John M. Frazier

The dynamics of metals in the American oyster, Crassostrea virginica. I. Seasonal effects

The seasonal dynamics of Mn, Fe, Zn, Cu and Cd were investigated in a genetically similar population of hatchery-reared American oysters, Crassostrea virginica, maintained in plastic trays in the Rhode River, a tributary of the Chesapeake Bay. Samples were collected monthly from September, 1971 through May, 1973. Annual cycles resulting in the turnover of large portions of the body burden were observed for all metals studied. Subtly different patterns of metal dynamics were observed as a result of the reduction in biological variations realized by employing genetically similar oysters. Metals are grouped into two classes according to their dynamics: (1) Mn and Fe concentrations in soft tissues are significantly correlated with shell deposition. A high rate of Mn turnover in soft tissues (approximately 2 times the body burden per day) occurs during the shell growth season. (2) Zn and Cu concentrations are not correlated with shell growth. Zn and Cu body burdens exhibit a gradual increase during the spring and early summer followed by a rapid loss during August–September in which 33% of the Zn and 50% of the Cu is lost in less than 4 weeks. Cd behavior is similar to zn and Cu with a 50% reduction in body burden during an 11 week period between July and October.

The dynamics of metals in the American oyster,Crassostrea virginica. II. Environmental effects

The dynamics of Mn, Fe, Zn, Cu and Cd in the shell and soft tissues of the American oyster,Crassostrea virginica, were observed in oysters exposedin situ to a metal-contaiminated environment from September, 1972 until August, 1973. Zn and Cu accumulated in soft tissues of exposed oysters reaching levels of 4100 μg Zn/gm and 450 μg Cu/gm compared to 1700 μg Zn/gm and 60 μg Cu/gm for controls (dry weight basis). The relative enhancement of metals in oyster soft tissues exposed to the contaminated environment over controls reflected the pattern of metal contamination in sediments. Although growth of the oysters, as measured by soft tissue dry weight and shell dimension, was identical, shells of exposed oysters were significantly thinner than controls (16%). Trace metal incorporation into shell was affected with Mn deposition surpressed and Fe, Zn and Cu slightly increased. Uptake of metals by oyster soft tissues was seasonally dependent with rapid uptake occurring in the summer and fall but delayed uptake occurring in the early spring.

Characterization of two molecular weight classes of cadmium binding proteins from the mussel, mytilus edulis (l.)

Inducible cadmium binding proteins (Cd-BP) in the mussel, Mytilus edulis, were resolved into two molecular weight components by gel permeation chromatography on Sephadex G-75. Each of these two molecular weight components was further resolved into four subcomponents by DEAE ion exchange chromatography. All eight subcomponents bound cadmium and exhibited significant u.v. absorption at 254 and little absorption at 280 nm. Based on amino acid composition analysis two classes of proteins were identified, one having higher cysteine (approximately 25 mole %) and lower serine and glutamic acid contents compared to the other class.

Kinetics of cadmium transport in the isolated perfused rat liver

Abstract The uptake of cadmium by the isolated perfused rat liver obeys first-order kinetics at each dose level tested, 0.178, 1.78, and 17.8 μmol cadmium as the chloride. The uptake rate constant is elevated at low cadmium doses indicating the possibility of a mediated transport process. Zinc is secreted into the perfusion medium by the isolated liver at a constant rate in control preparations (3.5 ± 1.2 nmol/min); however, this rate is significantly increased during cadmium exposure in a dose-dependent manner. At low doses the amount of excess zinc secreted by the isolated liver in response to cadmium exposure is approximately equal to the amount of cadmium taken up, on a molar basis. The data are consistent with the hypothesis that cadmium-zinc exchange is one mechanism for mediated cadmium transport in the liver. Cadmium rapidly appears in the bile following exposure, and biliary concentrations increase in relation to dose. The fraction of the cadmium dose excreted in the bile during 3 hr of cadmium exposure reached maximum levels at intermediate doses (6.9% excreted), indicating a threshold for biliary clearance at the low doses and supression of biliary excretion at the high doses due to reduced bile production.

Lateralization of zinc in rat brain and its relationship to a spatial behavior

Essential metals are differentially accumulated within the brain and have been related to normal neurotransmitter metabolism. Hippocampal glutaminergic pathways have the highest zinc levels in the brain, and lesions to these pathways disrupt behaviors with a spatial component. Zinc distribution may thus reflect glutaminergic activity or innervation and may have functional consequences for spatial behavior. The present data support this hypothesis, indicating that the lateral distribution of zinc between the right and left hippocampus is strongly correlated with the spatial preference of the animal (r = + 0.72). Other parameters tested but shown not to be significantly correlated with spatial preference were zinc in corpus striatum and cortex, and copper in hippocampus corpus striatum and cortex.

Dose dependence of cadmium kinetics in the rat liver following intravenous injection.

The objective of these experiments was to investigate the dose dependence of the kinetics of cadmium accumulation and redistribution within the rat liver over the period of 6 hr following iv administration. Male Wistar rats, 450–550 g, were injected with total doses of 20, 200, and 1000 μ g of cadmium as CdCl 2 . Total cadmium accumulated by the liver attained a steady state within 3 hr following injection. The fraction of the cadmium dose accumulated in the liver decreased from 76 ± 6% of the dose at the lowest exposure to 45 ± 7% at the highest exposure. These results indicate partial saturation of the capacity of the liver to accumulate cadmium. Cadmium accumulation in the 106,000 g 60-min centrifugation pellet was a linear function of dose while accumulation in the supernatant fraction was partially saturated at the highest dose. Gel permeation chromatography of supernatant indicated that this effect was due to partial saturation of the binding capacity of high molecular weight (>60,000) cytoplasmic macromolecules (HMWM). Although uptake into HMWM is complete by 2 hr, incorporation of cadmium into low molecular weight (6000–10,000) cadmium-binding proteins, metallothionein, continues linearly throughout the 6 hr. The rate of cadmium incorporation into metallothionein shows saturation kinetics with respect to dose. Cadmium exposure up to 6 hr had no effect on total liver zinc concentration; however, there are alterations in the distribution of zinc among hepatic cytoplasmic macromolecules. These data indicate that the dose dependence of cadmium accumulation by the liver can be accounted for by binding to HMWM while the dose dependence of intracellular redistribution can be explained by the saturation of the incorporation rate of cadmium into metallothionein.

The effect of sodium diethyldithiocarbamate treatment on copper and zinc concentrations in rat brain

Abstract Copper and zinc play important roles in the metabolic functions of the central nervous system. The effect of the chelating agent diethyl-dithiocarbamate (DDC) on the nervous system may be related to its effect on the kinetics and distribution of these essential metals. This study was designed to investigate the effects of DDC on copper and zinc concentrations in specific regions of the brain following both acute and repeated treatment. Acute treatment (250 mg/kg) had no effect on copper or zinc concentrations in brain regions at 24 hr while repeated treatment (250 mg/kg five times per week for 4 weeks) increased copper levels in the brain stem, cortex, hippocampus, and the rest of the brain but did not alter zinc concentrations in any brain regions. Many of the observed effects of DDC on distribution may be attributed to the formation of lipid-soluble DDC-metal complexes.

FRAMEWORK FOR VALIDATION AND IMPLEMENTATION OF IN VITRO TOXICITY TESTS

SummaryThe development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community—academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.

In vitro models for toxicological research and testing

The objective of this report is to discuss some of the issues involved in utilizing in vitro methods in toxicological research and testing. The subject is not new, in vitro methods have been used for many years in this context. However, there has been a significant increase in interest in the topic within the scientific community recently as witnessed by the increase in scientific journals dedicated to the topic, symposia held by scientific societies, and commitment of resources to in vitro toxicological research activities. Toxicologists should be aware of these developments as the future directions of the science will be influenced significantly by in vitro methodology.

Mathematical model for cadmium kinetics in the isolated perfused rat liver system

The isolated perfused rat liver (IPRL) preparation has previously been used to investigate cadmium kinetics. A mathematical model which has been developed to simulate cadmium kinetics in the IPRL is described. The model takes into consideration binding of cadmium to both intra- and extracellular proteins and the mechanisms of membrane transport. In addition, the competitive interaction of cadmium with endogenous zinc is incorporated into the model. Model simulations of the behavior of cadmium and zinc in the perfusion medium, liver, and bile are compared to results from IPRL experiments involving cadmium doses ranging from 0.29 to 15.6 mumol. A major contribution of this model is the identification, from a kinetic point of view, of two high-molecular-weight classes of intracellular cadmium-binding species which can be identified by two distinct peaks in Sephadex G-75 profiles of hepatic cytosol. This model can be utilized for the quantitation of kinetics based on specific mechanisms involved in cadmium hepatic kinetics.