The chemical and biogenotoxic characterization of organic xenobiotics in aquatic sediment materials 1. The application and comparison of chemically non-specific and biogenotoxic methods.

The aim of this work was to evaluate the Ames assay and mixed function oxidase (MFO)-Induct Test used in parallel with chemical group tests (ECD fingerprint and PAH estimation) for the characterization of the organic pollution of water sediment materials. Sediment materials were collected from "clean" and relatively heavily polluted locations in the Middle Adriatic Sea, and from some locations in continental Croatia polluted with wastewaters from different enterprises. Characterization of the organic extracts of the sediment materials investigated was performed chemically using UV spectrofluorometry for the determination polyaromatic hydrocarbons (PAH) and gas chromatography for the determination of volatile EC detector sensitive materials. Genotoxic analysis of the extracts was performed using the MFO-Induct Test and mutagenicity testing using the Standard Plate Incorporation Test as described by Maron and Ames with Salmonella typhimurium TA 98. Measurement of the BaPMO enzyme activity in the livers of carp treated i.p. with total extracts of the sediment investigated confirmed that the methanol extracts generally contained more inducing matter than the petroleum ether extracts. Ames assay showed that for all the samples following the elimination of the sulfur, there was an increase in the number of revertants in comparison to the control number, which indicates that the samples contained mutagenic substances. The larger doses of extracts generally demonstrated cytotoxicity, as evidenced by a reduced number of spontaneous revertants in the SalmonellalMicrosome Test. Investigation of the correlation of the chemical parameters with the biological parameter showed that the induction of BaPMO exhibited a statistically significant correlation with the level of the ECD fingerprint of the petroleum ether sediment extract.

Evaluation of the genotoxic and cytochrome P450 monooxygenase-inhibitory potential of Dicuran on procaryotic and eucaryotic test systems.

The effect of the herbicide Dicuran 500 FL (formulated product) on the phenotypical and genotypical changes in procaryotic and eucaryotic organisms was investigated using short-term tests for detecting genotoxins. Since pesticides discharged in the water environment can modulate the mixed-function monooxygenases (MFO) detoxification system of water organisms, the in vivo and in vitro effects of Dicuran on hepatic cytochrome P450 (cyt P450) monooxygenase activities were also examined in juvenile carp (Cyprinus carpio L.). By measuring the activities of MFO in experimental carp exposed to Dicuran an attempt was made to establish whether Dicuran could be bioactivated by MFO into ultimate mutagens. Our results on the bacterial strains Salmonella typhimurium TA100 and TA98 show that Dicuran does not possess either mutagenic or premutagenic characteristics. The micronucleus test on the erythrocytes of experimental carp did not establish any clastogenic effect either. However, Dicuran significantly inhibited the MFO activity of 7-ethoxyresorufin-O-deethylase (EROD) and benzo[a]pyrene monooxygenase (BaPMO) in the liver of experimental carp in vitro, as well as in in vivo conditions. These findings demonstrate the potentially damaging effect of Dicuran on the xenobiotic metabolizing enzyme systems of fish, and suggest the applicability of described methods for the prediction of the ecotoxicological significance of the presence of pesticides in the water environment.

The mutagenic potential of AH+ and AH26 by Salmonella/microsome assay.

The aim of this study was to examine the mutagenic potential of canal sealers AH+ and AH26 by Salmonella/microsome assay. The materials were tested immediately after mixing, 1 hr and 1 month later, respectively. The dimethyl sulfoxide extracts of sealers in amounts of 3.0, 1.5, and 0.75 microliters/plate were used. The plated bacterial strains of Salmonella were TA 98 and TA 100. The results showed that AH+ is mutagenic toward strain TA 100 1 hr after mixing. One month after mixing, mutagenic activity was expressed only in TA 98. Paste A showed strong mutagenicity toward TA 100. AH26 was more mutagenic to the TA 100 immediately after mixing, 1 hr later, and 1 month after it was polymerized. Also it was mutagenic toward TA 98 in the polymerized condition. Further examinations should be conducted to establish a definitive conclusion about mutagenic potential for these two endodontic materials.

DNA adducts in carp exposed to artificial diesel-2 oil slicks.

In attempts to mimic field exposure, oil slicks prepared from diesel-2 oil/water emulsions were poured onto the surface of water in tanks prepared fresh every day and liver DNA adducts were analyzed by 32P-postlabeling in carp free-swimming in these tanks. 'Clusters' of lipophilic DNA adducts were detected, with five major and numerous minor adducts. Essentially a similar adduct pattern was found in the liver DNA of carp exposed to crude oil-polluted water. Diesel-2 adduct induction was observed slowly with a steady increase to greater than 3000 amol/microgram DNA at day 12. After this time fish were transferred to clean water. Adduct levels continued to increase through day 17 (approximately 10,000 amol/microgram DNA) despite the cessation of exposure, but a 30% and 80% decline was evident at day 22 and day 27, respectively. All major adducts were distinct from the known benzo[a]pyrene diolepoxide-dG. These results indicate that diesel-2 oil can cause extensive DNA damage in carp in vivo and the damage accumulates proportionately with time of exposure.

Antimutagenic potency of the cytotoxic and anti-psoriatic compound anthralin (cignolin).

The anti-psoriatic compound anthralin (cignolin) was determined to exhibit a strong cytostatic activity on HeLa-Köln cells; an ED50 concentration of 1.2 microM are cytotoxic for the cells. These growth-inhibition data were confirmed by thymidine-uptake experiments. The drug anthralin was determined to be neither direct a mutagen nor a premutagen in the Ames test using Salmonella typhimurium strain TA 100 (anthralin-concentration = 5 microM). Moreover, this compound was a strong inhibitor of benzo(a)pyrene monooxygenase, an enzyme which causes the metabolic conversion of premutagens to mutagens. These data demonstrate anthralin to be an anti-psoriatic compound devoid of mutagenic property in vitro with regard to base-pair substitutions and provided at least with some antimutagenic potential.

Metabolism of some carcinogenic aromatic amines in four species of marine sponges.

Postmitochondrial fractions from marine sponges Geodia cydonium, Tethya aurantium, Verongia aerophoba and Pellina semitubulosa activate precarcinogenic aromatic amine 2-aminoanthracene, but not precarcinogenic polycyclic aromatic hydrocarbon benzo(a)pyrene, to Salmonella typhimurium TA 98 mutagens. All four sponge species lack a benzo(a)pyrene monooxygenase activity, but possesses the enzyme activity whose characteristics (selective activation of aromatic amines, NADPH-dependency, pH optimum at 8.4) are similar to FAD-containing monooxygenase. Tethya postmitochondrial fraction possesses an UDP-glucuronyl transferase activity which catalyzes the conjugation of a considerable part of metabolized 2-acetylamino [9-14C]fluorene to water soluble glucuronides. The possible ecological significance of exuded aromatic amine metabolites as well as the significance of the presence of the selective potential for the activation of aromatic amines to mutagens among sponges for our understanding of the fate and effects of carcinogens in the marine environment are discussed.

Selective activation of carcinogenic aromatic amines to bacterial mutagens in the marine mussel Mytilus galloprovincialis.

The postmitochondrial fraction of the marine mussel Mytilus galloprovincialis digestive gland activates selectively precarcinogenic aromatic amines, but not precarcinogenic benzo[a]pyrene, to Salmonella typhimurium TA 98 mutagens. This activation potential is NADPH-dependent, is not inducible by exposure to Diesel 2 oil and a polluted environment, and is inhibited by methimazole. The characteristics of this activation potential are consistent with the recent finding of the presence of FAD-containing-, and lack of cytochrome P-450 dependent-, monooxygenase activity in Mytilus edulis. The presence of such selective potential in marine invertebrate(s) may bring new insight into our understanding of the fate and the effects of carcinogens in the marine environment.

Antimutagenic activity of the novel antileukemic agents, avarone and avarol.

The two antileukemic agents, avarone and avarol, were determined to be neither direct nor indirect mutagenic agents in the Ames microsomal test. Moreover, the two sesquiterpenoid compounds drastically reduced the mutagenic effect of benzo[a]pyrene in the same system. Subsequent enzymic studies demonstrated that avarone and avarol are powerful inhibitors of benzo[a]pyrene monooxygenase.

Activity of benzo[a]pyrene monooxygenase in fish from the Sava River, Yugoslavia: correlation with pollution.

Benzo[a]pyrene monooxygenase (B[a]PMO) activity in non-migratory fish from a given river segment is highly correlated to the recent pollutional history of that part of the river. The enzyme activity level can serve as a relevant measure for the harmful pollutant potential in aquatic ecosystems. Caged experimental fish exposed for about 10 days in river segments show BaPMO activity changes with the same predictive validity as that of the natural population.

Consequences of detergent pollution of the sea: effects on regenerating sponge cubes of Geodia cydonium.

Regenerating cubes of the sponge Geodia cydonium cyconium were used as a model in the investigation of detergent pollution in the sea. The anionic detergent sodium dodecylsulphate (SDS) and a 1:1 mixture of Faks and Radion, two commercial laundry detergents, were used in the concentration range from 1 X 10(-9) g/ml (1 ppb) to 1 X 10(-5) g/ml. It is shown that SDS is taken up, weakly accumulated but not incorporated into the macromolecular fractions of the sponge. At concentrations of 0.1 ppm and above, SDS decreases the uptake of thymidine, uridine and phenylalanine into the acid-soluble sponge fraction. Their incorporation into the acid insoluble fractions, which have been isolated, was different from the controls at 10 ppb and higher levels. Faks and Radion were less active by a factor of 10. However, they showed similar effects. The chemical composition of the regenerating sponge cubes with respect to DNA, RNA and protein content has been evaluated. The alterations are less pronounced on detergent incubation than precursor uptake. The use of the cetyltrimethyl-ammoniumbromide-turbidity-dilution technique reveals drastic qualitative changes in the nucleic acid fractions. The relevant literature on biological effects of detergent is listed. It is shown that this investigation extends the scale of known effects far into the low and pollution-relevant concentration levels.