paraquat is accumulated into the lung by an energy-dependent uptake process (Rose et al., 1974) which appears to be specific to the lung, being absent in kidney and liver (Rose et al., 1976). However, it appears that the factor responsible for the lung toxicity is not the uptake of paraquat but rather the slow rate of efflux from the lung (Smith et al., 1978). Thus, if paraquat efflux from lung could be enhanced, then its toxicity might be reduced. Recent work in our laboratory examined the ability of a number of endogenous and exogenous compounds to inhibit uptake or to enhance efflux or paraquat in rat lung slices (Drew et al., 1979). Of the compounds investigated chlorpromazine was selected for further studies since this compound not only inhibited uptake but enhanced efflux of paraquat in lung slices. In the present study, the effect of chlorpromazine
443 All
0041-008X/79/120443-08$02.00/0 Copyright 0 1979 by Academic Press, Inc. rights of reproduction in any form reserved. Printed in Great Britain
444
SIDDIK,
DREW,
on paraquat accumulation and efflux was further examined and the possible amelioration of paraquat toxicity with chlorpromazine was investigated. METHODS Animals. Male Sprague-Dawley rats (200-250 g; Taconic Farms, Germantown, N.Y.) which had been allowed food and water ad Iibitum were used in all experiments. Lung slice studies. The methods used to prepare lung slices (20-50 mg) and study uptake and efflux of [Wlparaquat were described previously (Drew et al., 1979). Briefly, rats were anesthetized with pentobarbital (60 mg kg-‘, ip), lungs removed, and slices cut free hand. Lung slices were equilibrated for 20min at 37°C in Krebs-Ringer bicarbonate buffer (pH 7.4) supplemented with 5 mM glucose (KRB) and gassed with O2 : CO2 (95 : 5) before being transferred to the same medium (100 ml) containing paraquat or paraquat and chlorpromazine. Slices were removed at appropriate times, briefly rinsed, gently blotted, solubilized, and the radioactivity was determined by liquid scintillation spectrometry. For efflux studies, lung slices that were preincubated for 2 hr in 10 ,UM [Wlparaquat (100 ml) were briefly rinsed and transferred immediately to fresh KRB (100 ml) containing varying amounts of chlorpromazine. Slices were removed at appropriate times and their 14C content determined as above. The change in paraquat concentration in the medium during uptake study was negligible, and the medium paraquat concentration during efflux did not exceed 0.05 nmol ml-l. Paraquat toxicity. In order to investigate the effect of chlorpromazine on paraquat-induced lethality, chlorpromazine hydrochloride (25 mg kg-‘, SC) was injected either daily before and/or after oral administration of paraquat dichloride (235 mg kg-‘) or 30 min before intravenous administration of the herbicide (23 mg kg-‘). Saline (40 ml kg-r, po) was administered to some animals immediately before paraquat. Paraquat excretion and tissue distribution. The effect of chlorpromazine on urinary paraquat excretion was determined by injecting rats with chlorpromazine hydrochloride (25 mg kg-l, SC) 30 min before [“Clparaquat dichloride (23 mg kg-‘, iv) or [Wlparaquat dichloride and saline (40 ml kg-‘, po) and the urine collected for 24 hr and analyzed for radioactivity. At 24 hr the animals were anesthetized with ether, blood (from the severed axillary vessels collected in tubes containing heparin) and tissues were taken for i4C analyses. Radioactivity in aliquots of urine (0.5 1.0 ml), plasma (0.5 ml), and lung, kidney, liver, heart, and mesenchymal fat (lW150 mg) were determined by standard liquid scintillation techniques.
AND
GRAM
Chemicals. [‘%]Paraquat (specific activity 33 mCi mmol-i; Amersham-Searle, Arlington Heights, Ill.) nonradiolabeled paraquat and chlorpromazine (Sigma Chemical Co., St. Louis, MO.) were purchased commercially and used without further purification. The compounds were dissolved in saline for use in all experiments. Statistics. Statistical evaluations were made by using Student’s t test for significance at p < 0.05 level.
RESULTS Effect of chlorpromazine on uptake and eflux of paraquat in lung slices. The pul-
monary uptake of [i4C]paraquat (10 ,MM) was consistently reduced in the presence of chlorpromazine (Table 1). This inhibition was concentration dependent. At 100~~ chlorpromazine, the uptake of paraquat was only 40% of control. In the absence of chlorpromazine, the efflux of paraquat from lung slices was negligible during the 2-hr incubation period (Table 1, Uptake vs Efflux, top row). However, in the presence of chlorpromazine, efflux of paraquat from lung slices occurred and increased with increasing concentration of chlorpromazine. At 50 and 500 ,LLM chlorpromazine the concentration of remaining paraquat in lung slices was, respectively, 60 and 20% of that in slices incubated in absence of chlorpromazine. The temporal aspects of the uptake and efllux of paraquat by lung slices is presented in Fig. 1. The uptake of paraquat was linear with time for 60-90 min in the absence of chlorpromazine; however, in the presence of the inhibitor, the duration of linear paraquat accumulation decreased dramatically. In the presence of 300 ,LLM chlorpromazine, no further uptake of paraquat occurred beyond 60 min of incubation. As pointed out above, efflux of paraquat was observed only when the slices were incubated in media containing chlorpromazine (Fig. I). At 300~~ chlorpromzine, only about 30% of the initial paraquat remained in the lung slices after 2 hr. Lung slices were incubated for 30 min in varying concentrations of paraquat and the rate of uptake determined. The rate of
UPTAKE/EFFLUX
OF PARAQUAT
TABLE EFFECT OF CHLORPROMAZINE [W]PARAQUAT
1
ON THE UPTAKE AND EFFLUX IN RAT LUNG SLICES’
Chlorpromazine concentration (PM)
nmol g-lb
0 10 50 100 300 500
64.Ok4.4 51.0* 3.0 39.4 -+ 2.74 25.4+ 1.5” 19.9+0.54 14.2 +_0.9d
445
IN RAT LUNG
OF
Efflux
Uptake o/o’ 100 89.1 61.6 39.7 31.1 22.2
nmol g-lb 58.8 + 6.9 50.7 + 5.3 35.6* 5.64 38.9+ 1.3d 16.9* l.ld 11.5*0.76
%” 100 86.2 60.5 66.2 28.7 19.6
a Uptake was determined using 10 PM paraquat. Efflux studies were conducted using slices that had been incubated for 120 min at 37°C in KRB solution containing 10 PM paraquat only, and then transferred to fresh media containing the appropriate amounts of chlorpromazine. ’ Paraquat content (+ SE) of six lung slices incubated under conditions of uptake or efflux for 120 min. c Percentage of paraquat content of lung slices incubated with no chlorpromazine. d Significantly different from lung slices with no chlorpromazine (p < 0.05).
Uptake
EfflUX 1
d
I
1
uptake of paraquat was proportional to its concentration, in the range of 5-50 PM (Fig. 2). However, in the presence of 100 or 300 pM chlorpromazine, uptake was reduced but still proportional to the paraquat concentration. The extent of inhibition produced by 100 or 300 pM chlorpromazine was very similar. Efect of chlorpromazine on paraquat toxicity. Chlorpromazine was injected at 24-hr
0'
I 80
I 120 TIME lminl
I
I 180
I 240
1. The uptake and efflux in rat lung slices of [Wlparaquat in the presence and absence of 100 or 300~1~ chlorpromazine (CPZ). Efflux studies were conducted using slices that had been incubated for 120 min at 37°C in KRB solution containing 10~~ [Ylparaquat only, and then transferred to fresh media containing the appropriate amounts of CPZ. Data points represent the mean+ SE of six slices. An asterisk indicates statistical significance at p <0.05 compared to slices incubated in absence of CPZ. FIG. 10~~
intervals before and/or after oral administration of paraquat to rats. The results (Table 2) show that the rats injected with the indicated doses of either chlorpromazine or paraquat had, respectively, 0 or 38% lethality. However, pretreatment and/or post-treatment with chlorpromazine markedly potentiated the paraquat-induced lethality. This potentiation correlated well with body weight loss which confirmed the previous report by Sharp et al. (1972). The deaths were all due to pulmonary insufficiency as evidenced by preterminal dyspnea and hemorrhagic lungs. When paraquat was given orally a constant plasma concentration of the herbicide was
446
SIDDIK,
DREW,
AND
maintained for at. least 30 hr (Rose et al., 1976) and this may have been a complication in determining the effectiveness of chlorpromazine therapy. That is, at its peak
plasma concentration, chlorpromazine may have inhibited uptake and induced efflux of paraquat but as the concentration of the active phenothiazine drug declined then uptake of paraquat may have resumed. Thus, the possibility of protection was further investigated by studying the effect of subcutaneously injected chlorpromazine and/or orally administered saline on intravenous paraquat toxicity. Chlorpromazine again potentiated paraquat toxicity (Fig. 3) and by Day 4 there were no surviviors in the group given paraquat and chlorpromazine. However, if saline was also administered with paraquat and chlorpromazine, the lethality (70-75%) was about the same as in the group injected with paraquat alone. Saline alone had no effect on paraquat-induced lethality. Again, respiratory failure accounted for the deaths as indicated by dyspnea and grossly hemorrhagic lung seen on autopsy.
o No CPZ A 100 pM CPZ q 300 pM CPZ
PARAQUAT
CONCENTRATION
GRAM
ipM)
FIG. 2. Variation in the rate of uptake of [‘“Clparaquat with concentration in presence and absence of 100 or 300~~ chlorpromazine (CPZ). Slices were incubated for 30 min at 37°C in KRB solution containing the appropriate amounts of the two drugs. Data points represent the mean +_SE of six slices. An asterisk indicates statistical significance at p < 0.05 compared to slices incubated in absence of CPZ.
Effect of chlorpromazine on urinary excretion of paraquat. To account for the poten-
tiation by chlorpromazine of paraquat toxicity, urinary excretion and tissue distribution
TABLE
2
(CPZ) ON PARAQUAT (PQ) TOXICITY
THE EFFECT OF CHLORPROMAZINE
IN RATS
Percentage cumulative deaths Day -1
CPZb
PQ
-to
0 ->o
PQ + CPZ
PQ + CPZ
-0
0 . ..>o
PQ + CPZ -+O
0
+O
1
-+O
0
0
-to
+O
2
-+O
0
0
40
+14
-213
+57
20
3
-+O
0
57
4
0
13
86
25
6
0
13
38
8
0
25
10
0
38
100 100 loo
63 75
?>:o
100 100 100 100
0 Chlorpromazine (25 mg kg-l, SC)was injected at intervals of 24 hr on the indicated days (+). Paraquat (235 mgkg-‘, po) was administered as a single dose on Day 0 (+), and 30 min after the injection of chlorpromazine to rats receiving both drugs on the same day. Each group consisted of seven or eight animals. Ir CPZ = Chlorpromazine; PQ = paraquat.
UPTAKE/EFFLUX
OF PARAQUAT
loor 80
40 0 A 6
20
0
1
2 DAYS
3 AFTER
PQ + Saline PQ+CPZ PO + CPZ + Saline
4 5 PARAQUAT
6
7
FIG. 3. The effect of chlorpromazine (CPZ) and/or saline on paraquat (PQ)-induced lethality in rats. The animals were treated with PQ (23 mg kg-‘, iv) saline (40 ml kg-‘, po) and/or CPZ (25 mg kg-‘, SC) as deserihed in the text and mortality was assessed daily. Each group consisted of 15-22 animals.
0 PQ+Saline A PQ+CPZ A PQ+CPZ+Saline
0’
’
4
8
12 TIME lhr)
18
20
,
-.20
FIG. 4. The effect of chlorpromazine (CPZ) and/or saline on (A) voided urine and (B) urinary paraquat (PQ) excretion in rats. The animals were given [W]PQ (23 mg kg-r, iv), saline (4Ornl kg-r, PO), and/or CPZ (25 mg kg-r, SC) as described in the text and the. urine collected at the indicated time intervals and analyzed for 14C content. Data points represent the meanf SE of five or six animals. An asterisk indicates significance at p ~0.05 compared to rats administered PQ only.
of paraquat was investigated. In animals given both paraquat and chlorpromazine, the 24-hr urinary volume was only 20% that in animals injected with paraquat alone (Fig.
IN
RAT
447
LUNG
4A). Saline increased the rate of urine excretion at the early time intervals, and when administered in conjunction with paraquat and chlorpromazine, the cumulative voided urine volume in 24 hr increased threefold, compared to animals which were not hydrated. However, there was no significant difference in the cumulative urine volume in 24 hr between animals given paraquat or paraquat and saline. Temporally, the voided volume from animals given saline, saline and chlorpromazine, or chlorpromazine was very similar to that seen after administration of paraquat, paraquat and saline, or paraquat, chlorpromazine, and saline, respectively (data not presented). It is noteworthy that the urinary paraquat excretion was markedly lower (-50%) in animals receiving paraquat and chlorpromazine compared with the paraquat group (Fig. 4B). This difference was abolished if saline was also administered to animals injected with paraquat and chlorpromazine (Fig. 4B). Rats treated with paraquat and saline eliminated significantly more paraquat at only the 2- and 4-hr time points than rats injected with paraquat only. Thus, at 24 hr, rats given paraquat, paraquat and saline, or paraquat, chlorpromazine, and saline excreted &l--93% of the paraquat dose with SO-90% being excreted in the first 4 hr. E#ect of chlorpromazine on tissue concentrations of paraquat. Rats injected with
paraquat and chlorpromazine had significantly higher pulmonary (28%) and fat (108%) paraquat concentrations than animals given only paraquat (Fig. 5). In the rats given saline in conjunction with paraquat or paraquat and chlorpromazine, the tissue concentrations were generally comparable with those from rats given only paraquat. However, the cardiac paraquat content in rats given paraquat and saline was significantly, but inexplicably higher than in animals injected with paraquat alone. In all cases the tissue concentrations of paraquat greatly exceeded the plasma concentration with maximal
levels
being
present
in
the lung.
SIDDIK,
DREW,
OPQ n PQ+CPZ a W+Saline q PQ+CPZ+Saline
0-
PLASMA
KIDNEY
LIVER
HEART
FAT
FIG. 5. The effect of chlorpromazine (CPZ) and/or saline on the tissue distribution of paraquat (PQ) in rats. The animals were given [“C]PQ (23 mg kg-‘, iv), saline (40 ml kg-‘, PO), and/or CPZ (25 mg kg-‘, SC) as described in the text, sacrificed 24 hr later, and the tissues analyzed for 14C content. Data points represent mean+ SE of five or six animals. An asterisk indicates significance at p < 0.05 compared to rats administered PQ only.
There was no significant difference in the whole organ wet weights between the four treatment groups (data not presented). DISCUSSION Deaths due to paraquat poisoning are often caused by acute pulmonary edema or chronic fibrosis (Smith and Heath, 1976). The lung appears to be unusually susceptible because of its ability to actively accumulate paraquat (Rose et al., 1974). Although a number of compounds can inhibit this uptake of paraquat in lung slices (Lock et al., 1976; Drew et al., 1979), they do not necessarily enhance its efflux (Drew et al., 1979). In the present study, chlorpromazine not only inhibited accumulation of paraquat by lung slices but also enhanced its efflux. Both inhibition of uptake and enhancement of efflux of paraquat were dependent on the concentration of the phenothiazine. Furthermore, the inhibition of uptake and enhancement of the efflux did not remain constant with time, but increased rapidly after 1 hr of incubation. The reason for this may be that
AND
GRAM
during uptake of paraquat, chlorpromazine may initially compete directly for the paraquat binding sites on the carrier protein, but with time, as the binding of chlorpromazine to membrane increases (Di Francesco and Bickel, 1977), the drug may alter physicochemical properties of the membrane which may further inhibit paraquat uptake. Alterations in membrane properties by chlorpromazine have been reported previously (Gordon, 1967). Similar nonlinear timedependent inhibition of paraquat uptake in lung slices was also produced by imipramine (Drew et al., 1979), histamine, and betazole (Lock et al., 1976). The site of the energy-dependent carriermediated accumulation of paraquat has been proposed to be the type I and type II alveolar epithelial cells (Smith et al., 1976). Chlorpromazine is also accumulated in the lung against a concentration gradient but this process is probably by diffusion and nonspecific binding (Junod, 1975; Di Francesco and Bickel, 1977), and may involve the entire spectrum of pulmonary cell types which number in excess of 40 (Sorokin, 1970). Thus, during paraquat efflux studies, chlorpromazine accumulated in the lungs may initially bind to available sites, and only when these become saturated will competition for sites occupied by paraquat take place and displacement of the herbicide be induced. This may partially explain why efflux of paraquat in presence of chlorpromazine is time dependent and becomes substantial after 1 hr of incubation. Furthermore, since accumulation of chlorpromazine is probably not cell or organelle specific (Di Francesco and Bickel, 1977), a much greater pulmonary level of the phenothiazine than of paraquat would be needed to attain concentrations within the type I and type II cells sufficiently high to displace paraquat. This would be achieved either by a short- or long-term exposure to a high or low concentration of chlorpromazine respectively. Thus, incubation of slices for 2 hr with 10~~ chlorpromazine may not, have been sufficient to cause a significant
UPTAKE/EFFLUX
OF PARAQUAT
decrease in paraquat concentration during efflux studies. Inhibition of pulmonary drug uptake and/ or enhancement of efflux by chlorpromazine has been reported previously for imipramine and propranolol (Junod, 1975). However, the binding sites of chlorpromazine, imipramine, and propranolol are qualitatively the same (Huunan-Seppalti, 1972; Bickel, 1975) and competition for these sites would be expected. Although all three drugs inhibited uptake of paraquat to approximately the same extent, only chlorpromazine showed marked enhancement of efflux, with equimolar concentration of propranolol having no effect on this process (Drew et al., 1979). It is possible that the relative affinity for sites occupied by paraquat may be greater for chlorpromazine than imipramine or propranolol. However, ,other mechanisms such as cytotoxicity ;(Lahrichi et al., 1977), inhibition of ATPase, land alteration in membrane properties (Gordon, 1967) may all contribute toward the iinhibition of accumulation and enhancement iof efflux of paraquat by chlorpromazine. With the regular occurrence of accidental lor intentional paraquat poisoning, it has become increasingly important to identify an effective drug therapy for the poison. A compound could show protection merely by accelerating efflux of paraquat from the lungs. The in t&o interactions between paraquat and chlorpromazine indicated the possibility of using chlorpromazine in ameliorating the lethality due to paraquat. Although chlorpromazine also prevents pulmonary edema due to epinephrine and protects animals from oxygen toxicity (Gordon, 1967), it did not prevent paraquat toxicity. Administration of chlorpromazine (Jarvik, 1970) or paraquat (Lock and Ishmael, 1978) produces diuresis. Thus, voided urine volume in animals given chlorpromazine and saline or paraquat and saline was greater at early time points than in animals receiving saline alone. A combination of paraquat, chlorpromazine, and saline, on the other hand, resulted in lower urinary volume than after
IN
RAT
LUNG
449
coadministration of saline with either paraquat or chlorpromazine. Similarly, urinary volume in rats receiving both paraquat and chlorpromazine was significantly lower than in animals given paraquat or chlorpromazine alone. This indicated that an interaction between paraquat and chlorpromazine resulted in the unexpected decrease in urinary volume, which in turn reduced urinary paraquat excretion in animals receiving both paraquat and chlorpromazine, and thus resulting in increased pulmonary paraquat content. A similar effect of /3-adrenergic agonists on urinary excretion of paraquat also results in an increased herbicide concentration in the lung (Maling et al., 1978). Since Sharp et al. (1972) have reported a direct correlation between lethality and the concentration of paraquat in the lung, it is probable that potentiation of toxicity by chlorpromazine was a direct result of increased paraquat levels in this organ. Administration of saline as a diuretic increased paraquat excretion and reduced lung paraquat content and lethality to values obtained with rats injected with paraquat alone; that is, no beneficial effect of chlorpromazine was seen at the dose used. This dose of chlorpromazine produces a plasma concentration of 4-5 nmol chlorpromazine equivalents ml-’ between 4 and 24 hr, and lung concentrations of 99, 154, 191, and 76 nmol g-l at 0.5, 2, 4, and 24 hr after administration (unpublished observation). Pulmonary concentration of paraquat, on the other hand, remains fairly constant between 1 and 24 hr after its intravenous administration (Sharp et al., 1972). Thus, concentration of chlorpromazine in the lung was much greater than that of paraquat (30-40 nmol g-l ; Fig. 5) at all times up to 24 hr. However, chlorpromazine is highly bound to plasma proteins (Salzman and Brodie, 1956) and undergoes rapid metabolism (Minder et al., 1971), and thus the level of free chlorpromazine in the plasma, and consequently the lung, may not have been sufficient to inhibit uptake and enhance efflux of paraquat. In these experi-
450
SIDDIK,
DREW,
ments, the dose of chlorpromazine used was one which did not cause excessive impairment of food and water intake as a result of its depressant action. Higher doses of chlorpromazine in conjunction with saline were not attempted due to reduced food intake and toxicity due to chlorpromazine itself. It appears that although in vitro chlorpromazine is capable of reducing pulmonary paraquat content, in uiro it did not show protection against paraquat toxicity but indeed potentiated it. This potentiation was correlated with reduced urinary paraquat excretion, and increased pulmonary paraquat concentrations. ACKNOWLEDGMENTS The authors wish Adrienne Dishman assistance.
to thank Lillian C. Acosta and for their excellent technical
REFERENCES BICKEL, M. H. (1975). Binding of chlorpromazine and imipramine to red cells, albumin, lipoproteins and other blood components. .I. Pharm. Pharmacol. 27, 733-738. DREW, R., SIDDIK, Z. H., AND GRAM, T. E. (1979) The uptake and efflux of “‘C-paraquat by rat lung slices: The effect of imipramine and other drugs. Toxicol. AppI. Phdrmacol. 49,473-478. DI FRANCESCO, C., AND BICKEL, M. H. (1977). Membrane lipids as intracellular binders of chlorpromazine and related drugs. Chem.-Biol. Inter. 16,335-346. GORDON, M. (1967). Phenothiazines. In Psychopharmacological Agents (M. Gordon, ed.), Vol. 2. pp. l-198. Academic Press, New York. HUUNAN-SEPPALX, A. (1972). Binding of propranolol and chlorpromazine by mitochondrial membranes. Acta Chem. Stand. 26, 2712-2733. JARVIK, M. E. (1970). Drugs used in the treatment of psychiatric disorders. In The Pharmacological Basis of Therapeutics (L. S. Goodman and A. Gilman, eds.), 4th ed., pp. 151-203. Macmillan, New York. JUNOD, A. F. (1975). Mechanisms of drug accumulation in the lung. In Lung Metabolism (A. F. Junod and R. de Haller, eds.), pp. 219-227. Academic Press, New York.
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GRAM
LAHRICHI, M., HOUPERT, Y., TARALLO, P., LOPPINET, V., AND SIEST, G. (1977). Protein and enzyme release from human leukocytes: Influence of phenothiazine derivatives. Chetn-Biol. Inter. 19, 173183. LOCK, E. A., AND ISHMAEL, J. (1978). The effects of paraquat and diquat on rat kidney. Toxicol. Appl. Pharmacol. 45, 227. LOCK, E. A., SMITH, L. L., AND ROSE, M. S. (1976). Inhibition of paraquat accumulation in rat lung slices by a component of rat plasma and a variety of drugs and endogenous amines. Biochem. Pharmacoi. 25, 1769-l 772, MALING, H. M., SAUL, W., WILLIAMS, M. A., BROWN, E. A. B., AND GILLETTE, J. R. (1978). Reduced body clearance as the major mechanism of the potentiation by a-adrenergic agonists of paraquat lethality in rats. Toxicol. Appl. Pharmacol. 43, 57-72. MINDER, R., SCHNETZER, F., AND BICKEL, M. H. (1971). Hepatic and extrahepatic metabolism of the psychotropic drugs chlorpromazine, imipramine, and imipramine-N-oxide. Naunyn-Schmiedebergs Arch. Pharmakol. 268, 334-347. ROSE, M. S., SMITH, L. L., AND WYATT, I. (1974). Evidence for energy-dependent accumulation of 1 paraquat into rat lung. Nature (London) 252, / 314-315. ROSE, M. S., LOCK, E. A., SMITH, L. L., AND WYATT,, I. (1976). Paraquat accumulation: Tissue and species / specificity. Biochem. Pharmacol. 25, 419423. SALZMAN, N. P., AND BRODIE, B. B. (1956). Physiological disposition and fate of chlorpromazine and a, method for its estimation in biological material.’ J. Pharmacol. Exp. Ther. 118, 46-54. SHARP, C. W., OTTOLENGHI, A., AND POSNER, H. S. (1972). Correlation of paraquat toxicity with tissue concentration and weight loss of the rat. Toxicol. Appl. Pharmacol. 22, 241-25 1. SMITH, L. L., LOCK, E. A., AND ROSE, M. S. (1976). The relationship between S-hydroxytryptamine and paraquat accumulation into rat lung. Biochem. Pharmacol. 25, 2485-2487. SMITH, L. L., WYATT, I., AND ROSE, M. S. (1978). Factors effecting the efflux of paraquat from the lung. Toxicol. Appl. Pharmacol. 45, 301-302. SMITH, P., AND HEATH, D. (1976). Paraquat. CRC’ I Crif Reu. Toxicoi. 4, 4 f I-445. SOROKIN, S. P. (1970). The cells of the lungs. In Morphology of Experimental Respiratory Carcinogenesis (P. Nettesheim, M. G. Hanna, Jr. and J. W. Deatherage, Jr., eds.), pp, 3-41. U. S. Atomic’ Energy Commission, Division of Technical Information, Springfield, Va.
AND
APPLIED
PHARMACOLOGY
50,
443-450
(1979)
The Effect of Chlorpromazine on the Uptake and Efflux of Paraquat in Rat Lung Slices’ ZAHID H. SIDDIK, Laboratory
of Toxicology,
ROGER DREW, AND THEODORE E. GRAM
National Cancer Institute, National Bethesda, Maryland 20014
Received February The Effect of Chlorpromazine SIDDIK,
Z. H., DREW,
R.,
AND
Institutes of Health,
15, 1979; accepted May 18, 1979
on the Uptake and Efflux of Paraquat in Rat Lung Slices. GRAM,
T. E. (1979).
Toxicol.
Appl.
Pharmacol.
50,
443-450.
The uptake of paraquat by rat lung slices was inhibited by chlorpromazine in a concentration- and time-dependent manner. In addition, the efflux of paraquat from lung slices was enhanced by chlorpromazine in a similar fashion. These in citro findings suggested that chlorpromazine might be useful in ciao in reducing pulmonary paraquat content and, in turn, its pneumotoxicity. However, chlorpromazine potentiated the lethal toxicity of paraquat rather than ameliorating it. This potentiation by chlorpromazine was found to correlate with a reduction in the urinary excretion of paraquat and concomitant increase in pulmonary paraquat concentrations.
There is a growing concern over the continued widespread use of paraquat since the herbicide, when ingested, is known to cause fatalities in both man and animals (Smith and Heath, 1976). While paraquat produces toxic alterations in liver, kidney, and brain, death is more commonly the result of degenerative pulmonary changes, including interstitial and intraalveolar fibrosis, terminating in pulmonary insufficiency. Despite extensive research on the pathogenesis and biochemistry of the paraquat pulmonary lesion, an “antidote” for the herbicide is still not known. Clinical treatment of human cases is usually ineffective and consists essentially of removing the unabsorbed (by gastric lavage) and absorbed (by hemodialysis and forced diuresis) paraquat as rapidly as possible, followed by attempts at suppression ’ Presented in part at the joint meeting of the American Society for Pharmacology and Experimental Therapeutics and the Society of Toxicology, University of Houston, Tex., August 13-17, 1978.
of fibroblastic proliferation with corticosteroids or immunosuppressive drugs (Smith and Heath, 1976). Paraquat is accumulated into the lung by an energy-dependent uptake process (Rose et al., 1974) which appears to be specific to the lung, being absent in kidney and liver (Rose et al., 1976). However, it appears that the factor responsible for the lung toxicity is not the uptake of paraquat but rather the slow rate of efflux from the lung (Smith et al., 1978). Thus, if paraquat efflux from lung could be enhanced, then its toxicity might be reduced. Recent work in our laboratory examined the ability of a number of endogenous and exogenous compounds to inhibit uptake or to enhance efflux or paraquat in rat lung slices (Drew et al., 1979). Of the compounds investigated chlorpromazine was selected for further studies since this compound not only inhibited uptake but enhanced efflux of paraquat in lung slices. In the present study, the effect of chlorpromazine
443 All
0041-008X/79/120443-08$02.00/0 Copyright 0 1979 by Academic Press, Inc. rights of reproduction in any form reserved. Printed in Great Britain
444
SIDDIK,
DREW,
on paraquat accumulation and efflux was further examined and the possible amelioration of paraquat toxicity with chlorpromazine was investigated. METHODS Animals. Male Sprague-Dawley rats (200-250 g; Taconic Farms, Germantown, N.Y.) which had been allowed food and water ad Iibitum were used in all experiments. Lung slice studies. The methods used to prepare lung slices (20-50 mg) and study uptake and efflux of [Wlparaquat were described previously (Drew et al., 1979). Briefly, rats were anesthetized with pentobarbital (60 mg kg-‘, ip), lungs removed, and slices cut free hand. Lung slices were equilibrated for 20min at 37°C in Krebs-Ringer bicarbonate buffer (pH 7.4) supplemented with 5 mM glucose (KRB) and gassed with O2 : CO2 (95 : 5) before being transferred to the same medium (100 ml) containing paraquat or paraquat and chlorpromazine. Slices were removed at appropriate times, briefly rinsed, gently blotted, solubilized, and the radioactivity was determined by liquid scintillation spectrometry. For efflux studies, lung slices that were preincubated for 2 hr in 10 ,UM [Wlparaquat (100 ml) were briefly rinsed and transferred immediately to fresh KRB (100 ml) containing varying amounts of chlorpromazine. Slices were removed at appropriate times and their 14C content determined as above. The change in paraquat concentration in the medium during uptake study was negligible, and the medium paraquat concentration during efflux did not exceed 0.05 nmol ml-l. Paraquat toxicity. In order to investigate the effect of chlorpromazine on paraquat-induced lethality, chlorpromazine hydrochloride (25 mg kg-‘, SC) was injected either daily before and/or after oral administration of paraquat dichloride (235 mg kg-‘) or 30 min before intravenous administration of the herbicide (23 mg kg-‘). Saline (40 ml kg-r, po) was administered to some animals immediately before paraquat. Paraquat excretion and tissue distribution. The effect of chlorpromazine on urinary paraquat excretion was determined by injecting rats with chlorpromazine hydrochloride (25 mg kg-l, SC) 30 min before [“Clparaquat dichloride (23 mg kg-‘, iv) or [Wlparaquat dichloride and saline (40 ml kg-‘, po) and the urine collected for 24 hr and analyzed for radioactivity. At 24 hr the animals were anesthetized with ether, blood (from the severed axillary vessels collected in tubes containing heparin) and tissues were taken for i4C analyses. Radioactivity in aliquots of urine (0.5 1.0 ml), plasma (0.5 ml), and lung, kidney, liver, heart, and mesenchymal fat (lW150 mg) were determined by standard liquid scintillation techniques.
AND
GRAM
Chemicals. [‘%]Paraquat (specific activity 33 mCi mmol-i; Amersham-Searle, Arlington Heights, Ill.) nonradiolabeled paraquat and chlorpromazine (Sigma Chemical Co., St. Louis, MO.) were purchased commercially and used without further purification. The compounds were dissolved in saline for use in all experiments. Statistics. Statistical evaluations were made by using Student’s t test for significance at p < 0.05 level.
RESULTS Effect of chlorpromazine on uptake and eflux of paraquat in lung slices. The pul-
monary uptake of [i4C]paraquat (10 ,MM) was consistently reduced in the presence of chlorpromazine (Table 1). This inhibition was concentration dependent. At 100~~ chlorpromazine, the uptake of paraquat was only 40% of control. In the absence of chlorpromazine, the efflux of paraquat from lung slices was negligible during the 2-hr incubation period (Table 1, Uptake vs Efflux, top row). However, in the presence of chlorpromazine, efflux of paraquat from lung slices occurred and increased with increasing concentration of chlorpromazine. At 50 and 500 ,LLM chlorpromazine the concentration of remaining paraquat in lung slices was, respectively, 60 and 20% of that in slices incubated in absence of chlorpromazine. The temporal aspects of the uptake and efllux of paraquat by lung slices is presented in Fig. 1. The uptake of paraquat was linear with time for 60-90 min in the absence of chlorpromazine; however, in the presence of the inhibitor, the duration of linear paraquat accumulation decreased dramatically. In the presence of 300 ,LLM chlorpromazine, no further uptake of paraquat occurred beyond 60 min of incubation. As pointed out above, efflux of paraquat was observed only when the slices were incubated in media containing chlorpromazine (Fig. I). At 300~~ chlorpromzine, only about 30% of the initial paraquat remained in the lung slices after 2 hr. Lung slices were incubated for 30 min in varying concentrations of paraquat and the rate of uptake determined. The rate of
UPTAKE/EFFLUX
OF PARAQUAT
TABLE EFFECT OF CHLORPROMAZINE [W]PARAQUAT
1
ON THE UPTAKE AND EFFLUX IN RAT LUNG SLICES’
Chlorpromazine concentration (PM)
nmol g-lb
0 10 50 100 300 500
64.Ok4.4 51.0* 3.0 39.4 -+ 2.74 25.4+ 1.5” 19.9+0.54 14.2 +_0.9d
445
IN RAT LUNG
OF
Efflux
Uptake o/o’ 100 89.1 61.6 39.7 31.1 22.2
nmol g-lb 58.8 + 6.9 50.7 + 5.3 35.6* 5.64 38.9+ 1.3d 16.9* l.ld 11.5*0.76
%” 100 86.2 60.5 66.2 28.7 19.6
a Uptake was determined using 10 PM paraquat. Efflux studies were conducted using slices that had been incubated for 120 min at 37°C in KRB solution containing 10 PM paraquat only, and then transferred to fresh media containing the appropriate amounts of chlorpromazine. ’ Paraquat content (+ SE) of six lung slices incubated under conditions of uptake or efflux for 120 min. c Percentage of paraquat content of lung slices incubated with no chlorpromazine. d Significantly different from lung slices with no chlorpromazine (p < 0.05).
Uptake
EfflUX 1
d
I
1
uptake of paraquat was proportional to its concentration, in the range of 5-50 PM (Fig. 2). However, in the presence of 100 or 300 pM chlorpromazine, uptake was reduced but still proportional to the paraquat concentration. The extent of inhibition produced by 100 or 300 pM chlorpromazine was very similar. Efect of chlorpromazine on paraquat toxicity. Chlorpromazine was injected at 24-hr
0'
I 80
I 120 TIME lminl
I
I 180
I 240
1. The uptake and efflux in rat lung slices of [Wlparaquat in the presence and absence of 100 or 300~1~ chlorpromazine (CPZ). Efflux studies were conducted using slices that had been incubated for 120 min at 37°C in KRB solution containing 10~~ [Ylparaquat only, and then transferred to fresh media containing the appropriate amounts of CPZ. Data points represent the mean+ SE of six slices. An asterisk indicates statistical significance at p <0.05 compared to slices incubated in absence of CPZ. FIG. 10~~
intervals before and/or after oral administration of paraquat to rats. The results (Table 2) show that the rats injected with the indicated doses of either chlorpromazine or paraquat had, respectively, 0 or 38% lethality. However, pretreatment and/or post-treatment with chlorpromazine markedly potentiated the paraquat-induced lethality. This potentiation correlated well with body weight loss which confirmed the previous report by Sharp et al. (1972). The deaths were all due to pulmonary insufficiency as evidenced by preterminal dyspnea and hemorrhagic lungs. When paraquat was given orally a constant plasma concentration of the herbicide was
446
SIDDIK,
DREW,
AND
maintained for at. least 30 hr (Rose et al., 1976) and this may have been a complication in determining the effectiveness of chlorpromazine therapy. That is, at its peak
plasma concentration, chlorpromazine may have inhibited uptake and induced efflux of paraquat but as the concentration of the active phenothiazine drug declined then uptake of paraquat may have resumed. Thus, the possibility of protection was further investigated by studying the effect of subcutaneously injected chlorpromazine and/or orally administered saline on intravenous paraquat toxicity. Chlorpromazine again potentiated paraquat toxicity (Fig. 3) and by Day 4 there were no surviviors in the group given paraquat and chlorpromazine. However, if saline was also administered with paraquat and chlorpromazine, the lethality (70-75%) was about the same as in the group injected with paraquat alone. Saline alone had no effect on paraquat-induced lethality. Again, respiratory failure accounted for the deaths as indicated by dyspnea and grossly hemorrhagic lung seen on autopsy.
o No CPZ A 100 pM CPZ q 300 pM CPZ
PARAQUAT
CONCENTRATION
GRAM
ipM)
FIG. 2. Variation in the rate of uptake of [‘“Clparaquat with concentration in presence and absence of 100 or 300~~ chlorpromazine (CPZ). Slices were incubated for 30 min at 37°C in KRB solution containing the appropriate amounts of the two drugs. Data points represent the mean +_SE of six slices. An asterisk indicates statistical significance at p < 0.05 compared to slices incubated in absence of CPZ.
Effect of chlorpromazine on urinary excretion of paraquat. To account for the poten-
tiation by chlorpromazine of paraquat toxicity, urinary excretion and tissue distribution
TABLE
2
(CPZ) ON PARAQUAT (PQ) TOXICITY
THE EFFECT OF CHLORPROMAZINE
IN RATS
Percentage cumulative deaths Day -1
CPZb
PQ
-to
0 ->o
PQ + CPZ
PQ + CPZ
-0
0 . ..>o
PQ + CPZ -+O
0
+O
1
-+O
0
0
-to
+O
2
-+O
0
0
40
+14
-213
+57
20
3
-+O
0
57
4
0
13
86
25
6
0
13
38
8
0
25
10
0
38
100 100 loo
63 75
?>:o
100 100 100 100
0 Chlorpromazine (25 mg kg-l, SC)was injected at intervals of 24 hr on the indicated days (+). Paraquat (235 mgkg-‘, po) was administered as a single dose on Day 0 (+), and 30 min after the injection of chlorpromazine to rats receiving both drugs on the same day. Each group consisted of seven or eight animals. Ir CPZ = Chlorpromazine; PQ = paraquat.
UPTAKE/EFFLUX
OF PARAQUAT
loor 80
40 0 A 6
20
0
1
2 DAYS
3 AFTER
PQ + Saline PQ+CPZ PO + CPZ + Saline
4 5 PARAQUAT
6
7
FIG. 3. The effect of chlorpromazine (CPZ) and/or saline on paraquat (PQ)-induced lethality in rats. The animals were treated with PQ (23 mg kg-‘, iv) saline (40 ml kg-‘, po) and/or CPZ (25 mg kg-‘, SC) as deserihed in the text and mortality was assessed daily. Each group consisted of 15-22 animals.
0 PQ+Saline A PQ+CPZ A PQ+CPZ+Saline
0’
’
4
8
12 TIME lhr)
18
20
,
-.20
FIG. 4. The effect of chlorpromazine (CPZ) and/or saline on (A) voided urine and (B) urinary paraquat (PQ) excretion in rats. The animals were given [W]PQ (23 mg kg-r, iv), saline (4Ornl kg-r, PO), and/or CPZ (25 mg kg-r, SC) as described in the text and the. urine collected at the indicated time intervals and analyzed for 14C content. Data points represent the meanf SE of five or six animals. An asterisk indicates significance at p ~0.05 compared to rats administered PQ only.
of paraquat was investigated. In animals given both paraquat and chlorpromazine, the 24-hr urinary volume was only 20% that in animals injected with paraquat alone (Fig.
IN
RAT
447
LUNG
4A). Saline increased the rate of urine excretion at the early time intervals, and when administered in conjunction with paraquat and chlorpromazine, the cumulative voided urine volume in 24 hr increased threefold, compared to animals which were not hydrated. However, there was no significant difference in the cumulative urine volume in 24 hr between animals given paraquat or paraquat and saline. Temporally, the voided volume from animals given saline, saline and chlorpromazine, or chlorpromazine was very similar to that seen after administration of paraquat, paraquat and saline, or paraquat, chlorpromazine, and saline, respectively (data not presented). It is noteworthy that the urinary paraquat excretion was markedly lower (-50%) in animals receiving paraquat and chlorpromazine compared with the paraquat group (Fig. 4B). This difference was abolished if saline was also administered to animals injected with paraquat and chlorpromazine (Fig. 4B). Rats treated with paraquat and saline eliminated significantly more paraquat at only the 2- and 4-hr time points than rats injected with paraquat only. Thus, at 24 hr, rats given paraquat, paraquat and saline, or paraquat, chlorpromazine, and saline excreted &l--93% of the paraquat dose with SO-90% being excreted in the first 4 hr. E#ect of chlorpromazine on tissue concentrations of paraquat. Rats injected with
paraquat and chlorpromazine had significantly higher pulmonary (28%) and fat (108%) paraquat concentrations than animals given only paraquat (Fig. 5). In the rats given saline in conjunction with paraquat or paraquat and chlorpromazine, the tissue concentrations were generally comparable with those from rats given only paraquat. However, the cardiac paraquat content in rats given paraquat and saline was significantly, but inexplicably higher than in animals injected with paraquat alone. In all cases the tissue concentrations of paraquat greatly exceeded the plasma concentration with maximal
levels
being
present
in
the lung.
SIDDIK,
DREW,
OPQ n PQ+CPZ a W+Saline q PQ+CPZ+Saline
0-
PLASMA
KIDNEY
LIVER
HEART
FAT
FIG. 5. The effect of chlorpromazine (CPZ) and/or saline on the tissue distribution of paraquat (PQ) in rats. The animals were given [“C]PQ (23 mg kg-‘, iv), saline (40 ml kg-‘, PO), and/or CPZ (25 mg kg-‘, SC) as described in the text, sacrificed 24 hr later, and the tissues analyzed for 14C content. Data points represent mean+ SE of five or six animals. An asterisk indicates significance at p < 0.05 compared to rats administered PQ only.
There was no significant difference in the whole organ wet weights between the four treatment groups (data not presented). DISCUSSION Deaths due to paraquat poisoning are often caused by acute pulmonary edema or chronic fibrosis (Smith and Heath, 1976). The lung appears to be unusually susceptible because of its ability to actively accumulate paraquat (Rose et al., 1974). Although a number of compounds can inhibit this uptake of paraquat in lung slices (Lock et al., 1976; Drew et al., 1979), they do not necessarily enhance its efflux (Drew et al., 1979). In the present study, chlorpromazine not only inhibited accumulation of paraquat by lung slices but also enhanced its efflux. Both inhibition of uptake and enhancement of efflux of paraquat were dependent on the concentration of the phenothiazine. Furthermore, the inhibition of uptake and enhancement of the efflux did not remain constant with time, but increased rapidly after 1 hr of incubation. The reason for this may be that
AND
GRAM
during uptake of paraquat, chlorpromazine may initially compete directly for the paraquat binding sites on the carrier protein, but with time, as the binding of chlorpromazine to membrane increases (Di Francesco and Bickel, 1977), the drug may alter physicochemical properties of the membrane which may further inhibit paraquat uptake. Alterations in membrane properties by chlorpromazine have been reported previously (Gordon, 1967). Similar nonlinear timedependent inhibition of paraquat uptake in lung slices was also produced by imipramine (Drew et al., 1979), histamine, and betazole (Lock et al., 1976). The site of the energy-dependent carriermediated accumulation of paraquat has been proposed to be the type I and type II alveolar epithelial cells (Smith et al., 1976). Chlorpromazine is also accumulated in the lung against a concentration gradient but this process is probably by diffusion and nonspecific binding (Junod, 1975; Di Francesco and Bickel, 1977), and may involve the entire spectrum of pulmonary cell types which number in excess of 40 (Sorokin, 1970). Thus, during paraquat efflux studies, chlorpromazine accumulated in the lungs may initially bind to available sites, and only when these become saturated will competition for sites occupied by paraquat take place and displacement of the herbicide be induced. This may partially explain why efflux of paraquat in presence of chlorpromazine is time dependent and becomes substantial after 1 hr of incubation. Furthermore, since accumulation of chlorpromazine is probably not cell or organelle specific (Di Francesco and Bickel, 1977), a much greater pulmonary level of the phenothiazine than of paraquat would be needed to attain concentrations within the type I and type II cells sufficiently high to displace paraquat. This would be achieved either by a short- or long-term exposure to a high or low concentration of chlorpromazine respectively. Thus, incubation of slices for 2 hr with 10~~ chlorpromazine may not, have been sufficient to cause a significant
UPTAKE/EFFLUX
OF PARAQUAT
decrease in paraquat concentration during efflux studies. Inhibition of pulmonary drug uptake and/ or enhancement of efflux by chlorpromazine has been reported previously for imipramine and propranolol (Junod, 1975). However, the binding sites of chlorpromazine, imipramine, and propranolol are qualitatively the same (Huunan-Seppalti, 1972; Bickel, 1975) and competition for these sites would be expected. Although all three drugs inhibited uptake of paraquat to approximately the same extent, only chlorpromazine showed marked enhancement of efflux, with equimolar concentration of propranolol having no effect on this process (Drew et al., 1979). It is possible that the relative affinity for sites occupied by paraquat may be greater for chlorpromazine than imipramine or propranolol. However, ,other mechanisms such as cytotoxicity ;(Lahrichi et al., 1977), inhibition of ATPase, land alteration in membrane properties (Gordon, 1967) may all contribute toward the iinhibition of accumulation and enhancement iof efflux of paraquat by chlorpromazine. With the regular occurrence of accidental lor intentional paraquat poisoning, it has become increasingly important to identify an effective drug therapy for the poison. A compound could show protection merely by accelerating efflux of paraquat from the lungs. The in t&o interactions between paraquat and chlorpromazine indicated the possibility of using chlorpromazine in ameliorating the lethality due to paraquat. Although chlorpromazine also prevents pulmonary edema due to epinephrine and protects animals from oxygen toxicity (Gordon, 1967), it did not prevent paraquat toxicity. Administration of chlorpromazine (Jarvik, 1970) or paraquat (Lock and Ishmael, 1978) produces diuresis. Thus, voided urine volume in animals given chlorpromazine and saline or paraquat and saline was greater at early time points than in animals receiving saline alone. A combination of paraquat, chlorpromazine, and saline, on the other hand, resulted in lower urinary volume than after
IN
RAT
LUNG
449
coadministration of saline with either paraquat or chlorpromazine. Similarly, urinary volume in rats receiving both paraquat and chlorpromazine was significantly lower than in animals given paraquat or chlorpromazine alone. This indicated that an interaction between paraquat and chlorpromazine resulted in the unexpected decrease in urinary volume, which in turn reduced urinary paraquat excretion in animals receiving both paraquat and chlorpromazine, and thus resulting in increased pulmonary paraquat content. A similar effect of /3-adrenergic agonists on urinary excretion of paraquat also results in an increased herbicide concentration in the lung (Maling et al., 1978). Since Sharp et al. (1972) have reported a direct correlation between lethality and the concentration of paraquat in the lung, it is probable that potentiation of toxicity by chlorpromazine was a direct result of increased paraquat levels in this organ. Administration of saline as a diuretic increased paraquat excretion and reduced lung paraquat content and lethality to values obtained with rats injected with paraquat alone; that is, no beneficial effect of chlorpromazine was seen at the dose used. This dose of chlorpromazine produces a plasma concentration of 4-5 nmol chlorpromazine equivalents ml-’ between 4 and 24 hr, and lung concentrations of 99, 154, 191, and 76 nmol g-l at 0.5, 2, 4, and 24 hr after administration (unpublished observation). Pulmonary concentration of paraquat, on the other hand, remains fairly constant between 1 and 24 hr after its intravenous administration (Sharp et al., 1972). Thus, concentration of chlorpromazine in the lung was much greater than that of paraquat (30-40 nmol g-l ; Fig. 5) at all times up to 24 hr. However, chlorpromazine is highly bound to plasma proteins (Salzman and Brodie, 1956) and undergoes rapid metabolism (Minder et al., 1971), and thus the level of free chlorpromazine in the plasma, and consequently the lung, may not have been sufficient to inhibit uptake and enhance efflux of paraquat. In these experi-
450
SIDDIK,
DREW,
ments, the dose of chlorpromazine used was one which did not cause excessive impairment of food and water intake as a result of its depressant action. Higher doses of chlorpromazine in conjunction with saline were not attempted due to reduced food intake and toxicity due to chlorpromazine itself. It appears that although in vitro chlorpromazine is capable of reducing pulmonary paraquat content, in uiro it did not show protection against paraquat toxicity but indeed potentiated it. This potentiation was correlated with reduced urinary paraquat excretion, and increased pulmonary paraquat concentrations. ACKNOWLEDGMENTS The authors wish Adrienne Dishman assistance.
to thank Lillian C. Acosta and for their excellent technical
REFERENCES BICKEL, M. H. (1975). Binding of chlorpromazine and imipramine to red cells, albumin, lipoproteins and other blood components. .I. Pharm. Pharmacol. 27, 733-738. DREW, R., SIDDIK, Z. H., AND GRAM, T. E. (1979) The uptake and efflux of “‘C-paraquat by rat lung slices: The effect of imipramine and other drugs. Toxicol. AppI. Phdrmacol. 49,473-478. DI FRANCESCO, C., AND BICKEL, M. H. (1977). Membrane lipids as intracellular binders of chlorpromazine and related drugs. Chem.-Biol. Inter. 16,335-346. GORDON, M. (1967). Phenothiazines. In Psychopharmacological Agents (M. Gordon, ed.), Vol. 2. pp. l-198. Academic Press, New York. HUUNAN-SEPPALX, A. (1972). Binding of propranolol and chlorpromazine by mitochondrial membranes. Acta Chem. Stand. 26, 2712-2733. JARVIK, M. E. (1970). Drugs used in the treatment of psychiatric disorders. In The Pharmacological Basis of Therapeutics (L. S. Goodman and A. Gilman, eds.), 4th ed., pp. 151-203. Macmillan, New York. JUNOD, A. F. (1975). Mechanisms of drug accumulation in the lung. In Lung Metabolism (A. F. Junod and R. de Haller, eds.), pp. 219-227. Academic Press, New York.
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LAHRICHI, M., HOUPERT, Y., TARALLO, P., LOPPINET, V., AND SIEST, G. (1977). Protein and enzyme release from human leukocytes: Influence of phenothiazine derivatives. Chetn-Biol. Inter. 19, 173183. LOCK, E. A., AND ISHMAEL, J. (1978). The effects of paraquat and diquat on rat kidney. Toxicol. Appl. Pharmacol. 45, 227. LOCK, E. A., SMITH, L. L., AND ROSE, M. S. (1976). Inhibition of paraquat accumulation in rat lung slices by a component of rat plasma and a variety of drugs and endogenous amines. Biochem. Pharmacoi. 25, 1769-l 772, MALING, H. M., SAUL, W., WILLIAMS, M. A., BROWN, E. A. B., AND GILLETTE, J. R. (1978). Reduced body clearance as the major mechanism of the potentiation by a-adrenergic agonists of paraquat lethality in rats. Toxicol. Appl. Pharmacol. 43, 57-72. MINDER, R., SCHNETZER, F., AND BICKEL, M. H. (1971). Hepatic and extrahepatic metabolism of the psychotropic drugs chlorpromazine, imipramine, and imipramine-N-oxide. Naunyn-Schmiedebergs Arch. Pharmakol. 268, 334-347. ROSE, M. S., SMITH, L. L., AND WYATT, I. (1974). Evidence for energy-dependent accumulation of 1 paraquat into rat lung. Nature (London) 252, / 314-315. ROSE, M. S., LOCK, E. A., SMITH, L. L., AND WYATT,, I. (1976). Paraquat accumulation: Tissue and species / specificity. Biochem. Pharmacol. 25, 419423. SALZMAN, N. P., AND BRODIE, B. B. (1956). Physiological disposition and fate of chlorpromazine and a, method for its estimation in biological material.’ J. Pharmacol. Exp. Ther. 118, 46-54. SHARP, C. W., OTTOLENGHI, A., AND POSNER, H. S. (1972). Correlation of paraquat toxicity with tissue concentration and weight loss of the rat. Toxicol. Appl. Pharmacol. 22, 241-25 1. SMITH, L. L., LOCK, E. A., AND ROSE, M. S. (1976). The relationship between S-hydroxytryptamine and paraquat accumulation into rat lung. Biochem. Pharmacol. 25, 2485-2487. SMITH, L. L., WYATT, I., AND ROSE, M. S. (1978). Factors effecting the efflux of paraquat from the lung. Toxicol. Appl. Pharmacol. 45, 301-302. SMITH, P., AND HEATH, D. (1976). Paraquat. CRC’ I Crif Reu. Toxicoi. 4, 4 f I-445. SOROKIN, S. P. (1970). The cells of the lungs. In Morphology of Experimental Respiratory Carcinogenesis (P. Nettesheim, M. G. Hanna, Jr. and J. W. Deatherage, Jr., eds.), pp, 3-41. U. S. Atomic’ Energy Commission, Division of Technical Information, Springfield, Va.