Gerald R. Bratton

Effects of lead (Pb) exposure during gestation and lactation on female pubertal development in the rat.

Lead (Pb) can delay sexual maturation; however, the mechanism and critical time of insult are not clearly defined. Therefore, we assessed maternal Pb levels during low-level gestational and/or lactational exposure, as well as blood and tissue Pb in developing fetuses in relation to the subsequent detrimental effects of Pb on puberty-related hormones and the onset of female puberty. Adult Fisher 344 female rats were gavaged daily with either a 1-ml solution of PbAc containing 12 mg/ml Pb or an equal volume of sodium acetate (NaCl), for the controls, from 30 days prior to breeding until their pups were weaned at 21 days. By cross-fostering at the time of birth, the pups were either exposed to PbAc or NaAc during gestation only, lactation only, or during both gestation and lactation. Pb delayed the timing of puberty and this delay was associated with suppressed serum levels of insulin-like growth factor-1 (IGF-1), luteinizing hormone (LH), and estradiol (E(2)). Liver IGF-1 mRNA was not affected, suggesting that Pb altered translation and/or secretion of IGF-1. We reported previously that peripherally derived IGF-1 acts at the hypothalamic level to facilitate LH release at puberty; hence, we suggest that the action of Pb in decreasing circulating IGF-1 contributes to the delayed puberty. The detrimental effects occurred regardless of the developmental time of exposure, although gestational exposure appeared more sensitive to the effects of Pb. Also, the effects noted were with blood Pb levels less than previously reported and these levels are relevant to human health concerns.

CONCENTRATIONS AND INTERACTIONS OF SELECTED ESSENTIAL AND NON-ESSENTIAL ELEMENTS IN BOWHEAD AND BELUGA WHALES OF ARCTIC ALASKA

In this study, we evaluated concentrations of twelve essential and non-essential elements (As, Cd, Co, Cu, Pb, Mg, Mn, Hg, Mo, Se, Ag, and Zn) in tissues of bowhead (Balaena mysticetus) and beluga (Delphinapterus leucas) whales from arctic Alaska (USA) and northwestern Canada. Tissue samples were collected between 1983 and 1997, mostly in 1995–97. The essential elements are reported to develop reference ranges for health status determination, and to help assess known or suspected interactions affecting toxicoses of cadmium (Cd) and mercury (Hg). In some tissues, Cd, Hg, and selenium (Se) were present at concentrations that have been associated with toxicoses in some domestic animals. Nevertheless, tissue levels of all elements were within ranges that have been reported previously in marine mammals. While mean Ag concentrations in beluga whale liver were relatively high (15.91 μg/g ww), Ag was not associated with hepatic Se levels or age, contrary to previous findings. Significant associations included: Cd with age, Zn, or Cu; Cu with age, Zn or Ag; and Hg with age, Se, Zn, or Cu. This study found hepatic Hg:Se molar ratios to be consistently lower than unity and different between species. Possible explanations for observed elemental correlations (i.e., interactions) and ancillary mechanisms of Cd and Hg detoxification are discussed.

CONCENTRATIONS AND INTERACTIONS OF SELECTED ESSENTIAL AND NON-ESSENTIAL ELEMENTS IN RINGED SEALS AND POLAR BEARS OF ARCTIC ALASKA

In this study, we evaluated concentrations of twelve essential and non-essential elements (As, Cd, Co, Cu, Pb, Mg, Mn, Hg, Mo, Se, Ag, and Zn) in tissues of ringed seals (Phoca hispida) and polar bears (Ursus maritimus) of arctic Alaska (USA). All samples were collected between 1995–97 in conjunction with subsistence harvests. The essential elements are reported to help develop reference ranges for health status determination and to help assess known or suspected interactions affecting toxicoses of cadmium (Cd) and mercury (Hg). In some tissues, Cd, Hg, and selenium (Se) were present at concentrations that have been associated with toxicoses in some domestic animals. Nevertheless, tissue levels of all elements were within ranges that have been reported previously in other pinnipeds and polar bears. Significant associations included: Cd with Zn or Cu; Cu with Zn or Ag; and Hg with Se, Zn, or Cu. This study found hepatic Hg: Se molar ratios to be lower than unity and different between the two species. Based upon significant differences in mean tissue elemental concentrations for polar bear versus ringed seal, we concluded that biomagnification factors (bear/seal) were significant for: Cu in liver and muscle; Pb in kidney; Se in kidney and muscle; Zn in liver and muscle; and Hg in liver. Possible explanations for observed elemental correlations (i.e., interactions) and ancillary mechanisms of Cd and Hg detoxification are discussed.

Toxicological evaluation and metal bioavailability in pregnant rats following exposure to clay minerals in the diet.

Silicate clays are frequently added to animal feeds to bind and reduce the bioavailability of mycotoxins in the gastrointestinal tract. However, the bioavailability of trace metals in these clay feed additives has not been thoroughly investigated. Clays that act nonselectively may interact with nutrients, minerals, and other feedborne chemicals to pose significant hidden risks. In this study, a calcium montmorillonite clay (Novasil Plus, NSP) commonly used as an enterosorbent for aflatoxins and a sodium montmorillonite clay (Swy-2) (Source Clay Minerals Repository, Columbia, MO) were examined. Clays were supplemented in the balanced diet of Sprague-Dawley rats during pregnancy at a level of 2% (w/w). Evaluations of toxicity were performed on gestation d 16 and included maternal body weights, maternal feed intakes, litter weights, and embryonic resorptions. Liver, kidneys, tibia, brain, uterus, pooled placentas, and pooled embryonic mass were collected and weighed. Tissues were lyophilized and neutron activation analysis (NAA) was performed. Elements considered by NAA included Al, Ba, Br, Ca, Ce, Co, Cr, Cs, Cu, Dy, Eu, Fe, Hf, K, La, Lu, Mg, Mn, Na, Nd, Ni, Rb, S, Sb, Sc, Se, Sm, Sr, Ta, Tb, Te, Th, Ti, Tl, U, V, Yb, Zn, and Zr. Inductively coupled plasma–mass spectroscopy further confirmed that Al was below detection limits (<0.5 ppm) in the brain. Animals supplemented with either NSP or Swy-2 were similar to controls with respect to toxicity evaluations and metal analysis, with the exception of decreased brain Rb following clay supplementation. Overall, the results of this study suggest that neither NSP nor Swy-2, at relatively high dietary concentrations, influences mineral uptake or utilization in the pregnant rat.

Distribution of Inorganic Mercury in Liver and Kidney of Beluga and Bowhead Whales Through Autometallographic Development of Light Microscopic Tissue Sections

Inorganic mercury was localized through autometallography (AMG) in kidney and liver of free-ranging, subsistence-harvested beluga (Delphinapterus leucas; n = 20) and bowhead (Balaena mysticetus ; n = 5) whales. AMG granules were not evident in bowhead tissues, confirming nominal mercury (Hg) concentrations (range = 0.011 to 0.038 ug/g ww for total Hg). In belugas, total Hg ranged from 0.30 to 17.11 and from 0.33 to 82.47 ug/g ww in liver and kidney, respectively. AMG granules were restricted to cortical tubular epithelial cytoplasm in belugas with lower tissue burdens ; whales with higher tissue burdens had granules throughout the uriniferous tubular epithelium. In liver, AMG granular densities differed between lobular zones, concentrating in stellate macrophages and bile cannalicular domains of hepatocytes. AMG granules aggregated in periportal regions in belugas with lower hepatic Hg concentrations, yet among whales with higher Hg, AMG granule deposition extended to pericentral and midzonal regions of liver lobules. Mean areas occupied by AMG granules correlated well with hepatic Hg concentrations and age. In beluga livers, AMG staining density was not associated with lipofuscin quantity (an index of oxidative damage). Occasionally, AMG granules and lipofuscin were colocalized, but more often were not, implying that Hg was not a prominent factor in hepatic lipofuscin deposition in belugas.

DEVELOPMENT OF POROUS CLAY-BASED COMPOSITES FOR THE SORPTION OF LEAD FROM WATER

Lead contamination of water is a major health hazard, as illustrated by the fact that exposure to this metal has been associated with death and disease in humans, birds, and animals. The present research was aimed at the development of a porous, solid-phase sorbent that can be used in the remediation of lead-contaminated water. A suitable sorbent was identified by screening various clays and other materials for their ability to effectively bind lead. The clay was adhered to a solid support using an aqueous solution of carboxymethyl cellulose. The binary composite was then tested for its ability to bind lead from solution, while providing void volume, increased surface area, and considerably enhanced hydraulic conductivity. The results suggested that a combination of sodium montmorillonite clay and carbon exhibited enhanced sorption of lead compared to carbon alone, and also supported the potential application of various combinations of sorbent materials. This value-added combination of clay, solid support, and adhesive will allow for the construction of column filtration systems that are multifunctional and capable of purifying large volumes of contaminated water.

Effects of combined lead and cadmium exposure: changes in schedule-controlled responding and in dopamine, serotonin, and their metabolites

Adult male rats were maintained on 1 of 4 ad-lib diets: Group Control-Diet received a normal laboratory diet that contained no added chemicals: Group Lead-Diet received a diet containing 500 ppm (parts per million) lead: Group Cadmium-Diet received a diet containing 100 ppm cadmium: and Group Lead-Cadmium-Diet received a diet containing both 500 ppm lead and 100 ppm cadmium. After 60 days of exposure to their respective diets, animals were placed on restricted diets (15 g/day) of the identical food received during the exposure period. Each animal was trained to lever press on a fixed-interval 1-min schedule for 21 sessions (1 session day). The results of schedule training showed that lead alone or cadmium alone was associated with increased lever pressing relative to control diet. However, when lead and cadmium were exposed jointly, performance was not significantly different from control performance. Similar attenuation of effects were observed for central neurotransmitter functions. Specifically disturbances in dopamine and serotonin turnover that were produced by lead alone were attenuated by the cotreatment of cadmium and lead. Possible accounts of the apparent antagonism between cadmium and lead are discussed.

Effects of lead treatment on intracellular iron and copper concentrations in cultured astroglia

Astroglia are implicated in the pathogenesis of lead (Pb) neurotoxicity in two capacities: as a lead sink that sequesters lead and as a target for direct cellular damage. A proposed cellular mechanism of Pb neurotoxicity is the alteration of metal concentrations, particularly the intracellular accumulation of Cu2+. We measured Pb uptake and the effects of Pb acetate on intracellular trace metal concentrations in astroglial cultures prepared from 0-to 4-day-old rat cerebral hemispheres. Mature Sprague Dawley and immature Wistar rat astroglia in culture took up lead from the medium. This finding replicatesin vitro the finding reported by others that astroglia in the brain take up Pb. Intracellular Cu and Fe concentrations (micrograms per 2 x 106 cells) were increased fourfold or more by treatment with 100μMPb for 3 days in the cultures of immature astroglia. Cu levels were also increased twofold or more in mature astroglia treated for 1–3 days with 100μMPb. The significance of this finding is that Cu is a potent inhibitor of Na+,K+ -ATPase, an enzyme by which astroglia are thought to remove K+ from the extracellular fluid in the brain. Thus, this finding supports the hypothesis that elevated [Cu], and perhaps [Fe], is a subcellular mechanism of neurotoxicity.

Cellular targets of lead neurotoxicity: In vitro models

Four types of cells in culture were exposed to lead (Pb) acetate (0.1-1000 microM): astroglia-enriched, oligodendroglia-enriched, and meningeal fibroblast cultures prepared from neonatal rat brains; and human neuroblastoma cultures prepared from the SK-N-SH-SY5Y cell line. The viability (trypan blue dye exclusion and proliferation) of these cell types after Pb exposure was compared in order to identify cellular targets in the central nervous system that were directly susceptible to cytotoxicity. Of the 4 cell types tested, only oligodendroglia showed marked sensitivity to Pb treatment. However, proliferation of the SY5Y cells was temporarily inhibited if the cells were treated 1 day (but not 3 days) after seeding. The potential for thiamin, which is used to treat Pb intoxication in cattle, to prevent this effect was tested. Rather than preventing this toxic effect, however, thiamin (1 mM) exacerbated that inhibition of proliferation. Astroglia and meningeal fibroblasts, which were resistant to Pb toxicity, were shown by atomic absorption analysis to take up Pb from the culture medium and concentrate it intracellularly to at least 55X the extracellular concentration, thus supporting hypotheses that these cells act as Pb sinks in the brain.

Behavioral antagonism between lead and cadmium.

Adult male rats were exposed to one of four dietary conditions for a period of 60 days. Group Control-Diet received a diet containing no added lead or cadmium, group Lead-Diet received a diet that contained 500 ppm added lead, group Cadmium-Diet received a diet that contained 100 ppm added cadmium, and group Lead-Cadmium-Diet received a diet that contained both 500 ppm added lead and 100 ppm added cadmium. Subsequent to exposure, animals were tested in a Digiscan activity monitor. Animals were then sacrificed and metal concentrations were determined in blood and brain. The results from this experiment showed that lead alone increased movement and vertical activity. Cadmium alone decreased movement and increased rest time. Cotreatment with lead and cadmium failed to produce behavioral differences relative to controls; thus, it seems that the changes in activity caused by one metal are antagonized by the other. Results from the analyses of residues in tissues revealed that blood lead concentrations were lower in the cotreatment condition than the lead along condition. However, brain residue accumulations were not different for these two exposure conditions. There was no evidence that the presence of lead attenuated increases in cadmium residues in blood or brain. Overall, the residue data agree with a central, as contrasted with a peripheral, account of lead/cadmium interaction effects, at least as relates to behavior. Because lead and cadmium were additive with regard to producing decreased body weights, it seems that the toxic effect of these metals is antagonized by cotreatment in some instances, and augmented in others.