Designing Endocrine Disruption Out of the Next Generation of Chemicals.

A central goal of green chemistry is to avoid hazard in the design of new chemicals. This objective is best achieved when information about a chemical's potential hazardous effects is obtained as early in the design process as feasible. Endocrine disruption is a type of hazard that to date has been inadequately addressed by both industrial and regulatory science. To aid chemists in avoiding this hazard, we propose an endocrine disruption testing protocol for use by chemists in the design of new chemicals. The Tiered Protocol for Endocrine Disruption (TiPED) has been created under the oversight of a scientific advisory committee composed of leading representatives from both green chemistry and the environmental health sciences. TiPED is conceived as a tool for new chemical design, thus it starts with a chemist theoretically at "the drawing board." It consists of five testing tiers ranging from broad in silico evaluation up through specific cell- and whole organism-based assays. To be effective at detecting endocrine disruption, a testing protocol must be able to measure potential hormone-like or hormone-inhibiting effects of chemicals, as well as the many possible interactions and signaling sequellae such chemicals may have with cell-based receptors. Accordingly, we have designed this protocol to broadly interrogate the endocrine system. The proposed protocol will not detect all possible mechanisms of endocrine disruption, because scientific understanding of these phenomena is advancing rapidly. To ensure that the protocol remains current, we have established a plan for incorporating new assays into the protocol as the science advances. In this paper we present the principles that should guide the science of testing new chemicals for endocrine disruption, as well as principles by which to evaluate individual assays for applicability, and laboratories for reliability. In a 'proof-of-principle' test, we ran 6 endocrine disrupting chemicals (EDCs) that act via different endocrinological mechanisms through the protocol using published literature. Each was identified as endocrine active by one or more tiers. We believe that this voluntary testing protocol will be a dynamic tool to facilitate efficient and early identification of potentially problematic chemicals, while ultimately reducing the risks to public health.

Toxic chemicals: can what we don't know harm us?

The Chesapeake Bay Program began more than 20 years ago with assessments of a number of key areas, relying on measurements of habitats, plant and animal populations, and physical and chemical conditions. This approach used wildlife as indicators of Bay "health" and of potential threats to human health. The extent of toxic chemical contamination was one of the assessment endpoints in the original survey. When the initial assessment was completed in 1983, the results of Bay-wide surveys indicated that several specific waterways were contaminated. These waters, the Elizabeth River, Virginia, the James River, Virginia, and Baltimore Harbor, Maryland, were targeted for specific actions to address the problems of historical and ongoing pollution. Over the past 10 years or more, data on some toxic chemical releases into and levels in the environment have been collected, but these data are limited in scope. Furthermore, these data are not used to assess threats to human health or more generally to nonhuman endpoints. New and existing data on environmental levels of chemicals and effects at low concentrations provide evidence that toxic chemicals may threaten both human and nonhuman health in the wider Bay system.

Respiratory Responses of the Blue Crab Callinectes sapidus to Long-Term Hypoxia.

Blue crabs (Callinectes sapidus) were held in hypoxic (50-55 mm Hg) water for 7-25 days. Postbranchial blood PO2 fell by about 80% within 24 h and then remained unchanged. Postbranchial blood total CO2 increased within 24 h and remained elevated for the duration of the experiment. There was no change in postbranchial blood pH, osmolality, or Cl. Lactate, urate, and Ca+2 all raise the O2 affinity of blue crab hemocyanin; by 25 days, blood lactate and urate had risen slightly, but Ca+2 had increased dramatically. Hemocyanin concentration had also increased by 25 days. At both 7 and 25 days there was an intrinsic increase in hemocyanin-O2 affinity and a change in subunit composition. The highly adaptive homotropic change is believed to be due to an attendant shift in the proportions of two of the three variable monomeric hemocyanin subunits. Thus, both heterotropic and homotropic adaptations enhance blood oxygenation at the gill during long-term hypoxia.

Non-equilibrium acid-base status in C. productus: role of exoskeletal carbonate buffers.

Hemolymph acid-base variables (pH, PCO2 and CCO2), hemolymph Ca2+ and Na+ concentrations, and osmolality were measured in unrestrained crabs, Cancer productus, before, during and following 4 hr emersion and 43 hr hyperoxia (460-510 Torr), both at 10 degrees C. Emersion and hyperoxia provoked an acidosis associated with elevation of hemolymph CCO2 and PCO2, yet attempts to calculate PCO2 from measured pH and CCO2 always resulted in values greater than those measured directly. This discrepancy between measured and calculated PCO2, was associated with base excess, and was eliminated upon in vitro equilibration of the hemolymph and more slowly in vivo, suggesting that metabolic compensation for the acidosis occurred more rapidly than could acid-base equilibration. During emersion, increases of CCO2 and [Ca2+] provide evidence that the internal CaCO3 stores, possibly from the exoskeleton, were mobilized during acid-base compensation. Hyperoxia provoked no such increase in Ca2+, and branchial uptake of HCO3- may make a major contribution to the elevation of CCO2 during hyperoxia. It is suggested that shell buffering by aquatic crustaceans provides a means of compensation for acidosis under conditions during which branchial function is impaired.

A new operational approach to PCO2 determination in crustacean hemolymph.

A new method of calculating PCO2 based on mathematical expressions derived from a measured 'Davenport diagram' is described. The measured 'Davenport diagram' is constructed from in vitro buffer curves relating total CO2 to pH at PCO2 levels which adequately encompass the in vivo range of the acid-base status for the species in question. The 'Davenport diagram' is described by three linear equations such that PCO2 can be accurately calculated from in vivo measured CCO2 and pH. The equations are specific for a given species at a given temperature and hemolymph ionic strength, as are the constants in the more classical Henderson-Hasselbalch equation. The accuracy of the method is equal to calculations using the Henderson-Hasselbalch equation with corrected values for pK1' and alpha CO2 and in vivo PCO2 measured directly. This procedure is equally applicable to fluids with dissolved pigments such as hemolymph from freshwater and marine crustaceans and to human blood. The major benefit of this method of calculating PCO2 is that correction nomograms for the constants in the Henderson-Hasselbalch equation are not required.