Peroxisome Proliferation in the Digestive Epithelium of Mussels Exposed to the Water Accommodated Fraction of Three Oils

Comp. Biochem. Physiol. Vol. 117C, No. 3, pp. 233–242, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0742-8413/97/$17.00 PII S0742-8413(97)00057-1

Peroxisome Proliferation in the Digestive Epithelium of Mussels Exposed to the Water Accommodated Fraction of Three Oils M. P. Cajaraville, A. Orbea, I. Marigo´mez, and I. Cancio Zoologia eta Animali Zelulen Dinamika, Zientzi Fakultatea, Euskal Herriko Unibertsitatea/Universidad del Paı´s Vasco, 644 P.K., 48080 Bilbo, Basque Country, Spain ABSTRACT. Recent studies show that the peroxisomal marker enzyme catalase is induced in the digestive epithelium of mussels exposed to petroleum-derived hydrocarbons. The aim of the present study was to investigate whether this effect is accompanied by a proliferative response of the peroxisomes. Mussels were treated for 21, 49 and 91 days with three different doses of the water accommodated fraction (WAF) of two different crude oils (Ural and Maya types) and of one commercial lubricant oil. The volume density, surface density, surfaceto-volume ratio and numerical density of peroxisomes were quantified by stereology on cryostat sections of digestive glands stained for the histochemical demonstration of catalase activity. The results show that after 21 days, the three WAFs act as typical peroxisome proliferators, inducing a significant increase in the volume, surface and numerical densities of peroxisomes in the digestive epithelium. The response is diminished or completely abolished after 49 and 91 days of exposure to the WAFs. The ability to induce the proliferative response differs depending on the WAF type, the lubricant oil showing the maximal induction ability. These results suggest that peroxisome proliferation could be used as a diagnostic biomarker for both exposure and effects of petroleum hydrocarbons on marine biota. comp biochem physiol 117C;3:233–242, 1997.  1997 Elsevier Science Inc. KEY WORDS. Peroxisome proliferation, petroleum hydrocarbons, biomarkers, catalase, histochemistry, stereology, bivalve molluscs, Mytilus galloprovincialis

INTRODUCTION Peroxisomes are ubiquitous single membrane-bound organelles performing several key roles in the cell, particularly in relation with lipid metabolism and handling of reactive oxygen species. A variety of environmentally relevant xenobiotics, including hypolipidemic drugs, phthalate ester plasticizers, agrochemicals and polynuclear aromatic hydrocarbons, are known to induce peroxisome proliferation in the liver of sensitive species such as rats, mice and hamsters [for reviews, see (2,21,39)]. Peroxisome proliferation is accompanied by a heterogeneous induction of peroxisomal enzymes. Most sensitive are the β-oxidation enzymes that could increase their activity 20- to 30-fold whereas catalase activity only shows a 2- to 4-fold increase (19,28,34,37,39). Because peroxisome proliferators have been shown to induce hepatocarcinomas in rodents under chronic exposure but are non-mutagenic and non-genotoxic, the imbalance between oxyradical producing peroxisomal oxidases and Address reprint requests to: M. P. Cajaraville, Zoologia eta Animali Zelulen Dinamika, Zientzi Fakultatea, Euskal Herriko Unibertsitatea/Universidad del Pais Vasco, 644 P.K., 48080 Bilbo, Basque Country, Spain. Fax 34-44648500, Tel. 34-4-4647700, ext 3037; [email protected]. Abbreviations–PHC, petroleum-derived hydrocarbons; WAF: water accommodated fraction. Received 16 August 1996; accepted 19 December, 1996.

H2 O2-degrading catalase has been hypothesized to cause oxidative damage to DNA and initiate the neoplastic transformation of liver cells (38,40). The simultaneous decrease of the other major antioxidant enzyme superoxide dismutase with hypolipidemic drugs may further accentuate this effect (17). There are few studies concerning the effects of environmental xenobiotics on peroxisomes in non-mammalian organisms, and these have been recently reviewed by Fahimi and Cajaraville (20). Among the aquatic organisms, fish have received most attention. Intraperitoneal injection of hypolipidemic drugs (ciprofibrate and gemfibrozil) has been reported to induce hepatic peroxisome proliferation in rainbow trout as measured by increased levels of acyl-CoA oxidase, peroxisomal trifunctional enzyme, catalase and polypeptide PPA-80 and increased liver-to-body weight ratios (43,48). Morphometric analysis on livers of trout administered 35 mg/kg ciprofibrate for 3 weeks also shows a 2.3 fold increase in peroxisomal volume density but no significant difference in peroxisomal numerical density (48). Similar effects have been demonstrated in the Japanese medaka when exposed to gemfibrozil through the water for 2 weeks, with increases in peroxisomal trifunctional enzyme and acyl-CoA oxidase (43). Nevertheless, the peroxisome proliferation induced by hypolipidemic drugs in fish seems to

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be quite modest compared with that observed in rodents, more closely resembling the response of higher primates than that of rodents (43,48). After a 4-week exposure of the European eel Anguilla anguilla to different concentrations of the pesticide dinitroo-cresol, a stimulation of peroxisomal enzymes (catalase, allantoinase and urate oxidase) and a higher number of peroxisomes are observed in hepatocytes (4). On the other hand, in rainbow trout Oncorynchus mykiss, exposure to 0.2 and 1 mg/l of 4-chloroaniline for 4 weeks leads to a reduction in the number of hepatic peroxisomes and to a reduced catalase activity, respectively, whereas in vitro exposure of hepatocytes to the same toxicant at doses of 3–10 mg/l for 5 days provokes an increase in the number of peroxisomes (3). When rainbow trout is exposed simultaneously to 50 ng/l endosulfan (ES) plus 1 µg/l disulfoton (DS), there are significant increases in the total number of peroxisomes and mean peroxisomal diameter, which results in higher total and relative volumes of peroxisomes (1). In the same study, no changes in catalase activity were observed, whereas the specific activity of urate oxidase increased significantly after treatment with 50 ng/l ES plus 5 or 10 µg/l DS. On the other hand, Mather-Mihaich and Di Giulio (31) have recently studied the peroxisomal responses induced by bleached kraft pulp and paper mill effluent (BKME) in channel catfish Ictalarus punctatus. They have found significant dose-dependent increases in hepatic catalase, lauroyl-CoA oxidase and palmitoyl-CoA oxidase activities, maximal increases being 2- to 3-fold, 3-fold and 7-fold, respectively. From these results indicating peroxisomal enzyme induction, Mather-Mihaich and Di Giulio (31) suggest that exposure to BKME results in peroxisome proliferation in catfish hepatic tissue, although the BKME components responsible for this effect are unknown. In support of this idea, Bucher et al. (6) have reported a qualitative increase in the number of peroxisomes in hepatocytes of bullhead Cottus gobio from a site downstream of two BKME discharging paper mills, although catalase activity was unaltered (5). There are some reports suggesting that certain xenobiotics may also cause peroxisomal proliferation in molluscs. Thus, short-term treatment with the diaromatic hydrocarbon naphthalene causes an increase in peroxisome-like organelles in gastropod kidney cells (9). Numbers of peroxisomes are also increased in mussel duct epithelial cells after 91 days of exposure to the water accommodated fraction (WAF) of two crude oils (7,20). In addition, some studies have indicated that the activity of the peroxisomal marker enzyme catalase is increased transiently upon treatment of bivalve molluscs with the redox cycling quinones paraquat and menadione and with the polynuclear aromatic hydrocarbon benzo-a-pyrene (30,45,46). Moreover, catalase activity has been found to be increased in the peroxisomes of the digestive epithelium of mussels treated with the WAF of three different petroleum-derived hydrocarbons (PHC)

(14). The purpose of the present study is to investigate whether this PHC-induced increase in catalase activity is associated with a proliferative response of peroxisomes in the digestive epithelial cells of mussels. A portion of this work has been published previously in abstract form (35). MATERIALS AND METHODS Experimental Procedure Details of the experimental design have been described previously (11). Briefly, mussels Mytilus galloprovincialis were exposed, after a 10-day acclimatization period, to the WAF of two crude oils (Maya and Ural types) and of a commercial lubricant oil for 3 months in a thermostatized semicontinuous water flow system. Individuals (2.5–3.5 cm shell length) were exposed in two replicate series to three different doses of each oil (0.6%, low dose, 6%, intermediate dose and 40%, high dose). One hundred percent of the water was replaced every 2 days, and the different WAFs were dosed every day. Two replicate control sets were also carried out. Histochemistry Mussels were sampled following 21, 49 and 91 days of exposure. Five animals were removed from each replicate experimental group at each sampling period. Small pieces of freshly excised mussel digestive gland were frozen using Bright Cryo-Spray (dichlorodifluoromethane, 250/ 255°C) and embedded in Bright Cryo-M-Bed (UK). Sections (9 µm) were cut in a Bright’s cryostat (5030 microtome) at a cabinet temperature of 230°C. Sections were then collected onto glass slides brought from room temperature and stored at 270°C until required for staining. The histochemical detection of catalase activity was carried out in cryostat sections as described before (15). Sections were fixed in 4% formaldehyde in 0.1 M phosphate buffer, pH 7.4, with 1% calcium chloride at 4°C for 5 hr. After fixation, sections were rinsed in the same buffer and incubated in a freshly prepared medium containing 0.2% 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical Co. [St. Louis, MO, USA], no. D-5637), 0.3% H2 O2 and 0.01 M imidazole (Sigma, no. I-0125) in 0.05 M Tris–HCl buffer at pH 10.4. Incubation was performed in the dark at 42°C for 35 min with solutions and recipients preheated for 35 or 60 min. Sections were then rinsed in distilled water, dehydrated and mounted in Eukitt. Stereology The determination of the stereological parameters was carried out according to Weibel (44). A lattice with 168 test points (multipurpose test system P 168) was superimposed onto the preparations with the aid of a drawing tube attached to a Leitz Laborlux light microscope and the fraction of test points enclosed within peroxisomes was deter-

Peroxisome Proliferation in PHC-exposed Mussels

mined. The digestive epithelium was considered as the reference space, and test points falling on the connective or reproductive tissue were substracted from the total number of test points. Counts were made on five fields selected randomly on each section, one section being analyzed per animal (8–10 animals per experimental group). This minimum number of microscopical fields to be measured was established in preliminary studies in which the mean and SD values of the stereological parameters kept constant (data not shown). Additionally, the diameters of 90 peroxisomes were measured in each section using a measuring paper superimposed on the preparation using the camera lucida. Because of the small size of the organelles, a precise determination of their diameter was not possible, and approximate values of either 0.5, 1 or 1.5 mm were assigned to each peroxisome. One millimeter in the measuring paper was equivalent to 0.73 µm in the tissue section. The magnification used both for point counting and measurement of diameters was 1003. With all data obtained, the following stereological parameters were calculated: peroxisomal volume density (VD 5 Vp /V C ), peroxisomal surface density (SD 5 Sp/VC ), peroxisomal surface to volume ratio (SV 5 Sp /V p), and peroxisomal numerical density (ND 5 Np/VC ), where V 5 volume, S 5 surface, N 5 number, P 5 peroxisomes, C 5 digestive cell cytoplasm and D 5 density. Statistics Statistical analysis was carried out with the aid of the SPSS/ PC1 statistical package (SPSS Inc., Microsoft Co.) in a 486 PC computer. Data about peroxisomal VD and ND were logarithmically transformed previous to the statistical analyses because the variance within individuals depended on the mean. Three-way ANOVA were performed to detect the effects of the type of pollutant (W), the dose of WAF (D), and the exposure time (T). For each type of pollutant two-way ANOVAs were carried out to study the effect of D, T and their interaction (T3D). The test of Duncan for multiple comparison between paired means was further applied to detect significant (P , 0.05) differences between means. RESULTS Effects of Stabulation The one-way ANOVA performed to study the variability in the parameters VD, SD, SV and ND of peroxisomes with respect to the time of stabulation indicated that this factor did not present any significant effect on the parameters VD, SD and ND (Table 1). However, the values of these parameters tended to decrease slightly between days 21 and 49 and reached their highest values by day 91. On the other hand, the ANOVA indicated that SV changed significantly with stabulation time, mean values of SV decreasing significantly as estabulation was lengthened (Fig. 1).

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TABLE 1. Summary of one-way analysis of

variance (ANOVA) showing the effects of stabulation on the structure of digestive cell peroxisomes. VD: volume density; SD: surface density; SV: surface to volume ratio; and ND: numerical density; F: Fisher’s F ratio; df: degrees of freedom; P : probability of F. Statistically significant if P , 0.05. Data on peroxisomal VD and ND were logarithmically transformed before the ANOVA since variance depended on mean values F

P

2.2289 2.4326 5.1234 1.7890

0.1365 0.1161 0.0173 0.1956

Parameters VD SD SV ND

df (between) 5 2; df (residual) 5 18.

Effects of the Exposure to the WAFs The three-way ANOVA used to detect the effects of the time of exposure, dose of exposure and type of toxicant on the four stereological parameters indicated that the three factors produced significant changes on VD, SD and ND. SV was affected by exposure time and dose but not by the type of toxicant (Table 2). The two-way ANOVA for Ural WAF exposure indicated that both time and dose caused significant effects on VD, SD and ND (Table 3). For SV the exposure time exerted significant effects but not the exposure dose. The interaction between time and dose caused significant effects on all the parameters studied except on ND. As shown in the graphs (Fig. 2), the animals exposed for 21 days to the WAF of Ural showed higher values for peroxisomal VD, SD and ND than the control animals, but these changes were not dose dependent. Thus, the test of Duncan showed that animals treated with the low and high doses presented significantly higher values than those in the control group and in the group exposed to the intermediate dose (Fig. 2, A, B and D). Animals treated with the three different doses of Ural WAF for 21 days showed significantly lower values of SV than controls. After 49 days of exposure, no statistically significant changes were observed between control and treated animals except in the SV ratio, which was lower in Ural-treated organisms. Finally, after 91 days of exposure, significant differences were detected in VD, SD and ND, with animals exposed to the highest dose of Ural WAF showing higher values of volume, surface and number of peroxisomes than controls and the other two treated groups (Fig. 2). In the case of the exposure to Maya WAF, the two-way ANOVA showed that changes in the parameters SD and ND were significantly explained by changes in the exposure time (Table 3). On the other hand, the SV ratio was significantly affected by time and dose of exposure and by the interaction between both factors. VD varied significantly according to changes in the time of exposure and the inter-

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FIG. 1. Results of the stereological analysis of digestive cell peroxisomes in control mussels along the experimental period. (A) Volume density; (B) surface density; (C) surface to volume ratio and (D) numerical density. Animals were killed after 21, 49 and 91 days. Vertical segments show standard errors. Significant differences between pairs of mean values are indicated in the upper triangular matrix by an asterisk (P , 0.05). Statistical signification is based on the multiple range test of Duncan.

action (Table 3). The test of Duncan showed that the volume, surface and number of peroxisomes was significantly increased in animals treated with the highest dose of Maya WAF with respect to controls only at day 21 of exposure (Fig. 3). At day 49, no significant differences were detected, and at day 91, a significant reduction in the volume and size of peroxisomes was observed in animals treated with the highest dose of Maya WAF with respect to controls.

The two-way ANOVA for the lubricant oil WAF showed that both the time and dose of exposure had significant effects on VD, SD and ND but not on the SV ratio (Table 3). On the other hand, the interaction between the time and dose of exposure was very significant for all the parameters studied. According to the test of Duncan, mussels exposed to the three doses of the lubricant WAF showed a marked increase in VD, SD and ND after 21 days of expo-

TABLE 2. Summary of three-way ANOVA showing the effects of exposure to three different WAFs on the structure of digestive cell peroxisomes. VD: volume density; SD: surface density; SV: surface to volume ratio; and ND: numerical density; F: Fisher’s F ratio; df: degrees of freedom; P : probability of F; T: exposure time; D: exposure dose; and W: type of toxicant. Statistically significant if P , 0.05. Data on peroxisomal VD and ND were logarithmically transformed before the ANOVA since variance depended on mean values

T Parameters VD SD SV ND

D

W

F

P

F

P

F

P

26.296 41.319 19.240 51.874

,0.0001 ,0.0001 ,0.0001 ,0.0001

7.023 11.051 3.899 7.290

,0.0001 ,0.0001 0.009 ,0.0001

6.043 18.776 1.539 5.634

0.003 ,0.0001 0.217 0.004

df (T) 5 2; df (D) 5 3; df (W) 5 2; df (residual) 5 260.

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TABLE 3. Summary of two-way ANOVAs showing the effects of exposure time (T), exposure dose (D) and their interaction

(T 3 D) on the structure of digestive cell peroxisomes for each WAF treatment. VD: volume density; SD: surface density; SV: surface to volume ratio; and ND: numerical density; F: Fisher’s F ratio; df: degrees of freedom; P : probability of F. Statistically significant if P , 0.05. Data on peroxisomal VD and ND were logarithmically transformed before the ANOVA since variance depended on mean values T

T3D

D

Parameters

F

P

F

P

F

P

Ural WAF VD SD SV ND

9.054 18.107 8.028 15.469

,0.0001 ,0.0001 0.001 ,0.0001

12.203 15.885 2.133 12.503

,0.0001 ,0.0001 0.102 ,0.0001

4.024 4.856 4.457 2.011

0.001 ,0.0001 0.001 0.073

Maya WAF VD SD SV ND

3.420 4.452 14.646 12.008

0.037 0.014 ,0.0001 ,0.0001

1.523 1.036 3.192 0.668

0.214 0.381 0.028 0.574

4.277 2.130 4.519 2.115

0.001 0.058 0.001 0.060

Lubricant WAF VD SD SV ND

34.464 61.052 2.620 46.868

,0.0001 ,0.0001 0.081 ,0.0001

7.274 15.212 1.864 5.752

,0.0001 ,0.0001 0.145 0.002

7.725 10.242 2.684 5.145

,0.0001 ,0.0001 0.010 0.001

df (T) 5 2; df (D) 5 3; df (T 3 D) 5 6; df (residual)ural 5 85; df (residual)maya 5 86; df (residual)lubricant 5 63.

sure, with 4- to 5-fold increases over control values (Fig. 4). These changes were not dose dependent. In the same way, the three groups presented lower values for SV than controls. This would indicate that peroxisomes of digestive cells in animals treated with the three doses of the lubricant WAF were larger, more numerous and with a higher volume and surface than those present in controls, as illustrated also in Fig. 5. This response was not shown at days 49 and 91 of exposure, no significant differences being detected between control and treated animals at both sampling times (Fig. 4). Data from mussels treated with the highest dose of the lubricant WAF are not shown at days 49 and 91 because animals subjected to this treatment died by day 49 (11). DISCUSSION The results shown in the present work demonstrate that exposure of mussels for 21 days to the WAFs of two crude oils and one lubricant oil causes an increase in the volume, surface and numerical densities of peroxisomes in digestive epithelial cells. This response is variable depending on the type of WAF, the highest increase being found after exposure to the WAF of the lubricant oil. The exposure to the WAFs also causes an increase in the size of peroxisomes (decreased SV values), but it is much less pronounced than that found in the other parameters. This indicates that changes in peroxisome volume and surface densities are due mainly to changes in peroxisomal numbers. Therefore, we conclude that the response observed after exposure to the WAFs can be considered a true peroxisome proliferation, comparable with that described in rodents treated with per-

oxisome proliferators (39). Although a peroxisome proliferating effect of petroleum hydrocarbons has been suggested previously (9,14), this is the first study using quantitative tools to evaluate peroxisome proliferation in molluscs and showing that this effect is linked to the exposure to petroleum hydrocarbons. In accordance with our results, the field study carried out recently by Krishnakumar et al. (26) reports significant positive relationships between concentrations of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in mussels and the percentage of mussels showing catalase activity in more than 50% of the digestive tubules. One difference with the typical peroxisome proliferation response found in other organisms is that the response provoked by the three WAFs in mussels is not dose dependent. Thus, in the cases of Ural and lubricant WAFs, the values of peroxisomal volume, surface and numerical densities are similar in animals exposed to the low and high doses of WAF, whereas mussels exposed to the intermediate doses show generally lower values for the three parameters. Similar trends have been observed when studying the effects of the same WAFs on mussel catalase activity (14) and on measurements of other biological effects (11,13). Possible reasons to explain this disagreement between the dose of WAF and its biological effects have been discussed previously (14), but additionally it is tempting to speculate on the possibility that the peroxisome proliferation response is mediated by a saturable receptor(s) in mussels. A member of the nuclear hormone receptor superfamily, termed peroxisome proliferator activated receptor (PPAR), has been found to be involved in the induction of peroxisome prolif-

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FIG. 2. Results of the stereological analysis of digestive cell peroxisomes in mussels exposed to Ural WAF. (A) Volume density; (B) surface density; (C) surface to volume ratio and (D) numerical density. Animals were killed after 21, 49 and 91 days of exposure to three different concentrations of WAF (C, control; L, low dose; I, intermediate dose; H, high dose). Vertical segments show standard errors. Significant differences between pairs of mean values calculated for each WAF are indicated in the upper triangular matrix by an asterisk (P , 0.05). Statistical signification is based on the multiple range test of Duncan.

eration in mammalian systems (23). The PPAR forms heterodimers with other receptors of the same superfamily and binds to short DNA motifs on 5′-flanking promoters of various genes, including those of the peroxisomal β-oxidation enzymes then activating their transcription (22,39). We are unaware of any studies reporting the presence of PPAR in molluscs but these proteins have been already found in nonmammalian vertebrates such as Xenopus laevis (18). On the other hand, the peroxisome proliferation response observed in mussels after 21 days of exposure is not apparent after 49 and 91 days, except in mussels treated with the highest dose of Ural WAF after 91 days of exposure. Similarly, catalase activity is only transiently enhanced in digestive epithelium cells of mussels exposed to the low dose of the WAFs (14). Thus, it seems that the elevation of catalase activity and the peroxisome proliferation caused by WAFs is attenuated with exposure time in mussels but both changes are still evident after 3 months exposure in the case of mussels exposed to the high dose of Ural WAF. The increases in catalase and other antioxidant enzyme activities have also been reported to be transient in bivalves exposed to paraquat (46) or benzo(a)pyrene (30).

The WAF of the lubricant oil shows the highest capacity to induce peroxisomal proliferation (4- to 5-fold increase in VD), followed by the WAF of Ural (2- to 3-fold increase in VD) and finally the WAF of Maya. Interestingly, the different ability of the three WAFs to induce peroxisome proliferation agrees well with their different toxicities, measured in terms of effects on mortality and growth of mussels (11), changes in digestive and basophilic cell ratios (10) and reduction in digestive epithelial thickness (13). Indeed, the toxicity of hydrocarbons could be mechanistically linked to their ability to induce peroxisomal proliferation because peroxisome proliferation is known to be accompanied by a 20- to 30-fold increase in H2 O2 generating β-oxidation enzymes and only a 2-fold increase in H2 O2-scavenging catalase activity (19,21,28,34,39). Thus, the increases in antioxidant defenses could not be enough to cope with the increased generation of reactive oxygen radicals (ROIs) in cells showing peroxisome proliferation (38,40). There is increasing evidence linking these ROIs with cell damage in several pathological conditions (25) and also with xenobiotic-induced cell damage (14,30,46,47). Although WAF exposure appears to cause no marked ef-

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239

FIG. 3. Results of the stereo-

logical analysis of digestive cell peroxisomes in mussels exposed to Maya WAF. (A) Volume density; (B) surface density; (C) surface to volume ratio; and (D) numerical density. Symbols and statistical signification indicated as in Fig. 2.

fect on the size of peroxisomes, keeping mussels in laboratory conditions for 3 months induces a slight enlargement of the peroxisomes in the digestive epithelium. This change is not accompanied by an increase in the volume, surface or numerical densities of the peroxisomes. Nevertheless, we point out that the method used in the present work to measure the size of peroxisomes is not fully accurate. Because of the small size of peroxisomes in mussel digestive epithelial cells (16), a precise determination of their diameter is not possible in cryostat sections using manual stereological methods or automatic image analysis and, thus, only approximate size values can be assigned to each peroxisome. Although our stereological approach is adequate to measure the volume density of peroxisomes in mussel digestive tissue as an indication of peroxisome proliferation, semithin or ultrathin sections appear more appropriate to obtain accurate measures of peroxisomal size. On the other hand, the arbitrary approach used in the study by Krishnakumar et al. (26), in which ‘‘peroxisome proliferation was considered evident if .50% of the digestive tubules in five fields showed evidence of catalase activity’’ does not actually assess peroxisome proliferation but a conjunction of various possible ef-

fects such as changes in peroxisome size and/or numbers, alterations in catalase activity and changes in lipofuscin contents. With respect to lipofuscins, it is important to underline that these oxidized lipoprotein materials are unspecifically stained with DAB (15), and therefore, an overall low power visual inspection of DAB-stained cryostat sections could give rise to misconceptions regarding peroxisomal abundance. Recently, the use of biological markers measured at the molecular, biochemical or cellular level have been proposed as important early warning tools for the assessment of environmental quality (12,32,33). As defined by McCarthy and Shugart (32), biomarkers indicate that the organism has been exposed to pollutants (exposure biomarkers) and the magnitude of the organism’s response to the pollutant (effect biomarkers). The latter include lysosomal alterations (8,33), formation of DNA-xenobiotic adducts (29), induction of antioxidant enzymes (14,26,41) and induction of stress or heat-shock proteins (42) in a variety of organisms. However, these biomarkers are non-specific and respond to a variety of stressors, including organic and metallic pollutants and changes in several natural environmental vari-

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FIG. 4. Results of the stereological analysis of digestive cell peroxisomes in mussels exposed to lubricant oil WAF. (A) Volume density; (B) surface density; (C) surface to volume ratio; and (D) numerical density. Symbols and statistical signification indicated as in Fig. 2.

FIG. 5. Cryostat sections through digestive cells stained for catalase activity. (A) Control mussel sampled at day 21; (B) mussel exposed to the high dose of lubricant oil WAF for 21 days. Arrows point to peroxisomes reactive for catalase activity. Scale bars 5 10 mm.

Peroxisome Proliferation in PHC-exposed Mussels

ables. There are few ‘‘specific’’ biomarkers such as induction of metallothioneins by metals and induction of cytochrome P450 or mixed function oxidase enzymes by organic pollutants (29,36), and thus, there is a need to search for specific markers that could be used as diagnostic of a particular class of pollutants. It is in this context that peroxisome proliferation acquires importance as a potential biomarker because, at least in rodents, a characteristic feature of many peroxisome proliferators is the presence of an acidic function either in the parent molecule or in a metabolite (27). This acidic function is normally a carboxyl group, but tetrazole or sulphonamides can substitute. Several environmentally relevant xenobiotics or their metabolites appear to contain an acidic function, as, for example, some plasticizers (phthalate esters), herbicides (chlorophenoxy acetic acids), insecticides (dimethrin) and lubricants (perfluorocarboxylic acids), and they are all known to induce peroxisome proliferation in the liver of some experimental animals (2). In the case of the WAF of petroleum hydrocarbons, either some of their components could contain carboxyl groups or they could be metabolized through microsomal cytochrome P450 to molecules containing carboxyl groups, but this is largely unknown to our knowledge. In mammals, the peroxisome proliferator effect of petroleum hydrocarbons has been established by Khan et al. (24), who found that peroxisomal lipid β-oxidation is induced 2- to 5-fold in the liver of rats fed with crude oil. As suggested previously, there are certain similarities between the action of PHCs on mussel digestive gland and the effects of peroxisome proliferators on rodents, both causing a marked proliferation of peroxisomes, a moderate elevation of catalase activity and a transient suppression of the phase II enzyme gamma-glutamyl transpeptidase activity (14). The latter has also been emphasized recently by Krishnakumar et al. (26). It remains to be established whether PHCs induce the enzymes of the peroxisomal βoxidation pathway in mussels or other invertebrates as do typical peroxisome proliferators in fish and mammals (21,39,43,48). In conclusion, the results of the present work support that peroxisome proliferation could be a sensitive indicator of changes in environmental conditions and could be used as a specific biomarker of organic pollutants such as petroleum hydrocarbons. Further experiments are being carried out in the laboratory and in the field to test this hypothesis. This investigation has been supported by projects no. AMB93-0432 from CICYT, Ministry of Education and Science, and no. UPV 075.327-EB002/94 from the University of the Basque Country. Thanks are due to J. A. Uranga and I. Salutregi for helpful technical assistance.

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