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Postnatal toxicity of chronically administered paraquat in mice and interactions with oxygen and bromobenzene
TOXICOLOGY
Postnatal
AND
APPLIED
PHARMACOLOGY
33,461-470
(1975)
Toxicity of Chronically Administered Paraquat and Interactions With Oxygen and Bromobenzene
In Mice
JAMESS. Bus ANDJAMESE. GIBSON Department
of Pharmacology, Michigan State University, East Lansing, Michigan 48824 Received December 27,1974; accepted March 26, 1975
Postnatal Toxicity of Chronically Administered Paraquat in Mice and Interactions with Oxygen and Bromobenzene.Bus, J. S. AND GIBSON, J. E. (1975). Toxicol. Appl. Pharmacol. 33, 461-470. The purposeof this study wasto investigatethe effect of chronic paraquat administrationon developing mice and to examinethe interaction of chronic paraquatexposurewith 100% oxygen and the hepatotoxin bromobenzene.Paraquat was administeredat 50 and 100ppm in the drinking water to pregnantmice beginning at Day 8 of gestation,with continued exposureto the newbornsup to 42 days after birth. Neither paraquat treatment altered the postnatal growth rate; however, 100ppm but not 50 ppm paraquat significantly increased the postnatal mortality. Both 50- and lOO-ppmparaquat-treated mice weresensitizedto the onsetof oxygen toxicity, determinedby a significant reduction in the LT50 at 42 days after birth. An enhancedsensitivity to oxygen toxicity was also detectablein 100 ppm but not 50 ppm mice at Days 1 and 28 after birth. In 42-day-old mice, 50 and 100ppm paraquat treatment also significantly reducedthe LT50 after 3100mg/kg ip (LD85) bromobenzene.These observationssuggestthat the toxicity of paraquat may be mediatedthrough an interaction with oxygen and describepossible interactionsthat could occur with the environmentaluseof paraquat.
The herbicide paraquat (1,l ‘-dimethyl-4,4’-bipyridylium), which can be highly toxic to adult man (Bullivant, 1966) and animals (Clark et al., 1966; Murray and Gibson, 1971), induces only minor alterations in prenatal development of rats (Khera and Whitta, 1968)and mice (Bus et al., 1975a)in the absenceof maternal toxicity. The effect of paraquat on postnatal development, however, hasnot been investigated. The mechanism of paraquat toxicity in adult animals has been proposed to be mediated through an interaction of paraquat with oxygen which induces subsequent lipid peroxidation damage of cellular membranes(Bus et al., 1974). This investigation reports the effect of chronic administration of paraquat on the postnatal development of mice and the sensitivity of these animals to an oxygen environment. The interaction of chronic paraquat treatment with bromobenzene, a hepatotoxin which requires microsomal metabolic activation for expression of toxicity (Brodie et al., 1971) was also examined. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
461
462
BUS AND GIBSON MATERIALS
AND
METHODS
Developmental Studies
Virgin Swiss-Webster mice1 were mated by placing one male in a cage of five females for 1 hr starting at 8 AM. The day vaginal plugs were found was designated Day 1 of gestation. Paraquat dichloride2 was placed in the drinking water of pregnant females at concentrations of 0, 50, 100, and 150 ppm beginning on Day 8 of gestation, which is the onset of organogenesis in mice. Pregnant females were housed individually in clear plastic shoe box cages and allowed access to food and the paraquat treated water ad libitum. Following delivery of pups on Day 20 of gestation, litters were normalized to 10 mice. Total litter body weights and mortality were recorded at weekly intervals from Day 1 postnatally to termination of the experiment at 42 days postnatally. All litters were weaned on Day 28 postnatally and segregated by sex. Exposure of mice to paraquat-treated water was continuous from Day 8 of gestation to 42 days postnatally. In other experiments, litters were either exposed to 100 ppm paraquat from Day 8 of gestation to 28 days after birth (weaning) and then transferred to nonparaquat-treated water until 42 days postnatally or placed on 100 ppm paraquat only between Days 28 and 42 postnatally. Litter mortality rates were recorded at weekly intervals for each treatment group. The stability of the paraquat solutions was confirmed by calorimetric assay (Sharp et al., 1972), with no change in paraquat concentrations for up to 4 weeks after preparation. Histopathology
At 42 days, control, 50, and 100 ppm paraquat-treated mice were sacrificed and lung, liver, and kidneys fixed in 10% formalin. Five-micrometer paraffin sections were prepared, stained with hematoxylin-eosin, and examined by light microscopy. Interaction with Oxygen
Developing mice exposed to 0, 50, and 100 ppm paraquat beginning at Day 8 of gestation were examined for sensitivity to 100% oxygen at 1 atm on Days 1, 28, and 42 postnatally. Oxygen exposure was accomplished by placing both control and treated mice in a clear plastic cage approximately 12 liters in volume (12 x 24 x 44 cm) with a Plexiglas top and supplied with animal bedding, food, and tap water. The l-dayold group was normalized to 10 newborns per mother and the mothers replaced after 48 hr of oxygen exposure withmothers which had recently given birth to ensureadequate nursing of the newborns. Oxygen, ( 100°~), which was humidified by flow over a water source prior to entry into the chamber, was maintained at a flow rate of 1.65 liters/min into the chamber. Oxygen concentrations in the chamber were measured to be 95-100 %. The sensitivity of the 28- and 42-day-old treatment groups to 100% oxygen was measured by determination of the LT50 (median time to death) after initiation of oxygen treatment (Litchfield, 1949). The interaction of oxygen with newborn mice was determined by recording the number of mice dead after 120 hr of oxygen exposure. 1 Spartan Research Animals, Inc., Haslett, Michigan. z Paraquat concentrate (240 mg cation/ml), Chevron Chemical Co, Richmond, California.
PARAQUAT
POSTNATAL
TOXICITY
463
Lung concentrations of paraquat in 42-day-old 50- and lOO-ppm paraquat-treated mice were determined by a calorimetric modification of the methods of Ilett ef al. (1974) and Sharp et al. (1972). Interaction
with Bromobenzene
The toxicity of bromobenzene3 in 42-day-old 50- and lOO-ppm paraquat-treated mice was measured by determination of the LT50. Bromobenzene, dissolved in peanut oil, was administered ip at 3100 mg/kg (LD85 in Swiss-Webster mice, determined in this laboratory) and the time to death recorded. Liver reduced glutathione (GSH) was measured in other 42-day-old 50- and lOO-ppm mice after homogenization (Polytron@, Brinkman Instruments, Westbury, New York) of individual whole lungs in 4.0 ml cold 30 PM EDTA and subsequent fluorometric assay of GSH (Cohn and Lyle, 1966). Statistics
Analyses of data was performed by Student’s t test and analysis of variance, completely randomized design. The level of significance was chosen as p < 0.05. RESULTS
Paraquat and Development
Paraquat, when placed in the drinking water of pregnant mice and continued at 50 and 100 ppm from Day 8 of gestation to 42 days postnatally, did not alter the average litter body weight compared to controls at any time during the experiment (Fig. 1). Pregnant mice receiving 150 ppm paraquat starting at Day 8 of gestation died before delivery of newborns occurred (day of death was usually by Day 16 of gestation). Water consumption was not altered by these concentrations of paraquat. One hundred parts per million paraquat significantly increased postnatal mortality compared to 50 ppm paraquat and controls from Day 7 after birth, when mortality was 33.3x, up to a 42-day mortality of 66.7% (Fig. 2). Mortality among the 50-ppm paraquat group and controls was not significantly different and remained below 7% over the course of the experiment. Mortality induced by 100 ppm paraquat appeared biphasic, with an initial rapid increase to 33.3 % during the first 7 days after birth then reached a plateau up to Day 21 when mortality was 36.7%. The plateau phase was followed by a second increase prior to weaning on Day 28, reaching a final mortality of 66.7 oA on Day 35. Paraquat treatment did not affect the number of live fetuses born compared to controls. Experiments in which developing mice were exposed to 100 ppm paraquat from Day 8 of gestation to 28 days postnatally and then transferred to tap water until 42 days after birth resulted in a mortality of 26.7 &- 12.0% by Day 28. This mortality level was not significantly different from the 28-day mortality of mice continuously exposed to 100 ppm paraquat throughout development. Furthermore, transfer to tap water resulted in a 42-day mortality that was unchanged from Day 28 and thus eliminated the second rapid increase in lOO-ppm mortality depicted in Fig. 2. Exposure of developing mice to 100 ppm paraquat only from Days 28 to 42 postnatally resulted in 3 Aldrich Chemical Co., Inc., Milwaukee,
Wisconsin.
BUS AND GIBSON
1
1
7
14 DAYS
21 AFTER
28 BIRTH
35
42
FIG. 1. Effect of 50- and lOO-ppm paraquat in the drinking water from Day 8 of gestation to 42 days after birth on the postnatal growth weight of mice. Each point is the mean mouse body weight within litters for three to four litters. There were 10 mice/litter on Day 1.
o-0-0-0 1
7
14 DAYS
21 2s AFTER BIRTH
25
42
FIG. 2. Effect of 50- and lOO-ppm paraquat in the drinking water from Day 8 of gestation to 42 days after birth on the postnatal mortality rate of mice. Each point is the mean percent mortality within litters for three to four litters. There were 10 mice/litter on Day 1.
46.7&14.5x mortality, which was not significantly different from the 30% increase in mortality observed in the second lOO-ppm mortality phaseof Fig. 2. Histopathology Lung sections from 42-day-old lOO-ppm mice showed extensive alveolar consolidation and collapse, and areasof thickening of intraalveolar septa. Edema fluid was seen
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POSTNATAL
465
TOXICITY
in a few alveoli. Lung sections from 50-ppm and control mice did not show any significant pathological changes. Examination of liver and kidney sections of all treatment groups also did not reveal any significant pathological changes. Paraquat Interaction
with Oxygen
Paraquat significantly enhanced the sensitivity to oxygen toxicity of 42-day-old mice which received 50 and 100 ppm paraquat throughout development (Table 1). The LT50 for 50- and lOO-ppm paraquat-treated mice was 108 and 40 hr, respectively. compared to the control LT50 of 160 hr. The LT50 curves for both treatment groups were parallel to each other and controls, with calculated potency ratios of 1.5 for 50 ppm versus controls and 4.0 for 100 ppm versus controls. The concentration of paraquat in lung tissue of the 100-ppm mice was less than 0.2 ,ug/g lung tissue (wet weight) and was below detectable levels in 50-ppm lung tissue. At 28 days after birth, an increased sensitivity to 100% oxygen exposure was detected only in lOO-ppm paraquat-treated mice (Table 1). The LT50 of lOO-ppm mice was significantly reduced to 121 hr, compared to an LT50 of 180 hr for 50-ppm mice and 181 hr for control mice. A potency ratio of 1.5 was calculated for lOO-ppm mice versus controls. TABLE 1 EFFECT OF PARAQUAT IN THE WATER FROM DAY 8 OF GESTATION TO VARIOUS DAYS POSTNATALLY ON THE SURVIVAL OF MICE EXPOSED TO 100 % OXYGEN 95%
Treatment Control 50 mm 100ppm Control 5Owm
100ppm
Begin0, exposure(day)
LT50 (hr)
28 28 28
181 180 121 160 108
42 42 42
40
confidence limits -___
Potency ratio
..-
(156-210)
-
(142-229) (108-135)
1.0 1.5”
(126-203) (18-144) (3054)
1.5" 4.0"
-
’ Significantlydifferentfrom respectivecontrol,p < 0.05. One-day-old mice, which were exposed to paraquat only by prenatal placental transfer, also were sensitized to oxygen toxicity (Fig. 3). Mortality of l-day-old mice whose mothers received 100 ppm paraquat was significantly increased to 53.5% after 120 hr oxygen exposure compared to 25.8 and 24.2% mortality in 50-ppm and control mice, respectively. Paraquat Interaction with Bromobenzene The toxicity of bromobenzene, as measuredby the LT50, was determined in 42-dayold 50- and lOO-ppm paraquat-treated mice (Table 2). The LT50 for bromobenzene in control mice was 20.0 hr, in 50-ppm mice 4.2 hr, and 3.2 hr in lOO-ppm paraquattreated mice. The LTSOsof the 50- and lOO-ppmmice were significantly different from controls, but no different from each other. Liver GSH was assayedin 42-day-old 50- and lOO-ppmmice (Table 3). No significant differences were observed in the liver GSH of these mice compared to controls.
466
BUS AND
GIBSON
FIG. 3. Effect of 50- and lOO-ppm paraquat in the drinking water of pregnant mice from Day 8 of gestation to Day 19 of gestation on the survival of 1-day-old mice exposed to 100% oxygen for 120 hr. Each treatment represents the mean percent mortality for three to four litters with 10 mice/litter on Day 1. *Indicates significantly different from control and 50-ppm paraquat, p c 0.05. TABLE 2 EFFECT OF PARAQUAT IN THE WATER FROM DAY 8 OF GESTATION TO 42 DAYS POSTNATALLY ON THE SURVIVAL OF MICE TREATED WITH BROMOBENZENE, 3100 mg/kg ip (LD85)
Treatment
LT50 W-1
95% confidence limits
20.0 (12.631.6) 4.2 (2.6- 6.7) 50 pm 3.2 (1.9- 5.4) 100 ppm a Significantly different from control, p < 0.05.
Control
Potency ratio 4.8” 6.2*
TABLE 3 LIVER GSH CONCENTRATIONS IN MICE GIVEN PARAQUAT IN THE WATER FROM DAY 8 OF GESTATION ~0 42 DAYS POSTNATALLY~ Treatment
Liver GSH (mg GSH/g wet wt + SE)
Control 50 mm 100 mm
5.08 f 0.31 4.80 + 0.30 5.14 + 0.63
a Each treatment represents the mean k SE of three to six determinations. DISCUSSION
Chronic administration of paraquat at 50 and 100ppm in the drinking water did not alter the development of mice as reflected in the average body weight. Treated mice reaching 42 days of age not only weighed the sameas controls, but also did not exhibit
PARAQUAT
POSTNATAL
TOXICITY
467
any gross malformations. In contrast to the lack of any paraquat effect on growth rate, 100 ppm paraquat increased postnatal mortality in an apparent biphasic pattern. The first increase in lOO-ppm mortality occurring between Days 1 and 7 after birth may have been induced by the presence in the newborns of paraquat which remained from prenatal placental transfer of the herbicide to the fetuses, rather than from paraquat passage to the newborns through the mother’s milk. Bus et al. (1975a) have shown that paraquat administered to pregnant rats during the final 2 days of gestation was detectable in the newborns for up to 7 days postnatally. Furthermore, the 100-ppm mortality reached a plateau between 7 and 21 days, despite continued nursing by mothers exposed to paraquat in their drinking water. The possibility that paraquat ingested from the mother’s milk may have been responsible for the initial mortality appears unlikely as paraquat is poorly absorbed orally (Murray and Gibson, 1974) and thus only minute amounts of paraquat would be expected to be systemically absorbed in the newborns. The immediate postnatal increase in mortality of the IOO-ppm newborns may be linked to the initiation of breathing and exposure of lungs to higher oxygen tensions encountered after birth as 100 ppm paraquat did not cause any fetal mortality. Increased oxygen tensions increased paraquat toxicity in adult rats (Fisher et al., 1973) and oxygen has been linked to the mechanism of action of paraquat toxicity (Bus et al., 1974). Also, developing mice exposed to paraquat in the water were more sensitive than controls to oxygen toxicity which was detectable as early as the first day after birth. The second mortality increase in lOO-ppm paraquat-treated mice which began during the week prior to weaning on Day 28, may have been caused by the young animals beginning to ingest paraquat from the drinking water. When other paraquattreated mice were transferred to tap water at weaning, the second increase in mortality was eliminated. Furthermore, developing mice exposed to 100 ppm paraquat only between Days 28 and 42 postnatally yielded an increase in mortality that was not significantly different from the increase occurring between Days 21 and 42 in the continuously treated mice. Histologic examination of tissue from lung, liver, and kidney from 42-day-old 50and 100-ppm paraquat-treated mice revealed extensive lung alterations in the IOO-ppm treated group, despite low paraquat lung concentrations in these mice. The pulmonary lesions observed with chronic lOO-ppm paraquat treatment resembled the pulmonary alveolar congestion and collapse seen in animals and man after acute paraquat intoxication (Toner et al., 1970; Murray and Gibson, 1972). Similar pulmonary lesions were not observed in mice exposed to 50 ppm paraquat, the treatment in which mortality was not significantly elevated compared to controls. The lung appeared to be the site of chronic paraquat toxicity, as liver and kidney tissue sections were without significant pathological alterations. Fisher et al. (1973) reported that adult rats were sensitized to the development of oxygen toxicity after acute treatment with paraquat. The 42-day-old 50- and lOO-ppm chronically paraquat-treated mice in this study also were sensitized to lOOad oxygen exposure, as reflected by the significant reduction in LT50 values. The 50-ppm mice were more sensitive to oxygen toxicity than controls, even in the absence of any alteration in postnatal growth or mortality rate. Furthermore, the 42-day-old 50-ppm mice did not have any evidence of histopathological lesions and had no detectable amounts
468
BUS AND
GIBSON
of paraquat in lung tissue before being placed in 100% oxygen. Thus, the 42-day-old 50-ppm paraquat-treated mice were sensitized to oxygen despite being morphologically indistinguishable from controls. The 50-ppm paraquat-treated mice appeared to develop a sensitivity to oxygen toxicity between Days 28 and 42 postnatally, as the oxygen LT50 of 28-day-old 50-ppm mice was not altered from controls. One-day-old mice which received 50 ppm during gestation also were not sensitized to oxygen toxicity. In the IOO-ppm paraquat-exposed group, however, sensitivity to oxygen toxicity had developed by the first day after birth, even though these newborns could have only been exposed to paraquat prenatally. In 42-day-old mice chronically treated with 50 or 100 ppm paraquat, bromobenzene toxicity was significantly enhanced. Bromobenzene is metabolized to an hepatotoxic epoxide by microsomal enzymes (Brodie et al., 1971) and subsequently detoxified by either conjugation with GSH, nonenzymatic rearrangement to phenols, or enzymatic conversion to dihydrodiols by epoxide hydrase (Jollow et al., 1974). In acute studies in rats, bromobenzene toxicity was significantly enhanced by pretreatment with GSHdepleting agents due to decreased detoxification of the bromobenzene epoxide (Jollow et al., 1974). Although liver GSH concentrations in the chronically paraquat-treated mice were not altered compared to controls, it is possible that paraquat treatment affected the capability of mice to replenish GSH stores which are depleted by bromobenzene detoxification. Thus, less GSH would be available for epoxide conjugation with the result of increased bromobenzene toxicity. Evidence that paraquat may catalyze membrane lipid peroxidation (Bus et al., 1974; Bus et al., 1975b) suggests a second mechanism whereby paraquat treatment could decrease the bromobenzene LT50. Paraquat has been postulated to induce lipid peroxidation in vivo by undergoing cyclic oxidation-reduction with subsequent generation of superoxide anions. Superoxide anions may nonenzymatically dismutate to singlet oxygen which attack polyunsaturated lipids of cell membranes to form lipid hydroperoxides. Formation of lipid hydroperoxides initiates the chain reaction process of lipid peroxidation (Bus et al., 1975b). Several mechanisms function in mammalian systems to prevent the onset of lipid peroxidation, one of which is the enzymatic reduction of lipid hydroperoxides to stable lipid alcohols by GSH peroxidase with GSH providing the reducing equivalents (Chow and Tappel, 1974). The availability of GSH appears necessary to combat paraquat toxicity, as pretreatment of mice with the GSH-depleting agent diethylmaleate significantly enhanced acute paraquat toxicity (Bus et al., 1975b). Large doses of bromobenzene, like diethylmaleate, also decrease liver GSH for up to 20 hr in rats due to conjugation of the bromobenzene epoxide with GSH (Jollow et al., 1974). Thus, bromobenzene, by depleting GSH, may remove the activity of GSH peroxidase in protecting against paraquat-induced lipid peroxidation. Consequently, the decreased bromobenzene LT50 in paraquat-treated mice may be due to the added expression of paraquat toxicity in conjunction with bromobenzene toxicity. The possibility also exists that activation of bromobenzene to the epoxide may be affected by chronic paraquat treatment. Paraquat pretreatment in rats has been reported to decrease the in vitro metabolism of bromobenzene along with a destruction of microsomal cytochrome P-450 which functions to activate bromobenzene (Ilett et al., 1974). Lipid peroxidative damage has been implicated in the degradation of cytochrome
PARAQUAT
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TOXICITY
469
P-450 (Levin et aZ., 1973) with subsequent impairment of the metabolism of pentobarbital and acetanilide (Jacobson ef al., 1973). Alternatively, paraquat may disrupt the microsomal electron transport system (Ilett et al., 1974; Krieger et al., 1974) and thereby decrease bromobenzene metabolism. The enhancement of bromobenzene toxicity observed in our studies, however, may indicate that a possible inhibition of bromobenzene metabolism by chronic paraquat treatment was not sufficient to prevent the formation of epoxide and subsequent reduction in tissue GSH. The chronic administration of paraquat to developing animals has been shown to have expressed toxicity in the form of increased mortality and the development of pulmonary lesions in the lOO-ppm paraquat-treated mice. Moreover, susceptibility to the toxic agents oxygen and bromobenzene was detectable in 50-ppm exposed mice which showed no signs of toxicity from paraquat alone. These observations suggest possible toxic effects and interactions that might occur with the environmental use of paraquat. ACKNOWLEDGMENTS This study wassupportedby NIH Grant ES 00560,NIH Training Grant GM 01761,and by the Michigan Lung Association. REFERENCES BRODIE,B. B., REID, W., CHO,A. K., SIPES,G., KRISHNA,G. ANDGILLETTE,J. R. (1971). Possiblemechanismof liver necrosiscausedby aromatic organic compounds.Prac. Nut. Aead. Sci. Wash. 68, 160-164. BULLIVANT,C. M. (1966). Accidental poisoningby paraquat: Report of two casesin man. Brit. Med. J. 1, 1272-1273. Bus, J. S., AUST,S. D. ANDGIBSON,J. E. (1974). Superoxide-and singlet-oxygen-catalyzed lipid peroxidation asa possiblemechanismfor paraquat(methyl viologen)toxicity. Biochem. Biophys. Res. Commun. 58,749-755. Bus, J. S., PREACHE, M. M., CAGEN,S. Z., POSNER, H. S., ELIASON,B. C., SHARP,C. W. AND
GIBSON,J. E. (1975a).Fetal toxicity, teratogenicity and distribution of paraquat and diquat in miceand rats. Toxical. Appl. Pharmacol. 33,446-456. Bus, J. S., CAGEN,S. Z., ACJST, S.D. ANDGIBSON,J. E. (1975b).Lipid peroxidation: A possible mechanismfor paraquat toxicity. Toxicol. Appl. Pharmacol. in press. CHOW,C. K. ANDTAPPEL,A. L. (1974).Response of glutathioneperoxidaseto dietary selenium in rats. J. Nutr. 104, 444-451. CLARK,D. G., MCELLIGOTT,T. F. ANDHURST,E. W. (1966).The toxicity of paraquat. &it. f. Ind. Med. 23, 126-132.
COHN,V. H. ANDLYLE,J. (1966). A fluorometric assayfor glutathione. Anal. Biochem. 14, 434-440.
FISHER,H. K., CLEMENTS, J. A. ANDWRIGHT,R. R. (1973).Enhancementof oxygen toxicity by the herbicideparaquat. Ann. Rev. Resp. Dis. 107,246-252. ILETT,K. F., STRIPP,B., MENARD,R. H., REID,W. D. ANDGILLETTE,J. R. (1974).Studieson the mechanismof lung toxicity of paraquat: Comparisonof tissuedistribution and some biochemicalparametersin rats and rabbits. Toxicol. Appl. Pharmacol. 28, 216-226. JACOBSON, M., LEVIN,W., Lu, A. Y. H., MONNEY,A. H. ANDKUNTZMAN,R. (1973).The rate of pentobarbital and acetanilidemetabolism by liver microsomes:A function of lipid peroxidation and degradationof cytochromeP-450heme.Drug Metub. Dispos. 1,766-774. JOLLOW,D. J., MITCHELL,J. R., ZAMPAGLIONE, N. ANDGILLETTE, J. R. (1974).Bromobenzeneinduced liver necrosis.Protective role of glutathione and evidencefor 2,Cbromobenzene oxide asthe hepatotoxic metabolite.Pharmacology 11, 151-169.
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KHERA, K. S. ANDWHITTA, L. L. (1968).Embryopathic effectsof diquat and paraquat in the
rats. Ind. Med. Sat-g.37, 553. KRIEGER, R. I., LEE, P. W., BLACK, A. AND FUKUTO, T. R. (1973).Inhibition of microsomal
aldrin expoxidation by diquat and severalrelated bipyridylium compounds.Bull. Environ. Contam.Toxicol. 9, l-3. LEVIN, W., Lu, A. Y. H., JACOBSON, M., KUNTZMAN, R., POYER, J. L. ANDMCCAY, P. B. (1973). Lipid peroxidation and the degradationof cytochromeP-450heme.Arch. Biochem.Biophys. 158,842-852. LITCHHELD,J. T., JR. (1949). A method for rapid graphic solution of time-percent effect curves.J. Pharmacol.Exp. Ther. 97, 399-413. MURRAY, R. E. ANDGIBSON,J. E. (1971).Lethality and pharmacokineticsof paraquat in rats. Toxicol. Appl. Pharmacol.19,405. MURRAY, R. E. AND GIBSON, J. E. (1972).A comparative study of paraquat intoxication in rats, guineapigsand monkeys.Exp. Mol. Pathol. 17, 317-325. MURRAY, R. E. AND GIBSON,J. E. (1974). Paraquat disposition in rats, guinea pigs and monkeys.Toxicol. Appl. Pharmacol.27,283-291. SHARP, C. W., OTTOLENGHI, A. ANDPOSNER, H. S. (1972).Correlation of paraquat toxicity with tissueconcentrationsand weight lossin the rat. Toxicol. Appl. Pharmacol.22,241-251. TONER, P. G., VE~ERS, J. M., SPILG,W. G. S. ANDHARLAND, W. A. (1970).Fine structure of the lung lesionin a caseof paraquat poisoning.J. Pathol. 102, 182-185.
Postnatal
AND
APPLIED
PHARMACOLOGY
33,461-470
(1975)
Toxicity of Chronically Administered Paraquat and Interactions With Oxygen and Bromobenzene
In Mice
JAMESS. Bus ANDJAMESE. GIBSON Department
of Pharmacology, Michigan State University, East Lansing, Michigan 48824 Received December 27,1974; accepted March 26, 1975
Postnatal Toxicity of Chronically Administered Paraquat in Mice and Interactions with Oxygen and Bromobenzene.Bus, J. S. AND GIBSON, J. E. (1975). Toxicol. Appl. Pharmacol. 33, 461-470. The purposeof this study wasto investigatethe effect of chronic paraquat administrationon developing mice and to examinethe interaction of chronic paraquatexposurewith 100% oxygen and the hepatotoxin bromobenzene.Paraquat was administeredat 50 and 100ppm in the drinking water to pregnantmice beginning at Day 8 of gestation,with continued exposureto the newbornsup to 42 days after birth. Neither paraquat treatment altered the postnatal growth rate; however, 100ppm but not 50 ppm paraquat significantly increased the postnatal mortality. Both 50- and lOO-ppmparaquat-treated mice weresensitizedto the onsetof oxygen toxicity, determinedby a significant reduction in the LT50 at 42 days after birth. An enhancedsensitivity to oxygen toxicity was also detectablein 100 ppm but not 50 ppm mice at Days 1 and 28 after birth. In 42-day-old mice, 50 and 100ppm paraquat treatment also significantly reducedthe LT50 after 3100mg/kg ip (LD85) bromobenzene.These observationssuggestthat the toxicity of paraquat may be mediatedthrough an interaction with oxygen and describepossible interactionsthat could occur with the environmentaluseof paraquat.
The herbicide paraquat (1,l ‘-dimethyl-4,4’-bipyridylium), which can be highly toxic to adult man (Bullivant, 1966) and animals (Clark et al., 1966; Murray and Gibson, 1971), induces only minor alterations in prenatal development of rats (Khera and Whitta, 1968)and mice (Bus et al., 1975a)in the absenceof maternal toxicity. The effect of paraquat on postnatal development, however, hasnot been investigated. The mechanism of paraquat toxicity in adult animals has been proposed to be mediated through an interaction of paraquat with oxygen which induces subsequent lipid peroxidation damage of cellular membranes(Bus et al., 1974). This investigation reports the effect of chronic administration of paraquat on the postnatal development of mice and the sensitivity of these animals to an oxygen environment. The interaction of chronic paraquat treatment with bromobenzene, a hepatotoxin which requires microsomal metabolic activation for expression of toxicity (Brodie et al., 1971) was also examined. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
461
462
BUS AND GIBSON MATERIALS
AND
METHODS
Developmental Studies
Virgin Swiss-Webster mice1 were mated by placing one male in a cage of five females for 1 hr starting at 8 AM. The day vaginal plugs were found was designated Day 1 of gestation. Paraquat dichloride2 was placed in the drinking water of pregnant females at concentrations of 0, 50, 100, and 150 ppm beginning on Day 8 of gestation, which is the onset of organogenesis in mice. Pregnant females were housed individually in clear plastic shoe box cages and allowed access to food and the paraquat treated water ad libitum. Following delivery of pups on Day 20 of gestation, litters were normalized to 10 mice. Total litter body weights and mortality were recorded at weekly intervals from Day 1 postnatally to termination of the experiment at 42 days postnatally. All litters were weaned on Day 28 postnatally and segregated by sex. Exposure of mice to paraquat-treated water was continuous from Day 8 of gestation to 42 days postnatally. In other experiments, litters were either exposed to 100 ppm paraquat from Day 8 of gestation to 28 days after birth (weaning) and then transferred to nonparaquat-treated water until 42 days postnatally or placed on 100 ppm paraquat only between Days 28 and 42 postnatally. Litter mortality rates were recorded at weekly intervals for each treatment group. The stability of the paraquat solutions was confirmed by calorimetric assay (Sharp et al., 1972), with no change in paraquat concentrations for up to 4 weeks after preparation. Histopathology
At 42 days, control, 50, and 100 ppm paraquat-treated mice were sacrificed and lung, liver, and kidneys fixed in 10% formalin. Five-micrometer paraffin sections were prepared, stained with hematoxylin-eosin, and examined by light microscopy. Interaction with Oxygen
Developing mice exposed to 0, 50, and 100 ppm paraquat beginning at Day 8 of gestation were examined for sensitivity to 100% oxygen at 1 atm on Days 1, 28, and 42 postnatally. Oxygen exposure was accomplished by placing both control and treated mice in a clear plastic cage approximately 12 liters in volume (12 x 24 x 44 cm) with a Plexiglas top and supplied with animal bedding, food, and tap water. The l-dayold group was normalized to 10 newborns per mother and the mothers replaced after 48 hr of oxygen exposure withmothers which had recently given birth to ensureadequate nursing of the newborns. Oxygen, ( 100°~), which was humidified by flow over a water source prior to entry into the chamber, was maintained at a flow rate of 1.65 liters/min into the chamber. Oxygen concentrations in the chamber were measured to be 95-100 %. The sensitivity of the 28- and 42-day-old treatment groups to 100% oxygen was measured by determination of the LT50 (median time to death) after initiation of oxygen treatment (Litchfield, 1949). The interaction of oxygen with newborn mice was determined by recording the number of mice dead after 120 hr of oxygen exposure. 1 Spartan Research Animals, Inc., Haslett, Michigan. z Paraquat concentrate (240 mg cation/ml), Chevron Chemical Co, Richmond, California.
PARAQUAT
POSTNATAL
TOXICITY
463
Lung concentrations of paraquat in 42-day-old 50- and lOO-ppm paraquat-treated mice were determined by a calorimetric modification of the methods of Ilett ef al. (1974) and Sharp et al. (1972). Interaction
with Bromobenzene
The toxicity of bromobenzene3 in 42-day-old 50- and lOO-ppm paraquat-treated mice was measured by determination of the LT50. Bromobenzene, dissolved in peanut oil, was administered ip at 3100 mg/kg (LD85 in Swiss-Webster mice, determined in this laboratory) and the time to death recorded. Liver reduced glutathione (GSH) was measured in other 42-day-old 50- and lOO-ppm mice after homogenization (Polytron@, Brinkman Instruments, Westbury, New York) of individual whole lungs in 4.0 ml cold 30 PM EDTA and subsequent fluorometric assay of GSH (Cohn and Lyle, 1966). Statistics
Analyses of data was performed by Student’s t test and analysis of variance, completely randomized design. The level of significance was chosen as p < 0.05. RESULTS
Paraquat and Development
Paraquat, when placed in the drinking water of pregnant mice and continued at 50 and 100 ppm from Day 8 of gestation to 42 days postnatally, did not alter the average litter body weight compared to controls at any time during the experiment (Fig. 1). Pregnant mice receiving 150 ppm paraquat starting at Day 8 of gestation died before delivery of newborns occurred (day of death was usually by Day 16 of gestation). Water consumption was not altered by these concentrations of paraquat. One hundred parts per million paraquat significantly increased postnatal mortality compared to 50 ppm paraquat and controls from Day 7 after birth, when mortality was 33.3x, up to a 42-day mortality of 66.7% (Fig. 2). Mortality among the 50-ppm paraquat group and controls was not significantly different and remained below 7% over the course of the experiment. Mortality induced by 100 ppm paraquat appeared biphasic, with an initial rapid increase to 33.3 % during the first 7 days after birth then reached a plateau up to Day 21 when mortality was 36.7%. The plateau phase was followed by a second increase prior to weaning on Day 28, reaching a final mortality of 66.7 oA on Day 35. Paraquat treatment did not affect the number of live fetuses born compared to controls. Experiments in which developing mice were exposed to 100 ppm paraquat from Day 8 of gestation to 28 days postnatally and then transferred to tap water until 42 days after birth resulted in a mortality of 26.7 &- 12.0% by Day 28. This mortality level was not significantly different from the 28-day mortality of mice continuously exposed to 100 ppm paraquat throughout development. Furthermore, transfer to tap water resulted in a 42-day mortality that was unchanged from Day 28 and thus eliminated the second rapid increase in lOO-ppm mortality depicted in Fig. 2. Exposure of developing mice to 100 ppm paraquat only from Days 28 to 42 postnatally resulted in 3 Aldrich Chemical Co., Inc., Milwaukee,
Wisconsin.
BUS AND GIBSON
1
1
7
14 DAYS
21 AFTER
28 BIRTH
35
42
FIG. 1. Effect of 50- and lOO-ppm paraquat in the drinking water from Day 8 of gestation to 42 days after birth on the postnatal growth weight of mice. Each point is the mean mouse body weight within litters for three to four litters. There were 10 mice/litter on Day 1.
o-0-0-0 1
7
14 DAYS
21 2s AFTER BIRTH
25
42
FIG. 2. Effect of 50- and lOO-ppm paraquat in the drinking water from Day 8 of gestation to 42 days after birth on the postnatal mortality rate of mice. Each point is the mean percent mortality within litters for three to four litters. There were 10 mice/litter on Day 1.
46.7&14.5x mortality, which was not significantly different from the 30% increase in mortality observed in the second lOO-ppm mortality phaseof Fig. 2. Histopathology Lung sections from 42-day-old lOO-ppm mice showed extensive alveolar consolidation and collapse, and areasof thickening of intraalveolar septa. Edema fluid was seen
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in a few alveoli. Lung sections from 50-ppm and control mice did not show any significant pathological changes. Examination of liver and kidney sections of all treatment groups also did not reveal any significant pathological changes. Paraquat Interaction
with Oxygen
Paraquat significantly enhanced the sensitivity to oxygen toxicity of 42-day-old mice which received 50 and 100 ppm paraquat throughout development (Table 1). The LT50 for 50- and lOO-ppm paraquat-treated mice was 108 and 40 hr, respectively. compared to the control LT50 of 160 hr. The LT50 curves for both treatment groups were parallel to each other and controls, with calculated potency ratios of 1.5 for 50 ppm versus controls and 4.0 for 100 ppm versus controls. The concentration of paraquat in lung tissue of the 100-ppm mice was less than 0.2 ,ug/g lung tissue (wet weight) and was below detectable levels in 50-ppm lung tissue. At 28 days after birth, an increased sensitivity to 100% oxygen exposure was detected only in lOO-ppm paraquat-treated mice (Table 1). The LT50 of lOO-ppm mice was significantly reduced to 121 hr, compared to an LT50 of 180 hr for 50-ppm mice and 181 hr for control mice. A potency ratio of 1.5 was calculated for lOO-ppm mice versus controls. TABLE 1 EFFECT OF PARAQUAT IN THE WATER FROM DAY 8 OF GESTATION TO VARIOUS DAYS POSTNATALLY ON THE SURVIVAL OF MICE EXPOSED TO 100 % OXYGEN 95%
Treatment Control 50 mm 100ppm Control 5Owm
100ppm
Begin0, exposure(day)
LT50 (hr)
28 28 28
181 180 121 160 108
42 42 42
40
confidence limits -___
Potency ratio
..-
(156-210)
-
(142-229) (108-135)
1.0 1.5”
(126-203) (18-144) (3054)
1.5" 4.0"
-
’ Significantlydifferentfrom respectivecontrol,p < 0.05. One-day-old mice, which were exposed to paraquat only by prenatal placental transfer, also were sensitized to oxygen toxicity (Fig. 3). Mortality of l-day-old mice whose mothers received 100 ppm paraquat was significantly increased to 53.5% after 120 hr oxygen exposure compared to 25.8 and 24.2% mortality in 50-ppm and control mice, respectively. Paraquat Interaction with Bromobenzene The toxicity of bromobenzene, as measuredby the LT50, was determined in 42-dayold 50- and lOO-ppm paraquat-treated mice (Table 2). The LT50 for bromobenzene in control mice was 20.0 hr, in 50-ppm mice 4.2 hr, and 3.2 hr in lOO-ppm paraquattreated mice. The LTSOsof the 50- and lOO-ppmmice were significantly different from controls, but no different from each other. Liver GSH was assayedin 42-day-old 50- and lOO-ppmmice (Table 3). No significant differences were observed in the liver GSH of these mice compared to controls.
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FIG. 3. Effect of 50- and lOO-ppm paraquat in the drinking water of pregnant mice from Day 8 of gestation to Day 19 of gestation on the survival of 1-day-old mice exposed to 100% oxygen for 120 hr. Each treatment represents the mean percent mortality for three to four litters with 10 mice/litter on Day 1. *Indicates significantly different from control and 50-ppm paraquat, p c 0.05. TABLE 2 EFFECT OF PARAQUAT IN THE WATER FROM DAY 8 OF GESTATION TO 42 DAYS POSTNATALLY ON THE SURVIVAL OF MICE TREATED WITH BROMOBENZENE, 3100 mg/kg ip (LD85)
Treatment
LT50 W-1
95% confidence limits
20.0 (12.631.6) 4.2 (2.6- 6.7) 50 pm 3.2 (1.9- 5.4) 100 ppm a Significantly different from control, p < 0.05.
Control
Potency ratio 4.8” 6.2*
TABLE 3 LIVER GSH CONCENTRATIONS IN MICE GIVEN PARAQUAT IN THE WATER FROM DAY 8 OF GESTATION ~0 42 DAYS POSTNATALLY~ Treatment
Liver GSH (mg GSH/g wet wt + SE)
Control 50 mm 100 mm
5.08 f 0.31 4.80 + 0.30 5.14 + 0.63
a Each treatment represents the mean k SE of three to six determinations. DISCUSSION
Chronic administration of paraquat at 50 and 100ppm in the drinking water did not alter the development of mice as reflected in the average body weight. Treated mice reaching 42 days of age not only weighed the sameas controls, but also did not exhibit
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467
any gross malformations. In contrast to the lack of any paraquat effect on growth rate, 100 ppm paraquat increased postnatal mortality in an apparent biphasic pattern. The first increase in lOO-ppm mortality occurring between Days 1 and 7 after birth may have been induced by the presence in the newborns of paraquat which remained from prenatal placental transfer of the herbicide to the fetuses, rather than from paraquat passage to the newborns through the mother’s milk. Bus et al. (1975a) have shown that paraquat administered to pregnant rats during the final 2 days of gestation was detectable in the newborns for up to 7 days postnatally. Furthermore, the 100-ppm mortality reached a plateau between 7 and 21 days, despite continued nursing by mothers exposed to paraquat in their drinking water. The possibility that paraquat ingested from the mother’s milk may have been responsible for the initial mortality appears unlikely as paraquat is poorly absorbed orally (Murray and Gibson, 1974) and thus only minute amounts of paraquat would be expected to be systemically absorbed in the newborns. The immediate postnatal increase in mortality of the IOO-ppm newborns may be linked to the initiation of breathing and exposure of lungs to higher oxygen tensions encountered after birth as 100 ppm paraquat did not cause any fetal mortality. Increased oxygen tensions increased paraquat toxicity in adult rats (Fisher et al., 1973) and oxygen has been linked to the mechanism of action of paraquat toxicity (Bus et al., 1974). Also, developing mice exposed to paraquat in the water were more sensitive than controls to oxygen toxicity which was detectable as early as the first day after birth. The second mortality increase in lOO-ppm paraquat-treated mice which began during the week prior to weaning on Day 28, may have been caused by the young animals beginning to ingest paraquat from the drinking water. When other paraquattreated mice were transferred to tap water at weaning, the second increase in mortality was eliminated. Furthermore, developing mice exposed to 100 ppm paraquat only between Days 28 and 42 postnatally yielded an increase in mortality that was not significantly different from the increase occurring between Days 21 and 42 in the continuously treated mice. Histologic examination of tissue from lung, liver, and kidney from 42-day-old 50and 100-ppm paraquat-treated mice revealed extensive lung alterations in the IOO-ppm treated group, despite low paraquat lung concentrations in these mice. The pulmonary lesions observed with chronic lOO-ppm paraquat treatment resembled the pulmonary alveolar congestion and collapse seen in animals and man after acute paraquat intoxication (Toner et al., 1970; Murray and Gibson, 1972). Similar pulmonary lesions were not observed in mice exposed to 50 ppm paraquat, the treatment in which mortality was not significantly elevated compared to controls. The lung appeared to be the site of chronic paraquat toxicity, as liver and kidney tissue sections were without significant pathological alterations. Fisher et al. (1973) reported that adult rats were sensitized to the development of oxygen toxicity after acute treatment with paraquat. The 42-day-old 50- and lOO-ppm chronically paraquat-treated mice in this study also were sensitized to lOOad oxygen exposure, as reflected by the significant reduction in LT50 values. The 50-ppm mice were more sensitive to oxygen toxicity than controls, even in the absence of any alteration in postnatal growth or mortality rate. Furthermore, the 42-day-old 50-ppm mice did not have any evidence of histopathological lesions and had no detectable amounts
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of paraquat in lung tissue before being placed in 100% oxygen. Thus, the 42-day-old 50-ppm paraquat-treated mice were sensitized to oxygen despite being morphologically indistinguishable from controls. The 50-ppm paraquat-treated mice appeared to develop a sensitivity to oxygen toxicity between Days 28 and 42 postnatally, as the oxygen LT50 of 28-day-old 50-ppm mice was not altered from controls. One-day-old mice which received 50 ppm during gestation also were not sensitized to oxygen toxicity. In the IOO-ppm paraquat-exposed group, however, sensitivity to oxygen toxicity had developed by the first day after birth, even though these newborns could have only been exposed to paraquat prenatally. In 42-day-old mice chronically treated with 50 or 100 ppm paraquat, bromobenzene toxicity was significantly enhanced. Bromobenzene is metabolized to an hepatotoxic epoxide by microsomal enzymes (Brodie et al., 1971) and subsequently detoxified by either conjugation with GSH, nonenzymatic rearrangement to phenols, or enzymatic conversion to dihydrodiols by epoxide hydrase (Jollow et al., 1974). In acute studies in rats, bromobenzene toxicity was significantly enhanced by pretreatment with GSHdepleting agents due to decreased detoxification of the bromobenzene epoxide (Jollow et al., 1974). Although liver GSH concentrations in the chronically paraquat-treated mice were not altered compared to controls, it is possible that paraquat treatment affected the capability of mice to replenish GSH stores which are depleted by bromobenzene detoxification. Thus, less GSH would be available for epoxide conjugation with the result of increased bromobenzene toxicity. Evidence that paraquat may catalyze membrane lipid peroxidation (Bus et al., 1974; Bus et al., 1975b) suggests a second mechanism whereby paraquat treatment could decrease the bromobenzene LT50. Paraquat has been postulated to induce lipid peroxidation in vivo by undergoing cyclic oxidation-reduction with subsequent generation of superoxide anions. Superoxide anions may nonenzymatically dismutate to singlet oxygen which attack polyunsaturated lipids of cell membranes to form lipid hydroperoxides. Formation of lipid hydroperoxides initiates the chain reaction process of lipid peroxidation (Bus et al., 1975b). Several mechanisms function in mammalian systems to prevent the onset of lipid peroxidation, one of which is the enzymatic reduction of lipid hydroperoxides to stable lipid alcohols by GSH peroxidase with GSH providing the reducing equivalents (Chow and Tappel, 1974). The availability of GSH appears necessary to combat paraquat toxicity, as pretreatment of mice with the GSH-depleting agent diethylmaleate significantly enhanced acute paraquat toxicity (Bus et al., 1975b). Large doses of bromobenzene, like diethylmaleate, also decrease liver GSH for up to 20 hr in rats due to conjugation of the bromobenzene epoxide with GSH (Jollow et al., 1974). Thus, bromobenzene, by depleting GSH, may remove the activity of GSH peroxidase in protecting against paraquat-induced lipid peroxidation. Consequently, the decreased bromobenzene LT50 in paraquat-treated mice may be due to the added expression of paraquat toxicity in conjunction with bromobenzene toxicity. The possibility also exists that activation of bromobenzene to the epoxide may be affected by chronic paraquat treatment. Paraquat pretreatment in rats has been reported to decrease the in vitro metabolism of bromobenzene along with a destruction of microsomal cytochrome P-450 which functions to activate bromobenzene (Ilett et al., 1974). Lipid peroxidative damage has been implicated in the degradation of cytochrome
PARAQUAT
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469
P-450 (Levin et aZ., 1973) with subsequent impairment of the metabolism of pentobarbital and acetanilide (Jacobson ef al., 1973). Alternatively, paraquat may disrupt the microsomal electron transport system (Ilett et al., 1974; Krieger et al., 1974) and thereby decrease bromobenzene metabolism. The enhancement of bromobenzene toxicity observed in our studies, however, may indicate that a possible inhibition of bromobenzene metabolism by chronic paraquat treatment was not sufficient to prevent the formation of epoxide and subsequent reduction in tissue GSH. The chronic administration of paraquat to developing animals has been shown to have expressed toxicity in the form of increased mortality and the development of pulmonary lesions in the lOO-ppm paraquat-treated mice. Moreover, susceptibility to the toxic agents oxygen and bromobenzene was detectable in 50-ppm exposed mice which showed no signs of toxicity from paraquat alone. These observations suggest possible toxic effects and interactions that might occur with the environmental use of paraquat. ACKNOWLEDGMENTS This study wassupportedby NIH Grant ES 00560,NIH Training Grant GM 01761,and by the Michigan Lung Association. REFERENCES BRODIE,B. B., REID, W., CHO,A. K., SIPES,G., KRISHNA,G. ANDGILLETTE,J. R. (1971). Possiblemechanismof liver necrosiscausedby aromatic organic compounds.Prac. Nut. Aead. Sci. Wash. 68, 160-164. BULLIVANT,C. M. (1966). Accidental poisoningby paraquat: Report of two casesin man. Brit. Med. J. 1, 1272-1273. Bus, J. S., AUST,S. D. ANDGIBSON,J. E. (1974). Superoxide-and singlet-oxygen-catalyzed lipid peroxidation asa possiblemechanismfor paraquat(methyl viologen)toxicity. Biochem. Biophys. Res. Commun. 58,749-755. Bus, J. S., PREACHE, M. M., CAGEN,S. Z., POSNER, H. S., ELIASON,B. C., SHARP,C. W. AND
GIBSON,J. E. (1975a).Fetal toxicity, teratogenicity and distribution of paraquat and diquat in miceand rats. Toxical. Appl. Pharmacol. 33,446-456. Bus, J. S., CAGEN,S. Z., ACJST, S.D. ANDGIBSON,J. E. (1975b).Lipid peroxidation: A possible mechanismfor paraquat toxicity. Toxicol. Appl. Pharmacol. in press. CHOW,C. K. ANDTAPPEL,A. L. (1974).Response of glutathioneperoxidaseto dietary selenium in rats. J. Nutr. 104, 444-451. CLARK,D. G., MCELLIGOTT,T. F. ANDHURST,E. W. (1966).The toxicity of paraquat. &it. f. Ind. Med. 23, 126-132.
COHN,V. H. ANDLYLE,J. (1966). A fluorometric assayfor glutathione. Anal. Biochem. 14, 434-440.
FISHER,H. K., CLEMENTS, J. A. ANDWRIGHT,R. R. (1973).Enhancementof oxygen toxicity by the herbicideparaquat. Ann. Rev. Resp. Dis. 107,246-252. ILETT,K. F., STRIPP,B., MENARD,R. H., REID,W. D. ANDGILLETTE,J. R. (1974).Studieson the mechanismof lung toxicity of paraquat: Comparisonof tissuedistribution and some biochemicalparametersin rats and rabbits. Toxicol. Appl. Pharmacol. 28, 216-226. JACOBSON, M., LEVIN,W., Lu, A. Y. H., MONNEY,A. H. ANDKUNTZMAN,R. (1973).The rate of pentobarbital and acetanilidemetabolism by liver microsomes:A function of lipid peroxidation and degradationof cytochromeP-450heme.Drug Metub. Dispos. 1,766-774. JOLLOW,D. J., MITCHELL,J. R., ZAMPAGLIONE, N. ANDGILLETTE, J. R. (1974).Bromobenzeneinduced liver necrosis.Protective role of glutathione and evidencefor 2,Cbromobenzene oxide asthe hepatotoxic metabolite.Pharmacology 11, 151-169.
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KHERA, K. S. ANDWHITTA, L. L. (1968).Embryopathic effectsof diquat and paraquat in the
rats. Ind. Med. Sat-g.37, 553. KRIEGER, R. I., LEE, P. W., BLACK, A. AND FUKUTO, T. R. (1973).Inhibition of microsomal
aldrin expoxidation by diquat and severalrelated bipyridylium compounds.Bull. Environ. Contam.Toxicol. 9, l-3. LEVIN, W., Lu, A. Y. H., JACOBSON, M., KUNTZMAN, R., POYER, J. L. ANDMCCAY, P. B. (1973). Lipid peroxidation and the degradationof cytochromeP-450heme.Arch. Biochem.Biophys. 158,842-852. LITCHHELD,J. T., JR. (1949). A method for rapid graphic solution of time-percent effect curves.J. Pharmacol.Exp. Ther. 97, 399-413. MURRAY, R. E. ANDGIBSON,J. E. (1971).Lethality and pharmacokineticsof paraquat in rats. Toxicol. Appl. Pharmacol.19,405. MURRAY, R. E. AND GIBSON, J. E. (1972).A comparative study of paraquat intoxication in rats, guineapigsand monkeys.Exp. Mol. Pathol. 17, 317-325. MURRAY, R. E. AND GIBSON,J. E. (1974). Paraquat disposition in rats, guinea pigs and monkeys.Toxicol. Appl. Pharmacol.27,283-291. SHARP, C. W., OTTOLENGHI, A. ANDPOSNER, H. S. (1972).Correlation of paraquat toxicity with tissueconcentrationsand weight lossin the rat. Toxicol. Appl. Pharmacol.22,241-251. TONER, P. G., VE~ERS, J. M., SPILG,W. G. S. ANDHARLAND, W. A. (1970).Fine structure of the lung lesionin a caseof paraquat poisoning.J. Pathol. 102, 182-185.