Bioconcentration, biotransformation and elimination of pyrene in the arctic crustacean Gammarus setosus (Amphipoda) at two temperatures.

The influence of temperature on the bioaccumulation, toxicokinetics, biotransformation and depuration of pyrene was studied in the arctic marine amphipod Gammarus setosus. A two-compartment model was used to fit experimental values of total body burden, total metabolites and parent pyrene concentrations and to calculate toxicokinetic variables derived for two experimental treatments (2 and 8 °C). No statistically significant differences were observed with temperature for these toxicokinetic variables or bioconcentration factors. Contrarily, the Q10 values suggested that the toxicokinetic variables ke and km were temperature-dependent. This may be explained by the high standard deviation of the Q10 values. Q10 is the variation in the rate of a metabolic reaction with a 10 °C increase in temperature. Depuration rate constants were calculated from linear best fit equations applied to measured pyrene concentrations over time during the depuration phase of the experiment. During depuration, the parent pyrene was eliminated in two stages with faster elimination observed at 8 °C compared to 2 °C. This finding was also indicated by the Q10. No changes in total body burdens of metabolite concentrations were observed during the monitoring of depuration over a period of 96 h. The biotransformation pathway of pyrene in G. setosus was also investigated in this study with two main phase II biotransformation products discovered by liquid chromatography. These products are conditionally identified as the sulphate and glucose conjugates of 1-hydroxy-pyrene. Overall, the study contributes new knowledge to the understanding of the fate of PAHs in arctic biota. In particular, the study provides valuable insight into the bioaccumulation and biotransformation of an important PAH and its metabolites in a species that serves as both a predator and prey in the arctic ecosystem.

Genotoxicity of apomorphine and various catecholamines in the Salmonella mutagenicity test (Ames test) and in tests for primary DNA damage using DNA repair-deficient B. subtilis strains (rec assay).

Apomorphine, N-nor-N-propyl-apomorphine, dopamine, L-DOPA, 6-hydroxydopamine and adrenaline were evaluated for genotoxicity using the Ames test and DNA repair-deficient and DNA repair-proficient Bacillus subtilis strains (rec assay, H17/M45; HLL3g/HJ-15). In the absence of an S9 liver homogenate, apomorphine induced frame-shift mutations in Salmonella typhimurium, mainly in strain TA1537; no indication of DNA-damaging effects in B. subtilis was observed. N-Nor-N-propyl-apomorphine was tested using strain TA1537 only and found to be mutagenic. Dopamine, L-DOPA, 6-hydroxydopamine and adrenaline were non-mutagenic when tested without S9, whereas they were all more toxic for DNA repair-deficient than for DNA repair-proficient B. subtilis strains, indicating a DNA-damaging potential. In a second set of experiments the mode of action of apomorphine and the relevance of the positive Ames test data were investigated. Glutathione in physiological concentrations reduced the mutagenic effect of apomorphine in a dose-dependent way, both in the presence and the absence of S9. S9 also reduced the mutagenicity of apomorphine. By comparing the effects of a complete S9 mix with those of a preparation without glucose-6-phosphate and NADP, it became clear that S9 also had an activating effect, overshadowed under standard conditions by its deactivating activity. Apomorphine was not mutagenic under anaerobic conditions. Superoxide dismutase and catalase reduced the mutagenic effect of apomorphine. All test conditions which reduced the mutagenic effect also inhibited the dark discoloration of the tester plates, indicating a retardation of apomorphine oxidation. It can, therefore, be concluded that oxidation of apomorphine leads to mutagenic products which induce frame-shift mutations in Salmonella typhimurium. This oxidation was prevented both by glutathione in concentrations well below physiological levels and/or by catalase and superoxide dismutase. Under these conditions, apomorphine was non-mutagenic in therapeutic concentrations as well as at higher dose levels. The possibility of genotoxic side effects occurring in patients treated with apomorphine as an emetic drug is therefore considered to be very unlikely.

Mutagenicity evaluation of amino-oxazoline derivatives using in vitro and in vivo short-term tests.

During routine investigation of potential drugs it was found that 1-methyl-4-(2-oxazoline-2-ylamino)-indazole (1) compound 1 was mutagenic for Salmonella typhimurium TA1535 and TA100. The genotoxicity of compound 1 was confirmed by the following in vitro test systems: V79 Chinese hamster cells (HGPRT-locus), Saccharomyces cerevisiae D7 (mitotic gene conversion, mutations, aberrations), and Escherichia coli (rec-assay). On the other hand, when compound 1 was tested in vivo (micronucleus test in mice and sister chromatid exchange in Chinese hamsters) it did not show evidence of genotoxic activity. In order to study structure/activity relationships, different analogues of compound 1 were tested in Salmonella typhimurium TA1535. The tests showed that (1) most 2-amino-oxazolines were mutagenic for the test organism; (2) the 2-amino-imidazoline and 2-amino-thiazolidine derivatives tested were not mutagenic; (3) substituents bound to the extranuclear nitrogen of the 2-aminooxazoline ring had only a weak influence on the mutagenic potential; and (4) methylation of the oxazoline ring, most conspicuously at the carbon in position 5, strongly reduced the mutagenic effect. From these observations it is concluded that the reaction of nucleophilic DNA sites with the most electrophilic site of the oxazoline ring, ie, the carbon in position 5, is responsible for the genotoxicity of amino-oxazoline compounds. Due to the genotoxicity observed in these in vitro tests this class of compounds was no longer developed, but dropped, even without performing a long-term carcinogenicity study or considering the negative in vivo findings.

Comparative evaluation of different pairs of DNA repair-deficient and DNA repair-proficient bacterial tester strains for rapid detection of chemical mutagens and carcinogens.

The aim of the present study was to evaluate the usefulness of different pairs of DNA repair-deficient and DNA repair-proficient bacterial tester strains in a mutagenicity/carcinogenicity screen, possibly as complements to the Ames test. 70 carcinogenic and non-carcinogenic compounds, representing a variety of chemical structures, were tested for their DNA-damaging effects, using 6 different DNA-repair-deficient bacterial strains. 2 Bacillus subtilis systems, H17/M45 and HLL3g/HJ-15, were used. The susceptibility of Escherichia coli AB1157 was compared with the susceptibility of 4 recombination-deficient mutants, JC5547, JC2921, JC2926 and JC5519. The test compounds were applied onto paper disks (spot test, ST), or incorporated into a top agar layer (agar-incorporation test, AT). The 2 B. subtilis systems were generally found to be more sensitive and reliable than the assays using E coli. The incorporation of the test compounds in the agar increased the sensitivity of the test for polycyclic aromatic hydrocarbons and other poorly water-soluble compounds. Hydrazines and several other highly polar chemicals could be tested more efficiently when applied onto paper disks. About 30% of the test compounds did not induce any growth inhibition and so could not be tested properly. In order to evaluate the ability of these DNA-repair tests to complement the Ames Salmonella mutagenicity test in a genetic toxicology screening program, results from this study were compared with published data both on mutagenicity in the Ames test and on carcinogenicity. 8 carcinogens generally found to be non-mutagenic for Salmonella were tested: 2 showed DNA-damaging properties (mitomycin C, 1,2-dimethylhydrazine), 5 failed to do so (actinomycin D, griseofulvin, thioacetamide, diethylstilbestrol, safrole), and one (thiourea) was not toxic, so that no classification was possible. 2 non-carcinogenic bacterial mutagens were examined; one, sodium azide, was equitoxic for repair-proficient and -deficient strains, while the other, nitrofurantoin, primarily inhibited repair-deficient strains. The DNA-repair tests failed to indicate the mutagenic and carcinogenic properties of acridine orange. Nalidixic acid, a non-mutagenic DNA synthesis inhibitor, damaged bacterial DNA. Apart from the differences summarized above, carcinogenicity was indicated correctly by the Salmonella S9 assay and most sets of DNA-repair-deficient and DNA-repair-proficient tester strains evaluated in this study. Thus, several more carcinogens could be detected by performing the Ames test and the bacterial DNA-repair tests in tandem than by using either test alone. Nevertheless, the use of both bacterial in vitro systems in a battery of short-term tests for mutagenicity/carcinogenicity evaluation is not considered to be ideal, since the Ames test and the pairs of DNA-repair-deficient and DNA-repair-proficient tester strains used had several shortcomings in common under the conditions of this study.

Actions of an antispermatogenic, but non-mutagenic, indenopyridine derivative in mice and Salmonella typhimurium.

This paper describes a new antispermatogenic agent. Following single oral administration to mice, the indenopyridine derivative (4aRS,5SR,9bRS)-2-ethyl-1,3,4,4a,5,9b-hexahydro-7-methyl-5-p-tolyl-2H-indeno(1,2-c)pyridine hydrochloride, code No. 20-438, produced long-lasting inhibition of the spermatogenic process at dose levels of 10 mg/kg (1/40 of the lowest lethal dose) and higher. Testes weights were significantly reduced from days 2--217 after treatment, and no clear-cut evidence of a recovery was found during this time. The fertility of treated males was normal during the initial 2 weeks after treatment, followed by partial or total sterility in weeks 3--6, and incomplete recovery in weeks 7--29 after treatment. The antifertility effects were caused by maturation depletion of the germ cells, leading to oligospermia. The following rank of decreasing "susceptibility" of the germ cells was observed: Spermatocytes greater than early spermatids, intermediate spermatogonia greater than stem cells. Sperm and late spermatids were not affected. Despite the characteristic specific germ-cell pattern of antifertility effects, 20-438 showed neither indications of pre- and post-implantational dominant lethality, nor mutagenic potentiality as measured by cytogenetic analysis of spermatocytes or spermatogonia, the sperm abnormality assay, the micronucleus test, and the Salmonella assay. These data suggest that the action of 20-438, leading to oligospermia, does not involve genetic toxic effects.

Premature chromosome condensation, structural chromosome aberrations, and micronuclei in early mouse embryos after treatment of paternal postmeiotic germ cells with triethylenemelamine: possible mechanisms for chemically induced dominant-lethal mutatiions.

Cytogenetic effects in preimplantation 4-8-cell mouse embryos have been investigated after treating paternal postmeiotic germ cells with triethylenemelamine (TEM). Dose-levels of TEM which do not affect fertilization but yield high incidence of dominant-lethal mutations in sperm and spermatids were shown to produce relatively high frequencies of (a) premature chromosome condensation (PCC), (b) structural chromosome anomalies (breakage-reunion phenomena), and (c) micronuclei in these embryos. The results indicate that genetic death of embryos is mainly due to imbalance (i.e. loss) of genetic material, either from breaks leading to lagging fragments and micronuclei, or from the segregation of various types of exchange figures (dicentrics, rings etc.) resulting in mechanical disturbances of cleavage division. It is suggested that PCC, to some extent, is an expression of TEM-induced long-lived lesions which, transmitted into the egg, prevent the chromosomes in question from replicating and/or condensing normally. This phenomenon could well be associated with loss of chromosome material resulting in embryonic death.