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Altered glutathione S-transferase catalytic activities in female brown bullheads from a contaminated central Florida lake
Marine Environmental Research 50 (2000) 399±403 www.elsevier.com/locate/marenvrev
Altered glutathione S-transferase catalytic activities in female brown bullheads from a contaminated central Florida lake E.P. Gallagher *, K.M. Sheehy Department of Physiological Sciences, University of Florida, PO Box 110885, Gainesville, FL 32611, USA Received 29 April 1999; received in revised form 16 September 1999; accepted 20 January 2000
Abstract Brown bullheads (Ameriurus nebulosus) are a demersal freshwater species that can be found in a number of polluted ecosystems. The purpose of the present study was to determine the overall capacity for in vitro glutathione S-transferase (GST) detoxi®cation by brown bullheads, and to see if bullhead GST catalysis was altered in bullheads from a polluted site. Brown bullhead liver cytosolic GSTs catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) over a large range of substrate concentrations, with apparent Km and Vmax for CDNB at ®xed nucleophile (glutathione, GSH) concentrations of 1.8±1.9 mM and 12.1±14.6 mmol CDNB conjugated/min/mg, respectively. Bullhead GSTs were also highly active toward other substrates such as ethacrynic acid (ECA), 5-androstene-3,17-dione (ADI), and nitrobutyl chloride (NBC). Initial rate GST catalytic activities toward CDNB, NBC, ECA, and ADI were signi®cantly lower in female bullheads from a contaminated lake (Lake Apopka Marsh) as compared to female bullheads inhabiting a nearby control site (Lake Woodru). No site dierences were observed with respect to male bullhead GST activities. These studies suggest that brown bullheads eciently carry out GST conjugation of diverse electrophilic substrates. However, bullhead GST catalysis may be compromised in bullheads inhabiting polluted ecosystems. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Enzyme kinetics; Glutathione S-transferases; Liver; Organochlorines
1. Introduction The glutathione S-transferases (GSTs, EC 2.5.1.18) are a multigene family of enzymes involved in the detoxi®cation of a variety of reactive intermediates and * Corresponding author. Tel.: +1-352-392-4700 ext. 5545; fax: +1-352-392-4707. E-mail address: [email protected]¯.edu (E.P. Gallagher). 0141-1136/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(00)00041-6
400
E.P. Gallagher, K.M. Sheehy / Marine Environmental Research 50 (2000) 399±403
products of oxidative stress. Because certain GST subunits possess high speci®c catalytic activity toward epoxide carcinogens and pesticides, the environmental modulation of GST isozymes may aect the resistance or sensitivity of a particular species against chemical toxicities (Hayes & Pulford, 1996; Hayes, Judah & Neal, 1993). Fish GST activities are often assayed under initial rate conditions using 1chloro-2,4-dinitrobenzene (CDNB), a relatively non-speci®c GST reference substrate (Gallagher, Sheehy, Lame & Segall, 2000; George, 1994) GST±CDNB activity re¯ects the integration of GST isoenzyme activities (except theta class) and thus allows for cross-species comparisons of GST activity. However, the use of GST substrates such as 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), nitrobutyl chloride (NBC), and 5-androstene-3,17-dione (ADI) in conjunction with CDNB allows for a more complete biochemical characterization of GST isozyme activities (Gallagher et al., 1999). Our recent studies are evaluating the role of GST expression in protecting against environmental chemical toxicities in brown bullheads. Brown bullheads are found in reclaimed agricultural sites in central Florida that have been impacted by historical use of organochlorine pesticides. Brown bullheads and other aquatic species collected from these polluted sites have high tissue concentrations of persistent organochlorine pesticides and have generally exhibited poor reproduction (Guillette, Gross, Masson, Matter, Percival & Woodward, 1994). Because GSTs are involved in the conjugation and reductive halogenation of organochlorine pesticides (Hayes & Pulford, 1996), it is important to characterize GST expression and catalysis in this species. In the present study, we have examined the kinetics of bullhead GST conjugation and analyzed brown bullhead hepatic GST activities using a variety of class-speci®c GST reference substrates. Furthermore, we have determined that the GST catalytic pathway can be altered in bullheads inhabiting aquatic ecosystems impacted by agricultural chemicals. 2. Materials and methods All chemicals, buers, co-factors and GST substrates were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The ®eld sites for this study were selected based upon historical episodes of exposure to agricultural chemicals. Lake Woodru is an 890 ha lake located on Lake Woodru National Wildlife Refuge, and is a relatively unpolluted aquatic ecosystem with minimal disturbance from agricultural and industrial sources. Lake Apopka is a 12,500 ha lake located near the city of Winter Haven Florida and has received substantial agricultural and municipal runo (Guillette et al., 1994). Lake Apopka Marsh is located at the northern border of Lake Apopka and has received the greatest inputs of pesticide runo from farming. Brown bullheads (2±5 years old) were collected from Lake Apopka Marsh and Lake Woodru during January±July 1997 by electroshocking. The animals were killed by a blow to the head and sexed by visual inspection of the gonads. The livers were immediately excised, frozen in liquid nitrogen and transferred to ÿ80 C until preparation of cytosolic fractions. Cytosolic GST activities towards CDNB, DCNB,
E.P. Gallagher, K.M. Sheehy / Marine Environmental Research 50 (2000) 399±403
401
ECA, NBC, and ADI and apparent Km and apparent Vmax values for CDNB were determined as described (Gallagher et al., 1999). Michaelis parameters and statistics were determined for GST±CDNB activities in bullhead liver cytosols by non-linear regression analysis and proportional (1/y) weighting of the experimental data using ULTRAFIT software for Macintosh personal computer. Values reported represent the mean of quadruplicate reactions for n=4±6 ®sh of each sex for each site. Signi®cant dierences in GST activities by ®eld site were determined using two-tailed Student's t-test. A P value of 40.05 was regarded as signi®cant. 3. Results and discussion Non-linear regression analysis of the initial rate GST±CDNB data at ®xed nucleophile concentrations using the single enzyme Michaelis±Menten equation yielded excellent ®ts to the experimental data (R2=0.99; Table 1). The apparent Km and apparent Vmax for CDNB at ®xed [GSH] for male and female brown bullheads were 1.8±1.9 mM and 12,122±14,643 nmol CDNB conjugated/min/mg, respectively. Clearance values (Vmax/Km) calculated from the kinetic constants were 6380±8135 L/ nmol CDNB conjugated/min/mg/mmol, indicating rapid clearance of CDNB at low, physiologically relevant substrate concentrations. Accordingly, the results of the kinetic experiments indicate a high capacity of bullheads to carry out in vitro GSTdependent electrophile conjugation, at least toward the prototypical substrate CDNB. In addition, these data are indicative of rapid in vivo clearance of GST substrates by bullheads at environmentally relevant substrate concentrations (e.g. high nM to low mM range). To further examine GST-dependent conjugation by brown bullheads, initial rate in vitro GST activities toward three other GST substrates were measured using saturating substrate concentrations. The GST catalytic activities toward these substrates (ECA, ADI, NBC) for bullheads from a non-polluted site (Lake Woodru; Table 2) are in the upper range of GST activities reported for other aquatic species, including some other freshwater ®sh species present in these lakes (Gallagher et al., 2000; George, 1994). These data are consistent with our CDNB kinetics data suggesting Table 1 Apparent kinetic parameters for brown bullhead hepatic glutathione S-transferase±1-chloro-2,4-dinitrobenzene (GST±CDNB) conjugationa Substrate
Sex
Km (mM)
Vmax (nmol/min/mg)
Vmax/Km (L/nmol/min/mg/mmol)
R2
CDNBb
M F
1.8 (0.5) 1.9 (0.5)
14,643 (3027) 12,122 (2465)
8135 6380
0.99 0.99
a All animals (n=3 for each sex) used for kinetic experiments were from the non-polluted reference site (Lake Woodru). b Nucleophile (GSH) concentrations were ®xed at 50 mM and electrophile (CDNB) concentrations varied from 0.040 to 5.12 mM in this experiment.All data presented as mean (A.S.E., Asymptopic Standard Error).
402
E.P. Gallagher, K.M. Sheehy / Marine Environmental Research 50 (2000) 399±403
Table 2 Hepatic glutatione S-transferase (GST) activities in brown bullheads collected from ®eld sites in central Floridaa Site
Sex
CDNB
NBC
ECA
ADI
Lake Woodru Lake Apopka Marsh
Maleb
1969 (249) 1603 (181)
35 (4) 32 (2)
16 (4) 16 (2)
60 (11) 65 (7)
Lake Woodru Lake Apopka Marsh
Female
1909 (180) 1152 (73)*
36 (4) 18 (1)*
18 (1) 12 (1)*
65 (5) 37 (3)*
a
Summary of quadruplicate determinations conducted on n=4±6 individuals per site, mean (S.E.M.). All activities are in nmol substrate conjugated/min/mg. CDNB, 1-chloro-2,4-dinitobenzene; NBC, nitrobutyl chloride; ECA, ethacrynic acid; ADI, 5-androstene-3,17-dione. b There were no signi®cant dierences in mean GST catalytic activities among male bullheads from the sites. *Signi®cantly lower activity than in female bullheads the reference site (P40.05).
a high rate of clearance for multiple GST substrates by brown bullheads. A comparison of in vitro GST activities by ®eld site showed that GST catalytic activities were similar in male brown bullheads from the contaminated Lake Apopka Marsh and Lake Woodru (Table 2). In contrast, initial rate GST activities toward all four substrates tested (CDNB, NBC, ECA, ADI) were signi®cantly lower in female Lake Apopka Marsh bullheads. Speci®cally, GST±CDNB activities in female Lake Apopka Marsh bullheads were 40% lower than those activities in female Lake Woodru bullheads whereas GST±NBC activities in female Lake Apopka Marsh bullheads were 50% lower than GST±NBC activities in female Lake Woodru bullheads (Table 2). In addition, GST catalytic activities using ECA and ADI as substrates were 33 and 43% lower, respectively, in female Lake Apopka Marsh bullheads than in female bullheads from Lake Woodru (Table 2). Collectively, these data suggest that exposure to anthropogenic chemicals or natural compounds capable of inhibiting GST caused a loss of GST activity in female bullheads from Lake Apopka Marsh. A broad array of endogenous compounds as well as environmental xenobiotics have been shown to irreversibly inhibit GST activity (Mannervik & Danielson, 1988). This type of inhibition is characterized by a covalent modi®cation of an enzyme leading to an irreversible loss of activity. The list of inhibitors include bile acids, steroid hormone derivatives, drugs, metals, herbicides, pesticides, quinones, and chalcone derivatives (reviewed by Mannervik & Danielson, 1988). A number of compounds capable of inhibiting GST activity, including halogenated phenoxyacetic herbicides (Dierickx, 1985), alachlor (Moore et al., 1986) and atrazine (Egaas et al., 1995) have been historically used in the Lake Apopka Marsh area and have been detected in bullhead tissues. Alternatively, we are also investigating if the lower GST activities in female bullheads were caused by a decrease in the synthesis of GST protein(s) at the molecular level. In this regard, down-regulation of GST isoenzyme expression at the mRNA level and loss of GST catalytic activities have been demonstrated in rats exposed to the herbicide diquat (Gallagher et al., 1995).
E.P. Gallagher, K.M. Sheehy / Marine Environmental Research 50 (2000) 399±403
403
Why only the females were aected in the present study is also unknown. Although ®sh harvested during this time were not generally reproductively active, we cannot discount the possibility that a combination of chemical exposure and endocrine interactions may have aected GST pathways in the female bullheads. In summary, brown bullhead hepatic GSTs rapidly and eciently catalyze the conjugation of a number of GST reference substrates. However, female brown bullheads inhabiting a polluted site in central Florida have signi®cantly lower levels of hepatic GST activities when compared to female bullheads from a non-polluted site. Currently, the functional signi®cance of the loss of GST activity in ®sh from Lake Apopka Marsh and the mechanisms underlying the observed GST alterations in bullheads are unknown. Our laboratory is currently addressing these issues in the context of understanding GST regulation in aquatic animals. Acknowledgements This research was supported in part by an Intramural Research Award from the University of Florida College of Veterinary Medicine. The technical assistance of Dr. Tim Gross and Mr. William Johnson in the ®sh collections is gratefully appreciated. References Dierickx, P. J. (1985). Comp. Biochem. Physiol., 82C, 495±500. Egaas, E., Falls, J. G., Svendsen, N. O., Ramstad, H., Skaare, J. U., & Dauterman, W. C. (1995). Biochim. Biophys. Acta, 1243, 256±264. Gallagher, E. P., Sheehy, K. M., Lame, M. W., & Segall, H. J. (2000). Environ. Toxicol. Chem., 19, 319± 326. Gallagher, E. P., Buetler, T. M., Wang, C., Stapleton, P. L., Stahl, D. L., & Eaton, D. L. (1995). Toxicol. Appl. Pharmacol., 134, 81±91. George, S. (1994). In D. C. Malins, & G. K. Ostrander, Aquatic toxicology, molecular, biochemical and cellular perspectives (pp. 27±86). Ann Arbor: Lewis Publishers. Guillette Jr., L. J., Gross, T. S., Masson, G. R., Matter, J. M., Percival, H. F., & Woodward, A. R. (1994). Environ. Health Perspect, 102, 680±688. Hayes, J. D., & Pulford, D. J. (1996). CRC Crit. Rev. Biochem. Mol. Biol., 30, 445±600. Hayes, J. D., Judah, D. J., & Neal, G. E. (1993). Cancer Res., 53, 3887±3894. Mannervik, B., & Danielson, U. H. (1988). CRC Crit. Rev. Biochem., 23, 283±337. Moore, R. E., Davies, M. S., O' Connell, K. M., Harding, E. I., Wiegand, R. C., & Tiemeier, D. C. (1986). Nucleic Acids Res., 14, 7227±7335.
Altered glutathione S-transferase catalytic activities in female brown bullheads from a contaminated central Florida lake E.P. Gallagher *, K.M. Sheehy Department of Physiological Sciences, University of Florida, PO Box 110885, Gainesville, FL 32611, USA Received 29 April 1999; received in revised form 16 September 1999; accepted 20 January 2000
Abstract Brown bullheads (Ameriurus nebulosus) are a demersal freshwater species that can be found in a number of polluted ecosystems. The purpose of the present study was to determine the overall capacity for in vitro glutathione S-transferase (GST) detoxi®cation by brown bullheads, and to see if bullhead GST catalysis was altered in bullheads from a polluted site. Brown bullhead liver cytosolic GSTs catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) over a large range of substrate concentrations, with apparent Km and Vmax for CDNB at ®xed nucleophile (glutathione, GSH) concentrations of 1.8±1.9 mM and 12.1±14.6 mmol CDNB conjugated/min/mg, respectively. Bullhead GSTs were also highly active toward other substrates such as ethacrynic acid (ECA), 5-androstene-3,17-dione (ADI), and nitrobutyl chloride (NBC). Initial rate GST catalytic activities toward CDNB, NBC, ECA, and ADI were signi®cantly lower in female bullheads from a contaminated lake (Lake Apopka Marsh) as compared to female bullheads inhabiting a nearby control site (Lake Woodru). No site dierences were observed with respect to male bullhead GST activities. These studies suggest that brown bullheads eciently carry out GST conjugation of diverse electrophilic substrates. However, bullhead GST catalysis may be compromised in bullheads inhabiting polluted ecosystems. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Enzyme kinetics; Glutathione S-transferases; Liver; Organochlorines
1. Introduction The glutathione S-transferases (GSTs, EC 2.5.1.18) are a multigene family of enzymes involved in the detoxi®cation of a variety of reactive intermediates and * Corresponding author. Tel.: +1-352-392-4700 ext. 5545; fax: +1-352-392-4707. E-mail address: [email protected]¯.edu (E.P. Gallagher). 0141-1136/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(00)00041-6
400
E.P. Gallagher, K.M. Sheehy / Marine Environmental Research 50 (2000) 399±403
products of oxidative stress. Because certain GST subunits possess high speci®c catalytic activity toward epoxide carcinogens and pesticides, the environmental modulation of GST isozymes may aect the resistance or sensitivity of a particular species against chemical toxicities (Hayes & Pulford, 1996; Hayes, Judah & Neal, 1993). Fish GST activities are often assayed under initial rate conditions using 1chloro-2,4-dinitrobenzene (CDNB), a relatively non-speci®c GST reference substrate (Gallagher, Sheehy, Lame & Segall, 2000; George, 1994) GST±CDNB activity re¯ects the integration of GST isoenzyme activities (except theta class) and thus allows for cross-species comparisons of GST activity. However, the use of GST substrates such as 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), nitrobutyl chloride (NBC), and 5-androstene-3,17-dione (ADI) in conjunction with CDNB allows for a more complete biochemical characterization of GST isozyme activities (Gallagher et al., 1999). Our recent studies are evaluating the role of GST expression in protecting against environmental chemical toxicities in brown bullheads. Brown bullheads are found in reclaimed agricultural sites in central Florida that have been impacted by historical use of organochlorine pesticides. Brown bullheads and other aquatic species collected from these polluted sites have high tissue concentrations of persistent organochlorine pesticides and have generally exhibited poor reproduction (Guillette, Gross, Masson, Matter, Percival & Woodward, 1994). Because GSTs are involved in the conjugation and reductive halogenation of organochlorine pesticides (Hayes & Pulford, 1996), it is important to characterize GST expression and catalysis in this species. In the present study, we have examined the kinetics of bullhead GST conjugation and analyzed brown bullhead hepatic GST activities using a variety of class-speci®c GST reference substrates. Furthermore, we have determined that the GST catalytic pathway can be altered in bullheads inhabiting aquatic ecosystems impacted by agricultural chemicals. 2. Materials and methods All chemicals, buers, co-factors and GST substrates were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The ®eld sites for this study were selected based upon historical episodes of exposure to agricultural chemicals. Lake Woodru is an 890 ha lake located on Lake Woodru National Wildlife Refuge, and is a relatively unpolluted aquatic ecosystem with minimal disturbance from agricultural and industrial sources. Lake Apopka is a 12,500 ha lake located near the city of Winter Haven Florida and has received substantial agricultural and municipal runo (Guillette et al., 1994). Lake Apopka Marsh is located at the northern border of Lake Apopka and has received the greatest inputs of pesticide runo from farming. Brown bullheads (2±5 years old) were collected from Lake Apopka Marsh and Lake Woodru during January±July 1997 by electroshocking. The animals were killed by a blow to the head and sexed by visual inspection of the gonads. The livers were immediately excised, frozen in liquid nitrogen and transferred to ÿ80 C until preparation of cytosolic fractions. Cytosolic GST activities towards CDNB, DCNB,
E.P. Gallagher, K.M. Sheehy / Marine Environmental Research 50 (2000) 399±403
401
ECA, NBC, and ADI and apparent Km and apparent Vmax values for CDNB were determined as described (Gallagher et al., 1999). Michaelis parameters and statistics were determined for GST±CDNB activities in bullhead liver cytosols by non-linear regression analysis and proportional (1/y) weighting of the experimental data using ULTRAFIT software for Macintosh personal computer. Values reported represent the mean of quadruplicate reactions for n=4±6 ®sh of each sex for each site. Signi®cant dierences in GST activities by ®eld site were determined using two-tailed Student's t-test. A P value of 40.05 was regarded as signi®cant. 3. Results and discussion Non-linear regression analysis of the initial rate GST±CDNB data at ®xed nucleophile concentrations using the single enzyme Michaelis±Menten equation yielded excellent ®ts to the experimental data (R2=0.99; Table 1). The apparent Km and apparent Vmax for CDNB at ®xed [GSH] for male and female brown bullheads were 1.8±1.9 mM and 12,122±14,643 nmol CDNB conjugated/min/mg, respectively. Clearance values (Vmax/Km) calculated from the kinetic constants were 6380±8135 L/ nmol CDNB conjugated/min/mg/mmol, indicating rapid clearance of CDNB at low, physiologically relevant substrate concentrations. Accordingly, the results of the kinetic experiments indicate a high capacity of bullheads to carry out in vitro GSTdependent electrophile conjugation, at least toward the prototypical substrate CDNB. In addition, these data are indicative of rapid in vivo clearance of GST substrates by bullheads at environmentally relevant substrate concentrations (e.g. high nM to low mM range). To further examine GST-dependent conjugation by brown bullheads, initial rate in vitro GST activities toward three other GST substrates were measured using saturating substrate concentrations. The GST catalytic activities toward these substrates (ECA, ADI, NBC) for bullheads from a non-polluted site (Lake Woodru; Table 2) are in the upper range of GST activities reported for other aquatic species, including some other freshwater ®sh species present in these lakes (Gallagher et al., 2000; George, 1994). These data are consistent with our CDNB kinetics data suggesting Table 1 Apparent kinetic parameters for brown bullhead hepatic glutathione S-transferase±1-chloro-2,4-dinitrobenzene (GST±CDNB) conjugationa Substrate
Sex
Km (mM)
Vmax (nmol/min/mg)
Vmax/Km (L/nmol/min/mg/mmol)
R2
CDNBb
M F
1.8 (0.5) 1.9 (0.5)
14,643 (3027) 12,122 (2465)
8135 6380
0.99 0.99
a All animals (n=3 for each sex) used for kinetic experiments were from the non-polluted reference site (Lake Woodru). b Nucleophile (GSH) concentrations were ®xed at 50 mM and electrophile (CDNB) concentrations varied from 0.040 to 5.12 mM in this experiment.All data presented as mean (A.S.E., Asymptopic Standard Error).
402
E.P. Gallagher, K.M. Sheehy / Marine Environmental Research 50 (2000) 399±403
Table 2 Hepatic glutatione S-transferase (GST) activities in brown bullheads collected from ®eld sites in central Floridaa Site
Sex
CDNB
NBC
ECA
ADI
Lake Woodru Lake Apopka Marsh
Maleb
1969 (249) 1603 (181)
35 (4) 32 (2)
16 (4) 16 (2)
60 (11) 65 (7)
Lake Woodru Lake Apopka Marsh
Female
1909 (180) 1152 (73)*
36 (4) 18 (1)*
18 (1) 12 (1)*
65 (5) 37 (3)*
a
Summary of quadruplicate determinations conducted on n=4±6 individuals per site, mean (S.E.M.). All activities are in nmol substrate conjugated/min/mg. CDNB, 1-chloro-2,4-dinitobenzene; NBC, nitrobutyl chloride; ECA, ethacrynic acid; ADI, 5-androstene-3,17-dione. b There were no signi®cant dierences in mean GST catalytic activities among male bullheads from the sites. *Signi®cantly lower activity than in female bullheads the reference site (P40.05).
a high rate of clearance for multiple GST substrates by brown bullheads. A comparison of in vitro GST activities by ®eld site showed that GST catalytic activities were similar in male brown bullheads from the contaminated Lake Apopka Marsh and Lake Woodru (Table 2). In contrast, initial rate GST activities toward all four substrates tested (CDNB, NBC, ECA, ADI) were signi®cantly lower in female Lake Apopka Marsh bullheads. Speci®cally, GST±CDNB activities in female Lake Apopka Marsh bullheads were 40% lower than those activities in female Lake Woodru bullheads whereas GST±NBC activities in female Lake Apopka Marsh bullheads were 50% lower than GST±NBC activities in female Lake Woodru bullheads (Table 2). In addition, GST catalytic activities using ECA and ADI as substrates were 33 and 43% lower, respectively, in female Lake Apopka Marsh bullheads than in female bullheads from Lake Woodru (Table 2). Collectively, these data suggest that exposure to anthropogenic chemicals or natural compounds capable of inhibiting GST caused a loss of GST activity in female bullheads from Lake Apopka Marsh. A broad array of endogenous compounds as well as environmental xenobiotics have been shown to irreversibly inhibit GST activity (Mannervik & Danielson, 1988). This type of inhibition is characterized by a covalent modi®cation of an enzyme leading to an irreversible loss of activity. The list of inhibitors include bile acids, steroid hormone derivatives, drugs, metals, herbicides, pesticides, quinones, and chalcone derivatives (reviewed by Mannervik & Danielson, 1988). A number of compounds capable of inhibiting GST activity, including halogenated phenoxyacetic herbicides (Dierickx, 1985), alachlor (Moore et al., 1986) and atrazine (Egaas et al., 1995) have been historically used in the Lake Apopka Marsh area and have been detected in bullhead tissues. Alternatively, we are also investigating if the lower GST activities in female bullheads were caused by a decrease in the synthesis of GST protein(s) at the molecular level. In this regard, down-regulation of GST isoenzyme expression at the mRNA level and loss of GST catalytic activities have been demonstrated in rats exposed to the herbicide diquat (Gallagher et al., 1995).
E.P. Gallagher, K.M. Sheehy / Marine Environmental Research 50 (2000) 399±403
403
Why only the females were aected in the present study is also unknown. Although ®sh harvested during this time were not generally reproductively active, we cannot discount the possibility that a combination of chemical exposure and endocrine interactions may have aected GST pathways in the female bullheads. In summary, brown bullhead hepatic GSTs rapidly and eciently catalyze the conjugation of a number of GST reference substrates. However, female brown bullheads inhabiting a polluted site in central Florida have signi®cantly lower levels of hepatic GST activities when compared to female bullheads from a non-polluted site. Currently, the functional signi®cance of the loss of GST activity in ®sh from Lake Apopka Marsh and the mechanisms underlying the observed GST alterations in bullheads are unknown. Our laboratory is currently addressing these issues in the context of understanding GST regulation in aquatic animals. Acknowledgements This research was supported in part by an Intramural Research Award from the University of Florida College of Veterinary Medicine. The technical assistance of Dr. Tim Gross and Mr. William Johnson in the ®sh collections is gratefully appreciated. References Dierickx, P. J. (1985). Comp. Biochem. Physiol., 82C, 495±500. Egaas, E., Falls, J. G., Svendsen, N. O., Ramstad, H., Skaare, J. U., & Dauterman, W. C. (1995). Biochim. Biophys. Acta, 1243, 256±264. Gallagher, E. P., Sheehy, K. M., Lame, M. W., & Segall, H. J. (2000). Environ. Toxicol. Chem., 19, 319± 326. Gallagher, E. P., Buetler, T. M., Wang, C., Stapleton, P. L., Stahl, D. L., & Eaton, D. L. (1995). Toxicol. Appl. Pharmacol., 134, 81±91. George, S. (1994). In D. C. Malins, & G. K. Ostrander, Aquatic toxicology, molecular, biochemical and cellular perspectives (pp. 27±86). Ann Arbor: Lewis Publishers. Guillette Jr., L. J., Gross, T. S., Masson, G. R., Matter, J. M., Percival, H. F., & Woodward, A. R. (1994). Environ. Health Perspect, 102, 680±688. Hayes, J. D., & Pulford, D. J. (1996). CRC Crit. Rev. Biochem. Mol. Biol., 30, 445±600. Hayes, J. D., Judah, D. J., & Neal, G. E. (1993). Cancer Res., 53, 3887±3894. Mannervik, B., & Danielson, U. H. (1988). CRC Crit. Rev. Biochem., 23, 283±337. Moore, R. E., Davies, M. S., O' Connell, K. M., Harding, E. I., Wiegand, R. C., & Tiemeier, D. C. (1986). Nucleic Acids Res., 14, 7227±7335.