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Aromatic and heteroaromatic amines (ArNH(2)) represent a class of potential mutagens that after being metabolically activated covalently modify DNA. Activation of ArNH(2) in many cases starts with N-hydroxylation by P450 enzymes, primarily CYP1A2. Poor understanding of structure-mutagenicity relationships of ArNH(2) limits their use in drug discovery programs. Key factors that facilitate activation of ArNH(2) are revealed by exploring their reaction intermediates in CYP1A2 using DFT calculations. On the basis of these calculations and extensive analysis of structure-mutagenicity data, we suggest that mutagenic metabolites are generated by ferric peroxo intermediate, (CYP1A2)Fe(III)-OO(-), in a three-step heterolytic mechanism. First, the distal oxygen of the oxidant abstracts proton from H-bonded ArNH(2). The subsequent proximal protonation of the resulting (CYP1A2)Fe(III)-OOH weakens both the O-O and the O-H bonds of the oxidant. Heterolytic cleavage of the O-O bond leads to N-hydroxylation of ArNH(-) via S(N)2 mechanism, whereas cleavage of the O-H bond results in release of hydroperoxy radical. Thus, our proposed reaction offers a mechanistic explanation for previous observations that metabolism of aromatic amines could cause oxidative stress. The primary drivers for mutagenic potency of ArNH(2) are (i) binding affinity of ArNH(2) in the productive binding mode within the CYP1A2 substrate cavity, (ii) resonance stabilization of the anionic forms of ArNH(2), and (iii) exothermicity of proton-assisted heterolytic cleavage of N-O bonds of hydroxylamines and their bioconjugates. This leads to a strategy for designing mutagenicity free ArNH(2): Structural alterations in ArNH(2), which disrupt geometric compatibility with CYP1A2, hinder proton abstraction, or strongly destabilize the nitrenium ion, in this order of priority, prevent genotoxicity.

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The level of vitellogenin‐like (Vg‐like) proteins in the hemolymph of the soft‐shell clam (Mya arenaria) was indirectly determined using an alkali‐labile phosphate (ALP) measurement assay. Biochemical characterization of hemolymph from actively reproducing clams showed the presence of Zn, Ca and of substantial amounts of ALP, all of which are indicative of fish serum vitellogenin, reported to be a Zn and Ca containing glycolipophosphoprotein. ALP assays undertaken on different phosphate‐containing compounds (phosphate‐buffer saline, diphospholipids and 12,000×g supernatants of Vg‐rich trout ovary homogenates) showed little interference by the first two substrates and confirmed the specificity of the ALP assay as an indirect method of determining Vg‐like proteins in clam hemolymph. Moreover, clams injected with 17β‐estradiol and two known xenoestrogens (nonylphenol and pentachlorophenol) significantly induced hemolymph ALP levels. Field work revealed significant differences in hemolymph ALP levels from clams collected at specific intertidal stations of the Saguenay Fjord during temporal (May to October) and spatial (June) surveys in 1997, which appear to indicate that (anti‐)estrogenic chemicals may be present in the Saguenay Fjord. Our preliminary results thus show that this simple and rapid ALP assay holds promise as a biomarker of estrogen disruption with molluscan shellfish as bioindicators. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 455–465, 1999

The intensive development of industry and urban structures along the seashores of the world, as well as the immense increase in marine transportation and other activities, has resulted in the deposition of thousands of new chemicals and organic compounds, endangering the existence of organisms and ecosystems. The conventional single biomarker methods used in ecological assessment studies cannot provide an adequate base for environmental health assessment, management and sustainability planning. The present study uses a set of novel biochemical, physiological, cytogenetic and morphological methods to characterize the state of health of selected molluscs and fish along the shores of the German North Sea, as well as the Israeli Mediterranean and Red Sea. The methods include measurement of activity of multixenobiotic resistance-mediated transporter (MXRtr) and the system of active transport of organic anions (SATOA) as indicators of antixenobiotic defence; glutathione S-transferase (GST) activity as an indicator of biotransformation of xenobiotics; DNA unwinding as a marker of genotoxicity; micronucleus test for clastogenicity; levels of phagocytosis for immunotoxicity; cholinesterase (ChE) activity and level of catecholamines as indicators of neurotoxicity; permeability of external epithelia to anionic hydrophilic probe, intralysosomal accumulation of cationic amphiphilic probe and activity of non-specific esterases as indicators of cell/tissue viability. Complete histopathological examination was used for diagnostics of environmental pathology. The obtained data show that the activity of the defensive pumps, MXRtr and SATOA in the studied organisms was significantly higher in the surface epithelia of molluscs from a polluted site than that of the same species from control, unpolluted stations, providing clear evidence of response to stress. Enhanced frequency of DNA lesions (alkaline and acidic DNA unwinding) and micronucleus-containing cells was significantly higher in samples from polluted sites in comparison to those from the clean sites that exhibited genotoxic and clastogenic activity of the pollutants. In all the studied molluscs a negative correlation was found between the MXRtr levels of activity and the frequency of micronucleus-containing hemocytes. The expression of this was in accordance with the level of pollution. The complete histopathological examination demonstrates significantly higher frequencies of pathological alterations in organs of animals from polluted sites. A strong negative correlation was found between the frequency of these alterations and MXRtr activity in the same specimens. In addition to these parameters, a decrease in the viability was noted in molluscs from the polluted sites, but ChE activities remained similar at most sites. The methods applied in our study unmasked numerous early cryptic responses and negative alterations of health in populations of marine biota sampled from the polluted sites. This demonstrates that genotoxic, clastogenic and pathogenic xenobiotics a...