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Influence of oestradiol on induction of EROD activity in roach (Rutilus rutilus L.)
Chemosphere,Vol. 30, No. 8, pp. 1423-1428, 1995 Pclpgalnon
0045-6535(95)00035-6
Copyright O 1995 F.,lsevierScience Ltd Printed in Great Britain. All rights reserved 0045-6535/95 $9.50+0.00
INFLUENCE OF OESTRADIOL ON INDUCTION OF EROD ACTIVITY IN ROACH
(RUTILUSRUTILUSL.)
D.B. O'HARE, R. SIDDALL,P.W.J. ROBOTHAM& R.A. GILL
Centre for Research into Freshwater Pollution and Parasitology, University of Derby, Kedleston Road, Derby DE22 1GB, UK
(Received in C_~nnany 19 October 1994; accepted 23 December 1994)
Abstract
The induction ofethoxyresorufin-O-deethylase (EROD) activity by 13-naphthoflavone was examined in roach, Rutilus
rutilus, adrd~stered with oestradiol-1713 and in untreated fish. Constitutive levels of EROD activity were low and not significantly affected by steroid treatment hut oestradiol significantly suppressed the induction response. EROD induction following environmental exposure to PAils is therefore likely to be reduced in female fish, particularly during the spawning season. However, the response should still be of a sufficient magnitude to clearly demonstrate the presence of these xenobiotics in the freshwater environment.
Key words
Oestradiol; EROD induction, Rutilus rutilus
1. Introduction
The biotransformation of aquatic xenobiotics such as polycyclic aromatic hydrocarbons (PAHs) by hepatic cytochrome P-450-dependent mixed function oxygenase (MFO) systems is well documented in fish and provides a potentially valuable biomarker of environmental exposure to these contaminants (Payne et ai., 1987; O'I-Iare et al., 1994). However, cytochromes P-450 are also involved in the metabolism of steroids, including sex hormones, and each of these two classes of compounds could therefore influence the metabolism of the other (Stegeman & Woodin, 1984).
Several field studies have reported constitutive levels of cytochromes P-450 and MFO activity in fish to vary both seasonally (Koivusaari et al., 1981; LindstrOm-Seppii, 1985; Edwards et al., 1988) and between sexes (Stegeman & 1423
1424
Chevion, 1980; FOrlin & Haux, 1990; Snowberger Gray et aL, 1991), these differences being generally associated with levels of sex steroids. The female hormone oestradiol-1713, in particular, has been demonstrated to depress cytochrome P-450 content and monooxygenase activities (Frrlin & Hansson, 1982; Stegeman et al., 1982; Pajor et aL, 1990).
However, there is a paucity of information concerning the effects of the seasonal reproductive cycle of fish or of particular sex hormones on the level of induction of MFO activity following exposure to xenobiotics. Walton et at (1983) reported a depressed inducibility of aryl hydrocarbon hydroxylase (AHH) activity in cunners (Tautoglabrus adsperus) treated with crude oil during the spawning season when constitutive levels of I - I H activity were at a minimum. Oestradiol administration reduced the level of ethoxycoumarin- and ethoxyresorufin-O-deethylase (ECOD & EROD) activity induced in immature Rainbow trout (Oncorhynchus mykiss) by I~-naphthoflavone (Vodicnik & Leeh, 1983) and also reduced the induction of AHH activity by PCBs (F6rlin et al., 1984).
The roach, Rutilus rutilus L., is a common and widespread cyprinid in European freshwaters and the measurement of EROD activity in this species was proposed by O~-Iareet al. (1994) as a convenient biomonitor of PAH contamination. The present study aimed to determine experimentally whether the female sex hormone oestradiol-1713 can affect the induction response in roach and hence whether sex and seasonal factors are likely to limit the applicability of the biomarker in the field.
2. Materials and Methods
Roach (15-20 cm fork length) were obtained by dectroflshJng from the River Avon, England, in September 1994. They were maintained in dechlorinated tapwater at 12°C and fed on commercial pellet food for one week prior to experimental treatment and then divided between four treatment groups. Two groups received three successive intramuscular injections ofoestradiol-17[3 (3 mg/kg fish) in 0.1 ml polyethylene glycol (PEG) on alternate days, which has been reported to most effectively elicit gonadal effects and changes in the thyroid function in fish (Stegeman et at, 1982). The remaining two groups were injected with PEG alone.
Following the final oestradiol injection, EROD activity was induced in fish from one oestradiol pre-treated group and one control group by intraperitoneal injection with 13-naphthoflavone (100 mg.Kg"1) in 0.1 ml corn oil, the remaining two groups receiving corn oil alone. Three days post-induction all fish were sacrificed and their livers excised, wrapped in aluminium foil and flash frozen in liquid nitrogen. They were stored for a maximum of 3 days prior to biochemical analysis. The livers were thawed at room temperature, rinsed in 0.1 M phosphate buffer (pH 7.3) and weighed. EROD activity was assessed from the post-mitochondrial supernatent (PMS) at 25°C as described by O'Hare et al. (1994) and based
1425
on the method of Burke and Mayer (1974). Protein content was determined according to Lowry et al. (1951) and EROD activity expressed as picamoles per minute per milligram of protein. Statistical comparisons between groups were performed using Student's t-test. All chemicals were obtained from Sigma Chemical Company, except for 7ethoxyresorufin which was synthesised according to Klotz et al. (1984).
3. Results and Discussion
The mean constitutive level of EROD activity was lower in roach pre-treated with oestradiol-17[3 than in control fish but the two groups did not differ significantly from one another (P>0.05) (Table 1; Fig. 1). However, constitutive EROD activity was close to the lower limit of detection in this study which makes it difficult to identify any effects of steroid treatment.
Stegeman et al. (1982) were unable to detect EROD activity in immature brook trout, Salvelinusfontinalis, administered with oestradiol-17{3 or in untreated fish but benzo[a]pyrene hydroxylase activity was reported to be little affected by steroid treatment. In However, juvenile rainbow trout the administration of oestradiol-17[3 has been found to significantly decrease the liver microsomai metabolism ofbenzo[a]pyrene (F6rlin & Hansson, 1982). Snowberger Gray eta/. (1991) observed a depression in EROD activity of winter flounder, Pseudopleuronectes americanus, treated with oestradiol but the fish were collected from areas where they are known to be environmentally induced.
Table 1. Effect ofoestradiol-17[3 on constitutive levels of EROD activity and on EROD induction by 13-naphthoflavone (~-NF). No. fish (c:/¢)
Mean EROD activity (pmol/min/mg protein)
SE.
Control
1/4
0.345
0.06
Oestradiol
Control
2/2
0.192
0.02
Control
~-NF
3/3
4.350
0.25
Oestradiol
[3-NF
4/2
2.840
0.08
Pretreatment
Induction
Control
Induction of roach with [3-naphthoflavone ([3-NF) produced in a 13-fold increase in EROD activity above control levels. However, there was a significantly lower induction response among roach that were pretreated with oestradiol1713 (P<0.01), although EROD activity of these fish was still significantly higher than the control groups (P<0.01). The same effect was reported from Rainbow trout administered with oestradiol benzoate and induced with [3-NF by Vodicnik & Lech (1983) who also observed a decrease in the cytochrome P-450 content of these fsh. They suggested
1426
that the reduction in monooxygenase activity and cytochrome P-450 levels could represent a mechanism to ensure elevated oestrogen levels necessary to initiate and maintain vitellogenesis in pre-spawning female fish.
... o o o.
5
4
E E
3
0
E O. 2 _> gl
r-t 0
rvLU
0
I
Control
I
Oestredlol Treatment
I
BNF group
11
Oestradlol
+ BNF
Figure 1. Mean EROD activity (± standard error) in 4 treatment groups of roach: untreated, uninduced controls; oestradiol pretreated, uninduced; untreated, [3-NF induced; and oestradiol pretreated, [3-NF induced.
The experimental groups comprised both male and female fish but the extremely low variability in EROD activity within groups indicates that sex did not influence the constitutive EROD levels, the induction response or the effects of oestradiol- 1713 on EROD induction.
MFO induction in roach environmentally exposed to PAHs is therefore likely to be depressed in female fish, especially during the spawning season when elevated concentrations of oestradiol are present. Although this would complicate attempts to correlate the degree of induction with actual PAH exposure levels, the induction response would still be of a sufficient magnitude to clearly demonstrate the presence of these xenobiotics in the freshwater environment.
The mechanism(s) by which oestradiol-1713 depresses EROD induction and the implications of a reduced rate of metabolism of PAHs within fish tissues remain to be determined. However, the suitability ofF_ROD activity in roach as a biomonitor of PAH exposure despite sex and seasonal effects is currently being assessed in the field.
References Burke, D. & Mayer, R.T. (1974). Ethoxyresorufin: direct flurometric measurement of a microsomal O-dealkylation which is preferentially inducible by 3-methylcholanthrene. Drug Metob. Disp. 2, 223-239.
1427 Edwards, A.J., Addison, R.F., Willis, D.E. & Renton, K.W. (1988). Seasonal variation of hepatic mixed function oxidases in Winter flounder (Pseudopleuronectes americanus). Mar. Environ. Res. 26, 299-309.
Frrlin, L. & Hanssort, T. (1982). Effects of oestradiol-1713 and hypophysectomy on hepatic mixed function oxidases in rainbow trout. J. Endocrinol. 95, 245-252.
Frdin, L. & Haux, C. (1990). Sex differences in hepatic cytochrome P-450 monooxygenase activities in rainbow trout during an annual reproductive cycle. J. Endocrinol. 124, 207-213.
FOdin, L., Andersson, T., Koivusaari, U. & Hansson, T. (1984). Influence of biological and environmental factors on hepatic steroid and xenobiotic metabolism in fish: Interaction with PCB and 13-naphthoflavone. Mar. Environ. Res. 14, 47-58.
Klotz, A.V., Stegeman, J.J., Walsh, C. (1984). An alternative 7-ethoxyresorufin O-deethylase activity assay: A continuous visible speetrophotometdc method for measurement of eytochrome P-450 monooxygenase activity. Anal.
Biochem. 140, 138-145. Koivusaari, U., Hard, M. & Hanninen, O. (1981). Seasonal variation of hepatic biotransformation in female and male Rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 70C, 149-157.
Lindstr0m-Seppa, P. (1985). Seasonal variation of the xenobiotic metabolizing enzyme activities in the liver of male and female vendace (Coregonus albula L.). Aquat. Toxicol. 6, 323-331.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. (1951). Protein estimation with the Folin phenol reagent.
J. Biol. Chem. 193, 265-275. O'Hare, D.B., Robotham, P.J.W. & Gill, R.A. (1994). EROD measurement using post-mitochondrial supernatent (PMS) in roach (Rutilus rtttilus L.), a possible biomonitor for PAH contamination in the freshwater environment.
Chemosphere. In press. Pajor, A.M., Stegeman, J.J., Thomas, P. & Woodin, B.R. (1990). Feminization of the hepatic mirosomal cytochrome P-450 system in brook trout by estradiol, testosterone, and pituitary factors. J. Exper. Zoo/. 253, 51-60.
Payne, J.F., Fancey, L.L., Rahimtula, A.D. & Porter, E.L. (1987). Review and perspective on the use of mixed-function oxygenase enzymes in biological monitoring. Comp. Biochem. Physiol. 86C, 233-245.
1428 Snowberger Gray, E., Woodin, B.R. & Stegeman, J.J. (1991). Sex differences in hepatic monooxygenases in Winter flounder (Pseudopleuronectes americanus) and soup (Stenotomus chrysops) and the regulation of P450 forms by estradiol. ,/. Exper. Zool. 259, 330-342. Stegeman, J.J. & Chevion, M. (1980). Sex differences in cytochrome P-450 mixed-function oxygenase activity in gonadaUy mature trout. Biochem. Pharmacol. 29, 553-558. Stegeman, J.l. & Woodin, B.R. (1984). Differential regulation of hepatic xenobiotic and steroid metabolism in marine teleost species. Mar. Environ. Res. 14, 422-425. Stegeman, J.J., Pajor, A.M. & Thomas, P. (1982). Influence of estradiol and testosterone on cytochrome P-450 and monooxygenase activity in immature Brook trout, Salvelinusfontinalis. Biochem. Pharmacol. 51, 3979-3989. Vodicnik, M.J. & Lech, J.J. (1983). The effect of sex steroids and pregnenolone-16a-carbonitrile on the hepatic microsomal monooxygenase system of Rainbow trout (Salmo gairdneri). J. steroidBiochem. 18, 323-328. Walton, D.G., Fancey, L.L., Green, J.M., Kiceniuk, .J.W. & Penrose, W.R. (1983). Seasonal changes in aryi hydrocarbon hydroxylase activity of a marine fish Tcaaogolabrus adspersus (Walbaum) with and without petroleum exposure. Comp. Biochem. Physiol. 76C, 247-253.
0045-6535(95)00035-6
Copyright O 1995 F.,lsevierScience Ltd Printed in Great Britain. All rights reserved 0045-6535/95 $9.50+0.00
INFLUENCE OF OESTRADIOL ON INDUCTION OF EROD ACTIVITY IN ROACH
(RUTILUSRUTILUSL.)
D.B. O'HARE, R. SIDDALL,P.W.J. ROBOTHAM& R.A. GILL
Centre for Research into Freshwater Pollution and Parasitology, University of Derby, Kedleston Road, Derby DE22 1GB, UK
(Received in C_~nnany 19 October 1994; accepted 23 December 1994)
Abstract
The induction ofethoxyresorufin-O-deethylase (EROD) activity by 13-naphthoflavone was examined in roach, Rutilus
rutilus, adrd~stered with oestradiol-1713 and in untreated fish. Constitutive levels of EROD activity were low and not significantly affected by steroid treatment hut oestradiol significantly suppressed the induction response. EROD induction following environmental exposure to PAils is therefore likely to be reduced in female fish, particularly during the spawning season. However, the response should still be of a sufficient magnitude to clearly demonstrate the presence of these xenobiotics in the freshwater environment.
Key words
Oestradiol; EROD induction, Rutilus rutilus
1. Introduction
The biotransformation of aquatic xenobiotics such as polycyclic aromatic hydrocarbons (PAHs) by hepatic cytochrome P-450-dependent mixed function oxygenase (MFO) systems is well documented in fish and provides a potentially valuable biomarker of environmental exposure to these contaminants (Payne et ai., 1987; O'I-Iare et al., 1994). However, cytochromes P-450 are also involved in the metabolism of steroids, including sex hormones, and each of these two classes of compounds could therefore influence the metabolism of the other (Stegeman & Woodin, 1984).
Several field studies have reported constitutive levels of cytochromes P-450 and MFO activity in fish to vary both seasonally (Koivusaari et al., 1981; LindstrOm-Seppii, 1985; Edwards et al., 1988) and between sexes (Stegeman & 1423
1424
Chevion, 1980; FOrlin & Haux, 1990; Snowberger Gray et aL, 1991), these differences being generally associated with levels of sex steroids. The female hormone oestradiol-1713, in particular, has been demonstrated to depress cytochrome P-450 content and monooxygenase activities (Frrlin & Hansson, 1982; Stegeman et al., 1982; Pajor et aL, 1990).
However, there is a paucity of information concerning the effects of the seasonal reproductive cycle of fish or of particular sex hormones on the level of induction of MFO activity following exposure to xenobiotics. Walton et at (1983) reported a depressed inducibility of aryl hydrocarbon hydroxylase (AHH) activity in cunners (Tautoglabrus adsperus) treated with crude oil during the spawning season when constitutive levels of I - I H activity were at a minimum. Oestradiol administration reduced the level of ethoxycoumarin- and ethoxyresorufin-O-deethylase (ECOD & EROD) activity induced in immature Rainbow trout (Oncorhynchus mykiss) by I~-naphthoflavone (Vodicnik & Leeh, 1983) and also reduced the induction of AHH activity by PCBs (F6rlin et al., 1984).
The roach, Rutilus rutilus L., is a common and widespread cyprinid in European freshwaters and the measurement of EROD activity in this species was proposed by O~-Iareet al. (1994) as a convenient biomonitor of PAH contamination. The present study aimed to determine experimentally whether the female sex hormone oestradiol-1713 can affect the induction response in roach and hence whether sex and seasonal factors are likely to limit the applicability of the biomarker in the field.
2. Materials and Methods
Roach (15-20 cm fork length) were obtained by dectroflshJng from the River Avon, England, in September 1994. They were maintained in dechlorinated tapwater at 12°C and fed on commercial pellet food for one week prior to experimental treatment and then divided between four treatment groups. Two groups received three successive intramuscular injections ofoestradiol-17[3 (3 mg/kg fish) in 0.1 ml polyethylene glycol (PEG) on alternate days, which has been reported to most effectively elicit gonadal effects and changes in the thyroid function in fish (Stegeman et at, 1982). The remaining two groups were injected with PEG alone.
Following the final oestradiol injection, EROD activity was induced in fish from one oestradiol pre-treated group and one control group by intraperitoneal injection with 13-naphthoflavone (100 mg.Kg"1) in 0.1 ml corn oil, the remaining two groups receiving corn oil alone. Three days post-induction all fish were sacrificed and their livers excised, wrapped in aluminium foil and flash frozen in liquid nitrogen. They were stored for a maximum of 3 days prior to biochemical analysis. The livers were thawed at room temperature, rinsed in 0.1 M phosphate buffer (pH 7.3) and weighed. EROD activity was assessed from the post-mitochondrial supernatent (PMS) at 25°C as described by O'Hare et al. (1994) and based
1425
on the method of Burke and Mayer (1974). Protein content was determined according to Lowry et al. (1951) and EROD activity expressed as picamoles per minute per milligram of protein. Statistical comparisons between groups were performed using Student's t-test. All chemicals were obtained from Sigma Chemical Company, except for 7ethoxyresorufin which was synthesised according to Klotz et al. (1984).
3. Results and Discussion
The mean constitutive level of EROD activity was lower in roach pre-treated with oestradiol-17[3 than in control fish but the two groups did not differ significantly from one another (P>0.05) (Table 1; Fig. 1). However, constitutive EROD activity was close to the lower limit of detection in this study which makes it difficult to identify any effects of steroid treatment.
Stegeman et al. (1982) were unable to detect EROD activity in immature brook trout, Salvelinusfontinalis, administered with oestradiol-17{3 or in untreated fish but benzo[a]pyrene hydroxylase activity was reported to be little affected by steroid treatment. In However, juvenile rainbow trout the administration of oestradiol-17[3 has been found to significantly decrease the liver microsomai metabolism ofbenzo[a]pyrene (F6rlin & Hansson, 1982). Snowberger Gray eta/. (1991) observed a depression in EROD activity of winter flounder, Pseudopleuronectes americanus, treated with oestradiol but the fish were collected from areas where they are known to be environmentally induced.
Table 1. Effect ofoestradiol-17[3 on constitutive levels of EROD activity and on EROD induction by 13-naphthoflavone (~-NF). No. fish (c:/¢)
Mean EROD activity (pmol/min/mg protein)
SE.
Control
1/4
0.345
0.06
Oestradiol
Control
2/2
0.192
0.02
Control
~-NF
3/3
4.350
0.25
Oestradiol
[3-NF
4/2
2.840
0.08
Pretreatment
Induction
Control
Induction of roach with [3-naphthoflavone ([3-NF) produced in a 13-fold increase in EROD activity above control levels. However, there was a significantly lower induction response among roach that were pretreated with oestradiol1713 (P<0.01), although EROD activity of these fish was still significantly higher than the control groups (P<0.01). The same effect was reported from Rainbow trout administered with oestradiol benzoate and induced with [3-NF by Vodicnik & Lech (1983) who also observed a decrease in the cytochrome P-450 content of these fsh. They suggested
1426
that the reduction in monooxygenase activity and cytochrome P-450 levels could represent a mechanism to ensure elevated oestrogen levels necessary to initiate and maintain vitellogenesis in pre-spawning female fish.
... o o o.
5
4
E E
3
0
E O. 2 _> gl
r-t 0
rvLU
0
I
Control
I
Oestredlol Treatment
I
BNF group
11
Oestradlol
+ BNF
Figure 1. Mean EROD activity (± standard error) in 4 treatment groups of roach: untreated, uninduced controls; oestradiol pretreated, uninduced; untreated, [3-NF induced; and oestradiol pretreated, [3-NF induced.
The experimental groups comprised both male and female fish but the extremely low variability in EROD activity within groups indicates that sex did not influence the constitutive EROD levels, the induction response or the effects of oestradiol- 1713 on EROD induction.
MFO induction in roach environmentally exposed to PAHs is therefore likely to be depressed in female fish, especially during the spawning season when elevated concentrations of oestradiol are present. Although this would complicate attempts to correlate the degree of induction with actual PAH exposure levels, the induction response would still be of a sufficient magnitude to clearly demonstrate the presence of these xenobiotics in the freshwater environment.
The mechanism(s) by which oestradiol-1713 depresses EROD induction and the implications of a reduced rate of metabolism of PAHs within fish tissues remain to be determined. However, the suitability ofF_ROD activity in roach as a biomonitor of PAH exposure despite sex and seasonal effects is currently being assessed in the field.
References Burke, D. & Mayer, R.T. (1974). Ethoxyresorufin: direct flurometric measurement of a microsomal O-dealkylation which is preferentially inducible by 3-methylcholanthrene. Drug Metob. Disp. 2, 223-239.
1427 Edwards, A.J., Addison, R.F., Willis, D.E. & Renton, K.W. (1988). Seasonal variation of hepatic mixed function oxidases in Winter flounder (Pseudopleuronectes americanus). Mar. Environ. Res. 26, 299-309.
Frrlin, L. & Hanssort, T. (1982). Effects of oestradiol-1713 and hypophysectomy on hepatic mixed function oxidases in rainbow trout. J. Endocrinol. 95, 245-252.
Frdin, L. & Haux, C. (1990). Sex differences in hepatic cytochrome P-450 monooxygenase activities in rainbow trout during an annual reproductive cycle. J. Endocrinol. 124, 207-213.
FOdin, L., Andersson, T., Koivusaari, U. & Hansson, T. (1984). Influence of biological and environmental factors on hepatic steroid and xenobiotic metabolism in fish: Interaction with PCB and 13-naphthoflavone. Mar. Environ. Res. 14, 47-58.
Klotz, A.V., Stegeman, J.J., Walsh, C. (1984). An alternative 7-ethoxyresorufin O-deethylase activity assay: A continuous visible speetrophotometdc method for measurement of eytochrome P-450 monooxygenase activity. Anal.
Biochem. 140, 138-145. Koivusaari, U., Hard, M. & Hanninen, O. (1981). Seasonal variation of hepatic biotransformation in female and male Rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 70C, 149-157.
Lindstr0m-Seppa, P. (1985). Seasonal variation of the xenobiotic metabolizing enzyme activities in the liver of male and female vendace (Coregonus albula L.). Aquat. Toxicol. 6, 323-331.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. (1951). Protein estimation with the Folin phenol reagent.
J. Biol. Chem. 193, 265-275. O'Hare, D.B., Robotham, P.J.W. & Gill, R.A. (1994). EROD measurement using post-mitochondrial supernatent (PMS) in roach (Rutilus rtttilus L.), a possible biomonitor for PAH contamination in the freshwater environment.
Chemosphere. In press. Pajor, A.M., Stegeman, J.J., Thomas, P. & Woodin, B.R. (1990). Feminization of the hepatic mirosomal cytochrome P-450 system in brook trout by estradiol, testosterone, and pituitary factors. J. Exper. Zoo/. 253, 51-60.
Payne, J.F., Fancey, L.L., Rahimtula, A.D. & Porter, E.L. (1987). Review and perspective on the use of mixed-function oxygenase enzymes in biological monitoring. Comp. Biochem. Physiol. 86C, 233-245.
1428 Snowberger Gray, E., Woodin, B.R. & Stegeman, J.J. (1991). Sex differences in hepatic monooxygenases in Winter flounder (Pseudopleuronectes americanus) and soup (Stenotomus chrysops) and the regulation of P450 forms by estradiol. ,/. Exper. Zool. 259, 330-342. Stegeman, J.J. & Chevion, M. (1980). Sex differences in cytochrome P-450 mixed-function oxygenase activity in gonadaUy mature trout. Biochem. Pharmacol. 29, 553-558. Stegeman, J.l. & Woodin, B.R. (1984). Differential regulation of hepatic xenobiotic and steroid metabolism in marine teleost species. Mar. Environ. Res. 14, 422-425. Stegeman, J.J., Pajor, A.M. & Thomas, P. (1982). Influence of estradiol and testosterone on cytochrome P-450 and monooxygenase activity in immature Brook trout, Salvelinusfontinalis. Biochem. Pharmacol. 51, 3979-3989. Vodicnik, M.J. & Lech, J.J. (1983). The effect of sex steroids and pregnenolone-16a-carbonitrile on the hepatic microsomal monooxygenase system of Rainbow trout (Salmo gairdneri). J. steroidBiochem. 18, 323-328. Walton, D.G., Fancey, L.L., Green, J.M., Kiceniuk, .J.W. & Penrose, W.R. (1983). Seasonal changes in aryi hydrocarbon hydroxylase activity of a marine fish Tcaaogolabrus adspersus (Walbaum) with and without petroleum exposure. Comp. Biochem. Physiol. 76C, 247-253.