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Epidermal growth factor receptor-dependent mechanisms have been implicated in growth signal transduction pathways that contribute to cancer development, including dermal carcinogenesis. Detection of the extracellular domain of the epidermal growth factor receptor (EGFR ECD) in serum has been suggested as a potential biomarker for monitoring this effect in vivo. Arsenic is a known human carcinogen, producing skin and other malignancies in populations exposed through their drinking water. One such exposed population, which we have been studying for a number of years, is in Bangladesh. The purpose of this study was to examine the EGFR ECD as a potential biomarker of arsenic exposure and/or effect in this population. Levels of the EGFR ECD were determined by enzyme-linked immunosorbent assay in the serum samples from 574 individuals with a range of arsenic exposures from drinking water in the Araihazar area of Bangladesh. In multiple regression analysis, serum EGFR ECD was found to be positively associated with three different measures of arsenic exposure (well water arsenic, urinary arsenic and a cumulative arsenic index) at statistically significant levels (p

This study documents the response of 6500 rural households in a 25 km(2) area of Bangladesh to interventions intended to reduce their exposure to arsenic contained in well water. The interventions included public education, posting test results for arsenic on the wells, and installing 50 community wells. Sixty-five percent of respondents from the subset of 3410 unsafe wells changed their source of drinking water, often to new and untested wells. Only 15% of respondents from the subset of safe wells changed their source, indicating that health concerns motivated the changes. The geo-referenced data indicate that distance to the nearest safe well also influenced household responses.

A comparison of field and laboratory measurements of arsenic in groundwater of Araihazar, Bangladesh, indicates that the most widely used field kit correctly determined the status of 88% of 799 wells relative to the local standard of 50 microg/L As. Additional tests showthatthe inconsistencies, mainly underestimates in the 50-100 microg/L As range, can be avoided by increasing the reaction time from 20 to 40 min. Despite this limitation, the field data already compiled for millions of wells by the Bangladesh Arsenic Mitigation and Water Supply Project, in combination with information on well location and depth, should prove to be extremely useful to prioritize interventions in thousands of affected villages.

Iron homeostasis is tightly regulated, as cells work to conserve this essential but potentially toxic metal. The translation of many iron proteins is controlled by the binding of two cytoplasmic proteins, iron regulatory protein 1 and 2 (IRP1 and IRP2) to stem loop structures, known as iron-responsive elements (IREs), found in the untranslated regions of their mRNAs. In short, when iron is depleted, IRP1 or IRP2 bind IREs; this decreases the synthesis of proteins involved in iron storage and mitochondrial metabolism (e.g. ferritin and mitochondrial aconitase) and increases the synthesis of those involved in iron uptake (e.g. transferrin receptor). It is likely that more iron-containing proteins have IREs and that other IRPs may exist. One obvious place to search is in Complex I of the mitochondrial respiratory chain, which contains at least 6 iron-sulfur (Fe-S) subunits. Interestingly, in idiopathic Parkinson's disease, iron homeostasis is altered, and Complex I activity is diminished. These findings led us to investigate whether iron status affects the Fe-S subunits of Complex I. We found that the protein levels of the 75-kDa subunit of Complex I were modulated by levels of iron in the cell, whereas mRNA levels were minimally changed. Isolation of a clone of the 75-kDa Fe-S subunit with a more complete 5'-untranslated region sequence revealed a novel IRE-like stem loop sequence. RNA-protein gel shift assays demonstrated that a specific cytoplasmic protein bound the novel IRE and that the binding of the protein was affected by iron status. Western blot analysis and supershift assays showed that this cytosolic protein is neither IRP1 nor IRP2. In addition, ferritin IRE was able to compete for binding with this putative IRP. These results suggest that the 75-kDa Fe-S subunit of mitochondrial Complex I may be regulated by a novel IRE-IRP system.

In vivo bone lead measurements with 109Cd-based K-shell X-ray fluorescence (XRF) have been used to assess long-term lead exposure in adults. Tibia lead levels were measured in 210 children (106 boys, 104 girls) of 11-12(1/2) years of age in a lead smelter town and in a control (nonexposed) town. Tibia lead levels, methodological uncertainties, and models of some of the factors influencing them are presented. 109Cd-based K-shell XRF tibia lead methodological uncertainty in children is comparable to that in adults., (Copyright 2001 Academic Press.)

Maternal smoking during pregnancy elevates risk for later child behavior problems. Because prior studies considered only Western settings, where smoking co-occurs with social disadvantage, we examined this association in Yugoslavia, a different cultural setting. Mothers enrolled in pregnancy as the low-exposure group in a prospective study of lead exposure were interviewed about health, including smoking history. A total of 199 children were assessed on the Child Behavior Checklist (CBCL) at ages 4, 4 1/2, and 5 years. Average cumulative blood lead (BPb) was determined from serial samples taken biannually since delivery. Longitudinal analyses were derived from 191 children with available data on behavior and covariates. Smoking was unrelated to social adversity. Controlling for age, gender, birthweight, ethnicity, maternal education, and Home Observation for Measurement of the Environment (HOME) Acceptance, smoking was associated with worse scores on almost all subscales; BPb concentration was related to small increases in the Delinquency subscale. Daughters of smokers received significantly higher scores on Somatic Complaints compared to daughters of nonsmokers, consistent with other work relating biological factors and internalizing problems in young girls. Because the present smoking/child behavior associations persist after control for individual and social factors also related to behavior problems, possible biological mediators are considered.

To investigate associations between the timing of lead (Pb) exposure on early intelligence, we examined the results of psychometric evaluations at ages 3, 4, 5, and 7 years, from 442 children whose mothers were recruited during pregnancy from a smelter town and a non-lead-exposed town in Yugoslavia. We compared the relative contribution of prenatal blood lead (BPb) with that of relative increases in BPb in either the early (0-2 years) or the later (from 2 years on) postnatal period to child intelligence measured longitudinally at ages 3 and 4 (McCarthy GCI), 5 (Wechsler Preschool and Primary Scale of Intelligence-Revised, WPPSI-R IQ), and 7 (Wechsler Intelligence Scale for Children-version III, WISC-III IQ), controlling for: Home Observation for Measurement of the Environment (HOME) quality; maternal age, intelligence, education, and ethnicity; and birthweight and gender. Elevations in both prenatal and postnatal BPb were associated with small decrements in young children's intelligence.

Objective: To investigate associations between lead exposure and early motor development., Study Design: We conducted standardized assessments of motor function (Bruininks-Oseretsky Test of Motor Proficiency and Beery Developmental Test of Visual-Motor Integration) at age 54 months in 283 children whose mothers were recruited in pregnancy from a smelter town and a non-lead-exposed town in Yugoslavia and who have been monitored twice yearly since birth. Blood lead concentration (BPb) was summarized in a measure reflecting the average of the child's semiannual serial log BPbs through 54 months., Results: Multiple regression showed that taken together, anthropometric measures (birth weight, body mass index) and markers of a stimulating and organized home life (HOME scale, parental education and intelligence, availability of siblings) explained a significant 10% to 18% of the variance in motor functioning. Beyond these contributions, BPb was significantly associated with poorer fine motor and visual motor function but was unrelated to gross motor coordination., Conclusions: Modest associations between early lead exposure and fine motor and visual motor functioning appear even after statistical adjustment is done for other contributors to motor development. Associations with BPb are specific to these areas of motor skill; gross motor development was unaffected.

Recent studies suggest that manganese-induced neurodegenerative toxicity may be partly due to its action on aconitase, which participates in cellular iron regulation and mitochondrial energy production. This study was performed to investigate whether chronic manganese exposure in rats influenced the homeostasis of iron in blood and cerebrospinal fluid (CSF). Groups of 8-10 rats received intraperitoneal injections of MnCl2 at the dose of 6 mg Mn/kg/day or equal volume of saline for 30 days. Concentrations of manganese and iron in plasma and CSF were determined by atomic absorption spectrophotometry. Rats exposed to manganese showed a greatly elevated manganese concentration in both plasma and CSF. The magnitude of increase in CSF manganese (11-fold) was equivalent to that of plasma (10-fold). Chronic manganese exposure resulted in a 32% decrease in plasma iron (p<0.01) and no changes in plasma total iron binding capacity (TIBC). However, it increased CSF iron by 3-fold as compared to the controls (p<0.01). Northern blot analyses of whole brain homogenates revealed a 34% increase in the expression of glutamine synthetase (p<0.05) with unchanged metallothionein-I in manganese-intoxicated rats. When the cultured choroidal epithelial cells derived from rat choroid plexus were incubated with MnCl2 (100 microM) for four days, the expression of transferrin receptor mRNA appeared to exceed by 50% that of control (p<0.002). The results indicate that chronic manganese exposure alters iron homeostasis possibly by expediting unidirectional influx of iron from the systemic circulation to cerebral compartment. The action appears likely to be mediated by manganese-facilitated iron transport at brain barrier systems., (Copyright 1999 Elsevier Science B.V.)

The symptoms of Mn-induced neurotoxicity resemble those of Parkinson's diseases. Since iron (Fe) appears to play a pivotal role in pathophysiology of Parkinson's disease, we set out to test the hypothesis that alterations in Fe-requiring enzymes such as aconitase contribute to Mn-induced neurotoxicity. Mitochondrial fractions prepared from rat brain were preincubated with MnCl2 in vitro, followed by the enzyme assay. Mn treatment significantly inhibited mitochondrial aconitase activity (24% inhibition at 625 microM to 81% at 2.5 mM, p<0.05). The inhibitory effect was reversible and Mn-concentration dependent, and was reversed by the addition of Fe (0.05-1 mM) to the reaction mixture. In an in vivo chronic Mn exposure model, rats received intraperitoneal injection of 6 mg/kg Mn as MnCl2 once daily for 30 consecutive days. Mn exposure led to a region-specific alteration in total aconitase (i.e. , mitochondrial+cytoplasmic): 48.5% reduction of the enzyme activity in frontal cortex (p<0.01), 33.7% in striatum (p<0.0963), and 20.6% in substantia nigra (p<0.139). Chronic Mn exposure increased Mn concentrations in serum, CSF, and brain tissues. The elevation of Mn in all selected brain regions (range between 3.1 and 3.9 fold) was similar in magnitude to that in CSF (3.1 fold) rather than serum (6. 1 fold). The present results suggest that Mn alters brain aconitase activity, which may lead to the disruption of mitochondrial energy production and cellular Fe metabolism in the brain., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

A primary rat choroidal epithelial cell culture system was developed to investigate mechanisms of heavy metal toxicity on the blood-cerebrospinal fluid (CSF) barrier. Epithelial cells were dissociated from choroidal tissue by pronase digestion and cultured in standard DMEM culture media supplemented with 10% fetal bovine serum and 10 ng epithelial growth factor per ml. The procedure yielded 2-5 x 10(4) cells from pooled plexuses of three to four rats, and a viability of 77-85%. The cultures displayed a dominant polygonal type of epithelial cells, with a population doubling time of 2-3 d. The cultures were of distinct choroidal epithelial origins. For example, immunocytochemical studies using monospecific rabbit anti-rat TTR polyclonal antibody revealed a strong positive stain of transthyretin (TTR), a thyroxine transport protein exclusively produced by the choroidal epithelia. Also, reverse-transcriptase polymerase chain reaction (PCR) confirmed the presence of specific TTR mRNA in the cultures. The cultures were further adapted to grow on a freely permeable membrane sandwiched between two culture chambers. The formation of an impermeable confluent monolayer occurred within 5 d after seeding and was verified by the presence of a steady electrical resistance across the membrane (80 +/- 10 ohm per cm2). The epithelial barriers appeared to actively transport [125I]-thyroxine from the basal to apical chamber. These results suggest that this primary cell culture system possesses typical choroidal epithelial characteristics and appears to be a suitable model for in vitro mechanistic investigations of blood-CSF barrier.

For a prospective study of lead exposure and early development, we recruited pregnant women from a lead smelter town and from an unexposed town in Yugoslavia and followed their children through 7 years of age. In this paper we consider associations between lifetime lead exposure, estimated by the area under the blood lead (BPb) versus time curve (AUC7), and intelligence, with particular concern for identifying lead's behavioral signature. The Wechsler Intelligence Scales for Children-Version III (WISC-III) was administered to 309 7-year-old children, 261 of whom had complete data on intelligence, blood lead, and relevant sociodemographic covariates (i.e., Home Observation for the Measurement of the Environment (HOME), birth weight, gender, sibship size, and maternal age, ethnicity, intelligence, and education). These showed anticipated associations with 7-year intelligence, explaining 41-4% of the variance in Full Scale, Performance, and Verbal IQ. Before covariate adjustment, AUC7 was unrelated to intelligence; after adjustment, AUC7 explained a significant 2.8%-4.2% of the variance in IQ. After adjustment, a change in lifetime BPb from 10 to 30 micro/dl related to an estimated decrease of 4.3 Full Scale IQ points; estimated decreases for Verbal and Performance IQ were 3.4 and 4.5 points, respectively. AUC7 was significantly and negatively related to three WISC-III factor scores: Freedom from Distractibility, Perceptual Organization, and Verbal Comprehension; the association with Perceptual Organization was the strongest. Consistent with previous studies, the IQ/lead association is small relative to more powerful social factors. Findings offer support for lead's behavioral signature; perceptual-motor skills are significantly more sensitive to lead exposure than are the language-related aspects of intelligence.

The choroid plexus, which is responsible for the maintenance of the biochemical milieu of the cerebrospinal fluid (CSF), avidly sequesters Pb. In order to test the hypothesis that chronic Pb exposure may impair choroid plexus function, male weanling Sprague-Dawley rats were exposed to Pb in drinking water at doses of 0, 50, or 250 micrograms Pb/ml (as Pb acetate) for 30, 60, or 90 days. The function of the choroid plexus was assessed as reflected by CSF concentrations of transthyretin (TTR, a major CSF protein manufactured by brain choroid plexus) and CSF essential metal ions (Ca2+, Mg2+, K+, and Na+). TTR concentrations were determined by radioimmunoassay using a monospecific rabbit anti-rat TTR polyclonal antibody, and CSF metal ions analyzed by flame atomic absorption spectrophotometry. Two-way ANOVA of CSF TTR concentrations revealed highly significant dose (p < 0.0001), time (p < 0.0223), and dose-by-time effects (p < 0.0379). Moreover, the percentage of reduction of CSF TTR was directly correlated with Pb concentrations in the choroid plexus (r = 0.703, p < 0.05). Pb exposure significantly increased CSF concentrations of Mg2+, but did not markedly altered CSF concentrations of Ca2+, K+, and Na+. Histopathologic examination under the light microscope did not show distinct alterations of plexus structure in Pb-treated rats. Since TTR is responsible for transport of thyroid hormones to the developing brain, we postulate that the depression of choroid plexus TTR production (and/or secretion) by Pb may impair brain development in young animals by depriving the CNS of thyroid hormones.

Beverages stored in lead-crystal glass accumulate extraordinary concentrations of lead. We obtained a lead-crystal decanter manufactured with lead from Australia, where the ratio of 206Pb/207Pb is distinctly different from that in the United States. We sought to determine the bioavailability of crystal-derived lead, using the technique of stable isotope dilution in blood. We conducted a single-dose, nonrandomized cross-over study in which participants were admitted to the Clinical Research Center twice, 1 week apart. During the first admission, subjects ingested sherry obtained from the original bottle. During the second admission, they ingested sherry that had been stored in the crystal decanter and that had achieved a lead concentration of 14.2 mu mol/l. After ingesting decanter-stored sherry, mean blood lead rose significantly (p = 0.0003) from 0.10 to 0.18 mu mol/l, while mean 206Pb/207Pb fell from 1.202 to 1.137 (p = 0.0001). On average, 70% of the ingested dose of lead was absorbed. We conclude that lead derived from crystal glass is highly bioavailable; repeated ingestions could cause elevated blood lead concentration. The technique of stable isotope dilution lends itself to the study of the bioavailability of lead in other matrices, including soil.

Meso-2,3-dimercaptosuccinic acid (DMSA, or succimer) is an oral chelating agent for heavy-metal poisoning. While studying the urinary elimination of unaltered DMSA, altered DMSA (i.e., its mixed disulfides), and lead in children with lead poisoning, we observed a pattern of urinary drug elimination after meals suggestive of enterohepatic circulation. The excretion of lead in urine patterned the elimination of altered DMSA rather than the parent molecule. In addition, the half-life of elimination of DMSA via the kidney was positively associated with blood lead concentration. Two additional crossover studies of DMSA kinetics were conducted in normal adults to confirm the presence of enterohepatic circulation of DMSA after meals. In one, increases in plasma total DMSA concentration were observed after meals in all six subjects; these increases were prevented by cholestyramine administration 4, 8, and 12 hr after DMSA. In the second, the administration of neomycin also prevented increases in DMSA after meals. These studies indicate that 1) a metabolite(s) of DMSA undergoes enterohepatic circulation and that microflora are required for DMSA reentry; 2) in children, moderate lead exposure impairs renal tubular drug elimination; and 3) a metabolite of DMSA appears to be an active chelator.

Extensive research has been devoted to the development of biomarkers of environmental and occupational exposure to lead (Pb). This body of work can serve as a paradigm for biomarker development for other chemical exposures. Early efforts focused on indirect measurements of exposure by analyzing precursors and enzymes of a biosynthetic pathway (heme) in blood and urine. However, the direct measurement of Pb in blood has become increasingly simple and reliable and is now widely accepted for pediatric surveillance programs, in part because of known associations of Pb with adverse health outcomes. Other markers of exposure include measurements of Pb in important compartments: bone Pb, tooth Pb, and chelatable Pb. In addition, the technique of stable isotope dilution is available, since Pb exists in numerous nonradioactive isotopic forms. The strengths and weaknesses of all Pb biomarkers for confirming a diagnosis or for epidemiologic research vary widely depending upon the hypothesis under investigation.

For a prospective study of lead exposure and early development, we recruited pregnant women from a smelter town and a nonlead-exposed town in Yugoslavia and followed them and their children through age 4. For 332 children seen at age 4, mean scores on the McCarthy Scales General Cognitive Index (GCI) in the exposed and nonexposed towns were 81.3 and 86.6, respectively; geometric mean blood lead concentrations (BPb) were 39.9 and 9.6 micrograms/dl, respectively. Potential confounders included the quality of the HOME environment; maternal age, intelligence, education, and language; birthweight and gender. These showed predictable associations with 4-year intelligence, accounting for 42.7% of the variance in GCI. Following adjustment for these variables and for concurrent Hgb, we found significant independent adverse associations between GCI and BPb's, measured at 6-month intervals since birth. At age 4, BPb accounted for an incremental 3.5% of the variance in GCI, such that the estimated loss in GCI associated with an increase in BPb from 10-25 micrograms/dl was 3.8 points. The Perceptual-Performance subscale of the McCarthy was most sensitive to Pb exposure, a result consistent with findings from prospective studies in Boston and Port Pirie.

The purpose of this paper is to discuss issues which relate to the risks and benefits of pharmacologic intervention in children with elevated blood lead (BPb) concentrations. In January, 1991, 2,3-dimercaptosuccinic acid (DMSA; succimer or CHEMET) was approved by the FDA for administration to children with blood lead concentrations of 45 micrograms/dl or higher. Thus, it is now considerably simpler to treat children with elevated BPb's. DMSA is orally actively, relatively safe and specific, and can, in some situations, be used on an out-patient basis. The author is concerned that the recent CDC statement on lead poisoning, whose aim is to reduce (by various means) children's BPb's below 10 micrograms/dl, may unintentionally encourage pediatricians to treat children with relatively low BPb's with DMSA, both for medical and legal reasons. Should children with BPb's of less than 45 ug/dl be treated, and why? The risks and benefits of treatment in such children are uncertain. A randomized, placebo controlled, double-blind drug intervention trial is needed (and justified) in order to more carefully assess the benefits and the risks associated with the treatment of lower BPb's.

For a prospective study of lead exposure, iron status, and infant development, we recruited infants living in a smelter town and a non-lead-exposed town in Kosovo, Yugoslavia. Among 392 infants assessed at age 2 years, the mean Mental Development Index (MDI), Bayley Scales of Infant Development, was 105.2. At age 2 years, geometric mean blood lead concentrations were 35.5 and 8.4 micrograms/dl, respectively, among infants from the exposed and nonexposed towns. After controlling for variables associated with MDI, we found significant independent associations for both blood lead and hemoglobin concentrations. For example, a rise in blood lead concentration at age 2 years from 10 to 30 micrograms/dl was associated with an estimated 2.5 point decrement in MDI (p = 0.03); statistically nonsignificant decrements were associated with blood lead levels measured at birth and at 6, 12, and 18 months of age. A decrease in hemoglobin concentration at 18 months of age from 12 to 10 gm/dl was associated with an estimated 3.4 point decrement in MDI (p = 0.02); the latter association was present in both towns, suggesting that it was due to iron deficiency anemia independent of lead exposure. The findings suggest that the brain is vulnerable to the effects of both lead exposure and anemia before 2 years of age. On a global basis, the developmental consequences of anemia may exceed those of lead exposure.

It has been suggested that the accumulation of tobacco-derived cadmium (Cd) in the placenta is responsible for the adverse effect of cigarette smoking on infant birthweight. We chose to test this hypothesis; therefore, we studied a population of nonsmoking pregnant women who were exposed to low levels of smelter-derived Cd and a group of nonexposed women. A higher mean placental Cd concentration (p less than .0007) was found in the exposed women (n = 106), compared with those who were not exposed (n = 55); the observed Cd concentrations were comparable to concentrations reported previously for smoking and nonsmoking women, respectively. Least squares multiple regression (controlling for potentially confounding variables) revealed no association between placental Cd and birthweight. It was, therefore, concluded that the effect of smoking on birthweight was not mediated through Cd.

We examined the efficacy and safety of meso-2,3-dimercaptosuccinic acid (DMSA) in children with markedly elevated blood lead (BPb) concentrations. Among 19 children with BPb concentrations of 50 to 69 micrograms/dl (2.41 to 3.33 mumol/L) who received a 5-day inpatient oral course of DMSA (1050 mg/m2 per day), the mean BPb concentration decreased by 61%; in four who received calcium disodium ethylenediaminetetraacetic acid (CaNa2EDTA) (1000 mg/m2 per day intravenously), it decreased by 45% (p less than 0.0007). Urinary lead excretion was comparable in both groups. Treatment with DMSA was more effective than treatment with CaNa2EDTA in restoring metabolic activity to the heme pathway and was well tolerated even among nine patients who received concomitant iron supplementation and two who had homozygous deficiency of glucose-6-phosphate dehydrogenase. On discharge, these 19 children received either no chelation therapy or DMSA, 350 or 700 mg/m2 per day for 14 days on an outpatient basis. After 14 days the mean BPb values for the no-chelation, low-DMSA, and high-DMSA groups were 73%, 66%, and 50% of the pretreatment values, respectively. We conclude that a 5-day oral course of DMSA is effective in the treatment of children with severe lead poisoning. In addition, on an outpatient basis the administration of DMSA, 700 mg/m2 per day, is capable of delaying the typical rebound in BPb values and should ultimately reduce the need for repeated hospitalizations.

During the course of a prospective study of lead exposure and pregnancy outcome in 1,502 women, we tested the hypothesis that environmental lead exposure is associated with depressed serum erythropoietin concentration. At mid-pregnancy and at delivery, blood samples were stratified by hemoglobin concentration; within each hemoglobin stratum, sera of women with the lowest and highest whole blood lead concentrations were selected for serum erythropoietin analysis. Analysis of variance revealed that women with higher blood lead levels had inappropriately low serum erythropoietin at both mid-pregnancy and at delivery. Thus, depressed serum erythropoietin appears to indicate lead nephrotoxicity, and it may also be responsible for the anemia associated with lead poisoning.

This paper tests the hypothesis that exposure to lead during pregnancy is associated with reduced intrauterine growth and an increase in preterm delivery. The sample comprises women, recruited at mid-pregnancy, residing in Titova Mitrovica, a lead smelter town, or in Pristina, a non-exposed town 25 miles away. Both towns are in the province of Kosovo, Yugoslavia. Mean blood lead concentrations (BPb's) at mid-pregnancy were 0.92 mumol/L (+/- 0.38, N = 401) in the exposed town and 0.27 mumol/L (+/- 0.09, N = 506) in the comparison town. No differences were found between towns for either birthweight or length of gestation. Mean birthweight was 3308 (+/- 566) grams in Titova Mitrovica and 3361 (+/- 525) grams in Pristina. Mean length of gestation was 274 (+/- 18.8) days in Titova Mitrovica and 275 (+/- 15.6) days in Pristina. After adjustment for the effects of potential confounders, no significant relationships were found between maternal BPb measured at mid-pregnancy, at delivery or in the umbilical cord and either birthweight, length of gestation, or preterm delivery (less than 37 weeks). We conclude that exposure to environmental lead does not impair fetal growth or influence length of gestation.

In a study of the elution of lead (Pb) from crystal decanters and glasses, port containing 89 micrograms Pb/l was placed in decanters and the Pb content of the wine rose steadily to 3518 micrograms/l after 4 months. Wines and spirits stored in crystal decanters for a long time contained Pb at concentrations up to 21,530 micrograms/l. In a short-term experiment white wine eluted small amounts of Pb from crystal glasses within minutes.

We are prospectively examining the relation between environmental lead exposure and pregnancy outcome in cohorts of women exposed to a wide range of air lead concentrations. Titova Mitrovica, Yugoslavia, is the site of a large lead smelter, refinery, and battery factory. At midpregnancy, 602 women in T. Mitrovica and 900 women in Pristina, a non-lead-exposed control town, were interviewed. Blood was obtained for blood lead (PbB), hemoglobin, erythrocyte protoporphyrin, and serum ferritin measurements. Women were seen again at delivery, at which time maternal and umbilical cord blood samples were obtained. While many demographic and social characteristics were similar across the two towns, women in Pristina were more likely to report employment outside the home, cigarette smoking, and alcohol use during pregnancy. As expected, PbB levels were substantially higher in the smelter town. At midpregnancy, PbB geometric means were 17.1 micrograms/dL in T. Mitrovica and 5.1 micrograms/dL in Pristina; 86% of the pregnant women in T. Mitrovica, compared to 3.4% of those in Pristina, had PbB levels greater than 10 micrograms/dL. Within T. Mitrovica, distance between the home and the smelter was the most important predictor of PbB at mid-pregnancy and delivery. Husband's employment in the lead industry was associated with a significant increase in maternal PbB levels independent of place of residence. Higher maternal serum ferritin concentrations were associated with lower PbB levels, suggesting that dietary iron inhibits lead absorption. Overall, the placenta was a poor barrier to lead; the relationship between maternal PbB and umbilical cord PbB was linear across a wide range of PbB levels.

To study the effect of cadmium exposure on maternal and fetal zinc metabolism, rats were exposed to 0, 5, 50, or 100 ppm Cd in the drinking water on Days 6 through 20 of pregnancy. In comparison to controls, fetal and maternal weights were slightly reduced in the 50- and 100-ppm groups, but not the 5-ppm group. Multiple regression analysis revealed that in the 50-ppm group, but not in the 100-ppm group, the decrease in fetal weight was not solely a consequence of decreased maternal weight. Cd accumulated in a dose-dependent manner in both maternal organs and fetuses, although the absolute concentrations in fetuses were very low as compared to those of maternal tissues. In the 50- and 100-ppm groups, zinc concentrations were significantly increased in maternal liver and kidney, and significantly decreased in fetal liver. The changes in tissue Zn concentrations were accompanied by altered Zn-metalloenzyme activities in both maternal and fetal tissues. These findings support the hypothesis that Cd-induced maternal zinc retention is responsible for fetal Zn deprivation and impaired fetal growth.

This analysis compares the rates of spontaneous abortion among women living in the vicinity of a lead smelter with those of women living in a town where blood lead levels were low. Data derive from the obstetric histories of both groups of women obtained while seeking prenatal care for a later pregnancy. A total of 639 women (304 exposed, 335 unexposed) had at least one previous pregnancy and lived at the same address since their first pregnancy. The geometric mean blood lead concentrations in the sample at the time of the interviews were 0.77 mumol/L in the exposed town and 0.25 mumol/L in the unexposed town. The rates of spontaneous abortions in first pregnancies were similar, with 16.4 percent of women in the exposed town and 14.0 percent in the unexposed town reporting loss. The adjusted odds ratio relating town of residence to spontaneous abortion was 1.1 (95% CI = 0.9, 1.4). This analysis represents the first systematic attempt to seek an association between environmental lead exposure and spontaneous abortion. As such, the failure to find a positive association strongly suggests that at the levels of exposure represented in our sample, such an association does not exist.

The cardiorespiratory response to exercise was examined in 13 patients, 12 to 22 yr of age, who were in stable condition while receiving regular transfusions for thalassemia major. Before transfusion (Hgb, 10.8 g/dl), the patients had reduced peak oxygen consumption for body weight (by 25%, p less than 0.002) in comparison with 20 age- and sex-matched control subjects (Hgb, 13.8 g/dl). The ventilatory equivalent for carbon dioxide was significantly reduced at all work rates, and end-tidal PCO2 was abnormally high (46.3 versus 40.7 mm Hg, p less than 0.001), but ventilatory reserve, as estimated from pulmonary function tests, was normal. Both heart rate and cardiac output (as estimated by the indirect Fick method) were abnormally high during exercise; at an oxygen consumption of 19 ml/min/kg, heart rate was 13% higher (p less than 0.01) and, at 16 ml/min/kg, cardiac output was 28% higher (p less than 0.001) in the patients than in the control subjects. Nine patients were retested 3 to 8 days after transfusion (Hgb, 13.0 g/dl, p50, 26.4 mm Hg). Neither ventilatory nor circulatory abnormalities improved significantly. In patients 12 to 22 yr of age in stable condition with thalassemia major, we conclude: cardiac output, estimated by the CO2 rebreathing method, is high and arteriovenous O2 extraction is low during exercise; the high cardiac output during exercise is associated with hypoventilation; the high cardiac output is independent of short-term changes in hemoglobin concentration associated with transfusion.

In the rat, oral administration of 1 mg/kg arsenic (as As2O3) binds rapidly to mucosal glutathione such that effective glutathione concentration is reduced. In response to the binding of arsenic to glutathione, de novo synthesis of glutathione occurs in the mucosal cell, resulting in twice the normal concentrations of glutathione within 3 h. This finding may explain acquired tolerance to arsenic, as well as the protective effect of arsenic against selenium toxicity.

We have found bioassayable somatomedin activity to be subnormal in 20 of 32 children and adults with beta-thalassemia. The levels were comparable to values reported in growth hormone-deficient subjects. Since patients with thalassemia are not growth hormone deficient, the data suggest the possibility of defective hepatic biosynthesis of somatomedin. Increased iron stores in these patients, who have secondary hemosiderosis of many organs, including the liver, may depress somatomedin activity. Therapy for one year with daily subcutaneous infusions of the iron-chelating agent deferoxamine had no effect on mean bioassayable serum somatomedin activity.

2,3-Dimercaptosuccinic acid (DMSA) is a new orally active heavy metal chelator for the treatment of childhood Pb intoxication on an outpatient basis. The influence of DMSA, as well as other chelating agents, on gastrointestinal 203Pb absorption and whole-body 203Pb retention was examined. Groups of Sprague-Dawley rats (230-260 g) were gavaged with a solution containing approximately 25 mg/kg Pb [as Pb(NO3)2] plus 15 microCi 203Pb. Some groups were then immediately given 0.11 mmol/kg of either DMSA, CaNa2EDTA, D-penicillamine, or BAL by oral gavage, while other groups received the same drugs by ip injection. Control groups received solutions of the drug vehicles po or ip. Whole-body Pb retention and gastrointestinal Pb absorption (whole body retention + urinary Pb excretion) were significantly decreased in rats that received DMSA po. This finding implies that the use of DMSA to treat childhood lead intoxication on an outpatient basis is not associated with a risk for increased Pb absorption.

In an attempt to understand the variability of the hematologic response to oral sodium cyanate, iron metabolism was studied in a group of 39 patients with sickel cell disease. Eleven of the 39 patients were found to have no stainable iron in the marrow despite the fact that patients with sickle cell disease are generally considered to have hemosiderosis. The mean per cent saturation and total iron-binding capacity were in the low-normal range in sickle cell patients whether or not stainable iron was present in the bone marrow aspirate. Serum ferritin concentrations, on the other hand, were found to be high in both groups (greater than 500 mu g/liter) when compared to controls (60 mu g/liter). The high serum ferritin levels denoted significant total-body iron deposition which may be unavailable for normal metabolic processes. One patient with no stainable iron in the bone marrow aspirate did respond to iron therapy alone with an increase in hemoglobin concentration. Serum ceruloplasmin levels were also found to be high in sickle cell disease patients. The ability to respond to oral cyanate therapy was correlated with the amount of stainable iron in the bone marrow aspirate. These studies emphasize the necessity of a reevaluation of iron metabolism in the pathophysiology and treatment of sickle cell disease.

Both iron and precipitated haemoglobin may catalyse the formation of free radicals, which in turn react with the polyunsaturated fatty acids of membranes leading to membrane failure and cell death. 2,3-Dihydroxybenzoic acid (2,3-DHB), a recently identified, orally effective iron-chelating drug, inhibited membrane peroxidation in vitro in H2O2-stressed erythrocytes from patients with beta-thalassaemia major, beta-thalassaemia intermedia, haemoglobin Köln disease and sickle cell disease. We present evidence suggesting that the inhibition of peroxidation is due to ability of 2,3-DHB to scavenge free radicals via quinone formation, a mechanism analogous to that proposed for vitamin E.

2,3-Dimercaptosuccinic acid (DMSA) is an orally effective drug more specific and with a wider therapeutic index than currently available drugs for lead intoxication. Eighteen men with elevated blood lead (BPb) concentrations received either 30, 20, or 10 mg/kg DMSA for 5 days in three divided daily doses. The mean BPb level decreased 72.5%, 58.3%, and 35.5% of the pretreatment values, with a simultaneous elevation in urinary Pb excretion. Clinical symptoms and biochemical indices of lead toxicity also improved. Red blood cell d-aminolevulinic acid dehydratase activity increased, while urinary excretion of d-aminolevulinic acid and coproporphyrin fell. DMSA was well tolerated; the only observed adverse drug reaction was a mild, transient elevation of serum SGPT levels in two subjects. DMSA appears promising and may greatly simplify the treatment of lead intoxication.

For the past several years, we have searched for an orally effective iron-chelating drug and report here on several compounds which warrant further investigations based on their ability to promote iron excretion in the hypertransfused rat. Administrered orally, 2,3-dihydroxybenzyolglycine induced both urinary and fecal iron excretion, suggesting that a conjugate of 2,3-dihydroxybenzoic acid may be more efficacious than the parent compound. Tropolone, although rather toxic, stimulated fecal excretion of iron when given p.o. at low doses. Evaluation of less toxic derivatives of tropolone appears to be justifiable. L-Histidine may also be of use in chelatin therapy. Fecal iron excretion is significantly increased in response to oral doses of this essential amino acid. Lastly, cholylhydroxamic acid proved to be the most efficacious oral agent examined thus far. A marked increase in fecal iron excretion results from its administration.

2,3-Dimercaptosuccinic acid (DMSA) is an orally effective orphan drug that is more specific and has a wider therapeutic index than other currently available drugs used for lead intoxication. Its investigational use in the United States has been limited to the treatment of men with occupational plumbism. Twenty-one children with blood lead concentrations of 31 to 49 micrograms/dl, who also had a positive calcium disodium edetate (CaNa2EDTA) mobilization test result, were hospitalized for 7 days. Fifteen children were randomly assigned to three groups that received either 350, 700, or 1050 mg/m2/day, respectively, of DMSA in three divided doses daily. A fourth group of six children received conventional treatment with 1000 mg/m2/day of intravenously administered CaNa2EDTA in two divided doses daily. The 1050 mg/m2/day dose of DMSA was significantly more effective than lower doses of DMSA or intravenously administered CaNa2EDTA in reducing blood lead levels and restoring erythrocyte delta-aminolevulinic acid dehydratase activity. Intravenously administered CaNa2EDTA significantly increased the urinary excretion of several essential minerals (zinc, copper, iron, and calcium), whereas DMSA did not. The DMSA was well tolerated and appears extremely promising as a drug that will simplify the management of childhood lead poisoning.

A year-long double-blind study of 2,3-dihydroxybenzoic acid (2,3-DHB) given orally at a dose of 25 mg/kg four times per day was undertaken in 15 patients with beta-thalassemia major. 2,3-DHB and placebo (mannitol) were tolerated to an equal degree and there were no signs of drug toxicity at the end of 1 year. Efficacy in terms of retardation of iron accumulation could be documented using serial liver biopsies, serum ferritin determinations, or clinical laboratory assessment. Serum iron values increased, as did the iron binding capacity, in the group receiving 2,3-DHB. The increase in iron binding capacity was due to drug interference with the method of determination. Because of the greater efficacy of slow infusions of desferrioxamine in chelating iron when administered slowly, the clinic has shifted its emphasis toward further evaluation of that compound. Nevertheless, in view of the minimal toxicity of 2,3-DHB, further work appears warranted to define its role in the treatment of iron-overload.

2,3-Dimercaptosuccinic acid (D.M.S.), a new orally effective agent for the treatment of heavy-metal intoxication, was administered to five lead-poisoned smelter workers for six days at dosages ranging from 8.4--12.7 mg/kg/day on the first day to 28.1--42.2 mg/kg/day on the last day. Mean blood-lead concentration decreased significantly from an initial value of 97 +/- 6 microgram/dl to 43 +/- 4 microgram/dl on the last day. Urinary lead excretion was significantly raised. D.M.S. was very well tolerated with no signs of toxicity and no effect on urinary zinc, calcium, magnesium, or iron excretion. Urinary copper excretion was significantly increased, but the magnitude of that effect was not clinically important. D.M.S. seems to be safe and effective for the treatment of lead poisoning.

Our previous cross-sectional study of patients with thalassemia major suggested progressive lung changes characterized by low total lung capacity, hypoxemia, and elevated transfer factor for carbon monoxide. We reevaluated nine of the patients for three reasons: to determine the relationship of the previous findings to the immediate effects of blood transfusion; to assess the longitudinal progression of the lung changes; and to evaluate the effect of splenectomy on lung volume changes in these patients, all of whom underwent splenectomy in the interval between the two studies. We found that during the 5- to 6-yr period between studies total lung capacity had decreased significantly (p less than 0.05) from a mean 86% predicted to a mean 79% predicted. However, vital capacity increased significantly (p less than 0.05) from a mean 81% predicted to 88% with no significant change in functional residual capacity. There was no significant immediate effect of transfusion on total lung capacity, vital capacity, or functional residual capacity. However, the diffusion constant for carbon monoxide increased significantly (p less than 0.005) immediately following transfusion and there was a positive correlation between the increase and the amount of blood transfused (r = 0.74, p less than 0.05). Arterial oxygen saturation was below 95% in five of eight patients and increased significantly with transfusion (p less than 0.05). We conclude: 1) thalassemia major and/or its treatment is associated with hypoxemia and a progressive reduction in total lung capacity. 2) Despite the progressive reduction in total lung capacity, splenectomy in patients with thalassemia major increases expiratory reserve volume and thereby increases vital capacity.

The use of rhodotorulic acid (RA) as an iron-chelating drug was suggested by experiments in hypertransfused rats in which urinary and fecal iron excretion were significantly enhanced in response to RA. The toxicity of the drug appears to be minimal at a parenteral dose less than 250 mg/kg. An increased excretion of zinc was the only notable side effect of the drug at the doses used. When administered i.v. to humans, RA was only 16% more effective than desferrioxamine (DF). Pharmacokinetic studies showed that RA persisted in the bloodstream of dogs 6 times longer than desferrioxamine after an intravenous injection. Accordingly RA was evaluated as a potential repository drug. While animal experiments were encouraging, human subjects experienced a painful local reaction to RA administered either i.m. or s.c. as a suspension in physiological saline. Accordingly it appears that RA is best looked at as a second line drug, unless a means can be found to obviate local inflammatory reactions.

We have developed a rapid and simple method for the isolation of relatively young cells from ordinary fresh or previously frozen blood units, using the IBM-2991 Blood Cell Processor. Based on in vitro measurements of red cell pyruvate kinase activity, the resolution of the method is excellent, comparable to that of the stractan discontinuous gradient system. In splenectomized patients with thalassemia major, the administration of the "youngest" 50% of blood units yielded 51Cr survival times of 40 and 42 days for fresh packed red cells and 41 and 43 days for previously frozen red cells.