Aquatic Toxicology, 22 ( 1992) 61-68 @ 1992 Elseviet Science Publishers 3.V. All rights reserved 01~-~5X~92~~~.~
n vitro effects of
iram on liver antioxi burnsactivities of bait ok trout ( mykiss) S. Babo and P. Vasseur Cettrre des Sciettces de I’Enrirottrtetttettt, Met-. France (Received 8 July 1991: revision received 15 August 1991: accepted 19 November 1991) The effects of the dithiocarbamate Thiram on the activities of superoxide dismutase, catalase and glutathione peroxidase in young rainbow trout livers were studied in vitro. Catalase and glutathione peroxidase activities were evaluated with standardized methods. Superoxide dismutase was assayed by a method based on the inhibiting action of the enzyme on the rate of oxidation of NADH. The activities of superoxide dismutase, catalase and glutathione peroxidase were found to be 1I .3 pglmin ‘mg, 33.1 pmol: min’mg and 12.2 nmoli’min:mg protein respectively. in control animals. Incubation of liver su~r~atants with iO- h M, 10 5 M and 10 J M Thiram for I.5 h resulted in a total loss of superoxide dismutase activity at IO-J M. whereas catalase activity showed no significant reaction to the same range of toxic concentrations. The activity of glutathione peroxidase decreased at 10 h M of Thiram but was not further reduced at higher concentrations of dithiocar~mate. Key words: Superoxide dismutase; Catalase; Glutathione
peroxidase: In vitro; Rainbow trout
Oxygen reactive species are involved in useful biochemical processes in eukaryotic cells, such as fatty acid a-oxidation (Mead and Levis, 1963), alcohol oxidation (Oshino et al., 1973). thyroxine biosynthesis (Serif and Kirkwood, 1958). bioluminescence (Hastings, 1966) or phagocytosis (Roos, 1977). But their high reactivity also makes them responsible for non-specific oxidative cell damage. In order to protect themselves against oxygen toxicity. aerobic cells have a range of protective mechanisms, which include superoxide dismutase (SOD), catalase and glutathione peroxidase. Tissues containing superoxide dismutase also normally have catalase and/or g~utathione peroxidase. When an oxidative stress occurs, most of the superoxide anions released in the cell are transformed into hydrogen peroxide. H& molecules produced inside the peroxisomes are destroyed exclusively by catalase,
Cf~rrf?spottf~~J~rr~J fo: P. Vasseur, Centre des Sciences de I’Environnement,
57000 Metz, France.
whereas those present in the cytoplasm or in the mito~hondri~~ network react with g~ut~thio~~~peroxidase. There is competition for the hydrogen peroxide between these two enzymes in eukaryotic cells without sub&e~~ul~r~om~~rtrnents~ such as in erythro~yt~s. Many uf the ~~po~hi~~~ xemz&?ioticsproduce oxygen reactive species during their b~~transfor~~ation in the cell. This is the case for d~thi~~arbamate fun~~cide~*such as Thiram. This cupric chelator is known to inhibit cytosolic su~roxid~ dismut~s~ (~~ikkj~a et at., t976) and, in some cases, other antjoxid~nt enzymes such as gtutathiane peroxidase (Goldstein et al., 1979). Thimm was afsa found to induce mutagenicity in proka~yoti~ cells (~ann~g and ~~nnug, 1984). fn this report, we are des~~~bin~the in vitro effects of Thiram on the activities of superoxide dismutase, catalase and glutathion~ peroxidase of fish liver cells. The resuits can be used to study whether the metabolism of this dithiocarbamate may be responsible for mutagenicity in fish. fraction of fish liver cells; this fraction The investigations were based on the S ~~00~ is devoid of m~tocho~dria and ~on~ins the cytosolic and microsoma~ enzymes of the biotransformation processes which lead to the pr~u~ti~n of the oxygen reactive species. It must be pointed out that little has been done on this subject in aquatic species. Most of the studies have involved mammalian organisms.
Young immature r&bow trout (~~~u~~~~~?~~~~~ ~~~~~~~~ were obtained from an Aq~~~u~tu~~Centre in France. The fish weighed 5 to 8 g and were fed daily a commercial fry fodder (Trouvit ~~~,OO,O,~,France}. The water temp~~ture of the ponds did not exceed 20°C and the dissolved oxygen tension in the water was over 6 cm3/i,
NASH, NADPH and purified beef eryt~~r~~ytesuperoxide dismutase (no. 567 680) were obtained from ~oehr~~g~r-~annheim (~eyIan, France). MnCIz, EIITA and Feef serum ~~burn~nwere ~ur~has~d from Merck (~ug~nt-our-~arne~ France) and reduced gfutathione, yeast glutathione reductasc (no. C-475 I ). cumene hydropesoxide f~~rn Sigma (La V~rpi~I~~re,France).
The fish were taken out sf their ponds, killed and their livers removed ~pjdly* Each liver was washed three times in a saline solution and then ground in a phosphate buRer (~~~P~~/K~~P~~ 50 n&l, pH 7,&, EDTA I mM, mer~apt~ethan~lO.7
mM) using 50 ~1 of buffer per mg of liver. The homogenate was then centrifuged at 12000 x g for 5 min at 4°C. The resulting supernatant was used directly for assaying the glutathione peroxidase activity. To determine the superoxide dismutase activity, haemoglobin was removed from the supernatant because this haemoprotein is likely to interfere with superoxide dismutase for the consumption of superoxide anions. According to the extraction procedure, one volume of supernatant was treated with 0.3 volume of ethanol/chloroform (2/l vol/vol). The mixture was then vigorously mixed for 15 s (vortex) and centrifuged at 17600 x g for 15 min at 4°C. The resulting supernatant was used for the superoxide dismutase assay (Aksnes and Njaa, 198I ). For the catalase activity measurement, the supernatant was treated prior to the assay as follows: l/95 volume of pure ethanol was added to one volume of supernatant and left for 30 min at 4°C; l/8.9 volume of 10% Triton X-100 was then added to the assay medium (Cohen et al., 1970). The alcohol was used to prevent the formation of non-active but stable forms of catalase complexes with peroxides, or to destroy those preexisting in the biological samples. The detergent was useful in that it freed catalase molecules from any cellular particles bound to the enzyme during the homogenization of the samples.
Superoxide dismutase was measured by a method which is based on the inhibitory action of the enzyme on the rate of oxidation of NADH (Paoletti et al., 1986). The assay medium consisted of the phosphate buffer, 0.3 mM NADH, 2.3jl.15 mM EDTA/MnClz and 100 ~1 of the diluted supernatant. After 5 min of incubation at 25-C, 0.9 mM mercaptoethanol was added and the rate of NADH oxidation was measured at 340 nm, at 25°C. A blank, containing all reagents except the biological sample, was assayed in the same way. One unit was defined as the amount of enzyme which was able to inhibit the NADH oxidation rate by 50%. A standard NADH oxidation curve, using pure superoxide dismutase, was established in order to express the enzymatic unit in /tg SOD/min. Ca talase ama?
Catalase activity was measured by direct spectrophotometric assay described elsewhere (Beers and Sizer, 1952). The assay medium consisted of the phosphate buffer. 14 mM hydrogen peroxide and 100 ~1 of the diluted supernatant, at 25’C. A blank, which contained in addition 0.3 mM sodium azide. was assayed in the same way. One unit was expressed in pmol HzOz consumed per minute. A molar extinction coefficient of 43.5 M - ’ cm- ’ was used for the calculation.
The method of Lawrence and Burk ( 1976) was used for the determination of the glutathione peroxidase activity. The reaction mixture consisted of the phosphate buffer. 2 mM reduced glutathione. 120 PM NADPH, 3 units of yeast glutathione reductase and the supernatant. After 5 min incubation. the enzymatic reaction was initiated with 0.2 mM cumene hydroperoxide and the oxidation of NADPH was measured at 340 nm at 25-C. A blank without the biological sample was assayed under the same conditions. One unit was expressed in nanomoles of reduced glutathione consumed per minute. A molar extinction coefficient of 5550 M- tcm - ’ was used for the calculation. Prot4in df2vrrmitmtion The protein concentration in the biological samples was estimated orimetric method of Bradford (Bradford, 1976).
by the col-
Final concentrations of 10ph M, 10V5 M. LO-” M of Thiram (UCB ref TMBQ) and the appropriate blanks in 0.1 s DMSO were mixed with the supernatants and were left gently shaken in a water bath (Thermosi SB-16 TECAM) for 1.5 h, at 25’C. The measurements of superoside dismutase, catalase and glutathione peroxidase activities were performed as described above, in six assay series using six fish. RESULTS
In the first part of this study, the activities of superoxide dismutase, catalase and glutathione peroxidase were measured in the liver S lzooofraction of control fish. The mean values of these activities are summarized in Table I. Thereafter, the influence of DMSQ on these activities was investigated: 0.1% DMSQ appeared to be the optimal concentration as it was the minimal concentration needed to dissolve IO-” M Thiram in the test medium. The presence of 0.1% DMSO induced no significant decrease in enzyme activities. In vitro Thiranl incubation The values of the enzymatic activities found in rainbow trout liver supernatant incubated with Thiram in vitro are summarized in Table II. A slight difference was noted in the control enzyme activities compared to the results found previously, due
TABLE I Supcroxide dismutase. catalase and giutathione peroxidase activities of rainbow trout livers __.___~__ Enzyme
Specific acti*iity (mean + SD)
No. fish testedh
_ _,_ ii.3 + ‘3 33.1 + 7.6
1l.gmin:mg llrnoi min, mg nmoi min, mg
6 6 5
__.~____________.____ Superoxide dismutase rataiase Giutathionc peroxidase
JResults arc expressed in pg superoxide dismutase. min mg protein. pmoi hydrogen peroxide destroyed min mg protein and nmol reduced giutathione consumed,min:mp protein. respectively. hOne series of assays was carried out for each fish tested.
to the 1.S h incubation period at 25’C required for the in vitro exposure to Thiram. The superoxide dismutase activity was not influenced up to 10e5 M of Thiram in comparison with the controls, but was completely inhibited at 1UV3M Thiram. The catalase activity was only slightly affected by the whole range of toxicant concentrations tested. The inhibition WBSonly 12.4% at 1Oe3 M. in comparison with the controls. There was a great decrease in glutathione peroxidase activity at IO-” M Thiram. The inhibition was 36.2% at this concentration, compared with the controls. For higher toxicant concentrations, the inhibition did not increase: it did not exceed 37% at 10ee3 M Thiram. TABLE II In vitro effects of Thiram on superoxide dismutase. catalase and giutathione pcroxidase activities of rainbow trout liver supernatants Specific activitya Unit
Thiram (molJh IO hM
CAT firno1 min.;mg
GSH-Yx nmoi, min:‘mg
10~ 5 M 8.5 +O.Z
iO-J M OlU
22.5 + 0.6
Specific enzymatic activities expressed per mg protein. hSupernatanls were incubated with Thiram in DMSO 0. I $I for !.5 h at 25 C. Control experiments consisted of supernalant incubated with DMSO 0.1% alone. Number of fish tested for each toxicant concentration: 6.
TABLE III Comparison of the activities of fish liver superoxide dismutase, catalase and glutathione peroxidase of different species (expressed per gram tissue)
716 460 366
9003 6135 1173
M ackereP Saithe& Rainbow troutb
“Aksnes and Njaa, 198 1. bThis study.
Table III summarizes the values of superoxide dismutase, catalase and glutathione peroxidase activities found in fish livers of different species; specific activities being expressed per g tissue in Table III, in order to compare with the data given by Aksnes and Njaa (198 1). The enzyme activities we measured in the liver supernatant of control rainbow trout were lower than those found by Aksnes and Njaa ( 1981). This may be explained by differences between the species, the age of animals and the preparation of supernatant. The St2000fraction used in the present study was devoid of mitochondria. So mitochondrial manganese superoxide dismutase and glutathione peroxidase were not involved; these enzymes represent approximately 30% of the total amount of superoxide dismutase and glutathione-peroxidase in the whole rat liver cell (Weisiger and Fridovich, 1973a,b; Flohe and Schlegel, 1971). But as we were primarily interested in the metabolic pathways of the xenobiotic, the enzymes localized mitochondrially were not considered as they were less involved in the biotransformation processes than the cytosolic forms, due to their isolation inside the cell. In vitro incubation qf S~~ooc with Thiram We tested a range of Thiram concentrations from 10V6 M to low4 M in 0.1% DMSO. Enzyme assays with higher dithiocarbamate concentrations were not possible, due to the low solubility of this compound and its precipitation in the tested vial, resulting in an interference in the spectrophotometric measurements. So no comparison could be made with the results of ~eikkila et al. (1976) and Kelner and Alexander (1986), who used 10B3to 10e2 M dithiocarbamate. Glutathione peroxidase activity is exhibited by selenium-dependent and seleniumindependent glutathione peroxidases; the latter were identified mainly as glutathioneS-transferases (Laurence and Burk, 1976). We measured the total glutathione peroxi-
dase activity in the S 12000 fractions by initiating the assay with cumene hydroperoxide, which is a substrate for bot5 kinds of peroxidases. A great loss (over 40%) of glutathione peroxidase activity was registered at 10s6 M Thiram, compared with the control; no dose-effect correlation was observed between Thiram concentrations and the inhibition of enzymatic activity for higher dithiocarbamate levels. This suggests that Thiram acted specifically on some of the enzymes possessing glutathione peroxidase activity. As these enzymes seem to be important for the detoxification pathways, their further identi~cation would be of interest. The total inhibition of superoxide dismutase up to 10m4M Thiram is in agreement with the fact that dithiocarbamates are copper chelators. So when S12000fractions, which mostly contained CuZn superoxide dismutase, were incubated with 1O-4 M Thiram, the concentration was high enough for the toxicant to bind all prosthetic sites of the enzymes present in the supernatant, resulting in total inhibition of the activity. It would be interesting to confirm this hypothesis by adding an excess of copper to the medium containing the enzymes inhibited by Thiram, to check whether their enzymatic activity would be restored. We have no explanation for the lack of sensitivity of catalase to the levels of Thiram tested. The inhibition of superoxide dismutase and glutathione peroxidase may result in an increase in superoxide anions and hydrogen peroxide concentrations in the cell, which could be responsible for alterations of DNA structure and induction of mutagenic phenomena. It would be of interest to conduct in vivo experiments and to compare the enzyme activities of fish exposed to Thiram with the results obtained in vitro. ACKNOWLEDGEMENT
The authors thank Tracy Carmona and Kevin Connolly for their help with the language correction of this paper. REFERENCES Aksnes, A. and L.R. Njaa, 1981. Catalase. glutathione peroxidase and superoxide dismutase in different fish species. Comp. Biochcm. Physioi. 69 B, 893 -896. Beers, R.F. and 1-W. Sizer. 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195. 133-140. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72.248-254. Cohen, G., 0. Dembiec and J. Marcus, 1970. Measurement of cataiase activity in tissue extracts. Anal. Biochem. 34,3&38. FlohC, L. and W. Schlegel, 1971. Glutathion-Pcroxidase, IV. Intrazellullre Verteilung .Jes GlutathionPeroxidase-Systems in der Rattenleber. Hoppe-Seylers Z. Physiol. Chem. 352, 1401-1410. Hastings, J. W., 1966. The chemistry of bioluminescellce. Curr. Top. Bioenerg. 1, I 13- 152.
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