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Application of the SOS umu-test in detection of pollution using fish liver S9 fraction
0306~4492190$3.00 + 0.00
Camp. Biochem. Physioi.Vol. 95C, No. 1, pp. 15-18,1990
Pergamon Press plc
Printed in Great Britain
APPLICATION OF THE SOS umu-TEST IN DETECTION POLLUTION USING FISH LIVER S9 FRACTION
OF
NEVENKA BIHAR],* MILENA VUKMIROVI~, RENATO BATEL and RUDOLF K. ZAHN~ Center for Marine Research, “Rudjer BoSkoviC” Institute, 52 210 Rovinj, Yugoslavia and ~Commission for Molecular Biology, Academy of Science and Literature, 6500 Mainz, F.R.G. (Received 16 June 1989)
Abstract-l. The possibility of Aroclor 1254 and /I-naphthoflavone treated fish Mugil aura&s and fish sampled in low and high polluted areas to convert some premutagens to active intermediers in the SOS umu-test have been investigated. 2. Genotoxicity of Aflatoxin b, differed markedly upon activation with liver S9 fractions from fish with different pollution histories, with the highest activation potency in fish living near a fish cannery. 3. Inhibition of zwu gene expression by 7,8benzoflavone in vitro clearly demonstrates a cytochrome P-450 mediated activation of aflatoxin b, 4. 2-Aminoanthracene and 2-aminofluorene were weakly activated to genotoxic products and the induction of umu gene expression could be detected only in the presence of S9 fractions from fish treated with b-naphthoflavone and Aroclor 1254 in the laboratory. 5. The capability of S9 from fish living near a fish cannery to convert 2-aminoanthracene and 2-aminofluorene was not observed.
INTRODUCTION
purchased from Applied Science Labs, Inc., State Co!lege, USA, and 2-nitrophenyl-fi-D-galactopyranoside (ONPG) from Serva, Heidelberg, F.R.G. Commercial S9 fraction from Aroclor I254 induced rat liver was purchased from Organon, Tehnika N.V., Belgium. All other chemical reagents were of the highest commercial quality available.
The SOS umu-test has been used for the detection of DNA damaging agents (Oda et al., 1985). There are some indications that it can be useful for the detection of mutagenic environmental mixtures (Whong et al., 1986), and of airborne genotoxic agents (Ong et al., 1987). Comparison of Ames test and umu-test (Nakamura et al., 1987, Oda et al., 1988) suggested that the umu-test provides a practical advantage for the primary screening of environmental mutagens because of its simplicity and broad specificity. It is now well established that fish liver S9 is capable of transforming different compounds into mutagenic metabolites through cytochrome P-450mediated mixed function oxidase system. Conversion of premutagens to mutagens by the S9 fraction from the liver of rainbow trout Safmo gairdneri (Miyauchi, 1984) and the adaptation of fish liver homogenates to the SulmoneIIu/microsome test (Ames test) have been reported (Kurelec et al., 1979, Balk et al., 1982). The present study was therefore, undertaken to investigate the possibility of fish Mugil auratus liver acti-
Animals and treatment
The mullet Mugil auratus with an average weight of 200-300 g were collected at two different locations. Control fish from maricultured area were kept in basins with running sea water at 15”C, and served for induction expe~ments. After two weeks of adaptation to laboratory conditions fish were injected i.p. with inducers (Aro and fl-NF) dissolved in corn oil or with corn oil alone. One group of animals received 500mg/kg Aro and another group SOmg/kg /?-NF. Fish were sacrificed on the fourth day and their livers removed. Fish from the polluted area were processed immediately. Methods The S9 fractions were prepared according to Ames et al. (1975) from composite samples of at least three fish livers and stored under liquid nitrogen. The protein content of S9 fractions were measured according to Lowry et al. (1951). The S9 fractions from all treated and untreated fish had an average protein content of 14.4 i 2.5 (5) mg/ml. Benzo(afpyrene monooxygenase activity was measured according to Nebert and Gelboin (1968) using post-mitochondrial fractions. The umu-test (Oda et al., 1985) were performed using Salmonella typhimurium TAl53S/psKl002 strain, kindly supplied by Professor Oda, Japan. Briefly, the overnight culture was diluted 50-fold with fresh TGA medium and incubated at 37°C until the bacterial density of 600 nm reached 0.25-0.30. The standard incubation mix&e contained 1 ml bacteria-S9 mixture (1 ml 2% S9 mix and 0.3 ml bacterial suspension) and 10~1 test compound dissolved in DMSO. Bacteria were grown at 37°C for 2 hr with gentle shaking, and terminated by cooling in an ice-water bath. In some cases, the incubation time was prolonged to 5 hr and the bacteria were washed with TGA medium to prevent interference with measurements of /3-galactosidase
vation system to convert some premutagens to active intermediers in the umu-test. In order to develop fast
assays for aquatic genotoxicity testing with relevance to fish, we determined the potential of fish with different pollution histories to activate some premutagens. MATERIALS AND
METHODS
Chemicals
Afiatoxin b, (Ab,), 2-aminoanthracene (AA), 2-aminofluorene (AF), benzo(a)pyrene (BaP), 5,dbenzoflavone (/?naphthoflavone, /?-NF) and 7,8_benzoflavone (7,8-BF) were purchased from Sigma, St Louis, U.S.A. Aroclor 1254 was -.*To whom correspondence should be addressed. 15
NEVENKA BIHAR~et
16 Table
I. Benzo(a)pyrene
monooxygenase
and rat liver
7,8-BF 10m4M _ + + + +
s9 rat control fish fl-NF (50 mg/kg) Aro (500 mg/kg) ‘cannery’
in differently
nostmitochondrial
Results
(Welsch.
Table
with
number
control
activity
RESULTS
AND
was measured
S9
1.2*0.1
s9
2.Aminoanthracene (2-AA)
2-Aminofluorene (2-AF)
fish
9.3 * as a mean i
which
4. Inhibition
‘cannery’
Values
induced
++
2.0
+
+ __
4.0
0.0 38.1
10-1
1.8
61.9
5 X 10-J
1.1
95.2
by I for
represent
10.0
+
10.0
+
100.0
background t t +
induction
$SS were from
or the highest
inducuon
OrgAnon
and control
(rat), (C)
in the trmu-test
rat fish Aro
fish C, fl-NF, can. rat (5 h) fish C, b-NF, Aro ‘cannery’
fish
rat fish Aro fish can. fish
induced which
over the 3-fold,
of unto gene expression Aroclor
of
s9:
-.-
@-NF
which
concentration
fish liver.
with
fish C
6.0
chemicals
of umu gene expression
(+)
the 2-fold. induced.
level.
of at least 3 determinations.
6.0
_
values for induction
readings.
S9
(+i _ ++ + ++ +
1.0
of
from
blank
means
_ ++
4.0
% Inhibition
3.1
was calculated
1254 (Are),
UMU
gene
expression
did not induce +induction
1.8-2
2-fold
over
the
urns gene expression.
of umu gene expression
over
fold.
p-naphthoflavone
(It-NF)
and
in
in vitro
induction ratio
Source
0.1
b, mutagenicity
2.3
Inducibilityt
+
+
Aflatoxin
SOS
umu gene expression
sources of liver
0.3
concentration
of analysis
0
subtracted
0.1
lowest
1.3 (2)
10 4
% inhibition
3.0
*The
(3)
a number
S9 by 7,8-benzoflavone
(mol./l)
+
b,
SD with
of 6 pg/ml
fish liver
7,Gbenzoflavone
-~Piml
(Ab,)
ratio (6)
I.1 i0.l
are expressed
Table
100.0 Aflaloxm
liver
in
2-aminoanthracene and 2-aminofluorene for induction of SOS umu-gene expression in SaImoneNu typhimurium 1535/pSK 1002 was measured (Table 2). The mutagenicity of aflatoxin b, differed markedly upon activation with liver S9 from differently induced fish, while control fish S9 could not activate it (Table 3). Aroclor and ‘cannery’ fish were effective in converting Ab, to mutagens to a level comparable with liver preparations from rats treated with 500mg/kg Aroclor 1254. A high-fat diet that enhances Ab, hepatocarcinogenesis in rats also enhances the conversion of Abi to mutagens detectable in the Ames test (Suit et al., 1977). This is one of the possible explanations for the high potency of Ab, activation to mutagenic products in fish living near a fish cannery. The mutagenicity of Ab, was affected by adding 7,8-BF in vitro (Table 4). The addition of 7,8-BF to monooxygenase system from human liver markedly stimulates the metabolic activation of Ab, to mutagens (Buening et at., 1978). On the other hand 7,8-BF
according
of compounds
-__
rat
2.2 f 0.3 (5) 7.8 f 0.8 (3)
cont.’ ~__..
differently
with
parenthesis.
different
Compound _..-___
upon
4.9 + 0.2 (3)
Results
In order to check the capacity of Mugil auratu~ liver S9 to activate some premutagens in the SOS umu-test, the induction of benzo(a)pyrene monoxygenase (BPMO) activity was measured. A significant increase in BPMO enzyme activity was observed only in fish treated with ~-naphto~avone (Table I). The degree of enzyme activity in Aroclor 1254 treated fish was lower and the same as in control fish from maricultured area, as well as in fish living near a fish cannery. BPMO activity was inhibited by 7,8-benzflavone in t’itro, with the highest enzyme inhibition in ‘cannery’ fish. These results clearly demonstrate that induction of cytochrome P-450 isoenzymes occurred, although no significant difference in BPMO activity in all the cases was detected. To clarify what kind of induction has occurred, the capacity of fish liver S9 to activate aflatoxin bl, activity
b,
comparison
fish
‘cannery’
DISCUSSION
2. Genotoxic
aflatoxin in
fl-NF treated fish Aro treated fish
It is now well known that fish respond to pollution by increase in liver MFO activity (Payne, 1976, Kurelec et al., 1977, Stegeman, 1978, Payne et al., 1987). Such induced MFO system has higher capacity for conversion of premutagens to active intermediers, enhancing the genotoxic effects in exposed organisms.
Table
3/rg/ml
preparations
rat
parenthesis.
activity. &Galactosidase to Miller (1972).
of
S9
SOS Induction
without
control
samples in
of
liver
S9
386.8 i_ 52.0 (3) 109.2 i 10.8 (3) 154.8 -i: 3.2 (3) 60.8 rt 4.0 (3) 330.8 + 28.4 (3)* 106.0 + 16.4(3) 171.6i 138.0(8) 93.2 i 34.8 (5) 178.8 * 7.6 (3) 40.8 * 0.8 (3)
as a mean + SD
Mutagenicity fish
preparations
1977).
are expressed
3.
induced
BPMO pm01 3-OH BaP/mg/min
i*P < 0.05
fish
fractions
-
fish
induced
al.
caanner,~ (can.)
ratios
17
SOS umu-test Table 5. Mutagenicity of 2 pg/ml 2-aminoanthracene ently induced flash liver S9 preparations in comparison preparations s9 control without S9 rat control fish fi-NF treated fish Aro treated fish ‘cannery’ fish
SOS Induction
upon differwith rat liver ratio
1.1 +0.1 (5) 5.7 f 0.5 (3) 1.0*0.1 (3) 1.2i 0. I (3) 2.1 * 0.2 (3) I.3 * 0.3 (2)
Results are expressed as a mean f SD with a number of analysts in parenthesis.
does not stimulate the metabolic activation of Ab, to mutagens when rat liver is used as a source of monooxygenase (Suit et al., 1977). In our case 1O-3 M 7,8-BF strongly inhibited mutagenicity of Ab, when cannery fish S9 was used. Inhibition of MFO enzymes and umu gene expression by 7,8-BF in vitro clearly demonstrates a cytochrome P-450 mediated activation. Rat preparations were more competent in activating 2-aminoanthracene, than Aroclor treated fish (Table 5). It is known that S9 fraction from liver of rainbow trout treated with PCB can activate 2-AA to bacterial mutagens (Miyauchi, 1984). On the other hand, the capability of control, ‘cannery’ and b-NF fish S9 to activate 2-AA was not observed. In the case of 2_aminofluorene, rat preparations give mutagenic response only when the incubation time was prolonged from 2 hr to 5 hr. This is consistent with previous results (Nakamura et al., 1987). When laboratory induced fish preparations were employed as activation system, increase in umu gene expression could be observed after 2 hr (Table 6). However, control and ‘cannery’ fish preparations did not convert 2-AF to mutagens. Even more, induction of umu gene expression with ‘cannery’ fish preparations decreased under the background level. These results could be explained by different cytochrome P-450 forms involved in induction of fish with different pollutional histories. Five hepatic P-450 isozymes from rainbow trout have been purified and characterized and one of them is believed to be a constitutive isozyme which is correlated with aflatoxin metabolism (Williams and Buhler, 1983). Varanasi et al. (1986) reported the presence of at least two cytochromes P-450 in liver in English sole sampled from relatively uncontaminated areas, while Gilewicz et al. (1987) partially purified only one cytochrome P-450 in marine fish Mugil cephalus. This illustrates the complexity and differences in P-450 mediated metabolism of some compounds in
Table 6. Mutagenicity of IO pglml 2.aminotluorene upon differently induced fish hver S9 preparations in comparison with rat liver preparations s9 control without S9 rat control fish fi-NF treated fish Aro treated fish ‘cannery’ fish
SOS Induction l.OkO.1 3.4 + 0.4 1.0*0.1 2. I f 0.2 1.9kO.l 0.2 f 0. I
ratio (6) (3) (3) (3) (3) (5)
Results are expressed as a mean f SD with a number of analysis in parenthesis.
aquatic organisms. This complexity is directly connected with problems in determination of environmental pollution measured as MFO activity and induction. Until now, it is accepted that MFO induction in fish liver can be a useful index for assessing water quality around point sources of mixed domestic/industrial waste (Payne et al., 1987). On the other hand, the SOS umu-test gives better qualitative information about exposed organisms. Combination of these two tests could serve as a tool for studies on genotoxic risk assessment in marine fish due to mixed pollution. Acknowledgements-The
authors would like to express their
to Dr Yoshimitsu Oda from Osaka Prefectural Institute of Public Health, Japan, for his supply of Salmonella strain. This study was financially supported by the Self-Management Community for Scientific Research of the S.R. Croatia and by the GKSS, Forschungszentrum Geesthacht GmbH, F.R.G.
gratitude
REFERENCES
Ames B. N., McCann
J. and Yamasaki E. (1975) Methods for detecting carcinogens and mutagens with the Salmonrllu/mammalian microsome mutagenicity test. Mutation Res. 31, 347-364. Balk L., Depierre J. W., Sundvall A. and Rannung U. (1982) Formation of mutagenic metabolites from benzo(a)pyrene and 2-aminoanthacene by the S-9 fraction from the liver of the northern pike (&ox Lucius): inducibility with 3-methylcholanthrene. C&m. Biol. Interactions 4, l-13. Buening M. K., Fortner J. G., Kappas A. and Conney A. H. (1978) 7,8-benzoflavone stimulates the metabolic activation of aflatoxin b, to mutagens by human liver.
Biochem. hiophys. Res. Commun. 82, 348-355. Gilewicz M., Guillaume J. R. and Bertrand J. C. (1987) Characterization of the liver cytochrome P-450 in the marine fish Mugil cephalus and effects of some hydrocarbons on the mixed-function oxidase components. Mar. Biol. 96, 157-162. Kurelec B., MatijaSeviC Z., Rijavec M.. AlaEeviC M., BritviC S., Miiller W. E. G. and Zahn R. K. (1979) Induction of benzo(a)pyrene monooxygenase in fish and the Salmonrlla test as a tool for detecting mutagenic/ carcinogenic xenobiotics in the aquatic environment. Bull.
Envir. Contam. Toxicol. 21, 799-807. Kurelec B., BritviC S., Rijavec, M., Miiller W. E. G. and Zahn R. K. (1977) Benzo(a)pyrene monooxygenase induction in marine fish-molecular response to oil pollution. Mar. Biol. 44, 21 l-216. Lowry 0. H. Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with folin phenol reagent. J. biol. Chem. 193, 265-215. Miller J. H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, New York, pp. 352-355. Miyauchi M. (1984) Conversion of procarcinogens to mutagens by the S-9 fraction from the liver of rainbow trout (Salmo guirdneri): Inducibility with PCB, 3-methylcholanthrene and phenobarbital and inhibition by metyrapone and r-naphthoflavone. Comp. Biochem.
Physiol. 79C, 363-367. Nakamura S., Oda Y., Shimada T., Oki 1. and Sugimoto K. (1987) SOS-inducing activity of chemical carcinogens and mutagens in Salmonella typhimurium TA1535/pSK 1002: examination with 151 chemicals. Mutation Res. 192,
239-247. Nebert D. W. and Gelboin H. V. (1968) Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. I, Assay and properties of induced enzymes. J. biol. Chem.
243, 6242-6249.
18
NEVENKA BIHARI et al.
Oda Y., Nakamura S., Oki I., Kato T. and Shinagawa H. (1985) Evaluation of the new system &mu-test) for detection of environmental mutagens and carcinogens. Mutation Res. 147, 219-229. Oda Y., Nakamura S. and Oki I. (1988) Harman and norharman induce SOS responses and frameshift mutations in bacteria. Mutation Res. 208, 39-44. Ong T., Stewart J., Wen Y.-F. and Whong W.-Z. (1987) Application of SOS umu-test for the detection of genotoxic volatile chemicals and air pollutants, Envir. Mutagen. 9, 171-176. Payne J. F. (1976) Field evaluation of benzopyrene hydroxylase induction as a monitor for marine pollution. Science 191, 945-946. Payne J. F., Fancey L. L., Rahimtula A. D. and Porter E. L. (1987) Review and perspective on the use of mixedfunction oxygenase enzymes in biological monitoring. Comp. Biochem. Physiol. 86C, 233-245. Stegeman, J. J. (1978) Influence of environmental contamination of cytochrome P-450 mixed function oxygenases in
fish: implication for recovery in the Wild Harbour Marsh. J. Fish Res. Bd. Can. 35, 658-674. Suit J. L., Rogers, A. E., Jetten, M. E. R. and Luria, S. E. (1977) Effects of diet on conversion of aflatoxin b, to . bacterial mutagen(s) by rats in viva and by rat hepatic microsomes in vitro. Mutation Res. 46, 313-324. Varanasi U., Collier T. K., Williams D. E. and Buhler D. R. (1986) Hepatic cytochrome P-450 isozymes and aryl hydrocarbon hydroxylase in English sole (Parophrys vet&s). Biochem. Pharmac. 35, 2967-2971. Welsch R. E. (1977) Stepwise multiple comparation procedures. J. Am. Stat. Assoc. 72, 566-575. Williams D. E. and Buhler D. R. (1983) Purified form of cytochrome P-450 from rainbow trout with high activity toward conversion of Aflatoxin b, to Aflatoxin B,-2.3epoxide. Cancer Res. 43, 4752-4756. Whong W.-Z., Wen Y.-F., Stewart J. and Ong T. (1986) Validation of the SOS/umu-test with mutagenic complex mixtures. Mutation Res. 175, 139-144.
Camp. Biochem. Physioi.Vol. 95C, No. 1, pp. 15-18,1990
Pergamon Press plc
Printed in Great Britain
APPLICATION OF THE SOS umu-TEST IN DETECTION POLLUTION USING FISH LIVER S9 FRACTION
OF
NEVENKA BIHAR],* MILENA VUKMIROVI~, RENATO BATEL and RUDOLF K. ZAHN~ Center for Marine Research, “Rudjer BoSkoviC” Institute, 52 210 Rovinj, Yugoslavia and ~Commission for Molecular Biology, Academy of Science and Literature, 6500 Mainz, F.R.G. (Received 16 June 1989)
Abstract-l. The possibility of Aroclor 1254 and /I-naphthoflavone treated fish Mugil aura&s and fish sampled in low and high polluted areas to convert some premutagens to active intermediers in the SOS umu-test have been investigated. 2. Genotoxicity of Aflatoxin b, differed markedly upon activation with liver S9 fractions from fish with different pollution histories, with the highest activation potency in fish living near a fish cannery. 3. Inhibition of zwu gene expression by 7,8benzoflavone in vitro clearly demonstrates a cytochrome P-450 mediated activation of aflatoxin b, 4. 2-Aminoanthracene and 2-aminofluorene were weakly activated to genotoxic products and the induction of umu gene expression could be detected only in the presence of S9 fractions from fish treated with b-naphthoflavone and Aroclor 1254 in the laboratory. 5. The capability of S9 from fish living near a fish cannery to convert 2-aminoanthracene and 2-aminofluorene was not observed.
INTRODUCTION
purchased from Applied Science Labs, Inc., State Co!lege, USA, and 2-nitrophenyl-fi-D-galactopyranoside (ONPG) from Serva, Heidelberg, F.R.G. Commercial S9 fraction from Aroclor I254 induced rat liver was purchased from Organon, Tehnika N.V., Belgium. All other chemical reagents were of the highest commercial quality available.
The SOS umu-test has been used for the detection of DNA damaging agents (Oda et al., 1985). There are some indications that it can be useful for the detection of mutagenic environmental mixtures (Whong et al., 1986), and of airborne genotoxic agents (Ong et al., 1987). Comparison of Ames test and umu-test (Nakamura et al., 1987, Oda et al., 1988) suggested that the umu-test provides a practical advantage for the primary screening of environmental mutagens because of its simplicity and broad specificity. It is now well established that fish liver S9 is capable of transforming different compounds into mutagenic metabolites through cytochrome P-450mediated mixed function oxidase system. Conversion of premutagens to mutagens by the S9 fraction from the liver of rainbow trout Safmo gairdneri (Miyauchi, 1984) and the adaptation of fish liver homogenates to the SulmoneIIu/microsome test (Ames test) have been reported (Kurelec et al., 1979, Balk et al., 1982). The present study was therefore, undertaken to investigate the possibility of fish Mugil auratus liver acti-
Animals and treatment
The mullet Mugil auratus with an average weight of 200-300 g were collected at two different locations. Control fish from maricultured area were kept in basins with running sea water at 15”C, and served for induction expe~ments. After two weeks of adaptation to laboratory conditions fish were injected i.p. with inducers (Aro and fl-NF) dissolved in corn oil or with corn oil alone. One group of animals received 500mg/kg Aro and another group SOmg/kg /?-NF. Fish were sacrificed on the fourth day and their livers removed. Fish from the polluted area were processed immediately. Methods The S9 fractions were prepared according to Ames et al. (1975) from composite samples of at least three fish livers and stored under liquid nitrogen. The protein content of S9 fractions were measured according to Lowry et al. (1951). The S9 fractions from all treated and untreated fish had an average protein content of 14.4 i 2.5 (5) mg/ml. Benzo(afpyrene monooxygenase activity was measured according to Nebert and Gelboin (1968) using post-mitochondrial fractions. The umu-test (Oda et al., 1985) were performed using Salmonella typhimurium TAl53S/psKl002 strain, kindly supplied by Professor Oda, Japan. Briefly, the overnight culture was diluted 50-fold with fresh TGA medium and incubated at 37°C until the bacterial density of 600 nm reached 0.25-0.30. The standard incubation mix&e contained 1 ml bacteria-S9 mixture (1 ml 2% S9 mix and 0.3 ml bacterial suspension) and 10~1 test compound dissolved in DMSO. Bacteria were grown at 37°C for 2 hr with gentle shaking, and terminated by cooling in an ice-water bath. In some cases, the incubation time was prolonged to 5 hr and the bacteria were washed with TGA medium to prevent interference with measurements of /3-galactosidase
vation system to convert some premutagens to active intermediers in the umu-test. In order to develop fast
assays for aquatic genotoxicity testing with relevance to fish, we determined the potential of fish with different pollution histories to activate some premutagens. MATERIALS AND
METHODS
Chemicals
Afiatoxin b, (Ab,), 2-aminoanthracene (AA), 2-aminofluorene (AF), benzo(a)pyrene (BaP), 5,dbenzoflavone (/?naphthoflavone, /?-NF) and 7,8_benzoflavone (7,8-BF) were purchased from Sigma, St Louis, U.S.A. Aroclor 1254 was -.*To whom correspondence should be addressed. 15
NEVENKA BIHAR~et
16 Table
I. Benzo(a)pyrene
monooxygenase
and rat liver
7,8-BF 10m4M _ + + + +
s9 rat control fish fl-NF (50 mg/kg) Aro (500 mg/kg) ‘cannery’
in differently
nostmitochondrial
Results
(Welsch.
Table
with
number
control
activity
RESULTS
AND
was measured
S9
1.2*0.1
s9
2.Aminoanthracene (2-AA)
2-Aminofluorene (2-AF)
fish
9.3 * as a mean i
which
4. Inhibition
‘cannery’
Values
induced
++
2.0
+
+ __
4.0
0.0 38.1
10-1
1.8
61.9
5 X 10-J
1.1
95.2
by I for
represent
10.0
+
10.0
+
100.0
background t t +
induction
$SS were from
or the highest
inducuon
OrgAnon
and control
(rat), (C)
in the trmu-test
rat fish Aro
fish C, fl-NF, can. rat (5 h) fish C, b-NF, Aro ‘cannery’
fish
rat fish Aro fish can. fish
induced which
over the 3-fold,
of unto gene expression Aroclor
of
s9:
-.-
@-NF
which
concentration
fish liver.
with
fish C
6.0
chemicals
of umu gene expression
(+)
the 2-fold. induced.
level.
of at least 3 determinations.
6.0
_
values for induction
readings.
S9
(+i _ ++ + ++ +
1.0
of
from
blank
means
_ ++
4.0
% Inhibition
3.1
was calculated
1254 (Are),
UMU
gene
expression
did not induce +induction
1.8-2
2-fold
over
the
urns gene expression.
of umu gene expression
over
fold.
p-naphthoflavone
(It-NF)
and
in
in vitro
induction ratio
Source
0.1
b, mutagenicity
2.3
Inducibilityt
+
+
Aflatoxin
SOS
umu gene expression
sources of liver
0.3
concentration
of analysis
0
subtracted
0.1
lowest
1.3 (2)
10 4
% inhibition
3.0
*The
(3)
a number
S9 by 7,8-benzoflavone
(mol./l)
+
b,
SD with
of 6 pg/ml
fish liver
7,Gbenzoflavone
-~Piml
(Ab,)
ratio (6)
I.1 i0.l
are expressed
Table
100.0 Aflaloxm
liver
in
2-aminoanthracene and 2-aminofluorene for induction of SOS umu-gene expression in SaImoneNu typhimurium 1535/pSK 1002 was measured (Table 2). The mutagenicity of aflatoxin b, differed markedly upon activation with liver S9 from differently induced fish, while control fish S9 could not activate it (Table 3). Aroclor and ‘cannery’ fish were effective in converting Ab, to mutagens to a level comparable with liver preparations from rats treated with 500mg/kg Aroclor 1254. A high-fat diet that enhances Ab, hepatocarcinogenesis in rats also enhances the conversion of Abi to mutagens detectable in the Ames test (Suit et al., 1977). This is one of the possible explanations for the high potency of Ab, activation to mutagenic products in fish living near a fish cannery. The mutagenicity of Ab, was affected by adding 7,8-BF in vitro (Table 4). The addition of 7,8-BF to monooxygenase system from human liver markedly stimulates the metabolic activation of Ab, to mutagens (Buening et at., 1978). On the other hand 7,8-BF
according
of compounds
-__
rat
2.2 f 0.3 (5) 7.8 f 0.8 (3)
cont.’ ~__..
differently
with
parenthesis.
different
Compound _..-___
upon
4.9 + 0.2 (3)
Results
In order to check the capacity of Mugil auratu~ liver S9 to activate some premutagens in the SOS umu-test, the induction of benzo(a)pyrene monoxygenase (BPMO) activity was measured. A significant increase in BPMO enzyme activity was observed only in fish treated with ~-naphto~avone (Table I). The degree of enzyme activity in Aroclor 1254 treated fish was lower and the same as in control fish from maricultured area, as well as in fish living near a fish cannery. BPMO activity was inhibited by 7,8-benzflavone in t’itro, with the highest enzyme inhibition in ‘cannery’ fish. These results clearly demonstrate that induction of cytochrome P-450 isoenzymes occurred, although no significant difference in BPMO activity in all the cases was detected. To clarify what kind of induction has occurred, the capacity of fish liver S9 to activate aflatoxin bl, activity
b,
comparison
fish
‘cannery’
DISCUSSION
2. Genotoxic
aflatoxin in
fl-NF treated fish Aro treated fish
It is now well known that fish respond to pollution by increase in liver MFO activity (Payne, 1976, Kurelec et al., 1977, Stegeman, 1978, Payne et al., 1987). Such induced MFO system has higher capacity for conversion of premutagens to active intermediers, enhancing the genotoxic effects in exposed organisms.
Table
3/rg/ml
preparations
rat
parenthesis.
activity. &Galactosidase to Miller (1972).
of
S9
SOS Induction
without
control
samples in
of
liver
S9
386.8 i_ 52.0 (3) 109.2 i 10.8 (3) 154.8 -i: 3.2 (3) 60.8 rt 4.0 (3) 330.8 + 28.4 (3)* 106.0 + 16.4(3) 171.6i 138.0(8) 93.2 i 34.8 (5) 178.8 * 7.6 (3) 40.8 * 0.8 (3)
as a mean + SD
Mutagenicity fish
preparations
1977).
are expressed
3.
induced
BPMO pm01 3-OH BaP/mg/min
i*P < 0.05
fish
fractions
-
fish
induced
al.
caanner,~ (can.)
ratios
17
SOS umu-test Table 5. Mutagenicity of 2 pg/ml 2-aminoanthracene ently induced flash liver S9 preparations in comparison preparations s9 control without S9 rat control fish fi-NF treated fish Aro treated fish ‘cannery’ fish
SOS Induction
upon differwith rat liver ratio
1.1 +0.1 (5) 5.7 f 0.5 (3) 1.0*0.1 (3) 1.2i 0. I (3) 2.1 * 0.2 (3) I.3 * 0.3 (2)
Results are expressed as a mean f SD with a number of analysts in parenthesis.
does not stimulate the metabolic activation of Ab, to mutagens when rat liver is used as a source of monooxygenase (Suit et al., 1977). In our case 1O-3 M 7,8-BF strongly inhibited mutagenicity of Ab, when cannery fish S9 was used. Inhibition of MFO enzymes and umu gene expression by 7,8-BF in vitro clearly demonstrates a cytochrome P-450 mediated activation. Rat preparations were more competent in activating 2-aminoanthracene, than Aroclor treated fish (Table 5). It is known that S9 fraction from liver of rainbow trout treated with PCB can activate 2-AA to bacterial mutagens (Miyauchi, 1984). On the other hand, the capability of control, ‘cannery’ and b-NF fish S9 to activate 2-AA was not observed. In the case of 2_aminofluorene, rat preparations give mutagenic response only when the incubation time was prolonged from 2 hr to 5 hr. This is consistent with previous results (Nakamura et al., 1987). When laboratory induced fish preparations were employed as activation system, increase in umu gene expression could be observed after 2 hr (Table 6). However, control and ‘cannery’ fish preparations did not convert 2-AF to mutagens. Even more, induction of umu gene expression with ‘cannery’ fish preparations decreased under the background level. These results could be explained by different cytochrome P-450 forms involved in induction of fish with different pollutional histories. Five hepatic P-450 isozymes from rainbow trout have been purified and characterized and one of them is believed to be a constitutive isozyme which is correlated with aflatoxin metabolism (Williams and Buhler, 1983). Varanasi et al. (1986) reported the presence of at least two cytochromes P-450 in liver in English sole sampled from relatively uncontaminated areas, while Gilewicz et al. (1987) partially purified only one cytochrome P-450 in marine fish Mugil cephalus. This illustrates the complexity and differences in P-450 mediated metabolism of some compounds in
Table 6. Mutagenicity of IO pglml 2.aminotluorene upon differently induced fish hver S9 preparations in comparison with rat liver preparations s9 control without S9 rat control fish fi-NF treated fish Aro treated fish ‘cannery’ fish
SOS Induction l.OkO.1 3.4 + 0.4 1.0*0.1 2. I f 0.2 1.9kO.l 0.2 f 0. I
ratio (6) (3) (3) (3) (3) (5)
Results are expressed as a mean f SD with a number of analysis in parenthesis.
aquatic organisms. This complexity is directly connected with problems in determination of environmental pollution measured as MFO activity and induction. Until now, it is accepted that MFO induction in fish liver can be a useful index for assessing water quality around point sources of mixed domestic/industrial waste (Payne et al., 1987). On the other hand, the SOS umu-test gives better qualitative information about exposed organisms. Combination of these two tests could serve as a tool for studies on genotoxic risk assessment in marine fish due to mixed pollution. Acknowledgements-The
authors would like to express their
to Dr Yoshimitsu Oda from Osaka Prefectural Institute of Public Health, Japan, for his supply of Salmonella strain. This study was financially supported by the Self-Management Community for Scientific Research of the S.R. Croatia and by the GKSS, Forschungszentrum Geesthacht GmbH, F.R.G.
gratitude
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