Interaction of paraquat with the pulmonary microsomal fatty acid desaturase system

TOXICOLOGY

AND

Interaction

APPLIED

PHARMACOLOGY

of Paraquat Fatty Acid

36,

543-554 (1976)

with the Pulmonary Desaturase System

Microsomal

MARK R. MONTGOMERY Pulmonary-Toxicology Laboratory and Research Services, Veterans Administration Hospital, Minneapolis, Minnesota 55417: and Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455 Received October 22,1975; accepted January 12,1976

Interaction of Paraquat with the Pulmonary Microsomal Fatty Acid DesaturaseSystem.MONTGOMERY, M. R. (1976).Toxicol. Appl. Pharmcaol. 36,543-554. Administration of paraquat(methyl viologen) to rats resulted in an acute inhibition of the pulmonary fatty acid desaturaseenzyme systemwhichmay beexplainedby the anorexiceffect of paraquat.However, this sameenzyme system in paraquat-treated rats appearsto be more strongly inhibited by in vitro addition of paraquatthan control preparations. This effect doesnot appearto berelated to an increasedrate of microsomal hydrogen peroxide or superoxideproduction. This enzymatic sensitivity to paraquat is partially reversedby treatment of rats with phenobarbital which also mildly protects rats against the toxic effects of paraquat as expressedby a 50% increasein time to death at 35 mg/kg paraquat. Pulmonary lesions in man or experimental animals may result from accidental or intentional exposure to various chemicals,including the commercial herbicide, paraquat (methyl viologen, l,l’-dimethyl-4-4’-bipyridinium dichloride). Paraquat is one of a family of bipyridinium compounds which has been described in numerous experimental investigations and clinical casereports to produce specificlung lesions.Ingestion of small doses (4 mg/kg) of this compound in man often follows a fatal course (Almog and Tal, 1967; Bullivant, 1966; Campbell, 1968; Copland et al., 1974; Toner et al., 1970), with the initial injury occurring within IO-24 hr after ingestion, even though pulmonary edema and ultimately death may not occur until 7-l 5 days later. Even in the presence of vigorous treatment, the true fatality rate is probably 33-50x (Sharp et al., 1972; Ilett et al., 1974). The observed pathological changesin paraquat toxicity include pulmonary edema followed by vascular infiltration, fibrosis and alveolar collapse. Numerous pathological and histochemical studies have suggestedthat acutely altered pulmonary surfactant and alveolar membranesmay be implicated in the early development of this particular lesion (Fisher et al., 1973; Fletcher and Wyatt, 1972; Malmqvist et al., 1973; Robertson et ol., 1970). The role of pulmonary lipid metabolism in the development of these lesionsis not known. However, biochemical studies on this aspect of this toxicity have been lacking. This lack may partially result from the fact that a specific, pulmonary enzyme system responsible for pulmonary lipid metabolism has not been readily available for experimental investigations and manipulations. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain

543

544

MARKR.MONTGOMERY

We have recently characterized several of the properties of such an enzyme system which is present in the microsomal fraction of lung tissue homogenate from rats (Montgomery, 1976). This enzyme system, the fatty acid desaturase, is a microsomal mixed function oxidase which catalyzes the conversion of fatty acyl-CoA to the corresponding d9 monoenoic acid ester. The electrons required for this desaturase are transferred from NADH or NADPH through cytochrome b, to a cyanide sensitive factor. While the role of the lipid desaturation process in overall pulmonary lipid metabolism is not yet defined, it may be involved in the metabolism of pulmonary membranes and/or alveolar surfactant. This study reports an investigation on the effects of the pulmonary toxin, paraquat, on the pulmonary lipid metabolism system. METHODS Preparation of microsomes. Male, Charles River rats (Charles River Co., North Willmington, MASS), 150-275 g, were sacrificed by cervical fracture between 9 : 00 and 10:00 AM. The lungs were removed and immediately minced with scissors. The minced lungs were washed three times with cold Tris-KC1 buffer (0.02 M Tris0.15 M KCl, pH 7.4) and filtered through cheese cloth. This washing procedure removes much of the contaminating hemoglobin. The washed mince was homogenized in a motor-driven Teflon glass homogenizer with 0.10-O. 15-mm clearance in three volumes of the cold Tris-HCI buffer. The mincing procedure also greatly facilitates this homogenization. The homogenate was centrifuged at 9OOOgfor 15 min and the resulting supernatant was centrifuged at 165,000 g for 38 min. The microsomal pellet was suspended in Tris-KC1 buffer and resedimented at 165,000 g for 38 min (equals 6 x 106/gmin). The pellet was resuspended in a Tris-KC1 buffer (0.02 M Tris-O.15 M KCI, pH 7.25) to yield a final concentration of IO-I 5 mg of microsomal protein per ml. This washing procedure removes contaminating hemoglobin from the microsomes as evidenced by the presence of a strong Soret band at 420 nm in unwashed microsomes and the disappearance of this Soret band in the washed microsomal preparation as determined by the method of Falk (1964). Microsomal protein was determined by the method of Sutherland et al. (1949). Determination of fatty acid desaturation. The desaturation of [I-“C]stearoyl-CoA was determined as previously described (Montgomery, 1976). The standard incubation mixture contained 1.0-l .5 mg of microsomal protein, 1 mM NADH, and 70 pM [l-‘4C]stearoyl-CoA in a final volume of 0.50 ml. Incubations were performed for 6 min at 37°C in a shaking water bath during which time product formation was a linear function of time. [l-14C]Stearoyl-CoA was obtained from New England Nuclear (Boston, Mass.) (sp act of 51.8 mCi/mmol, Lot #678-279). Two-tenths of a mg of [ 1J4C]stearoyl-CoA (0.010 mCi) was added to 10 mg of the cold stearoyl-CoA (Sigma Company, St. Louis, MO.) in a Tris-KC1 buffer, pH 7.25, for use in the incubation mixtures. All incubations were performed in duplicate or triplicate. The reaction was stopped by the addition of 1.Oml of 10 y0 KOH in methanol followed by saponification at 80°C for 30 min. The mixture was then acidified with 2.0 ml of 4 N HCI, the fatty acids extracted with 30 ml of petroleum ether or hexane and converted to methyl esters with 4 ml of 14 % boron trifluoride in methanol. The methyl esters were extracted into petroleum ether or hexane and separated by thin-layer chromatography on silica gel

PARAQUAT

AND

PULMONARY

LIPID

METABOLISM

545

GF plates (Analtech Inc., Newark, De.), impregnated with 10 % AgN03 by developing in ether-hexane (1:9 v/v). Separation of mono-unsaturated esters from saturated esters was markedly facilitated by prewashing the plates with ethylacetate prior to spraying with the AgNO, solution. The spots were identified under ultraviolet light by comparison with authentic standards following spraying of 0.050 % Rhodamine B (in methanol). The spots were scraped and counted in a toluene scintillator (4 g PPO and 0.25 g POPOP/liter of toluene) in a Beckman LC-100 scintillation counter. Desaturase activity was determined by dividing the radioactivity found in the mono-unsaturated ester by the sum of the radioactivities in both the saturated and unsaturated esters; this ratio was then converted to nanomoles of product formed/mg microsomal protein/ min. Incubation blanks were obtained by addition of the microsomes after addition of the KOH solution. Estimation of hydrogen peroxide generation. The microsomal generation of hydrogen peroxide was measured by the conversion of methanol to formaldehyde as previously reported (Ilett et al., 1974). Each reaction mixture (1.0 ml) contained 4 mg of lung microsomal protein, 1800 units of catalase (Sigma Company), paraquat (10V4 M), Tris buffer, and an NADPH-generating system as previously described (Stripp et al., 1971). Methanol (final concentration 0.25 M) was added to start the reaction. The mixtures were incubated in air for 15 min at 37°C. The reaction was stopped by the sequential addition of 0.3 ml of an 8.9% w/v ZnSO, solution and 0.7 ml of a freshly preprared mixture (3 : 1) of saturated Ba(OH), and saturated sodium borate solutions. The reaction mixtures were then centrifuged at 2000 g for 15 min and the formaldehyde in the supernatant was determined by the method of Nash (1953). Estimation of superoxide generation. The microsomal generation of the superoxide anion radical, 02-, was followed by measuring the conversion of epinephrine to adrenochrome at 480 nm. The l-ml incubation mixtures contained 300 PM epinephrine, 1 mM NADPH, pH 7.4, Tris buffer, and 0.5-1.0 mg of lung microsomal protein and were incubated in air for 15 min at 37°C. The linear phase of adrenochrome appearance was expressed as dOD,,,/min per mg of microsomal protein. Negligible changes (<0.005) in absorbance were obtained over the entire incubation period in the absence of either microsomal protein or NADPH. The epinephrine conversion was reversible by addition of superoxide dismutase. Estimation of lipid peroxidation. Lipid peroxidation was followed by the in vitro production of malonaldehyde. The 3.0-ml incubation system contained 3-5 mg of lung microsomal protein, 12 PM FeCl,, 4 mM ADP, and 0.5 mM NADPH. The incubation time was 20 min at 37°C in air and the reaction was terminated by the addition of 1 ml of 30 “//, w/v trichloroacetic acid. Malonaldehyde was determined as described by Ernster and Nordenbrand (1967). No malonaldehyde formation was detected when FeCl, was omitted from the incubations. Dietary treatment. When desaturase activity was studied, the rats were maintained on a fat free diet (“Fat Free” Test Diet, Nutritional Biochemicals, Cleveland, Ohio) for 4 days priors to sacrifice. This dietary treatment was previously shown to double the activity of the pulmonary desaturase system when compared to animals maintained on standard rat diet (Purina Rat Chow, Ralston-Purina, Ralston, 11)(Montgomery, 1976). Body weight changes were determined as the percentage increase or decrease during the 4 days of dietary, phenobarbital, and/or paraquat treatment prior to sacrifice,

546

MARK R. MONTGOMERY

Statistical significancewas selectedasp < 0.05 by the Student’s t test for between group analysisand paired analysisor one way analysisof variance for multiple group analysis. Source of chemicals. Paraquat hydrochloride was generously supplied as a gift by Imperial Chemical Industries Ltd., Cheshire, England. The paraquat was dissolved in 0.9 % NaCl solution at 5, 10, and 20 mg/ml for ip injection at 20, 35, and 50 mg/kg, respectively. Phenobarbital sodium (Sigma Company) wasadministered in the drinking water (1 mg/ml) for 4 days prior to sacrifice where indicated. RESULTS

When paraquat (20 mg/kg) was administered to rats and the animals sacrified 24 hr later, a 68 % decreasewasobservedin the activity of the lung fatty acid desaturasesystem (Table 1). However, it was also noted that those animals receiving paraquat became markedly anorexic, consuming only 25% as much food or water during the 24 hr TABLE 1 NUTRITIONAL INFLUENCES OF PARAQUAT ON LUNG FATTY ACID DESATURASE ACTIVITY

Treatment’ Control Paraquat-Group Ib Pair-fed’ Paraquat-Group IId

Desaturase activity (rim01oleic formedlmg proteimmin) 0.33 + 0.03

Percentage of control 100

0.11 f 0.03’ 0.10f 0.02’

32.7 f 10.6

0.47 f 0.05f

142.2 f 21 .l

31.2 f

6.2

’ N = Sixratspergroup;data are shown as .?+ SE for duplicate determinations. AI1 animals, except pair-fed, received fat-free diet adIibitumfor 4 days prior to sacrifice. b One dose, 20 mg/kg ip, given 24 hr prior to sacrifice. c Received fat-free diet adZibitumfor 3 days prior to sacrifice, then 5 g of diet on Day 4. d One dose, 20 mg/kg ip, given 14 days prior to sacrifice. pp c 0.01 compared to control. fp = 0.05 compared to control. following injection asuntreated or saline-injectedcontrols. This type of feeding behavior has been previously noted to cause a decreasein hepatic desaturase activity after administration of various drugs and chemicals (Montgomery and Holtzman, 1975). Since the pulmonary desaturase system is very sensitive to nutritional disturbances (Montgomery, 1976), animals were pair-fed to control this variable. These animals showed a similar decreasein lung desaturaseactivity (Table 1). This would suggestthat the acute effect of paraquat on this lung enzyme systemmay simply be a reflection of a nutritional alteration causedby the paraquat-induced anorexia. Animals which were injected once with the same dose of paraquat (20 mg/kg) and sacrified 14 days later showedan apparent stimulation of desaturaseactivity. Theseanimals were nutritionally

PARAQUAT AND PULMONARY LIPID METABOLISM

547

comparable to the controls, both groups consuming identical quantities of the fat-free diet for all 4 days prior to sacrifice. No deaths occurred in the paraquat group killed at 24 hr and 2/9 animals in the second paraquat group (for assay on day 14) died on Day 4 and Day 7 after injection. When rat lung microsomes were preincubated in the absence of NADPH at 37°C in air, a slight decrease (15 %) in desaturase activity was observed (Fig. 1). This activity was markedly decreased (64 %) when the microsomes were preincubated in the presence of NADPH. This marked decrease in desaturase activity was presumed to be due to NADPH-supported lipid peroxidation of the microsomal enzymes during the pre-, incubation period. Incubation of mouse lung microsomes in the presence of paraquat added in vitro has been previously shown by Bus et al. (1974) to result in an increase in microsomal lipid peroxidation. The effectiveness of both in vitro and in z&o paraquat

Fwcent

ofControl Activity

0

1 2 3 4

5 6 7

Preincubation

8 9 10

Tim&

FIG. 1. Effect of preincubation of lung microsomes in the presence (O---O) and absence (O--O) of NADPH on fatty acid desaturase activity. Microsomes prepared from pooled lungs of eight rats were preincubated for the indicated periods at 37°C in the absence or presence of an NADPH generating system [NADP (0.33 mM), glucose-dphosphate (5 mM), and glucose-6-phosphate dehydrogenase (0.67 units/ml)]. Desaturase activity then determined following addition of 70 PM [1-“C]stearoyl-CoA and 1 mM NADH. Data represent average of triplicate determinations at each time point.

on desaturase activity was, therefore, examined in the presence and absence of this preincubation period. Treatment of rats with paraquat (20 mg/kg, 24 hr or 14 days before assay) did not alter the response of the desaturase activity to a 5-min preincubation treatment (Table 2). Both paraquat groups resulted in a 20-30 % decrease in the fatty acid desaturase activity, identical to the response seen with microsomes from control animals. Preincubation in the presence of 0.5 mM paraquat (added in vitro) further depressed desaturase activity in all groups (Table 2). However, the decrease in desaturase activity was significantly greater in the paraquat group killed 24 hr after paraquat administration (5.8 %) than either the control (31%) or the paraquat group killed 14 days after paraquat (23%). Thus, it would appear that, acutely, lung microsomes are very sensitive to the destructive effects of the presence of reduced paraquat. This sensitivity is significantly greater than that seen in either control preparations or preparations obtained 14 days after paraquat treatment and is specific for paraquat in the reduced form. Preincubation of microsomes for up to 10 min with low4 M paraquat in the absence of NADPH resulted in no significant alteration in desaturase activity (Table 2). In all experiments, maximal desaturase activity was insured by initiation of the desaturase

548

MARK

R. MONTGOMERY

TABLE EFFECT OF PREINCUBATION

5-min Preincubation”

Sources of microsomes

2

IN THE PRESENCE AND ABSENCE DESATIJRASE ACTIVITY

Controls

-

Paraquat-Group

Ic

+ + + +

Paraquat-Group

Ild

+ +

0.5 rnM Paraquat + + + +

OF PARAQUAT

ON LUNG

formed* min/mg protein

nmol

oleic

0.33 0.31 0.21 0.10

FATTY

ACID

Percentage of

nonpr&cubated activity

+ 0.03 f 0.03 f 0.03 _+ 0.02

100 93.9 f: 4.5 75.2 f 0.7 31.2 f 7.8

0.11 + 0.03 0.09 + 0.03 0.01 f 0.01

100 79.2 jI 3.8 5.8 f 4.0’

0.47 + 0.05 0.37 + 0.04 0.08 + 0.01

100 70.4 31 6.3 23.1 + 3.8

e Preincubation was carried out at 37°C in air with NADPH-generating system containing NADP (0.33 mM), glucose&phosphate (5 mM), and glucose-6-phosphate dehydrogenase (0.67 units/ml). Microsomal protein concentration was approximately 1 .O mg/ml. * Desaturase reaction initiated with 70~~ [1-Wlstearoyl-CoA and 1.0 mM NADH. Data are shown asf+SEforN=6. c One dose, 20 mg/kg ip, 24 hr prior to sacrifice. d One dose, 20 mg/kg ip, 14 days prior to sacrifice. ep < 0.02 compared to similarly treated control or chronic paraquat preparations.

with substrate and I mM NADH. There was no observable effect of paraquat when present only during the desaturase reaction. This supports the absence of paraquat reduction in the presence of NADH. To investigate the increased sensitivity of microsomes from acutely treated animals, the microsomal generation of H,O, was measured. Rats were injected with either 20 or 50 mg/kg of paraquat and the H,O, production determined in the presence or absence of 10M4M paraquat (Table 3). The addition of 1Oe4M paraquat resulted in a three to fourfold increase in the rate of H,O, production. However, acute in vivo pretreatment with paraquat did not significantly alter the basal rate of HzOz production or the degree of stimulation in the presence of paraquat added in vitro. Thus, an increased rate of microsomal H,O, generation does not appear to be responsible for the difference seen in Table 2 for the microsomes from acutely paraquat-treated animals. The toxicity of paraquat may be related to the formation of the highly reactive species, superoxide anion radical, O,- (Ilett et al., 1974; Bus et al., 1974) which may dismutate to form singlet oxygen. Singlet oxygen is known to react with unsaturated fatty acids to produce fatty acid hydroperoxides (Rawls and van Lanten, 1970). The superoxide anion-dependent conversion of epinephrine to adrenochrome was found to require the presence of both NADPH and microsomal protein (Table 4). This assay for superoxide generation is not supported by NADP and minimally supported by NADH. The addition of 1 or 5 mM paraquat caused a marked stimulation of the apparent 02- generation (Table 4).

reaction

549

PARAQUAT AND PULMONARY LIPID METABOLISM

EFFECT OF

TABLE 3 in oioo AND in vitro PARAQUAT ON M~CRO.WMAL HzOz PRODUCTION

Sources of microsomes

0.10 rnM Paraquat + + +

Controls Paraquat

(20 mg/kg)b

Paraquat

(50 mg/kg)b

Formaldehyde formation’ bmW-w/h) 18.88 64.27 15.23 63.22 12.65 70.30

f + f f k -t

1.51 6.5gc 0.66 2.14c 1.81 9.49’

a Data are shown as 2 f SE for N = three groups of lungs pooled from four rats per group. b Administered ip at indicated dosage 24 hr prior to sacrifice. =p < 0.001 compared to incubations not containing added paraquat. TABLE EFFECT

Incubation

4

OF COFACTORS ON SUPEROXIDE GENERATION BY WASHED RAT LUNG MICROSOMES’

mixture

Completd Minus epinephrine Minus NADPH Minus microsomes Minus NADPH, plus 1 mM NADP Minus NADPH, plus 1 mM NADH Complete plus 1 mM phenobarbital Complete plus 1 mM paraquat Complete plus 5 mM paraquat

dOD.,s,/mg/min 0.037 0.001 0.003 0.002 0.003 0.006 0.041 0.079 0.126

Percentage of control 100 3.0 7.3 6.2 a.2 15.5 109.5 212.0 338.6

a Data shown are average values for duplicate incubations for microsomes prepared from lungs pooled from four rats. * 300 ,UM epinephrine, 1 mM NADPH, and 0.5 mg of microsomes in 1.0-ml incubation volume.

This microsomal generation of hydrogen peroxide and superoxide anion suggests an alternative explanation to lipid peroxidation for the inhibition of enzymatic activity which was observed following preincubation of the microsomes in the presence of NADPH (Fig. 1). In these studies, lipid peroxidation could not be observed in lung or liver microsomal preparations in the absence of exogenously added inorganic iron. This observation has been recently confirmed (D. Cinti, personal communication). Thus, in these studies involving microsomal incubations in which exogenous iron was not added, lipid peroxidation would appear not to be responsible for the enzymatic inhibition.

550

MARKR.MONTGOMERY

The microsomal production of superoxide following in vivo treatment with paraquat was also determined (Table 5). The apparent generation of superoxide by control microsomes was inhibited by superoxide dismutase, suggesting that the assay does TABLE INFLUENCE

OF PARAQUAT

AND S~~PEROXIDE

Sourceof microsomes” Control

Paraquatd

10 units SODb

5 (SOD) ON MICROSOMAL

DISMUTASE SUPEROXIDE

0.5 rnM

paraquat

-

-

+ +

+ +

+

-

+

+ +

dODdsomg protein min 13.2 6.4 22.7 14.5

L- 2.5 + 2.8 f 6.3 + 3.8

18.1?- 1.6 3.6 + 1.1 33.7 + 1.2 18.7 + 2.3

GENERATION

OF

Percentage of control 100 45 + 11’ 168 k 16’ 108 + 10

100 20 f 4’ 188 * 11’ 104L- 4

a N = four rats per group, data represent X + SE. b One unit of superoxide dismutase (SOD) will cause a 50% inhibition of the spontaneous conversion of epinephrine to adrenochrome at pH = 10.2. cp < 0.01 compared to control not containing SOD or paraquat, paired analysis. d One dose, 20 mg/kg, 24 hr prior to sacrifice.

indeed indicate the presenceof the O,- anion. The addition of 0.5 mM paraquat to the control microsomal incubations significantly increased the production of superoxide which was again reversible upon the addition of superoxide dismutase. Acute treatment of rats with paraquat (20 mg/kg) did not alter the formation of O,as compared to the control preparation (p > 0.05, Table 5). Furthermore, the in vitro addition of paraquat to microsomesisolated from either control or paraquat-treated rats stimulated the generation of O,-. The addition of superoxide dismutase again inhibited the O,- formation both in the absenceand presenceof 0.5 mM paraquat. The data from Tables 3 and 5 would then suggestthat neither the generation of O,- nor H,O, is significantly altered in lung microsomal incubations by acute pretreatment of rats with paraquat. It hasbeenpreviously reported that the pulmonary lesionsassociatedwith exposure of experimental animals to high concentrations of oxygen can be partially reversed by pretreatment of the animals with barbiturates (Jamieson and van den Bienk, 1964). Thus the effect of phenobarbital on the toxicity of paraquat wasexamined. The administration of phenobarbital did not significantly alter the feeding and drinking behavior. This was reflected by a normal rate of growth during this period (Table 6). Furthermore, phenobarbital treatment did not alter the anorexic responseto paraquat administration (Table 6). The effect of phenobarbital treatment on pulmonary microsomal desaturaseactivity was also studied. Phenobarbital alone did not alter either the basal desaturaseactivity of the lung microsomesor the responseto the 5-min preincubation (Table 7). Preincubation of microsomes prepared from animals which were treated in viva with paraquat

PARAQUAT

AND

PULMONARY

LIPID

551

METABOLISM

TABLE 6 EFFECT

OF PHENOBARBITAL AND PARAQUAT WEIGHT CHANGE IN RATS

ON E~IDY

Body weight change”

Treatment

17.7+ 14.1* -3.7 * -6.0 it

Control Phenobarbital* Paraquat’

Phenobarbitalplus paraquatd

1.5%

1.2% 1.4% 2.1%

a Expressed as percentage change over Cday period while receiving fat free diet ad libitum. Data are shown as f + SE for six rats per group. * One milligram per milliliter in drinking water for 4 days of treatment. c One dose, 20 mg/kg ip, 24 hr prior to sacrifice. d Combination of b and c.

TABLE 7 EFFECT

OF PHENOBARBITAL

AND

Treatment Control Phenobarbital’ Paraquatd Phenobarbitalplus paraquaF

PARAQUAT ACID

TREATMENT DESATURASE

ON RAT

LUNG

5-Min preincubation’

Desaturaseactivity* (nmol oleic formed/min/mg)

+ + + +

0.269+ 0.027 0.209* 0.025 0.296f 0.016 0.204+ 0.027 0.146f 0.033 0.042f 0.005 0.122f 0.014 0.069f 0.008

MICRO~~MAL

FA~Y

Decreaseby preincubation(%)

22.4 f 12.1 31.1f

9.8

71.3+ 7.3f 43.5 f

9.2

a Procedure same as in Table 2. * Data are shown as Z f SE, N = six rats per group. c Phenobarbital administered 1 mg/ml in drinking water for 4 days perior to sacrifice. d Paraquat given ip 20 mg/kg, 24 hr prior to sacrifice. e Combination of c and d. fp < 0.05 compared to other preincubation treatments, paired analysis.

resulted in a significantly depresseddesaturaseactivity. The addition of0.5 mM paraquat to microsomesisolated from in viuo paraquat-treated rats resulted in total inhibition of desaturaseactivity (data not shown). Pretreatment of rats with phenobarbital prior to paraquat administration resulted in a partial reversal of the preincubation effect. The percentage decreaseof desaturaseactivity of 43.5 % following the combined treatment was not significantly different from the controls or the phenobarbital treatment alone and represented a significant protection (p < 0.05) over the 71.3 % inhibition observed with paraquat treatment alone.

552

MARKR.MONTGOMERY

Phenobarbital wasthen examined for a possibleinfluence on in vivo paraquat toxicity. Separate groups of 30 rats were injected with either 50 or 35 mg/kg of paraquat. Onehalf of the animals in each group were pretreated with phenobarbital (1 mg/ml) in their drinking water for 4 days prior to injection and then continued to receive the phenobarbitalin the drinking water for the duration of the experiment. Total deathsper group and and time to death were then determined for 14days following paraquat treatment. It was observed that phenobarbital treatment offered no protection against the lethal effects of paraquat at either dosagenor was the time to death altered at the higher dose(Table 8). However, for the lower paraquat dose, a significant (p < 0.05) delay in the lethal effect was noted; the phenobarbital-treated group survived 49 % longer than the untreated group. TABLE EFFECT

OF PHENOBARBITAL

8

ON PARAQUAT-INDUCED

LETHALITY~

Paraquat dosage PhenobarbitaP Died/tested Time to death (hr)” hx/k) 50 13/15 27 f 3 lljl5 + 32 f 4 35 lljl5 45 + 2 +

lo/l5

67 + 5d

n Lethality determined for 14 days after administration of paraquat. b One milligram per milliliter in drinking water for 4 days prior to administration of paraquat and then continued in drinking water for duration of experiment. ’ Data are shown as 2 + SE. Animals which died between 10 PM and 6 AM were scored dead at 6 AM. d p c 0.01 compared to same dosage but not receiving phenobarbital.

DISCUSSION In vivo studieswhich investigate the interaction of chemicalswith fatty acid desaturase activity are complicated by the sensitivity of this enzyme system to alterations in nutritional status of the experimental animal (Montgomery and Holtzman, 1975; Montgomery, 1976).Failure to recognize and account for this nutritional effect may lead to false conclusions concerning the inhibitory or stimulatory mechanism of a given substance. As shown here, paraquat treatment produced a marked inhibition of the pulmonary lipid desaturaseactivity. However, this acute effect may be fully explained by the concomitant paraquat-induced anorexia. The adaptive significance of the slight stimulation of desaturaseactivity observed in animals 14 days after paraquat treatment is currently under investigation. It hasbeen suggestedthat the in vitro effects of paraquat on the microsomal enzymes may be related to an increasedrate of lipid peroxidation (Bus et al., 1975). However, this observation was not supported by the study of Ilett et al. (1974) who actually reported a decreasedrate of microsomal lipid peroxidation in the presence of paraquat. Lipid peroxidation would appear not to be the cause of the decreasedmicrosomal activity observed in this study sincelipid peroxidation could not be observed in the absenceof

PARAQUAT

AND

PULMONARY

LIPID

METABOLISM

553

exogenously added iron. The decrease in enzymatic activity following preincubation of microsomes in the presence of NADPH may be due to the oxidizing influence of the superoxide and peroxide species on microsomal cytochromes and membranes. The severe inhibition of desaturase activity observed with paraquat-preincubated microsomes prepared from animals treated acutely in uivo with paraquat is intriguing, particularly in light of the well-recognized, early manifestations of pulmonary membrane damage which is associated with paraquat exposure. Current theories of paraquat-induced superoxide generation and subsequent hydrogen peroxide formation were supported by the apparent stimulation in formation of both species from lung microsomes in the presence of paraquat added in vitro. However, no stimulation in the generation of either species was observed from animals treated in viva with paraquat. Also, neither 02- nor H,Oz generation was apparently increased when microsomes, prepared from acutely paraquat-treated rats, were incubated in the presence of added paraquat in vitro. This in vitro investigation does not, of course, rule out the possibility of an increased in vivo, intracellular production of these toxic ,oxidants. The partial protection against inhibition of desaturase and prolongation in time to death found with phenobarbital in this study is similar to the protection afforded by pentobarbital against oxidant-induced lung damage seen in oxygen toxicity (Jamieson and van den Bienk, 1964). In both cases treatment with the barbiturate did not prevent the ultimate development of pulmonary toxicity, but rather prolonged the time for development. This type of protection is similar to that offered by intravenous administration of a solution of superoxide dismutase to rats pretreated with paraquat (Autor, 1974). While the mechanism of the protective effect of phenobarbital is not defined, it .apparently is not related to a nutritional influence. The phenobarbital treatment did not alter either the paraquat-induced anorexia or protect against the resultant loss in body weight. Microsomal enzyme induction by phenobarbital is probably not involved, since fatty acid desaturase proceeds through the cytochrome b5 system which is not induced by phenobarbital (Ernster and Orrenius, 1965; Holloway and Katz, 1972; Oshino and Omura, 1973). These studies suggest that paraquat may interact with the pulmonary, microsomal fatty acid desaturase system in a detrimental fashion and that this inhibition of desaturase activity may be separated from the nutritional effects of the paraquat treatment, as detailed in the experiments utilizing preincubations and the experiments with phenobarbital treatment. The possible role of the desaturase system in overall lung biochemistry and maintenance is unclear. However, the observation of in vitro and in vivo protective effects of phenobarbital on desaturase activity and time to death from the paraquat-induced pulmonary toxicity suggests that a relationship may exist but its significance remains to be established.

REFERENCES C., AND TAL, E. (1967).Death from paraquatafter subcutaneous injection. &it. Med. J. 3, 721. AUTOR, A. (1974) Reduction of paraquat toxicity by superoxidedismutase.Life Sci. 14, 1309-1319. ALMOG,

MARKR. MONTGOMERY

554 BULLWANT,

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