Toxwology, 72 (1992) 1-16 Elsewer Scientific Publishers Ireland Ltd

Paraquat damage of rat liver mitochondria by superoxide production depends on extramitochondrial NADH Kei-Ichi Hiral a, Kazuo Ikeda b and Guo-Ying Wang a'* aDepartments of Anatomy and i~Anesthestology, Kanazawa Medwal Umverstty, Uchmada, lshlkawa 920-02 (Japan) (Recewed January 7th, 1991, accepted December 10th, 1991)

Summary Pure rat hver heavy mltochondrlal fractions, m which the absence of s~gmficant m~crosomal contamination was confirmed by electron microscopy and by the lack of glucose-6-phosphatase actLvlty, were used to demonstrate the effect of paraquat on mltochondnal ultrastructure m the presence of external N A D H Starved m l t o c h o n d n a (orthodox conformation) &d not show 02 uptake or structural injury from either paraquat alone or N A D H alone Marked 02 uptake and structural breakage occurred only when paraquat and N A D H were added in combination These alterations were resistant to rotenone and malate plus glutamate or N A D P H could not substitute for N A D H Paraquat was reduced anaerobically by the mltochondrm m the presence of N A D H , but not of N A D P H The addition of superoxlde dlsmutase, ferncytochrome c or p-benzoqulnone protected against the breakage of mltochondrla caused by paraquat plus N A D H These results demonstrate that mltochondrla may produce paraquat radicals in the presence of extramltochondnal N A D H and thus generate superoxlde anion radicals, resulting m structural injury to the mltochondrm, by m e c h a m s m s that may revolve the mltOchondrml outer membrane rather than the electron transfer chain These mltochondrml mechanisms in paraquat toxicity seemed to be more probable m VlVOthan are m~crosomal mechanisms, the latter are postulated to function m detox~cat~on because phenobarbital &tarnished paraquat toxicity and SKF 525-A or cobaltous Jons enhanced the toxicity

Key words Paraquat, Liver mltochondna, Mltochondnal injury by paraquat, Free ra&cal product:on

Introduction The toxicity of paraquat (1,1 '-dlmethyl-4,4'-btpyrtdihum dichloride) to mammals, is mediated by oxygen free radicals produced by its cyclic oxidation-reduction reaction [1]. When paraquat is converted into a radical form, the paraquat radicals produce superoxlde anions (02-) which may or the derivatives of which may cause hpid peroxidation, probably resulting m cell death. Gage [2] and Bus et al. [3-5] Correspondence to Kel-lchl H~ral, Department of Anatomy. Kanazawa Medical University. Uchlnada, Ishlkawa 920-02, Japan *Present address Department of Anatomy, The First Mlhtary Medical Umverslty, Guangzhou, China 0300-48"~X/92/$05 00 © 1992 Elsevier Scientific Pubhshers Ireland Ltd Printed and Published m Ireland

reported that N A D P H - c y t o c h r o m e c reductase anaerobically reduces paraquat m the presence of N A D P H in vitro and, therefore the mlcrosomal fraction (corresponding to the endoplasmlc reticulum) has generally been believed to be the intracellular site of the toxic mechanism [6]. Indeed, several substances are metabolized in the endoplasmic reticulum, such as CC14 [7], naphthalene [8,9], phenobarbital [10], 3-methylindole [11] 1,1-dichloroethylene [12] and bromobenzene [13]. These substances cause hypertrophy or dilation of the endoplasmic reticulum in hver or lung cells, frequently resulting in cell death. Similar structural alterations in the endoplasmic reticulum are speculated to occur in the case of paraquat as well. However, with paraquat this does not seem to be the case, since it does not influence the endoplasmic reticulum structure in vivo as a primary cytotoxic event [14,15] It is known that paraquat accumulates in alveolar type II cells of the lung [16]. Keeling et al. found that paraquat predominantly damages alveolar type II cells [17] and Harai et al. further demonstrated that the initial ultrastructural alterations occurred only in the mltochondria, with swelling and disappearance of mitochondrlal crlstae in type II cells [15]. Mitochondrial alterations were also observed in rat hepatocytes, although only with larger doses of paraquat than those needed to produce alterations In lung cells [18]. It is relevant, therefore, to investigate the mechanism of mitochondriat damage by paraquat. Gage previously reported that the combination of paraquat and N A D H stimulates 02 uptake by mitochondrlal fragments [2]. He, however, concluded that a more probable explanation for the increased 02 uptake is microsomal oxidation, since paraquat alone produces only slight stimulation of respiration with intact hepatic mitochondrla and does not penetrate the mitochondrlal membranes. Despite the inactivity of both external N A D H and paraquat to mitochondrla, in the present study, we found a rotenone insensitive N A D H oxidation resulting in the formation of oxygen free radicals by intact mltochondria only in the presence of paraquat. This oxidation seemed to result in mitochondrial destruction The mechanisms involved were, thus, investigated using isolated rat hepatic mltochondraal fractions only slightly contaminated with mlcrosomes. Materials and methods

Preparauon of mttochondria Male SPF Wistar rats (approximately 250 g) were deeply anesthetized with pentobarbltal. The liver was removed and minced with a small scissors in a cold mannitol solution containing 0.225 M D-mannltol, 75 mM sucrose and 0 2 mM ethylenedlaminetetraacetlc acid (EDTA) [19]. The minced liver (30 g) was gently homogenized in a glass homogenizer with a teflon pestle and then centrifuged at 700 × g for 10 mln at 4°C to remove nuclei, unbroken cells and other non-subcellular tissues. The supernatants were centrifuged at 7000 × g for 20 min These second supernatants were pooled as the crude microsomal fraction and the pale loose upper layer, which was rich in swollen or broken mltochondria, lysosomes and some microsomes, of sediments was washed away. The dark packed lower layer (heavy matochondrial fraction) was resuspended in the mannatol solution and recentrlfuged twice at 7000 × g for 20 min. The heavy mitochondrlal sediments were suspended

m a Tris solution containing 0.05 M Tris-HC1 buffer (pH 7.4), 0.25 M sucrose, 20 m M KCI, 2.0 m M MgC12 and 1.0 m M Na2HPO4, starved for 20 min at 37°C to consume endogenous substrates and cooled again before assay.

Electron microscopy Intact mitochondrla diluted in 1.0 ml Tris solution in a small conical tube were reacted with or without paraquat (methyl viologen, Sigma, St. Lores, MO) for 15-30 min at 37°C in the presence of N A D H , N A D P H (Oriental Yeast, Tokyo, Japan) or malate plus glutamate with agitation. Various free radical scavengers were evaluated: superoxide dismutase (Sigma), p-benzoquinone (Nakarai, Kyoto, Japan), ferricytochrome c (Sigma) and L-ascorbic acid (Kanto Chem., Tokyo). Incubations were stopped by the addition of 3 ml cold buffer. The mitochondria were immediately centrifuged at 7000 × g for 10 min and the packed sediments were covered with 3 ml cold 2% glutaraldehyde in phosphate-buffered saline (PBS) (pH 7.4) and fixed for 6 h. The fixed clots were diced and washed in PBS followed by refixation with 1% OsO 4 for 1 h and block-stained with 0.1 M uranyl acetate for 20 mln. The dice were dehydrated and embedded in Quetol 653 resin. Ultrathln sections were stained with uranyl acetate and lead citrate and examined using a JEM-100C electron microscope.

02 uptake analysts The reaction mixture contained intact mltochondrla or frozen-and-thawed mitochondrla (2-5 mg protein) in the Tris solution located in a closed reaction well (1.2 ml) of an MD-1000 oximeter equipped with a G U - B M oxygen electrode (hjlmaSeimitsu Kogyo, Aichi, Japan). 02 uptake was measured at 37°C in the presence of paraquat, sodium succinate, malate plus glutamate, N A D H , or N A D P H . Inhibition tests were carried out with rotenone (Sigma) [20], Inhibitor-1 (I-l; courtesy of Dr. I. Takagawara, Central Research Institute, Oriental Yeast, Tokyo) [21] and cibacron (Polysciences, PA) [22]. Each reagent (10 /zl) was injected Into the well with a microsyringe.

Anaerobic paraquat reduction The mitochondrial solution was mixed with 2.0 mM N A D H or N A D P H and paraquat at 37°C m the presence of 5.0/xM rotenone, in a Tunberg tube-type reaction cell with a 1.0-cm light pass filled with pure nitrogen gas which was passed through a pyrogallol-KOH solution to absorb 02 contaminants. Changes in absorbance at 395 nm were recorded in a Hitachi U-3200 spectrophotometer by the method of Bus et al. [3].

Glucose-6-phosphatase analysts The assay was based on the method of Nordlie and Araon [23]. The assay mixture contains 40 mM cacodylate buffer, pH 6 5, 30 /zmoles of sodium glucose-6phosphate (Sigma) and 10-50 #g of enzymes In a 1.5-mi aliquot. Incubation for assay was carried out for 10 mln at 30°C and terminated by the addition of trichloroacetic acid. The phosphate content of the supernatant after sedimentation was determined colorlmetrically by a molybdate method.

In vivo liver injury Rats (n= 12) were injected lntrapentoneally (i.p) once with 150 mg/kg paraquat an saline, with 4 controls receiwng saline alone. Three surwving rats receiwng paraquat were deeply anesthetized at 12 h and the livers were fixed by a portal vemperfusion with 2% glutaraldehyde as described previously [18]. Further preparaUons of electron microscopic specimens were carried out in an HEM-480 automatic processmg device (Sakura Fmetechnlcal, Tokyo, Japan). The role of mlcrosomal systems m paraquat toxtcity in vivo We investigated the role of mlcrosomal drug-metabolizing enzyme systems in the paraquat toxicity in VlVO. Male SPF ICR mice (n = 20, approximately 20 g) recewed i p. injections of 100 mg/kg sodium phenobarbital once a day for 2 days. Another 20 mice recewed 50 mg/kg S K F 525-A (Smith Kline Beecham, Middlesex, U.K.) twace a day from 12 h before paraquat administration to 36 h after. The third 20-mice group was gwen 50 mg/kg cobaltous chloride twice a day for 3 days. A normal control group of 20 mice recewed saline. Three groups of 10 mice each were rejected with phenobarbital, S K F 525-A and cobaltous chloride, respectively, as addmonal controls. After (24 h) these injections all animals were injected i.p. with 50 mg/kg paraquat (IDs0 = 45 mg/kg) m sahne. Survival rates were noted for 1 week.

Fig 1 Ultrastructures of rat hver m wvo (a) Control hepatocyte with normal mltochondna (b) Hepatocyte 12 h after paraquat (150 mg/kg, i p ) rejection M. mltochondna, ER, endoplasmlc retlculum × 10 000


Fig 2 Electron mlcrographs of~solated rat hver m l t o c h o n d n a and paraquat damage after 30 m m incubation at 37°C (a) Control starved mltochondna, (b) 3 0 m M paraquat, (c) 2 0 m M N A D H , (d) paraquat m the presence of N A D H , (e) protection by 2800 IU/ml superoxlde dlsmutase against p a r a q u a t - N A D H dependent damage × l0 000


In vzvo injury of hver mttochondria The h e p a t o c y t e s o f the rats receiving 150 m g / k g p a r a q u a t Lp. showed ultrastructural a l t e r a t i o n s in some m i t o c h o n d r i a , n a m e l y a decrease m the electron density o f the matrix, d i s i n t e g r a t i o n o f cristae a n d swelling (Fig. 1). The e n d o p l a s m i c reticulum a n d o t h e r structures were u n c h a n g e d . The results were identical to those prevxously r e p o r t e d [18]. However, these m i t o c h o n d n a l changes were less severe than those in the lungs o f the rats receiving 40 mg/kg p a r a q u a t [15]

The purity of mttochondrzal fractions Electron microscopy The high purity o f the m l t o c h o n d r l a l fractions was

Fig 3 Electron mlcrographs of isolated mltochondna effects of NADPH and malate plus glutamate on paraquat toxlclty (a) 2 0 mM NADPH, (b) 3 0 mM paraquat w~th NADPH, (c) 5 0 mM malate plus glutamate, (d) 3 0 mM paraquat with malate plus glutamate × 10 000

demonstrated by electron m i c r o s c o p y (see Figs. 2 - 4 ) . Contamination by m l c r o s o m a l vesicles appeared quite m i n o r with only a few small rough endoplasm~c reticulum vesicles counted against 50 or m o r e mitochondria. Glucose-6-phosphatase activity N o significant glucose-6-phosphatase activity was detected in the heavy mltochondrial fractions c o m p a r e d with the supernatants which were predominantly rich m m l c r o s o m e s (Table I), suggesting m l m m a l mlcrosomal contamination.

ii~ I ~


~i ¸ ~ ~


ii~i¸¸i:i¸~I!~II~ ~ ~ ~i~il¸i~i~/ ~

Fig. 4 Electron mlcrographs of isolated mltochondrla and effect of rotenone on paraquat damage after 15 mm incubation at 37°C (a) Control starved mltochondrla, (b) 2 0 mM NADH in the presence of 5 0 ttM rotenone, (c) 3 0 mM paraquat plus NADH with rotenone, (d) protection against rotenone msenslUve paraquat-NADH dependent damage by superoxlde dlsmutase × l0 000


TABLE I G L U C O S E - 6 - P H O S P H A T A S E (G-6-Pase) ACTIVITY O F M I T O C H O N D R I A L F R A C T I O N S A N D SUPERNATANTS M~tochondrml fractions and supernatants were incubated w~th glucose-6-phosphate for 10 mm at 37°C Values represent means ± S D of 4 rats (4 experiments)

G-6-Pase specific actwlty (tzmoles G-6-P/mg per mm)

Mltochondrlal fractions


-0 018

0 027 + 0 008

Structural alterations of mitochondrla Effects of N A D ( P ) H and malate plus glutamate on paraquat toxictty.


mitochondria, prevtously starved for 20 mln at 37°C and incubated for 30 min as a blank, showed an orthodox conformation (93%) and contained approximately 6% broken

and swollen mltochondrla

( F i g . 2, T a b l e

II). W h e n

was added to the mitochondria

for 30 min, the conformation



of swollen and broken

endogenous vere breakage


toxicity was markedly

of the mltochondria

increased enhanced

3.0 mM



did not change but the

slightly to 11%. Th~s shght with NADH,

resulting m se-

a t r a t e s o f 2 4 % a t 15 m l n a n d t h e n 6 5 % a t 30 m l n ,

TABLE 11 EFFECTS OF N A D H A N D M A L A T E PLUS G L U T A M A T E ON M I T O C H O N D R I A L P A R A Q U A T TOXICITY Mltochondrla were incubated for 30 mm at 37°C w~th agitation Values represent means + S D of 5 rats Treatments

Control NADH (20mM) Paraquat ( 3 0 mM) N A D H + paraquat N A D H + paraquat (15 mln) N A D P H (20 mM) N A D P H + paraquat Malate + glutamate ( 5 0 mM) Malate + glutamate + paraquat

Orthodox (%)

Condensed (%)

Swollen or broken mltochondna ('V,,)

927 63 89 1 11 2 40 93 9 907 93 8 889

07 ± 05 875 ± 113 04 ± 06 239 + 30 715 ± 53 18± 14 0 7 ± 16 03 ± 05 05 ± 06

57 61 106 647 245 51 86 59 106

Intact m~tochondrm

aSlgmficant difference from control bSlgmficant difference from paraquat alone

± ± + + ± ± ± ± ±

16 04 29 12 I9 54 74 18 12

± 19 ± 15 ± 3 Id ± 42 b ± 53 b ±24 ± 24 ± 19 ± 16 a

TABLE III MITOCHONDRIAL NADH-PARAQUAT TOXICITY IN THE PRESENCE OF ROTENONE Mltochondna were incubated for 15 mm at 37°C Values represent average of 2 rats Treatments

Control NADH + rotenone (50/xM) Paraquat (3 0 mM) + rotenone NADH + paraquat + rotenone



Swollen or broken mltochondrla




927 42 785 180

15 916 138 523

13 42 77 29 7

Intact m~tochondrm

a t whxch t i m e , t h e m a j o r i t y o f i n t a c t m l t o c h o n d r t a e x h i b i t e d a c o n d e n s e d c o n f o r m a tion. NADH alone &d not destroy the mitochondria, but changed them into a condensed conformation. The addmon of superoxlde dismutase sigmficantly protected against NADH-paraquat induced N A D P H (2.0 m M ) a n d m a l a t e mttochondrial structures and did Effect ofrotenone R o t e n o n e a t

mitochondrial destruction. p l u s g l u t a m a t e (5.0 m M e a c h ) d i d n o t a f f e c t t h e n o t e n h a n c e p a r a q u a t t o x i c i t y (Fig. 3, T a b l e II). a c o n c e n t r a t i o n o f 5 . 0 / ~ M , w h i c h is s u f f i c i e n t t o

i n h i b i t t h e o x i d a t i o n o f N A D H b y c o m p l e x I, d i d n o t i n f l u e n c e t h e N A D H - m e d i a t e d m i t o c h o n d n a l c h a n g e s (Fig. 4, T a b l e III). E v e n m t h e p r e s e n c e o f r o t e n o n e , t h e c o m bination of NADH and paraquat caused destruction of mitochondria.

TABLE IV PREVENTION OF MITOCHONDRIAL NADH-PARAQUAT TOXICITY BY SUPEROXIDE SCAVENGERS Mltochondrla were incubated for 15 mm at 37°C Values represent means 4- S D of 4 rats Treatments

Control NADH (2.0 mM) NADH + paraquat (30 mM) + benzoqumone (0 I mM) + SOD (2800 IU/ml) + cytochrome c (0 1 mM) + Vttamm C (30 mM)

Intact m~tochondna Orthodox (%)

Condensed (%)

Swollen or broken mltochondna (%)

6 0 ± 29 294 4- 6 0 483 4- 66 423 4- 62 196 4- 25 4244-95

910 4- 93 407 4- 4 2 467 4- 6 0 493 • 5 6 775 4- 35 305-)-44

30 299 50 84 56 271

aSlgmficant &fference from NADH + paraquat

+ 16 ± 38 4- 2 0 a 4- 3 4 a 4- 17 a 4-53





.o,/ ~



/ - ~ ' /

Fig 5 Paraquat-NADH dependent respiration of intact rat hver m~tochondna NADH or NADPH, 4 0 mM, PQ, 3 0 mM paraquat, ROT, 5 0 #M rotenone

Effects of superoxide scavengers. Mitochondrla were markedly protected against NADH-paraquat injury by free radical scavengers such as superoxide dtsmutase (SOD) (Figs. 2 and 4), p-benzoquinone or ferricytochrome c, but not ascorblc acid (Table IV).





M +G








/ PQ

















F~g 6 Effectof paraquat on NADH respiration of rat hver mltochondna fragments SUC, 5 0 mM succreate; M + G, 5 0 mM malate plus 5 0 mM glutamate, NADH, 2 0 mM, PQ, 3 0 mM paraquat, ROT, 5 0/zM rotenone, I-l, 2 0 raM, CIB, 0 5 mM clbacron, NADPH, 2 0 mM

02 uptake by intact mitochondria 02 uptake by mitochondria was not induced by either N A D H alone or paraquat alone (Fig. 5). This fact that N A D H was not oxidized in these fractions proved the intactness of the isolated mltochondnal structures. In contrast, the combination of N A D H and paraquat apparently induced O 2 consumption. In this case, when 02 was completely consumed, the reaction mixture appeared blue suggesting the productlon of paraquat radicals. Rotenone did not inhibit the reaction. N A D P H in combination with paraquat did not stimulate 02 uptake.

02 uptake by mltochondrial fragments Paraquat did not affect the oxidation of succinate and malate plus glutamate in mitochondrial fragments (Fig. 6), N A D H induced 02 uptake, which was inhibited by rotenone, cibacron and I-1. Another N A D H oxidation resistant to these inhibitors occurred with paraquat. When N A D H was replaced by N A D P H , no 02 uptake occurred. The mltochondria preheated for 1 min at 100°C showed a complete loss of reactivity

Anaerobic reduction of paraquat Intact mitochondria anaerobically reduced paraquat with N A D H but not with N A D P H (Fig. 7). The former reduction reaction was not sensitive to rotenone.



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Paraquat damage of rat liver mitochondria by superoxide production depends on extramitochondrial NADH.

Pure rat liver heavy mitochondrial fractions, in which the absence of significant microsomal contamination was confirmed by electron microscopy and by...
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