Branko Kurelec

The genotoxic disease syndrome

Abstract Since lower animals develop neoplasia rarely or not at all, the DNA- and chromosomal-alterations observed in them after exposure to genotoxic chemicals should be correlated to some genotoxic endpoint other than neoplasia. On the basis of good correlations between induction of DNA- and chromosomal-alterations and the induction of gene mutations, this paper argues that in natural species a variety of manifestations of the mutational event may actually prove to be of much greater biological and ecological importance than induction of neoplasia. These manifestations include impairments in enzyme function, altered protein turnover, impairments in general metabolism, production of initiators of cytotoxic injuries, inhibition of growth, degenerative processes and atrophy in tissues and organs, decreased scope for growth in organisms, faster ageing, decreased fitness and well-being, impairments in immunoresponse and reproduction, increased frequency of diseases and neoplasia, impairments in adaptation, survival, and succession, and finally, extinction of species. Taken together, these ‘symptoms’ constitute a syndrome of a disease which etiologically could be named the genotoxic disease syndrome.

Evidence for a multixenobiotic resistance mechanism in the mussel Mytilus galloprovincialis

Abstract Membrane vesicles isolated from the gills, mantle, and digestive gland of the marine mussel Mytilus galloprovincialis possess a potential similar to that involved in multidnig resistance (MDR) described in resistant tumor cell lines: they bind 2-acetylaminofluorene (AAF) in saturable and trypsin- and verapamil-sensitive manner. Cytosolic fractions from mussel tissue homogenates reveal a relatively high glulathione- S -transferase (GST) and glutathione-peroxidase (GSH-Px) activities with substrate affinities similar to the isoenzyme of anionic GST and its intrinsic GSH-Px involved in MDR. In mussel these biochemical elements of the MDR mechanism seem to be inherent, rather than induced, since they were found in specimens living in the pristine aquarium of Limski Kanal, Northern Adriatic. The enhancement in the accumulation of AAF in laboratory exposure experiments in the presence of verapamil indicates that the MDR-like mechanism functions in mussel also in vivo. The recognition that the MDR-like mechanism is operative in mussel may explain the relative resistance of this species to pollution. The knowledge that the presence of one xenobiotic may block the pumping out, and hence accelerating accumulation, of other(s), may help us to understand and interpret our present and past data on different parameters obtained with this most exploited indicator organism.

Distinct glutathione-dependent enzyme activities and a verapamil-sensitive binding of xenobiotics in a fresh-water mussel Anodonta cygnea.

A fresh-water mussel Anodonta cygnea, an aquatic invertebrate resistant to pollution, possesses an inherent high potential to bind 2-acetylaminofluorene onto membrane vesicles. This binding is saturable and trypsin- and verapamil-sensitive. Simultaneously, this mussel reveals a relatively high inherent activity of glutathione-dependent enzyme activities with a distinct spectrum of substrate affinities. Both these activities are similar to the elements of the molecular mechanism involved in the acquired multi-drug resistance phenomenon described in tumor cell-lines. The recognition that in organisms exposed to polluted waters a multi-xenobiotic resistance mechanism may be involved is essential for understanding both the biological impact of pollution and the development of methods for rational risk assessment in regulatory policy.

Induction of benzo(a)pyrene monooxygenase in fish and the Salmonella test as a tool for detecting mutagenic/carcinogenic xenobiotics in the aquatic environment

From the ecological point of view and on account of the interest in the environmental assessment of biological effects of xenobiotics on water organisms, it would be highly desirable to detect their presence by an equally revealing biochemical method in water samples. Considering the induction of BPMO as a means of detoxification in fish, we studied the consequences of i/p application of polluted water extracts from different marine sites to young carp. Aliquots of these extracts served in Salmonella/microsome mutagenicity tests using liver homogenates of pollution-induced fish as activating systems. The results we obtained so far are presented in this paper.

Inhibition of multixenobiotic resistance mechanism in aquatic organisms: ecotoxic consequences

Abstract Recent evidence indicates that inherent presence of multixenobiotic resistance (MXR) mechanism in sponges, mussel and fish represents a general biological defence mechanism for their protection against both endogenous and environmental toxins. In aquatic organisms exposed to polluted waters, this P170 glycoprotein pump removes out of cells also ‘new’, man-made toxic chemicals. Many chemical agents, the ‘chemosensitizers’, may alter the function of this fragile mechanism. The knowledge that the presence of one xenobiotic may block the pumping out of other xenobiotic(s), and hence accelerate their accumulation, may have important implications on environmental parameters like exposure, uptake, bioaccumulation, and toxicity. In this report we present the results of measurements of MXR-phenotype expression using the ‘accumulation’ technique and the modulators of MXR, verapamil and staurosporine, demonstrating (1) the interpopulation differences in the level of MXR expression in a marine snail Monodonta turbinata and its induction in specimens exposed to polluted waters, (2) the characteristics of the MXR-expression in a mussel Mytilus galloprovincialis , and (3) how model- or natural-inhibitors of MXR can modulate the no observed effect concentrations (NOEC) of xenobiotics in a fresh water clam Corbicula fluminea and a carp Cyprinus carpio to the observed effect concentrations (OEC).

Expression of multixenobiotic resistance mechanism in a marine mussel Mytilus galloprovincialis as a biomarker of exposure to polluted environments

Abstract The gills of mussel Mytilus galloprovincialis express a multixenobiotic resistance mechanism (MXRM) mediated by P-glycoprotein activity, as evidenced by the rate of (G-3H) vincristine accumulation and its sensitivity to verapamil, as well as by the direct immunochemical detection of P-glycoprotein by Western blotting analysis. The MXRM is induced at polluted sites: gills from mussels living at polluted sites accumulate less vincristine, the vincristine accumulation is less sensitive to verapamil, and in most cases express higher levels of P-glycoprotein. The state of induction of functional activity of MXRM is proportional to the level of pollution, as demonstrated at sites along the well defined pollution gradient in the aquatorium of Rovinj, Northern Adriatic. Since the immunological expression of P-glycoprotein may be compromised by high interindividual differences, season, and some as yet unknown factors, it could not be used as a single marker of exposure. However, the rate of vincristine accumulation and its sensitivity to verapamil offer a new, informatively powerful, molecular biomarker of exposure.

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.

Postlabeling analysis of carcinogen-DNA adducts in mussel, Mytilus galloprovincialis

Abstract The 32P-postlabeling analysis of DNA adducts formed in mussel digestive gland homogenates incubated with 2-aminofluorene (32 μ m ) showed one major and one minor adduct in the range of 1 adduct per 1–4 × 108 nucleotides. The predominant adduct was found to be chromatographically similar to the known C8-aminofluorene-substituted guanine adduct. The metabolic activation of 2-aminofluorene was blocked in the presence of methimazole (470 μ m ), a competitive inhibitor of FAD-containing monooxygenase. In contrast to 2-aminofluorene, benzo(a)pyrene-modified DNA either did not show any adduct or a very weak adduct spot (1 adduct per 1–4 × 109 nucleotides). The samples of DNA from mussel, carp and bream, which had no obvious exposure to pollutants, showed one to several weak ‘natural adducts’. The origin of these adducts is as yet undetermined. Since postlabeling assay enables measurement of carcinogen-DNA adducts formed with any, even unknown, chemicals from the composite environmental mixture of genotoxic pollutants, it may become a valuable tool for risk assessment also in marine environments.

Inhibitory effects of extracts from the marine alga Caulerpa taxifolia and of toxin from Caulerpa racemosa on multixenobiotic resistance in the marine sponge Geodia cydonium

The invasive growth of the introduced green alga Caulerpa taxifolia, already affecting the richness and diversity of the littoral ecosystems, has become a major ecological problem in the Mediterranean Sea. Previously, we demonstrated that the water pollutant tributyltin induces apoptosis in tissue of the marine sponge Geodia cydonium at concentrations of 3 μM and higher. Here we show that exposure of G. cydonium to low (non-toxic) concentrations of Caulerpa extract or purified caulerpin (10 μg/ml) together with low doses of tributyltin (1 μM; non-toxic), results in a strong apoptotic effect. Evidence is presented that the enhancement of toxicity of tributyltin by Caulerpa extract is at least partially caused by inhibition of the multixenobiotic resistance (MXR) pump by the algal toxin. Caulerpa extract, as well as caulerpin, strongly enhance the accumulation of the test substrate of MXR, rhodamine B, in the gills of the mussel Dreissena polymorpha, used as a model system for testing MXR-inhibiting potential.

Reversion of the multixenobiotic resistance mechanism in gills of a marine mussel Mytilus galloprovincialis by a model inhibitor and environmental modulators of P170-glycoprotein

Abstract In gills of the marine mussel Mytilus galloprovincialis there is a P-glycoprotein-mediated multixenobiotic resistance mechanism (MXRM) which is similar to the multidrug resistance (MDR) mechanism in tumor cells resistant to cytotoxic drugs. This mechanism in mussel gills could be inhibited by verapamil, a known competitive inhibitor of P-glycoprotein, as measured by the enhanced accumulation of (G- 3 H)vincristine (VCR). The activity of MXRM is indicated to be regulated in mussel by protein kinase C (PKC), since the PKC-specific inhibitor, bisindolylmaleimide, inhibits its activity. Similar inhibitory effects were exerted by a water-extract of Diesel-2 oil, or xenobiotics from the XAD-2 concentrates of river water or river sediment pore-water. MXRM was elevated in mussel specimens living at, or transplanted to, a polluted site, as demonstrated by the striking decrease in the rate of VCR accumulation. These results indicate that MXRM may be used as a biomarker of exposure to pollutants, and stress the importance of a new type of hazardous xenobiotic — the ‘chemosensitizers’ of MXRM.