Application of the SOS umu-test in detection of pollution using fish liver S9 fraction

doi:10.1016/0742-8413(90)90075-K

Comparative Biochemistry and Physiology Part C: Comparative Pharmacology

Volume 95, Issue 1, 1990, Pages 15-18

https://doi.org/10.1016/0742-8413(90)90075-K Get rights and content

Abstract

1.
1. The possibility of Aroclor 1254 and β-naphthoflavone treated fish Mugil auratus 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.
2. Genotoxicity of Aflatoxin b₁ 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.
3. Inhibition of umu gene expression by 7,8-benzoflavone in vitro clearly demonstrates a cytochrome P-450 mediated activation of aflatoxin b₁.
4.
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 β-naphthoflavone and Aroclor 1254 in the laboratory.
5.
5. The capability of S9 from fish living near a fish cannery to convert 2-aminoanthracene and 2-aminofluorene was not observed.

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Harman and norharman induce SOS responses and frameshift mutations in bacteria
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Hepatic cytochrome P-450 isozymes and aryl hydrocarbon hydroxylase in English sole (Parophrys vetulus)
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Validation of the SOS/umu-test with mutagenic complex mixtures
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Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test

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Cited by (15)

PAH content, toxicity and genotoxicity of coastal marine sediments from the Rovinj area, Northern Adriatic, Croatia
2006, Science of the Total Environment
Surface marine sediments collected from 8 sampling sites within the Rovinj coastal area, Northern Adriatic, Croatia, were used for determining priority pollutant polycyclic aromatic hydrocarbons (PAHs) and toxic/genotoxic potential of sediment organic extracts. Total PAH concentrations ranged from 32 μg/kg (protected area) to 13.2 mg/kg dry weight (harbor) and showed clear differences between pristine, urban industrial and harbor areas. PAHs distribution revealed their pyrogenic origin with some biogenic influence in harbor. At all sampling sites sediment extracts showed toxic potential that was consistent with the sediment type. No correlation between toxicity measured by Microtox assay and concentrations of individual or total PAHs was found. Noncytotoxic dose of sediment extracts showed no genotoxic potential in bacterial umu-test. DNA damage is positively related to total PAHs at 4 sampling sites (S-1, S-2, S-3, S-6), but the highest DNA damage was not observed at the site with the highest total sediment PAH content (S-5).
Adriatic coast as a microcosm for global genotoxic marine contamination - A long-term field study
2005, Marine Pollution Bulletin
Global changes in the marine environment and the continuing disposal of genotoxic xenobiotics are increasing the importance of environmental pollution monitoring and of biomonitoring programs. Current approaches focus on investigations at regional and local levels in an attempt to precisely define the nature and extent of any potential environmental crisis. We have initiated, for the first time, a long-term biomonitoring program focusing on the Croatian coast of the Adriatic Sea to contribute to a more detailed understanding of marine genotoxic effects using the mussels Mytilus galloprovincialis Lam., collected along the eastern Adriatic coast over a period of five years (1998–2002), as a key test organism. The integrity of DNA in its gill homogenate was examined by the Fast Micromethod^®. The strand scission factor (SSF) values, as a measure of DNA integrity, DNA damage or incomplete repair have been used for the ranking of sampling sites with respect to significant genotoxic stress due to the influence or effects of genotoxic xenobiotics. The region of Split (Kaštela Bay) proved to be the area with the heaviest load of genotoxic agents. The investigation of harmful effects in the ecosystem based on biomonitoring of genetic and other agents, not only on local levels but also on a wider scale, is considered as an important step in marine environmental management.
Stress-70 proteins in marine mussel Mytilus galloprovincialis as biomarkers of environmental pollution: A field study
2004, Environment International
In the present work we have investigated levels of stress-70 proteins in the gills of mussel Mytilus galloprovincialis collected seasonally from subtidal rocky shores at 6 different sites of the Rovinj coastal area (Northern Adriatic, Croatia). 1-D analysis (SDS-PAGE) using monoclonal mouse antibodies anti-HSP70 detected two bands of stress-70 proteins, 70 and 72 kDa constitutively present during the year. 2-D analysis (IEF+SDS-PAGE) proved that the antibodies used detected HSP70 (pI 5.7–5.9) and HSP72 (pI 5.5–5.6). The quantification of stress-70 proteins was possible using 200 ng of external HSP70 protein standard included on every blot. Maximal levels of HSP72 and HSP70 were observed in mussels in summer (September), and minimal levels in winter (December), and only HSP70 showed significant correlation with the sea temperature (r=+0.822, p<0.05). Acclimatization of mussels to a different lower salinity under experimental conditions proved that small changes in sea salinity (Δ=2 psu) could not cause significant stress-70 proteins induction. Results indicated that there are significant differences in HSP70 and HSP72 content in mussels from the control site (S-1) and mussels from other sampling sites with urban and industrial pollution. The usefulness of stress-70 proteins as biomarkers of environmental pollution is discussed.
Flow cytometric detection of DNA cell cycle alterations in hemocytes of mussels (Mytilus galloprovincialis) off the Adriatic coast, Croatia
2003, Aquatic Toxicology
Studies were carried out to determine the alteration in DNA cell cycle characteristics of hemocytes of the mussel Mytilus galloprovincialis collected at 17 different locations (146 individuals) along the Adriatic coast, Croatia. In order to connect possible genomic manifestation to urban and/or industrial waste flow cytometry was used. We studied incidence of altered DNA profile reflective of chromosomal fragmentation phenomena or aneuploid mosaicism, coefficient of variation (CV) in DNA fluorescence as a measure of intraindividual genome size variability and DNA index (DI) as a measure of ploidy. The different classes of DNA cell cycle alterations found in this study mirror either acute or cumulative genotoxic effects of the surrounding environment on mussel hemocyte DNA. These are intraindividual genome size variability (CV>8, seven individuals from four sites), aneuploidy (altered DNA profile and DI<0.9, 45 individuals from 14 sites) and accidental apoptotic processes (altered DNA profile and presence of apoptotic cells, two individuals from two sites). Normal cell cycle DNA profiles were obtained for 89 (60.9%) individuals from all 17 sites and for 146 examined samples polyploids were absent. Flow cytometry proved to be a powerful technique for the determination of alterations in cell cycle characteristics in mussel hemocyte DNA. Therefore, it may be used in pollution control measurements to distinguish affected or vulnerable populations from healthy populations living in the presence of a wide variety of marine environmental contaminants.
Benzo[a]pyrene and β-naphthoflavone mutagenic activation by European eel (Anguilla anguilla L.) S9 liver fraction
2002, Ecotoxicology and Environmental Safety
Is Anguilla anguilla L. (eel) liver ethoxyresorufin O-deethylase (EROD) induction absolutely necessary in order to convert promutagens as benzo[a]pyrene (BaP) into a mutagenic compound? Eels were exposed for 8 h to clean (control) and 0.3 μM β-naphthoflavone (BNF)-contaminated water. The 8-h exposure to 0.3 μM BNF brought about a very high EROD induction (10 pmol/min/mg protein) compared to control animals (1 pmol/min/mg protein). The Ames test (Maron and Ames, 1983) was carried out with Salmonella typhimurium TA 98 strain (TA98 His⁻) and eel isolated S9 liver fraction was used as a metabolic BaP activator. The BaP and BNF dose range concentrations tested were 0 (blank), 0.015, 0.08, 0.15, 0.38, 0.75, 1.5, 3.8, and 7.5 μM/plate and 0 (blank), 0.412, 1.235, 3.704, 11.1, 33.0, and 100 nM BNF, respectively. A dose–response relationship between BaP concentration and mutagenic activity was observed in the presence of S9 fractions in control and 0.3 μM BNF-exposed eels. Significant positive results, as TA98 His⁺ revertants, were observed at 0.38, 0.75, 1.5, 3.8, and 7.5 μM BaP/plate induced by BNF S9 fractions. Significant BaP mutagenic activation by liver control S9 was detected only at 1.5, 3.8, and 7.5 μM/plate. The BaP 1.5, 3.8, and 7.5 μM/plate mutagenic activation by BNF S9 and control S9 were not significantly different. Relative to BNF activation, it was only possible to detect His⁺ reversion at 11.1 nM BNF concentration with 0.3 μM BNF-induced S9. The above results demonstrate that the eel S9 liver fraction has the capacity to biotransform high BaP concentrations and convert it into a mutagenic compound with or without previous liver BNF biotransformation induction. The same does not apply to low BaP concentrations, where liver S9 induction by BNF is necessary to promote mutagenesis.
Evaluation of the SOS/umu-test post-treatment assay for the detection of genotoxic activities of pure compounds and complex environmental mixtures
2000, Mutation Research - Genetic Toxicology and Environmental Mutagenesis
This study presents an evaluation of the SOS/umu-test after introducing an additional dilution and incubation in the post-treatment assay. This treatment reduces the influence of coloured test compounds that otherwise affect the colorimetric determination of the β-galactosidase activity and the bacterial growth measurement during the testing of complex environmental samples. The post-treatment assay significantly increased the β-galactosidase activity and consequently the enzyme induction ratios at higher doses of model genotoxins 4-nitroquinoline-N-oxide, N-methyl-N′-nitro-N-nitrosoguanidine, 2-aminoanthracene, benzo(a)pyrene with low or no effect on the sensitivity of the test itself. On the other hand tests of environmental extracts indicated significant increases in sensitivity after additional incubation. 4-Nitroquinoline-N-oxide treatments of bacteria in the test affected cell division and caused filamentous growth. The size of filamentous bacteria and incidence rate of the length categories was positively correlated with the concentrations of genotoxins. Presence of filamentous tester bacteria proved induction of SOS response and genotoxic activity of environment samples in SOS/umu-test.

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Comparative Biochemistry and Physiology Part C: Comparative Pharmacology

Abstract

References (22)

Formation of mutagenic metabolites from benzo(a)pyrene and 2-aminoanthacene by the S-9 fraction from the liver of the northern pike (Esox lucius): inducibility with 3-methylcholanthrene

Chem. Biol. Interactions

7,8-benzoflavone stimulates the metabolic activation of aflatoxin b₁ to mutagens by human liver

Biochem. biophys. Res. Commun.

Protein measurement with folin phenol reagent

J. biol. Chem.

Conversion of procarcinogens to mutagens by the S-9 fraction from the liver of rainbow trout (Salmo gairdneri): Inducibility with PCB, 3-methyl-cholanthrene and phenobarbital and inhibition by metyrapone and α -naphthoflavone

Comp. Biochem. Physiol.

Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. I, Assay and properties of induced enzymes

J. biol. Chem.

Evaluation of the new system (umu-test) for detection of environmental mutagens and carcinogens

Mutation Res.

Harman and norharman induce SOS responses and frameshift mutations in bacteria

Mutation Res.

Review and perspective on the use of mixed-function oxygenase enzymes in biological monitoring

Comp. Biochem. Physiol.

Hepatic cytochrome P-450 isozymes and aryl hydrocarbon hydroxylase in English sole (Parophrys vetulus)

Biochem. Pharmac.

Validation of the SOS/umu-test with mutagenic complex mixtures

Mutation Res.

Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test

Mutation Res.

Cited by (15)

PAH content, toxicity and genotoxicity of coastal marine sediments from the Rovinj area, Northern Adriatic, Croatia

Adriatic coast as a microcosm for global genotoxic marine contamination - A long-term field study

Stress-70 proteins in marine mussel Mytilus galloprovincialis as biomarkers of environmental pollution: A field study

Flow cytometric detection of DNA cell cycle alterations in hemocytes of mussels (Mytilus galloprovincialis) off the Adriatic coast, Croatia

Benzo[a]pyrene and β-naphthoflavone mutagenic activation by European eel (Anguilla anguilla L.) S9 liver fraction

Evaluation of the SOS/umu-test post-treatment assay for the detection of genotoxic activities of pure compounds and complex environmental mixtures