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The effects of naphthalene on glucose metabolism in the European flat oyster Ostrea edulis
(',,rap Bu~thera. Phvxiol Vol 70('. pp. 13 to 20. 1981 Printed in Great Britain All rights reserved
0306.-4492.81;050013-0810200/0 Copyright :C 1981 Pergamon Press Lid
THE E F F E C T S O F N A P H T H A L E N E O N G L U C O S E M E T A B O L I S M IN THE E U R O P E A N FLAT OYSTER O S T R E A E D U L I S R. T. RILEY t and M. C. MIx 2 ~Toxicology and Biological Constituents Research Unit, Russell Research Center, P.O. Box 5677, Athens, GA 30613: and 2Department of General Science, Oregon State University, Corvallis. OR 97331, U S A . (Receiced 26 January 1981)
Abstract--- 1. The pool sizes and redistribution of ~4C-label from D-[U-l'tC]glucose into ethanol insoluble polysaccharides, total protein, total polar lipids, total neutral lipids, free alanine, aspartate, glutamate and total organic acids were determined for control and naphthalene-treated oysters Ostrea edulix. 2. The following effects were attributed to naphthalene treatment: a decrease in the percentage of protein and polar lipid: an increase in the total amino acids (sum of alanine, aspartate and glutamateS; an increase in organic acids: and an increase in the mean specific activity Igeometric) for all measured pools. 3. These results suggested that naphthalene stimulated protein and polar lipid catabolism, while simultaneously stimulating the flow of glucose-carbon into all measured pools.
I NTRODU('TION
ullaneously exposing oysters to three different treatments in a flow-through system containing Millipore filtered synthetic seawater with 1 mM glucose and 100ppm streptomycin. The first group [8 oystersl was not exposed to naphthalene [control-treated (CtJl. the second group 18 oysters) was exposed to unlabeled naphthalene [naphthalene treated (Ntl) and the third group 18-12 oysters) was exposed to [I-14C]naphthalene at the same concentration as the second group. The gills from oysters in the former two groups were utilized for measuring the pool sizes of the major precursors, intermediates and end products of glucose metabolism, and for an in ril~o kinetic analysis of glucose metabolism. Oysters from the latter group were used to determine the tissue concentration of [l-t'tC]naph thalene after 72 hr exposure to an average naphthalene concentration of 65.7ppb: uptake and accumulation of [I-~*C]naphthalene was the topic of a previous publication [Riley et al., 19811. A total of 4 runs were conducted [R-I-R-4). Run R-I was concerned primarily with testing the flow system and the uptake of [14C]naphthalene. Runs R-2-R-4 determined the effects of naphthalene exposure on the pool sizes and on glucose metabolism; the results of these 3 runs are the subject of this paper. All oysters were collected at the same time and then stored in a closed holding aquarium. Since the runs were conducted sequentially, the total storage time Istarvation period) increased between runs: 67, 85 and 105 days for R-2-R-4. respectively.
There have been few studies of sublethal petroleum toxicity in bivalves which have investigated metabolic effects at levels more basic than oxygen consumption. Gilfillan [1975) calculated the c a r b o n budgets for two species of mussels exposed to oil in seawater extracts. In 10",, mixtures, the carbon flux [carbon assimilated minus carbon respired) was reduced by 50",i and at higher concentrations the c a r b o n flux became negative. Gilfillan 09751 theorized that negative carbon flux would reduce the a m o u n t of c a r b o n available for growth and reproduction. The most basic metabolic effect is at the level of the enzymes involved in metabolism. There have been few published reports of the effects of oil on enzyme activity in bivalves (Heitz et al., 1974; Bayne et al., 1979; Stekoll et al., 1980). A study of the c a r b o n flux through intermediary metabolism and into metabolic end products in a bivalve mollusk seemed warranted. Metabolic perturbation either precedes or is simultaneous with physiologic alteration. Theoretically, alterations in enzyme activity should be rapidly and accurately determined by in t'iro kinetic analysis using ~aC-labeled precursors. The effect of a chemical perturbant at one point in a metabolic pathway should be conveyed to other pathways via stoichiometric linkage. The purpose of the present study was to utilize an in rit'o analysis of glucose metabolism to elucidate the mechanism of toxicity of n a p h t h a l e n e in oysters O.strea edulix. Naphthalene was chosen for this study because it is representative of one of the more toxic organic components [aromatic hydrocarbons) of the water soluble fraction of crude and refined oils (Neff et al., 1976).
Experimental protocol
At the end of each 72-hr dosing run IR-2-R-41 each Ct and Nt oyster was removed and placed into individual incubation vessels containing 25 ml of filtered synthetic seawater to which was added 4#Ci of D-[U-~'*C]glucose 1274 mCi/mmol: Amersham Corporation, Illinois, U.S.A.). Each vessel was sealed with a Teflon gasket and aerated. Oysters were removed at intervals up to 240 inin and quick frozen on a bed of dry ice. The gill tissue and mantle edge were then removed, weighed, freeze-dried and stored under N2 at - 2 0 C in a desicator over anhydrous sodium sulfate. The [~4C]glucose uptake experiments were conducted at 15 C + 0 . 2 C . Pool sizes and specific radioactivity time curves for ethanol insoluble polysaccharides [assumed to be primarily
MATERIALS AND METHODS The experimental protocol for dosing oysters O. edulis with naphthalene has been described previously IRiley et al., 1981). Briefly. each experiment IRunl consisted of sire13
14
R . T . RItFY and M. C. Mix INCUBATION
I
FREEZE-DRY, HOMOGENIZE IN 80% ETOH
I
I
FOLCH EXTRACTION- PELLET
I
I
I
I
CHC 13 I I LIPIDS F
PPT DISSOLVE HC I
TCA PPT
SUPER- CHC I3 EXTRACT AND ql WATER WASH
I AQUEOUS INCLUDES WATER WASH OF FOLCi EXTRACT
SILICIC ACID COLUMN
1
MEOH
DOW 50
1
CHI~I5 WASH
SUPER
(GLYCOGEN)
PELLET
Dew I
(PROTEIN) WASH
;
NH4OH
AA' S
FORMIC TL~
NEUTRAL ~COMPOUNOS ORGANIC ACIDS
TLC Fig. 1. Fractionation scheme for the metabolic pools: ethanol insoluble polysaccharides Iglycogen), protein, neutral lipids, polar lipids, amino acids, organic acids and neutral compounds. glycogenL total protein, total polar lipids, total neutral lipids, free alanine (Ala), aspartate (Aspl, glutamate IGlu) and total organic acids were determined for Ct and Nt oysters. Radioactivity-time curves for malate, succinate and neutral substances were also determined. Qutmtitatire methods
Each freeze-dried gill was fractionated according to the procedure outlined in Fig. I. Water. organic solvents and all other reagents were obtained and purified as described in Riley et al. (1981). The tissue was weighed, put into a 10-ml Vitro-tissue grinding tube. liquid N , added and the tissue pulverized into a fine uniform powder. The powdered tissue was homogenized in 80°,, ethanol with a Potter-Elvehjem type homogenizer and the homogenate was allowed to stand I hr at 4 C: it was then centrifuged at 4340.q at 4 C for 10 rain and the supernatant pipeted into a screw-cap culture tube. The homogenization and centrifugation procedure was repeated twice and the final precipitate.was washed with 80",, ethanol. The supernatants and washes were combined. 2vol chloroform and 2 ml of water added, and then mixed. The upper aqueous phase was pipeted into an Erlenmeyer flask and the water washes repeated 3 times, and all washes combined in the Erlenmeyer flask. The precipitate from the original ethanol homogenization stop was extracted by the method of Folch et u/. (1957). The .,upernatant was pipeted into a screw-cap culture tube and the 2:1 chloroform-methanol extract was water washed. After phase separation, the aqueous phase was pipeted into the Erlenmeyer flask containing the water washes from the chloroform extract of the ethanol supernatant. The chloroform and chloroform-methanol extracts
were pooled, made to one phase by the addition of methanol and then evaporated to dryness under N2 at 50 C. The residue was brought up in chloroform and separated into polar and neutral lipids by the method of Dittmer & Wells (1969). An aliquot of the neutral lipids dissolved in chloroform was placed in a scintillation vial and the chloroform evaporated under N:. The scintillation vial was filled with 5 ml of toluene scintillation fluor and counted on a Packard Tri-Carb LS Spectrometer. Counting efficiencies were determined by internal standardization. An aliquot of the polar lipids dissolved in methanol was evaporated in a scintillation vial to remove any residual chloroform and then redissolved in methanol and counted in PCS fluor (Amersham Corporation). The total lipids present in each fraction were quantified by the charring technique of Marsh & Weinstein (1966) with tripalmitin used as a standard. The combined aqueous extracts prepared previously, were partially evaporated under a flow of N2 at 50 C and then evaporated to dryness over sodium sulfate in a sleevetype Pyrex desiccator under '.acuum using a water aspirator vacuum pump. An infra-red heat lamp was used to speed evaporation. The dried residue was stored under N: at - 2 0 C in a desiccator over sodium sulfate until ready for fractionation into amino acids, organic acids and neutral compounds. The lipid-free pellet from the Folch extraction was partially dried under a flow of N:, homogenized in 0.01 N hydrochloric acid (HCI) and transferred to a corex centrifuge tube. An equal volume of 10% trichloroacetic acid (TCA) was added and the mixture held at 4 C for I hr followed by centrifugation at 12.100g at 4 C for 10rain. The TCA supernatant was pipeted into a graduated tube and the precipitate washed twice with 5"° TCA and recen-
Naphthalene in oysters trifuged between washes. The washes were combined with the supernatant in the graduated tube. An aliquot of the TCA soluble substances flotal polysaccharidesl was counted in PCS fluor and another aliquot was analyzed for total reducing sugars after acid hydrolysis by the method of Dubois et al. (1956) using glucose as a standard. The TCA precipitate (total protein) was washed with two aliquots of 0.1 N potassium acetate in 80". ethanol. The washes were discarded and the precipitate dried under a flow of N , at room temperature. The dried precipitate was then dissolved in 0.1 N sodium hydroxide (NaOH) at 50 C. O n e aliquot was counted in PCS and a second was analyzed for total protein by the method of Lowry et al. (1951~ using bovine serum albumin as a standard. The dried residue from the water wash of the combined lipid extracts was dissolved in 0.01 N formic acid and then passed through a column of Dowex 50 x 4, 200 400 mesh resin in the H" form. The column was washed 3 times with 0.01 N formic acid. The sample solvent and washes were collected in an Erlenmeyer flask. Amino acids were eluted with 4 N a m m o n i u m hydroxide and the eluate was collected in another Erlenmeyer flask. The eluate and wash were frozen and then freeze-dried. Recoveries of L-[U-~'*C]glutamate and L-[U-~4C]leucine carried through from the homogenization step were 90.8'!o (standard deviation (SD = 5.5, n = 4 ) and 90.7°,, (SD = 1.2, n = 4 ) respectively. The freeze-dried Dowex 50 eluate lamino acids) was brought up in 0.01 N HCI and transferred to a graduated tube along with two rinses of the Erlenmeyer flask. An aliquot was then counted in PCS and the eluate reevaporated to dryness. The residue was dissolved in a small volume of 0.01 N HCI. and an aliquot counted in PCS. The exact volume of the concentrated eluate was calculated. based on the previously determined volume and activity. Aliquots of the concentrated Dowex 50 eluate were streaked in 2 cm scribed lanes on thin-layer plates prepared as described by Turner & Redgwell (19661. The plates were developed in glass-distilled phenol water (80:20w,vl and air-dried overnight. The two end lanes were sprayed with 0.5",, ninhydrin in 95". ethanol. In the remaining lanes the areas corresponding to the amino acids Ala, Asp and Glu, were removed and counted by the method of Redgwell et ,/. 11974l. Ala, Asp and Glu were the only amino acids with detectable counts and they were well resolved in one dimension by the phenol--water solvent system. Aspartate and asparagine were not resolved by the one-dimensional method. The Dowex 50 amino acids were quantified by thin-layer chromatography ITLCI. Aliquots of the e l u a t e were streaked in I cm scribed lanes adjacent to known standards of Ala, Asp and Glu. The plates were scanned with a Schoeffel SF 3000 spectrodensitometer using an illumination wavelength of 585 n m and a reflectance wavelength of 510 n m The standard curves were generated for each plate in order to compensate for variations between plates. The reproducibility of this method was +4.0"0 In = 421 for Ala. -,-4.6". (n = 421 for Asp, and +4.40. (n = 44) for Glu. The freeze-dried Dowex 50 wash was dissolved in pH 6.0, 0.01 N Na formate buffer and then passed through a column of Dowex 1 × 8. 100--200 mesh in the OH form. All the original solvent and rinses were collected in a graduated tube. and subsequently referred to as "'neutral compounds". Organic acids were eluted with 6 N formic acid and the eluate collected and freeze-dried. An aliquot of the neutral c o m p o u n d s was counted in PCS fluor. The freeze-dried Dowex 1 eluate containing organic acids was dissolved in 50°,, acetone, transferred to a graduated tube and an aliquot counted in PCS after evaporating the acetone. After the addition of phenol red indicator an aliquot was titrated to the end point with 0.01 N N a O H . The results of the titration were expressed as milliequivalents H * , g dry wt. The remaining Dowex l eluate was
15
evaporated to dryness, and then dissolved in a small volume of 50°. acetone. The recovery of [l,4-~'*C]succinic acid and [2-14C]acetic acid carried through from the homogenization step were 88.8°0 (SD = 4.6, n = 4) and 1.3°o (SD = 1.8, n = 4), respectively. An aliquot was counted and then 10jul were spotted in the lower left corner of a mixed layer plate and the organic acids separated by twodimensional T L C (Riley & Mix, 19801. Stuti.~tical method,~
The following abbreviations are used for convenience: .~ = mean; SD = standard deviations; C1 = confidence intervals: r 2 = coefficient of determination; P = probability. An analysis of variance INeter & Wasserman, 1974) was conducted to determine differences in the pools sizes (response variables) as a function of sample time, run and treatment 0ndependent variablesl. The 5°. significance level (a = 0.05) was set as the level for rejecting the null hypothesis. Curve fitting was accomplished by linear regression analysis. Data were fitted to a model of the form: In Yo = a./ + hi(In x) + e~(ln x ) 2. Comparison of specific radioactivity, time curves (Ct to Nt) was accomplished as described by Neter & W a s s e r m a n (1974l. The alternative conclusions were: C~ : equality of regression parameters (a~ b~ and cfl. C2 : inequality of regression parameters (aj, b~ or cj). Visual inspection of the regression curves suggested that the mean specific activities were always greater in naphthalene-treated oysters. In order to demonstrate this difference statistically an analysis of the residuals was conducted. Residuals lei, i = 1, 2 . . . . . n) are defined as the difference between the observed values (Y0 and the corresponding fitted values (~) from the reduced regression equations. The mean residual (~) can be calculated as follows: ~=
~" lnY,,,'n- ~" In~,'n i
1
i
1
and (geometric) = antilog e = Y (geometric)/Y(geometric); t h u s . . ~ (geometric) is the mean specific activity (geometric) for each treatment expressed as a fraction of the mean specific activity (geometric) of the reduced models. Confidence intervals of X (geometric) can be calculated as follows: antilog(~; _+ t S D n - ° s); where SD is the standard deviation of e and t is the t-statistic for the appropriate degrees of freedom. RESU LTS" T h e p e r c e n t d r y wt of t h e gill tissue o f O. e d u l i s d e c r e a s e d b e t w e e n r u n s ; 17.0, 16.3 a n d 15.6°,o for R-2, R-3 a n d R-4, respectively (Table 1). T h e difference w a s statistically significant b e t w e e n R u n s 2 a n d 4 (P = 0.025: S t u d e n t ' s t-testj. T h e m e a n wet wt o f gill tissue w a s 133.4 + 10.8 m g ( + 9 5 ° CI, n = 48), a b o u t 47"0 of t h e total tissue wet wt. Since e a c h r u n was d o n e s e q u e n t i a l l y , t h e total l e n g t h o f s t a r v a t i o n inc r e a s e d b e t w e e n e a c h run. D u r i n g t h e [ t ' * C ] g l u c o s e u p t a k e e x p e r i m e n t for R-2, t h e t e m p e r a t u r e c o n t r o l m e c h a n i s m failed a n d t h e t e m p e r a t u r e fell to a p p r o x 12.8"C for the c o n t r o l oysters. T h e specific a c t i v i t y - t i m e c u r v e s for this r u n were n o t c o m p a r a b l e since t h e t e m p e r a t u r e effect w o u l d be p r i m a r i l y a rate effect. H o w e v e r , t h e pool sizes for C t a n d N t o y s t e r s f r o m R-2 were a s s u m e d to
R. T. Rn.ev and M. C. Mtx
16
Table I. Dry wt of the gill tissue expressed as percent of the wet wt for Runs R-2-R-4
Starvation (days) ",, Dry wt 95",, CI t)
R-2
R-3
R-4
67 17.0 +0.9 16
85 16.3 +0.8 16
105 15.6 +0.9 16
The R-4 value was significantly less than the R-2 (Student's t-test: P = 0.025~. Both Ct and Nt gills were pooled to obtain the combined means.
be comparable. It was reasoned that the total carbon flux over the short period of the glucose uptake experiment (-,< 240 min) would have been insufficient to affect the absolute pool sizes which were a result of the carbon flux during the prior 72-hr dosing period in the flow system. The results of the analysis of variance indicated that there was no relationship between sample time and the pool sizes of glycogen, protein, polar and neutral lipids, total Ala, Asp and Glu and total organic acids. Thus, verifying that these pools were in a steady-state during the [14C]glucose uptake experiments. However, there were significant effects related to run and n a p h t h a l e n e treatment.
Percent neutral lipids, total Ala, Asp and Glu and total organic acids were all significantly related to run ~P < 0.001). Percent polar lipids were close to being significantly related to run (P = 0.10). The percent total lipids Ipolar plus neutral) increased between runs (Table 2) while amino acids and organic acids decreased (Table 3). The Student's t-test was used to test for significant differences, by treatment, in those pools which were shown to be significantly related to the increasing storage (starvation) between runs. For Ct and Nt oysters neutral lipids were significantly greater for R-4 compared to R-2 and R-3, and total lipids were significantly greater for R-4 compared to R-2. The total amino acids (sum of Ala, Asp and Glu) decreased significantly between each run for both treatments and organic acids decreased significantly between runs 2 and 4 for both treatments. Percent glycogen and protein were not significantly affected by the increasing storage lstarvation) between runs. The percent protein and percent polar lipids were b o t h significantly affected by naphthalene treatment (P < 0.01). Protein and polar lipids were always less in Nt oysters (Table 2). The total Ala, Asp and Glu Isum of Ala. Asp and Glu) and the total organic acids were close to being significantly affected by n a p h t h a lene treatment (P ~< 0.10~. The total Ala. Asp and Glu and total organic acids were always greater in Nt oysters (Table 3). The Student's t-test was used to test for significant differences, by run. in those pools
Table 2. Comparison of the percent glycogen, protein, neutral lipids, polar lipids and total lipids between runs in the gills of control (Ct) and naphthalene-treated (Ntl oysters R-2 Pool
R-3
Ct
Nt
Ct
Glycogen
5.3
5.7
Protein*
59.1
58.3
Polar lipids*
5.0
4.9
Neutral lipids't
4.3
4.2
Total lipids*'t
9.3
9.1
5.6 SD 63.2 SD 5.4 SD 4.0 SD 9.4 SD
R-4 Nt
5.2 = 0.641 55.4 = 4.147 4.9 = 0.296 4.4 = 0.366 9.3 = 0.546
Ct
Nt
5.6
5.6
61.9
60.2
5.4
5.0
5.1
4.9
10.5
9.9
SD. (mean square error) °5. from the analysis of variance model for the full three-way model using each sample time as a replicate. * Significant difference between treatments (P < 0.01); 1, 42 degrees of freedom, F-distribution. "l"Significant difference between runs (P < 0.001); 2, 42 degrees of freedom, F-distributiOn. Table 3. Comparison of the total Ala, Asp and Glu (#M/g dry wt) and total organic acids (Mequiv..,g dry wt) between runs in the gills of control (Ct) and naphthalene treated (Nt) oysters R-2 Pool Total Ala, Asp and Glu* Total organic acids*
R-3
R-4
Ct
Nt
Ct
Nt
Ct
Nt
49.1
54.2
42.1
44.9 SD = 7.693
19.0
22.5
0.194
0.219
0.188 0.198 SD = 0,050
SD, (mean square error) °5, from the analysis of variance model. * Significant difference between runs (P < 0.001): d.f. 2, 42, F-distribution.
0.117
0.156
17
Naphthalene in oysters which were shown to be significantly affected by naphthalene treatment based on the results of the analysis of variance, in Nt oysters the percent protein was significantly less, compared to Ct oysters, in R-3 and significantly less for polar lipids in R-3 and R-4. Naphthalene treatment had no significant effect on the pool sizes of either glycogen or neutral lipids (Table 2). The flow of "=C-label was directed primarily into metabolic end products (averaged for all runs). Ethanol insoluble polysaccharides accounted for 62.30",. (SD = 9.99, n = 48), total protein 5.63", (SD = 2.94, n = 48). total polar lipids 0.20". (SD = 0.10, n = 47) and total neutral lipids 0.03 (SD = 0.02, n = 47) of the total "~C-label recovered in the gill. Amino acids and organic acids accounted for 9.16°. (SD = 4.81, n = 48) and 5.4", (SD = 4.6, n = 48), respectively and neutral compounds accounted for 17.28".. (SD = 12.16, n = 48). The radioactivity time data for R-3 (Ct) are presented as an example of the manner in which glucose-carbon was redistributed to the measure pools
o
I00
• Glycogen (0 60) o Protein (0 98) X Polar Lip=ds (0.93) A Neutral L=p=ds (0.92)
I0 1.0 Ol 0.01 llJ I/1 0 0
b X AIo ( 0 8 1 )
I0 X
0 w _J
x xx.~--x~
.
---IV-
I0
./
U u i, 0
~;
o Glu (056) • Asp (0 68)
in the gill (Fig. 2). The pattern of redistribution was the same for all runs and all treatments. The percent accumulated dose (percent of the total glucose--carbon recovered from gills) in end products (Figs 2a & 2b) increased with time, neutral compounds (precursors) decreased (Fig. 2c) and organic acids remained constant (Fig. 2c). Almost all of the activity from the Dowex 50 eluate was recovered in Aia, Asp and (3lu (X = 96.80., + 5.0, 95",, CI, n = 47). T L C of the Dowex 1 eluate revealed that the majority of the activity in this fraction was in unidentified organic acids and taurine (.~ = 71.56",~, + 5.45, 95°. C1, n = 16). Malate and succinate were the only Krebs cycle intermediates that were labeled in detectable quantities ()f = 2.1°,, + 0.80, 95°, CI. n = 15). About 26'!, of the activity in the Dowex 1 fraction could not be accounted for. 5pec~hc rad,oacttv~ty t,me curves lot ethanol insoluble polysaccharides, (assumed to be primarily glycogen), total protein, total polar lipids, total neutral lipids, free Ala, Asp and Glu, were determined. In order to allow a comparison of the relative importance of the pools with respect to pool size, the data for R-3 (Ct) are normalized to carbon equivalents (Fig. 3). Glucose-carbon was diluted into relatively large glycogen and protein pools and relatively small amino acid pools. Therefore, even though the percent of the accumulated dose recovered in Ala was low, the specific activity was very high. Because the activities in malate and succinate were so low, no radioactivity-time curves are given. Comparison of the specific activity- time curves for Ct and Nt oysters revealed significant differences for protein, polar lipids, Ala, and Glu (Table 4). Visual inspection of the fitted regression curves suggested that in every case the total area under each Nt curve was greater than the Ct (for example, Figs 4a & 4b). The residual analysis confirmed this observation. For every pool and run the mean specific activity (geometric) was greater for the Nt oysters (Table 5).
Ol
I
I
I
I
c
I00
X Neutral Substances (0 82 ) • Orgamc Aods (000)
X ~ X , ~X I0
~
IO
O
X ~ ~ ~
i
A
60
120
I
IBO TIME (rain)
x
I 240
Fig. 2. Percent of total recovered dose in the metabolic pools for Run R-3 (CI). The r z values are given in parentheses. The data are calculated as a percent of the total "*C-label recovered from all measured pools in the gill tissue and are presented as examples of the pattern of redistribution of glucose-carbon in oyster gill ]'or all runs and treatments. (B.P(()
70. I
B
DISCUSSION
There were two types of effects observed; effects attributed to starvation and those attributed to naphthalene treatment. Examples of the former were the decreasing percent dry wt, decreasing concentrations of total amino acids and total organic acids and the increasing concentration of total lipids. Increased water content of the tissues and decreased free amino acids are common manifestations of prolonged starvation in oysters (Riley, 1976; Riley, 19801. Increased total lipids was correlated with increased bioaccumulation of naphthalene (Riley et a/., 1981), however, the increase in total lipids could not. by itself, account for the increased naphthalene accumulation. The bioaccumulation factor ([ l-"=C]naphthalene treatment) for R-4 was 26". greater than R-2 while the percentage of total lipids [unlabeled naphthalene treated) was only 8". greater. The effects attributed to naphthalene treatment were decreased protein and polar lipid concentration, increased total Ala, Asp and Glu, increased total Dowex 1 acids and increased mean specific activity (geometric) for all measured pools. These results suggested that naphthalene stimulated protein and polar
R.T. RILEY and M. C. Mtx
18
a
I OO"
10 8 .
~
in 7.
AIo (.96)
Ct, AIo (96) Glu (.92)
07
NI
(.88) (.911
n
_...=1"~nx_f__.=
,
I 06. !
iI
1 ¢_) cn
I0 6
05. ~
E ~E 104 •
X
,, ~¢"
...~---~:"~,,
~,
Protein1.971 Polar Lipid$
105
O.
103 . I
"~) ~
i0 4
'
.~
108
Ct
,
b
02.
] In t 0
60
ten i80 TIME (rain)
Z40
a
Fig. 3. Fitted specific activity-time curves for the metabolic pools for Run R-3 (Ctl. The r 2 values are given in parentheses. Specific activities are expressed per mg carbon; thus. allowing comparison of the relative specific activities. Conversion factors used were (in percent carbon) glycogen = 44°0, protein = 50"0, lipid (neutral and polar) = 76",, Ala = 40°0, Asp = 36"., Glu = 41"o.
C941 '(.95)
107 t
/
(.g t) (.93) ~ ~_ _ .~- n ,.t°--
,,;/
..a .....
o
X /t
1051 lipid catabolism while simultaneously stimulating the flow of glucose-carbon into all measured pools. Heitz et a/. 119741 demonstrated increased activity of leucine aminopeptidase in Crassostrea giyas exposed to petroleum hydrocarbons. They also d e m o n s t r a t e d significant alterations in the activities of other key enzymes associated with intermediary metabolism: glutamateoxaloacetate transaminase and malate dehydrogenase. Catabolism of stored energy reserves and disturbed protein metabolism are responses which partially characterize the "'stress syndrome" of mussels (Bayne, 19731. The fact that glycogen reserves were not reduced by n a p h t h a l e n e treatment may be due to the ample dissolved glucose available for glycogen synthesis. The increased flow of glucose--carbon into all pools may have resulted from increased transepilhelial glucose transport. Riley 11981) found that in the whole body of starved oysters, maintained and preincubated under similar conditions as the present study, gluc o s e - c a r b o n was redistributed mostly to ethanol insoluble polysaccharides and amino acids. It was
AIo Glu
NI
iO 4 L 0
zx
60
120
180
240
T I M E (rnin) Fig. 4. Fitted specific activity-time curves for Ala and Glu for R-3 (a] and R-4 (bl. The fitted curves and data for Ala and Glu are presented to demonstrate graphically the nature of the differences between the mean specific activities for Ct and Nt oysters. The mean specific activities (geometric) for all the pools, calculated from the residuals generated from the regression analysis, are given in Table 5 The r 2 value is gi'.en in parentheses. Nt, - -: Ct. = - . Ala:Ct = ×. Nt = E, G l u : C t = A, Nt = O.
hypothesized that the m a x i m u m disposal rate (oneway transport) was limited by the m a x i m u m rate of transepithelial transport. The relationship between cellular metabolic rate and the rate of glucose uptake has been wel; demonstrated in m a m m a l i a n systems (Elbrink & Bihler, 19751. For example, in rat adipose
Table 4. Results of the comparison ,ff the regression curves for Runs R-3 and R-4
P d.f.
Glycogen
Protein
Polar lipids
Neutral lipids
Ala
Asp
Glu
NS 6. 20
0.025 6, 20
0.050 6. 19
NS 6, 19
01305 6, 20
NS 6, 19
0.050 6, 20
Curves were significantly different (P < 0.05) if there was inequality of regression parameters. NS not significant (P > 0.05) d.f.. degrees of freedom (F-distribution).
19
Naphthalene in oysters
I
tissue the rate of glucose oxidation is limited by the rate of glucose uptake (Ho & Jeanrenaud, 1967). Recent work {Riley, 1979) indicated that n a p h t h a lene stimulated the transport of methyl(a-o-gluco)pyranoside (a-MG) by isolated gill tissue. ~ - M G is a nonmetabolized glucose transport analog. Roubal {1974) demonstrated that n a p h t h a l e n e interacts primarily with m e m b r a n e surfaces, a fact which makes interaction with m e m b r a n e b o u n d enzyme systems an attractive hypothesis. Stekoll et al. (1980j demonstrated that aromatic h y d r o c a r b o n s are effective inhibitors of MgZ+-ATPase in crude homogenates of the clam Macoma balthica. The observed n a p h t h a l e n e effects occurred at relatively low dosages. The mean n a p h t h a l e n e concentration m the gills after 72-hr expo~u,c ~, 66 pp~ ¢,,ts 2.69 ppm and 2.84 p p m for R-3 and R-4, respectively {Riley et al., 1981). Whether or not elevated mean specific activities represent a deleterious effect is not certain. Depuration would probably result in a return to the normal metabolic state. However, chronic exposure to low concentrations of aromatic hydrocarbons would lead to reduced growth efficiency if increased carbon metabolism was not accompanied by increased carbon assimilation. Increased mean specific activities were not accompanied by increased glycogen, protein, or neutral lipids, thus suggesting that more glucose c a r b o n was being used for maintenance in naphthalene-treated oysters.
--
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I
o
t-~
I
e-
--
At'knowledyements This research was supported by a grant to MCM from: the Oregon State University Sea Grant Program, supported by NOAA Office of Sea Grant, U.S. Department of Commerce. under Grant No. 04-5-158-2: and the U.S. Environmental Protection like to thank Marcia Gumpertz for her assistance with the statistical analysis and review of the manuscript and Dr Richard Tetley for his review of the manuscript.
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--
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-
REFERENCES BAYNE B. (1973) Aspects of the metabolism of Mytilus edulis during starvation. Neth..I. Sea Re.s. 7, 399. 410. BAYNE B. L., MOORE M. N., WtDOOWS J., LIVINGSTONE n . R. •
m
t-
~m
i I
7:
--
g~g
b--
~N ~m m~ I
.._• _,~ --
~.~_
SALKEID P. (1979) M e a s u r e m e n t
of the response
of individuals to environmental stress and pollution: studies with bivalve molluscs. Phil. Trans R. Soc. Lxmd. B. 286, 563-581. Dlrrmt!r J. C. & WFt,t.s M. A. (1969) Quantitative and qualitative analysis of lipids and lipid components. In Method.s oJ En:.vmolo,qy. Vol. XIV (Edited by Lov,I!r~srLiN J. M.L pp. 482-530. Academic Press, New York. Duaots M., GILLES K., HaMILrO~ P., RFVrRS P. & SsnrH F. 119561 Colorimetric method for determination of sugars and related substances. Amdvt. Chem. 28, 350.356. Et BRINK J. & BUtI.ER I. 119751 Membrane transport: the relation to cellular metabolic rates. Science 188, 1177 1184. FoLctl J., LeES N. & SLOANE-SIANLEY H. (19571 A simple method for the isolation and purification of total lipids from animal tissues. 3. biol. Chem. 226, 497-509. GIt.HLLAN E. S. 119751 Decrease in net carbon flux in two species of mussels caused by extracts of crude oil. Mar. Biol. 29, 53-57. HI-ITZ J. R., LEWIS L.. CHAMBERSJ. & YARBROUGH J. D. (1974) T h e
acute
effects o f e m p i r e
mix
crude
oil o n
enzymes in oysters, shrimp and mullet. In Pollution and Physiolog}' of Marine organisms (Edited by VERNBERG
20
R.T. RII.I~Yand M. C. Mix
F. J. & VERNBERG W. B.}, pp. 311-328. Academic Press, New York. Ho R. J. & JEaNRE~aUD B. 11967~ Insulin-like action of o u a b a i n - I. Effect on carbohydrate metabolism. Biochim biophys. Acta 144. 61-73. LOWRY O. H., ROSEBROUCm N., FARR A. L. & RAnDALl. R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193. 265--.275. M a r s h J. B. & Wemstet~ D. B. (19661 Simple charring method for determination of lipids. J. Lipid Res. 7, 574-576." Nut.t: J. M.. ANDI-RSON j. W., COX B. A.. LAUGHLIN R. B. &
ROSSl S. S. 11976) Effects of petroleum on survival, respiration and grov.th of marine animals. Prin. Syrup. on So,rce.~. Elh'~ t, ,nd Sink.~ ~!] II ith'o(tn'hon,~ in lilt" ,4q,ttlic Em'ironmcnl. pp. 515-.539. American Institute of Biological Sciences. Arlington. Virginia. U . S A NI:TER J. t~ WASSI!RMAN W. (1974) .4pplied l, incar Stati.stical Model,. Irwin. Illinois. REI)G~AI!LL R. J.. TtRNI:R N. A. & BIEKIiSKI r . L. 11974~ Stripping thin layers from chromatographic plates for radiotracer measurements..I. Chromut. 88, 25 31. RtLFV R. T. (1976~ Changes in the total protein, lipid, carbohydrates and extracellular body fluid free amino acids of the Pacitic oyster, ('r~t~so.~trca gi~la.~, during starxation. Proc. hath. Shel!lish. Ass. 57, 84.-90. RH.Fv R. T. (19791 Stimulatory effect of naphthalene on glucose transport in the gill tissuc of the oyster (Abstract). Phurnrt~olo~li.~t 21,251.
RILEY R. T. (1980) The effect of prolonged starvation on the relative free amino acid composition of the extracellular body fluids and protein bound amino acids in the oyster, ('ru.~.~o.~trea t¢i~l(t.~. Comp. Biochem. Phv.~iol. 67A. 279 28 I. RILEY R. T. (1981) Aerobic glucose disposal in the oyster: an in cico kinetic analysis. Comp. Biochem Phy.~iol. 68A, 253..260. RILEY R. T. & MIx M. C. 119801 Thin-layer separation of citric acid cycle intermediates, lactic acid and the amino acid taurine. J. Chromat. Ig9, 286 288. RII.I!Y r . T.. Mix M. C., SCHAF'FKRR. L. & BUNTING D. t,. (1981) Uptake and accumulation of naphthalcne by the oyster Oxtreu edulis in a flow-through system. Mar. Biol. 61,267-276. Rot:Bat W. T. (1974) Spin-labeling of living tissue, a method for investigating pollutant-host interaction. In Poll,lion and Phvsiolo.qv of Marine Or~t~lni.~m.s(Edited by Vt!RNBIR(J F. J. & VERNBER(_I W. D.). pp. 367 379. Academic Press, New York. SCHt~CHrI-.R Y. & KARt_tSH S. J. D. (1980) Insulin-like stimulation of glucose oxidation in rat apidocytcs by vanadyl (IV) ions. Nature 284, 556 558. STEKOI.I.M.S.. ('I.I'MENI [-. E. ~. SHAW D. G. (1980) Sublethal effects of chronic oil exposure on the intertidal clam Mucomu hahhic,. Mur. Biol. 57, 51 60. Tt:RNI.R N. A. & RI-I×IWEt.I.R.J. 119661 A mixed layer for separation of amino acids by thin-layer chromatography+ J. ('hromat. 21, 129 132.
0306.-4492.81;050013-0810200/0 Copyright :C 1981 Pergamon Press Lid
THE E F F E C T S O F N A P H T H A L E N E O N G L U C O S E M E T A B O L I S M IN THE E U R O P E A N FLAT OYSTER O S T R E A E D U L I S R. T. RILEY t and M. C. MIx 2 ~Toxicology and Biological Constituents Research Unit, Russell Research Center, P.O. Box 5677, Athens, GA 30613: and 2Department of General Science, Oregon State University, Corvallis. OR 97331, U S A . (Receiced 26 January 1981)
Abstract--- 1. The pool sizes and redistribution of ~4C-label from D-[U-l'tC]glucose into ethanol insoluble polysaccharides, total protein, total polar lipids, total neutral lipids, free alanine, aspartate, glutamate and total organic acids were determined for control and naphthalene-treated oysters Ostrea edulix. 2. The following effects were attributed to naphthalene treatment: a decrease in the percentage of protein and polar lipid: an increase in the total amino acids (sum of alanine, aspartate and glutamateS; an increase in organic acids: and an increase in the mean specific activity Igeometric) for all measured pools. 3. These results suggested that naphthalene stimulated protein and polar lipid catabolism, while simultaneously stimulating the flow of glucose-carbon into all measured pools.
I NTRODU('TION
ullaneously exposing oysters to three different treatments in a flow-through system containing Millipore filtered synthetic seawater with 1 mM glucose and 100ppm streptomycin. The first group [8 oystersl was not exposed to naphthalene [control-treated (CtJl. the second group 18 oysters) was exposed to unlabeled naphthalene [naphthalene treated (Ntl) and the third group 18-12 oysters) was exposed to [I-14C]naphthalene at the same concentration as the second group. The gills from oysters in the former two groups were utilized for measuring the pool sizes of the major precursors, intermediates and end products of glucose metabolism, and for an in ril~o kinetic analysis of glucose metabolism. Oysters from the latter group were used to determine the tissue concentration of [l-t'tC]naph thalene after 72 hr exposure to an average naphthalene concentration of 65.7ppb: uptake and accumulation of [I-~*C]naphthalene was the topic of a previous publication [Riley et al., 19811. A total of 4 runs were conducted [R-I-R-4). Run R-I was concerned primarily with testing the flow system and the uptake of [14C]naphthalene. Runs R-2-R-4 determined the effects of naphthalene exposure on the pool sizes and on glucose metabolism; the results of these 3 runs are the subject of this paper. All oysters were collected at the same time and then stored in a closed holding aquarium. Since the runs were conducted sequentially, the total storage time Istarvation period) increased between runs: 67, 85 and 105 days for R-2-R-4. respectively.
There have been few studies of sublethal petroleum toxicity in bivalves which have investigated metabolic effects at levels more basic than oxygen consumption. Gilfillan [1975) calculated the c a r b o n budgets for two species of mussels exposed to oil in seawater extracts. In 10",, mixtures, the carbon flux [carbon assimilated minus carbon respired) was reduced by 50",i and at higher concentrations the c a r b o n flux became negative. Gilfillan 09751 theorized that negative carbon flux would reduce the a m o u n t of c a r b o n available for growth and reproduction. The most basic metabolic effect is at the level of the enzymes involved in metabolism. There have been few published reports of the effects of oil on enzyme activity in bivalves (Heitz et al., 1974; Bayne et al., 1979; Stekoll et al., 1980). A study of the c a r b o n flux through intermediary metabolism and into metabolic end products in a bivalve mollusk seemed warranted. Metabolic perturbation either precedes or is simultaneous with physiologic alteration. Theoretically, alterations in enzyme activity should be rapidly and accurately determined by in t'iro kinetic analysis using ~aC-labeled precursors. The effect of a chemical perturbant at one point in a metabolic pathway should be conveyed to other pathways via stoichiometric linkage. The purpose of the present study was to utilize an in rit'o analysis of glucose metabolism to elucidate the mechanism of toxicity of n a p h t h a l e n e in oysters O.strea edulix. Naphthalene was chosen for this study because it is representative of one of the more toxic organic components [aromatic hydrocarbons) of the water soluble fraction of crude and refined oils (Neff et al., 1976).
Experimental protocol
At the end of each 72-hr dosing run IR-2-R-41 each Ct and Nt oyster was removed and placed into individual incubation vessels containing 25 ml of filtered synthetic seawater to which was added 4#Ci of D-[U-~'*C]glucose 1274 mCi/mmol: Amersham Corporation, Illinois, U.S.A.). Each vessel was sealed with a Teflon gasket and aerated. Oysters were removed at intervals up to 240 inin and quick frozen on a bed of dry ice. The gill tissue and mantle edge were then removed, weighed, freeze-dried and stored under N2 at - 2 0 C in a desicator over anhydrous sodium sulfate. The [~4C]glucose uptake experiments were conducted at 15 C + 0 . 2 C . Pool sizes and specific radioactivity time curves for ethanol insoluble polysaccharides [assumed to be primarily
MATERIALS AND METHODS The experimental protocol for dosing oysters O. edulis with naphthalene has been described previously IRiley et al., 1981). Briefly. each experiment IRunl consisted of sire13
14
R . T . RItFY and M. C. Mix INCUBATION
I
FREEZE-DRY, HOMOGENIZE IN 80% ETOH
I
I
FOLCH EXTRACTION- PELLET
I
I
I
I
CHC 13 I I LIPIDS F
PPT DISSOLVE HC I
TCA PPT
SUPER- CHC I3 EXTRACT AND ql WATER WASH
I AQUEOUS INCLUDES WATER WASH OF FOLCi EXTRACT
SILICIC ACID COLUMN
1
MEOH
DOW 50
1
CHI~I5 WASH
SUPER
(GLYCOGEN)
PELLET
Dew I
(PROTEIN) WASH
;
NH4OH
AA' S
FORMIC TL~
NEUTRAL ~COMPOUNOS ORGANIC ACIDS
TLC Fig. 1. Fractionation scheme for the metabolic pools: ethanol insoluble polysaccharides Iglycogen), protein, neutral lipids, polar lipids, amino acids, organic acids and neutral compounds. glycogenL total protein, total polar lipids, total neutral lipids, free alanine (Ala), aspartate (Aspl, glutamate IGlu) and total organic acids were determined for Ct and Nt oysters. Radioactivity-time curves for malate, succinate and neutral substances were also determined. Qutmtitatire methods
Each freeze-dried gill was fractionated according to the procedure outlined in Fig. I. Water. organic solvents and all other reagents were obtained and purified as described in Riley et al. (1981). The tissue was weighed, put into a 10-ml Vitro-tissue grinding tube. liquid N , added and the tissue pulverized into a fine uniform powder. The powdered tissue was homogenized in 80°,, ethanol with a Potter-Elvehjem type homogenizer and the homogenate was allowed to stand I hr at 4 C: it was then centrifuged at 4340.q at 4 C for 10 rain and the supernatant pipeted into a screw-cap culture tube. The homogenization and centrifugation procedure was repeated twice and the final precipitate.was washed with 80",, ethanol. The supernatants and washes were combined. 2vol chloroform and 2 ml of water added, and then mixed. The upper aqueous phase was pipeted into an Erlenmeyer flask and the water washes repeated 3 times, and all washes combined in the Erlenmeyer flask. The precipitate from the original ethanol homogenization stop was extracted by the method of Folch et u/. (1957). The .,upernatant was pipeted into a screw-cap culture tube and the 2:1 chloroform-methanol extract was water washed. After phase separation, the aqueous phase was pipeted into the Erlenmeyer flask containing the water washes from the chloroform extract of the ethanol supernatant. The chloroform and chloroform-methanol extracts
were pooled, made to one phase by the addition of methanol and then evaporated to dryness under N2 at 50 C. The residue was brought up in chloroform and separated into polar and neutral lipids by the method of Dittmer & Wells (1969). An aliquot of the neutral lipids dissolved in chloroform was placed in a scintillation vial and the chloroform evaporated under N:. The scintillation vial was filled with 5 ml of toluene scintillation fluor and counted on a Packard Tri-Carb LS Spectrometer. Counting efficiencies were determined by internal standardization. An aliquot of the polar lipids dissolved in methanol was evaporated in a scintillation vial to remove any residual chloroform and then redissolved in methanol and counted in PCS fluor (Amersham Corporation). The total lipids present in each fraction were quantified by the charring technique of Marsh & Weinstein (1966) with tripalmitin used as a standard. The combined aqueous extracts prepared previously, were partially evaporated under a flow of N2 at 50 C and then evaporated to dryness over sodium sulfate in a sleevetype Pyrex desiccator under '.acuum using a water aspirator vacuum pump. An infra-red heat lamp was used to speed evaporation. The dried residue was stored under N: at - 2 0 C in a desiccator over sodium sulfate until ready for fractionation into amino acids, organic acids and neutral compounds. The lipid-free pellet from the Folch extraction was partially dried under a flow of N:, homogenized in 0.01 N hydrochloric acid (HCI) and transferred to a corex centrifuge tube. An equal volume of 10% trichloroacetic acid (TCA) was added and the mixture held at 4 C for I hr followed by centrifugation at 12.100g at 4 C for 10rain. The TCA supernatant was pipeted into a graduated tube and the precipitate washed twice with 5"° TCA and recen-
Naphthalene in oysters trifuged between washes. The washes were combined with the supernatant in the graduated tube. An aliquot of the TCA soluble substances flotal polysaccharidesl was counted in PCS fluor and another aliquot was analyzed for total reducing sugars after acid hydrolysis by the method of Dubois et al. (1956) using glucose as a standard. The TCA precipitate (total protein) was washed with two aliquots of 0.1 N potassium acetate in 80". ethanol. The washes were discarded and the precipitate dried under a flow of N , at room temperature. The dried precipitate was then dissolved in 0.1 N sodium hydroxide (NaOH) at 50 C. O n e aliquot was counted in PCS and a second was analyzed for total protein by the method of Lowry et al. (1951~ using bovine serum albumin as a standard. The dried residue from the water wash of the combined lipid extracts was dissolved in 0.01 N formic acid and then passed through a column of Dowex 50 x 4, 200 400 mesh resin in the H" form. The column was washed 3 times with 0.01 N formic acid. The sample solvent and washes were collected in an Erlenmeyer flask. Amino acids were eluted with 4 N a m m o n i u m hydroxide and the eluate was collected in another Erlenmeyer flask. The eluate and wash were frozen and then freeze-dried. Recoveries of L-[U-~'*C]glutamate and L-[U-~4C]leucine carried through from the homogenization step were 90.8'!o (standard deviation (SD = 5.5, n = 4 ) and 90.7°,, (SD = 1.2, n = 4 ) respectively. The freeze-dried Dowex 50 eluate lamino acids) was brought up in 0.01 N HCI and transferred to a graduated tube along with two rinses of the Erlenmeyer flask. An aliquot was then counted in PCS and the eluate reevaporated to dryness. The residue was dissolved in a small volume of 0.01 N HCI. and an aliquot counted in PCS. The exact volume of the concentrated eluate was calculated. based on the previously determined volume and activity. Aliquots of the concentrated Dowex 50 eluate were streaked in 2 cm scribed lanes on thin-layer plates prepared as described by Turner & Redgwell (19661. The plates were developed in glass-distilled phenol water (80:20w,vl and air-dried overnight. The two end lanes were sprayed with 0.5",, ninhydrin in 95". ethanol. In the remaining lanes the areas corresponding to the amino acids Ala, Asp and Glu, were removed and counted by the method of Redgwell et ,/. 11974l. Ala, Asp and Glu were the only amino acids with detectable counts and they were well resolved in one dimension by the phenol--water solvent system. Aspartate and asparagine were not resolved by the one-dimensional method. The Dowex 50 amino acids were quantified by thin-layer chromatography ITLCI. Aliquots of the e l u a t e were streaked in I cm scribed lanes adjacent to known standards of Ala, Asp and Glu. The plates were scanned with a Schoeffel SF 3000 spectrodensitometer using an illumination wavelength of 585 n m and a reflectance wavelength of 510 n m The standard curves were generated for each plate in order to compensate for variations between plates. The reproducibility of this method was +4.0"0 In = 421 for Ala. -,-4.6". (n = 421 for Asp, and +4.40. (n = 44) for Glu. The freeze-dried Dowex 50 wash was dissolved in pH 6.0, 0.01 N Na formate buffer and then passed through a column of Dowex 1 × 8. 100--200 mesh in the OH form. All the original solvent and rinses were collected in a graduated tube. and subsequently referred to as "'neutral compounds". Organic acids were eluted with 6 N formic acid and the eluate collected and freeze-dried. An aliquot of the neutral c o m p o u n d s was counted in PCS fluor. The freeze-dried Dowex 1 eluate containing organic acids was dissolved in 50°,, acetone, transferred to a graduated tube and an aliquot counted in PCS after evaporating the acetone. After the addition of phenol red indicator an aliquot was titrated to the end point with 0.01 N N a O H . The results of the titration were expressed as milliequivalents H * , g dry wt. The remaining Dowex l eluate was
15
evaporated to dryness, and then dissolved in a small volume of 50°. acetone. The recovery of [l,4-~'*C]succinic acid and [2-14C]acetic acid carried through from the homogenization step were 88.8°0 (SD = 4.6, n = 4) and 1.3°o (SD = 1.8, n = 4), respectively. An aliquot was counted and then 10jul were spotted in the lower left corner of a mixed layer plate and the organic acids separated by twodimensional T L C (Riley & Mix, 19801. Stuti.~tical method,~
The following abbreviations are used for convenience: .~ = mean; SD = standard deviations; C1 = confidence intervals: r 2 = coefficient of determination; P = probability. An analysis of variance INeter & Wasserman, 1974) was conducted to determine differences in the pools sizes (response variables) as a function of sample time, run and treatment 0ndependent variablesl. The 5°. significance level (a = 0.05) was set as the level for rejecting the null hypothesis. Curve fitting was accomplished by linear regression analysis. Data were fitted to a model of the form: In Yo = a./ + hi(In x) + e~(ln x ) 2. Comparison of specific radioactivity, time curves (Ct to Nt) was accomplished as described by Neter & W a s s e r m a n (1974l. The alternative conclusions were: C~ : equality of regression parameters (a~ b~ and cfl. C2 : inequality of regression parameters (aj, b~ or cj). Visual inspection of the regression curves suggested that the mean specific activities were always greater in naphthalene-treated oysters. In order to demonstrate this difference statistically an analysis of the residuals was conducted. Residuals lei, i = 1, 2 . . . . . n) are defined as the difference between the observed values (Y0 and the corresponding fitted values (~) from the reduced regression equations. The mean residual (~) can be calculated as follows: ~=
~" lnY,,,'n- ~" In~,'n i
1
i
1
and (geometric) = antilog e = Y (geometric)/Y(geometric); t h u s . . ~ (geometric) is the mean specific activity (geometric) for each treatment expressed as a fraction of the mean specific activity (geometric) of the reduced models. Confidence intervals of X (geometric) can be calculated as follows: antilog(~; _+ t S D n - ° s); where SD is the standard deviation of e and t is the t-statistic for the appropriate degrees of freedom. RESU LTS" T h e p e r c e n t d r y wt of t h e gill tissue o f O. e d u l i s d e c r e a s e d b e t w e e n r u n s ; 17.0, 16.3 a n d 15.6°,o for R-2, R-3 a n d R-4, respectively (Table 1). T h e difference w a s statistically significant b e t w e e n R u n s 2 a n d 4 (P = 0.025: S t u d e n t ' s t-testj. T h e m e a n wet wt o f gill tissue w a s 133.4 + 10.8 m g ( + 9 5 ° CI, n = 48), a b o u t 47"0 of t h e total tissue wet wt. Since e a c h r u n was d o n e s e q u e n t i a l l y , t h e total l e n g t h o f s t a r v a t i o n inc r e a s e d b e t w e e n e a c h run. D u r i n g t h e [ t ' * C ] g l u c o s e u p t a k e e x p e r i m e n t for R-2, t h e t e m p e r a t u r e c o n t r o l m e c h a n i s m failed a n d t h e t e m p e r a t u r e fell to a p p r o x 12.8"C for the c o n t r o l oysters. T h e specific a c t i v i t y - t i m e c u r v e s for this r u n were n o t c o m p a r a b l e since t h e t e m p e r a t u r e effect w o u l d be p r i m a r i l y a rate effect. H o w e v e r , t h e pool sizes for C t a n d N t o y s t e r s f r o m R-2 were a s s u m e d to
R. T. Rn.ev and M. C. Mtx
16
Table I. Dry wt of the gill tissue expressed as percent of the wet wt for Runs R-2-R-4
Starvation (days) ",, Dry wt 95",, CI t)
R-2
R-3
R-4
67 17.0 +0.9 16
85 16.3 +0.8 16
105 15.6 +0.9 16
The R-4 value was significantly less than the R-2 (Student's t-test: P = 0.025~. Both Ct and Nt gills were pooled to obtain the combined means.
be comparable. It was reasoned that the total carbon flux over the short period of the glucose uptake experiment (-,< 240 min) would have been insufficient to affect the absolute pool sizes which were a result of the carbon flux during the prior 72-hr dosing period in the flow system. The results of the analysis of variance indicated that there was no relationship between sample time and the pool sizes of glycogen, protein, polar and neutral lipids, total Ala, Asp and Glu and total organic acids. Thus, verifying that these pools were in a steady-state during the [14C]glucose uptake experiments. However, there were significant effects related to run and n a p h t h a l e n e treatment.
Percent neutral lipids, total Ala, Asp and Glu and total organic acids were all significantly related to run ~P < 0.001). Percent polar lipids were close to being significantly related to run (P = 0.10). The percent total lipids Ipolar plus neutral) increased between runs (Table 2) while amino acids and organic acids decreased (Table 3). The Student's t-test was used to test for significant differences, by treatment, in those pools which were shown to be significantly related to the increasing storage (starvation) between runs. For Ct and Nt oysters neutral lipids were significantly greater for R-4 compared to R-2 and R-3, and total lipids were significantly greater for R-4 compared to R-2. The total amino acids (sum of Ala, Asp and Glu) decreased significantly between each run for both treatments and organic acids decreased significantly between runs 2 and 4 for both treatments. Percent glycogen and protein were not significantly affected by the increasing storage lstarvation) between runs. The percent protein and percent polar lipids were b o t h significantly affected by naphthalene treatment (P < 0.01). Protein and polar lipids were always less in Nt oysters (Table 2). The total Ala, Asp and Glu Isum of Ala. Asp and Glu) and the total organic acids were close to being significantly affected by n a p h t h a lene treatment (P ~< 0.10~. The total Ala. Asp and Glu and total organic acids were always greater in Nt oysters (Table 3). The Student's t-test was used to test for significant differences, by run. in those pools
Table 2. Comparison of the percent glycogen, protein, neutral lipids, polar lipids and total lipids between runs in the gills of control (Ct) and naphthalene-treated (Ntl oysters R-2 Pool
R-3
Ct
Nt
Ct
Glycogen
5.3
5.7
Protein*
59.1
58.3
Polar lipids*
5.0
4.9
Neutral lipids't
4.3
4.2
Total lipids*'t
9.3
9.1
5.6 SD 63.2 SD 5.4 SD 4.0 SD 9.4 SD
R-4 Nt
5.2 = 0.641 55.4 = 4.147 4.9 = 0.296 4.4 = 0.366 9.3 = 0.546
Ct
Nt
5.6
5.6
61.9
60.2
5.4
5.0
5.1
4.9
10.5
9.9
SD. (mean square error) °5. from the analysis of variance model for the full three-way model using each sample time as a replicate. * Significant difference between treatments (P < 0.01); 1, 42 degrees of freedom, F-distribution. "l"Significant difference between runs (P < 0.001); 2, 42 degrees of freedom, F-distributiOn. Table 3. Comparison of the total Ala, Asp and Glu (#M/g dry wt) and total organic acids (Mequiv..,g dry wt) between runs in the gills of control (Ct) and naphthalene treated (Nt) oysters R-2 Pool Total Ala, Asp and Glu* Total organic acids*
R-3
R-4
Ct
Nt
Ct
Nt
Ct
Nt
49.1
54.2
42.1
44.9 SD = 7.693
19.0
22.5
0.194
0.219
0.188 0.198 SD = 0,050
SD, (mean square error) °5, from the analysis of variance model. * Significant difference between runs (P < 0.001): d.f. 2, 42, F-distribution.
0.117
0.156
17
Naphthalene in oysters which were shown to be significantly affected by naphthalene treatment based on the results of the analysis of variance, in Nt oysters the percent protein was significantly less, compared to Ct oysters, in R-3 and significantly less for polar lipids in R-3 and R-4. Naphthalene treatment had no significant effect on the pool sizes of either glycogen or neutral lipids (Table 2). The flow of "=C-label was directed primarily into metabolic end products (averaged for all runs). Ethanol insoluble polysaccharides accounted for 62.30",. (SD = 9.99, n = 48), total protein 5.63", (SD = 2.94, n = 48). total polar lipids 0.20". (SD = 0.10, n = 47) and total neutral lipids 0.03 (SD = 0.02, n = 47) of the total "~C-label recovered in the gill. Amino acids and organic acids accounted for 9.16°. (SD = 4.81, n = 48) and 5.4", (SD = 4.6, n = 48), respectively and neutral compounds accounted for 17.28".. (SD = 12.16, n = 48). The radioactivity time data for R-3 (Ct) are presented as an example of the manner in which glucose-carbon was redistributed to the measure pools
o
I00
• Glycogen (0 60) o Protein (0 98) X Polar Lip=ds (0.93) A Neutral L=p=ds (0.92)
I0 1.0 Ol 0.01 llJ I/1 0 0
b X AIo ( 0 8 1 )
I0 X
0 w _J
x xx.~--x~
.
---IV-
I0
./
U u i, 0
~;
o Glu (056) • Asp (0 68)
in the gill (Fig. 2). The pattern of redistribution was the same for all runs and all treatments. The percent accumulated dose (percent of the total glucose--carbon recovered from gills) in end products (Figs 2a & 2b) increased with time, neutral compounds (precursors) decreased (Fig. 2c) and organic acids remained constant (Fig. 2c). Almost all of the activity from the Dowex 50 eluate was recovered in Aia, Asp and (3lu (X = 96.80., + 5.0, 95",, CI, n = 47). T L C of the Dowex 1 eluate revealed that the majority of the activity in this fraction was in unidentified organic acids and taurine (.~ = 71.56",~, + 5.45, 95°. C1, n = 16). Malate and succinate were the only Krebs cycle intermediates that were labeled in detectable quantities ()f = 2.1°,, + 0.80, 95°, CI. n = 15). About 26'!, of the activity in the Dowex 1 fraction could not be accounted for. 5pec~hc rad,oacttv~ty t,me curves lot ethanol insoluble polysaccharides, (assumed to be primarily glycogen), total protein, total polar lipids, total neutral lipids, free Ala, Asp and Glu, were determined. In order to allow a comparison of the relative importance of the pools with respect to pool size, the data for R-3 (Ct) are normalized to carbon equivalents (Fig. 3). Glucose-carbon was diluted into relatively large glycogen and protein pools and relatively small amino acid pools. Therefore, even though the percent of the accumulated dose recovered in Ala was low, the specific activity was very high. Because the activities in malate and succinate were so low, no radioactivity-time curves are given. Comparison of the specific activity- time curves for Ct and Nt oysters revealed significant differences for protein, polar lipids, Ala, and Glu (Table 4). Visual inspection of the fitted regression curves suggested that in every case the total area under each Nt curve was greater than the Ct (for example, Figs 4a & 4b). The residual analysis confirmed this observation. For every pool and run the mean specific activity (geometric) was greater for the Nt oysters (Table 5).
Ol
I
I
I
I
c
I00
X Neutral Substances (0 82 ) • Orgamc Aods (000)
X ~ X , ~X I0
~
IO
O
X ~ ~ ~
i
A
60
120
I
IBO TIME (rain)
x
I 240
Fig. 2. Percent of total recovered dose in the metabolic pools for Run R-3 (CI). The r z values are given in parentheses. The data are calculated as a percent of the total "*C-label recovered from all measured pools in the gill tissue and are presented as examples of the pattern of redistribution of glucose-carbon in oyster gill ]'or all runs and treatments. (B.P(()
70. I
B
DISCUSSION
There were two types of effects observed; effects attributed to starvation and those attributed to naphthalene treatment. Examples of the former were the decreasing percent dry wt, decreasing concentrations of total amino acids and total organic acids and the increasing concentration of total lipids. Increased water content of the tissues and decreased free amino acids are common manifestations of prolonged starvation in oysters (Riley, 1976; Riley, 19801. Increased total lipids was correlated with increased bioaccumulation of naphthalene (Riley et a/., 1981), however, the increase in total lipids could not. by itself, account for the increased naphthalene accumulation. The bioaccumulation factor ([ l-"=C]naphthalene treatment) for R-4 was 26". greater than R-2 while the percentage of total lipids [unlabeled naphthalene treated) was only 8". greater. The effects attributed to naphthalene treatment were decreased protein and polar lipid concentration, increased total Ala, Asp and Glu, increased total Dowex 1 acids and increased mean specific activity (geometric) for all measured pools. These results suggested that naphthalene stimulated protein and polar
R.T. RILEY and M. C. Mtx
18
a
I OO"
10 8 .
~
in 7.
AIo (.96)
Ct, AIo (96) Glu (.92)
07
NI
(.88) (.911
n
_...=1"~nx_f__.=
,
I 06. !
iI
1 ¢_) cn
I0 6
05. ~
E ~E 104 •
X
,, ~¢"
...~---~:"~,,
~,
Protein1.971 Polar Lipid$
105
O.
103 . I
"~) ~
i0 4
'
.~
108
Ct
,
b
02.
] In t 0
60
ten i80 TIME (rain)
Z40
a
Fig. 3. Fitted specific activity-time curves for the metabolic pools for Run R-3 (Ctl. The r 2 values are given in parentheses. Specific activities are expressed per mg carbon; thus. allowing comparison of the relative specific activities. Conversion factors used were (in percent carbon) glycogen = 44°0, protein = 50"0, lipid (neutral and polar) = 76",, Ala = 40°0, Asp = 36"., Glu = 41"o.
C941 '(.95)
107 t
/
(.g t) (.93) ~ ~_ _ .~- n ,.t°--
,,;/
..a .....
o
X /t
1051 lipid catabolism while simultaneously stimulating the flow of glucose-carbon into all measured pools. Heitz et a/. 119741 demonstrated increased activity of leucine aminopeptidase in Crassostrea giyas exposed to petroleum hydrocarbons. They also d e m o n s t r a t e d significant alterations in the activities of other key enzymes associated with intermediary metabolism: glutamateoxaloacetate transaminase and malate dehydrogenase. Catabolism of stored energy reserves and disturbed protein metabolism are responses which partially characterize the "'stress syndrome" of mussels (Bayne, 19731. The fact that glycogen reserves were not reduced by n a p h t h a l e n e treatment may be due to the ample dissolved glucose available for glycogen synthesis. The increased flow of glucose--carbon into all pools may have resulted from increased transepilhelial glucose transport. Riley 11981) found that in the whole body of starved oysters, maintained and preincubated under similar conditions as the present study, gluc o s e - c a r b o n was redistributed mostly to ethanol insoluble polysaccharides and amino acids. It was
AIo Glu
NI
iO 4 L 0
zx
60
120
180
240
T I M E (rnin) Fig. 4. Fitted specific activity-time curves for Ala and Glu for R-3 (a] and R-4 (bl. The fitted curves and data for Ala and Glu are presented to demonstrate graphically the nature of the differences between the mean specific activities for Ct and Nt oysters. The mean specific activities (geometric) for all the pools, calculated from the residuals generated from the regression analysis, are given in Table 5 The r 2 value is gi'.en in parentheses. Nt, - -: Ct. = - . Ala:Ct = ×. Nt = E, G l u : C t = A, Nt = O.
hypothesized that the m a x i m u m disposal rate (oneway transport) was limited by the m a x i m u m rate of transepithelial transport. The relationship between cellular metabolic rate and the rate of glucose uptake has been wel; demonstrated in m a m m a l i a n systems (Elbrink & Bihler, 19751. For example, in rat adipose
Table 4. Results of the comparison ,ff the regression curves for Runs R-3 and R-4
P d.f.
Glycogen
Protein
Polar lipids
Neutral lipids
Ala
Asp
Glu
NS 6. 20
0.025 6, 20
0.050 6. 19
NS 6, 19
01305 6, 20
NS 6, 19
0.050 6, 20
Curves were significantly different (P < 0.05) if there was inequality of regression parameters. NS not significant (P > 0.05) d.f.. degrees of freedom (F-distribution).
19
Naphthalene in oysters
I
tissue the rate of glucose oxidation is limited by the rate of glucose uptake (Ho & Jeanrenaud, 1967). Recent work {Riley, 1979) indicated that n a p h t h a lene stimulated the transport of methyl(a-o-gluco)pyranoside (a-MG) by isolated gill tissue. ~ - M G is a nonmetabolized glucose transport analog. Roubal {1974) demonstrated that n a p h t h a l e n e interacts primarily with m e m b r a n e surfaces, a fact which makes interaction with m e m b r a n e b o u n d enzyme systems an attractive hypothesis. Stekoll et al. (1980j demonstrated that aromatic h y d r o c a r b o n s are effective inhibitors of MgZ+-ATPase in crude homogenates of the clam Macoma balthica. The observed n a p h t h a l e n e effects occurred at relatively low dosages. The mean n a p h t h a l e n e concentration m the gills after 72-hr expo~u,c ~, 66 pp~ ¢,,ts 2.69 ppm and 2.84 p p m for R-3 and R-4, respectively {Riley et al., 1981). Whether or not elevated mean specific activities represent a deleterious effect is not certain. Depuration would probably result in a return to the normal metabolic state. However, chronic exposure to low concentrations of aromatic hydrocarbons would lead to reduced growth efficiency if increased carbon metabolism was not accompanied by increased carbon assimilation. Increased mean specific activities were not accompanied by increased glycogen, protein, or neutral lipids, thus suggesting that more glucose c a r b o n was being used for maintenance in naphthalene-treated oysters.
--
E
<
I
o
t-~
I
e-
--
At'knowledyements This research was supported by a grant to MCM from: the Oregon State University Sea Grant Program, supported by NOAA Office of Sea Grant, U.S. Department of Commerce. under Grant No. 04-5-158-2: and the U.S. Environmental Protection like to thank Marcia Gumpertz for her assistance with the statistical analysis and review of the manuscript and Dr Richard Tetley for his review of the manuscript.
7.=
E
I
--
~ N 2
o ct~ +~
i
o
-
REFERENCES BAYNE B. (1973) Aspects of the metabolism of Mytilus edulis during starvation. Neth..I. Sea Re.s. 7, 399. 410. BAYNE B. L., MOORE M. N., WtDOOWS J., LIVINGSTONE n . R. •
m
t-
~m
i I
7:
--
g~g
b--
~N ~m m~ I
.._• _,~ --
~.~_
SALKEID P. (1979) M e a s u r e m e n t
of the response
of individuals to environmental stress and pollution: studies with bivalve molluscs. Phil. Trans R. Soc. Lxmd. B. 286, 563-581. Dlrrmt!r J. C. & WFt,t.s M. A. (1969) Quantitative and qualitative analysis of lipids and lipid components. In Method.s oJ En:.vmolo,qy. Vol. XIV (Edited by Lov,I!r~srLiN J. M.L pp. 482-530. Academic Press, New York. Duaots M., GILLES K., HaMILrO~ P., RFVrRS P. & SsnrH F. 119561 Colorimetric method for determination of sugars and related substances. Amdvt. Chem. 28, 350.356. Et BRINK J. & BUtI.ER I. 119751 Membrane transport: the relation to cellular metabolic rates. Science 188, 1177 1184. FoLctl J., LeES N. & SLOANE-SIANLEY H. (19571 A simple method for the isolation and purification of total lipids from animal tissues. 3. biol. Chem. 226, 497-509. GIt.HLLAN E. S. 119751 Decrease in net carbon flux in two species of mussels caused by extracts of crude oil. Mar. Biol. 29, 53-57. HI-ITZ J. R., LEWIS L.. CHAMBERSJ. & YARBROUGH J. D. (1974) T h e
acute
effects o f e m p i r e
mix
crude
oil o n
enzymes in oysters, shrimp and mullet. In Pollution and Physiolog}' of Marine organisms (Edited by VERNBERG
20
R.T. RII.I~Yand M. C. Mix
F. J. & VERNBERG W. B.}, pp. 311-328. Academic Press, New York. Ho R. J. & JEaNRE~aUD B. 11967~ Insulin-like action of o u a b a i n - I. Effect on carbohydrate metabolism. Biochim biophys. Acta 144. 61-73. LOWRY O. H., ROSEBROUCm N., FARR A. L. & RAnDALl. R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193. 265--.275. M a r s h J. B. & Wemstet~ D. B. (19661 Simple charring method for determination of lipids. J. Lipid Res. 7, 574-576." Nut.t: J. M.. ANDI-RSON j. W., COX B. A.. LAUGHLIN R. B. &
ROSSl S. S. 11976) Effects of petroleum on survival, respiration and grov.th of marine animals. Prin. Syrup. on So,rce.~. Elh'~ t, ,nd Sink.~ ~!] II ith'o(tn'hon,~ in lilt" ,4q,ttlic Em'ironmcnl. pp. 515-.539. American Institute of Biological Sciences. Arlington. Virginia. U . S A NI:TER J. t~ WASSI!RMAN W. (1974) .4pplied l, incar Stati.stical Model,. Irwin. Illinois. REI)G~AI!LL R. J.. TtRNI:R N. A. & BIEKIiSKI r . L. 11974~ Stripping thin layers from chromatographic plates for radiotracer measurements..I. Chromut. 88, 25 31. RtLFV R. T. (1976~ Changes in the total protein, lipid, carbohydrates and extracellular body fluid free amino acids of the Pacitic oyster, ('r~t~so.~trca gi~la.~, during starxation. Proc. hath. Shel!lish. Ass. 57, 84.-90. RH.Fv R. T. (19791 Stimulatory effect of naphthalene on glucose transport in the gill tissuc of the oyster (Abstract). Phurnrt~olo~li.~t 21,251.
RILEY R. T. (1980) The effect of prolonged starvation on the relative free amino acid composition of the extracellular body fluids and protein bound amino acids in the oyster, ('ru.~.~o.~trea t¢i~l(t.~. Comp. Biochem. Phv.~iol. 67A. 279 28 I. RILEY R. T. (1981) Aerobic glucose disposal in the oyster: an in cico kinetic analysis. Comp. Biochem Phy.~iol. 68A, 253..260. RILEY R. T. & MIx M. C. 119801 Thin-layer separation of citric acid cycle intermediates, lactic acid and the amino acid taurine. J. Chromat. Ig9, 286 288. RII.I!Y r . T.. Mix M. C., SCHAF'FKRR. L. & BUNTING D. t,. (1981) Uptake and accumulation of naphthalcne by the oyster Oxtreu edulis in a flow-through system. Mar. Biol. 61,267-276. Rot:Bat W. T. (1974) Spin-labeling of living tissue, a method for investigating pollutant-host interaction. In Poll,lion and Phvsiolo.qv of Marine Or~t~lni.~m.s(Edited by Vt!RNBIR(J F. J. & VERNBER(_I W. D.). pp. 367 379. Academic Press, New York. SCHt~CHrI-.R Y. & KARt_tSH S. J. D. (1980) Insulin-like stimulation of glucose oxidation in rat apidocytcs by vanadyl (IV) ions. Nature 284, 556 558. STEKOI.I.M.S.. ('I.I'MENI [-. E. ~. SHAW D. G. (1980) Sublethal effects of chronic oil exposure on the intertidal clam Mucomu hahhic,. Mur. Biol. 57, 51 60. Tt:RNI.R N. A. & RI-I×IWEt.I.R.J. 119661 A mixed layer for separation of amino acids by thin-layer chromatography+ J. ('hromat. 21, 129 132.