Biological Trace Element Research Volume 51 Issue 1 1996 Laura Tallandini; Rodolfo Cecchi; Silvia de...

7/29/2019 Biological Trace Element Research Volume 51 Issue 1 1996 Laura Tallandini; Rodolfo Cecchi; Silvia de Boni; Silvio

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~C o p y r i g h t 1 9 96 b y H u m an a P re s s Inc .A l l r ig h t s o f an y n a t u re w h a t s o ev e r r e s e rv ed .0163-4984/96/5101-0097 $07.50

T o xic L e v e ls o f S e l e n i u m in E n z y m e sa n d S e l e n i u m U p t a k e in T i s s u e so f a M a r i n e F i s hLAUR A TALLANDINI,1 RO DO LFO CECCHI, 2 SILVIA D E BONI, 1

SILVIO GA LA SSINI,3 GRA ZIA GHERMANDI,G I A N C A R L O G I A L A N E L L A , 5 N IA N Q I N G L I u , 6 R E N A T A M O R O , *'5

M A R G H E R I T A T U R C H E T T O , 1 A N D Y U A N X U N G Z H A N G 7'Dipar t imento di Biologia , Univers i t~ d i Padova, Padova, I ta ly;2Dipar t imento di Sc ienza de l l ' lngegner ia , Univers i td l d i Modena,Modena, I taly; 3ls t i tuto di Chimica Biologica, Universi t~di Ve ron a, Ve ron a, I taly; 40 sserv ato rio Geofis ico, Universit ,~di M ode na, M ode na, I ta ly; 5Dipart imento di Sc ien ze Fisiche ,U niversi t~ di N ap oli "Federico II ," Napoli , I taly; 6LaboratoriNaz ional i de l l ' lNFN, Legnaro (Padova) , I ta ly and Ins t i tu te

o f H i g h E n e r g y P h y s i c s, A c a d e m i a S i n ic a , B e i ji ng , C h in a ;and 7ls t i tu to Naz ionale d i Fis ica Nucleare , Sez ione di Napol i ,I ta l y a n d S h a n g a i In s ti tu t e o f N u c l e a r R e s e a r c h ,S h a n g a i , C h i n aA B S T R A C T

A c u t e t o x i c i t y o f s e l e n i u m a s s e l e n i t e i n Zosterisessor ophio-cephalus b y i p i n j e c t i o n w a s s t u d i e d . T h e 5 0 % l e t h a l d o s e a n d 5 0 %l e th a l t im e w e r e m e a s u r e d t o b e 0 .2 9 p p m a n d 9 6 h , r e sp e c ti v e ly . S ec o n c e n t r a t i o n s i n l iv e r , g i ll , s k i n a n d m u s c l e , a n d C y t . P 4 5 0 l e v e l, Se -G P x , a n d T o t a l G P x e n z y m e a c t i v i t i e s i n l i v e r w e r e a l s o a s s e s s e d a td i f f e r e n t d o s e s a n d t i m e s a f t e r in j e c ti o n . S t a r t i n g a t 0.3 p p m i n j e c t e dd o s e , e n z y m e a c t i v i t i e s a n d S e c o n c e n t r a t i o n i n t i s s u e s , b u t n o t i nm u s c l e , s h o w e d s i g n if i c an t d i ff e r e n c e s f r o m t h e c o n t r o l g r o u p . At h r e sh o l d b e h a v i o r w a s i n fe r re d . N o r m a l c o n d i ti o n s o f e n z y m e a c ti v-i t i e s a n d S e c o n c e n t r a t i o n i n t i s s u e s w e r e r e s t o r e d a b o u t 1 w k a f t e ri n je c t io n . B i o lo g i c a l e l i m i n a t i o n h a l f - l iv e s w e r e a b o u t 2 d f o r l iv e r a n dgi l l , and 5 d fo r sk in .

I nde x Ent r ie s : S e l e n i u m t o x i c it y ; f is h ; 5 0 % l e t h a l t i m e a n d l e t h a ld o s e ; e f f e c t o n C y t . P 45 0; S e G P x a n d t o t a l G P x e n z y m e s ; S e ti s s u ec o n t e n t s .

* A u t h o r t o w h o m a l l c o r r e s p o n d e n c e a n d r e p r in t r e q u e s t s s h o u l d b e a d d r e s s e d .B i o l o g ic a l T r a c e E l e m e n t R e s e a r c h 97 Vol. 51 , 1996


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9 8 T a l l a n d i n i e t a l .I N TRODUCT ION

S e l e n i u m i s b o t h a t o x i c a n t a n d a n e s s e n t i al m i c r o n u t r i e n t f o r f is h asi t i s f o r m o s t an im a l s . Th e d i sco v e r y ( 1 ) t h a t S e i s a n i n t e g r a l c o m p o n e n to f t h e s e l e n iu m e n z y m e g l u t a t h i o n e p e r o x id a s e p r o v i d e d a n e x p l a n a t io nf o r t h e p h y s io lo g ica l r o l e o f t h i s e l em en t . Ho wev e r , e l ev a ted l ev e l s o f Sew e r e f o u n d t o b e l i n k e d t o d r a s t ic d e c l i n e s i n fi s h p o p u l a t i o n s ( 2 ) . I t w a sh y p o t h e s i z e d t h a t S e b i o a c c u m u l a t i o n t h r o u g h t h e f o o d c h a i n l e d t o S el e v e l s h i g h e n o u g h t o i m p a i r r e p r o d u c t i o n . L a t e r , t h i s r e s u l t w a s c o n -f i r m e d i n f i s h ( 3 ) a n d i n a q u a t i c b i r d s ( 4 ) . F u r t h e r m o r e , i t w a s o b s e r v e dt h a t S e i n a q u a t ic e c o s y s t e m s b e c o m e s t ox ic a t s l i g h t l y h i g h e r t h a n n u t r i -t i o n a l l ev e l s ( 5 ) .T h e a b s o r p t i o n , d i s t ri b u t i o n , a n d e l i m i n a t i o n o f Se r e c en t ly h a v e b e e ni n v e s t i g a t e d i n b o t h f ie l d a n d l a b o r a t o r y s tu d i e s. A d a m s a n d J o h n s o n ( 6 )f o u n d h ig h e r b io co n cen t r a t io n f ac to r s f o r Se in f i sh ex p o sed in n a tu r a le n v i r o n m e n t a s c o m p a r e d t o t h o s e i n l a b o r a t o r y s t u d i e s , p r o b a b l yb e c a u s e o f t h e p r e s e n c e o f o r g a n i c s e l e n i u m c o m p o u n d s , r a t h e r t h a n o fin o r g an ic se l en i t e o r se l en a te , i n n a tu r a l sy s t em s . Lab o r a to r y s tu d ie s we r ed ev e lo p ed f o r wa te r b o r n e ex p o su r e s , i p i n j ec t io n , an d d i e t a r y r o u te s o fa d m i n i s t r a t i o n . I n w a t e r b o r n e e x p o s u r e , s e l e n i u m w a s f o u n d t o b e l e t h a li n r a i n b o w t r o u t a t c o n c e n t r a t i o n s r a n g i n g f r o m 2 t o 1 6 m g / L a n d S er e s id u es we r e f o u n d in c r ea sed in th e f i sh t i s su es a s Se ex p o su r e in c r ea sed(7 ). I n th e sam e f ish , t h e t i s su e d i s t r ib u t io n o f Se ip ad m in i s t e r ed w as d i f-f e r en t f r o m th a t o b se r v ed f o r Se ad m in i s t e r ed in th e d i e t ( 8 ) . Selen i ter e su l t ed s ig n i f i can t ly m o r e to xic th an se l en a te ( 9 ) . Mo st o f t h e se s tu d ie sh a v e s h o w n t h e l iv e r a n d k i d n e y t o b e m a j o r s i t es o f s e l e n it e d e p o si t io n .S t u d i e s o n t h e m e t a l e l i m i n a t i o n f r o m t i s s u e s h a v e b e e n c a r r i e d o u ta f te r o r al a d m i n i s t r a t i o n a n d i p i n j e ct io n a t s l ig h t l y h i g h e r t h a n n u t r i -t i o n a l l ev e l s ( 1 0 , 1 1 ) . Af te r o r a l ad m in i s t r a t io n , e l im in a t io n o f Se f r o ml i v e r a n d k i d n e y w a s f o u n d t o b e b i p h a s i c f or m o s t c o m p o u n d s . B o t h t h el i v e r a n d k i d n e y e x h i b i t e d a p e r i o d o f r a p i d e l i m i n a t i o n , f o l l o w e d b y ap e r i o d o f m o r e g r a d u a l e l i m i n a t i o n ( 1 0 ) . T h e b i p h a s i c n a t u r e o f t h e e l im -i n a t i o n s u g g e s t s t h a t m o r e t h a n o n e m e t a b o l i c p o o l i s i n v o l v e d .L i tt le is k n o w n a b o u t t h e m e c h a n i s m s o f S e r e d i s t r ib u t i o n a n d e l im -in a t io n a t se l en i t e acu te an d su b acu te d o ses . Th e r e f o r e , we d ev e lo p ed th ep r e s e n t w o r k i n o r d e r t o :

1 . d e t e r m in e th e acu te to x ic i ty o f Se a s se l en i t e i n Z o s t e r i s e s s o ro p h i o c e p h a l u s by ip in jec t ion ;2 . m e a s u r e t h e S e t i s s u e d i s t r i b u t i o n a n d e l i m i n a t i o n r a t e a td i f f e r en t Se d o ses ;3 . observe the Se e ffec ts on d e tox i fy in g enz ym at ic sys tem s; and4 . ch eck th e r eco v e r y o f p h y s io lo g ic a l a c t iv i ti e s i n p a r a l l e l w i thSe e l im in a t io n .

T h e c h o i c e o f t h e o r g a n i s m t o b e e m p l o y e d w a s m o t i v a t e d b y t h ein c r ea s in g u se o f f i sh a s t e s t o r g an i sm s , i n t h a t t h e i r m e tab o l i c r e sp o n sesB i o l o g ic a l T r a c e E l e m e n t R e s e a r c h V o l. 5 1 , 1 9 9 6


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S e l e n i u m T o x ic i ty i n F i s h 9 9c lo s e ly re s e m b l e t h o s e i n m a m m a l s . I n a d d i t io n , e x t en s iv e k n o w l e d g e o ft h e b i o l o g y a n d b i o c h e m i s t r y o f t h i s f i s h w a s a c q u i r e d a t t h e B i o l o g yD e p a r t m e n t o f th e U n i v e r s it y of P a d u a o v e r t h e p a s t y e a r s.

M A T E R IA L S A N D M E T H O D SA d u l t Z. ophiocephalus (80% ma le s ) w i th m ea n w e igh t o f 41.7 _+ 1 .2 ga n d m e a n l e n g t h o f 1 5 . 6 +_ 0 .2 c m , w e r e c o l le c t ed i n t h e N o r t h e r n b a s i no f th e l a g o o n o f V e n ic e (l o w p o l l u t i o n l ev e l a re a ) a n d k e p t a t t h e H y d r o -b i o l o g i c a l S t a t io n o f t h e U n i v e r s i t y o f P a d u a i n C h i o g g i a . F is h w e r e a c cl i-m a ted i n g l a s s t ank s w i th rec i rcu l a t i ng mar in e w a te r (T = 21 .0 _+ 0 .2 ~pH = 8 .2-8 .3 , 32 pp t sa l i n i t y ) fo r 15 d be fore t he exp e r im ent an d fed d a i l yw i t h m o l l u s ca n c e p h a l o p o d s (Se pia o f f ic inal is) .A f t e r a c c li m a t i o n , f i sh w e r e t r a n s f e r r e d t o n e w t a n k s w i t h c o n t r o l le dw a t e r b e f o r e b e i n g i n j e c t e d w i t h S e . F i s h p r e v i o u s l y a n e s t h e t i s e d w i t hMS 2 22 S i g m a w e r e t r e a t e d w i t h a s i n g le i p i n j e c ti o n o f s o d i u m s e l e n it e(Na2SeO3) d i s so lved i n ph ys io log i c a l so lu t i on a t s even do ses f ro m 0 .1 t o1 .0 p p m (~ tg S e / g b o d y w t ) f o r s t u d y i n g l e t h a l e f fe c ts a n d u p t o 0 .5 p p mf o r e n z y m e - r e c o v e r y a n d t i s s u e - d i s t r i b u t i o n a n a l y s i s . C o n t r o l s w e r ei n je c t e d w i t h p h y s i o l o g i c a l s o l u t i o n o n l y. A b o u t 2 0 f is h w e r e s a m p l e d a teach i n j ec t ed dose .F i s h tr e a t e d a t d i f f e re n t d o s e s w e r e k e p t i n s e p a r a t e w a t e r t a n k s . T h ef is h s u r v i v i n g t h e i n j e c ti o n w e r e s a c r if i ce d b y s p i n a l t r a n s e c t i o n a t t h e

base o f the h ead a t 4, 8 , an d 28 d a f t e r t he i n j ec t i on t o a s say P450 l eve l ,e n z y m e a c t iv i ti e s a n d S e c o n c e n t r a t i o n i n t i ss u e s. T h e f i sh e x h i b i t i n gc l ea r i nd i ca t i ons o f suf fe r i ng a f te r i n j ec t i on we re co l l ec t ed be fore d ea th i no r d e r t o a s s a y e n z y m e l e v e l s . T h i s g r o u p w i l l b e m e n t i o n e d h e r e a f t e r a s" n o n s u r v i v i n g f is h . " T h e s a m p l e s f r o m l iv e r, g i ll , s k i n , a n d m u s c l e w e r ef r o z e n a t - 1 8 0 ~ a n d s t o re d a t - 8 0 ~ u n t i l a s s a y e d .In t he l i ve r, t he Cyt . P450 l eve l s w e re m eas ure d acco rd ing t o re f. (12)a n d t h e S e- a n d n o n - S e - d e p e n d e n t G l u t a t h i o n e P e r o x i d a s e ( G Px ), s p e c if icac t i v i t i e s we re a s sayed accord ing t o re f s . (13,14).S e c o n c e n t r a t io n s in g i ll , m u s c l e , a n d s k i n w e r e m e a s u r e d b y t h eP r o t o n - I n d u c e d X - ra y E m i s s io n ( P IX E ) m e t h o d u s i n g t h e 2 -M e V p r o t o nb e a m d e l iv e r e d b y t h e 3 -M V t a n d e m a c c el er a to r f ro m t h e U n i v e r s it y o fN a p l e s . S a m p l e p r e p a r a t i o n a n d m e a s u r e m e n t p r o c e d u r e w e r e s i m i la r t ot h o s e u s e d f o r o t h e r b io l o g i c a l s a m p l e s ( 1 5 ) . In b r i e f , t he t i s sues wereh o m o g e n i z e d , l y o p h i l i z e d , a n d ( fo r ea c h ti ss u e) a n a m o u n t o f a b o u t 5 0m g ( d r y w t ) w a s d i g e s t e d i n 0 .3 m L o f H N O 3 a t 7 0~ p e r 5 m i n . P a ll a -d i u m ( 1 0 0 ~ tg ) w a s a d d e d a s i n t e r n a l s t a n d a r d . A d r o p (4 0 ~ tL ) o f th e f i n a ls o l u ti o n w a s d e p o s i te d o n a k a p t o n b a c k i n g a n d d r ie d u n d e r v a c u u m a tr o o m t e m p e r a t u re . S a m p l e s w e r e v a c u u m s t o re d u n t i l as s ay e d .A d i f f e re n t p r o c e d u r e , c o n s i s t i n g i n t h e p r e c o n c e n t r a t i o n t e c h n i q u ed e v e l o p e d b y B u so e t a l. (16) w a s u s e d f o r l i v e r s a m p l e s . A f t e r d i g e s t i o nby n i t r i c an d pe rch lor i c ac ids , Se w as prec ip i t a t ed o n M i l l ipore f i lt e r s (0.8

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1O0 TaU andini et al.~tm porosity) after addition of Te as internal standard and hydrazine-dihyd rochlo ride as coprecipitant. The Se concentrations were dete rminedby PIXE at the AN2000 Van de Graaff accelerator of the INFN NationalLaboratory in Legnaro.The results are expressed as mean + SEM. Correlations and differ-ences between mean values were analyzed by the r-test and Wilcoxon-test at a significance level of p < 0.05.

RESULTSL e t h a l D o se

Acute toxic effects of Se were studied by measuring the relation bothbetween Se dose and 50% lethal time (LTs0), and between the percen tageof lethality at 96 h and dose. The resulting data are shown in Fig 1. Eachpoint was obtained by sampling 20 fish.The LT50 plot indicates that, for doses greater than 0.3 ppm, the LT50was smaller than 24 h. The 50% lethal dose (LD50) at 96 h was 0.29 ppm,as determined by graphic interpolation in the % mortality diagram.E f f ec ts o f S e o n E n z y m e s

At doses of 0.1 and 0.2 ppm, almost all fish survived the treatment.They were sacrificed 4, 8, and 28 d after injection. No significant differ-ences among the values of liver Cyt. P450, Se-GPx, and total GPx, mea-sured at different sampling times for each treatment, were detected.Values averaged on sampling times are given in Table 1 togetherwith the corresponding values for the control group. The Cyt. P450,Se-GPx, and total GPx values at 0.1 and 0.2 ppm doses do not differ eachother. Therefore, data were pooled to get dose averaged values of 0.122+_ 0.008, 479 + 15, and 582 + 18, respectively. The values for Cyt. P450 andSe-GPx well agree wi th control values.At 0.3, 0.4, and 0.5 ppm doses, the number of fish dead within 48 hafter injection was increasing. The enzyme concentration or activity inthe nonsurviving fish, as assessed within 1 h from death, are reported inTable 2.Concerning the Cyt. P450 and Se-GPx, no significant differences inthe enzyme levels or activities of the nonsurv iving fish at the three doseswere detected. The dose averaged values resulted to be 0.047 _+ 0.004 and353 _+ 15, respectively, which are significantly different from the corre-sponding values averaged on 0.1 and 0.2 ppm doses. On the contrary, adecrease in the total GPx activity with increasing dose was observed.Table 3 reports the enzyme concentration or activity in fish surviv-ing after 0.3 or 0.4 ppm injection and sacrificed 4 or 28 d after injection.No data are reported for the 0.5 ppm dose because all fish were deadwithin 48 h after injection.B i o l o g ic a l T r a c e E l e m e n t R e s e a r c h V o l. 5 1 , 1 9 9 6


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S e l e n i u m T o x ic it y in F i s h

o .o .

1.0 -~

0 .9 -~0 . 8 - -0 . 7 - -0 .6 - -

0 . 4 - -0 . 3 - "0 , 2 - "0 . 1 - -

0 , 0

I 0 0

9 0

8 0

7 0

6 0

5 0

4 0

3 0

2 0

t0

0

1 0 1

' I ' I ' I ' I I I ' I ~ I ' 11 0 2 0 3 0 4 0 0 .0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0

L T 5 0 ( h o u r s ) s . (ppm)a ) b )

F ig . 1. ( a) Se d o s e a s a f u n c t i o n o f t h e 5 0 % l e t h a l t i m e ; ( b) P e r c e n t m o r -t a l it y a t 9 6 h a s a f u n c t i o n o f t h e S e d o s e .

Tab l e 1L i v e r E n z y m e C o n c e n t r a t i o n s o r A c t i v i t i e s , A v e r a g e d o n t h e S a m p l i n g T i m e ,i n t h e C o n t r o l G r o u p a n d i n F is h I n j e c t e d 0 .1 o r 0.2 p p mC o n t r o l g r o u p D o s e 0 .1 p p m D o s e 0 . 2 p p m

E n z y m e N L e v e l N L e v e l N L e v e lCy t . P450a 13 0 . 1 1 0 + 0.012 12 0.125 + 0.012 14 0.119 _+ 0.012Se -GP xb 16 42 8 _+ 28 12 46 3 _+ 34 15 48 3 _+ 17Tot -GPxc 16 675 _ 50 12 602 _+ 40 15 577 _+ 20

a C o n c e n t ra t io n i n n m o l / m g p r o te i n .b S pe cif ic a c ti v it y in n m o l N A D P H o x . / m i n m g p r o t ei n , s u b s t r at e H 2 0 2 .c S p e c i f i c a c t i v i t y i n n m o l N A D P H o x . / m i n m g p r o t e i n , s u b s t r a t e C H P ( C u m e n eH y d r o p e r o x i d e ) .

S e U p t ak e a n d E l i m i n a t i o n i n T i s su e sT h e S e t i s s u e c o n c e n t r a t i o n s i n f is h i n j e c te d 0 .1 o r 0 .2 p p m a r e

r e p o r t e d i n T ab le 4 . A t t h e s e d o s e s , s e l e n i u m c o n c e n t r a t i o n s w e r e f o u n du n c h a n g e d w i t h r e s p e c t t o t h e c o n t ro l v a l u e s a t a ll s a m p l i n g t im e s ,e x c e p t f o r g i l l w h i c h s h o w a s li g h t in c r e a s e .I n T a b l e 5 , t h e S e t i s s u e c o n c e n t r a t i o n s i n f i s h i n j e c t e d 0 . 3 o r 0 . 4 p p ma r e r e p o r t e d , b o t h f o r n o n s u r v i v i n g f is h a n d f o r t h e s a c r if ic e d o n e s a t 4 ,

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1 0 2 T a l l a n d i n i e t a l.T ab le 2L i v e r E n z y m e C o n c e n t r a t i o n s o r A c t i v i t i e s i n N o n s u r v i v i n g F i s hA fter 0 .3 , 0.4, o r 0 .5 pp m in ject io n

D o s e 0 .3 p p m D o s e 0 .4 p p m D o s e 0 . 5 p p mE n z y m e N L e v e la N L e v e la N L e v e laC y t . P 4 5 0S e* G P xT o t a l G P x

5 0.044 + 0.005 5 0.055 + 0.011 5 0.049 _+ 0.0087 387 + 25 9 293 + 36 6 351 + 217 58 2 _+ 75 9 44 0 _+ 93 6 43 2 _+ 29aFor un i t s , r e f e r t o Tab le 1 .

T ab le 3L i v e r E n z y m e C o n c e n t r a t i o n s o r A c t i v i t i e s , A v e r a g e do n 0 .3 o r 0 .4 In j ec t ed D o ses , i n F i sh S ac r i f i ceda t D i f f e r e n t T i m e s A f t e r I n j e c t i o n

T im e 4 d T ime 2 8 dE n z y m e N L e v e la N L e v e laC yt . P4 50 4 0.102 + 0.015 3 0.144 _+ 0.007Se -G Px 4 378 _+ 28 3 418 + 104Total G Px 4 680 + 106 3 513 + 141

aFor uni ts , refer to Table 1 .

T ab le 4T i s s ue S e C o n c e n t ra t io n , a A v e r a g e d o n t h e S a m p l i n g T i m e ,i n F i s h I n j e c t e d 0 . 1 a n d 0 . 2 p p m a n d i n t h e C o n t r o l G r o u pC o n t r o l s D o s e 0 .1 p p m D o s e 0 .2 p p m

T issu e N S e N S e N S eL i v e rGi l lS k inM u s c l e

10 2.4 +_ 0.4 - - - - 13 2.6 _+ 0.420 3.9 _+ 0.3 14 4. 7 _+ 0.6 20 4.8 _+ 0.320 2.0 _+ 0.2 14 2.1 + 0.2 20 2.2 _+ 0.220 2.0 + 0.2 14 1.9 _+ 0.1 20 1.8 _+ 0.1a Se c o n c e n t r a t i o n v a l u e s i n ~ t g / g d r y w e i g h t .

8 , a n d 2 8 d a f t e r th e i n je c t io n . S e c o n c e n t r a t i o n s i n t h e n o n s u r v i v i n g f i s hw e r e f o u n d s i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l s i n l i v e r , g i l l , a n d s k i n .I n t h e s u r v i v i n g f is h , S e l e v e l s in li ve r , g il l, a n d s k i n w e r e l o w e r i n g w i t ht im e . S e c o n c e n t r a t i o n i n m u s c l e c a n b e c o n s i d e r e d u n a f f e c t e d b y t h e S ei n je c t io n . S e t i s s u e c o n c e n t r a t i o n v e r s u s s a m p l i n g t i m e i s s h o w n i n Fi g. 2 .I n li ve r , g il l, a n d s k i n t h e S e c o n c e n t r a t i o n s a s a f u n c t i o n o f t i m ea f te r in j ec t io n , a re w e l l r e p r e s e n t e d b y a n e x p o n e n t i a l d e c a y t o w a r d t h e

B i o l o g i ca l T r ac e E l e m e n t R e s e a r c h V o l. 5 1 , 1 9 9 6


7/10

Selenium Toxicity in Fish 1 0 3T ab le 5T i s s ue S e C o n c e n t r a t io n s a i n B o t h N o n s u r v i v i n g F i s h a n d F i s h S a c ri f ic e da t D i f f e r e n t T i m e s A f t e r I n j e c t i o n o f 0 . 3 o r 0 . 4 p p m

N o n s u r v i v i n g T i m e 4 d T i m e 8 d T i m e 2 8 dT i s s u e N S e N S e N S e N S eL iv e r 14 6.2 + 0.5 2 3.4 _+ 0.9 1 2.6 3 2.6 _+ 0.6G ill 28 14.1 _+ 1.0 5 6.9 _+ 0.5 3 4.1 _+ 0.3 4 4.8 + 0.8S ki n 28 4.8 _+ 0.4 5 4.0 _+ 0.4 3 2.7 _+ 0.5 4 2.2 + 0.3M u s c le 28 2.3 _+ 0.1 5 2.3 + 0.3 3 2.2 _+ 1.5 4 2.2 ___ 0.2

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10 20T ime a f ter in jec t ion (days)

t3 0

F ig . 2 . T i s s u e S e c o n c e n t r a t i o n s i n f is h i n je c t e d w i t h 0 .3 o r 0 .4 p p m , a s af u n c t i o n o f t h e s a m p l i n g t i m e i n f i s h i n j e c t e d w i t h 0 .3 o r 0. 4 p p m . T h e 0 - d v a l -u e s w e r e m e a s u r e d i n n o n s u r v i v i n g f i s h . T h e h o r i z o n t a l l i n e s r e p r e s e n t t h e n o r -m a l v a l u e s ( N V ) o f t h e c o n t r o l g r o u p .

n o r m a l v a l u e ( N V ) . T h e a s s i g n m e n t o f t h e v a l u e s f o r t h e n o n s u r v i v i n gf i sh a t z e r o t i m e i s , t o a c e r t a i n e x t e n t , a r b i t r a ry . I t i s m o t i v a t e d b y t h el i m i t e d n u m b e r o f f i s h a n d i s r e a s o n a b l e f o r e s t i m a t i n g t h e e l im i n a t i o nh a l f - t i m e s a n d m a k i n g c o m p a r i s o n s b e t w e e n t i s s ue s . B y f i tt in g th e co n -c e n t r a t i o n ( y) a s a f u n c t i o n o f t i m e ( t) t h r o u g h t h e m o d e l

y = N V + A ' e x p ( - B t ) (1 )w e o b t a i n e d t h e v a l u e s o f t h e p a r a m e t e r s ( A a n d B ) r e p o r t e d i n Ta b le 6. I nt h e l a s t c o l u m n , t h e b i o l o g i c a l e l i m i n a t i o n h a l f- l if e , T1/2 = 0 . 6 9 3 / B , i s g i v e n .B i o l o g ica l T r ace E l em en t R es ea r ch V o l. 5 1 , 1 9 9 6


8/10

1 0 4 T a U a n d i n i e t a l .Table 6P a r a m e t e r s C a l c u l a te d by F i tt ing t he S e T is sue Con c e n t r a t ionw i t h t he E xpone n t i a l M ode l ( 1 ). T1/2 i s the Biological Half-Life

Tissue NV, ~tg/g A, ~tg/g B, d-1 T1/2, dL iv er 2.4 + 0.4 3.70 _+ 0.12 0.33 _+ 0.03 2.1 _+ 0.2G ill 3.9 _+ 0.3 10 .2 _+ 0.6 0.33 + 0.05 2.1 +_ 0.3S k in 2.0 + 0.2 2.88 _+ 0.25 0.13 _+ 0.03 5.3 _+ 1.2

DISCUSS IONT h e s h a r p i n c r e a s e o f t h e l e t h a l i t y c u r v e s u g g e s t s t h e p r e s e n c e o f a

t h r e s h o l d i n t h e a c t i o n o f S e i p i n j e c t e d . T h i s e v i d e n c e i s a l s o s u p p o r t e db y t h e b e h a v i o r o f C y t. P 45 0 c o n c e n t r a t i o n i n l iv e r m i c r o s o m e s a n d o f t h es p e c i f i c a c t i v i t y o f l i v e r S e - G P x . I n d e e d , t h e s e e n z y m e s r e m a i n e d a t n o r -m a l v a l u e s i n f is h i n j e c t e d 0.1 o r 0 .2 p p m , w h i l e t h e y f el l s i g n i f i c a n t ly i nf i sh i n j e c t e d w i t h d o s e s g r e a t e r t h a n t h e 5 0 % l e t h a l d o s e (L D s0 = 0.2 9p p m ) . I n p a r t i c u l a r , a d e c r e a s e o f a f a c t o r a b o u t 2 . 5 i n C y t . P 4 5 0 c o n c e n -t r a ti o n w a s o b s e r v e d . T h e d e c r e a s e o f t h e e n z y m e a c ti v it ie s c o u l d b ea t t r i b u t e d e i t h e r t o a d e c r e a s e d e n z y m e s y n t h e s i s o r t o i t s d i r e c t i n h i b i -t i o n b y t h e S e i n je c t e d .

T h e b e h a v i o r o f to t a l G P x a c t i v i t y a p p e a r s s l i g h t ly d i f fe r e n t . I t s h o w sa d e p e n d e n c e o n d o s e s w h i c h h a s t o b e a t t r ib u t e d t o th e n o n S e - G P xa c t i v i t y . T h i s f i n d i n g c o u l d i n d i c a t e t h a t t h e e n z y m a t i c t u r n o v e r w a si n h i b i t e d b y c o m p r o m i s e d c e l l u l a r p r o c e s s e s .

T h e S e c o n c e n t r a t io n i n a l l ( ex c e p t th e m u s c l e ) t h e e x a m i n e d t is s u e ss h o w e d a si m i l a r b e h a v i o r . A l a r g e d i ff e r en c e w a s o b s e r v e d b e t w e e n l ev -e ls a t 0.1 a n d 0 .2 p p m a n d t h e l e v e l s a t 0.3 a n d 0 .4 p p m d o s e s .

T h e L D 5 0 v a l u e w a s m u c h l o w e r t h a n t h e t i s s u e n a t u r a l c o n c e n t r a -t i o n ( 2 . 2 ~ t g / g i n m u s c l e ) . T h i s f a c t i n d i c a t e s a h e a v y a c c u m u l a t i o n o f S ei n t h e v i t a l o r g a n s , w h i c h i n h i b i t s e s s e n t i a l f u n c t i o n s . I n d e e d , w e f o u n dt h a t S e c o n c e n t r a t i o n s i n l i v e r , s k i n , a n d g i l l o f n o n s u r v i v i n g f i s h w e r et h r e e t i m e s h i g h e r t h a n t h o s e i n c o n t r o l s .

T h e S e a b s o r p t i o n i n l i v e r a n d g i l l s h o u l d o c c u r i n a v e r y s h o r t t i m e( l e s s t h a n 2 4 h ) , a s i n d i c a t e d b y t h e h i g h S e c o n c e n t r a t i o n f o u n d i n n o n -s u r v i v i n g f is h. T h e g o o d n e s s o f t h e f it t h r o u g h a n e x p o n e n t i a l e l i m i n a t i o nf u n c t i o n s e e m s t o i n d i c at e th a t o n l y o n e m e t a b o l i c p o o l w a s i n v o l v e d f o rt h e s e o rg a n s . T h e b ip h a s i c b e h a v i o r f o u n d i n f a t h e a d m i n n o w (11) c a n b ee x p l a i n e d a s o w i n g t o t h e v e r y l o w d o s e i n j e c t e d ( 2 0 p p b ) .

T h e b i o l o g i c a l e l i m i n a t i o n h a l f - l i v e s o f S e c o n c e n t r a t i o n i n l i v e r a n dg i ll w e r e n e a r l y i d e n t i c al , w h e r e a s t h e s k i n e x h i b i t e d a h a lf - li fe 2.5 t i m e sg r e a t e r .T h e e l e v a t e d c o n c e n t r a t i o n i n g il l a n d i ts d e c r e a s e w i t h t i m e c o n f i r mt h e e f f e c t i v e n e s s o f t h i s e l i m i n a t i o n r o u t e . A s i m i l a r r o l e c o u l d a l s o b e

B i o l o g ic a l T r ac e E l e m e n t R e s e a r c h V o l. 5 1 , 1 9 9 6


9/10

S el e n i u m T o x i c i t y i n F i sh 105a t tr i b u te d t o t h e s k i n o w i n g t o t h e h i g h S e c o n c e n t r a t io n f o u n d i n t h is t is -s u e a n d t h e d e c r e a s e w i t h t i m e . T h i s l a t t e r p o s s i b i l i t y w o u l d a p p e a r i nc o n t r a s t w i t h t h e k n o w n i m p e r m e a b i l i t y o f t h e s k i n o f a h e a l t h y t e l e o s tf i s h u n d e r n o r m a l c o n d i t i o n s . H o w e v e r , b e c a u s e t h e S e c o n c e n t r a t i o nm e a s u r e m e n t s w e r e c a r r i e d o u t o n t h e s k i n d e p r i v e d o f s c a l e s , o t h e rm e c h a n i s m s i n v o l v i n g S e p a r ti ti on a m o n g s k in , s c al es , a n d m u c u s s h o u l db e c o n s i d e r e d . T h e h i g h c o n c e n tr a t i o n o f S e i n l i v e r a f t e r i n j e c t i o n c o n -f i r m s t h e r o l e o f t h i s o r g a n i n s t o r a g e , m e t a b o l i s m , a n d e l i m i n a t i o n (8 ) .

I n t h e s u r v i v i n g f i s h , t h e e n z y m e a c t i v it i es s h o w e d a c o m p l e t e r e c o v -e r y d u r i n g t h e o b s e r v a t i o n p e r i o d , i n c o n c o m i t a n c e w i t h t h e S e l e v e ld e c r e a s e i n t h e l i v e r t o t h e o r i g i n a l c o n t r o l v a l u e s . N o r m a l c o n d i t i o n s o fa l l m e a s u r e d q u a n t i t i e s w e r e r e s to r e d w i th i n a w e e k a f t e r i n j e c t i o n .

R E F E R E N C E S1 . J . T . Ro t ruck , A. L . Pope , H. E . Gan the r , A. B. Swanson , D. G. Hafeman , and W. G.H o e k s t r a , S e le n i u m : b i o c h e m i c a l ro l e a s a c o m p o n e n t o f g l u t a t h i o n e p e r o x i d a s e , Sci-ence 179, 588-590 (1973).2 . P. M . Cu m b i e a n d S . L . V a n H o r n , S e l e n i u m a c c u m u l a t i o n a s s o c ia t e d w i t h f i sh m o r -t a l it y a n d r e p r o d u c t i v e f a i l ur e , Proc. A. Conf. S.E. Ass. Fish Wildl. Agencies 32, 612-624(1978).3 . R . S . O g l e a n d A . W . K n i g h t , E f fe c ts o f e le v a t e d f o o d b o r n e s e l e n i u m o n g r o w t h a n dr e p r o d u c t i o n o f t h e f a t h e a d m i n n o w (Pimephales promelas), Arch. Env iron. Contam.Toxicol. 18, 795-803 (1989).4 . H. M. O h lend or f , D . J. Ho f fm an , M. K. Sa ik i, an d T. W. Ald r ich , Em bryo n ic mor ta l -i t y a n d a b n o r m a l i t i e s o f a q u a t i c b ir d s : a p p a r e n t i m p a c t s o f s e l e n i u m f r o m i r r i g a t i o nd r a i n w a t e r , Sci. Total Environ. 52, 49-63 (1986).5 . P . Ki f fney and A. Kn igh t , The tox ic i ty and b ioa ccu m ula t io n o f se lena te , se len i te ands e l e n o - L- m e t h i o n i n e i n t h e c y a n o b a c t e r i u m Anabaena .flos aquae, Arch. Environ. Con-tam. Toxicol. 19, 488-495 (1990).6. W . J. A d a m s a n d H . E . J o h n s o n , S e l e n i um : a h a z a r d a s s e s s m e n t a n d a w a t e r q u a l i t yc r i te r ion ca lcu la t ion , in Aquatic Toxicology and Hazard Assessm ent: Fou rth Conference,A S T M S T P 7 3 7 , D. R. Bran son an d K. L . Dickson , eds . , Am er ican Soc ie ty fo r Tes t ingand Mater ia ls , pp . 124-137 (1981) .7 . P . V. Ho dso n , D. J. Sp ry , and B. R. Blun t , Ef fect s on ra inb ow t rou t (Salmo gairdneri)o f a c h r o n i c e x p o s u r e t o w a t e r b o r n e s e l e n i u m , Can. J. Fish. Aquat. Sci. 37, 233-240(1980).8 . J . W. Hil ton, P . V. Hodson, and S. J . S l inger , Absorpt ion, d is tr ibut ion, half- l i fe andp o s s i b l e r o u t e s o f e l i m i n a t i o n o f d i e t a r y s e l e n i u m i n j u v e n i l e r a i n b o w t r o u t (Salmogairdneri), Comp. B iochem. Physiol. 71C, 49-55 (1982).9 . S . J. H a m i l t o n a n d K . J . Bu h l , A c u t e t o x i c it y o f b o r o n , m o l y b d e n u m , a n d s e l e n i u mt o f ry o f C h i n o o k s a l m o n a n d C o h o s a lm o n , Arch. Environ. Contain. Toxicol. 19 ,366-373 (1990).10. K . M . K l e i n o w a n d A . S . B r o o k s , S e l e n i u m c o m p o u n d s i n t h e f a t h e a d m i n n o w(Pimephales promelas) I . U p t a k e , d i s t r i b u t i o n , a n d e l i m i n a t i o n o f o r a l l y a d m i n i s t e r e ds e l e n at e , s e le n i t e a n d L - s e l e n o m e t h i o n i n e , Com p. Biochem. Physiol. 83C, 61-69 (1986).11. K . M . K l e i n o w a n d A . S. B r o o k s , S e l e n i u m c o m p o u n d s i n t h e f a t h e a d m i n n o w(Pimephales p romelas) I I. Q u a n t i t a t i v e a p p r o a c h t o g a s t r o i n te s t i n a l a b s o r p t i o n , r o u t e so f e l i m i n a t i o n a n d i n f l u e n c e o f d i e t a r y p r e t r e a t m e n t , Comp. Biochem. Physiol. 83C,71-76 (1986) .12. R. W . Es t a b r o o k a n d J. W e r r i ng l o e r, Th e m e a s u r e m e n t s o f d i f f e re n c e s pe c t ra : a p p l i -c a t i o n t o t h e c y t o c h r o m e o f m i c r o s o m e s , i n Methods in Enzymology, S. F le i she r and L.Packer , eds . , Academ ic , NY, pp . 212-220 (1978).

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106 T a l lan d in i e t a l .13. A. L . Tappe l, Glu ta th ione pe rox idase and hydrop erox idases , in M e t h o d s i n E n z y m o l -ogy, S. Fleischer an d L . Packer, eds., pp. 506-517, Aca dem ic, Ne w York (1978).14. F. Ursini , M. Major ino, M. Valente , L . Ferr i , and C. Gregol in , Pur i f ica t ion f rom pigl i v e r o f a p r o t e i n wh i c h p r o t e c t s l i p o s o me s a n d b i o me mb r a n e s f r o m p e r o x i d a t i v edegrada t ion and exh ib i t s g lu ta th ione pe rox idase ac t iv i ty on phospha t id i l cho l ineh y d r o p e r o x i d e s , Biochem. Biophys. Acta 710 , 197-211 (1982).15. R. Moro, G. Gialanel la , Y. X. Zhang, L. Perrone, and R. Di Toro, Trace e lements infu l l - te rm ne ona te ha i r , J . Trace E lem. E lec t ro ly tes Hea l th Dis . 6, 27-31 (1992).16. G. P . Buso, P. Co laut t i , G. M oschini , H. X usheng , and B. M. St ievano, H igh sensi t iv-i t y P I XE d e t e r mi n a t i o n o f s e l e n i u m i n b i o l og i ca l s a mp l e s u s i n g a p r e c o n c e n t r a ti o ntechn ique , N u c l . I n s t r . Me t h . B3, 177 -180 (1984).

Bio log ical Trace E le m ent Re sear ch Vo l. 51 , 1996

Biological Trace Element Research Volume 51 Issue 1 1996 Laura Tallandini; Rodolfo Cecchi; Silvia de...

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Transcript of Biological Trace Element Research Volume 51 Issue 1 1996 Laura Tallandini; Rodolfo Cecchi; Silvia de...