Estimating the Prevalence of Toxic Waste Sites in Low- and Middle-Income Countries.

BACKGROUND: Exposure to heavy metals at contaminated industrial and mining sites, known also as hot spots, is a significant source of toxic exposure and adverse health outcomes in countries around the world. The Toxic Sites Identification Program (TSIP) developed by Pure Earth, a New York-based nongovernmental organization, is the only systematic effort to catalogue contaminated sites globally. To date, TSIP has identified and catalogued 3282 sites in low- and middle-income countries. The TSIP methodology is not designed to survey all contaminated sites in a country. Rather sites are prioritized based on their perceived impact on human health, and only a limited number of the most highly hazardous sites are surveyed. The total number of contaminated sites globally and the fraction of contaminated sites captured by TSIP is not known. OBJECTIVE: To determine the TSIP site capture rate, the fraction of contaminated sites in a country catalogued by TSIP. METHODS: Ghana was selected for this analysis because it is a rapidly industrializing lower middle income country with a heterogeneous industrial base, a highly urban population (51%), and good public records systems. To develop an estimate of the fraction of sites in Ghana captured by TSIP, assessors targeted randomly selected geographic quadrats for comprehensive assessment using area and population statistics from the Ghana Statistical Service. Investigators physically walked all accessible streets in each quadrat to visually identify all sites. Visual identification was supplemented by field-based confirmation with portable x-ray fluorescence instruments to test soils for metals. To extrapolate from survey findings to develop a range of estimates for the entire country, the investigators used 2 methodologies: a "bottom-up" approach that first estimated the number of waste sites in each region and then summed these regional subtotals to develop a total national estimate; and a "top-down" method that estimated the total number of sites in Ghana and then allocated these sites to each region. Both methods used cluster random sampling principles. FINDINGS: The investigators identified 72 sites in the sampled quadrats. Extrapolating from these findings to the entire country, the first methodology estimated that there are 1561 sites contaminated by heavy metals in Ghana (confidence interval [CI]: 1134-1987), whereas the second estimated 1944 sites (CI: 812-3075). The estimated total number of contaminated sites in Ghana is thus 7-9 times the number of sites captured through TSIP. On a population basis, it was estimated that there are between 31 and 115 contaminated sites per million inhabitants in Ghana. CONCLUSIONS: The findings of this study indicate that the TSIP methodology provides a sound statistical basis for policy formulation. The statistical approaches used in this study can be replicated in other countries to improve estimates of the prevalence of contaminated sites. This information provides important input to calculations of the global burden of disease attributable to hazardous exposures at contaminated sites.

Blood lead levels in Mexico and pediatric burden of disease implications.

BACKGROUND: Although there has been success in reducing lead exposure with the phase-out of leaded gasoline, exposure to lead in Mexico continues to threaten the health of millions, much of which is from lead-based glazes used in pottery that leaches into food. OBJECTIVES: An extensive historical review and analysis of available data on blood lead levels in Mexican populations was conducted. We used a calculated geometric mean to evaluate the effect of lead on the pediatric burden of disease. METHODS: An extensive bibliographic search identified 83 published articles from 1978 to 2010 with blood lead level (BLL) data in Mexican populations representing 150 data points from more than 50,000 study participants. Values from these publications were categorized into various groupings. We then calculated the incidence of disease and disability-adjusted life-years resulting from these BLLs using the World Health Organization's burden of disease spreadsheets for mild mental retardation. RESULTS: Reviewing all relevant studies, the geometric means of Mexican BLLs in urban and rural areas were found to be 8.85 and 22.24 ug/dL, respectively. Since the phase-out of leaded gasoline, the mean in urban areas was found to be 5.36 ug/dL and the average in rural areas is expected to be much higher. The U.S. Centers for Disease Control and Prevention's (CDC) upper limit of blood lead in children under the age of 6 years is 5 ug/dL and the current U.S. average is 1.2 ug/dL. Our results indicate that more than 15% of the population will experience a decrement of more than 5 IQ points from lead exposure. The analysis also leads us to believe that lead is responsible for 820,000 disability-adjusted life-years for lead-induced mild mental retardation for children aged 0 to 4 years. CONCLUSION: Lead continues to threaten the health of millions and remains a significant cause of disability in Mexico. Additional interventions in reducing or managing lead-based ceramic glazes are necessary to protect the public health.

Effects of exercise training on calf tissue oxygenation in men with intermittent claudication.

OBJECTIVE: To determine the effects of exercise training on calf tissue oxygenation in men with peripheral arterial disease and intermittent calf claudication. DESIGN: This pilot study was prospective and longitudinal and used a one-group, pretest-posttest design. SETTING: Tertiary care medical center for veterans. PARTICIPANTS: Fifteen male veterans (mean age 69 years) with Fontaine stage IIa peripheral arterial disease and classic intermittent claudication. MAIN OUTCOME MEASUREMENTS: Before and after intervention, participants performed graded treadmill exercise tests while medial calf tissue oxygenation (StO(2), % oxyhemoglobin saturation) was monitored continuously with near-infrared spectroscopy. INTERVENTION: The intervention consisted of a 3-month exercise training program involving 3 sessions per week at the clinic (treadmill walking, calf ergometry) and 2 sessions per week at home (free walking, standing heel raises). RESULTS: After completion of the intervention, participants significantly increased their maximal treadmill exercise time from 7.19 to 11.27 minutes. Mean exercise StO(2) decreased from 29% to 19% saturation, StO(2) x time area increased from 421% * min to 730% * min StO(2) nadir, and StO(2) recovery time did not change significantly. CONCLUSIONS: After the exercise intervention, the improved treadmill walking performance was accompanied by greater calf tissue deoxygenation during exercise. Given the continued presence of ischemia, this finding may represent increased capillarization and diffusion-based enhancement of arteriovenous O(2) extraction.

Investigation of thin filament near-neighbour regulatory unit interactions during force development in skinned cardiac and skeletal muscle.

Ca(2+)-dependent activation of striated muscle involves cooperative interactions of cross-bridges and thin filament regulatory proteins. We investigated how interactions between individual structural regulatory units (RUs; 1 tropomyosin, 1 troponin, 7 actins) influence the level and rate of demembranated (skinned) cardiac muscle force development by exchanging native cardiac troponin (cTn) with different ratio mixtures of wild-type (WT) cTn and cTn containing WT cardiac troponin T/I + cardiac troponin C (cTnC) D65A (a site II inactive cTnC mutant). Maximal Ca(2+)-activated force (F(max)) increased in less than a linear manner with WT cTn. This contrasts with results we obtained previously in skeletal fibres (using sTnC D28A, D65A) where F(max) increased in a greater than linear manner with WT sTnC, and suggests that Ca(2+) binding to each functional Tn activates < 7 actins of a structural regulatory unit in cardiac muscle and > 7 actins in skeletal muscle. The Ca(2+) sensitivity of force and rate of force redevelopment (k(tr)) was leftward shifted by 0.1-0.2 -log [Ca(2+)] (pCa) units as WT cTn content was increased, but the slope of the force-pCa relation and maximal k(tr) were unaffected by loss of near-neighbour RU interactions. Cross-bridge inhibition (with butanedione monoxime) or augmentation (with 2 deoxy-ATP) had no greater effect in cardiac muscle with disruption of near-neighbour RU interactions, in contrast to skeletal muscle fibres where the effect was enhanced. The rate of Ca(2+) dissociation was found to be > 2-fold faster from whole cardiac Tn compared with skeletal Tn. Together the data suggest that in cardiac (as opposed to skeletal) muscle, Ca(2+) binding to individual Tn complexes is insufficient to completely activate their corresponding RUs, making thin filament activation level more dependent on concomitant Ca(2+) binding at neighbouring Tn sites and/or crossbridge feedback effects on Ca(2+) binding affinity.

Thin-filament regulation of force redevelopment kinetics in rabbit skeletal muscle fibres.

Thin-filament regulation of isometric force redevelopment (k(tr)) was examined in rabbit psoas fibres by substituting native TnC with either cardiac TnC (cTnC), a site I-inactive skeletal TnC mutant (xsTnC), or mixtures of native purified skeletal TnC (sTnC) and a site I- and II-inactive skeletal TnC mutant (xxsTnC). Reconstituted maximal Ca(2+)-activated force (rF(max)) decreased as the fraction of sTnC in sTnC: xxsTnC mixtures was reduced, but maximal k(tr) was unaffected until rF(max) was <0.2 of pre-extracted F(max). In contrast, reconstitution with cTnC or xsTnC reduced maximal k(tr) to 0.48 and 0.44 of control (P < 0.01), respectively, with corresponding rF(max) of 0.68 +/- 0.03 and 0.25 +/- 0.02 F(max). The k(tr)-pCa relation of fibres containing sTnC: xxsTnC mixtures (rF(max) > 0.2 F(max)) was little effected, though k(tr) was slightly elevated at low Ca(2+) activation. The magnitude of the Ca(2+)-dependent increase in k(tr) was greatly reduced following cTnC or xsTnC reconstitution because k(tr) at low levels of Ca(2+) was elevated and maximal k(tr) was reduced. Solution Ca(2+) dissociation rates (k(off)) from whole Tn complexes containing sTnC (26 +/- 0.1 s(-1)), cTnC (38 +/- 0.9 s(-1)) and xsTnC (50 +/- 1.2 s(-1)) correlated with k(tr) at low Ca(2+) levels and were inversely related to rF(max). At low Ca(2+) activation, k(tr) was similarly elevated in cTnC-reconstituted fibres with ATP or when cross-bridge cycling rate was increased with 2-deoxy-ATP. Our results and model simulations indicate little or no requirement for cooperative interactions between thin-filament regulatory units in modulating k(tr) at any [Ca(2+)] and suggest Ca(2+) activation properties of individual troponin complexes may influence the apparent rate constant of cross-bridge detachment.

Cardiac length dependence of force and force redevelopment kinetics with altered cross-bridge cycling.

We examined the influence of cross-bridge cycling kinetics on the length dependence of steady-state force and the rate of force redevelopment (k(tr)) during Ca(2+)-activation at sarcomere lengths (SL) of 2.0 and 2.3 microm in skinned rat cardiac trabeculae. Cross-bridge kinetics were altered by either replacing ATP with 2-deoxy-ATP (dATP) or by reducing [ATP]. At each SL dATP increased maximal force (F(max)) and Ca(2+)-sensitivity of force (pCa(50)) and reduced the cooperativity (n(H)) of force-pCa relations, whereas reducing [ATP] to 0.5 mM (low ATP) increased pCa(50) and n(H) without changing F(max). The difference in pCa(50) between SL 2.0 and 2.3 microm (Delta pCa(50)) was comparable between ATP and dATP, but reduced with low ATP. Maximal k(tr) was elevated by dATP and reduced by low ATP. Ca(2+)-sensitivity of k(tr) increased with both dATP and low ATP and was unaffected by altered SL under all conditions. Significantly, at equivalent levels of submaximal force k(tr) was faster at short SL or increased lattice spacing. These data demonstrate that the SL dependence of force depends on cross-bridge kinetics and that the increase of force upon SL extension occurs without increasing the rate of transitions between nonforce and force-generating cross-bridge states, suggesting SL or lattice spacing may modulate preforce cross-bridge transitions.

Familial hypertrophic cardiomyopathy mutations in troponin I (K183D, G203S, K206Q) enhance filament sliding.

A major cause of familial hypertrophic cardiomyopathy (FHC) is dominant mutations in cardiac sarcomeric genes. Linkage studies identified FHC-related mutations in the COOH terminus of cardiac troponin I (cTnI), a region with unknown function in Ca(2+) regulation of the heart. Using in vitro assays with recombinant rat troponin subunits, we tested the hypothesis that mutations K183Delta, G203S, and K206Q in cTnI affect Ca(2+) regulation. All three mutants enhanced Ca(2+) sensitivity and maximum speed (s(max)) of filament sliding of in vitro motility assays. Enhanced s(max) (pCa 5) was observed with rabbit skeletal and rat cardiac (alpha-MHC or beta-MHC) heavy meromyosin (HMM). We developed a passive exchange method for replacing endogenous cTn in permeabilized rat cardiac trabeculae. Ca(2+) sensitivity and maximum isometric force did not differ between preparations exchanged with cTn(cTnI,K206Q) or wild-type cTn. In both trabeculae and motility assays, there was no loss of inhibition at pCa 9. These results are consistent with COOH terminus of TnI modulating actomyosin kinetics during unloaded sliding, but not during isometric force generation, and implicate enhanced cross-bridge cycling in the cTnI-related pathway(s) to hypertrophy.

Thin filament near-neighbour regulatory unit interactions affect rabbit skeletal muscle steady-state force-Ca(2+) relations.

The role of cooperative interactions between individual structural regulatory units (SUs) of thin filaments (7 actin monomers : 1 tropomyosin : 1 troponin complex) on steady-state Ca(2+)-activated force was studied. Native troponin C (TnC) was extracted from single, de-membranated rabbit psoas fibres and replaced by mixtures of purified rabbit skeletal TnC (sTnC) and recombinant rabbit sTnC (D27A, D63A), which contains mutations that disrupt Ca(2+) coordination at N-terminal sites I and II (xxsTnC). Control experiments in fibres indicated that, in the absence of Ca(2+), both sTnC and xxsTnC bind with similar apparent affinity to sTnC-extracted thin filaments. Endogenous sTnC-extracted fibres reconstituted with 100 % xxsTnC did not develop Ca(2+)-activated force. In fibres reconstituted with mixtures of sTnC and xxsTnC, maximal Ca(2+)-activated force increased in a greater than linear manner with the fraction of sTnC. This suggests that Ca(2+) binding to functional Tn can spread activation beyond the seven actins of an SU into neighbouring units, and the data suggest that this functional unit (FU) size is up to 10-12 actins. As the number of FUs was decreased, Ca(2+) sensitivity of force (pCa(50)) decreased proportionally. The slope of the force-pCa relation (the Hill coefficient, n(H)) also decreased when the reconstitution mixture contained < 50 % sTnC. With 15 % sTnC in the reconstitution mixture, n(H) was reduced to 1.7 +/- 0.2, compared with 3.8 +/- 0.1 in fibres reconstituted with 100 % sTnC, indicating that most of the cooperative thin filament activation was eliminated. The results suggest that cooperative activation of skeletal muscle fibres occurs primarily through spread of activation to near-neighbour FUs along the thin filament (via head-to-tail tropomyosin interactions).

Proliferative Dynamics and the Role of FGF2 During Myogenesis of Rat Satellite Cells on Isolated Fibers.

Myogenic precursors in adult skeletal muscle (satellite cells) are mitotically quiescent but can proliferate in response to a variety of stresses including muscle injury. To gain further understanding of adult myoblasts, we are analyzing myogenesis of satellite cells on fibers isolated from adult rat muscle. In this culture model, satellite cells are maintained in their in situ position underneath the fiber basement membrane. Employing two different approaches to monitor proliferation of satellite cells on isolated fibers (autoradiography following 3H-thymidine incorporation and immunofluorescence of cells positive for proliferating cell nuclear antigen (PCNA)), we show in the present study that satellite cells initiate cell proliferation at 12 to 24 hours following fiber culture establishment and that cell proliferation is reduced to minimal levels by 60 to 72 hours in culture. Maximal number of proliferating cells is seen at 36 to 48 hours in culture. These PCNA+ satellite cells transit into the differentiated, myogenin+ state following about 24 hours in the proliferative state. Continuous exposure of the fiber culture to FGF2 (basic FGF; added at the time of culture establishment) leads to a 2 fold increase in the number of PCNA+ cells by 48 hours in culture but the overall schedule of proliferation and transition into the myogenin+ state is not affected. Delaying the addition of FGF2 until 15 to 18 hours following the initiation of the fiber culture does not reduce its effect. However, the addition of FGF2 at 24 hours or later results in a progressive reduction in the number of proliferating satellite cells. Exposure of fiber cultures to transforming growth factor ß (TGFß1) leads to a reduction in the number of proliferating cells in both the absence or presence of FGF2. We propose that FGF2 enhances the number of proliferating cells by facilitating the recruitment of additional satellite cells from the quiescent state. However, satellite cells on isolated fibers conform to a highly coordinated program and rapidly transit from proliferation to differentiation regardless of the presence of FGF. The identification of agents that can prolong the proliferative state of satellite cells when the cells undergo myogenesis in their native position by the intact myofiber might be useful in improving myoblast transplantation into skeletal muscle for cell-mediated gene therapy.