Comparative studies of paraquat in teractions with fish and bovine oxy- and deoxyhemoglobins

A C T A U N I V E R S I T A T I S L O D Z I E N S I S

F O L IA B IO C H IM IC A E T B IO P H Y S IC A 9, 1992

W irgiliusz Duda

C O M P A R A T IV E S T U D IE S O F P A R A Q U A T IN T E R A C T IO N S

W IT H F IS H A N D B O V IN E OX Y - A N D D E O X Y H E M O G L O B IN S

In this stu dy it h a s been d e m o n s tra ted th a t in o xy he m og lo bin s olu tio n s p a r a q u a t in duces a d ecreas e in th e c o n te n t o f this fo rm o f he m og lo bin follow ed, a fte r som e tim e d elay, by a n incre ase in th e m e tH b c o n te n t. T hese d ifferen ces ha ve been tentativ ely e xp lain ed till now by differences in th e c o o p era tiv e p ro p ertie s o f h em o glob in s (e.g. c arp an d bovine H b). C a rp he m og lo bin ex hib its the so-called R o o t e ffe c t‘w hile bovine he m og lo bin (like o th e r m a m m alia n H bs) is ch arac te riz ed by the so-called B oh r effect. H ow ever, a t th e p re se nt sta te o f in vestigations, such a n e x p la n a tio n c an a cc o u n t only fo r a sm all p a rt o f the effect o f p a ra q u a t on he m o glob in solution s . T h e m o s t im p o rta n t res ult o f th is stu dy is the d e m o n s tra tio n th a t the o x y H b so lu tio ns e xam ine d in the presen ce o f p a ra q u a t a re ab le to co n v ert n o t only in to the m e tH b fo rm b u t th a t o xy H b (H b F e 2 + ) u nd e r the influe nce o f g en era ted by p a ra q u a t active fo rm s o f oxygen an d ra dicals (in clud ing 0 2) m ay be co n verted in to a n o th e r u ns ta b le o x y H b fo rm (ferrylH b fo rm , H b F e 4 + , de scribe d als o as the p o rp h y rin Fe 4 + -rr-cation). Such an in te rp re ta tio n o f th e discussed effect is c on firm e d by th e o b ta in e d s pe ctra o f h em og lo bin s olu tions , especially by sp e c tra o f oxy-, deoxy -, m e tH b a n d R a m a n sp ectra .

1. IN T R O D U C T IO N

P ara q u a t (P Q, 1,1 ’-dim ethy l-4,4’-bipyridylium ion) is a pow erful herbicide

which shows co n tac t an d system ic activity. M ichaelis an d Hill [9] described in

1933 its redox pro p erties an d since th at tim e it has been used as a „redox

in d ic ato r” b etter kn ow n for chem ists u nd er the nam e o f „m eth yl viologen” .

O ne electron reductio n o f the PQ m olecule leads to the relatively stable catio n

radical form ation . This process is related to the tra nsfer o f one o f the p air o f

electrons fro m the bip yridylium group. As a result, the reso nan ce fo rm o f the

co m p o u n d is created. These free radicals have p oten tial ability to react with

oxygen (w hat leads p ro b a b ly to generatio n o f superoxide anion radical) with

the sim ultaneou s reo xid ation o f its ionic form .

P r i m a r y m e c h a n i s m o f P a r a q u a t t o x i c i t y . P ara q u at has the

ability to un dergo a one-electron redu ction from the cation to form a stable

blue colo u red free radical in the absence o f oxygen [9], In the presence o f

oxygen the radical will im m ediately reform the catio n with the co nc om itan t

p ro d u ctio n o f superoxide anion (O

2

). This reactio n between p a r a q u a t radical

an d oxygen is so rapid th a t it is diffusion-lim ited [5]. Th us, prov ided there is

a co n tin u o u s su pply o f electrons to PQ, an d oxygen is present, PQ will rapidly

cycle fro m its oxidized to reduced form w ith the co n tinu o u s p ro d u c tio n o f O

2

.

G age [

6

] first repo rted th a t in cells un der ana ero b ic cond ition s, N A D P H

to g ether w ith a flavo protein could reduce PQ from its cation to radical. U n d er

aero bic con d ition s the radical is reoxidized and this redox cycling co ntin ues

until available N A D P H is consum ed.

Ist step

0

;

IInd step

°2 + °2

then ^2^2 + O9

2H

- > H 2° 2

-> 0 H ‘ + OH" + 0 ,

II I rd step

R eaction o f H

2

O

2

a n d /o r free radicals (i.e. m ainly O

2

, O H )

with hem oglobin.

The stu dies o f G a ge [

6

] were extended by Baldwin et al. [1] w ho

d em o n stra ted th a t m icrosom al p re p ara tio n s from liver, lung an d kidney were

able to generate rad icals o f PQ and eventu ally in the presence o f H b prod uce

H

2

O

2

. Th e p ro d u ctio n o f O

2

an d H

2

O

2

m ay then lead to the for m a tio n o f m ore

reactive oxygen radicals, which in tu rn m ay be m ore toxic to the cell and

functio n o f respiratory p rotein s (hem og lo bins) [2]. T hu s the cycling o f PQ

from its reduced to reoxidized form provid es a plausible p rim ary m echanism o f

its toxicity th at is entirely ana log o u s with the p ro posed m echanism o f the

hem og lobin toxicity o f p a raq u at. PQ toxicity on m am m alian organ ism s is

d ifferentiated w hat depend s on an absorb ed dose and a kind o f tissue on which

it acts. H owever, a toxic dose, regardless o f the m ethod o f ingestion (per os or

intrav enou s), causes a sim ilar com plex o f toxic sym ptom s. The presence o f PQ

a t a dose o f 0.2 ¿tg/ml in hu m an serum is a lethal dose (d uring 24 h). F o r PQ

doses o f over

10“ 5

m ol

/1

in hib ition o f m itoch on drial o xid ation was observed.

M ichael and Levis [

8

] rep orted th a t the m ost significant cum u lative effects

were given by th e range o f 1 0 ~ 5- 10

~ 4

mol/1 o f PQ co n cen tration w hich were

the best visible from h a lf an h o u r till two h o urs afte r th e dose ad m inistratio n

in to an isolated biological system.

2. M A T E R IA L S A N D M E T H O D S

1. Bovine H bA was iso lated using th e m etho d o f Rigss, B ona Ventura and

B o nav en tu ra [14] an d purified acco rdin g to T en to ri et al. [17].

2. Fish (C arp ) H b was isolated using the m etho d o f Lin et al. [7].

3. The protein s stud ied were oxidized with ab o u t two fold excess o f

K.

3

[Fe(CN )(J w hich w as sub sequently rem oved by a S ephadex G -25 colum n

ch rom atog rap hy .

4. P Q p.a. was p urchased from Sigm a (U .S.A .).

5. All o th e r chem icals w ere o f analytical grade an d used w itho u t furth er

p urificatio n, an d double-distilled, deionized w ater w as used th ro u g h o u t the

experim ents.

R eaction conditions:

- co n cen tration o f H b 1.8 m g/m l in respect to on e hem e,

- p a ra q u at co n cen tration o f 5,10, 25 an d 50 /ig/m l o f H b,

- in cu batio n d u rin g 0 -5 hrs in 0.2 M p ho sph ate buffer, p H 7 .0 -7.1, at

tem p eratu re 37° C.

A nalytical m ethods:

- sp ectrop ho tom etry,

-

m easu rem ents o f ab so rp tio n spectra (cycle) in a UV-Vis range (SPE-

C O R D U V-V IS an d S P E C O R D M 4 0 C arl Zeiss Jen a),

---R am an resonance sp ectra were p erfo rm ed th a nk s to kindnes o f D oc.

J. Tw ardow ski fro m Jagiello nian U niversity - C rac ow a n d Prof.

K. G e rson de from R heinisch-W estfälische Technische H och schu le

- A achen.

3. R E S U L T S A N D D IS C U S S IO N

The influence o f PQ on chan ges o f ca rp (C iprin us carp io) and bovine (Bos

ta u ru s

the Low land B lack-an d-W h ite cows) oxy- an d deox yhem oglobin was

investigated. Th e reactio n m ixture co n tain ed oxy- o r deox yH b (co ncentration :

0.0018 ± 0.001 g H b /m l in N a H

2

P 0

4

: N a

2

H P 0

4

buffer, pH 7.1 ± 0.02 an d

different doses o f P(^ - 5, 10, 25 and 50 ppm ). The in teractio n s o f PQ with Hb

were analysed spec tro ph o to m etrically where changes in the visible range o f

ab so rp tio n sp ectra o f H b in the presence o f PQ were com p ared w ith these o f

co n tro l sam ples w ith ou t PQ. T he solu tio n o f oxyH b con tain ing PQ show ed the

decreasing ab so rp tio n m ax im a a t 540 an d 570 nm an d the parallel increasing at

500 an d 630 nm w hat seems to be due to m etH b form ation (Fig. I and Tab le

1). T he q u an titativ e enh ancem en t o f the m etH b level un der these co nd itio ns

was alw ays observed con siderab ly later in the relation to „th e loss” o f th e oxy

form . T he degree o f the d isap perance o f ox yH b was also positively correlated

w ith th e increase o f doses o f PQ. Instead, fo r the sam e doses o f PQ, the m etH b

T a b l e I

P erc en t de cre as e o f A ) c a rp o x y H b a n d B) bovine o x y H b ind uce d by p a ra q u a t (25 a n d 50 ppm ). All c o n d itio n s the sam e as in Fig. 1

A ) B) 25 ppm PQ 50 ppm PQ X % X % O' — 4.6 5’ 2.2 8.6 15’ 3.3 12.8 30’ 5.0 18.7 1 h 8.3 24.5 2 h 17.2 32.3 3 h 22.7 36.8 4 h 2 6 .5 ' 42.2 5 h 28.0 47.7 25 p p m PQ 50 pp m PQ X % X % 0’ _ _5.5 5’ 2.0 14.5 15’ 6.2 23.5 30’ 9.8 34.6 1 h 14.5 45.0 2 h 24.6 55.0 3 h 34.5 62.0 4 h 37.5 67.9 5 h 39.6

-X % - M e an values fo r n = 20 sam ples, p erce n t w ith re spec t to th e c o n tro l H b = 100% oxy H b .

form ation in the case o f bovine H b was tim e-depen dent and linear in the

ch arac te r, co n tra ry to the fo rm atio n o f fish m etH b which du rin g the sam e time

increased ex ponen tially (T ab le 2 an d Fig. 2). C arp H b form tran sfo rm a tio n

Fig. 1. P a ra q u a t - in du ce d c h an ge s in a b s o rp tio n s pe ctra o f o xy he m og lo bin in the X re gion o f 22 0 0 0 -1 4 000 cm i. O x y H b c o n ce n tra tio n : 0.0018 g/rnl in 0.2 M p ho s p h ate buffer. pH 7.1. P a ra q u a t c o n c e n tra tio n : 50 p pm . A ) C a rp ox y H b , tim e: 1 - 0 ’ (o x y H b ). 2 - 0 ’ (o xy H b + PQ), 3 - 5 ' (o x yH b + PQ ), 4 15’ (o x y H b + PQ ), 5 30' (o xy H b + PQ ), 6 - 1 h (o x y H b + PQ ), 7 2 h (o x yH b + PQ ), 8 3 h (o x yH b + PQ ); B) Bovine o x yH b , tim e: 1 0’ (o x yH b), 2 0 ’ (o xy H b + PQ ), 3 - 5 ’ (o xy H b + PQ ), 4 15’ (o x yH b + PQ), 5 - 30’ (o xy H b + PQ ). 6 - 1 h (o xy H b + PQ ), 7 2 h (o xy H b + PQ ), 8 - 3 h (o x yH b + PQ ), 9 4 h (o xy H b + PQ ), 10

- 5 h (o x y H b + PQ)

app eared to be m o re susceptible to the influence o f PQ th a n bovine H b. So, the

m o re consid erable level o f m e tH b occured in solu tio n o f ca rp H b th a n ot

bovine H b. This m ay be explained thro u gh the existence o f the differences

T a b l e 2

Pe rcen t incre ase o f A ) c a rp m e tH b a nd B) b ov in e m e tH b ind uced by p a r a q u a t (25 a nd 50 ppm ). All c o n d itio n s the sam e as in Fig. 1

A) B) 25 ppm PQ 50 pp m PQ X % X % O’ - -15’ 6.6 10.8 30’ 12.7 25.3 1 h 22.8 44.7 2 h 32.0 63.2 3 h 42.0 86.3 4 h 47.0 -5 h - -25 ppm PQ 50 pp m PQ X % X % 0 ’ _ _ 15’ - -30’ - 10.1 1 h 4.9 18.7 2 h 14.5 25.6 3 h 24.6 35.2 4 h 29.1 43.3 5 h 33.9 54.4

X % M ea n values fo r n = 20 sam ples, p erc en t with re spec t to th e c o n tro l H b = 100% m etH b .

Fig. 2. G ra p h ic re p re se n ta tion o f th e incre ase in A ) c a rp m e tH b a n d B) b ov in e m e tH b for a p a ra q u a t d os e o f 25 a n d 50 pp m

Fig. 3. P a ra q u a t + 0 2 in du ced ch ange s in a b s o rp tio n spe ctra o f de ox yh em og lo bin in the

X region o f 22 000 14 000 cm '. D e o xy H b c o nc e n tra tio n: 0.0018 g/m l in 0.2 M p h o s p h ate buffer, pH 7.1. P a ra q u a t c o n ce n tra tio n : 25 ppm . A) C a rp de oxy H b : 1 o xy H b, 2 - de ox yH b, 3 - d e o xy H b + PQ , 4 d e o xy H b + PQ + 2 c m 3 0 2, 5 d e o xy H b + P Q + 4 c m 3 0 2. 6 d e o x yH b + PQ + 6 c m 3 0 2, 7 d e o x yH b + PQ + 8 c m 1 0 2, 8 d e o xy H b + PQ + 10 cm-’ 0 2, 9 - d e o x yH b + PQ + 12 c m 3 0 2, 10 m etH b ; B) Bovine de ox yH b : 1 - o x y H b , 2 - de ox yH b , 3 d e ox y H b + PQ , 4 d e o x y H b + PQ + 5 c m 3 0 2, 5 d e ox y H b + PQ + 10 c m 3 0 2, 6 d e o xy H b + PQ + 15 c m 3 0 2, 7 d e o xy H b + PQ + 20 c m 3 0 2, 8 d e o x yH b + P Q + 25 c m 3 0 2, 9 d e o x y H b + PQ + 30 c m 3 0 2, 10 m e tH b

between th e co op erative effects m ech anism in these tw o H b s [4, 12, 13, 15]. It

was also fou nd th at P Q inhibited the deoxy- to o xy H b tra nsitio n (Fig. 3). The

am o u n t o f created o xy H b was alw ays significantly low er than in the case o f

co n tro l sam ples. The inh ibitio n o f this tran sition in the p re se n ce ,o f oxygen

d ep ended on the doses used in th e ex perim ent (un der the co n stan t H b

con cen tration ). The degree o f this in hibitio n was again better visible for fish

H b, even in th e presence o f m uch low er doses o f PQ. V alues o f P

50

O

2

p ar am e ter were also calculated as th e in d icator o f changes o f oxygen affinity

induced by P Q in th e ran ge o f doses o f 5-50 ppm (Table 3). It was show n th a t

T a b l e 3

D e creas e o f pso O j value s (o xy g en ation p a ram e te r) ind uc ed by p a ra q u a t. All c o n d itio n s th e sam e as in Fig . 4

A ) C a rp H b Pso O2 (m m H g ) C o n tro l 5 ppm PQ 10 ppm PQ 25 pp m PQ X 5.75 3.05 1.56 0.78 S.D. ± 1 .0 6 ± 0 .7 5 ± 0 .6 6 ± 0 .3 8 B) B ovine H b Pso 0 2 (m m H g ) C o n tro l 10 ppm PQ 25 pp m PQ 50 pp m PQ X 18.62 15.36 12.57 9.04 S.D . ± 1 .3 4 ± 0 .8 0 ± 0 .9 8 ± 1 .01 X - M e a n values; S.D . - S ta n d a rd de viation .

values o f this p aram eter increased w ith the increase o f th e dose. These results

indicate th a t tran sfer o f ox yH b to its m et form goes th ro u g h add ition al

in term ed iate(s) p ro ba bly ferry lH b [3, 10, 20]. A s it has been show n the action

o f PQ on H b solu tion (as well as on o th e r h em op ro tein s [16, 18]) con tain ing

high a m o u n t o f dissolved oxygen, is con nected w ith th e p rod u c tio n o f m ore

active species o f oxygen, especially O

2

radicals. It is suggested th at du rin g this

ra th e r com plex and com plicated reaction an u nstab le p orp h y rin F e

4

+-

7

i-cation

(i.e. ferryl derivative) is form ed [11], W alters an d F rederick [19] in their w ork

have even hypothesised th e direct o xy d ation o f M b (m ioglobin) resu ltin g PQ

14000 cm’1

\ =

22

\ = 18000 cm

Fig. 4. Visible sp ec tra in the ra n ge o f 22 000 14 000 cm 1 o f 1) 0 .5 % bovin e m etH b , 2) 0.5 % bo vin e m e tH b + 10 ppm PQ , 1 h inc u b a tio n (m eas ure d with respec t to c o ntro l: p h o s p h a te buffers,

pH 7.0, plus 10 ppm p a ra q u a t), a n d 3) differen ce sp ec tru m o f (2) w ith respec t to (1)

Fig. 5. R e so na nc e R a m a n s pe ctra o f A ) m e tH b a n d B) ferry lH b , us ing 406.7 nm e x cita tion in the 250 to 600

cm I

action as a tran sfer reaction M b F e 2+ -* M b F e 4+ alth o ug h the details o f

m echanism o f this reactio n rem ains to be explained. On the o th er hand o u r

pilot studies using R am an spectroscopy m etho ds d em o n strate evidences o f the

existence o f the ferryl deriv ative in so lutio n co ntain in g PQ (Fig. 4 and Fig. 5).

This stu dy does n o t ex hau st the problem and requires fu rther investigations o f

the behav io r o f p a ra q u at itself in hem oglobin solu tions. It seems th at the m ore

im p o rta n t result o f this w ork is th a t p a ra q u a t com m only applied in so lu tio ns

as a herbicide p re p ara tio n (u nd er the nam e G ram o xo n e) is a co m p o u nd

severely toxic fo r anim als an d hum ans.

This w ork was su p p o rted by Research G r a n t - Subject II. 11.1.1.

4. R E F E R E N C E S

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C am e in ed itorial office C h a ir o f Biophysics

„ F o lia biochim ic a e t b io p h ys ica ” U niv ersity o f Ł ó dź 25.06.1991 .

W irgiliusz Duda

B A D A N IA P O R Ó W N A W C Z E IN T E R A K C J I P A R A K W A T U Z O K SY - I D E O K S Y - H E M O G L O B IN Ą R Y B Y I B Y D Ł A

W y ko na ne b a d a n ia pozw oliły w yk azać , że p a ra k w a t w ro z tw o ra c h hem og lo biny po w o du je zm nie jszenie się p oz io m u fo rm y o k sy H b , z jedn o cze sn ym (w tym sa m ym czasie) w zrostem po zio m u form y m etH b . Stw ie rd zo no rów nie ż is to tn e różnice w od d zia ły w an iu p a ra k w a tu z hem oglobin;! k arpia i kro w y, co w części zw iązan e je s t z k o op eraty w ny m i w łaściw ościam i tych hem oglo bin . K o op e ra ty w n oś ć he m og lo biny k a rp ia zac ho dzi zgodnie z efektem R o o ta , a ko o p era- tyw no ść hem og lo biny krow y (p o d ob n ie ja k u po z os tałych ssak ów ) op isy w a na jes t po przez efek t B ohra. T a k ie w yjaśnienie, na p od staw ie ob ecneg o s ta n u b a d a ń, tłu m acz y tylko niew ielką część wpływu p a ra k w a tu na he m og lobinę. N ajw aż niejsz ym w ynikiem o bec nyc h b a d ań je s t w yk aza nie , że o k s y H b w ro z tw o ra ch w o bec noś ci p a ra k w a tu prze ks z tałc an a je s t nie tylk o w fo rm ę m e tH b , ale rów nież o k s y H b (F e2 + ) m oże p rz ech od zić p od wpływ em ak tyw n ych form tlen u i ge nero w a nyc h przez p a ra k w a t rod nikó w , d o in nych „wyżej u tlen ion y ch ” n ies tabilnyc h fo rm h em oglo bin y zaw ierającej tlen, np. d o w yk azan ej fo rm y ferry lH b ( F e 4+ ), opisyw an ej tak że ja k o p orfiry na F e 4+-7t-kation. T a k a in te rp re ta cja pow yższego efektu w pływ u p a rak w a tu na he m o glo b in ę jest p os tulo w a n a na po d sta w ie b a da n yc h w idm ro z tw o ró w oksy- deo ksy - i m e tH b o ra z w idm a R a m a n a.