Neurodevelopmental effects: making the case for biologic plausibility.

It has been suggested that the most critical missing link between science and policy is causality; that is, the establishment of a definite cause-effect relationship between exposure and adverse health effects. As has been clearly demonstrated by the decades-long tobacco debate, causality is extremely difficult to establish with absolute certainty, particularly in the minds of scientists. Because of this, it has been suggested that a "weight of evidence" approach based on biologic plausibility should be used as a surrogate for causality when translating science into policy and public health practice. In the case of neurodevelopmental effects, the case for biologic plausibility is supported by scientific findings from three broad areas consisting of wildlife biology, toxicology, and epidemiology. A striking example of this is provided by research findings from the Great Lakes Basin, an area which has been the focus of significant scientific research for the last thirty years in these three broad areas. In this paper, we examine relevant findings from the Great Lakes Basin and elsewhere as they relate to establishing and supporting the biologic plausibility of neurodevelopmental effects associated with environmental exposures to persistent toxic substances.

Key environmental human health issues in the Great Lakes and St. Lawrence River basins.

In May 1997, Health Conference '97-Great Lakes/St. Lawrence, an international conference on the effects of the environment on human health in the Great Lakes and St. Lawrence River basins, was held in Montreal, Québec, Canada. This was the third international conference on this topic sponsored by agencies in the United States and Canada. More than 120 platform and poster presentations were given by scientists of different disciplines from the Great Lakes region and elsewhere. The presentations represented the most current research findings on the effects of the Great Lakes environment on human health. The reports covered environmental contaminant levels of persistent toxic substances (PTSs), routes and pathways of exposure, exposure assessment and human tissue levels of PTSs, human health outcomes, risk communication and assessment, and approaches to scientific collaboration. Reports indicate that levels of contaminants in the Great Lakes and St. Lawrence River basins have generally declined since the 1970s, although certain contaminants have plateaued or slightly increased. The findings include elevated body burden levels of contaminants in persons who consume large amounts of some Great Lakes sport fish, developmental deficits and neurologic problems in children of some fish-consuming parents, nervous system dysfunction in adults, and disturbances in reproductive parameters. The findings underscore the need for better public health intervention strategies.

The Great Lakes: a historical overview.

The Great Lakes are collectively the largest inland body of freshwater on this planet. For more than two hundred years, the Great Lakes basin has been used as a resource for industry, agriculture, shipping, and recreation. The physical characteristics of the basin and the long retention time of chemicals in the lakes combine to make this huge freshwater resource a repository for chemical by-products of these activities. Many of the more than one thousand chemicals detected in the waters, sediment, or biota of the Great Lakes have known toxic effects. This overview will identify the 11 most persistent toxic chemicals known as "critical" Great Lakes pollutants. It also will describe some of the adverse health effects that have been observed in fish and other wildlife because of exposure to these pollutants. Finally, it will discuss some of the early human health studies that 1) have demonstrated a correlation between increased body burdens and fish consumption, and 2) suggest an association between consumption of contaminated Great Lakes fish and adverse human health effects.

Harmonizing human health studies in the Great Lakes.

Epidemiological studies of exposed human populations can provide valuable evidence of human health effects. Information has been sparse on human health effects associated with consumption of contaminated Great Lakes fish. As part of its Great Lakes Human Health Effects Research Program, the Agency for Toxic Substances and Disease Registry (ATSDR) has funded ten projects. Of these studies, eight are epidemiologic investigations of human exposure and potential health effects from consumption of contaminated fish. To strengthen and to enhance the findings and comparability across the health studies, ATSDR has initiated several activities. These activities include harmonizing questionnaires, analytical protocols, human health end points, and contaminants tested. Also included is the establishment of a quality assurance and quality control (QA/QC) program and tissue bank. These activities will allow ATSDR to enhance exposure assessment in the Great Lakes basin. In addition, these research activities allow ATSDR to evaluate and to interpret data across all the projects, including a basin-wide health risk analysis on exposure, levels of contaminants or body burden, and the potential for human health effects from exposure to Great Lakes contaminants.

Differences in morphology, growth rate, and protein synthesis between cultured arterial and venous endothelial cells.

Intrinsic differences between aortic and venous endothelial cells were demonstrated by studies of morphology, growth rates, and protein synthesis. Endothelial cells from bovine thoracic aortas (AECs) and inferior vena caval (VECs) were harvested, maintained in cell culture, and characterized. VECs were consistently larger (mean cell diameter 14.3 micron vs 12.7 micron; p less than 0.001) and more pleomorphic than were AECs. Scanning electron microscopy revealed that VECs were thinner than AECs and that VECs had numerous fine, cellular processes that were much less abundant on the AECs. Enzymatic disaggregation of confluent cells resulted in a reduced generation time by the AECs compared with VECs. Moreover, VECs responded to disaggregation by a significantly larger increase in cell size than the AECs. Protein synthesis was quantitated by computer analysis of autoradiograph of two-dimensional gel eletrophoresis and separation of 998 35S-methionine-labeled proteins from cell lysates. Synthesis of 257 proteins was significantly different (p less than 0.05); synthesis of 239 proteins by VECs was either decreased (219) or undetectable (20) compared with those of AECs. In contrast, only 18 proteins were produced in significantly greater quantity by VECs than by AECs. The cytoskeletal proteins actin and alpha- and beta-tubulins were produced in significantly greater quantity by AECs than VECs. These results indicate that cultured endothelial cells of arterial origin are substantially different from those of the venous circulation. These phenotypic differences are maintained in vitro despite eliminating variability in hemodynamic stress and interaction with other cellular elements of the vessel wall.

The in vivo biosynthesis of embryonic proteins after maternal administration of phenytoin in the mouse.

Pregnant A/J mice received 60 mg phenytoin/kg body weight on Day 10 of gestation. Eighteen hours after phenytoin injection, animals were injected (ip) with 20 microCi/g of [35S]methionine. After 6 hr of incorporation animals were sacrificed and the embryos were removed. Protein synthesis in the embryo, as measured by [35S]methionine incorporation into trichloroacetic-precipitable protein, was analyzed by SDS-PAGE and quantitation of autoradiograms. The results of gel electrophoresis indicate that in embryonic primary palates and limb buds from phenytoin-treated mothers there is an increase in synthesis of 66-, 50-, 44-, and 13-kDa proteins and a decrease in synthesis of an 18-kDa protein compared with values for the control counterpart. No role has been assigned to the 66-, 44-, or 13-kDa proteins but the 50-kDa band comigrates with tubulin and the 18-kDa band comigrates with calmodulin. Palatal cells in vitro stained positively with specific antibody to both these proteins. An adverse effect of the anticonvulsant drug phenytoin, when administered to pregnant A/J mice is an increase in the incidence of cleft lip with or without cleft palate [CL(P)] in their offspring. These alterations in protein synthesis may be a direct or secondary result of maternal phenytoin treatment and may play a role in CL(P) formation in vivo.

The water, DNA, collagen and noncollagen protein contents in embryos after maternal administration of a teratogenic dose of phenytoin.

The growth of developing A/J mouse embryos was studied after maternal administration of phenytoin (Dilantin), an anticonvulsant drug. Wet weight, dry weight, protein and DNA contents of the embryos were quantitated 24 h after drug administration. Collagen content was investigated because of its importance in cellular differentiation. The wet and dry weight of embryos from phenytoin-treated mothers were 52.3 and 57.5%, respectively, of that of embryos of control mothers. DNA and protein contents were also decreased in embryos from phenytoin-treated mothers. Collagen represented only 0.07% of the protein present in day 11 control embryos, but was increased 4.9-fold in embryos from phenytoin-treated mothers in comparison to controls. These results suggest that phenytoin reduces overall embryonic growth but stimulates collagen synthesis.

Maternal phenytoin administration affects DNA and protein synthesis in embryonic primary palates.

Recent studies have shown that phenytoin (Dilantin) administration to pregnant A/J mice on day 10 causes reduced growth in embryonic primary palates. The current investigation concentrates on biochemical and autoradiographic changes toward the end of primary palate formation (gestational day 11), which coincides with the developmental period used for the previously conducted morphological studies. On gestational day 10, one group of pregnant A/J mice was injected intraperitoneally (IP) with 60 mg/kg phenytoin and the other group with vehicle. Twenty-three hours after phenytoin administration, all animals were injected (IP) with either [3H]-thymidine or [3H]-leucine. After one hour of incorporation, animals were sacrificed, embryos removed and placed in ice-cold Eagle's minimum essential medium containing 0.02% NaN3 for biochemical assay or fixed immediately in Bouin's solution for autoradiography. For biochemical analyses, palates and limb buds were removed, homogenized, TCA precipitated, lyophilized, and acid hydrolyzed. Examination of the data revealed that DNA synthesis in control palates was 3.8-fold greater than in primary palates from embryos of phenytoin-treated mothers. Results were similar for limb buds from control embryos and from embryos of phenytoin-treated mothers. Experiments utilizing [3H]-leucine indicated that protein synthesis was 2.6-fold greater in primary palates from phenytoin-treated mothers than in control primary palates. Similar results were obtained for protein synthesis in limb-bud tissue from controls and embryos of phenytoin-treated mothers. Autoradiographic data supported the biochemical findings. DNA synthesis in primary palates from embryos of phenytoin-treated mothers decreased 3-fold; protein synthesis increased 2.2-fold compared with control primary palates.(ABSTRACT TRUNCATED AT 250 WORDS)