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Cytochrome P450 observations in Gulf fish
Marine Pollution Bulletin, Volume 27, pp. 293-296, 1993. Printed in Great Britain.
0025-326X/93 56.00+0.00 O 1993 Pergamon Press Ltd
Cytochrome P450 Observations in Gulf Fish JONNY BEYER and ANDERS GOKSOYR
Laboratory of Marine Molecular Biology (LMM), Universityof Bergen, HIB, N-5020 Bergen, Norway
Samples from nine fish species caught at different sites in the Gulf during leg IV of the Mt Mitchell cruise in the spring of 1992 were analysed for levels of cytochrome P450 1A enzymes. P450 monooxygenases catalyse the first step in the biotransformation of lipophilic xenobiotics. The P450 1A enzyme subfamily is induced by planar, organic compounds, and is used as a biomarker for exposure to oil compounds and PAH-type xenobiotics. The P450 1A analyses of the samples included both a catalytic method (EROD activity measurements) and immunochemical methods (Western blotting and an indirect ELISA). Polyclonal antibodies against P450 1A1 from cod (Gadus morhua) were used in the immunochemical assays. The Western blotting results demonstrated strong crossreaction between polyclonal anti-cod P450 1A1 IgG and a single protein band with an M~ of approximately 58 kDa in eight species examined. Elevated EROD levels were found in fish caught at the northern stations compared to individuals of the same species caught further south. The results from the P450 1AI ELISA analyses supported these findings, although with some differences between species. Overall, conclusive statements about the P450 1A status in fish from the survey area are difficult to make from this investigation. This is mainly due to diverse species composition and the small size of the catches at the different fishing locations.
source of the oil spill in Kuwait after the 1991 Gulf War. If a gradient in P450 1A1 levels was observed, then petroleum would appear to be responsible for induction of P450 1A1 in the fish. This would indicate that the fish from the oil exposed area have been accumulating biochemically significant levels of petroleum-derived compounds. The relative size of the P450 1A1 protein in different Gulf species was analysed by Western blotting using a polyclonal anti-cod P450 1A1 antibody. The P450 1A1 levels in individual liver samples were measured catalytically by EROD activity and immunologically by an indirect, semiquantitative P450 1A1 ELISA. The immunochemical method was used to control effects of the sample preservation and preparation techniques on the P450 enzyme activity. Materials and M e t h o d s
Stations and fish During leg IV of the 1992 NOAA ship Mt Mitchell cruise, fish were caught at eight of the nine stations A-I (Table 1). No fish were caught at station C (Dawhat al Asli). The distribution of the fishing locations is shown in Fig. 1. The approximate distances in km between Kuwait City harbour (station A) and the other fishing locations are included in Table 1. Catfish and groupers, which are known to be of suitable size, able to survive in water tanks prior to sampling and relatively nonmigratory and demersal, were defined as target species for the P450 sampling. However, due to small catches a number of new target species had to be defined (Table 2). For the same reason two closely related species of groupers (Epinephelus tauvina and Epinephelus areolatus) are treated as one group in this report. The fish were caught mainly using hook and line and also small bottom trawls operated from Jensen launchers. Live trawled fish were transported in small water tanks back to Mt Mitchell and transferred to bigger tanks until sampling.
Exposure of ecosystems to oil hydrocarbons after major oil spills, is known to cause a number of environmental effects. Classically, these are monitored in either ecological or chemical terms. More recently, molecular biomarkers have come into use (e.g. Stegeman et al., 1992), elucidating whether the exposed organisms are biochemically or physiologically affected by the oil compounds. Among the biomarkers best described are monooxygenases in the cytochrome P450 system and especially the 1A subfamily, in which P450 1A1 is the single known member in fish. P450 1A1 is induced by Sampling and tissue preparation PAHs, which are present in crude oils. P450 1A1 as a The fish were killed by a blow to the head. Tissue biomarker for exposure of fish to marine hydrocarbon samples were kept on ice during sample preparation. pollution is reviewed in Payne et al. (1987) and The liver was the main organ of concern, but to Goksoyr & F6rlin (1992). evaluate other target organs, kidney, heart, gills and The objective of this study was to test whether the spleen were sampled from some of the fish. Tissues level of P450 1A1 in fish from the coastal area of were quickly cut into small pieces and frozen at -80°C Kuwait to Qatar was associated with proximity to the in 87% glycerol. After transport on dry ice to the 293
Marine Pollution t3~aiietm
'
IRAN
I Fig. 1 Distribution of the nine fishing locations (station A-I) during leg
IV of the 1992 NOAA ship Mt Mitchell cruise in the Gulf. TABLE 1
The nine fishing locations during leg IV of the 1992 NOAA ship Mt Mitchellcruise in the Gulf. Station A B C D E F G H 1
Date
Location
24 April 25 April 26 April 27 April 28 April 29 April 30 April 1 May 2 May
Jun al Kuwait RasalQualayah Dawhat al Asli Manifah Abu Ali North Dawal Abu All Rennie Shoals Bahrain Qatar
Approximate area
Km
Statistical analyses
29°29'N 28°58'N 28°22'N 27°42'N 27°2YN 27°19'N 27°02'N 26°08'N 25°56'N
0 65 140 240 290 310 390 480 550
Correlation analyses of the EROD/P450 1A1 observations were performed on log-transformed individual data of three fish species and by using the JMP Software for Statistical Visualization (SAS Institute) for Macintosh. The correlation of the two dependent variables was obtained by X and Y plots with fitting bivariate normal elipses at P=0.950. The correlation is reported by the r 2. The level of significance is in all cases set to p < 0.05.
48°02"E 48°19'E 48°37'E 49°15'E 49°36'E 49°5 I'E 50°4 I'E 50°48'E 51°39'E
Km = Approximate distance from Kuwait City harbour. No fish were caught at station C.
laboratory, the glycerol was removed by flushing with 0.1 M phosphate-buffer (pH 7.4). The buffer also included KCI (0.15 M), EDTA (1 mM), DTT (1 raM) and glycerol (10%). The tissue samples were then homogenized in four volumes of buffer using a Potter Elvehjem type glass homogenizer with teflon pestle (five strokes). Supernatants were obtained by a centrifugation for 20 min at 10 000 g in an Eppendorf centrifuge. The 10 000 g supernatants were used in all P450 1A1 determinations. Biochemical analyses
The protein content of the samples were measured by the method of Bradford using BSA as a standard (Bradford, 1976). The Western blotting was carried out according to Goksoyr et aL (1991). The EROD activity was measured by the method described by F6rlin et aL (1993). The EROD assay was not optimized for each species tested, The detection limit for the EROD assay was set to 1 pmol resorufin min -1 mg protein -1. The 294
P450 1A1 ELISA absorbances were measured by the method given by Goksoyr (1991).
Results and D i s c u s s i o n The total number of individual fish sampled was 68, with the number of each species varying between 1 and 20 (Table 2). None of the fish species targeted for P450 TABLE 2 Fish species and numbers (n) of individuals sampled for P450 analyses during leg IV of the 1992 NOAA ship Mt Mitchell cruise in the Gulf. The two species of groupers are treated as one group in the data presentation. Latin name
Local name
Arius thalassinus Epinephelus tauvina Epinephelus areolatus Nemipterus tolu Lethrinus kallopterus Argyropsfilarnetosus Acanthopagrus bifasciatus Acanthopagrus latus Plectorhynchus pictus
Chim Hamoor Gataw Bassi Sheiry Andag Fasker Sheim Fersh
nn-- Not named.
English name
(n)
Sea catfish Grouper Areolatus grouper Treadfin bream Pigface bream Sea bream Two-banded porgy Sea bream nn
3 3 6 20 20 2 3 1 10
V o l u m e 27
sampling (catfish and groupers) were caught at all stations of the cruise (stations A-I). Hence new target species had to be defined during the cruise, bringing the number of species sampled up to nine (Table 2). Still, the number of individuals caught and sampled varied much, giving the investigation a difficult statistical basis. It was also difficult to keep trawled fish alive during transport on Jensen launchers back to Mt Mitchell. Polyclonal anti-cod P450 1A1 IgG was tested for cross-reaction against proteins in liver samples from eight individuals of different target species. The Western blot (Fig. 2) displayed single bands in all samples examined. All the bands corresponded to the size of P450 1A1 in cod (Mr-- 58 kDa). The coastal area from Kuwait to Abu Ali in Saudi Arabia was most affected by the 1991 oil spill. Hence the hepatic P450 1A1 levels in fish from this area were expected to be higher than in the reference areas further south (coast of Qatar). For each species, the highest EROD values were observed in fish caught at the northern stations (Table 3). Samples from extrahepatic tissues (kidney, heart, spleen and gills) displayed very low EROD activity compared to the observed levels in the liver (extrahepatic activities not shown). The P450 1A1 ELISA results showed more or less the same trend as the EROD results, finding the highest values at the northern stations (Table 4). The bivariate correlation analysis (Fig. 3) showed the EROD activity levels and the P450 1A1 ELISA absorbances (using logtransformed data) to be significantly correlated (p< 0.05) in groupers and sheiry (r2=0.63, p=0.011, n = 9 and r2=0.59, p=0.0001, n = 2 0 , respectively),
1
2
3
4
5
6
7
8
m
9
10
Samples Fig. 2 Western blot of S D S - P A G E using liver 10 0 0 0 g s u p e r u a t a n t s of individuals from eight fish species from leg I V of the 1992 N O A A ship Mt Mitchell cruise in the Gulf. The eight species are: fersh (sample 1), sea catfish (2), g r o u p e r (3), fasker (4), andag (5), sheim (6), bassi (7), sheiry (8). The individuals of each species displaying highest P 4 5 0 1A1 levels were used in the analysis. The blot was incubated with polyclonal anti-cod P 4 5 0 1A1 IgG. Samples 9 and 10 are 10 0 0 0 g s u p e r n a t a n t s of
liver from control cod and 13-naphthoflavone (I~NF) exposed cod, respectively. 13NF is a p o t e n t P 4 5 0 1A1 inducer, m - B i n R a d prestained molecular weight standard (low range). Ten ixg
sample protein were loaded in each well. The single protein band in sample well 1-10 correlates with an M r of a p p r o x i m ately 58 kDa.
whereas bassi showed a borderline significance with r 2 = 0.19, p-- 0.056, n = 20. In summary, the Western blotting demonstrated cross-reaction between the polyclonal anti-cod P450 1A1 IgG and a single protein band with an Mr of approximately 58 kDa in all eight species examined. An
TABLE 3
Mean pmol resorufin min -l m g protein -1 ± SD (n) of 7-ethoxy resorufin O-deethylase activity ( E R O D ) in liver 10 0 0 0 g s u p e m a t a n t s of fish species caught at eight fishing locations during leg IV of the 1992 N O A A ship Mt Mitchell cruise in the Gulf. The detection limit was set to 1 p m o l resorufin min -1 nag protein -I.
Station
Andag
A
-
B
-
O E F G H I
136+112(2) . .
Bassi
Catfish
-
151 (1) . . . 56 (1)
170 + 1 (2) 68 + 54 (15) 26±19(2) . . 25 (1) . .
Fasker
Fersh
. .
. . -
. .
271±89(3) . . .
.
Groupers 180 (1) . .
Sheim
-
-
515(1) 170+76(3) 209:1:68 (6)
67(1) -
108±86(10)
-
. .
16±29(5) 26+10(2)
. . 372+127(10)
Sheiry
.
. 1(1)
- - N o s a m p l i n g of the given species at that station. TABLE 4
Mean P 4 5 0 1A1 E L I S A a b s o r b a n c e s + SD (n) measured in liver 10 0 0 0 g s u p e r n a t a n t s of fish species caught at eight fishing locations d u r i n g leg IV of the 1992 N O A A ship Mt Mitchell cruise in the Gulf. T h e semiquantitative E L I S A was p e r f o r m e d by using polyclonal antibodies against P 4 5 0 1A1 from cod
Station
Andag
Bassi*
Catfish
Fasker -
A
-
-
0.43 (1)
B
-
0.028 + o.ooo (2)
.
D E F G H I
0.70+0.03(2) . .
0.021 + 0.012 (15) 0.011+0.004(2) . . 0.016 (1) . . .
. . 0.26 (1)
(Gadus morhua). Fersh
Groupers* 0.17 (1)
.
. 0 . 7 4 + 0 . 1 0 (3) . .
.
.
. 1.12 + 0.25
Sheiry*
Sheim
-
-
0.083(1) 0 . 0 5 1 + 0 . 0 1 1 (3) 0.055+0.018(6)
0.27(1) -
.
. 0.032+0.012(5) 0 . 0 4 2 + 0 . 0 1 4 (2) .
. 0.034 (1)
0.047 + 0.016 (10)
-
- -- N o s a m p l i n g of the given species at that station. *The results presented for these species are from a separate assay using antibodies purified b y magnetic beads (Dynabeads, Dynal) conjugated to c o d P 4 5 0 1A1, hence the lower absorbances observed. 295
<
B. Bassi
A. Groupers
-0,6
m
~2
i°~ -1,0 -0,8 -1,2
[]
1
-1,4 -1,6
tt3
-1,4
m
-1,8
m nl
Off
-1,6 -1,8
<
-1,2
El
1 []
-2,0 []
!
!
1
2
Log EROD activity
-2,2
()
!
[]
!
1
1
2
~
Log EROD activity
C. Sheiry
-1,0 i
-1,2 ms Fig. 3 Correlation plots of EROD activity vs. P450 IA1 EL1SA
-1,4 1=!ii
[]
-1,6 -1,8
!
2 Log EROD activity
increase with proximity to the area most affected by the 1991 oil spill was observed for the EROD activity in fish livers, supported also by the P450 1A1-ELISA measurements. However, conclusive statements about the P450 1A1 status in fish from the region investigated cannot be made. This is mainly because the catches of live fish were too small and diverse, resulting in large differences in species composition between stations. The study was supported by grants from the Norwegian State Pollution Control Authority (SFT). We want to express our gratitude to Sissel O. Olsen for assistance during analyses and to Bernard K.-M. Gadagbui for assistance during analyses and writing.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Analyt. Biochem. 72,248-254. F6rlin, L., Goksoyr, A. & Husoy, A.-M. (1993). Cytochrome P450 monooxygenase as indicator of PCB/dioxin like compounds in fish.
296
absorbances in liver samples (10 000 g supernatants) of fish from leg IV of the 1992 NOAA ship Mt Mitchell cruise in the Gulf. Individuals of three fish species (A) groupers, (B) bassi and (C) sheiry are used in the test and all values are logtransformed prior to testing. The p-value display the level of significance of the correlation while 'n' is the number of individuals tested of each species. The results obtained were: groupers, r2=0.63, p=0.011, n=9. Bassi, r2=0.19, p=0.056, n = 20. Sheiry, r 2= 0.59, p = 0.0001, n = 20.
In Biological Monitoring of Estuarine and Coastal Waters (K. Kramer, ed.). Boca Raton, Florida, CRC Press (in press). Goks~yr, A. (1991). A semi-quantitative cytochrome P4501A1 ELISA: A simple method for studying the monooxygenase inducation response in environmental monitoring and ecotoxicological testing offish. Sci. Tot. Envir. 101,255-262. Goksoyr, A., Andersson, T., Buhler, D. R., Stegeman, J. J., Williams, D. E. & F6rlin, L. (1991). Immunochemical cross-reactivity of 13naphthotlavone-inducible cytochrome P-450 (P4501A)in liver microsomes from different fish species and rat. FL~h. l'h>~iol. Biochem. 9, 1-13. Goks~yr, A. & Ffrlin, L. (1992). The cytochrome P450 system in fish, aquatic toxicology, and environmental monitoring. Aquat. ToxicoL 22,287-312. Payne, J. E, Fancey, L. L., Rahimtula, A. D. & Porter, E. Ix. (1987). Review and perspective on the use of mixed-function oxygenase enzymes in biological monitoring. Comp. Biochem. Physiol. 86C, 233-245. Stegeman, J. J., Brouwer, M., Richard, T. D. G., F6rlin, [.., Fowler, B. A., Sanders, B. M. & Van Veld, P. A. (t992). Molecular responses to environmental contamination: Enzyme and protein systems as indicators of chemical exposure and effect. In Biornarkers: Biochemical Physiological and Histological Markers of Anthropogenic Stress (R. J. Huggett, R. A. Kimerle, P. M. Mehrte .It. & H. L Bergman, eds), pp. 235-335. Lewis Publishers, Chelsea, MI, USA.
0025-326X/93 56.00+0.00 O 1993 Pergamon Press Ltd
Cytochrome P450 Observations in Gulf Fish JONNY BEYER and ANDERS GOKSOYR
Laboratory of Marine Molecular Biology (LMM), Universityof Bergen, HIB, N-5020 Bergen, Norway
Samples from nine fish species caught at different sites in the Gulf during leg IV of the Mt Mitchell cruise in the spring of 1992 were analysed for levels of cytochrome P450 1A enzymes. P450 monooxygenases catalyse the first step in the biotransformation of lipophilic xenobiotics. The P450 1A enzyme subfamily is induced by planar, organic compounds, and is used as a biomarker for exposure to oil compounds and PAH-type xenobiotics. The P450 1A analyses of the samples included both a catalytic method (EROD activity measurements) and immunochemical methods (Western blotting and an indirect ELISA). Polyclonal antibodies against P450 1A1 from cod (Gadus morhua) were used in the immunochemical assays. The Western blotting results demonstrated strong crossreaction between polyclonal anti-cod P450 1A1 IgG and a single protein band with an M~ of approximately 58 kDa in eight species examined. Elevated EROD levels were found in fish caught at the northern stations compared to individuals of the same species caught further south. The results from the P450 1AI ELISA analyses supported these findings, although with some differences between species. Overall, conclusive statements about the P450 1A status in fish from the survey area are difficult to make from this investigation. This is mainly due to diverse species composition and the small size of the catches at the different fishing locations.
source of the oil spill in Kuwait after the 1991 Gulf War. If a gradient in P450 1A1 levels was observed, then petroleum would appear to be responsible for induction of P450 1A1 in the fish. This would indicate that the fish from the oil exposed area have been accumulating biochemically significant levels of petroleum-derived compounds. The relative size of the P450 1A1 protein in different Gulf species was analysed by Western blotting using a polyclonal anti-cod P450 1A1 antibody. The P450 1A1 levels in individual liver samples were measured catalytically by EROD activity and immunologically by an indirect, semiquantitative P450 1A1 ELISA. The immunochemical method was used to control effects of the sample preservation and preparation techniques on the P450 enzyme activity. Materials and M e t h o d s
Stations and fish During leg IV of the 1992 NOAA ship Mt Mitchell cruise, fish were caught at eight of the nine stations A-I (Table 1). No fish were caught at station C (Dawhat al Asli). The distribution of the fishing locations is shown in Fig. 1. The approximate distances in km between Kuwait City harbour (station A) and the other fishing locations are included in Table 1. Catfish and groupers, which are known to be of suitable size, able to survive in water tanks prior to sampling and relatively nonmigratory and demersal, were defined as target species for the P450 sampling. However, due to small catches a number of new target species had to be defined (Table 2). For the same reason two closely related species of groupers (Epinephelus tauvina and Epinephelus areolatus) are treated as one group in this report. The fish were caught mainly using hook and line and also small bottom trawls operated from Jensen launchers. Live trawled fish were transported in small water tanks back to Mt Mitchell and transferred to bigger tanks until sampling.
Exposure of ecosystems to oil hydrocarbons after major oil spills, is known to cause a number of environmental effects. Classically, these are monitored in either ecological or chemical terms. More recently, molecular biomarkers have come into use (e.g. Stegeman et al., 1992), elucidating whether the exposed organisms are biochemically or physiologically affected by the oil compounds. Among the biomarkers best described are monooxygenases in the cytochrome P450 system and especially the 1A subfamily, in which P450 1A1 is the single known member in fish. P450 1A1 is induced by Sampling and tissue preparation PAHs, which are present in crude oils. P450 1A1 as a The fish were killed by a blow to the head. Tissue biomarker for exposure of fish to marine hydrocarbon samples were kept on ice during sample preparation. pollution is reviewed in Payne et al. (1987) and The liver was the main organ of concern, but to Goksoyr & F6rlin (1992). evaluate other target organs, kidney, heart, gills and The objective of this study was to test whether the spleen were sampled from some of the fish. Tissues level of P450 1A1 in fish from the coastal area of were quickly cut into small pieces and frozen at -80°C Kuwait to Qatar was associated with proximity to the in 87% glycerol. After transport on dry ice to the 293
Marine Pollution t3~aiietm
'
IRAN
I Fig. 1 Distribution of the nine fishing locations (station A-I) during leg
IV of the 1992 NOAA ship Mt Mitchell cruise in the Gulf. TABLE 1
The nine fishing locations during leg IV of the 1992 NOAA ship Mt Mitchellcruise in the Gulf. Station A B C D E F G H 1
Date
Location
24 April 25 April 26 April 27 April 28 April 29 April 30 April 1 May 2 May
Jun al Kuwait RasalQualayah Dawhat al Asli Manifah Abu Ali North Dawal Abu All Rennie Shoals Bahrain Qatar
Approximate area
Km
Statistical analyses
29°29'N 28°58'N 28°22'N 27°42'N 27°2YN 27°19'N 27°02'N 26°08'N 25°56'N
0 65 140 240 290 310 390 480 550
Correlation analyses of the EROD/P450 1A1 observations were performed on log-transformed individual data of three fish species and by using the JMP Software for Statistical Visualization (SAS Institute) for Macintosh. The correlation of the two dependent variables was obtained by X and Y plots with fitting bivariate normal elipses at P=0.950. The correlation is reported by the r 2. The level of significance is in all cases set to p < 0.05.
48°02"E 48°19'E 48°37'E 49°15'E 49°36'E 49°5 I'E 50°4 I'E 50°48'E 51°39'E
Km = Approximate distance from Kuwait City harbour. No fish were caught at station C.
laboratory, the glycerol was removed by flushing with 0.1 M phosphate-buffer (pH 7.4). The buffer also included KCI (0.15 M), EDTA (1 mM), DTT (1 raM) and glycerol (10%). The tissue samples were then homogenized in four volumes of buffer using a Potter Elvehjem type glass homogenizer with teflon pestle (five strokes). Supernatants were obtained by a centrifugation for 20 min at 10 000 g in an Eppendorf centrifuge. The 10 000 g supernatants were used in all P450 1A1 determinations. Biochemical analyses
The protein content of the samples were measured by the method of Bradford using BSA as a standard (Bradford, 1976). The Western blotting was carried out according to Goksoyr et aL (1991). The EROD activity was measured by the method described by F6rlin et aL (1993). The EROD assay was not optimized for each species tested, The detection limit for the EROD assay was set to 1 pmol resorufin min -1 mg protein -1. The 294
P450 1A1 ELISA absorbances were measured by the method given by Goksoyr (1991).
Results and D i s c u s s i o n The total number of individual fish sampled was 68, with the number of each species varying between 1 and 20 (Table 2). None of the fish species targeted for P450 TABLE 2 Fish species and numbers (n) of individuals sampled for P450 analyses during leg IV of the 1992 NOAA ship Mt Mitchell cruise in the Gulf. The two species of groupers are treated as one group in the data presentation. Latin name
Local name
Arius thalassinus Epinephelus tauvina Epinephelus areolatus Nemipterus tolu Lethrinus kallopterus Argyropsfilarnetosus Acanthopagrus bifasciatus Acanthopagrus latus Plectorhynchus pictus
Chim Hamoor Gataw Bassi Sheiry Andag Fasker Sheim Fersh
nn-- Not named.
English name
(n)
Sea catfish Grouper Areolatus grouper Treadfin bream Pigface bream Sea bream Two-banded porgy Sea bream nn
3 3 6 20 20 2 3 1 10
V o l u m e 27
sampling (catfish and groupers) were caught at all stations of the cruise (stations A-I). Hence new target species had to be defined during the cruise, bringing the number of species sampled up to nine (Table 2). Still, the number of individuals caught and sampled varied much, giving the investigation a difficult statistical basis. It was also difficult to keep trawled fish alive during transport on Jensen launchers back to Mt Mitchell. Polyclonal anti-cod P450 1A1 IgG was tested for cross-reaction against proteins in liver samples from eight individuals of different target species. The Western blot (Fig. 2) displayed single bands in all samples examined. All the bands corresponded to the size of P450 1A1 in cod (Mr-- 58 kDa). The coastal area from Kuwait to Abu Ali in Saudi Arabia was most affected by the 1991 oil spill. Hence the hepatic P450 1A1 levels in fish from this area were expected to be higher than in the reference areas further south (coast of Qatar). For each species, the highest EROD values were observed in fish caught at the northern stations (Table 3). Samples from extrahepatic tissues (kidney, heart, spleen and gills) displayed very low EROD activity compared to the observed levels in the liver (extrahepatic activities not shown). The P450 1A1 ELISA results showed more or less the same trend as the EROD results, finding the highest values at the northern stations (Table 4). The bivariate correlation analysis (Fig. 3) showed the EROD activity levels and the P450 1A1 ELISA absorbances (using logtransformed data) to be significantly correlated (p< 0.05) in groupers and sheiry (r2=0.63, p=0.011, n = 9 and r2=0.59, p=0.0001, n = 2 0 , respectively),
1
2
3
4
5
6
7
8
m
9
10
Samples Fig. 2 Western blot of S D S - P A G E using liver 10 0 0 0 g s u p e r u a t a n t s of individuals from eight fish species from leg I V of the 1992 N O A A ship Mt Mitchell cruise in the Gulf. The eight species are: fersh (sample 1), sea catfish (2), g r o u p e r (3), fasker (4), andag (5), sheim (6), bassi (7), sheiry (8). The individuals of each species displaying highest P 4 5 0 1A1 levels were used in the analysis. The blot was incubated with polyclonal anti-cod P 4 5 0 1A1 IgG. Samples 9 and 10 are 10 0 0 0 g s u p e r n a t a n t s of
liver from control cod and 13-naphthoflavone (I~NF) exposed cod, respectively. 13NF is a p o t e n t P 4 5 0 1A1 inducer, m - B i n R a d prestained molecular weight standard (low range). Ten ixg
sample protein were loaded in each well. The single protein band in sample well 1-10 correlates with an M r of a p p r o x i m ately 58 kDa.
whereas bassi showed a borderline significance with r 2 = 0.19, p-- 0.056, n = 20. In summary, the Western blotting demonstrated cross-reaction between the polyclonal anti-cod P450 1A1 IgG and a single protein band with an Mr of approximately 58 kDa in all eight species examined. An
TABLE 3
Mean pmol resorufin min -l m g protein -1 ± SD (n) of 7-ethoxy resorufin O-deethylase activity ( E R O D ) in liver 10 0 0 0 g s u p e m a t a n t s of fish species caught at eight fishing locations during leg IV of the 1992 N O A A ship Mt Mitchell cruise in the Gulf. The detection limit was set to 1 p m o l resorufin min -1 nag protein -I.
Station
Andag
A
-
B
-
O E F G H I
136+112(2) . .
Bassi
Catfish
-
151 (1) . . . 56 (1)
170 + 1 (2) 68 + 54 (15) 26±19(2) . . 25 (1) . .
Fasker
Fersh
. .
. . -
. .
271±89(3) . . .
.
Groupers 180 (1) . .
Sheim
-
-
515(1) 170+76(3) 209:1:68 (6)
67(1) -
108±86(10)
-
. .
16±29(5) 26+10(2)
. . 372+127(10)
Sheiry
.
. 1(1)
- - N o s a m p l i n g of the given species at that station. TABLE 4
Mean P 4 5 0 1A1 E L I S A a b s o r b a n c e s + SD (n) measured in liver 10 0 0 0 g s u p e r n a t a n t s of fish species caught at eight fishing locations d u r i n g leg IV of the 1992 N O A A ship Mt Mitchell cruise in the Gulf. T h e semiquantitative E L I S A was p e r f o r m e d by using polyclonal antibodies against P 4 5 0 1A1 from cod
Station
Andag
Bassi*
Catfish
Fasker -
A
-
-
0.43 (1)
B
-
0.028 + o.ooo (2)
.
D E F G H I
0.70+0.03(2) . .
0.021 + 0.012 (15) 0.011+0.004(2) . . 0.016 (1) . . .
. . 0.26 (1)
(Gadus morhua). Fersh
Groupers* 0.17 (1)
.
. 0 . 7 4 + 0 . 1 0 (3) . .
.
.
. 1.12 + 0.25
Sheiry*
Sheim
-
-
0.083(1) 0 . 0 5 1 + 0 . 0 1 1 (3) 0.055+0.018(6)
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.
. 0.032+0.012(5) 0 . 0 4 2 + 0 . 0 1 4 (2) .
. 0.034 (1)
0.047 + 0.016 (10)
-
- -- N o s a m p l i n g of the given species at that station. *The results presented for these species are from a separate assay using antibodies purified b y magnetic beads (Dynabeads, Dynal) conjugated to c o d P 4 5 0 1A1, hence the lower absorbances observed. 295
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B. Bassi
A. Groupers
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C. Sheiry
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increase with proximity to the area most affected by the 1991 oil spill was observed for the EROD activity in fish livers, supported also by the P450 1A1-ELISA measurements. However, conclusive statements about the P450 1A1 status in fish from the region investigated cannot be made. This is mainly because the catches of live fish were too small and diverse, resulting in large differences in species composition between stations. The study was supported by grants from the Norwegian State Pollution Control Authority (SFT). We want to express our gratitude to Sissel O. Olsen for assistance during analyses and to Bernard K.-M. Gadagbui for assistance during analyses and writing.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Analyt. Biochem. 72,248-254. F6rlin, L., Goksoyr, A. & Husoy, A.-M. (1993). Cytochrome P450 monooxygenase as indicator of PCB/dioxin like compounds in fish.
296
absorbances in liver samples (10 000 g supernatants) of fish from leg IV of the 1992 NOAA ship Mt Mitchell cruise in the Gulf. Individuals of three fish species (A) groupers, (B) bassi and (C) sheiry are used in the test and all values are logtransformed prior to testing. The p-value display the level of significance of the correlation while 'n' is the number of individuals tested of each species. The results obtained were: groupers, r2=0.63, p=0.011, n=9. Bassi, r2=0.19, p=0.056, n = 20. Sheiry, r 2= 0.59, p = 0.0001, n = 20.
In Biological Monitoring of Estuarine and Coastal Waters (K. Kramer, ed.). Boca Raton, Florida, CRC Press (in press). Goks~yr, A. (1991). A semi-quantitative cytochrome P4501A1 ELISA: A simple method for studying the monooxygenase inducation response in environmental monitoring and ecotoxicological testing offish. Sci. Tot. Envir. 101,255-262. Goksoyr, A., Andersson, T., Buhler, D. R., Stegeman, J. J., Williams, D. E. & F6rlin, L. (1991). Immunochemical cross-reactivity of 13naphthotlavone-inducible cytochrome P-450 (P4501A)in liver microsomes from different fish species and rat. FL~h. l'h>~iol. Biochem. 9, 1-13. Goks~yr, A. & Ffrlin, L. (1992). The cytochrome P450 system in fish, aquatic toxicology, and environmental monitoring. Aquat. ToxicoL 22,287-312. Payne, J. E, Fancey, L. L., Rahimtula, A. D. & Porter, E. Ix. (1987). Review and perspective on the use of mixed-function oxygenase enzymes in biological monitoring. Comp. Biochem. Physiol. 86C, 233-245. Stegeman, J. J., Brouwer, M., Richard, T. D. G., F6rlin, [.., Fowler, B. A., Sanders, B. M. & Van Veld, P. A. (t992). Molecular responses to environmental contamination: Enzyme and protein systems as indicators of chemical exposure and effect. In Biornarkers: Biochemical Physiological and Histological Markers of Anthropogenic Stress (R. J. Huggett, R. A. Kimerle, P. M. Mehrte .It. & H. L Bergman, eds), pp. 235-335. Lewis Publishers, Chelsea, MI, USA.