- Email: [email protected]
In vivo effects of phenolic compounds on blood parameters of a marine fish (Dicentrarchus labrax)
Comparative Biochemistry and Physiology Part C 125 (2000) 345 – 353
www.elsevier.com/locate/cbpc
In vivo effects of phenolic compounds on blood parameters of a marine fish (Dicentrarchus labrax) He´le`ne Roche a,*, Ge´rard Boge´ b a
Uni6ersity of Paris-Sud, Laboratory of Ecology and Zoology, CNRS UPRESA 8079, Baˆt. 442, F91405 Orsay cedex, France b LEPI, Department of Biology, Uni6ersity of Toulon et du Var, BP 132, F-83957 La Garde cedex, France Received 14 April 1999; received in revised form 3 December 1999; accepted 6 December 1999
Abstract Sea bass (Dicentrarchus labrax) were injected intraperitoneally once (single dose) or three times (fractionated dose) with phenol or OH-phenols (hydroquinone, resorcinol, and pyrocatechol). On the basis of the lethal doses, OH-phenols were more toxic than phenol, and pyrocatechol was the most powerful compound. Hematological, metabolic and antioxidant blood parameters were measured 3 days after the end of the treatment. Metabolic variations as specific effects on erythrocytes were revealed and differences between single and fractionated doses were observed. OH-phenolstreated fish showed disorders in the metabolic toxicity indicators as hypoglycemia, low blood urea nitrogen level (BUN) and decrease of alkaline phosphatase activity (ALP). In addition, quantitative structure-activity relationships were developed using the n-octanol:water partition coefficient (log Kow). Positive correlations were found with ALP, plasma glucose and hemoglobin. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Phenolic compounds; Sea bass; Blood parameters; Erythrotoxicity; Metabolic effects; Antioxidant enzymes; Log Kow; QSAR
1. Introduction Phenolic compounds are good models of widespread xenobiotics. These lipophilic compounds have numerous industrial applications, which enhance the risk to the environment and to human health (Bradbury et al., 1989). They are commonly found in the marine environment and in fish tissues (Mukherjee et al., 1990) where they induce acute or chronic toxicities. Their actions are multiple and often antagonistic. They are genotoxic (Jagetia and Aruna, 1997), carcinogenic (Tsutsui et al., 1997), immunotoxic (Taysse et al., 1995), but they also convey protection against * Corresponding author. Tel.: + 33-1-69157312; fax: +331-69157947. E-mail address: [email protected] (H. Roche)
genetic damage or development of cancers induced by other carcinogens (Stich, 1991). Some of them are scavengers for free radical species, while others are considered as reactive oxygen species generating agents (Winston, 1991). The present study analyses the toxic effects of phenol and hydroxyphenols (OH-phenols) — hydroquinone, resorcinol and pyrocatechol (o-, m-, p-OH-phenol) — on fish blood parameters of sea bass, Dicentrarchus labrax. Fish blood parameters are suitable biomarkers for evaluating the potential risk of chemicals (Roche and Boge´, 1996). Since chemical intoxication may be considered as a potential source of stress, plasma hormones, metabolic substrates, cell volumes, or enzyme activities were first measured (Adams et al., 1989; Panduranga-Rao et al., 1990). Stress indicators such as levels of plasma cortisol and glucose,
0742-8413/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 2 - 8 4 1 3 ( 9 9 ) 0 0 1 1 9 - X
346
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
by means of intra-abdominal injections of the xenobiotics and blood sample collection.
2. Experimental procedures
2.1. Organisms Fig. 1. Chemical structures of some simple monosubstituted phenols considered as models of xenobiotics for sea bass in this study.
blood hemoglobin (Hb), packed cell volume (PVC), mean corpuscular hemoglobin concentration (MCHC), total plasma proteins, blood urea nitrogen (BUN), and alkaline phosphatase (ALP) activity. The specific cellular responses of enzymes involved in the protection of erythrocytes against reactive oxygen species were also investigated. Phenolics are frequently considered as reactive oxygen species-generating agents leading to major cell damage, such as oxidation of membrane polyunsaturated lipids (Pradhan et al., 1990). These radicals and other activated forms contain the superoxide anion (O− 2 ), hydrogen peroxide (H2O2), hydroxyl radical ( − OH) and singlet oxygen (1O2) (Winston, 1991). Fish blood cells have relatively high antioxidant defence levels (Wilhelm-Filho, 1996) including enzymatic and nonenzymatic systems (Winston and Di Giulio, 1991). The key enzymes in the protection against the ‘oxidative stress’ consist of mitochondrial and cytosolic superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) (Roche and Boge´, 1993). The evaluation of the toxicological properties of these chemicals was carried out
Sea bass (D. labrax; approximately 125g) were obtained from DEVA-Sud, experimental Farm of IFREMER (French Institute of Research and Exploitation of the Sea, Palavas-les-flots, France). They were kept for at least 15 days at 159 1°C before use in experiment. During this conditioning period, they were fed daily with commercial sea bass food pellets (Aqualim), even during the course of the intoxication period. They were starved 48 h before blood sampling.
2.2. Chemicals Xenobiotics were phenol and monosubstituted OH-phenols (Fig. 1). Phenol (C6H4OH), hydroquinone (1,4-dihydroxybenzene), resorcinol (1,3-dihydroxybenzene) and pyrocatechol (1,2-dihydroxybenzene) were purchased from Riedel-deHae¨n (Seelze, Germany).
2.3. Chemical administration Stock solutions were prepared in isotonic NaCl (170 mM). The administered doses of chemicals were calculated on the basis of preliminary lethal toxicity studies and ranged between 1/10 and 1/5 of the lethal dose (Table 1). Therefore 125 ml of phenolic stock solutions were injected intra-abdominally per 100 g fish once (single dose) or three times (fractionated doses). In this case, a
Table 1 Lethal and administered doses (in mg per 100 g body weight (BW)) of phenolics in D. labrax Substituent
Hydroxy-
Chemical
Phenol Resorcinol Hydroquinone Pyrocatechol
Log Kow Measured
Bibliography
1.46 0.8 0.59 1.0
1.6490.03 0.529 0.03 0.739 0.03 0.899 0.09
Lethality (mg·100 g BW−1 (100% in 2 h))
Experimental doses (mg·100 g BW−1)
50 5.8 5.8 0.6
4.8–6.9 0.55–1.05 0.56–0.69 0.06–0.08
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
4-day delay was kept between each injection. Control groups received equivalent volume of isotonic NaCl. Any lethal case occurring during treatment was recorded.
2.4. Blood samples Blood was collected by caudal puncture. Samples were taken from the tail vein with a heparinized syringe 3 days after the last injection. The blood was centrifuged in order to separate erythrocytes from plasma. Red blood cells were washed using an isotonic solution of NaCl (170 mM), then treated in such a way obtain a homogenous hemolysate. PVC and total Hb level were determined immediately.
2.5. Blood parameters Hemoglobin contents (Hb) were determined using cyanomethemoglobin with Drabkin’s solution. PVC was determined by the hematocrit method. MCHC was calculated by dividing Hb concentration (g l − 1) by Hct (l l − 1). Plasma glucose (Glu) was measured by the glucose oxidase method (Hugget and Nixon, 1957). Plasma cortisol concentrations (Cort) were assessed by radioimmuno assay (RIA) (Foster and Dunn, 1974) and ALP activity in accordance with Bessey et al. (1946). Other metabolic indicators were estimated using Sigma diagnostic kits: BUN was determined by the Berthelot method (Sigma ref 67-50) and proteins content by the Biuret method (Sigma ref 541-2). Antioxidant activities (Total peroxidase (Px), CAT, GPx, and SOD) were determined as previously reported (Roche and Boge´, 1996). Enzymatic activities were related to cell Hb content. All values reported in the text represent the average of determinations for five animals (means 9 standard error).
2.6. Quantitati6e structure acti6ity relationship (QSAR) This QSAR is generally studied to correlate the biological effects of xenobiotics with physicochemical and structural properties determined by molecular modeling such as lipophilicity. We were especially interested in the octanol-water partition coefficients (Kow) of the phenolic compounds which were found in the literature (Leo et al., 1971; Schultz, 1987; Xu et al., 1994) or specifically measured (Table 1).
347
2.7. Statistical analysis ANOVA was performed to compare the means of the different using on a Statview program (Statview 4.02 for Macintosh Abacus concepts, California). Significant differences (PB 0.05) were estimated on the basis of Fisher, Scheffe, Dunnett or Student’s test for unpaired data. For QSAR analysis, parametric tests were carried and the statistical signification was given by Pearson’s coefficient. 3. Results
3.1. Lethal effects An intra-abdominal injection of 50 mg phenol per 100 g fish killed all the fish in 2 h. Five milligrams per 100 g fish did not, even after 1 week. Injections of either 5.8 mg hydroquinone or resorcinol per 100 g fish were fatal in 2 h or less, whereas an injection of 0.58 mg was not fatal. Fish injected with 0.58 mg per 100 g pyrocatechol died but those injected with 0.06 mg per 100 g did not (Table 1).
3.2. Non lethal effects The non-lethal effects of the phenolic compounds on the blood parameters of fish were monitored after one injection (single dose) or three injections (fractionated dose) of 1/10 of the administrated maximal lethal dose. The detailed results are expressed in Figs. 2–4. Three kinds of effects were selected: metabolic effects detected throughout measurements of BUN, ALP, Glu and Cort; erythrotoxic effects detected throughout Hb and PVC; and oxidative stress detected through detoxification enzyme activity measurements.
3.2.1. Phenol Metabolic and erythrotoxic markers rather than antioxidant enzyme activities were sensible to phenol. The administration of single or fractionated doses inhibited ALP activity and stimulated cortisol release. Hb contents were more elevated in fish treated by single doses, whereas fractionated doses had no significant effects (Fig. 3). In contrast, BUN was depressed on account of injections of the fractionated dose (Fig. 2).
348
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
Fig. 2. Metabolic effects on plasma parameters in sea bass treated with phenol and OH-phenols as a function of administration process. Single dose ; Fractionated dose . Prot., proteinemia.
Fig. 3. Erythrotoxic effects in sea bass treated with phenol and OH-phenols as a function of toxic administration process. Single dose
; Fractionated dose .
The antioxidant enzyme activities changed after phenol treatment. The main observation concerned the superoxide dismutase manganese-dependent (MnSOD) activity, which was slightly depressed after administration of single doses. In addition, the activity of peroxidase and GPx were increased by the fractionated doses (Fig. 4).
3.2.2. OH-phenols OH-phenols exerted a larger influence on metabolic and erythrotoxic markers than on enzyme activities. Glucose concentrations were significantly decreased after single dose injections of hydroquinone, resorcinol, and pyrocatechol. This hypoglycemic effect was intensified in case of frac-
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
tionated injections. Such modifications were not correlated with changes of cortisol levels, which were significantly increased in fish having received a fractionated dose of resorcinol. Plasma ALP activity was also sensitive to OH-phenols and lower activities were shown in sea bass treated by fractionated doses of resorcinol and pyrocatechol or single doses of hydroquinone and resorcinol (Fig. 2). A single injection of these phenolics caused a slight increase of hematological parameters as Hb content and PCV, which was not observed after fractionated injections. Such a procedure generated a low decrease of Hb blood concentration with pyrocatechol and a PVC reduction in fish treated with resorcinol, whereas MCHC increased after repeated injections of hydroquinone and resorcinol (Fig. 3). Concerning antioxidant activities, resorcinol inhibited of the total SOD and MnSOD activities and stimulated peroxidase activity. The injection of fractionated doses caused a particular stress in fish with possible consequence on blood parameters. In fact, the classical plasma stressors such as cortisol or glucose were not higher
349
in control fish having received several injections of isotonic NaCl (Fig. 2). Erythrocyte parameters were not affected with the exception of MnSOD activity, which was lower in fish having received fractionated injections (Fig. 4).
3.3. Structure acti6ity relationships Structure-activity relationships were developed using the log n-octanol: water partition coefficient (log Kow) as a descriptor (Table 1). The log Kow decreased in the following order: phenol (1.46); pyrocatechol (1.0); resorcinol (0.8); hydroquinone (0.59). Pearson’s test revealed that among nonlethal effects, glycemia was highly correlated with the partition coefficient of all the phenolic compounds, the chemicals having lower partition coefficients being more active (OH-phenols and especially hydroquinone). In the case of OH-phenols, ALP activities and Hb were significantly correlated following fractionated dose injection whereas no correlation has been found after a single dose (Fig. 5).
Fig. 4. Effect on enzymatic activities of erythrocytes in sea bass treated with phenol and OH-phenols as a function of toxic administration process. Single dose ; Fractionated dose .
350
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
Fig. 5. Quantitative structure-activity relationships. Pearson’s correlations between log of biological parameters and log Kow of phenolic compounds. Single dose ; Fractionated dose .
4. Discussion This work showed considerable differences in the lethal and non-lethal effects of phenolic compounds on marine fish and provided information about the structure-activity relationships of such chemicals. On the basis of the lethal doses, OHphenols and phenol were more toxic than alkyland NO2-phenols. Moreover, OH-phenols were more toxic than phenol. Among OH-phenols, pyrocatechol was the strongest compound. It is at least 100-times more toxic than phenol, and ten times more toxic than hydroquinone and resorcinol. Most of the monosubstituted phenols tested in this study are major metabolites of benzene as phenol, catechol and hydroquinone (Tsutsui et al., 1997). They are responsible for physiological effects of benzene in particular hematotoxicity (Briviba et al., 1993). The metabolic mechanisms of this toxicity include both oxidation and conjugation with a potential competition among various implicated enzymes. It also includes interactions among several metabolites and eventually by other intermediates including reactive oxygen species (Witz et al., 1996). Phenolics are involved in oxidative mechanisms either in different steps in the pathway for their biodegradation as agents generating reactive oxygen species or as antioxidants (Gut et al., 1996; Hiraku and Kawanishi, 1996). The non-lethal effects of these compounds have been characterized on blood parameters suitable to reveal metabolic disturbance, or specific effects on erythrocytes. This study shows that phenol and OH-phenols have metabolic effects. On the basis of cortisol levels, phenol is a more active
stressor than OH-phenols; in fact, the levels were higher after single or fractionated doses (Fig. 2). This observation is in accordance with Gluth and Hanke (1976) who found a significant elevation of serum cortisol in carp exposed to higher concentrations of phenol (1–10 mg l − 1). Unexpectedly in D. labrax, this stimulation is not connected to an increase of plasma glucose, as is usually the case. Numerous authors consider that, in fish, acute stress induces hypercortisolemia, hyperglycemia and hyperproteinemia whereas chronic stress induces hypoglycemia, as a consequence of the sugar reserves (Hrubec et al., 1997a). This could be the case in sea bass, particularly if successive handling stress is liable for a modification in feeding status. In addition, OH-phenols which are structurally analogous to catecholamines and they could therefore compete with adrenaline to lower glycemia (Kaila, 1982; CajinaQuezada and Schultz, 1990). Others metabolic responses to phenols such as the decrease in BUN level and in ALP activity (Fig. 2) could be a possible consequence of their nephrotoxicity and a more important elimination in urine (Peters et al., 1997). In fish, BUN is generally low (Hrubec et al., 1997b), we have observed that, in sea bass, the values were only three times lower than in mammals. Decrease of BUN levels after phenolic intoxication indicates possible effect on the protein metabolism. Such effects were suggested by Gupta et al. (1983) on Notopterus notopterus. These authors observed a stimulatory effect of phenolic compounds on the activity of transaminases, which implies disturbance of protein metabolism. However, we never have found change on plasma protein content in D. labrax.
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
Increased plasma Hb concentrations are a sign of cell disruption (Fig. 3). Significant effects were shown after single injections of phenol and OHphenols which confirm the erythrotoxicity of these compounds (Boge´ and Roche, 1996). In return, phenol was less toxic on the base of this Hb release. The modifications in free radical detoxification activities of fish treated with phenol and OH-phenols were rather limited. Such an observation is unexpected since these compounds generate reactive oxygen species. Single injections of phenol and OH-phenols failed to affect SOD activity (Fig. 4). The absence of effect of hydroquinone is surprising since this primary benzene metabolite exhibits an oxygen species generating potential and then may cause DNA damage through H2O2 generation in cells (Hiraku and Kawanishi, 1996; Yu and Anderson, 1997). In sea bass, this absence of change in SOD activities could strengthen the specific effects of H2O2 on DNA. Fractionated injections had more effect on SOD activity notably resorcinol (Fig. 4). Resorcinol is an inducer of monooxygenase synthesis and a powerful methemoglobinizing compound (Hachet, 1989). In D. labrax, it greatly reduces SOD activities. However, the deleterious consequences on cell protection could be offset by a stimulation of total peroxidase activity. Previous experiments carried out on erythrocytes incubated with resorcinol for 24 h did not confirm such a drastic decrease of the total SOD activity (Boge´ and Roche, 1996). These discrepancies could be ascribed to an in vivo biotransformation and interactions with metabolites (Lemaire et al., 1990; Medinsky et al., 1996; Bratton et al., 1997). Antioxidant activities were much more sensible to alkylphenols and nitrophenols than to OH-phenols or phenol. Total SOD activity was increased following injections of alkylphenols (o-cresol and o-ethylphenol and o-propylphenol), and nitrophenols (o-, m- and p-nitrophenol; personal observations). Specific single dose effects were observed notably on Hb with phenol and OH-phenols (Fig. 3). Specific fractionated dose effects were also disclosed, notably on peroxidase activity and BUN (Figs. 2 and 4). Differences between single and fractionated doses affected particularly glucose and PCV with OH-phenols, Hb, BUN, peroxidase and SOD activities with all compounds (Figs. 2 – 4). These observations indicate that biotrans-
351
formation reactions could be efficient for these compounds especially in fractionated doses. These reactions are well known for OH-phenols (Sawahata and Neal, 1983; Wallin and Morgenstern, 1990; Cravedi et al., 1999). They lead to intermediate products which could be more reactive that the initial compound as in the case of hydroquinone (Bratton et al., 1997; Peters et al., 1997). Phenolic compounds are generally concentrated through the food chain due to their accumulation in lipids (Mukherjee et al., 1990). Their lower partition coefficients indicate that OH-phenols could be less accumulated than phenol and are more bioavailable (Table 1). We established a structure activity relationship with glucose for phenol and OH-phenols and with ALP and Hb for OH-phenols with fractionated doses (Fig. 5). Consequently, the lipophilic character of phenol and OH-phenols (Ancerewicz et al., 1998) could influence these parameters. For other parameters, in particular BUN, which was highly depressed with fractionated doses of all compounds, the toxicity could be more dependent upon other chemical properties and perhaps with a possible relationship with catecholamines in the case of OH-phenols. In conclusion, this work confirmed that the toxicity of phenols depends greatly on their structure. Their major effect is on some plasma parameters, but interactions with metabolism of free radicals could contribute to their toxicity.
Acknowledgements This study was supported by the French Ministry of Environment, grant no. 90 131 and 91 192. The experiments were performed in Michel Pacha Institute, maritime laboratory of Physiology of University of Lyon, France.
References Adams, S.M., Shepard, K.L., Greeley, M.S., Ryon, M.G., Jimenez, B.D., Shugart, L.R., McCarthy, J.F., Hinton, D.E., 1989. The use of bioindicators for assessing the effects of pollutant stress in fish. Mar. Environ. Res. 28, 459 – 464. Ancerewicz, J., Migliavacca, E., Carrupt, P.A., Testa, B., Bree, F., Zini, R., Tillement, J.P., Labidalle, S.,
352
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
Guyot, D., Chauvet-Monges, A.M., Crevat, A., Le Ridant, A., 1998. Structure-property relationships of trimetazidine derivatives and model compounds as potential antioxidants. Free Radic. Biol. Med. 25, 113–120. Bessey, O.A., Lowry, O.H., Brock, M.J., 1946. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. Biol. Chem. 164, 321–329. Boge´, G., Roche, H., 1996. Cytotoxicity of phenolic compounds in Dicentrarchus labrax erythrocytes. Bull. Environ. Toxicol. Contamin. 57, 171–178. Bradbury, S.P., Henry, T.R., Niemi, G.J., Carlson, R.W., Snarski, V.M., 1989. Use of respiratory-cardiovascular response of rainbow trout (Salmo gairdneri ) in identifying acute toxicity syndromes in fish. Part 3. Polar narcosis. Environ. Toxicol. Chem. 8, 247–262. Bratton, S.B., Lau, S.S., Monks, T.J., 1997. Identification of quinol thioethers in bone marrow of hydroquinone/phenol-treated rats and mice and their potential role in benzene-mediated hematotoxicity. Chem. Res. Toxicol. 10, 859–865. Briviba, K., Devasagayam, T.P.A., Sies, H., Steenken, S., 1993. Selective para hydroxylation of phenol and aniline by singlet molecular oxygen. Chem. Res. Toxicol. 6, 548–553. Cajina-Quezada, M., Schultz, W., 1990. Structure-toxicity relationships for selected weak acid respiratory uncouplers. Aquat. Toxicol. 17, 239–252. Cravedi, J.P., Lafuente, A., Baradat, M., Hillenweck, A., Perdu-Durand, E., 1999. Biotransformation of pentachlorophenol, aniline and biphenyl in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes: comparison with in vivo metabolism. Xenobiotica 29, 499–509. Foster, L.B., Dunn, R.T., 1974. Single antibody technique for radioimmuno-assay of cortisol unextracted serum of plasma. Clin. Chem. 20, 365–368. Gluth, G., Hanke, W., 1976. The effect of temperature on physiological changes in carp, Cyprinus carpio L., induced by phenol. Ecotoxicol. Environ. Saf. 7, 373–389. Gupta, S., Dalela, R.C., Saxena, P.K., 1983. Effect of phenolic compounds on in vivo activity of transaminases in certain tissues of the fish, Notopterus notopterus. Environ. Res. 32, 8–13. Gut, I., Nedelcheva, V., Soucek, P., Stopka, P., Tichavska, B., 1996. Cytochromes P450 in benzene metabolism and involvement of their metabolites and reactive oxygen species in toxicity. Environ. Health Perspect. 104, 1211–1218. Hachet, J.C., 1989. Toxicologie d’Urgence. Produits Chimiques Industriels, Collection Abre´ge´s de Me´decine. Masson, Paris.
Hiraku, Y., Kawanishi, S., 1996. Oxidative DNA damage and apoptosis induced by benzene metabolites. Cancer Res. 56, 5172 – 5178. Hrubec, T.C., Robertson, J.L., Smith, S.A., 1997a. Effects of temperature on hematologic and serum biochemical profiles of hybrid striped bass (Morone chrysops×Morone saxatilis). Am. J. Vet. Res. 58, 126 – 130. Hrubec, T.C., Robertson, J.L., Smith, S.A., 1997b. Effects of ammonia and nitrate concentration on hematologic and serum biochemical profiles of hybrid striped bass (Morone chrysops × Morone saxatilis). Am. J. Vet. Res. 58, 131 – 135. Hugget, A.S.G., Nixon, D.A., 1957. Use of glucose oxidase, peroxidase and o-dianisidine in determination of blood and urinary glucose. Lancet 2, 368 – 379. Jagetia, G.C., Aruna, R., 1997. Hydroquinone increases the frequency of micronuclei in a dose-dependent manner in mouse bone marrow. Toxicol. Lett. 93, 205 – 213. Kaila, K., 1982. Cellular neurophysiological effects of phenol derivatives. Comp. Biochem. Physiol. [C] 73C, 231 – 241. Lemaire, P., Mathieu, A., Carrie`re, S., Drai, P., Giudicelli, J., Lafaurie, M., 1990. The uptake metabolism and biological half-life of benzo(a)pyrene in different tissues of sea bass Dicentrarchus labrax. Ecotox. Environ. 20, 223 – 233. Leo, A., Hansch, C., Elkins, D., 1971. Partition coefficients and their uses. Chem. Rev. 71, 525 – 616. Medinsky, M.A., Kenyon, E.M., Seaton, M.J., Schlosser, P.M., 1996. Mechanistic considerations in benzene physiological model development. Environ. Health Perspect. 104, 1399 – 1404. Mukherjee, D., Bhattacharya, S., Kumar, V., Moitra, J., 1990. Biological significance of [14C]phenol accumulation in different organs of a murrel, Channa punctatus, and the common carp, Cyprinus carpio. Biomed. Environ. Sci. 3, 337 – 342. Panduranga-Rao, D., Ram-Bhaskar, B., Srinivasa-Rao, K., Durga-Prasad, Y.V.K., Someswara-Rao, N., Venkateswara-Rao, T.N.V., 1990. Haematological effects in fishes from complex polluted waters of Visakhapatnam harbour. Mar. Environ. Res. 30, 217 – 231. Peters, M.M., Jones, T.W., Monks, T.J., Lau, S.S., 1997. Cytotoxicity and cell-proliferation induced by the nephrocarcinogen hydroquinone and its nephrotoxic metabolite 2,3,5-(tris-glutathion-S-yl)hydroquinone. Carcinogenesis 18, 2393 – 2401. Pradhan, D., Weiser, M., Lumley-Sapanski, K., Frazier, D., Kemper, S., Williamson, P., Schlegel, R.A., 1990. Peroxidation-induced perturbations of erythrocyte lipid organization. Biochim. Biophys. Acta 1023, 398 – 404.
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
Roche, H., Boge´, G., 1993. In vitro effects of Cu, Zn and Cr salts on antioxidant enzyme activities of red blood cells of a marine fish Dicentrarchus labrax. Toxicol. In Vitro 7, 623–629. Roche, H., Boge´, G., 1996. Fish blood parameters as a potential tool for identification of stress caused by environmental factors or chemical intoxication. Mar. Environ. Res. 41, 27–44. Sawahata, T., Neal, R.A., 1983. Biotransformation of phenol to hydroquinone and catechol by rat liver microsomes. Mol. Pharmacol. 23, 453–460. Schultz, T.W., 1987. Relative toxicity of para-substituted phenols: Log Kow and pKa-dependent structure-activity relationships. Bull. Environ. Contam. Toxicol. 38, 994–999. Stich, H.F., 1991. The beneficial and hazardous effects of simple phenolic compounds. Mut. Res. 259, 307– 324. Taysse, L., Troutaud, D., Khan, N.A., Deschaux, P., 1995. Structure-activity relationship of phenolic compounds (phenol, pyrocatechol and hydroquinone) on natural lymphocytotoxicity of carp (Cyprinus carpio). Toxicology 98, 207–214. Tsutsui, T., Hayashi, N., Maizumi, H., Huff, J., Barrett, J.C., 1997. Benzene-, catechol-, hydroquinoneand phenol-induced cell transformation, gene mutations, chromosome aberrations, aneuploidy, sister chromatid exchanges and unscheduled DNA synthe-
.
353
sis in Syrian hamster embryo cells. Mutat. Res. 373, 113 – 123. Wallin, H., Morgenstern, R., 1990. Activation of microsomal glutathione transferase activity by reactive intermediates formed during the metabolism of phenol. Chem. Biol. Interact. 75, 185 – 199. Wilhelm-Filho, D., 1996. Fish antioxidant defenses — a comparative approach. Braz. J. Med. Biol. Res. 29, 1735 – 1742. Winston, G.W., 1991. Mini-review. Oxidants and antioxidants in aquatic animals. Comp. Biochem. Physiol. [C] 100C, 173 – 176. Winston, G.W., Di Giulio, T., 1991. Prooxidant and antioxidant mechanisms in aquatic organisms. Aquat. Toxicol. 19, 137 – 161. Witz, G., Zhang, Z., Goldstein, B.D., 1996. Reactive ring-opened aldehyde metabolites in benzene hematotoxicity. Environ. Health Perspect. 104, 1195 – 1199. Xu, L., Ball, J.W., Dixon, S.L., Jurs, P.C., 1994. Quantitative structure-activity relationships for toxicity of phenols using regression analysis and computational neural networks. Environ. Toxicol. Chem. 13, 841 – 851. Yu, T.W., Anderson, D., 1997. Reactive oxygen species-induced DNA damage and its modification: a chemical investigation. Mutat. Res. 379, 201 – 210.
www.elsevier.com/locate/cbpc
In vivo effects of phenolic compounds on blood parameters of a marine fish (Dicentrarchus labrax) He´le`ne Roche a,*, Ge´rard Boge´ b a
Uni6ersity of Paris-Sud, Laboratory of Ecology and Zoology, CNRS UPRESA 8079, Baˆt. 442, F91405 Orsay cedex, France b LEPI, Department of Biology, Uni6ersity of Toulon et du Var, BP 132, F-83957 La Garde cedex, France Received 14 April 1999; received in revised form 3 December 1999; accepted 6 December 1999
Abstract Sea bass (Dicentrarchus labrax) were injected intraperitoneally once (single dose) or three times (fractionated dose) with phenol or OH-phenols (hydroquinone, resorcinol, and pyrocatechol). On the basis of the lethal doses, OH-phenols were more toxic than phenol, and pyrocatechol was the most powerful compound. Hematological, metabolic and antioxidant blood parameters were measured 3 days after the end of the treatment. Metabolic variations as specific effects on erythrocytes were revealed and differences between single and fractionated doses were observed. OH-phenolstreated fish showed disorders in the metabolic toxicity indicators as hypoglycemia, low blood urea nitrogen level (BUN) and decrease of alkaline phosphatase activity (ALP). In addition, quantitative structure-activity relationships were developed using the n-octanol:water partition coefficient (log Kow). Positive correlations were found with ALP, plasma glucose and hemoglobin. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Phenolic compounds; Sea bass; Blood parameters; Erythrotoxicity; Metabolic effects; Antioxidant enzymes; Log Kow; QSAR
1. Introduction Phenolic compounds are good models of widespread xenobiotics. These lipophilic compounds have numerous industrial applications, which enhance the risk to the environment and to human health (Bradbury et al., 1989). They are commonly found in the marine environment and in fish tissues (Mukherjee et al., 1990) where they induce acute or chronic toxicities. Their actions are multiple and often antagonistic. They are genotoxic (Jagetia and Aruna, 1997), carcinogenic (Tsutsui et al., 1997), immunotoxic (Taysse et al., 1995), but they also convey protection against * Corresponding author. Tel.: + 33-1-69157312; fax: +331-69157947. E-mail address: [email protected] (H. Roche)
genetic damage or development of cancers induced by other carcinogens (Stich, 1991). Some of them are scavengers for free radical species, while others are considered as reactive oxygen species generating agents (Winston, 1991). The present study analyses the toxic effects of phenol and hydroxyphenols (OH-phenols) — hydroquinone, resorcinol and pyrocatechol (o-, m-, p-OH-phenol) — on fish blood parameters of sea bass, Dicentrarchus labrax. Fish blood parameters are suitable biomarkers for evaluating the potential risk of chemicals (Roche and Boge´, 1996). Since chemical intoxication may be considered as a potential source of stress, plasma hormones, metabolic substrates, cell volumes, or enzyme activities were first measured (Adams et al., 1989; Panduranga-Rao et al., 1990). Stress indicators such as levels of plasma cortisol and glucose,
0742-8413/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 2 - 8 4 1 3 ( 9 9 ) 0 0 1 1 9 - X
346
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
by means of intra-abdominal injections of the xenobiotics and blood sample collection.
2. Experimental procedures
2.1. Organisms Fig. 1. Chemical structures of some simple monosubstituted phenols considered as models of xenobiotics for sea bass in this study.
blood hemoglobin (Hb), packed cell volume (PVC), mean corpuscular hemoglobin concentration (MCHC), total plasma proteins, blood urea nitrogen (BUN), and alkaline phosphatase (ALP) activity. The specific cellular responses of enzymes involved in the protection of erythrocytes against reactive oxygen species were also investigated. Phenolics are frequently considered as reactive oxygen species-generating agents leading to major cell damage, such as oxidation of membrane polyunsaturated lipids (Pradhan et al., 1990). These radicals and other activated forms contain the superoxide anion (O− 2 ), hydrogen peroxide (H2O2), hydroxyl radical ( − OH) and singlet oxygen (1O2) (Winston, 1991). Fish blood cells have relatively high antioxidant defence levels (Wilhelm-Filho, 1996) including enzymatic and nonenzymatic systems (Winston and Di Giulio, 1991). The key enzymes in the protection against the ‘oxidative stress’ consist of mitochondrial and cytosolic superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) (Roche and Boge´, 1993). The evaluation of the toxicological properties of these chemicals was carried out
Sea bass (D. labrax; approximately 125g) were obtained from DEVA-Sud, experimental Farm of IFREMER (French Institute of Research and Exploitation of the Sea, Palavas-les-flots, France). They were kept for at least 15 days at 159 1°C before use in experiment. During this conditioning period, they were fed daily with commercial sea bass food pellets (Aqualim), even during the course of the intoxication period. They were starved 48 h before blood sampling.
2.2. Chemicals Xenobiotics were phenol and monosubstituted OH-phenols (Fig. 1). Phenol (C6H4OH), hydroquinone (1,4-dihydroxybenzene), resorcinol (1,3-dihydroxybenzene) and pyrocatechol (1,2-dihydroxybenzene) were purchased from Riedel-deHae¨n (Seelze, Germany).
2.3. Chemical administration Stock solutions were prepared in isotonic NaCl (170 mM). The administered doses of chemicals were calculated on the basis of preliminary lethal toxicity studies and ranged between 1/10 and 1/5 of the lethal dose (Table 1). Therefore 125 ml of phenolic stock solutions were injected intra-abdominally per 100 g fish once (single dose) or three times (fractionated doses). In this case, a
Table 1 Lethal and administered doses (in mg per 100 g body weight (BW)) of phenolics in D. labrax Substituent
Hydroxy-
Chemical
Phenol Resorcinol Hydroquinone Pyrocatechol
Log Kow Measured
Bibliography
1.46 0.8 0.59 1.0
1.6490.03 0.529 0.03 0.739 0.03 0.899 0.09
Lethality (mg·100 g BW−1 (100% in 2 h))
Experimental doses (mg·100 g BW−1)
50 5.8 5.8 0.6
4.8–6.9 0.55–1.05 0.56–0.69 0.06–0.08
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
4-day delay was kept between each injection. Control groups received equivalent volume of isotonic NaCl. Any lethal case occurring during treatment was recorded.
2.4. Blood samples Blood was collected by caudal puncture. Samples were taken from the tail vein with a heparinized syringe 3 days after the last injection. The blood was centrifuged in order to separate erythrocytes from plasma. Red blood cells were washed using an isotonic solution of NaCl (170 mM), then treated in such a way obtain a homogenous hemolysate. PVC and total Hb level were determined immediately.
2.5. Blood parameters Hemoglobin contents (Hb) were determined using cyanomethemoglobin with Drabkin’s solution. PVC was determined by the hematocrit method. MCHC was calculated by dividing Hb concentration (g l − 1) by Hct (l l − 1). Plasma glucose (Glu) was measured by the glucose oxidase method (Hugget and Nixon, 1957). Plasma cortisol concentrations (Cort) were assessed by radioimmuno assay (RIA) (Foster and Dunn, 1974) and ALP activity in accordance with Bessey et al. (1946). Other metabolic indicators were estimated using Sigma diagnostic kits: BUN was determined by the Berthelot method (Sigma ref 67-50) and proteins content by the Biuret method (Sigma ref 541-2). Antioxidant activities (Total peroxidase (Px), CAT, GPx, and SOD) were determined as previously reported (Roche and Boge´, 1996). Enzymatic activities were related to cell Hb content. All values reported in the text represent the average of determinations for five animals (means 9 standard error).
2.6. Quantitati6e structure acti6ity relationship (QSAR) This QSAR is generally studied to correlate the biological effects of xenobiotics with physicochemical and structural properties determined by molecular modeling such as lipophilicity. We were especially interested in the octanol-water partition coefficients (Kow) of the phenolic compounds which were found in the literature (Leo et al., 1971; Schultz, 1987; Xu et al., 1994) or specifically measured (Table 1).
347
2.7. Statistical analysis ANOVA was performed to compare the means of the different using on a Statview program (Statview 4.02 for Macintosh Abacus concepts, California). Significant differences (PB 0.05) were estimated on the basis of Fisher, Scheffe, Dunnett or Student’s test for unpaired data. For QSAR analysis, parametric tests were carried and the statistical signification was given by Pearson’s coefficient. 3. Results
3.1. Lethal effects An intra-abdominal injection of 50 mg phenol per 100 g fish killed all the fish in 2 h. Five milligrams per 100 g fish did not, even after 1 week. Injections of either 5.8 mg hydroquinone or resorcinol per 100 g fish were fatal in 2 h or less, whereas an injection of 0.58 mg was not fatal. Fish injected with 0.58 mg per 100 g pyrocatechol died but those injected with 0.06 mg per 100 g did not (Table 1).
3.2. Non lethal effects The non-lethal effects of the phenolic compounds on the blood parameters of fish were monitored after one injection (single dose) or three injections (fractionated dose) of 1/10 of the administrated maximal lethal dose. The detailed results are expressed in Figs. 2–4. Three kinds of effects were selected: metabolic effects detected throughout measurements of BUN, ALP, Glu and Cort; erythrotoxic effects detected throughout Hb and PVC; and oxidative stress detected through detoxification enzyme activity measurements.
3.2.1. Phenol Metabolic and erythrotoxic markers rather than antioxidant enzyme activities were sensible to phenol. The administration of single or fractionated doses inhibited ALP activity and stimulated cortisol release. Hb contents were more elevated in fish treated by single doses, whereas fractionated doses had no significant effects (Fig. 3). In contrast, BUN was depressed on account of injections of the fractionated dose (Fig. 2).
348
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
Fig. 2. Metabolic effects on plasma parameters in sea bass treated with phenol and OH-phenols as a function of administration process. Single dose ; Fractionated dose . Prot., proteinemia.
Fig. 3. Erythrotoxic effects in sea bass treated with phenol and OH-phenols as a function of toxic administration process. Single dose
; Fractionated dose .
The antioxidant enzyme activities changed after phenol treatment. The main observation concerned the superoxide dismutase manganese-dependent (MnSOD) activity, which was slightly depressed after administration of single doses. In addition, the activity of peroxidase and GPx were increased by the fractionated doses (Fig. 4).
3.2.2. OH-phenols OH-phenols exerted a larger influence on metabolic and erythrotoxic markers than on enzyme activities. Glucose concentrations were significantly decreased after single dose injections of hydroquinone, resorcinol, and pyrocatechol. This hypoglycemic effect was intensified in case of frac-
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
tionated injections. Such modifications were not correlated with changes of cortisol levels, which were significantly increased in fish having received a fractionated dose of resorcinol. Plasma ALP activity was also sensitive to OH-phenols and lower activities were shown in sea bass treated by fractionated doses of resorcinol and pyrocatechol or single doses of hydroquinone and resorcinol (Fig. 2). A single injection of these phenolics caused a slight increase of hematological parameters as Hb content and PCV, which was not observed after fractionated injections. Such a procedure generated a low decrease of Hb blood concentration with pyrocatechol and a PVC reduction in fish treated with resorcinol, whereas MCHC increased after repeated injections of hydroquinone and resorcinol (Fig. 3). Concerning antioxidant activities, resorcinol inhibited of the total SOD and MnSOD activities and stimulated peroxidase activity. The injection of fractionated doses caused a particular stress in fish with possible consequence on blood parameters. In fact, the classical plasma stressors such as cortisol or glucose were not higher
349
in control fish having received several injections of isotonic NaCl (Fig. 2). Erythrocyte parameters were not affected with the exception of MnSOD activity, which was lower in fish having received fractionated injections (Fig. 4).
3.3. Structure acti6ity relationships Structure-activity relationships were developed using the log n-octanol: water partition coefficient (log Kow) as a descriptor (Table 1). The log Kow decreased in the following order: phenol (1.46); pyrocatechol (1.0); resorcinol (0.8); hydroquinone (0.59). Pearson’s test revealed that among nonlethal effects, glycemia was highly correlated with the partition coefficient of all the phenolic compounds, the chemicals having lower partition coefficients being more active (OH-phenols and especially hydroquinone). In the case of OH-phenols, ALP activities and Hb were significantly correlated following fractionated dose injection whereas no correlation has been found after a single dose (Fig. 5).
Fig. 4. Effect on enzymatic activities of erythrocytes in sea bass treated with phenol and OH-phenols as a function of toxic administration process. Single dose ; Fractionated dose .
350
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
Fig. 5. Quantitative structure-activity relationships. Pearson’s correlations between log of biological parameters and log Kow of phenolic compounds. Single dose ; Fractionated dose .
4. Discussion This work showed considerable differences in the lethal and non-lethal effects of phenolic compounds on marine fish and provided information about the structure-activity relationships of such chemicals. On the basis of the lethal doses, OHphenols and phenol were more toxic than alkyland NO2-phenols. Moreover, OH-phenols were more toxic than phenol. Among OH-phenols, pyrocatechol was the strongest compound. It is at least 100-times more toxic than phenol, and ten times more toxic than hydroquinone and resorcinol. Most of the monosubstituted phenols tested in this study are major metabolites of benzene as phenol, catechol and hydroquinone (Tsutsui et al., 1997). They are responsible for physiological effects of benzene in particular hematotoxicity (Briviba et al., 1993). The metabolic mechanisms of this toxicity include both oxidation and conjugation with a potential competition among various implicated enzymes. It also includes interactions among several metabolites and eventually by other intermediates including reactive oxygen species (Witz et al., 1996). Phenolics are involved in oxidative mechanisms either in different steps in the pathway for their biodegradation as agents generating reactive oxygen species or as antioxidants (Gut et al., 1996; Hiraku and Kawanishi, 1996). The non-lethal effects of these compounds have been characterized on blood parameters suitable to reveal metabolic disturbance, or specific effects on erythrocytes. This study shows that phenol and OH-phenols have metabolic effects. On the basis of cortisol levels, phenol is a more active
stressor than OH-phenols; in fact, the levels were higher after single or fractionated doses (Fig. 2). This observation is in accordance with Gluth and Hanke (1976) who found a significant elevation of serum cortisol in carp exposed to higher concentrations of phenol (1–10 mg l − 1). Unexpectedly in D. labrax, this stimulation is not connected to an increase of plasma glucose, as is usually the case. Numerous authors consider that, in fish, acute stress induces hypercortisolemia, hyperglycemia and hyperproteinemia whereas chronic stress induces hypoglycemia, as a consequence of the sugar reserves (Hrubec et al., 1997a). This could be the case in sea bass, particularly if successive handling stress is liable for a modification in feeding status. In addition, OH-phenols which are structurally analogous to catecholamines and they could therefore compete with adrenaline to lower glycemia (Kaila, 1982; CajinaQuezada and Schultz, 1990). Others metabolic responses to phenols such as the decrease in BUN level and in ALP activity (Fig. 2) could be a possible consequence of their nephrotoxicity and a more important elimination in urine (Peters et al., 1997). In fish, BUN is generally low (Hrubec et al., 1997b), we have observed that, in sea bass, the values were only three times lower than in mammals. Decrease of BUN levels after phenolic intoxication indicates possible effect on the protein metabolism. Such effects were suggested by Gupta et al. (1983) on Notopterus notopterus. These authors observed a stimulatory effect of phenolic compounds on the activity of transaminases, which implies disturbance of protein metabolism. However, we never have found change on plasma protein content in D. labrax.
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
Increased plasma Hb concentrations are a sign of cell disruption (Fig. 3). Significant effects were shown after single injections of phenol and OHphenols which confirm the erythrotoxicity of these compounds (Boge´ and Roche, 1996). In return, phenol was less toxic on the base of this Hb release. The modifications in free radical detoxification activities of fish treated with phenol and OH-phenols were rather limited. Such an observation is unexpected since these compounds generate reactive oxygen species. Single injections of phenol and OH-phenols failed to affect SOD activity (Fig. 4). The absence of effect of hydroquinone is surprising since this primary benzene metabolite exhibits an oxygen species generating potential and then may cause DNA damage through H2O2 generation in cells (Hiraku and Kawanishi, 1996; Yu and Anderson, 1997). In sea bass, this absence of change in SOD activities could strengthen the specific effects of H2O2 on DNA. Fractionated injections had more effect on SOD activity notably resorcinol (Fig. 4). Resorcinol is an inducer of monooxygenase synthesis and a powerful methemoglobinizing compound (Hachet, 1989). In D. labrax, it greatly reduces SOD activities. However, the deleterious consequences on cell protection could be offset by a stimulation of total peroxidase activity. Previous experiments carried out on erythrocytes incubated with resorcinol for 24 h did not confirm such a drastic decrease of the total SOD activity (Boge´ and Roche, 1996). These discrepancies could be ascribed to an in vivo biotransformation and interactions with metabolites (Lemaire et al., 1990; Medinsky et al., 1996; Bratton et al., 1997). Antioxidant activities were much more sensible to alkylphenols and nitrophenols than to OH-phenols or phenol. Total SOD activity was increased following injections of alkylphenols (o-cresol and o-ethylphenol and o-propylphenol), and nitrophenols (o-, m- and p-nitrophenol; personal observations). Specific single dose effects were observed notably on Hb with phenol and OH-phenols (Fig. 3). Specific fractionated dose effects were also disclosed, notably on peroxidase activity and BUN (Figs. 2 and 4). Differences between single and fractionated doses affected particularly glucose and PCV with OH-phenols, Hb, BUN, peroxidase and SOD activities with all compounds (Figs. 2 – 4). These observations indicate that biotrans-
351
formation reactions could be efficient for these compounds especially in fractionated doses. These reactions are well known for OH-phenols (Sawahata and Neal, 1983; Wallin and Morgenstern, 1990; Cravedi et al., 1999). They lead to intermediate products which could be more reactive that the initial compound as in the case of hydroquinone (Bratton et al., 1997; Peters et al., 1997). Phenolic compounds are generally concentrated through the food chain due to their accumulation in lipids (Mukherjee et al., 1990). Their lower partition coefficients indicate that OH-phenols could be less accumulated than phenol and are more bioavailable (Table 1). We established a structure activity relationship with glucose for phenol and OH-phenols and with ALP and Hb for OH-phenols with fractionated doses (Fig. 5). Consequently, the lipophilic character of phenol and OH-phenols (Ancerewicz et al., 1998) could influence these parameters. For other parameters, in particular BUN, which was highly depressed with fractionated doses of all compounds, the toxicity could be more dependent upon other chemical properties and perhaps with a possible relationship with catecholamines in the case of OH-phenols. In conclusion, this work confirmed that the toxicity of phenols depends greatly on their structure. Their major effect is on some plasma parameters, but interactions with metabolism of free radicals could contribute to their toxicity.
Acknowledgements This study was supported by the French Ministry of Environment, grant no. 90 131 and 91 192. The experiments were performed in Michel Pacha Institute, maritime laboratory of Physiology of University of Lyon, France.
References Adams, S.M., Shepard, K.L., Greeley, M.S., Ryon, M.G., Jimenez, B.D., Shugart, L.R., McCarthy, J.F., Hinton, D.E., 1989. The use of bioindicators for assessing the effects of pollutant stress in fish. Mar. Environ. Res. 28, 459 – 464. Ancerewicz, J., Migliavacca, E., Carrupt, P.A., Testa, B., Bree, F., Zini, R., Tillement, J.P., Labidalle, S.,
352
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
Guyot, D., Chauvet-Monges, A.M., Crevat, A., Le Ridant, A., 1998. Structure-property relationships of trimetazidine derivatives and model compounds as potential antioxidants. Free Radic. Biol. Med. 25, 113–120. Bessey, O.A., Lowry, O.H., Brock, M.J., 1946. A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. Biol. Chem. 164, 321–329. Boge´, G., Roche, H., 1996. Cytotoxicity of phenolic compounds in Dicentrarchus labrax erythrocytes. Bull. Environ. Toxicol. Contamin. 57, 171–178. Bradbury, S.P., Henry, T.R., Niemi, G.J., Carlson, R.W., Snarski, V.M., 1989. Use of respiratory-cardiovascular response of rainbow trout (Salmo gairdneri ) in identifying acute toxicity syndromes in fish. Part 3. Polar narcosis. Environ. Toxicol. Chem. 8, 247–262. Bratton, S.B., Lau, S.S., Monks, T.J., 1997. Identification of quinol thioethers in bone marrow of hydroquinone/phenol-treated rats and mice and their potential role in benzene-mediated hematotoxicity. Chem. Res. Toxicol. 10, 859–865. Briviba, K., Devasagayam, T.P.A., Sies, H., Steenken, S., 1993. Selective para hydroxylation of phenol and aniline by singlet molecular oxygen. Chem. Res. Toxicol. 6, 548–553. Cajina-Quezada, M., Schultz, W., 1990. Structure-toxicity relationships for selected weak acid respiratory uncouplers. Aquat. Toxicol. 17, 239–252. Cravedi, J.P., Lafuente, A., Baradat, M., Hillenweck, A., Perdu-Durand, E., 1999. Biotransformation of pentachlorophenol, aniline and biphenyl in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes: comparison with in vivo metabolism. Xenobiotica 29, 499–509. Foster, L.B., Dunn, R.T., 1974. Single antibody technique for radioimmuno-assay of cortisol unextracted serum of plasma. Clin. Chem. 20, 365–368. Gluth, G., Hanke, W., 1976. The effect of temperature on physiological changes in carp, Cyprinus carpio L., induced by phenol. Ecotoxicol. Environ. Saf. 7, 373–389. Gupta, S., Dalela, R.C., Saxena, P.K., 1983. Effect of phenolic compounds on in vivo activity of transaminases in certain tissues of the fish, Notopterus notopterus. Environ. Res. 32, 8–13. Gut, I., Nedelcheva, V., Soucek, P., Stopka, P., Tichavska, B., 1996. Cytochromes P450 in benzene metabolism and involvement of their metabolites and reactive oxygen species in toxicity. Environ. Health Perspect. 104, 1211–1218. Hachet, J.C., 1989. Toxicologie d’Urgence. Produits Chimiques Industriels, Collection Abre´ge´s de Me´decine. Masson, Paris.
Hiraku, Y., Kawanishi, S., 1996. Oxidative DNA damage and apoptosis induced by benzene metabolites. Cancer Res. 56, 5172 – 5178. Hrubec, T.C., Robertson, J.L., Smith, S.A., 1997a. Effects of temperature on hematologic and serum biochemical profiles of hybrid striped bass (Morone chrysops×Morone saxatilis). Am. J. Vet. Res. 58, 126 – 130. Hrubec, T.C., Robertson, J.L., Smith, S.A., 1997b. Effects of ammonia and nitrate concentration on hematologic and serum biochemical profiles of hybrid striped bass (Morone chrysops × Morone saxatilis). Am. J. Vet. Res. 58, 131 – 135. Hugget, A.S.G., Nixon, D.A., 1957. Use of glucose oxidase, peroxidase and o-dianisidine in determination of blood and urinary glucose. Lancet 2, 368 – 379. Jagetia, G.C., Aruna, R., 1997. Hydroquinone increases the frequency of micronuclei in a dose-dependent manner in mouse bone marrow. Toxicol. Lett. 93, 205 – 213. Kaila, K., 1982. Cellular neurophysiological effects of phenol derivatives. Comp. Biochem. Physiol. [C] 73C, 231 – 241. Lemaire, P., Mathieu, A., Carrie`re, S., Drai, P., Giudicelli, J., Lafaurie, M., 1990. The uptake metabolism and biological half-life of benzo(a)pyrene in different tissues of sea bass Dicentrarchus labrax. Ecotox. Environ. 20, 223 – 233. Leo, A., Hansch, C., Elkins, D., 1971. Partition coefficients and their uses. Chem. Rev. 71, 525 – 616. Medinsky, M.A., Kenyon, E.M., Seaton, M.J., Schlosser, P.M., 1996. Mechanistic considerations in benzene physiological model development. Environ. Health Perspect. 104, 1399 – 1404. Mukherjee, D., Bhattacharya, S., Kumar, V., Moitra, J., 1990. Biological significance of [14C]phenol accumulation in different organs of a murrel, Channa punctatus, and the common carp, Cyprinus carpio. Biomed. Environ. Sci. 3, 337 – 342. Panduranga-Rao, D., Ram-Bhaskar, B., Srinivasa-Rao, K., Durga-Prasad, Y.V.K., Someswara-Rao, N., Venkateswara-Rao, T.N.V., 1990. Haematological effects in fishes from complex polluted waters of Visakhapatnam harbour. Mar. Environ. Res. 30, 217 – 231. Peters, M.M., Jones, T.W., Monks, T.J., Lau, S.S., 1997. Cytotoxicity and cell-proliferation induced by the nephrocarcinogen hydroquinone and its nephrotoxic metabolite 2,3,5-(tris-glutathion-S-yl)hydroquinone. Carcinogenesis 18, 2393 – 2401. Pradhan, D., Weiser, M., Lumley-Sapanski, K., Frazier, D., Kemper, S., Williamson, P., Schlegel, R.A., 1990. Peroxidation-induced perturbations of erythrocyte lipid organization. Biochim. Biophys. Acta 1023, 398 – 404.
H. Roche, G. Boge´ / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 345–353
Roche, H., Boge´, G., 1993. In vitro effects of Cu, Zn and Cr salts on antioxidant enzyme activities of red blood cells of a marine fish Dicentrarchus labrax. Toxicol. In Vitro 7, 623–629. Roche, H., Boge´, G., 1996. Fish blood parameters as a potential tool for identification of stress caused by environmental factors or chemical intoxication. Mar. Environ. Res. 41, 27–44. Sawahata, T., Neal, R.A., 1983. Biotransformation of phenol to hydroquinone and catechol by rat liver microsomes. Mol. Pharmacol. 23, 453–460. Schultz, T.W., 1987. Relative toxicity of para-substituted phenols: Log Kow and pKa-dependent structure-activity relationships. Bull. Environ. Contam. Toxicol. 38, 994–999. Stich, H.F., 1991. The beneficial and hazardous effects of simple phenolic compounds. Mut. Res. 259, 307– 324. Taysse, L., Troutaud, D., Khan, N.A., Deschaux, P., 1995. Structure-activity relationship of phenolic compounds (phenol, pyrocatechol and hydroquinone) on natural lymphocytotoxicity of carp (Cyprinus carpio). Toxicology 98, 207–214. Tsutsui, T., Hayashi, N., Maizumi, H., Huff, J., Barrett, J.C., 1997. Benzene-, catechol-, hydroquinoneand phenol-induced cell transformation, gene mutations, chromosome aberrations, aneuploidy, sister chromatid exchanges and unscheduled DNA synthe-
.
353
sis in Syrian hamster embryo cells. Mutat. Res. 373, 113 – 123. Wallin, H., Morgenstern, R., 1990. Activation of microsomal glutathione transferase activity by reactive intermediates formed during the metabolism of phenol. Chem. Biol. Interact. 75, 185 – 199. Wilhelm-Filho, D., 1996. Fish antioxidant defenses — a comparative approach. Braz. J. Med. Biol. Res. 29, 1735 – 1742. Winston, G.W., 1991. Mini-review. Oxidants and antioxidants in aquatic animals. Comp. Biochem. Physiol. [C] 100C, 173 – 176. Winston, G.W., Di Giulio, T., 1991. Prooxidant and antioxidant mechanisms in aquatic organisms. Aquat. Toxicol. 19, 137 – 161. Witz, G., Zhang, Z., Goldstein, B.D., 1996. Reactive ring-opened aldehyde metabolites in benzene hematotoxicity. Environ. Health Perspect. 104, 1195 – 1199. Xu, L., Ball, J.W., Dixon, S.L., Jurs, P.C., 1994. Quantitative structure-activity relationships for toxicity of phenols using regression analysis and computational neural networks. Environ. Toxicol. Chem. 13, 841 – 851. Yu, T.W., Anderson, D., 1997. Reactive oxygen species-induced DNA damage and its modification: a chemical investigation. Mutat. Res. 379, 201 – 210.