Identification, assessment and management of "endocrine disruptors" in wildlife in the EU substance legislation--discussion paper from the German Federal Environment Agency (UBA).

A discussion paper was developed by a panel of experts of the German Federal Environment Agency (UBA) contributing to the on-going debate on the identification, assessment and management of endocrine disruptors with a view to protect wildlife according to the EU substance legislation (plant protection products, biocides, industrial chemicals). Based on a critical synthesis of the state-of-the-art regarding regulatory requirements, testing methods, assessment schemes, decision-making criteria and risk management options, we advise an appropriate and consistent implementation of this important subject into existing chemicals legislation in Europe. Our proposal for a balanced risk management of endocrine disruptors essentially advocates transparent regulatory decision making based on a scientifically robust weight of evidence approach and an adequate risk management consistent across different legislations. With respect to the latter, a more explicit consideration of the principle of proportionality of regulatory decision making and socio-economic benefits in the on-going debate is further encouraged.

Redox regulation: a new challenge for pharmacology.

Redox signaling is evolving as a new field of biochemical and pharmacological research. Unlike oxidative stress which is characterized by a macroscopic shift in cellular redox potentials and usually accompanied by oxygen radical induced damage, redox regulation involves subtle and more chemically defined oxidations of short duration. Most important is the reductive component as a necessary part of a reversible regulatory process. Examples of redox regulation occur during early stages of the immune response, in hypoxia or in endothelial dysfunction. Persistent oxidative events together with a decline in the cellular reduction potential lead to oxidative stress as is seen in the pathophysiology of sepsis, reperfusion damage, atherosclerosis and diabetes. Oxidative signals involve superoxide and nitric oxide as the main players which form a system of oxidizing, nitrating or nitrosating species leading to posttranslational modifications of proteins. Modern techniques of immunohistochemistry and mass spectrometry allow a correlation of protein modification, e.g., disulfide, S-oxide, S-nitroso or nitrotyrosine formation, with enzyme activities and cellular responses. In this commentary, examples of the control of prostanoid synthesis by the NO/O2- system are described. Redox regulation represents an interesting challenge for the development of drugs that modulate the oxidative trigger mechanisms or enforce the reductive pathways.

A new pitfall in detecting biological end products of nitric oxide-nitration, nitros(yl)ation and nitrite/nitrate artefacts during freezing.

The present study shows that when freezing nitrite containing biological samples in the presence of sodium and phosphate, a process of tyrosine nitration and S-nitrosocysteine formation is observed. The underlying mechanism is obviously based on the already described pH decrease in sodium phosphate buffered solutions during the freezing process and probably involves nitrous acid as an intermediate. However, in pure potassium phosphate buffer freeze-artefacts were absent. The yield of 3-nitrotyrosine from albumin-bound or free tyrosine depends not only on the concentration of nitrite, tyrosine or protein, and sodium phosphate but also on the velocity of the freezing process. Nitrite and nitrate were quantified by the Griess/nitrate reductase assay. 3-nitrotyrosine formation was quantitatively measured by HPLC analysis with optical and electrochemical detection as well as qualitatively investigated by immunohistochemistry and slot blot analysis using 3-nitrotyrosine specific antibodies. The formation of S-nitrosocysteine was detected by S-nitrosothiol specific antibodies and quantified by a fluorometric assay. Irrespective of the mechanism and although the here presented results cannot be generalized, the data warrant caution for the analysis of nitration or nitros(yl)ation products following freezing of nitrite containing biological material.

Superoxide as inhibitor of calcineurin and mediator of redox regulation.

The concept of NO as a redoxactive messenger has to be broadened by including superoxide as an antagonistic messenger. Superoxide alone was found to inhibit calcineurin by interacting with the FeII-ZnII binuclear site. This links oxidative stress conditions with a Ca-dependent phosphorylation/dephosphorylation cascade. When NO and superoxide are generated at equal fluxes the resulting peroxynitrite can cause tyrosine nitrations (e.g. prostacyclin synthase inhibition) or oxidations of zinc-fingers in proteins, indicating a new messenger function. Finally, if generated in excess, NO can convert peroxynitrite to N(2)O(3) as a nitrosating agent. Thus, the NO/superoxide system provides four different messengers affecting important regulatory pathways.

Oxidation and nitrosation in the nitrogen monoxide/superoxide system.

Based on the previous report of McCord and co-workers (Crow, J. P., Beckman, J. S., and McCord, J. M. (1995) Biochemistry 34, 3544-3552), the zinc dithiolate active site of alcohol dehydrogenase (ADH) has been studied as a target for cellular oxidants. In the nitrogen monoxide ((*NO)/superoxide (O(2)) system, an equimolar generation of both radicals under peroxynitrite (PN) formation led to rapid inactivation of ADH activity, whereas hydrogen peroxide and ( small middle dot)NO alone reacted too slowly to be of physiological significance. 3-Morpholino sydnonimine inactivated the enzyme with an IC(50) value of 250 nm; the corresponding values for PN, hydrogen peroxide, and (*NO) were 500 nm, 50 microm, and 200 microm. When superoxide was generated at low fluxes by xanthine oxidase, it was quite effective in ADH inactivation (IC(50) (XO) approximately 1 milliunit/ml). All inactivations were accompanied by zinc release and disulfide formation, although no strict correlation was observed. From the two zinc thiolate centers, only the zinc Cys(2)His center released the metal by oxidants. The zinc Cys(4) center was also oxidized, but no second zinc atom could be found with 4-(2-pyridylazo)resorcinol (PAR) as a chelating agent except under denaturing conditions. Surprisingly, the oxidative actions of PN were abolished by a 2-3-fold excess of (*)NO under generation of a nitrosating species, probably dinitrogen trioxide. We conclude that in cellular systems, low fluxes of (*)NO and O(2) generate peroxynitrite at levels effective for zinc thiolate oxidations, facilitated by the nucleophilic nature of the complexed thiolate group. With an excess of (*)NO, the PN actions are blocked, which may explain the antioxidant properties of (*)NO and the mechanism of cellular S-nitrosations.