Prasad V.S.V. Neti

Elevated blood lead concentrations and vitamin D deficiency in winter and summer in young urban children

Background It is widely recognized that blood lead concentrations are higher in the summer than in winter. Although the effects of some environmental factors such as lead in dust on this phenomenon have been studied, relationships to sunlight-induced vitamin D synthesis have not been adequately investigated. Vitamin D status is influenced by the diet, sunlight exposure, age, skin pigmentation, and other factors, and may modify gastrointestinal lead absorption or release of lead stored in bones into the bloodstream. Objective and Methods We collected paired blood samples from 142 young, urban African-American and Hispanic children in the winter and summer to study the seasonal increase in blood lead and its relationships to vitamin D nutrition, age, and race. Results A winter/summer (W/S) increase in blood lead concentrations of 32.4% was found for children 1–3 years of age. There was a smaller W/S increase of 13.0% in children 4–8 years of age. None of the 51 Hispanic children had an elevated blood lead concentration (≥ 10 μg/dL) during the winter, and only one had an elevated summertime concentration. In contrast, elevated blood lead concentrations were frequent in the 91 African-American children, especially those 1–3 years of age. For the latter, the percentage with elevated blood lead levels increased from 12.2% in winter to 22.5% in summer. A 1.2% W/S increase in serum 25-hydroxy-vitamin D (serum 25-OH-D) concentrations was found for children 1–3 years of age. However, in children 4–8 years of age the W/S increase in serum 25-OH-D was much larger—33.6%. The percentages of children with low (< 16 μg/L) serum 25-OH-D concentrations were 12.0% in winter and 0.7% in summer and were consistently greater in African-American than in Hispanic children. The seasonal increases in blood lead and serum 25-OH-D in children 4–8 years of age were significantly associated. Conclusion The higher summertime serum 25-OH-D concentrations for the 4- to 8-year-old children are likely caused by increased sunlight-induced vitamin D synthesis and may contribute to the seasonal increase in blood lead. Age and race are key factors that affect blood lead and vitamin D nutrition, as well as their interactions, in young urban children.

A Multi-port Low-Fluence Alpha-Particle Irradiator: Fabrication, Testing and Benchmark Radiobiological Studies

Abstract Neti, P. V. S. V., de Toledo, S. M., Perumal, V., Azzam, E. I. and Howell, R. W. A Multi-port Low-Fluence Alpha-Particle Irradiator: Fabrication, Testing and Benchmark Radiobiological Studies. Radiat. Res. 161, 732–738 (2004). A new multi-port irradiator, designed to facilitate the study of the effects of low fluences of α particles on monolayer cultures, has been developed. The irradiator consists of four individual planar 241Am α-particle sources that are housed inside a helium-filled Lucite chamber. Three of the radioactive sources consist of 20 MBq of 241Am dioxide foil. The fourth source, used to produce higher dose rates, has an activity of 500 MBq. The four sources are mounted on rotating turntables parallel to their respective 1.5-μm-thick Mylar exit windows. A stainless steel honeycomb collimator is placed between the four sources and their exit windows by a cantilever attachment to the platform of an orbital shaker that moves its table in an orbit of 2 cm. Each exit window is equipped with a beam delimiter to optimize the uniformity of the beam and with a high-precision electronic shutter. Opening and closing of the shutters is controlled with a high-precision timer. Custom-designed stainless steel Mylar-bottomed culture dishes are placed on an adapter on the shutter. The α particles that strike the cells have a mean energy of 2.9 MeV. The corresponding LET distribution of the particles has a mean value of 132 keV/μm. Clonogenic cell survival experiments with AG1522 human fibroblasts indicate that the RBE of the α particles compared to 137Cs γ rays is about 7.6 for this biological end point.

Pharmacokinetic Analysis of Polyamide Nucleic-Acid-Cell Penetrating Peptide Conjugates Targeted against HIV-1 Transactivation Response Element

We have demonstrated that polyamide nucleic acids complementary to the transactivation response (TAR) element of HIV-1 LTR inhibit HIV-1 production when transfected in HIV-1 infected cells. We have further shown that anti-TAR PNA (PNA(TAR)) conjugated with cell-penetrating peptide (CPP) is rapidly taken up by cells and exhibits strong antiviral and anti-HIV-1 virucidal activities. Here, we pharmacokinetically analyzed (125)I-labeled PNA(TAR) conjugated with two CPPs: a 16-mer penetratin derived from antennapedia and a 13-mer Tat peptide derived from HIV-1 Tat. We administered the (125)I-labeled PNA(TAR)-CPP conjugates to male Balb/C mice through intraperitoneal or gavage routes. The naked (125)I-labeled PNA(TAR) was used as a control. Following a single administration of the labeled compounds, their distribution and retention in various organs were monitored at various time points. Regardless of the administration route, a significant accumulation of each PNA(TAR)-CPP conjugate was found in different mouse organs and tissues. The clearance profile of the accumulated radioactivity from different organs displayed a biphasic exponential pathway whereby part of the radioactivity cleared rapidly, but a significant portion of it was slowly released over a prolonged period. The kinetics of clearance of individual PNA(TAR)-CPP conjugates slightly varied in different organs, while the overall biphasic clearance pattern remained unaltered regardless of the administration route. Surprisingly, unconjugated naked PNA(TAR) displayed a similar distribution and clearance profile in most organs studied although extent of its uptake was lower than the PNA(TAR)-CPP conjugates.

Modeling Multicellular Response to Nonuniform Distributions of Radioactivity: Differences in Cellular Response to Self-Dose and Cross-Dose

Abstract Howell, R. W. and Neti, P. V. S. V. Modeling Multicellular Response to Nonuniform Distributions of Radioactivity: Differences in Cellular Response to Self-Dose and Cross-Dose. Radiat. Res. 163, 216–221 (2005). Radiopharmaceuticals are distributed nonuniformly in tissue. While distributions of radioactivity often appear uniform at the organ level, in fact, microscopic examination reveals that only a fraction of the cells in tissue are labeled. Labeled cells and unlabeled cells often receive different absorbed doses depending on the extent of the nonuniformity and the characteristics of the emitted radiations. The labeled cells receive an absorbed dose from radioactivity within the cell (self-dose) as well as an absorbed dose from radioactivity in surrounding labeled cells (cross-dose). Unlabeled cells receive only a cross-dose. In recent communications, a multicellular cluster model was used to investigate the lethality of microscopic nonuniform distributions of 131I iododeoxyuridine (131IdU). For a given mean absorbed dose to the tissue, the dose response depended on the percentage of cells that were labeled. Specifically, when 1, 10 and 100% of the cells were labeled, a D37 of 6.4, 5.7 and 4.5 Gy, respectively, was observed. The reason for these differences was recently traced to differences in the cellular response to the self- and cross-doses delivered by 131IdU. Systematic isolation of the effects of self-dose resulted in a D37 of 1.2 ± 0.3 Gy. The cross-dose component yielded a D37 of 6.4 ± 0.5 Gy. In the present work, the overall survival of multicellular clusters containing 1, 10 and 100% labeled cells is modeled using a semi-empirical approach that uses the mean lethal self- and cross-doses and the fraction of cells labeled. There is excellent agreement between the theoretical model and the experimental data when the surviving fraction is greater than 1%. Therefore, when the distribution of 131I in tissue is nonuniform at the microscopic level, and the cellular response to self- and cross-doses differs, multicellular dosimetry can be used successfully to predict biological response, whereas the mean absorbed dose fails in this regard.

Changes in Lognormal Shape Parameter Guide Design of Patient-Specific Radiochemotherapy Cocktails

Uptake of radiopharmaceuticals and chemotherapeutic drugs is nonuniform at the microscopic level. Their distributions are typically lognormal, suggesting that failure in chemotherapy and targeted radionuclide therapy may be attributable, in part, to the characteristics of this biologically ubiquitous distribution. The lognormal problem can be overcome by using cocktails of 2 or more agents, tailored such that at least 1 agent is strongly incorporated by every cell in the target population. Therefore, critical assessment of the tissue uptake of each cocktail component is warranted. Methods: Cellular incorporation of the α-particle–emitting radiochemical (210Po-citrate) and 2 anticancer drugs (daunomycin and doxorubicin) was determined using flow cytometry. The role of their lognormal distribution in clonogenic cell survival was evaluated. Results: The shape parameter of the lognormal distribution was found to be correlated to both intracellular agent concentration and cell survival. Although no difference emerged between the shape parameters for citrate within the first 2 logs of cell kill, those for daunomycin and doxorubicin changed significantly. Conclusion: Changes in the value of the lognormal shape parameter and slope of the cellular drug uptake curves can be used to rapidly screen radiopharmaceuticals and other cytotoxic agents to formulate more effective cocktails for cancer therapy.

Biological Response to Nonuniform Distributions of 210Po in Multicellular Clusters

Abstract Neti, P. V. S. V. and Howell, R. W. Biological Response to Nonuniform Distributions of 210Po in Multicellular Clusters. Radiat. Res. 168, 332–340 (2007). Radionuclides are distributed nonuniformly in tissue. The present work examined the impact of nonuniformities at the multicellular level on the lethal effects of 210Po. A three-dimensional (3D) tissue culture model was used wherein V79 cells were labeled with 210Po-citrate and mixed with unlabeled cells, and multicellular clusters were formed by centrifugation. The labeled cells were located randomly in the cluster to achieve a uniform distribution of radioactivity at the macroscopic level that was nonuniform at the multicellular level. The clusters were maintained at 10.5°C for 72 h to allow α-particle decays to accumulate and then dismantled, and the cells were seeded for colony formation. Unlike typical survival curves for α particles, two-component exponential dose–response curves were observed for all three labeling conditions. Furthermore, the slopes of the survival curves for 100, 10 and 1% labeling were different. Neither the mean cluster absorbed dose nor a semi-empirical multicellular dosimetry approach could accurately predict the lethal effects of 210Po-citrate.

Lognormal Distribution of Cellular Uptake of Radioactivity: Statistical Analysis of α-Particle Track Autoradiography

Recently, the distribution of radioactivity among a population of cells labeled with 210Po was shown to be well described by a lognormal (LN) distribution function (J Nucl Med. 2006;47:1049–1058) with the aid of autoradiography. To ascertain the influence of Poisson statistics on the interpretation of the autoradiographic data, the present work reports on a detailed statistical analysis of these earlier data. Methods: The measured distributions of α-particle tracks per cell were subjected to statistical tests with Poisson, LN, and Poisson-lognormal (P-LN) models. Results: The LN distribution function best describes the distribution of radioactivity among cell populations exposed to 0.52 and 3.8 kBq/mL of 210Po-citrate. When cells were exposed to 67 kBq/mL, the P-LN distribution function gave a better fit; however, the underlying activity distribution remained lognormal. Conclusion: The present analysis generally provides further support for the use of LN distributions to describe the cellular uptake of radioactivity. Care should be exercised when analyzing autoradiographic data on activity distributions to ensure that Poisson processes do not distort the underlying LN distribution.

Marked changes in endogenous antioxidant expression precede vitamin A, C and E-protectable, radiation-induced reductions in small intestinal nutrient transport

Rapidly proliferating epithelial crypt cells of the small intestine are susceptible to radiation-induced oxidative stress, yet there is a dearth of data linking this stress to expression of antioxidant enzymes and to alterations in intestinal nutrient absorption. We previously showed that 5-14 days after acute γ-irradiation, intestinal sugar absorption decreased without change in antioxidant enzyme expression. In the present study, we measured antioxidant mRNA and protein expression in mouse intestines taken at early times postirradiation. Observed changes in antioxidant expression are characterized by a rapid decrease within 1h postirradiation, followed by dramatic upregulation within 4h and then downregulation a few days later. The cell type and location expressing the greatest changes in levels of the oxidative stress marker 4HNE and of antioxidant enzymes are, respectively, epithelial cells responsible for nutrient absorption and the crypt region comprising mainly undifferentiated cells. Consumption of a cocktail of antioxidant vitamins A, C, and E, before irradiation, prevents reductions in transport of intestinal sugars, amino acids, bile acids, and peptides. Ingestion of antioxidants may blunt radiation-induced decreases in nutrient transport, perhaps by reducing acute oxidative stress in crypt cells, thereby allowing the small intestine to retain its absorptive function when those cells migrate to the villus days after the insult.

Radiation-induced reductions in transporter mRNA levels parallel reductions in intestinal sugar transport

More than a century ago, ionizing radiation was observed to damage the radiosensitive small intestine. Although a large number of studies has since shown that radiation reduces rates of intestinal digestion and absorption of nutrients, no study has determined whether radiation affects mRNA expression and dietary regulation of nutrient transporters. Since radiation generates free radicals and disrupts DNA replication, we tested the hypotheses that at doses known to reduce sugar absorption, radiation decreases the mRNA abundance of sugar transporters SGLT1 and GLUT5, prevents substrate regulation of sugar transporter expression, and causes reductions in sugar absorption that can be prevented by consumption of the antioxidant vitamin A, previously shown by us to radioprotect the testes. Mice were acutely irradiated with (137)Cs gamma rays at doses of 0, 7, 8.5, or 10 Gy over the whole body. Mice were fed with vitamin A-supplemented diet (100x the control diet) for 5 days prior to irradiation after which the diet was continued until death. Intestinal sugar transport was studied at days 2, 5, 8, and 14 postirradiation. By day 8, d-glucose uptake decreased by approximately 10-20% and d-fructose uptake by 25-85%. With increasing radiation dose, the quantity of heterogeneous nuclear RNA increased for both transporters, whereas mRNA levels decreased, paralleling reductions in transport. Enterocytes of mice fed the vitamin A supplement had > or = 6-fold retinol concentrations than those of mice fed control diets, confirming considerable intestinal vitamin A uptake. However, vitamin A supplementation had no effect on clinical or transport parameters and afforded no protection against radiation-induced changes in intestinal sugar transport. Radiation markedly reduced GLUT5 activity and mRNA abundance, but high-d-fructose diets enhanced GLUT5 activity and mRNA expression in both unirradiated and irradiated mice. In conclusion, the effect of radiation may be posttranscriptional, and radiation-damaged intestines can still respond to dietary stimuli.

High Levels of Dietary Supplement Vitamins A, C and E are Absorbed in the Small Intestine and Protect Nutrient Transport Against Chronic Gamma Irradiation.

We examined nutrient transport in the intestines of mice exposed to chronic low-LET 137Cs gamma rays. The mice were whole-body irradiated for 3 days at dose rates of 0, 0.13 and 0.20 Gy/h, for total dose delivery of 0, 9.6 or 14.4 Gy, respectively. The mice were fed either a control diet or a diet supplemented with high levels of vitamins A, C and E. Our results showed that nutrient transport was perturbed by the chronic irradiation conditions. However, no apparent alteration of the macroscopic intestinal structures of the small intestine were observed up to day 10 after initiating irradiation. Jejunal fructose uptake measured in vitro was strongly affected by the chronic irradiation, whereas uptake of proline, carnosine and the bile acid taurocholate in the ileum was less affected. D-glucose transport did not appear to be inhibited significantly by either 9.6 or 14.4 Gy exposure. In the 14.4 Gy irradiated groups, the diet supplemented with high levels of vitamins A, C and E increased intestinal transport of fructose compared to the control diet (day 10; t test, P = 0.032), which correlated with elevated levels of vitamins A, C and E in the plasma and jejunal enterocytes. Our earlier studies with mice exposed acutely to 137Cs gamma rays demonstrated significant protection for transport of fructose, glucose, proline and carnosine. Taken together, these results suggest that high levels of vitamins A, C and E dietary supplements help preserve intestinal nutrient transport when intestines are irradiated chronically or acutely with low-LET gamma rays.