C L I N I C A L TOXICOLOGY, 23(4-6), 299-308 (1985)
EFFECTS OF DISSOLVED CARBON M O N O X I D E ON THE RESPIRATORY ACTIVITY OF PERFUSED NEURONAL AND MUSCLE CELL CULTURES
Er ik Walum, Inger Varnbo and Anders Pe terson Unit of Neurochemistry and Neurotoxicology
Univers i ty of Stockholm Enkapingsv 126
S-172 46 Sundbyberg, Sweden
ABSTRACT
In order t o address the ques t ion of whether small amounts of d i s s o l v e d CO may i n h i b i t c e l l u l a r r e s p i r a t i o n , c u l t u r e d mouse n e u r o b l a s t o m a c e l l s and p r imary c u l t u r e s of c h i c k n e u r o n s , r a t a s t r o c y t e s and chick s k e l e t a l muscle and h e a r t c e l l s were exposed t o EO c o n t a i n i n g b u f f e r s o l u t i o n s i n a c l o s e d p e r f u s i o n system. Oxygen uptake was measured s imul taneous ly wi th two po la rograph ic oxygen e l e c t r o d e s a s t h e d i f f e r e n c e i n p a r t i a l p ressure of oxygen between t h e i n l e t and o u t l e t of t h e p e r f u s i o n chamber. A f t e r r e g i s t r a t i o n of t h e b a s a l r e s p i r a t o r y a c t i v i t y , p e r f u s i o n s o l u t i o n s c o n t a i n i n g 5 u l 0 2 / m l were b u b b l e d w i t h CO o r N 2 a t a r a t e of 200 ml /min f o r 120 sec . By t h i s p r o c e d u r e t h e p a r t i a l p re s su re of O2 was decreased t o r each a v a l u e of about 50% of t h e i n i t i a l O2 content f o r both gases. Per fus ion was then cont inued f o r 30 min a t a r a t e of 0.5 ml /min . The r e s p i r a t o r y a c t i v i t y of a l l the perfused c e l l c u l t u r e s , except chick neurons, was found t o be i n h i b i t e d (13-292) by pe r fus ion s o l u t i o n s bubbled f o r 120 sec wi th CO a s compared t o Nq con t ro l s . Of the c e l l s from the nervous system, a s t r o c y t e s were more s e n s i t i v e than neurons. Apparent ly , sma l l amounts of d i s s o l v e d CO can i n h i b i t c e l l u l a r r e s p i r a t i o n i n the presence of a p h y s i o l o g i c a l l y adequate amount of oxygen.
INTRODUCTION
Impai rment of b r a i n f u n c t i o n s due t o c a r b o n monoxide (CO)
i n t o x i c a t i o n may a r i s e a s a consequence of d e c r e a s e d oxygen
supply, i n h i b i t i o n of cytochrome oxidase a c t i v i t y and hemodynamic
d i s t u r b a n c e s . It i s g e n e r a l l y a g r e e d t h a t CO t o x i c i t y m a i n l y
depends on i t s competi t ion f o r oxygen binding s i t e s on hemoglobin,
299
Copyright 0 1985 by Marcel Dekker, Inc. 073 1-38 10/85/2304-0299$3.50/0
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300 WALUM, VARNBO, AND PETERSON
t h u s impeding oxygen d e l i v e r y t o t i s s u e s . F u r t h e r m o r e , CO i s
known t o bind t o cytochrome oxidase (1) bu t i t i s l a r g e l y unknown
i f t h i s b i n d i n g i s r e l a t e d t o t h e t o x i c a c t i o n of CO. As i t
a p p e a r s , t h e r e s p i r a t o r y c h a i n , p a r t i a l l y i n h i b i t e d by C O , c a n
m a i n t a i n i t s a c t i v i t y t h r o u g h compensa tory mechanisms (2 ) .
Never the less , Przybylsk i and Krasnicka (3) found t h a t exposure of
organotypic c u l t u r e s of r a t cerebe l lum t o a i r conta in ing 10% CO
f o r 30 min caused s i g n i f i c a n t changes i n the e l e c t r o p h y s i o l o g i c a l
p r o p e r t i e s of t h e c o n s t i t u e n t c e l l s . Due t o t h e i n h e r e n t
d e f i c i e n c i e s of t r a d i t i o n a l c u l t u r i n g techniques , c e l Is i n c u l t u r e
most o f t e n s u f f e r from hypoxia (4). Therefore a per fus ion system,
which makes i t p o s s i b l e t o s t u d y c u l t u r e d c e l l s unde r good
p h y s i o l o g i c a l c o n d i t i o n s has been d e v e l o p e d ( 5 ) and used t o
address more d i r e c t l y the ques t ion whether d i s s o l v e d CO i n h i b i t s
c e l l u l a r r e s p i r a t i o n i n one continuous neuroblastoma c e l l l i n e and
primary c u l t u r e s of neurons, g l i a , muscle and h e a r t c e l l s .
MATERIALS AND METHODS
Cell c u l t u r e s
The mouse neuroblastoma c e l l l i n e C1300 c l o n e NlE115 ( 6 ) was
grown a s d e s c r i b e d p r e v i o u s l y ( 7 ) . Pr imary c u l t u r e s o f 8 day
c h i c k embryo b r a i n n e u r o n s and of newborn r a t b r a i n a s t r o c y t e s
were prepared according t o Booher and Sensenbrenner (8) and grown
i n 21 cm2 p l a s t i c t i s s u e c u l t u r e d i s h e s i n Ham's F10 medium
containing 10% Clex and Dulbecco's mod i f i ca t ion of Eagle's medium
supp lemen ted w i t h 15% f e t a l c a l f serum r e s p e c t i v e l y . P r imary
c u l t u r e s of 12 day chick embryo l e g and b r e a s t muscle c e l l s were
p r e p a r e d and grown a s d e s c r i b e d by S h a i n b e r g e t a 1 ( 9 1 , b u t w i t h
minor m o d i f i c a t i o n s . I n b r i e f , m u s c l e s were d i s s e c t e d and s k i n
a n d bone w e r e c a r e f u l l y r e m o v e d . The m u s c l e s w e r e t h e n
m e c h a n i c a l l y d i s s o c i a t e d i n g rowth medium (Ham's F10 medium
supp lemen ted w i t h 10% newborn c a l f serum and 2% c h i c k embryo
e x t r a c t ) . The c e l l s u s p e n s i o n was p r e - p l a t e d t o r e m o v e
f i b r o b l a s t s and t h e n p l a t e d on c o l l a g e n c o a t e d 21 cm2 p l a s t i c
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DISSOLVED CO ON THE RESPIRATORY ACTlVITY 301
t i s s u e c u l t u r e dishes. Af te r 48h the growth medium was r ep laced
wi th one conta in ing M cy tos ine arabinoside. Fol lowing a n t i -
me tabo l i t e t reatment f o r 96h, t he c u l t u r e s were changed t o f r e s h
growth medium. Hea r t c e l l s f rom 10-12 day c h i c k embryos were
prepared a s p rev ious ly descr ibed f o r embryonic l i v e r c e l l s (10)
w i th the except ion t h a t Dulbecco's modi f ica t ion of Eagle's medium
containing 9% newborn c a l f serum and 4% f e t a l c a l f serum was used
a s p r o c e s s i n g and growth medium. Fur the rmore , t h e h e a r t c e l l
c u l t u r e s were t r e a t e d w i t h c y t o s i n e a r a b i n o s i d e a s d e s c r i b e d
above f o r s k e l e t a l muscle c e l l s . A l l c u l t u r e s were maintained a t
3 7 O C i n a h u m i d i f i e d a tmosphe re of 4% COP i n a i r . Growth media
were changed t w i c e a week and t h e n e u r o b l a s t o m a c e l I s , n e u r o n s ,
a s t rocy te s , muscle and hea r t c e l l s were used f o r experiments 2-17,
3-5 , 16-18, 14-17, and 6-9 days a f t e r p l a t i n g r e s p e c t i v e l y .
Cul ture Per fus ion
The method used f o r continuous per fus ion of c e l l c u l t u r e s has
been d e s c r i b e d i n d e t a i l e l s e w h e r e ( 5 ) . B r i e f l y , t h e p l a s t i c
t i s s u e c u l t u r e d i sh conta in ing t h e a t tached c e l l s was pressed on
t o a cylinder-shaped, polycarbonate p l a s t i c per fus ion b lock wi th
an outer diameter equal t o the inner diameter of t he c u l t u r e dish.
I n l e t and o u t l e t channels f o r t he per fus ion s o l u t i o n were d r i l l e d
through the per iphery of the top of t he block. A c losed per fus ion
chamber i s t h u s formed from t h e d i s h and t h e b l o c k . From a
r e s e r v o i r a c o n t i n u o u s f l o w of b u f f e r (150 mM NaC1, 3.0 mM K C 1 ,
1.0 mM CaC12, 0.6 mM MgC12, 1.6 mM KH2P04, 4.3 mM Na2HP04, 7.0 mM
g l u c o s e , 300 m O s m , pH 7.3) was d i r e c t e d t h r o u g h t h e chamber a t a
r a t e of 0.5 ml /min by means of a p e r i s t a l t i c pump. A three-way
v a l v e e n a b l e d t h e f l o w t o be changed t o come f rom a n o t h e r
r e s e r v o i r c o n t a i n i n g b u f f e r which had been b u b b l e d f o r 120 s e c
w i t h CO o r N2 a t a r a t e of 200 ml /min ( F i g 1).
Determination of oxmen consumption
The assembled per fus ion system was p laced i n a water ba th a t
37OC. I n i t i a l l y , bu f fe r f low was d i r ec t ed through a c a l i b r a t i o n
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302 WALUM, VARNBO, AND P E T E R S O N
FIGURE 1
S c h e m a t i c p r e s e n t a t i o n o f t h e p e r f u s i o n s y s t e m u s e d f o r determinat ion of r e s p i r a t o r y a c t i v i t y i n c u l t u r e d c e l l s . From the r e s e r v o i r s , CO o r Nq b u b b l e d (A) o r c o n t r o l b u f f e r (B) was d i r e c t e d t h r o u g h t h e p e r f u s i o n chamber (C) by means o f a p e r i s t a l t i c pump (D). Standard oxygen e l e c t r o d e s were i n s e r t e d a t t he i n l e t (E) and o u t l e t (F) of t he chamber. These were operated by two separa te a m p l i f i e r s (G, H) connected t o a two channel cha r t r e c o r d e r (I). Oxygen u p t a k e of c e l l s (J) a t t a c h e d t o o r d i n a r y p l a s t i c t i s s u e c u l t u r e d i shes (K) was measured a s d i f f e r e n c e i n 02 c o n t e n t of t h e b u f f e r (L) between in- and o u t l e t s . C o n t r o l l e d add i t ion of CO o r N p t o t he t e s t bu f fe r r e s e r v o i r (B) was achieved by the use of two sepa ra t e gas f low meters (M,N).
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DISSOLVED CO ON THE RESPIRATORY ACTIVITY 303
loop and r e s u l t i n g e l e c t r o d e readings were def ined as 100% oxygen
content. The medium was then d i r e c t e d thrdough t h e chamber. The
r e s u l t i n g reading gave an i n i t i a l peak fo l lowed by a s t a b l e l e v e l
( F i g 2). The d i f f e r e n c e between i n l e t ( F i g 2 E) and o u t l e t ( F i g
2F) e l e c t r o d e r e a d i n g s , r e p r e s e n t s t h e oxygen d e p l e t i o n of t h e
p e r f u s i o n b u f f e r a s a r e s u l t of c e l l u l a r r e s p i r a t o r y a c t i v i t y .
A f t e r a c o n s t a n t r e a d i n g f o r a b o u t 1 5 min, c o n t r o l b u f f e r was
changed t o b u f f e r b u b b l e d w i t h CO o r N2. Oxygen consumpt ion ( r )
was c a l c u l a t e d according t o the fo l lowing s i m p l i f i e d equat ion:
t x VP (a-b) x 5.2 r = a
where t r e p r e s e n t s t he time, Vp the pe r fus ion r a t e (0.5ml/min), a
i s the i n l e t and b the o u t l e t readings and 5.2 the oxygen conten t
of 1 m l of bu f fe r a t an atmospheric p re s su re of 760 nun Hg.
Chemicals
Ham's F10 medium, Dulbecco 's m o d i f i c a t i o n of Eag le s medium,
f e t a l bovine serum and newborn c a l f serum were purchased from Flow
L a b o r a t o r i e s L t d , I r v i n e , UK. C l e x i s a supp lemen t f o r t i s s u e
c u l t u r e media s o l d by Dextran Products Ltd, Scarborough, Ontar io ,
Canada. C y t o s i n e a r a b i n o s i d e was o b t a i n e d f rom Sigma Chemica l
Company, S t Lousi, Montana, USA.
RESULTS AND DISCUSSION
I n a c c o r d a n c e w i t h p r e v i o u s o b s e r v a t i o n s (11) t h e
neuroblastoma NlE115 c e l l s were found t o extend n e u r i t e s under the
p r e s e n t s t a n d a r d c u l t u r e c o n d i t i o n s . The c e l l s h a v e a l s o been
shown t o possess a high degree of neuronal matura t ion w i t h r e spec t
t o e l ec t rophys io logy (12) and n e u r o c h e m i s t r y (13). I n t h e c h i c k
b r a i n hemisphe re n e r v e c e l l c u l t u r e s b o t h c o l o n y fo rming and
ind iv idua 1 neur i te -bear ing neurones were present . The p r o p e r t i e s
of t h e s e c u l t u r e s h a v e been d e s c r i b e d by S e n s e n b r e n n e r (14) .
Newborn r a t a s t r o c y t e s formed dense monolayers of po lygonal c e l l s
w i t h h i g h a c t i v i t y o f g l u t a m i n e s y n t h e t a s e ( u n p u b l i s h e d
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DlSSOLVED CO ON THE RESPIRATORY ACTIVITY 305
TABLE 1
E f f e c t s of D i s s o l v e d Carbon Monoxide on t h e R e s p i r a t o r y Ac t iv i ty of Perfused Cell Cu l tu re s
02 uptake ( % of c o n t r o l )
co n N2 n
Mouse neuroblastoma NlE115 85 2 6 5 9 8 % 1 4 Chick neurons 71 2 13 3 74 2 15 3
Chick s k e l e t a l muscle 60 2 11 4 8 3 2 4 3 Chick h e a r t c e l l s 7 1 % 4 5 8 6 2 1 4
............................
Rat a s t r o c y t e s 7 5 5 4 5 9 7 5 2 5
C e l l c u l t u r e s were p e r f u s e d f o r 30 min w i t h b u f f e r b u b b l e d w i t h CO f o r 120 s e c a t a r a t e of 200 ml /min . Buffer bubbled w i t h N2 under the same cond i t ions was used a s a n i n e r t h y p o x i c c o n t r o l . Oxygen u p t a k e was d e t e r m i n e d a s d e s c r i b e d i n t h e t e x t and i n F i g s 1 and 2. Each v a l u e r ep resen t s t he mean 2 SEM of t h e number (n) of independent exper iments ind ica ted .
observa t ions) . Embryonic ch ick s k e l e t a l muscle c e l l s fused i n t o
ne tworks of l o n g s p o n t a n e o u s l y c o n t r a c t i n g myotubes w i t h h i g h
l e v e l s of n i c o t i n i c a c e t y l c h o l i n e r e c e p t o r s (15) . Heart c e l l s
f rom c h i c k embryos formed m o n o l a y e r s w i t h l a r g e a r e a s of c e l l s
b e a t i n g s y n c h r o n o u s l y a t an a v e r a g e r a t e of 20 b e a t s / m i n . The
r a t e of con t r ac t ion could be a f f e c t e d by a d r e n a l i n and p r o p r a n o l o l
( un pu b 1 i s hed ob s e r v a t i ons) . B u b b l i n g t h e p e r f u s i o n b u f f e r w i t h CO o r N 2 ( F i g s 1 and 2 ) ,
r educed t h e p a r t i a l p r e s s u r e o f 02- T h i s r e d u c t i o n c o u l d be
measured w i t h t h e r e f e r e n c e e l e c t r o d e ( F i g s 1 E, 2 E) and
amounted, a f t e r 30 Qin per fus ion wi th bubbled bu f fe r , t o 44+1 % (n
= 22) f o r C O and 4152 % (n = 19) f o r N 2 . The u s e of N 2 t h u s made
it p o s s i b l e t o d i s t i a g u i s h e f f e c t s on the r e s p i r a t o r y a c t i v i t y due
t o CO-poisoning from t h o s e due t o r e d u c e d oxygen t e n s i o n . A s o l u b i l i t y c o e f f i c i e n t of 0.024 f o r O 2 i n a p h y s i o l o g i c a l l y b a l a n c e d s a l t s o l u t i o n g i v e s a p a r t i a l p r e s s u r e of O 2 i n t h e
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306 WALUM, V A R N B O , A N D PETERSON
p e r f u s i o n s o l u t i o n of 160 mm Hg under t h e p r e s e n t e x p e r i m e n t a l
c o n d i t i o n s . On t h e a s sumpt ion t h a t t h e c u l t u r e d c e l l s were i n
e q u i l i b r i u m w i t h t h e p e r f u s i o n s o l u t i o n , t h e O 2 t e n s i o n i n t h e
c e l l s would be 90 and 94 Hg a f t e r 30 min per fus ion wi th CO and
N2 bubbled s o l u t i o n s r e s p e c t i v e l y . Consequently, t h e c e l l s never
experienced seve re hypoxia during measurements. Due t o the des ign
of t h e p e r f u s i o n sys tem a s a c l o s e d u n i t d e t e r m i n a t i o n of CO
concentrat ions a t t he c e l l monolayer were not made. Therefore the
dose of C O u sed i n t h e e x p e r i m e n t s i s d e f i n e d o n l y by t h e d a t a
g i v e n under M a t e r i a l s and Methods and i n t h e T a b l e and F i g u r e s .
The CO concent ra t ion i n the per fus ion chamber was s lowly b u i l t up
concomi tan t t o t h e d e c r e a s e i n O 2 p r e s s u r e (F ig . 2) t o r e a c h a v a l u e a f t e r 30 min which, on t h e o r e t i c a l grounds, can be est imated
t o be of the order of 10-5M and comparable t o t h a t used by Chance
and coworkers i n t h e i r experiments on i s o l a t e d mitochondria (2).
As can be s e e n from T a b l e 1, CO i n h i b i t e d t h e O 2 u p t a k e i n t h e neu rob la s toma c e l l s by 13% b u t was w i t h o u t e f f e c t i n t h e
pr imary c u l t u r e s of neu rons a s compared t o t h e N 2 c o n t r o l s .
However, the l a t t e r r e s u l t i s h ighly unce r t a in a s ind ica ted by t h e
p v a l u e >0.05. Of the neuronal c e l l s , a s t r o c y t e s were t h e most
s e n s i t i v e t o CO exposure w i t h a r e d u c t i o n i n r e s p i r a t i o n of 23%
(p<O.O1) compared t o t h e N2 experiments. S imi l a r i n h i b i t i o n s were
o b t a i n e d f o r h e a r t c e l l s (18%; p<0.05) whereas t h e e f f e c t on
s k e l e t a l m u s c l e c e l l s (29%) was n o t s i g n i f i c a n t . None of t h e
t e s t ed c e l l types showed any s i g n i f i c a n t s e n s i t i v i t y t o r educ t ion
i n O2 t ens ion per se.
A p p a r e n t l y , s m a l l amounts of d i s s o l v e d CO can i n h i b i t
c e l l u l a r r e s p i r a t i o n i n the presence of a p h y s i o l o g i c a l l y adequate
amount of oxygen. The p r e s e n t r e s u l t s f u r t h e r i n d i c a t e t h a t of
c e l l s from the nervous system a s t r o c y t e s a r e more s e n s i t i v e t o CO
t h a n neurons.
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DISSOLVED CO O N THE RESPIRATORY ACTIVITY 307
ACKNOWLEDGEMENTS
T h i s work was s u p p o r t e d by g r a n t s f rom t h e Swedish Work Environment Fund (Grant No. 83-0156). Thanks a r e due t o Dr B r i g i t t a Werner , The Swedish P o i s o n I n f o r m a t i o n C e n t e r f o r i n i t i a t i n g t h i s work and t o Ms A n n - C h a r l o t t e E r i c s s o n f o r preparing chick primary cu l tu re s .
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