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Effects of pulp bleach plant effluents on hepatic xenobiotic biotransformation enzymes in fish: Laboratory and field studies
Marine Environmental.Research 17 (1985) 109-112
Effects of Pulp Bleach Plant Effluents on Hepatic Xenobiotic Biotransformation Enzymes in Fish: Laboratory and Field Studies L. F6rlin,* T. Anderssoon,* B.-E. Bengtsson,t J. Hiirdig++ & A. L a r s s o n t * Department of Zoophysiology,Universityof G6teborg, Sweden t Swedish Environment Protection Board, Brackish Water Toxicology Laboratory, Studsvik, Nyk6ping, Sweden .+ Institution of Zoophysiology,Universityof Uppsala, Sweden
The present preliminary report on studies of the influence of pulp bleach plant effluents on hepatic biotransformation enzymes in fish is a part of a major characterisation of biochemical and physiological effects of effluents from pulp industries in Sweden. Our studies on the xenobiotic biotransformation enzymes demonstrate, so far, that the pulp effluents have the potential to strongly induce 7-ethoxyresorufin-O-deethylase activity (EROD) infish liver and that fish living in the receiving water of a n effluent from a pulp bleach plant have induced hepatic EROD activities as well as higher hepatic UDP glucuronyltransferase activities than those living in an unpolluted area. It is thus apparent that measures of induction of biotransformation enzymes in fish liver may be a useful approach in monitoring the presence of potential hazardous compounds released from pulp industries. The removal of lipid soluble organic compounds (xenobiotics) in fish, through urine and bile excretion, is facilitated by enzyme systems converting the compounds to water soluble products. 1 The initial xenobiotic biotransformation in fish is often associated with the hepatic mixed function oxidase system (MFO) (cytochrome P-450 monooxygenase system). Cytochrome P-450 monooxygenases introduce functional groups into the substrate which facilitate the further biotransformation mediated by postoxidation enzyme systems such as hepatic glucuronidation enzymes. 109 Marine Environ. Res. 0141-1136/85/$03.30 © ElsevierApplied Science Publishers Ltd, England, 1985. Printed in Great Britain
L. F6rlin et al.
110
Induction of fish cytochrome P-450 activities in response to treatment and exposure to polycyclic aromatic hydrocarbons (PAH) and its possible use in biological monitoring programmes have received a great deal of attention. :-5 Inasmuch as induction of M F O is a detoxication response it may be regarded as quite sensitive, possibly occurring before gross changes such as growth abnormalities and reproduction disturbances are apparent. Induction of M F O in feral fish can assist in monitoring point sources of certain pollutants and possibly delineating larger polluted areas. 5 Induction of postoxidation enzymes in fish has been less thoroughly investigated. However, we recently observed increased hepatic U D P glucuronyltransferase activities in rainbow trout pretreated with PAH. 6, v In the present study, fourhorn sculpin (Myoxocephahts quadricornis) were exposed to a pulp bleach plant effluent for 9 months in the laboratory. The hepatic microsmal E R O D activity was markedly elevated (5-fold) in response to the exposure. In field investigations it was found that perch (Percafluviatilis)caught at a distance of 2, 4.5 and 8 km from a nrnol/mg
oror/train
EROO +
O6
i
04
+
Ii 22
2~ 22 I
~ 24
I
o N
II 111
R
I II III
t
III
OCT MAY AUG Fig. 1. Liver microsomal 7-ethoxyresorufin-O-deethylase activity ( E R O D ) in perch. Perch were caught at a distance of 2 (III), 4-5 (II) and 8 (I) km from a pulp bleach plant in May (MAY), August ( A U G ) and October (OCT). The reference (R) was located in an unpolluted area approximately 85 km south of the pulp industry. Number offish is given within or above each pile. *P < 0.05 when compared to R; - P < 0-05 when compared to I.
Effects O! pulp blcach plant ~Tfftuents on enzymes in fish UOP GLUCURONYLTR~NSFERASE
111
(PNP}
nmol/rng prot/rnin
04
'~ + "it"+ FLA.
+.L
*1 O2
26 26 27 o
R I II Ill
MAY
Fig. 2.
R
I II III
AUG
I21 22 I II III OCT
Liver microsoma| UD P glucuronyltransferaseactivity towards paranitrophenol (PNP) in perch. Key as for Fig. 1.
pulp industry (sulphate pulp bleach plant) on the Swedish east-coast had markedly higher hepatic microsomal EROD activities (6-19 times) than those caught in an unpolluted area (Fig. l). SDS-PAGE profiles of the hepatic microsomes seemed to indicate that certain protein(s) were also introduced by the pulp effluent. The elevated EROD activities in the fish caught in the vicinity of the pulp bleach plant were accompanied by increased hepatic U D P glucuronyltransferase activities in the May and October studies (Fig. 2). The results indicate the presence of PAH-inducers in the effluent from a pulp bleach plant. In addition, a 2-fold larger volume of pulp effluents was produced during spring than in summer and autumn. Therefore the apparent higher induction of the biotransformation enzyme activities seen in May may reflect a high exposure concentration of the wastewater from the bleached pulp in the receiving body of water during this period. The diagnostic value of the response parameters is indicated by the similarity between the effects noted in fish exposed to pulp effluents under natural field conditions and those found in laboratory experiments, and also by the fact that the same responses are present at different times of the year. REFERENCES . Bend, J. R. & James, M. O. In Biochemical and Biophysical Perspecti~'es in Marine Biology, (D, C. Malins & J. R. Sargent, eds). Academic Press. New York, pp. 126-88, 1978.
112
L. Forlin et at.
2. Stegman, J. J. In Polycyclic Hydrocarbons and Cancer. (P. O. P. Ts'o & H. V. Gelboin, eds). Academic Press, New York. Chapter 3, pp. 1-47, 1981. 3. FOrlin. L., Andersson, T., Koivusaari, U. & Hansson. T. Marine Environ. Res., 14, 47-58 (1984). 4. Binder, R. L., Melancon, M. J. & Lech, J. J. Drug Metab. Ret'., 15. 697-724. 1984. 5. Payne, J. F. In Ecological TestingJor the Marine Ent'ironment. (G. Persoone, E. Jaspers & C. Claus, eds). State Univ. Ghent and inst. Mar. Scient. Res., Bredene, Belgium, Chapter 1, pp. 625-55, 1984. 6. FOrlin, L. & Haux, C. Aquat. Toxicol., in press. 7. Andersson, T., Personen, M. &Johansson, C. Biochem. Pharmacol., in press.
Effects of Pulp Bleach Plant Effluents on Hepatic Xenobiotic Biotransformation Enzymes in Fish: Laboratory and Field Studies L. F6rlin,* T. Anderssoon,* B.-E. Bengtsson,t J. Hiirdig++ & A. L a r s s o n t * Department of Zoophysiology,Universityof G6teborg, Sweden t Swedish Environment Protection Board, Brackish Water Toxicology Laboratory, Studsvik, Nyk6ping, Sweden .+ Institution of Zoophysiology,Universityof Uppsala, Sweden
The present preliminary report on studies of the influence of pulp bleach plant effluents on hepatic biotransformation enzymes in fish is a part of a major characterisation of biochemical and physiological effects of effluents from pulp industries in Sweden. Our studies on the xenobiotic biotransformation enzymes demonstrate, so far, that the pulp effluents have the potential to strongly induce 7-ethoxyresorufin-O-deethylase activity (EROD) infish liver and that fish living in the receiving water of a n effluent from a pulp bleach plant have induced hepatic EROD activities as well as higher hepatic UDP glucuronyltransferase activities than those living in an unpolluted area. It is thus apparent that measures of induction of biotransformation enzymes in fish liver may be a useful approach in monitoring the presence of potential hazardous compounds released from pulp industries. The removal of lipid soluble organic compounds (xenobiotics) in fish, through urine and bile excretion, is facilitated by enzyme systems converting the compounds to water soluble products. 1 The initial xenobiotic biotransformation in fish is often associated with the hepatic mixed function oxidase system (MFO) (cytochrome P-450 monooxygenase system). Cytochrome P-450 monooxygenases introduce functional groups into the substrate which facilitate the further biotransformation mediated by postoxidation enzyme systems such as hepatic glucuronidation enzymes. 109 Marine Environ. Res. 0141-1136/85/$03.30 © ElsevierApplied Science Publishers Ltd, England, 1985. Printed in Great Britain
L. F6rlin et al.
110
Induction of fish cytochrome P-450 activities in response to treatment and exposure to polycyclic aromatic hydrocarbons (PAH) and its possible use in biological monitoring programmes have received a great deal of attention. :-5 Inasmuch as induction of M F O is a detoxication response it may be regarded as quite sensitive, possibly occurring before gross changes such as growth abnormalities and reproduction disturbances are apparent. Induction of M F O in feral fish can assist in monitoring point sources of certain pollutants and possibly delineating larger polluted areas. 5 Induction of postoxidation enzymes in fish has been less thoroughly investigated. However, we recently observed increased hepatic U D P glucuronyltransferase activities in rainbow trout pretreated with PAH. 6, v In the present study, fourhorn sculpin (Myoxocephahts quadricornis) were exposed to a pulp bleach plant effluent for 9 months in the laboratory. The hepatic microsmal E R O D activity was markedly elevated (5-fold) in response to the exposure. In field investigations it was found that perch (Percafluviatilis)caught at a distance of 2, 4.5 and 8 km from a nrnol/mg
oror/train
EROO +
O6
i
04
+
Ii 22
2~ 22 I
~ 24
I
o N
II 111
R
I II III
t
III
OCT MAY AUG Fig. 1. Liver microsomal 7-ethoxyresorufin-O-deethylase activity ( E R O D ) in perch. Perch were caught at a distance of 2 (III), 4-5 (II) and 8 (I) km from a pulp bleach plant in May (MAY), August ( A U G ) and October (OCT). The reference (R) was located in an unpolluted area approximately 85 km south of the pulp industry. Number offish is given within or above each pile. *P < 0.05 when compared to R; - P < 0-05 when compared to I.
Effects O! pulp blcach plant ~Tfftuents on enzymes in fish UOP GLUCURONYLTR~NSFERASE
111
(PNP}
nmol/rng prot/rnin
04
'~ + "it"+ FLA.
+.L
*1 O2
26 26 27 o
R I II Ill
MAY
Fig. 2.
R
I II III
AUG
I21 22 I II III OCT
Liver microsoma| UD P glucuronyltransferaseactivity towards paranitrophenol (PNP) in perch. Key as for Fig. 1.
pulp industry (sulphate pulp bleach plant) on the Swedish east-coast had markedly higher hepatic microsomal EROD activities (6-19 times) than those caught in an unpolluted area (Fig. l). SDS-PAGE profiles of the hepatic microsomes seemed to indicate that certain protein(s) were also introduced by the pulp effluent. The elevated EROD activities in the fish caught in the vicinity of the pulp bleach plant were accompanied by increased hepatic U D P glucuronyltransferase activities in the May and October studies (Fig. 2). The results indicate the presence of PAH-inducers in the effluent from a pulp bleach plant. In addition, a 2-fold larger volume of pulp effluents was produced during spring than in summer and autumn. Therefore the apparent higher induction of the biotransformation enzyme activities seen in May may reflect a high exposure concentration of the wastewater from the bleached pulp in the receiving body of water during this period. The diagnostic value of the response parameters is indicated by the similarity between the effects noted in fish exposed to pulp effluents under natural field conditions and those found in laboratory experiments, and also by the fact that the same responses are present at different times of the year. REFERENCES . Bend, J. R. & James, M. O. In Biochemical and Biophysical Perspecti~'es in Marine Biology, (D, C. Malins & J. R. Sargent, eds). Academic Press. New York, pp. 126-88, 1978.
112
L. Forlin et at.
2. Stegman, J. J. In Polycyclic Hydrocarbons and Cancer. (P. O. P. Ts'o & H. V. Gelboin, eds). Academic Press, New York. Chapter 3, pp. 1-47, 1981. 3. FOrlin. L., Andersson, T., Koivusaari, U. & Hansson. T. Marine Environ. Res., 14, 47-58 (1984). 4. Binder, R. L., Melancon, M. J. & Lech, J. J. Drug Metab. Ret'., 15. 697-724. 1984. 5. Payne, J. F. In Ecological TestingJor the Marine Ent'ironment. (G. Persoone, E. Jaspers & C. Claus, eds). State Univ. Ghent and inst. Mar. Scient. Res., Bredene, Belgium, Chapter 1, pp. 625-55, 1984. 6. FOrlin, L. & Haux, C. Aquat. Toxicol., in press. 7. Andersson, T., Personen, M. &Johansson, C. Biochem. Pharmacol., in press.