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Toxicology, 27 (1983) 301--313 Elsevier Scientific Publishers Ireland Ltd.
A S C O R B I C A C I D T U R N O V E R IN T H E M O U S E F O L L O W I N G A C U T E OZONE EXPOSURE*
MICHAEL A. DUBICK**, JAMES W. CRITCHFIELD, JEROLD A. LAST, CARROLL E. CROSS and ROBERT B. RUCKER
Department of Nutrition and Internal Medicine and California Primate Research Center, University of California, Davis, CA 95616 (U.S.A.)
(Received September 10th, 1982) (Accepted December 9th, 1982)
SUMMARY
Swiss Webster mice were c o n t i n u o u s l y exposed to an a t m o s p h e r e con- ta ining 1.5 p p m o z o n e (03) for 5 days. Con t ro l mice b rea thed f i l tered air. I m m e d i a t e l y fo l lowing the expos u re pe r iod each m o u s e was t hen in jec ted with [ 1-14C] ascorbic acid. The ra te of d i sappea rance o f [ 1-14C] ascorbic acid and the levels o f to ta l ascorbic acid were d e t e r m i n e d in serum, lung, liver, and the r ema in ing carcass over a 9-day per iod. 03 e x p o s u r e caused t rans ien t decreases in the ascorbic acid levels in liver and se rum, whereas lung ascorbic acid levels increased. The a p p a r e n t biological half-l ife o f a scorba te in carcasses (minus lung and liver) f r o m O3-exposed mice , was s ignif icant ly p ro longed . Signif icant changes were also obse rved in the ne t f lux o f ascorbic acid in all t issues examined . The resul ts ind ica ted t ha t the change in ascorbic acid levels in a given t issue a p p e a r e d to re f lec t changes in the ra te o f ascorbic acid degrada t ion , its mob i l i za t i on or t issue c o m p a r t m e n t a l i z a t i o n , r a the r t han increased synthes is in r e sponse to 03 exposure .
Key words: Ascorb ic acid; Lung; Liver; Ozone; Mouse
INTRODUCTION
Many of the phys io log ica l func t ions o f ascorbic acid r emain i n c o m p l e t e l y u n d e r s t o o d . In par t icu lar , m o r e i n f o r m a t i o n is needed regarding the role o f
*Supported in part by a grant from the California Research and Medical Education Fund of the American Lung Association of California and NIH Grants HL-26620, HL-15965 and ES-00628. **Address correspondence to: Dr. Michael A. Dubick, Department of Nutrition, University of California, Davis, CA 95616, U.S.A.
0300-483 x/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
301
ascorbic acid in the lung where it is normally found in relatively high tissue concentrations [ 1--3]. Ascorbic acid is believed to have a protective function in lung by acting in part as an antioxidant [1--6]. For example, injection of ascorbic acid into the mouse offered some protection from otherwise lethal levels of ozone [4--6]. In addition, lung ascorbic acid levels were reduced by exposure to hyperbaric oxygen [1,7], nitrogen dioxide [8] and other conditions that induced pulmonary edema and signs of oxidative damage [9,101.
In the present study we examined ascorbic acid metabolism in animals exposed to the oxidant, ozone, It was of interest to examine ascorbate metabolism in an animal that synthesizes ascorbic acid, since a marked increase in ascorbic acid synthesis or turnover in response to an oxidant would strengthen the suggestion for ascorbic acid supplementation in man under such conditions. Ozone was selected as the oxidant gas in this study because of its strong oxidizing potential, its ability to induce edema and other inflammatory responses in the lung, and its significance as a major environmental pollutant [11]. Mice were chosen because the effects of ozone have been characterized in this species and some information is available regarding ascorbic acid metabolism in the mouse.
Our results indicate that following a 5-day exposure to i .5 ppm ozone, ascorbic acid levels decrease in serum and liver, but are slightly elevated in lung for the first 4 days following exposure. Of particular significance, there was no apparent increase in ascorbic acid synthesis in response to ozone. Although ascorbic acid turnover in serum, lung, and liver was not changed in response to ozone, the half-life of ascorbate in the remaining carcass was significantly prolonged. In addition, the data indicate a shift in the com- partmentalization of ascorbic acid in response to ozone that, in part, may be due to decreased ascorbic acid catabolism.
MATERIALS AND METHODS
A n i m a l s and o z o n e exposure p ro toco l Adult, Swiss-Webster mice (Chronic Respiratory Disease-Free) were
obtained from Hilltop Laboratory Animals, Inc. (Chatsworth, CA) and were fed ad libitum a mouse diet containing no detectable ascorbic acid [12]. After a 3-day pre-conditioning period to the exposure facilities, half of the mice were exposed to atmospheres of 1.5 ppm ozone continuously for 5 days. The remaining animals served as controls and breathed filtered air. Other details regarding the exposure to ambient ozone and the detection and quanti tat ion procedures have been described elsewhere [ 13].
Immediately following the exposure, each animal received an intraperi- toneal injection of 4 pCi of freshly dissolved [1-14C]ascorbic acid (New England Nuclear, Boston, MA) in saline. Animals from both the air- and ozone-exposed groups were killed 20 min, 3 h, 6 h, 12 h, 24 h, 2 days, 3 days, 6 days, or 9 days following the injection. At these times body, lung, and liver weights were determined. All reagents used for biochemical determinations were of analytical grade or of the highest purity commercially available.
302
Ascorbic acid and radioactivity determinations At each sampling period, ascorbic acid concentrations were determined
in serum, lung, liver, and carcass according to a modification of the method of Day et al. [14]. The protein in one volume of serum was precipitated with 2 vols. of ice-cold 10% trichloroacetic acid (TCA) and centrifuged at 2000 g for 10 min. For tissues, a 10% homogenate was prepared in deionized water. Protein in a 1-ml aliquot was then precipitated with 2 ml of cold 10% TCA and centrifuged (2000 g, 15 min). Ascorbic acid was determined in the resulting supernatant fractions.
To determine radioactivity as ascorbic acid in tissues and serum, an aliquot of the supernatant fraction (100--200 pl) was introduced to a microfuge tube packed with AG-MP1 resin in the acetate form (Biorad Laboratories, Richmond, CA). The tubes were thoroughly agitated and then centrifuged. Radioactivity as [1-14C]ascorbic acid was determined by subtracting the amount of 14C-labelled material in the supernatant from that introduced into each tube. Preliminary studies indicated that 98--100% of radiolabelled ascorbic acid binds to the resin and is not released nor displaced by millimolar concentrations of glucuronic acid, or similar bio- logically important organic acids. The radioactive samples were counted in Aquasol LSC cocktail (New England Nuclear, Boston, MA) using a Packard 2660 Tri-Carb liquid scintillation counter.
In vitro studies Rates of ascorbic acid synthesis in whole liver homogenates were deter-
mined in vitro as described by Grollman and Lehninger [15] using D- glucuronic acid as substrate, except that ascorbic acid was measured by the dipyridyl method [ 16].
To ascertain the ability of liver and lung to metabolize the 1-14C-moiety of radiolabelled ascorbic acid, mice from the ozone exposed and control groups were killed immediately after the exposure period. Lungs and liver were minced into l-ram 3 sections and incubated in Medium 199 (Grand Island Biochemical, GIBCO, Grand Island, NY) in stoppered flasks with a center well. This medium contained 0.29 pmol/ml of ascorbic acid. To each flask was added 1 pCi/ml (essentially carrier-free) of the freshly dissolved [1-14C] ascorbic acid, and the tissue was incubated for 2 h at 37°C (in a shaken water bath). After 2 h, 0.2 ml of hyamine hydroxide was injected into the center well and 2 ml of 2 N sulfuric acid was injected into the medium. The flasks were incubated an additional hour. The center wells were then removed and dropped into glass scintillation vials containing Aquasol LSC cocktail and their radioactivity was determined.
Statistics and modeling techniques An amended BMDP non-linear regression statistical program was used to
develop a kinetic model (Fig. 1) for studies on ascorbic acid compartmentali- zation [17]. The radioisotope dilution technique was employed to evaluate pharmacokinetic parameters of ascorbic acid metabolism. Data were analyzed initially assuming each tissue represented a separate ascorbic acid pool. How-
303
MODEL
Y~7 ~" K~°
K4
Fig. t . Model used in the s tudy of [1-~4C]ascorbic acid compar tmen ta l i za t ion . K~--K~ re- present rate cons tan ts into and ou t of the various c o m p a r t m e n t s as indicated by the arrows. Values for K~ th rough K~ in (h) -~ are given in Table III. Values K~ (presumed to represent [ 1-~4C ] ascorbic acid deca rboxy la t ion) and K 8 (presumed to represent urinary excre t ion) are given in the text . K 9 and K1¢ , did no t appear to con t r ibu te significantly to the es t imat ion of ascorbic acid flux. Values for the net flux of ascorbic acid are given in Table IV.
ever, the best bit of the data was described by a 2-compartment model de- fined by the equation: y = Ae-J~, t + Be-h, t . The best fit for the [1-14C] ascorbic acid disappearance curves were analyzed by least squares linear regression [18]. All ascorbic acid fluxes calculated were of the form: Flux (I) = K (I) × (amount in pool of origin). Other statistical considerations utilized Student 's t-test for the difference between means [18].
RESULTS
Tissue and body weights Acute exposure to 1.5 ppm of ozone for 5 days resulted in an increase in
the lung wet weight and in the lung wet weight to body weight ratio for at least 6 days following the exposure period (Table I). Liver weights were only affected for the first several hours following exposure to ozone. Ex- posure to ozone affected food intake for only the first 24 h and resumption of food intake was rapid following removal from the exposure chambers.
Ascorbic acid levels As shown in Fig. 2, serum ascorbic acid levels were reduced for the first
24 h following ozone exposure in comparison to serum values for the con- trols breathing filtered air. Likewise, liver ascorbic acid levels, expressed as mg/100 g of tissue or per whole liver, were consistently reduced (approx. 15--25%) in the mice recovering from ozone exposure (Fig. 3). A similar trend was observed in the ascorbic acid content of the carcass (Fig. 3).
Lung ascorbic acid levels expressed as mg/100 g of tissue, however, were elevated for the first 3--4 days following recovery from ozone exposure (Fig. 4). The increase in lung ascorbic acid content was more pronounced when data were expressed as total lung ascorbic acid content , i.e. approxi- mately 2-fold the control values.
304
TA
BL
E I
TIS
SU
E A
ND
BO
DY
WE
IGH
T C
HA
NG
ES
FO
LL
OW
ING
OZ
ON
E E
XP
OS
UR
E a
Tis
sue
tre
atm
en
t H
ours
0.3
3
3 6
12
24
48
7
2
144
21
6
Lu
ng
wt
Air
0
.19
7
0.1
92
0
.18
3
0.1
99
0
.21
4
0.2
12
0
.20
6
0.2
08
0
.26
0
(g)
±0
.01
2
±0
.01
2
±0
.00
7
±0
.00
6
±0
.00
6
±0
.00
6
±0
.00
5
+0
.00
6
_+0.
020
O3
0.2
53
0
.30
7
0.2
60
0
.30
3
0.2
98
0
.28
4
0.2
94
0
.26
1
0.2
77
+
0.0
08
b
±0
.01
2 b
-+
0.00
95
+0
.01
75
+
_0.0
08 b
±
0.0
16
b
±0
.01
4 b
_+
0.01
7 ±
0.0
01
Liv
er w
t A
ir
1.5
4
1.4
0
1.7
9
1.9
0
1.7
3
1.9
1
1.9
1
2.1
2
(g)
-+0.
08
±0
.06
-+
0.06
+
0.1
3
-+0.
13
-+0.
13
±0
.14
+
0.1
3
03
1
.01
1
.22
1
.39
1
.94
1
.87
1
.77
1
.83
1
.91
±
0.0
9 b
-+
0.06
±
0.1
3
±0
.16
_+
0.13
±
0.1
6
±0
.12
+
0.1
4
2.0
0
-+0.
19
2.0
3
+0
.12
Bo
dy
wt
Air
2
9.9
2
7.8
2
9.6
3
2.6
2
8.5
3
1.3
3
0.7
3
5.1
(g
) +
0.5
±
1.1
±
0.6
±
1.8
±
1.0
±
1.6
±
1.3
+
1.3
0
3
19
.6
22
.2
24
.6
28
.5
27
.0
29
.7
27
.3
28
.8
_+0.
55
+1
.4
-+1.
3 -+
2.1
±0
.7
±2
.0
-+1.
6 ±
2.2
35
.8
+0
.9
31
.2
±0
.9
Lu
ng
wt/
A
ir
6.8
9
6.9
1
6.2
1
6.2
5
7.5
4
6.8
7
6.7
5
5.9
3
7.3
0
Bo
dy
wt
±0
.45
-+
0.36
±
0.3
4
±0
.35
+
0.3
0
±0
.56
±
0.3
5
+0
.19
±
0.6
5
(mg
/g)
03
1
2.5
7
12
.96
1
0.6
5
10
.83
1
1.0
6
9.6
0
10
.94
9
.21
8
.90
±
0.6
4 b
±
1.1
5 b
±
0.5
3 b
_+
0.86
5 ±
0.4
4 b
±
0.2
25
-+
0.96
5 ±
0.6
75
±
0.2
7
Liv
er w
t/
Air
5
.17
5
.08
6
.05
5
.88
6
.09
6
.07
6
.26
6
.03
5
.57
B
od
y w
t ±
0.3
0
±0
.32
±
0.1
2
+0
.35
±
0.5
1
±0
.22
+
0.4
1
+0
.25
+
0.4
4
(g/1
00
g)
03
5
.13
5
.59
5
.63
6
.83
6
.91
5
.94
6
.70
6
.65
6
.50
±
0.3
8
+0
.50
±
0.3
1
+0
.31
±
0.4
5
±0
.35
+
0.1
7
÷0
.11
±
0.3
5
aMea
n -
+ S
.E.M
. fo
r 5
det
erm
inat
ion
s.
¢~
bS
ign
ific
antl
y d
iffe
ren
t fr
om
th
e c
orr
esp
on
din
g c
on
tro
l at
P <
0
.05
. o
w I O / /
¢n - ~ - rY 0 (D co < O 5
LIJ o9
0 i I . _ _ L I I I l 0 : '4 48 72 96 i20 I44 168 192 2'16 240
HOURS
F i l t e r - A i r
Ozone
Fig . 2. C h a n g e s in s e r u m a s c o r b i c a c i d l e v e l s f o l l o w i n g a 5 - d a y e x p o s u r e t o o z o n e o r t o f i l t e r e d a i r . E a c h v a l u e r e p r e s e n t s t h e m e a n -+ S . E . M . f o r a t l e a s t f i ve d e t e r m i n a t i o n s .
S e r u m a s c o r b a t e l e v e l s in o z o n e - e x p o s e d a n i m a l s a r e s i g n i f i c a n t l y d i f f e r e n t ( P < 0 . 0 5 )
f r o m c o n t r o l v a l u e s f o r f i r s t 24 h.
5 <I (D El3 fit O ~
< S
< ' x {D {:~ rr E
O
<I
O H-
@ 08 o <
m 0 6
o9 --- 4- . . . 0 4
13:: w E _ > - o2
d <I I..- 0 I--
2° I 4 ~5
5 I0
!t L 5
© I I 1 ~ 1 I 1 I I 0 24 48 72 96 120 144168 192216 240
HOURS
C5
<I (.9 C13 cr O ~ (D c~
<I (D
I-- 0 I.-
G) m g o co
rr m > _J _..1
0
Fig. 3. Changes in liver and carcass ascorbic acid
~0
25
0~. 15 t .
E iO
5
1 I 1 i l ~ 1 J I - J O0 24 48 72 96 120 144 168 192 216 240
HOURS
l e v e l s f o l l o w i n g a 5 - d a y e x p o s u r e t o o z o n e
(= . . . . . ~ ) o r t o f i l t e r e d a i r (a L). E a c h v a l u e r e p r e s e n t s t h e m e a n ± S . E . M . f o r a t
l e a s t 5 d e t e r m i n a t i o n s . S t a t i s t i c a l s i g n i f i c a n c e (P < 0 . 0 5 ) f o r o z o n e e x p o s e d vs. c o n t r o l s : A s c o r b a t e / 1 0 0 g c a r c a s s - - i n i t i a l t i m e p o i n t ; t o t a l c a r c a s s a s c o r b a t e - f i r s t 1 4 4 h ; a s c o r -
b a t e / 1 0 0 g l i v e r - - f i r s t 24 h ; t o t a l l i v e r a s c o r b a t e - - f i r s t 6 h .
306
3 0
2 5
c~ 2 0
o O -- b5
r-~ ~-
O E P0
<D -- 5 m
O 0 <D
8O <_9 Z D
6O
0 ~ 40
2 O
5
I I I I m l l l l l J
0 I i I [ I I I J 1 I 0 24 48 72 96 120 144 168 192 216 240
HOURS
Fig. 4. Changes in the ascorbic acid levels in lung over the course of the experiment: Ozone-exposed, (: . . . . . D); filtered-air ( ~ - - : ~ ) . Each value represents the mean + S.E.M. for 5 determinations. Total ascorbate/lung in the ozone-exposed animals are significantly different P < 0.05 from controls for first 72 h.
[1-]4C] Ascorbic acid metabolism N o s ignif icant d i f ferences , h o w e v e r , were de tec t ed wi th respect to the
d e c a y curves for [1-14C] ascorbic acid d isappearance in serum, lung, or liver, w h e t h e r the data were expressed as speci f ic act iv i ty or as to ta l act iv i ty o f ascorbic ac id /organ or ml o f s erum versus t i m e (Fig. 5). H o w e v e r , the rate o f [1-14C] ascorbic acid was s igni f icant ly reduced in the carcass o f the O3- e x p o s e d mice . Direc t ca l cu la t ion o f the various turnover t imes f rom the data also indicated n o statist ical d i f f erence in the half-l ife o f ascorbic acid in serum, liver, or lung. In carcass, h o w e v e r , the half- l i fe for [ 1 J 4 C ] a s c o r b i c acid appeared t o be a b o u t 40% longer in the o z o n e - e x p o s e d m i c e (Table II).
To further evaluate p h a r m a c o k i n e t i c parameters , the radioact ive d e c a y data were also a n a l y z e d in the c o n t e x t o f the m o d e l s h o w n in Fig. 1. The ca lcu la ted k inet ic parameters f r o m this s tudy suggested that the best fit was t o a 2 - c o m p a r t m e n t m o d e l cons i s t ing o f a rapidly e x c h a n g i n g p o o l b e t w e e n serum and the liver and lung and a s lower exchang ing p o o l b e t w e e n serum and the remain ing carcass. The rate c o n s t a n t s ca lcu la ted f r o m the m o d e l are given in Table III. The rate c o n s t a n t s k2, k3 and k4 were s igni f icant ly dif- ferent in the c o n t r o l and o z o n e - e x p o s e d m i c e (cf. Fig. 3). The o ther c o n s t a n t s for t i ssue e l i m i n a t i o n that proved t o be i m p o r t a n t in describing the m o d e l
307
6
[ 0 5z Z
a_ 3 121 ~2
- LUNG
l l l l l l l l l l
6
-.1
"~ 4 n
3
- - 2
L I V E R
1 1 1 1 1 1 1 1 1 1
7
(1) 6 I
u 5
u 4
Q
~ 2 o
" C A R C A S S ~ . SERUM
I
0 i I i I i I I I I I 0 24 48 7'2 96 120 144 168 192 216 240
3
~' 2 o
0 I I I 1 I I I 1 I I 2 4 4-8 7"2 9 6 120 144 168 192 216 24.0
H O U R S
Fig. 5. Change in [ 1 J 4 C ] a s c o r b i c acid specif ic act ivi ty in lung, liver, se rum, and carcass fo l lowing in t r ape r i t onea l in j ec t ion : ozone (~ . . . . '~); f i l tered-air (:~ L). Each value r ep resen t s the m e a n of 5 de t e r m i na t i ons . S t a n d a r d er ror bars are c o n t a i n e d wi th in the b o u n d a r i e s of the symbols .
T A B L E II
ASCORBIC ACID H A L F - L I V E S IN S E L E C T E D TISSUES a
Tissue tl/2 (h) for t r e a t m e n t s b
Air Ozone
S e r u m 67.3 80.6 ( 5 8 . 2 - - 7 9 . 7 ) ( 6 4 . 2 - - 1 0 8 . 3 )
Liver 67.7 80.2 ( 6 0 . 8 - - 7 7 . 0 ) (67 .9 - -99 .0 )
Lung 69.8 69.3 ( 63 .4 - - 77 .6 ) (59 .7 - -80 .6 )
Carcass 85.7 120.1 c ( 82 .6 - - 89 .0 ) ( 99 .4 - -151 .6 )
aDecay curves given in Fig. 5. b M e a n s and (95% c o n f i d e n c e intervals) . cp < 0.01.
308
T A B L E III
R A T E C O N S T A N T S AND V O L U M E OF D I S T R I B U T I O N D E R I V E D F R O M THE M O D E L I N G S T U D Y a
Paramete r s* T r e a t m e n t s
Air Ozone
K~ 12 .6972 ± 1 .2712 7 .4039 + 0 .7691 K 2 1 .2822 ± 0 .1124 0 .3479 ± 0 .0540 b K 3 0 .8555 ± 0 .0488 1 .0290 ± 0 .09615 K~ 0 .1356 ± 0 .0085 0 .1461 + 0 .0151 b K~ 4 .4162 ± 0 .3037 5 .0816 + 0 .4635 K~ 0 .2447 ± 0 .0159 0 .1862 ± 0 .0164 V d 8.50 ml ± 0 .03 4.81 ml ± 0.03 b
aMean ± S.E. Each of ra te c o n s t a n t s is def ined in Fig. 1 and is expressed as (h) -~ . b Signif icant ly d i f fe ren t at P < 0 .05 (ozone vs. f i l tered-air) .
w e r e k7 ( 0 . 2 0 3 ( h ) -1) a n d k s ( 1 . 2 1 ( h ) -~) f o r o z o n e - e x p o s e d a n d f i l t e r e d
a i r - b r e a t h i n g r a t s , r e s p e c t i v e l y .
G i v e n i n T a b l e I V a r e t h e n e t a s c o r b i c a c i d f l u x e s e x p r e s s e d as m g / m l ×
h -~ f o r t h e s e l e c t e d c o m p a r t m e n t s . T h e s e v a l u e s w e r e d e r i v e d f r o m t h e r a t e
c o n s t a n t s a n d e s t i m a t e s o f t h e a s c o r b i c a c i d c o n t e n t i n t h e t i s s u e c o m p a r t -
m e n t s a t e a c h o f t h e t i m e s i n d i c a t e d .
T h e n e t f l u x o f a s c o r b i c a c i d b a c k i n t o l i v e r a c c o u n t e d f o r a p p r o x i m a t e l y
o n e - h a l f o f t h e t o t a l d i s a p p e a r a n c e o f a s c o r b i c a c i d f r o m s e r u m ( a s s u m i n g a
t o t a l s e r u m v o l u m e o f 1 . 5 - - 2 m l ) . L u n g a c c o u n t e d f o r a p p r o x i m a t e l y 2 0 %
T A B L E IV
NET ASCORBIC ACID F L U X F R O M S E R U M AND INTO S E L E C T E D TISSUE C O M P A R T M E N T S a
T ime Tissue ( m g / o r g a n or ml of s e r u m / h )
Liver Lung Carcass Se rum × 102 × 102 x 103 × 102
Air O~ Air 0 3 Air O, Air O,
0 .22 13.0 2.4 4.91 1.97 8 .25 3.24 - -20 .0 --5.0 3 12.0 4.7 4 .70 3.56 7.93 6.28 - -19 .0 --9.3 6 8.0 3.0 3 .15 2.30 4 .98 3.50 - -13 .0 --6.0
12 9.8 5.0 3 .80 3.76 6.31 6 .58 - -15 .0 9.8 48 7.2 4.0 3.14 3.11 4 .75 5.31 - -12 .0 --8.0 72 9.2 8.8 3 .59 6 .29 6 .46 6.16 - -15 .0 - -17 .0
216 11.0 6.6 4.51 4.97 7.66 6.37 18.0 - -13 .0
aDer ived f rom the mode l given in Fig. 1 and the rate c o n s t a n t s def ined in Table III and the text . Negat ive sign for t he se rum values indica tes d i sappearance of ascorbic acid f rom serum. The posi t ive values for liver, lung, and carcass indica tes ne t u p t a k e of ascorbic acid.
3 0 9
TABLE V
E F F E C T OF OZONE E X P O S U R E ON THE D E C A R B O X Y L A T I O N OF [ 1 -I 'C ] ASCORBIC ACID AND HEPATIC SYNTHESIS OF ASCORBIC ACID
Trea tmen t Rate of deca rboxy la t ion as ~4CO 2 (DPM/g X 10 .6 )
Liver synthesis b (umoles /g /h)
Lung Liver
Fil tered-air 1.23 ± 0.24 (8) 7.79 ± 0.81 (8) 0.23 ± 0.0~i (6) Ozone-Exposed 0.19 *~ 0.07 (4) c 3.45 ± 0.70 (4) d 0.13 ± 0.04 (7) d
aData expressed as the means ± S.E.M. (nu mb er of de terminat ions) . De te rmined im- media te ly fol lowing exposure .
b R e p r e s e n t s the mean + S.E.M. f rom samples ob ta ined over the 216-h exper imenta l period.
cp < 0.05. d p < 0.01.
and the carcass accounted for 3--5% of the flux from serum. Exposure to ozone appeared to inhibit ascorbic acid uptake by liver and lung, particularly during the first 48 h following ozone exposure.
In vitro studies Since the ascorbic acid used in this study was labelled at the 1-carbon
position, it was determined whether ozone exposure caused changes in the metabolism of [1-14C] ascorbic acid to 14CO2, a significant pathway of ascor- bic acid metabolism in the rodent. As shown in Table V, ~4CO2 production from [1-~4C] ascorbic acid in vitro was reduced to 15% (in the lung) and 44% (in the liver} of the control values for mice exposed to ozone. The decrease in this catabolic pathway for ascorbic acid was also accompanied by an apparent decrease in the rate of net synthesis of ascorbic acid by the liver in vitro (Table V).
DISCUSSION
The increase in lung wet weight that was observed following exposure to ozone is typical of effects previously seen in the mouse [19] and reflects the edematous and inf lammatory response of the lung to ozone. This level of ozone is the maximum tolerated dose for mice and of sufficient magnitude to clearly stimulate mechanisms involving endogenous anti-inflammatory and anti-oxidant factors. Indeed, it is clear that ozone caused changes in tissue levels of ascorbic acid, at least on a whole organ basis. The changes were observed not only in lung, but in liver, serum and the carcass as well.
As indicated by the radioactive decay curves, intraperitoneal injection of [1-14C] ascorbic acid into mice resulted in an early equilibration of ascorbic acid into tissues. Peak radioactivity levels appeared almost immediately in the serum, liver, and carcass and by 3 h in the lung. These results agree with
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the observations of Hammarstroem [20] following the intravenous injection of radiolabelled ascorbic acid into mice. From these data, kinetic modeling indicated that disappearance of [1-'4C] ascorbic acid was most easily inter- preted as a 2-compartment model consisting of a rapidly exchanging ascorbic acid pool between serum and liver and lung and a slowly exchanging pool between serum and other tissues. With respect to liver, the observed lower flux of ascorbic acid from serum into liver in ozone-exposed mice compared to the controls, as well as reduced hepatic synthesis explained in part the lower liver ascorbic acid levels observed in the ozone treated mice. The observation that the turnover of ascorbic acid was not changed by ozone exposure most probably reflected the concommitant reduction in the rate of hepatic ascorbic acid catabolism in mice exposed to ozone.
The lower flux of ascorbic acid into lung following ozone exposure was not reflected by lower tissue values. The observed increase in lung ascorbic acid levels differs from the reported decrease in lung ascorbic acid following acute ozone exposure [8]. Willis and Kratzing [1,10] observed a decrease in lung ascorbate following injections of nonepinephrine or epinephrine or acute exposure to hyperbaric 02. Leung and Morrow [9] reported a decrease in guinea pig lung ascorbate following acute exposure to NO2. However, lung ascorbate was not significantly reduced in rats exposed to hyperbaric 02 for 2 days [7]. The increase in lung ascorbic acid after ozone exposure was most probably due to edema from serum infiltration, decreased catabolism, and undoubtedly infiltration of leukocytes and macrophages and changes in the lung cell population that occurs in response to ozone [24]. Wright et al. [3] have demonstrated selective uptake of ascorbic acid into lung type II cells and alveolar macrophages. A shift in the lung cell population due to ozone exposure could also result in a net increase in the lung ascorbic acid level.
The results of the present study may reflect an acquired "metabolic adaptat ion" with respect to lung ascorbate metabolism in animals chronically exposed to 03. A similar discrepancy was observed in lung glutathione levels following O3-exposure. DeLucia [21,22] saw decreased lung glutathione levels in rats acutely exposed to Oa, but if the exposure period was extended to 7 days, glutathione levels increased above control lung levels [ 23].
The question of whether ascorbic acid synthesis is stimulated in response to an oxidant may also be addressed. Synthesis by whole liver homogenates was reduced in mice exposed to ozone and the net flux of ascorbic acid from the serum of ozone-exposed mice into tissue compartments appeared reduced. If it is assumed the flux from serum is L~lanced by hepatic syn- thesis and release, then it would appear that ozone expasure does not cause positive changes in the rate of ascorbate synthesis; a somewhat surprising finding in view of ascorbic acid's proposed function as an anti-oxidant. Estimates from the data for the negative flux from serum, and for liver synthesis in vitro, indicate that roughly 2--5 mg (for filtered air-breathing mice) or 1--3 mg (for ozone-exposed mice) of ascorbic acid were synthesized per day by the mice in this s tudy [also cf. 25]. Although the estimates in the ozone-exposed mice are possibly compromised by the mild inanition that
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o c c u r s d u r i n g o z o n e e x p o s u r e , t h e d a t a c e r t a i n l y d o n o t sugges t s t i m u l a t i o n o f a s c o r b a t e s y n t h e s i s . R a t h e r t h a n i n c r e a s i n g s y n t h e s i s , i t w o u l d a p p e a r t h a t t i s sue levels o f a s c o r b i c a c i d in t h e m o u s e a r e m a i n t a i n e d b y m o d u l a t i n g t h e r a t e o f d e g r a d a t i o n .
Th i s t y p e o f r e s p o n s e m a y b e i m p o r t a n t f r o m a r e g u l a t i o n s t a n d p o i n t . I t has a l so b e e n o b s e r v e d t h a t t h e a s c o r b i c a c i d c o n c e n t r a t i o n in ce l l a n d t i s s u e c u l t u r e m e d i a is an i m p o r t a n t m o d u l a t o r o f c o l l a g e n s y n t h e s i s . I n f i b r o b l a s t a n d s m o o t h m u s c l e ce l l c u l t u r e s , h igh c o n c e n t r a t i o n s o f a s c o r b i c ac id cause a d i s p r o p o r t i o n a t e i n c r e a s e in c o l l a g e n s y n t h e s i s r e l a t i v e t o t h e s y n t h e s i s o f o t h e r p r o t e i n s [ 2 6 ] . Even t h o u g h a s c o r b i c ac id m a y b e an i m p o r t a n t an t i - o x i d a n t , i t m a y be o f s o m e a d v a n t a g e n o t t o m a r k e d l y i n c r e a s e t h e a s c o r b i c a c i d c o n c e n t r a t i o n in l ung e s p e c i a l l y in c o n d i t i o n s i n d u c e d b y O3 e x p o s u r e k n o w n t o p r o m o t e p u l m o n a r y f i b r o s i s [ 2 7 ] .
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