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Biodegradation and enzymatic responses in the marine diatom Skeletonema costatum upon exposure to 2,4-dichlorophenol
Aquatic Toxicology 59 (2002) 191– 200
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Biodegradation and enzymatic responses in the marine diatom Skeletonema costatum upon exposure to 2,4-dichlorophenol Shao Yang, Rudolf S.S. Wu *, Richard Y.C. Kong Department of Biology and Chemistry, Centre for Coastal Pollution and Conser6ation, City Uni6ersity of Hong Kong, 83 Tat Chee A6enue, Kowloon, Hong Kong Special Administrati6e Region, Hong Kong, China Received 3 June 2001; received in revised form 30 September 2001; accepted 1 October 2001
Abstract The biodegradation and responses of selected detoxification and antioxidant enzymes in the marine diatom, Skeletonema costatum, upon exposure to sublethal concentrations of 2,4-dichlorophenol (2,4-DCP) were investigated. Results show that 2,4-DCP was readily metabolised, but bioaccumulation and adsorption were negligible. Glutathione S-transferase, ascorbate peroxidase and superoxide dismutase activities were increased markedly after exposure to 2,4-DCP for 96 h, while no appreciable change in peroxidase activity was observed. The addition of exogeneous glutathione to diatom culture enhanced the degradation of 2,4-DCP, and promoted diatom growth. The inhibition of glutathione synthesis enhanced the toxicity of 2,4-DCP. These results suggest that glutathione conjugation was one of the principal mechanisms involved in the degradation of 2,4-DCP in this diatom. © 2002 Elsevier Science B.V. All rights reserved. Keywords: 2,4-Dichlorophenol; Degradation; Diatom; Detoxification; Enzyme
1. Introduction Phytoplankton are abundant in the marine environment and play a pivotal role in both primary production and nutrient recycling. Therefore, any adverse effects of pollutants on phytoplankton may lead to serious ecological consequences. In addition, if xenobiotics are bioconcentrated by phytoplankton, this may potentially lead to food * Corresponding author. Tel.: + 852-2788-7401; fax: + 8522788-7406 E-mail address: [email protected] (R.S.S. Wu).
chain transfer or biomagnification (Riisgaard and Hansen, 1990). Investigating the degradation and biotransformation of xenobiotics in phytoplankton is hence important in assessing the environmental fate and risk of pollutants in marine ecosystems. While the mechanisms of biotransformation and biodegradation of xenobiotics have been studied extensively in bacteria (Kobayashi and Rittmann, 1982), higher plants (Komoßa et al., 1995) and animals ( James et al., 1977), there is a paucity of data on biodegradation and biotransformation of xenobiotics in algae. A number of studies have shown that certain freshwater
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algae (e.g. Chlorella sp., Scenedesmus obliquus, Selenastrum capricornutum and Ochromonas danica), are able to degrade a variety of xenobiotics, such as the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) (Butler et al., 1975), phenolic compounds (Ellis, 1977; Klekner and Kosaric, 1992; Semple and Cain, 1996; Tikoo et al., 1997) and polyaromatic hydrocarbons (PAHs) (Cerniglia et al., 1980; Warshawsky et al., 1988, 1995). Despite this, limited studies have been conducted with marine phytoplankton. Previous studies have shown that certain marine microalgae (including pennate diatoms such as Phaeodactylum tricornutum, Na6icula sp., Nitzschia sp. and Synedra sp.) are able to degrade phloroglucinol (Craigie et al., 1965) and napthalene (Cerniglia et al., 1982). The potential of marine phytoplankton to degrade xenobiotics however, remains unclear. In animals and higher plants, selected xenobiotics are degraded or biotransformed through phase I (cytochrome P-450) and phase II (conjugation) enzymes. The cytochrome P-450 system is believed to be involved in the biotransformation of herbicides in the unicellular green algae Chlorella sorokiniana and Chlorella fusca (Thies et al., 1996), but degradation of the PAH benzo(a)pyrene (B[a]P) in the freshwater green alga, S. capricornutum, was mediated through dioxygenase and the metabolites were then conjugated to sulfate and glucose (Warshawsky et al., 1988, 1990). In freshwater algae, glutathione S-transferase (GST) was suggested to play an important role in the metabolism of atrazine (Tang et al., 1998). Information on the degradation and biotransformation of xenobiotics by diatoms, however, is virtually unknown. 2,4-dichlorophenol (2,4-DCP) is used in the production of the herbicide 2,4-D, and it is one of the most abundant chlorophenols in the aquatic environment (House et al., 1997). The deleterious effects of 2,4-DCP on estuarine and coastal ecosystems have raised considerable concern. Biodegradation and biotransformation of 2,4-DCP have been reported in the fungus Phanerochaete chrysosporium (Valli and Gold, 1991), a human cytochrome P-450 containing yeast (Mehmood et al., 1997), and the aquatic
angiosperm Lemna gibba (Ensley et al., 1994). Although the study by Klekner and Kosaric (1992) showed that the green algae Chlorella sp. and S. obliquus were unable to degrade or biotransform 2,4-DCP, it is not known whether or not other algae could degrade or biotranform 2,4-DCP. The diatom, Skeletonema costatum, is abundant in coastal and oceanic waters world-wide (Grahame, 1987). The objectives of this study are: (a) to investigate whether this centric diatom is able to degrade or biotransform 2,4DCP; and (b) to study the responses of selected conjugation and antioxidant enzymes of S. costatum to 2,4-DCP.
2. Material and methods
2.1. Chemicals 2,4-dichlorophenol (99% purity), 1-aminobenzotriazole (ABT) and piperonyl butoxide (PBO) were purchased from Aldrich Chemical Co. ahexa-chlorocyclohexane (HCH), hexane, nitroblue tetrazolium (NBT), chloro-2,4-dinitrobenzene (CDNB), guaiacol, ascorbate, hydrogen peroxide, glutathione (GSH), phenylmethylsulphonyl fluoride (PMSF), DL-buthionine-(S,R)-sulfoximine (BSO), L-methionine and riboflavin were purchased from Sigma Chemical Co. Triton X100 was purchased from Amersham-Phamacia. All other chemicals used were of analytical grade.
2.2. Culture conditions An axenic culture of S. costatum (CCMP 1332) was obtained from the Provasoli-Guillard National Center for the Culture of Marine Phytoplankton, Maine, USA. The diatom was cultured in sterile f/2 medium (Guillard, 1975) in 250 ml Erlenmeyer flasks at 20 °C with a photon flux density of 45 mE m − 2 s − 1 with a 12:12 h light:dark cycle in an environmental chamber (Conviron, CMP3244). pH of the medium ranged from 8 to 9, and was not adjusted throughout the experiment.
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2.3. Remo6al of 2,4 -DCP Exponential phase S. costatum cells were inoculated into f/2 medium (100 ml) to a cell density of 5× 104 cells ml − 1. 2,4-DCP was added to a final concentration of 6 mg l − 1 to: (a) control medium without diatom cells and (b) medium with diatom cells, and 2,4-DCP concentrations in the media were measured at different time intervals. Three replicates of each were set up for both the control and treatment samples. All replicates were incubated for 10 days under the same condition as described above. During the incubation period, 2-ml samples were collected from each replicate at days 0, 4, 6, 8 and 10 for analysis. The concentration of 2,4-DCP was measured using the following protocol. Culture media samples (1 ml) were centrifuged at 14 000 rpm for 5 min to remove the diatom cells and 2,4-DCP was extracted from 100 ml of the supernatant fluid with 1.0 ml of hexane. The loss of 2,4-DCP due to adsorption on, and absorption by, the diatom cells was estimated by filtering : 1.5 ×108 cells onto a glass fibre filter (pretreated with hexane) and the cells rinsed with clean sea water, and extracted with 5 ml of hexane by ultrasonication. 2,4-DCP was analysed by gas chromatography with an electron capture detector (HP 5890, column Ultra-2, 25 m×0.2 mm ×0.33 mm film thickness). a-HCH was used as an internal standard. The density of diatom cells in the culture was counted using a hematocytometer, and growth rates were determined over time. In order to investigate the effects of exogenous glucose (a substrate for Phase II conjugation reactions) and GSH on the removal of 2,4-DCP in S. costatum culture, 150 mM glucose and 72 mM GSH were added to the diatom culture at day 4. The concentrations of 2,4-DCP and cell densities in each treatment were determined using the protocols described previously.
2.4. Toxicity of 2,4 -DCP on S. costatum in the presence of GSH synthesis and cytochrome P-450 inhibitors In order to determine whether GSH and cytochrome P-450 system are involved in the bio-
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transformation of 2,4-DCP, the effect of 2,4-DCP on the growth of the diatom in the presence of two specific cytochrome P-450 inhibitors (i.e. ABT, and PBO), and the GSH synthesis inhibitor (BSO) were investigated. Four replicates of the following treatments were set up: 6 mg l − 1 2,4 DCP; 6 mg l − 1 2,4-DCP plus 500 mM BSO; 6 mg l − 1 2,4-DCP plus 50 mM ABT; and 6 mg l − 1 2,4-DCP plus 3 mM PBO. Each treatment contained an initial cell density of 1× 105 cells ml − 1 and was incubated for 96 h.
2.5. Enzyme assays The results of the previous experiment showed that the growth rate of the diatom were affected by 6.0 mg l − 1 2,4-DCP, but not 1.0 mg l − 1 2,4DCP. Subsequently, the activities of the three antioxidant enzymes (i.e. superoxide dismutase (SOD), peroxidase (Pox), ascorbate peroxidase (AsPox)) and two conjugation enzymes (GST and UDP-glucosyltransferase) were, therefore, determined in the diatom cells after 96 h exposure to 0, 1.0, 3.0, 6.0 mg l − 1 2,4-DCP. S. costatum cultures from the control and treatment samples were centrifuged at 8000×g for 10 min at 4 °C. The cell pellet was re-suspended in 1.0 ml of 10 mM potassium phosphate buffer (pH 7.5) containing 0.1 mM PMSF, and was sonicated for 5 min (Braun Ultrasonicator) in an ice bath. The cell debris were removed by centrifugation at 6000× g for 2 min at 4 °C. The supernatant was used for the determination of SOD, Pox, AsPox, GST and UDP-glucosyltransferase activities. Spectrophotometric measurements were performed with a UV/VIS Shimadzu spectrophotometer (Model UV-3100). SOD (EC1.15.1.1) was assayed using the method of Beyer and Fridovich (1987). Reduction of NBT was determined at 560 nm, and 1 unit of SOD activity was defined, as the amount of enzyme required to inhibit NBT reduction by 50%. Pox (EC1.11.1.7) activity was determined using the method of Putter (1975). The rate of increase in absorbance at 470 nm was measured at 25 °C. Enzyme activity was calculated with an extinction coefficient of 26.6 mM − 1 cm − 1 for tetraguaiacol.
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AsPox (EC1.11.1.11) activity was assayed using the method of Nakano and Asada (1981). The decrease in absorbance at 290 nm was measured at 25 °C and enzyme activity was calculated with an extinction coefficient of 2.8 mM − 1 cm − 1. GST (EC2.5.1.18) activity was determined using CDNB as the substrate (Habig and Jakoby, 1981). The increase in absorbance at 340 nm was measured at 25 °C and the enzyme activity was calculated with an extinction coefficient of 9.6 mM − 1 cm − 1. UDP-glucosyltransferase (EC2.4.1) activity was determined using the method of Schlenk and Buhler (1988). Absorption at 405 nm was measured. Protein content of the homogenates was determined according to Bradford (1976), using crystalline bovine serum albumin as the standard.
2.6. Statistical analysis An analysis of variance was used to test the null hypothesis that there was no significant difference in 2,4-DCP concentration and/or enzyme activities (P 00.05). If there was significance between treatment groups, Dunnett’s test was used for
pair-wise comparison between the control and individual treatment groups (Zar, 1984).
3. Results
3.1. Remo6al of 2,4 -DCP In the present study, the removal of 2,4-DCP over a 10-day period in the control medium and diatom culture was 45.8 and 65%, respectively (Fig. 1). The removal of 2,4-DCP in the control over time may be attributed to photodecomposition and evaporation. The additional removal of 2,4-DCP in the diatom culture (19.2%) may be due to factors such as the adsorption of the chemical to the cell surface, 2,4-DCP uptake, or by biodegradation or biotransformation of the chemical by the diatom. To differentiate the possibilities of 2,4-DCP being: (a) adsorbed and stored; and (b) degraded/biotransformed by the diatom cells, residual concentrations of 2,4-DCP were measured in diatom cells. These results showed that the amount of 2,4-DCP adsorbed or stored by diatom cells (0.0190.00017 mg ml − 1) was
Fig. 1. Removal of 2,4-DCP in culture medium (control) and diatom culture with various treatments (Mean 9SD, n =3).
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Fig. 2. Growth of diatom and removal of 2,4-DCP by diatom. (Mean 9 SEM, n =3). Arrows indicate addition of glucose or GSH.
minimal, and only accounted for 0.9% of the total loss of 2,4-DCP. Thus, it can be concluded that up to 18.3% of the 2,4-DCP loss in the diatom culture was due to biotransformation or biodegradation by the S. costatum culture. In this study, the loss of 2,4-DCP in the diatom culture was also positively correlated with the exponential growth of the diatom (Fig. 2), and therefore, further supports the hypothesis that diatom adsorption, uptake and biodegradation of 2,4-DCP resulted in a loss of the chemical from the test solution. The addition of 75 mM GSH enhanced the removal of 2,4-DCP to 73.7% of the total 2,4DCP in 10 days (Fig. 3), and the removal of 2,4-DCP by the diatom was increased by 45.7% (Fig. 2). In contrast, removal of 2,4-DCP was not significantly enhanced after addition of glucose (Fig. 3).
3.2. Effects of GSH, glucose and enzyme inhibitors on the toxicity of 2,4 -DCP In this study, growth rate was used as the
endpoint to evaluate the toxicity of 2,4-DCP to S. costatum. GSH enhanced the degradation of 2,4DCP, and detoxification was accompanied by a significant increase in growth rate (7.2%) of the diatom. However, no significant effect was found in cell density at day 10 for all treatment (Fig. 4A). Growth rate was significantly reduced (− 17.3%) upon addition of BSO (a specific inhibitor for GSH synthesis)(Fig. 4B). These results show that GSH was involved in the degradation and detoxification of 2,4-DCP in the diatom. However, the addition of exogenous glucose had no effect on the degradation of 2,4-DCP or diatom growth (Figs. 3 and 4A). The above results indicate an increase in degradation of 2,4-DCP will increase growth rate of the diatom. Compared with diatom culture with 2,4-DCP only, no significant change in growth rate was observed upon addition of ABT and PBO (cytochrome P-450 inhibitors) in the presence of 2,4-DCP (Fig. 4B), suggesting that cytochrome P-450 does not play a major role in the detoxification of 2,4-DCP in the diatom.
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3.3. Responses of conjugation and antioxidant enzymes A short-term (96 h) experiment was conducted to investigate responses of conjugation and antioxidant enzyme when diatom were exposed to 2,4-DCP. Activity of GST markedly increased ( : 2.5 times) when the diatom was exposed to 6 mg l − 1 2,4-DCP. However, an increase in GST activity was not observed at lower test concentrations (Table 1). UDP-glucosyltransferase activity was not detectable at any of the test concentrations. Significant increases in activities of SOD (+ 150%) and AsPox ( + 172%) were observed after the diatom was exposed to 6 mg l − 1 2,4-DCP. Both antioxidant enzymes did not respond at lower concentrations, and Pox did not respond to 2,4-DCP at any of the test concentrations.
4. Discussion Klekner and Kosaric (1992) found that the green algae Chlorella and Scenedesmus were un-
able to degrade 2,4-DCP, despite phenolic compounds such as 2-chlorophenol, 2,4-dimethylphenol and 2,4-dinitrophenol were readily degraded. In the present study, the removal of 2,4-DCP was significantly higher in an axenic culture of S. costatum, as compared to the control medium, and up to 65% of 2,4-DCP disappeared in diatom culture within 10 days. Furthermore, the removal of 2,4-DCP increased with an increase in diatom biomass, while accumulation and adsorption of 2,4-DCP within diatom cells was negligible. The experimental evidence presented in this study, therefore, demonstrated that S. costatum was able to metabolize 2,4-DCP. The low bioaccumulation in diatom cells may be due to the low log Kow (3.06 in freshwater) of 2,4-DCP. High pH of the media (8–10) would also reduce the undissociated forms of 2,4-DCP and hence bioaccumulation potential. Ensley et al. (1994) found that 95% of 2,4-DCP at 2.45 mg l − 1 disappeared within 6 days after exposure to the aquatic plant, L. gibba. In this study, the proposed 18.3% reduction of 2,4-DCP by the diatom may be an underestimate, as the shading effect caused by high cell density of the culture towards
Fig. 3. Concentration of 2,4-DCP in diatom culture and various treatments after 10-days (Mean 9 SEM, n = 3). An asterisk indicates a significant difference from the control (PB 0.05).
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Fig. 4. A. Effect of exogenous GSH and glucose on growth rate and cell density (at day 10) of S. costatum in the presence of 2,4-DCP (Mean 9SEM, n= 3). B. Effect of various inhibitors on growth rate of S. costatum in the presence of 2,4-DCP (Mean9 SEM, n =4). Values significantly different from the control are indicated by asterisk (*PB0.05, **PB 0.01).
the end of the experiment could have reduced the light exposure, thereby lowering the photodecomposition rate of 2,4-DCP in the diatom culture. Peaks of 2,4-DCP metabolites could not be revealed in the organic (hexane) phase in the GC spectra, indicating that 2,4-DCP was readily
transformed into water-soluble compounds. It has been shown that a cell line of cotton can take up GSH directly from culture medium (Gossett et al., 1996). In the present study, the addition of GSH significantly enhanced 2,4-DCP removal as well as growth of the diatom. Three possibili-
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ties may exist: (1) GSH might directly enhance biodegradation of 2,4-DCP in the diatom; (2) GSH might stimulate diatom growth, and biodegradation was enhanced indirectly as a result of increase in diatom biomass; and (3) a combination of both 1 and 2. The fact that diatom biomass showed no significant increase while removal of 2,4-DCP was enhanced by 45.7% after addition of GSH suggest that removal of 2,4-DCP after GSH addition was due to the enhancement of degradation/biotransformation of 2,4-DCP by exogenous GSH. The observed increase in toxicity of 2,4-DCP upon addition of a GSH synthesis inhibitor lend further support to the hypothesis that GSH plays a significant role in degradation of 2,4-DCP in the diatom. Tikoo et al. (1997) showed that addition of 2.0 g l − 1 glucose increased degradation of pentachlorophenol (PCP) as well as growth rate of the microalga VT-1. They attributed the enhanced degradation to an increase in algal biomass. In the present study, however, growth enhancement was not observed after glucose addition. Glucose serves as an important substrate in phase II conjugation reactions. For example, glucoside conjugations of 2,4-DCP and PCP have been reported in the aquatic angiosperm L. gibba (Ensley et al., 1994), soybean and wheat (Schmitt et al., 1985). In this study, despite the concentration of glucose being twice that of the concentration of GSH supplied to the diatom in culture, no significant decrease in 2,4-DCP was observed. This result indicates that glucose did not enhance the metabolism of 2,4-DCP in S. costatum.
In higher plants, xenobiotics are generally metabolized through sequential phases of degradation, conjugation and compartmentation (Komoßa et al., 1995). Although the cytochrome P-450 system is considered to be the principal enzyme complex involved in phase I transformation, Pox has also been reported to play a significant role in phase I reactions in higher plants, such as Lemna minor (Roy et al., 1995). Recently, cytochrome P-450 activities have been demonstrated in the marine chlorophytes, chromophytes and rhodophytes (Pflugmacher and Sandermann, 1998). Metabolism of 2,4-DCP was demonstrated in the yeast Saccharomyces cere6isiae modified to contain the human cytochrome P450 3A4 (Mehmood et al., 1997). Cytochrome P450 system in rat liver was inducible by PCP and 2,4,5trichlorophenol (Vizethum and Goerz, 1979). In wheat cells, PCP was metabolized by phase I reactions, in which PCP is hydroxylated and a chlorine atom is displaced (Komoßa et al., 1995). Thies et al. (1996) reported that addition of 20 mM PBO and 20 mM ABT reduced the P450catalysed N-demethylation of the herbicide merflurazon by 46% and 43% in green algae (C. sorokiniana). In this experiment, however, 3 mM PBO and 50 mM ABT showed no effect on diatom growth. The results therefore imply that the cytochrome P-450 system is not important in the detoxification of 2,4-DCP in S. costatum. In another experiment, we also found no significant induction in cytochrome P-450 gene expression when S. costatum were exposed to 2,4-DCP (Yang et al., unpublished data).
Table 1 Induction of antioxidant enzyme activities (nmol min−1 mg per protein) upon exposure to 2,4-DCP (Mean 9 SD, n =4). Values with asterisks are significantly different from their respective controls (*PB0.05; **PB0.01) Enzymes
GST SODa AsPox Pox a
Concentration of 2,4-DCP 0 mg l−1 (control)
1 mg l−1
3 mg l−1
6 mg l−1
5.23 91.3 41.79 2.15 504.19 82.4 4.979 0.86
7.71 92.87 42.39 9 0.75 486.79 57.1 6.31 9 1.88
6.6 9 1.84 44.85 9 2.06 577.7 9 65.3 4.56 91.08
13.24 9 4.33** 64.36 9 9.06** 867.2 9 268.2* 4.14 90.32
SOD unit: units per mg protein.
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Although Pox is considered to be an important enzyme in metabolizing benzo-[a]-pyrene in many marine chlorophytes (Enteromorpha intestnalis, Cladophora glomerata) and chara (Chara aspera) (Kirso and Irha, 1998), Pox activity was not induced by 2,4-DCP in S. costatum. The result indicates Pox is not involved in the metabolism of 2,4-DCP in the diatom. Sulfate and glucuronide conjugates are metabolic products of chlorophenols in fish (Ahlbory and Thunberg, 1980), while methylation of chlorophenols usually occur in bacteria (Allard et al., 1987). In higher plants, glucose conjugation is the major form of chlorophenol metabolites. For example, soybean and wheat cell suspension metabolize PCP into b-D-glucoside conjugate of PCP (Schmitt et al., 1985). 2,4-DCP was metabolized in L. gibba, and the principal metabolite was 2,4-dichlorophynyl--D-glucopyranoside (Ensley et al., 1994). In this study, no activity of the conjugation enzyme UDP-glucosyltransferase was detected, although high levels of glucosyltransferase and GST activities have been reported in the marine macroalgae (Pflugmacher and Sandermann, 1998). Likewise, GST activity has also been detected in freshwater algae, and was suggested to play a role in atrazine metabolism in the algal species (Tang et al., 1998). An induction of GST activity and decreased intracellular GSH level were also found in the aquatic angiosperm, Eichhornia crassipes, exposed to PCP (Roy and Hanninen, 1994). In this study, a 2.5 fold increase in GST activity was found when S. costatum was exposed to 2,4-DCP for 96 h. This, together with the evidence that degradation was enhanced by exogenous GSH, suggest that degradation of 2,4DCP in the diatom is mainly mediated through GSH conjugation. Significant increases in the activities of antioxidant enzymes (i.e. SOD and AsPox) were evident, after S. costatum was exposed to 2,4-DCP for 96 h. The results suggest that there was an increase in the production of oxyradicals by the diatom. Roy and Hanninen (1994) reported the induction of AsPox and SOD in the aquatic plant, E. crassipes, after exposure to PCP. This was related to the generation of oxyradicals by hydroxyquinones and catechols produced via biotransformation of
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PCP. In the yeast, S. cere6isiae, modified to contain the human cytochrome P-450 3A4, the compounds 2-chloro-1,4-hydroxyquinone and 2-chloro-1,4-benzoquinone were found as the major metabolites of 2,4-DCP (Mehmood et al., 1997). Vesar (1979) showed that polymer of 2-hydroxy benzoquinone was formed as a product of photolysis of 2,4-DCP. It is likely that similar redox-active metabolites were produced by S. costatum upon exposure to 2,4-DCP, resulting in the induction of SOD and AsPox activities. In summary, we found that S. costatum is able to degrade and detoxify 2,4-DCP. As such, bioaccumulation of 2,4-DCP in diatom is negligible and hence the possibility of food chain transfer via this diatom is unlikely to be important. GSH appears to play a significant role in the degradation of 2,4-DCP. In contrast, cytochrome P-450 and Pox enzymes appear to be unimportant in these processes. Acknowledgements We thank Dr Carmel Pollino for reading a draft of this paper. References Ahlbory, U.G., Thunberg, T.M., 1980. Chlorinated phenols: occurrence, toxicity, metabolism, and environmental impact. Crit. Rev. Toxicol. 7, 1 – 36. Allard, A.S., Remberger, M., Neilson, A.H., 1987. Bacterial O-methylation of halogen-substituted phenols. Appl. Environ. Microbiol. 53, 839 – 845. Beyer, W.F. Jr., Fridovich, I., 1987. Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Anal. Biochem. 161, 559 – 566. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248 – 254. Butler, G.L., Deason, T.R., O’Kelley, J.C., 1975. Loss of five pesticides from cultures of twenty-one planktonic algae. Bull. Environ. Contam. Toxicol. 13, 149 – 152. Cerniglia, C.E., Gibson, D.T., Van Baalen, C., 1980. Oxidation of Naphthalene by cyanobacteria and microalgae. J. Gen. Microbiol. 116, 495 – 500. Cerniglia, C.E., Gibson, D.T., Van Baalen, C., 1982. Naphthalene metabolism by diatoms isolated from Kachemak Bay region of Alaska. J. Gen. Microbiol. 128, 987 – 990.
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in a marine grazing food-chain: algal cells Phaeodactylum tricornutum, mussels Mytilus edulis and flounders Platichthys flesus studied by means of a stepwise-reduction-CVAA method. Mar. Ecol. Prog. Ser. 62, 259 – 270. Roy, S., Hanninen, O., 1994. Pentachlorophenol: uptake/elimination kinetics and metabolism in an aquatic plant, Eichhornia crassipes. Environ. Toxicol. Chem. 13, 763 – 773. Roy, S., Lindstroem-Seppae, P., Huuskonen, S., Haenninen, O., 1995. Responses of biotransformation and antioxidant enzymes in Lemna minor and Oncorhynchus mykiss exposed simultaneously to hexachlorobenzene. Chemosphere 30, 1489 – 1498. Schlenk, D., Buhler, D.R., 1988. Cytochrome P-450 and phase II activities in the gumboot chiton Cryptochiton stelleri. Aquat. Toxicol. 13, 167 – 182. Schmitt, R., Kaul, J., Trenck, T.V.D., Schaller, E., Sandermann, H., 1985. b-D-Glucosyl and O-malonyl-b-D-glucosyl conjugates of pentachlorophenol in soybean and wheat: identification and enzymatic synthesis. Pestic. Biochem. Physiol. 24, 77 – 85. Semple, K.T., Cain, R., 1996. Biodegradation of phenols by the alga Ochromonas danica. Appl. Environ. Microbiol. 62, 1265 – 1273. Tang, J., Siegfried, B.D., Hoagland, K.D., 1998. Glutathione S-transferase and in vitro metabolism of atrazine in freshwater algae. Pest. Biochem. Physiol. 59, 155 – 161. Tikoo, V., Scragg, A.H., Shales, S.W., 1997. Degradation of pentachlorophenol by microalgae. J. Chem. Tech. Biotech. 68, 425 – 431. Thies, F., Backhaus, T., Bossmann, B., Grimme, L.H., 1996. Xenobiotic biotransformation in unicellular green algae. Plant Physiol. 112, 361 –370. Valli, K., Gold, M.H., 1991. Degradation of 2,4-dichlorophenol by lignin degrading fungus Phanerochaete chrysosporium. J. Bacteriol. 173, 345 – 352. Vesar, N., 1979. Water-related environmental fate of 129 priority pollutants. vol. II. US Environmental Protection Agency, Publication No. EPA-440/4-79-029b, Washington, DC. Vizethum, W., Goerz, G., 1979. Induction of the hepatic microsomal and nuclear cytochrome P-450 system by hexachlorobenzene, pentachlorophenol and trichlorophenol. Chem. Biol. Interact. 28, 291 – 300. Warshawsky, D., Radike, M., Jayasimhulu, K., Cody, T., 1988. Metabolism of benzo(a)pyrene by a dioxygenase enzyme system of the freshwater green alga Selenastrum capricornutum. Biochem. Biophys. Res. Commun. 152, 540 – 544. Warshawsky, D., Keenan, T.H., Reilman, R., 1990. Conjugation of benzo(a)pyrene metabolites by freshwater green alga Selenastrum capricornutum. Chem. Biol. Interact. 74, 93 – 105. Warshawsky, D., Cody, T., Radike, M., Reilman, R., Schumann, B., LaDow, K., Schneider, J., 1995. Biotransformation of benzo(a)pyrene and other polycyclic aromatic hydrocarbons and heterocyclic analogs by several green algae and other algal species under gold and white light. Chem. Biol. Interact. 97, 131 – 148. Zar, J., 1984. Biostatistics. Prentice Hall, New York.
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Biodegradation and enzymatic responses in the marine diatom Skeletonema costatum upon exposure to 2,4-dichlorophenol Shao Yang, Rudolf S.S. Wu *, Richard Y.C. Kong Department of Biology and Chemistry, Centre for Coastal Pollution and Conser6ation, City Uni6ersity of Hong Kong, 83 Tat Chee A6enue, Kowloon, Hong Kong Special Administrati6e Region, Hong Kong, China Received 3 June 2001; received in revised form 30 September 2001; accepted 1 October 2001
Abstract The biodegradation and responses of selected detoxification and antioxidant enzymes in the marine diatom, Skeletonema costatum, upon exposure to sublethal concentrations of 2,4-dichlorophenol (2,4-DCP) were investigated. Results show that 2,4-DCP was readily metabolised, but bioaccumulation and adsorption were negligible. Glutathione S-transferase, ascorbate peroxidase and superoxide dismutase activities were increased markedly after exposure to 2,4-DCP for 96 h, while no appreciable change in peroxidase activity was observed. The addition of exogeneous glutathione to diatom culture enhanced the degradation of 2,4-DCP, and promoted diatom growth. The inhibition of glutathione synthesis enhanced the toxicity of 2,4-DCP. These results suggest that glutathione conjugation was one of the principal mechanisms involved in the degradation of 2,4-DCP in this diatom. © 2002 Elsevier Science B.V. All rights reserved. Keywords: 2,4-Dichlorophenol; Degradation; Diatom; Detoxification; Enzyme
1. Introduction Phytoplankton are abundant in the marine environment and play a pivotal role in both primary production and nutrient recycling. Therefore, any adverse effects of pollutants on phytoplankton may lead to serious ecological consequences. In addition, if xenobiotics are bioconcentrated by phytoplankton, this may potentially lead to food * Corresponding author. Tel.: + 852-2788-7401; fax: + 8522788-7406 E-mail address: [email protected] (R.S.S. Wu).
chain transfer or biomagnification (Riisgaard and Hansen, 1990). Investigating the degradation and biotransformation of xenobiotics in phytoplankton is hence important in assessing the environmental fate and risk of pollutants in marine ecosystems. While the mechanisms of biotransformation and biodegradation of xenobiotics have been studied extensively in bacteria (Kobayashi and Rittmann, 1982), higher plants (Komoßa et al., 1995) and animals ( James et al., 1977), there is a paucity of data on biodegradation and biotransformation of xenobiotics in algae. A number of studies have shown that certain freshwater
0166-445X/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 4 5 X ( 0 1 ) 0 0 2 5 2 - 1
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algae (e.g. Chlorella sp., Scenedesmus obliquus, Selenastrum capricornutum and Ochromonas danica), are able to degrade a variety of xenobiotics, such as the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) (Butler et al., 1975), phenolic compounds (Ellis, 1977; Klekner and Kosaric, 1992; Semple and Cain, 1996; Tikoo et al., 1997) and polyaromatic hydrocarbons (PAHs) (Cerniglia et al., 1980; Warshawsky et al., 1988, 1995). Despite this, limited studies have been conducted with marine phytoplankton. Previous studies have shown that certain marine microalgae (including pennate diatoms such as Phaeodactylum tricornutum, Na6icula sp., Nitzschia sp. and Synedra sp.) are able to degrade phloroglucinol (Craigie et al., 1965) and napthalene (Cerniglia et al., 1982). The potential of marine phytoplankton to degrade xenobiotics however, remains unclear. In animals and higher plants, selected xenobiotics are degraded or biotransformed through phase I (cytochrome P-450) and phase II (conjugation) enzymes. The cytochrome P-450 system is believed to be involved in the biotransformation of herbicides in the unicellular green algae Chlorella sorokiniana and Chlorella fusca (Thies et al., 1996), but degradation of the PAH benzo(a)pyrene (B[a]P) in the freshwater green alga, S. capricornutum, was mediated through dioxygenase and the metabolites were then conjugated to sulfate and glucose (Warshawsky et al., 1988, 1990). In freshwater algae, glutathione S-transferase (GST) was suggested to play an important role in the metabolism of atrazine (Tang et al., 1998). Information on the degradation and biotransformation of xenobiotics by diatoms, however, is virtually unknown. 2,4-dichlorophenol (2,4-DCP) is used in the production of the herbicide 2,4-D, and it is one of the most abundant chlorophenols in the aquatic environment (House et al., 1997). The deleterious effects of 2,4-DCP on estuarine and coastal ecosystems have raised considerable concern. Biodegradation and biotransformation of 2,4-DCP have been reported in the fungus Phanerochaete chrysosporium (Valli and Gold, 1991), a human cytochrome P-450 containing yeast (Mehmood et al., 1997), and the aquatic
angiosperm Lemna gibba (Ensley et al., 1994). Although the study by Klekner and Kosaric (1992) showed that the green algae Chlorella sp. and S. obliquus were unable to degrade or biotransform 2,4-DCP, it is not known whether or not other algae could degrade or biotranform 2,4-DCP. The diatom, Skeletonema costatum, is abundant in coastal and oceanic waters world-wide (Grahame, 1987). The objectives of this study are: (a) to investigate whether this centric diatom is able to degrade or biotransform 2,4DCP; and (b) to study the responses of selected conjugation and antioxidant enzymes of S. costatum to 2,4-DCP.
2. Material and methods
2.1. Chemicals 2,4-dichlorophenol (99% purity), 1-aminobenzotriazole (ABT) and piperonyl butoxide (PBO) were purchased from Aldrich Chemical Co. ahexa-chlorocyclohexane (HCH), hexane, nitroblue tetrazolium (NBT), chloro-2,4-dinitrobenzene (CDNB), guaiacol, ascorbate, hydrogen peroxide, glutathione (GSH), phenylmethylsulphonyl fluoride (PMSF), DL-buthionine-(S,R)-sulfoximine (BSO), L-methionine and riboflavin were purchased from Sigma Chemical Co. Triton X100 was purchased from Amersham-Phamacia. All other chemicals used were of analytical grade.
2.2. Culture conditions An axenic culture of S. costatum (CCMP 1332) was obtained from the Provasoli-Guillard National Center for the Culture of Marine Phytoplankton, Maine, USA. The diatom was cultured in sterile f/2 medium (Guillard, 1975) in 250 ml Erlenmeyer flasks at 20 °C with a photon flux density of 45 mE m − 2 s − 1 with a 12:12 h light:dark cycle in an environmental chamber (Conviron, CMP3244). pH of the medium ranged from 8 to 9, and was not adjusted throughout the experiment.
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2.3. Remo6al of 2,4 -DCP Exponential phase S. costatum cells were inoculated into f/2 medium (100 ml) to a cell density of 5× 104 cells ml − 1. 2,4-DCP was added to a final concentration of 6 mg l − 1 to: (a) control medium without diatom cells and (b) medium with diatom cells, and 2,4-DCP concentrations in the media were measured at different time intervals. Three replicates of each were set up for both the control and treatment samples. All replicates were incubated for 10 days under the same condition as described above. During the incubation period, 2-ml samples were collected from each replicate at days 0, 4, 6, 8 and 10 for analysis. The concentration of 2,4-DCP was measured using the following protocol. Culture media samples (1 ml) were centrifuged at 14 000 rpm for 5 min to remove the diatom cells and 2,4-DCP was extracted from 100 ml of the supernatant fluid with 1.0 ml of hexane. The loss of 2,4-DCP due to adsorption on, and absorption by, the diatom cells was estimated by filtering : 1.5 ×108 cells onto a glass fibre filter (pretreated with hexane) and the cells rinsed with clean sea water, and extracted with 5 ml of hexane by ultrasonication. 2,4-DCP was analysed by gas chromatography with an electron capture detector (HP 5890, column Ultra-2, 25 m×0.2 mm ×0.33 mm film thickness). a-HCH was used as an internal standard. The density of diatom cells in the culture was counted using a hematocytometer, and growth rates were determined over time. In order to investigate the effects of exogenous glucose (a substrate for Phase II conjugation reactions) and GSH on the removal of 2,4-DCP in S. costatum culture, 150 mM glucose and 72 mM GSH were added to the diatom culture at day 4. The concentrations of 2,4-DCP and cell densities in each treatment were determined using the protocols described previously.
2.4. Toxicity of 2,4 -DCP on S. costatum in the presence of GSH synthesis and cytochrome P-450 inhibitors In order to determine whether GSH and cytochrome P-450 system are involved in the bio-
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transformation of 2,4-DCP, the effect of 2,4-DCP on the growth of the diatom in the presence of two specific cytochrome P-450 inhibitors (i.e. ABT, and PBO), and the GSH synthesis inhibitor (BSO) were investigated. Four replicates of the following treatments were set up: 6 mg l − 1 2,4 DCP; 6 mg l − 1 2,4-DCP plus 500 mM BSO; 6 mg l − 1 2,4-DCP plus 50 mM ABT; and 6 mg l − 1 2,4-DCP plus 3 mM PBO. Each treatment contained an initial cell density of 1× 105 cells ml − 1 and was incubated for 96 h.
2.5. Enzyme assays The results of the previous experiment showed that the growth rate of the diatom were affected by 6.0 mg l − 1 2,4-DCP, but not 1.0 mg l − 1 2,4DCP. Subsequently, the activities of the three antioxidant enzymes (i.e. superoxide dismutase (SOD), peroxidase (Pox), ascorbate peroxidase (AsPox)) and two conjugation enzymes (GST and UDP-glucosyltransferase) were, therefore, determined in the diatom cells after 96 h exposure to 0, 1.0, 3.0, 6.0 mg l − 1 2,4-DCP. S. costatum cultures from the control and treatment samples were centrifuged at 8000×g for 10 min at 4 °C. The cell pellet was re-suspended in 1.0 ml of 10 mM potassium phosphate buffer (pH 7.5) containing 0.1 mM PMSF, and was sonicated for 5 min (Braun Ultrasonicator) in an ice bath. The cell debris were removed by centrifugation at 6000× g for 2 min at 4 °C. The supernatant was used for the determination of SOD, Pox, AsPox, GST and UDP-glucosyltransferase activities. Spectrophotometric measurements were performed with a UV/VIS Shimadzu spectrophotometer (Model UV-3100). SOD (EC1.15.1.1) was assayed using the method of Beyer and Fridovich (1987). Reduction of NBT was determined at 560 nm, and 1 unit of SOD activity was defined, as the amount of enzyme required to inhibit NBT reduction by 50%. Pox (EC1.11.1.7) activity was determined using the method of Putter (1975). The rate of increase in absorbance at 470 nm was measured at 25 °C. Enzyme activity was calculated with an extinction coefficient of 26.6 mM − 1 cm − 1 for tetraguaiacol.
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AsPox (EC1.11.1.11) activity was assayed using the method of Nakano and Asada (1981). The decrease in absorbance at 290 nm was measured at 25 °C and enzyme activity was calculated with an extinction coefficient of 2.8 mM − 1 cm − 1. GST (EC2.5.1.18) activity was determined using CDNB as the substrate (Habig and Jakoby, 1981). The increase in absorbance at 340 nm was measured at 25 °C and the enzyme activity was calculated with an extinction coefficient of 9.6 mM − 1 cm − 1. UDP-glucosyltransferase (EC2.4.1) activity was determined using the method of Schlenk and Buhler (1988). Absorption at 405 nm was measured. Protein content of the homogenates was determined according to Bradford (1976), using crystalline bovine serum albumin as the standard.
2.6. Statistical analysis An analysis of variance was used to test the null hypothesis that there was no significant difference in 2,4-DCP concentration and/or enzyme activities (P 00.05). If there was significance between treatment groups, Dunnett’s test was used for
pair-wise comparison between the control and individual treatment groups (Zar, 1984).
3. Results
3.1. Remo6al of 2,4 -DCP In the present study, the removal of 2,4-DCP over a 10-day period in the control medium and diatom culture was 45.8 and 65%, respectively (Fig. 1). The removal of 2,4-DCP in the control over time may be attributed to photodecomposition and evaporation. The additional removal of 2,4-DCP in the diatom culture (19.2%) may be due to factors such as the adsorption of the chemical to the cell surface, 2,4-DCP uptake, or by biodegradation or biotransformation of the chemical by the diatom. To differentiate the possibilities of 2,4-DCP being: (a) adsorbed and stored; and (b) degraded/biotransformed by the diatom cells, residual concentrations of 2,4-DCP were measured in diatom cells. These results showed that the amount of 2,4-DCP adsorbed or stored by diatom cells (0.0190.00017 mg ml − 1) was
Fig. 1. Removal of 2,4-DCP in culture medium (control) and diatom culture with various treatments (Mean 9SD, n =3).
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Fig. 2. Growth of diatom and removal of 2,4-DCP by diatom. (Mean 9 SEM, n =3). Arrows indicate addition of glucose or GSH.
minimal, and only accounted for 0.9% of the total loss of 2,4-DCP. Thus, it can be concluded that up to 18.3% of the 2,4-DCP loss in the diatom culture was due to biotransformation or biodegradation by the S. costatum culture. In this study, the loss of 2,4-DCP in the diatom culture was also positively correlated with the exponential growth of the diatom (Fig. 2), and therefore, further supports the hypothesis that diatom adsorption, uptake and biodegradation of 2,4-DCP resulted in a loss of the chemical from the test solution. The addition of 75 mM GSH enhanced the removal of 2,4-DCP to 73.7% of the total 2,4DCP in 10 days (Fig. 3), and the removal of 2,4-DCP by the diatom was increased by 45.7% (Fig. 2). In contrast, removal of 2,4-DCP was not significantly enhanced after addition of glucose (Fig. 3).
3.2. Effects of GSH, glucose and enzyme inhibitors on the toxicity of 2,4 -DCP In this study, growth rate was used as the
endpoint to evaluate the toxicity of 2,4-DCP to S. costatum. GSH enhanced the degradation of 2,4DCP, and detoxification was accompanied by a significant increase in growth rate (7.2%) of the diatom. However, no significant effect was found in cell density at day 10 for all treatment (Fig. 4A). Growth rate was significantly reduced (− 17.3%) upon addition of BSO (a specific inhibitor for GSH synthesis)(Fig. 4B). These results show that GSH was involved in the degradation and detoxification of 2,4-DCP in the diatom. However, the addition of exogenous glucose had no effect on the degradation of 2,4-DCP or diatom growth (Figs. 3 and 4A). The above results indicate an increase in degradation of 2,4-DCP will increase growth rate of the diatom. Compared with diatom culture with 2,4-DCP only, no significant change in growth rate was observed upon addition of ABT and PBO (cytochrome P-450 inhibitors) in the presence of 2,4-DCP (Fig. 4B), suggesting that cytochrome P-450 does not play a major role in the detoxification of 2,4-DCP in the diatom.
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3.3. Responses of conjugation and antioxidant enzymes A short-term (96 h) experiment was conducted to investigate responses of conjugation and antioxidant enzyme when diatom were exposed to 2,4-DCP. Activity of GST markedly increased ( : 2.5 times) when the diatom was exposed to 6 mg l − 1 2,4-DCP. However, an increase in GST activity was not observed at lower test concentrations (Table 1). UDP-glucosyltransferase activity was not detectable at any of the test concentrations. Significant increases in activities of SOD (+ 150%) and AsPox ( + 172%) were observed after the diatom was exposed to 6 mg l − 1 2,4-DCP. Both antioxidant enzymes did not respond at lower concentrations, and Pox did not respond to 2,4-DCP at any of the test concentrations.
4. Discussion Klekner and Kosaric (1992) found that the green algae Chlorella and Scenedesmus were un-
able to degrade 2,4-DCP, despite phenolic compounds such as 2-chlorophenol, 2,4-dimethylphenol and 2,4-dinitrophenol were readily degraded. In the present study, the removal of 2,4-DCP was significantly higher in an axenic culture of S. costatum, as compared to the control medium, and up to 65% of 2,4-DCP disappeared in diatom culture within 10 days. Furthermore, the removal of 2,4-DCP increased with an increase in diatom biomass, while accumulation and adsorption of 2,4-DCP within diatom cells was negligible. The experimental evidence presented in this study, therefore, demonstrated that S. costatum was able to metabolize 2,4-DCP. The low bioaccumulation in diatom cells may be due to the low log Kow (3.06 in freshwater) of 2,4-DCP. High pH of the media (8–10) would also reduce the undissociated forms of 2,4-DCP and hence bioaccumulation potential. Ensley et al. (1994) found that 95% of 2,4-DCP at 2.45 mg l − 1 disappeared within 6 days after exposure to the aquatic plant, L. gibba. In this study, the proposed 18.3% reduction of 2,4-DCP by the diatom may be an underestimate, as the shading effect caused by high cell density of the culture towards
Fig. 3. Concentration of 2,4-DCP in diatom culture and various treatments after 10-days (Mean 9 SEM, n = 3). An asterisk indicates a significant difference from the control (PB 0.05).
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Fig. 4. A. Effect of exogenous GSH and glucose on growth rate and cell density (at day 10) of S. costatum in the presence of 2,4-DCP (Mean 9SEM, n= 3). B. Effect of various inhibitors on growth rate of S. costatum in the presence of 2,4-DCP (Mean9 SEM, n =4). Values significantly different from the control are indicated by asterisk (*PB0.05, **PB 0.01).
the end of the experiment could have reduced the light exposure, thereby lowering the photodecomposition rate of 2,4-DCP in the diatom culture. Peaks of 2,4-DCP metabolites could not be revealed in the organic (hexane) phase in the GC spectra, indicating that 2,4-DCP was readily
transformed into water-soluble compounds. It has been shown that a cell line of cotton can take up GSH directly from culture medium (Gossett et al., 1996). In the present study, the addition of GSH significantly enhanced 2,4-DCP removal as well as growth of the diatom. Three possibili-
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ties may exist: (1) GSH might directly enhance biodegradation of 2,4-DCP in the diatom; (2) GSH might stimulate diatom growth, and biodegradation was enhanced indirectly as a result of increase in diatom biomass; and (3) a combination of both 1 and 2. The fact that diatom biomass showed no significant increase while removal of 2,4-DCP was enhanced by 45.7% after addition of GSH suggest that removal of 2,4-DCP after GSH addition was due to the enhancement of degradation/biotransformation of 2,4-DCP by exogenous GSH. The observed increase in toxicity of 2,4-DCP upon addition of a GSH synthesis inhibitor lend further support to the hypothesis that GSH plays a significant role in degradation of 2,4-DCP in the diatom. Tikoo et al. (1997) showed that addition of 2.0 g l − 1 glucose increased degradation of pentachlorophenol (PCP) as well as growth rate of the microalga VT-1. They attributed the enhanced degradation to an increase in algal biomass. In the present study, however, growth enhancement was not observed after glucose addition. Glucose serves as an important substrate in phase II conjugation reactions. For example, glucoside conjugations of 2,4-DCP and PCP have been reported in the aquatic angiosperm L. gibba (Ensley et al., 1994), soybean and wheat (Schmitt et al., 1985). In this study, despite the concentration of glucose being twice that of the concentration of GSH supplied to the diatom in culture, no significant decrease in 2,4-DCP was observed. This result indicates that glucose did not enhance the metabolism of 2,4-DCP in S. costatum.
In higher plants, xenobiotics are generally metabolized through sequential phases of degradation, conjugation and compartmentation (Komoßa et al., 1995). Although the cytochrome P-450 system is considered to be the principal enzyme complex involved in phase I transformation, Pox has also been reported to play a significant role in phase I reactions in higher plants, such as Lemna minor (Roy et al., 1995). Recently, cytochrome P-450 activities have been demonstrated in the marine chlorophytes, chromophytes and rhodophytes (Pflugmacher and Sandermann, 1998). Metabolism of 2,4-DCP was demonstrated in the yeast Saccharomyces cere6isiae modified to contain the human cytochrome P450 3A4 (Mehmood et al., 1997). Cytochrome P450 system in rat liver was inducible by PCP and 2,4,5trichlorophenol (Vizethum and Goerz, 1979). In wheat cells, PCP was metabolized by phase I reactions, in which PCP is hydroxylated and a chlorine atom is displaced (Komoßa et al., 1995). Thies et al. (1996) reported that addition of 20 mM PBO and 20 mM ABT reduced the P450catalysed N-demethylation of the herbicide merflurazon by 46% and 43% in green algae (C. sorokiniana). In this experiment, however, 3 mM PBO and 50 mM ABT showed no effect on diatom growth. The results therefore imply that the cytochrome P-450 system is not important in the detoxification of 2,4-DCP in S. costatum. In another experiment, we also found no significant induction in cytochrome P-450 gene expression when S. costatum were exposed to 2,4-DCP (Yang et al., unpublished data).
Table 1 Induction of antioxidant enzyme activities (nmol min−1 mg per protein) upon exposure to 2,4-DCP (Mean 9 SD, n =4). Values with asterisks are significantly different from their respective controls (*PB0.05; **PB0.01) Enzymes
GST SODa AsPox Pox a
Concentration of 2,4-DCP 0 mg l−1 (control)
1 mg l−1
3 mg l−1
6 mg l−1
5.23 91.3 41.79 2.15 504.19 82.4 4.979 0.86
7.71 92.87 42.39 9 0.75 486.79 57.1 6.31 9 1.88
6.6 9 1.84 44.85 9 2.06 577.7 9 65.3 4.56 91.08
13.24 9 4.33** 64.36 9 9.06** 867.2 9 268.2* 4.14 90.32
SOD unit: units per mg protein.
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Although Pox is considered to be an important enzyme in metabolizing benzo-[a]-pyrene in many marine chlorophytes (Enteromorpha intestnalis, Cladophora glomerata) and chara (Chara aspera) (Kirso and Irha, 1998), Pox activity was not induced by 2,4-DCP in S. costatum. The result indicates Pox is not involved in the metabolism of 2,4-DCP in the diatom. Sulfate and glucuronide conjugates are metabolic products of chlorophenols in fish (Ahlbory and Thunberg, 1980), while methylation of chlorophenols usually occur in bacteria (Allard et al., 1987). In higher plants, glucose conjugation is the major form of chlorophenol metabolites. For example, soybean and wheat cell suspension metabolize PCP into b-D-glucoside conjugate of PCP (Schmitt et al., 1985). 2,4-DCP was metabolized in L. gibba, and the principal metabolite was 2,4-dichlorophynyl--D-glucopyranoside (Ensley et al., 1994). In this study, no activity of the conjugation enzyme UDP-glucosyltransferase was detected, although high levels of glucosyltransferase and GST activities have been reported in the marine macroalgae (Pflugmacher and Sandermann, 1998). Likewise, GST activity has also been detected in freshwater algae, and was suggested to play a role in atrazine metabolism in the algal species (Tang et al., 1998). An induction of GST activity and decreased intracellular GSH level were also found in the aquatic angiosperm, Eichhornia crassipes, exposed to PCP (Roy and Hanninen, 1994). In this study, a 2.5 fold increase in GST activity was found when S. costatum was exposed to 2,4-DCP for 96 h. This, together with the evidence that degradation was enhanced by exogenous GSH, suggest that degradation of 2,4DCP in the diatom is mainly mediated through GSH conjugation. Significant increases in the activities of antioxidant enzymes (i.e. SOD and AsPox) were evident, after S. costatum was exposed to 2,4-DCP for 96 h. The results suggest that there was an increase in the production of oxyradicals by the diatom. Roy and Hanninen (1994) reported the induction of AsPox and SOD in the aquatic plant, E. crassipes, after exposure to PCP. This was related to the generation of oxyradicals by hydroxyquinones and catechols produced via biotransformation of
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PCP. In the yeast, S. cere6isiae, modified to contain the human cytochrome P-450 3A4, the compounds 2-chloro-1,4-hydroxyquinone and 2-chloro-1,4-benzoquinone were found as the major metabolites of 2,4-DCP (Mehmood et al., 1997). Vesar (1979) showed that polymer of 2-hydroxy benzoquinone was formed as a product of photolysis of 2,4-DCP. It is likely that similar redox-active metabolites were produced by S. costatum upon exposure to 2,4-DCP, resulting in the induction of SOD and AsPox activities. In summary, we found that S. costatum is able to degrade and detoxify 2,4-DCP. As such, bioaccumulation of 2,4-DCP in diatom is negligible and hence the possibility of food chain transfer via this diatom is unlikely to be important. GSH appears to play a significant role in the degradation of 2,4-DCP. In contrast, cytochrome P-450 and Pox enzymes appear to be unimportant in these processes. Acknowledgements We thank Dr Carmel Pollino for reading a draft of this paper. References Ahlbory, U.G., Thunberg, T.M., 1980. Chlorinated phenols: occurrence, toxicity, metabolism, and environmental impact. Crit. Rev. Toxicol. 7, 1 – 36. Allard, A.S., Remberger, M., Neilson, A.H., 1987. Bacterial O-methylation of halogen-substituted phenols. Appl. Environ. Microbiol. 53, 839 – 845. Beyer, W.F. Jr., Fridovich, I., 1987. Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Anal. Biochem. 161, 559 – 566. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248 – 254. Butler, G.L., Deason, T.R., O’Kelley, J.C., 1975. Loss of five pesticides from cultures of twenty-one planktonic algae. Bull. Environ. Contam. Toxicol. 13, 149 – 152. Cerniglia, C.E., Gibson, D.T., Van Baalen, C., 1980. Oxidation of Naphthalene by cyanobacteria and microalgae. J. Gen. Microbiol. 116, 495 – 500. Cerniglia, C.E., Gibson, D.T., Van Baalen, C., 1982. Naphthalene metabolism by diatoms isolated from Kachemak Bay region of Alaska. J. Gen. Microbiol. 128, 987 – 990.
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