Thiocyclam does not induce structural chromosome aberrations in human lymphocytes in vitro

Thiocyclam (trade name Evisect) is a broad-spectrum nereistoxin analogue insecticide used widely for agricultural applications. The aim of this investigation was to determine its genotoxic effects in the chromosome aberration (CA) test and determining of mitotic index (MI), using lymphocytes from peripheral blood samples of healthy human donors. A negative and a positive control (MMC) were also included. Chromosomal analyses of the metaphase plates of the samples treated with 14 different concentrations (from 0.1 to 120 μg/ml) of thiocyclam, indicating the lack effect on chromosomes. Thus thiocyclam is not genotoxic but highly toxic on cell proliferation in human lymphocytes.     

Radiolabeling of HYNIC-annexin V with technetium-99m for in vivo imaging of apoptosis

Apoptosis is a critical factor in AIDS and other viral illnesses, cerebral and myocardial ischemia, autoimmune and neurodegenerative states, organ and bone marrow transplant rejection, and tumor response to chemotherapy and radiation. Improved methods to identify sites of apoptosis are increasing our understanding of the pathophysiology and treatment of these and numerous other human disorders. Here we describe the most used method for labeling annexin V, a protein with a high affinity for apoptotic cells in vitro, with technetium-99m (99mTc) as a radionuclide imaging agent that can localize and non-invasively quantify apoptosis in vivo when coupled with single-photon emission tomography. In this method, annexin V is first attached to the bifunctional chelator molecule hydrazino nicotinate (HYNIC). Once prepared, HYNIC-annexin V can be labeled with 99mTc, a widely available gamma-radiation-emitting radionuclide, for intravenous injection in as little as 30 min without the need for specialized reagents or equipment.     

Maternal exposure to multi-wall carbon nanotubes does not induce embryo-fetal developmental toxicity in rats

BACKGROUND: Although the potential risk of carbon nanotubes (CNTs) to humans has recently increased due to expanding production and widespread use, the potential adverse effects of CNTs on embryo–fetal development have not yet been determined. METHODS: This study investigated the potential effects of multi‐wall CNTs (MWCNTs) on pregnant dams and embryo–fetal development in rats. MWCNTs were administered to pregnant rats by gavage at 0, 40, 200, and 1,000 mg/kg/day. All dams were subjected to Cesarean section on day 20 of gestation, and the fetuses were examined for any morphological abnormalities. RESULTS: All animals survived to the end of the study. A decrease in thymus weight was observed in the high dose group in a dose‐dependent manner. However, maternal body weight, food consumption, and oxidant–antioxidant balance in the liver were not affected by treatment with MWCNTs. No treatment‐related differences in gestation index, fetal deaths, fetal and placental weights, or sex ratio were observed between the groups. Morphological examinations of the fetuses demonstrated no significant difference in incidences of abnormalities between the groups. CONCLUSIONS: The results show that repeated oral doses of MWCNTs during pregnancy induces minimal maternal toxicity and no embryo–fetal toxicity at 1,000 mg/kg/day in rats. The no‐observed‐adverse‐effect level of MWCNTs is considered to be 200 mg/kg/day for dams and 1,000 mg/kg/day for embryo–fetal development. In this study, the dosing formulation was not analyzed to determine the degree of reaggregation (or not), nor were blood levels of CNT's measured in the dosed animals to verify or characterize absorption. Birth Defects Res (Part B) 92:69–76, 2011. © 2011 Wiley‐Liss, Inc.     

Evaluation of technetium-99m decay on Escherichia coli inactivation: effects of physical or chemical agents.

Technetium-99m (99mTc) has been used in nuclear medicine and in biomedical research to label molecular and cellular structures employed as radiotracers. Here, we have evaluated, on a DNA repair proficient Escherichia coli strain, the 99mTc decay inactivation and the influence of the (i) pre-treatment with metal ion chelators or of the (ii) treatment with a free radical scavenger on the protection of the cells against the lethal effect of the 99mTc. As SnCl2 is frequently used as a reducing agent in the 99mTc-labeling process, we have also studied the capability of SnCl2 to alter the biological effects induced by the 99mTc decay. As we are exposed to either chemical or physical agents in the nature, we have decided to study a possible influence of the ultraviolet solar radiation in the biological phenomena induced by the 99mTc decay. Our data point out (i) a very important role of the Auger and/or conversion electrons in the cytotoxicity induced by the 99mTc decay; (ii) SnCl2, the metal ion chelators and the free radical scavenger protect the cells against the lethal effect of the 99mTc; and (iii) near-UV does not alter the lethal effect of the 99mTc decay.     

Continuous estrogen exposure in the rat does not induce loss of uterine estrogen receptor

Cytosol estrogen receptor (ERc) and nuclear estrogen receptor (ERN) levels were investigated in rat uteri under different conditions of hormonal exposure. The amount of directly assayable receptor was closely related to the serum concentration of 17 beta-[2,4,6,7-3H] estradiol ( [3H]E2). A double injection technique was established to maintain serum levels of [3H]E2 which were sufficient to saturate receptor sites. Under these conditions, stable ERC and ERN levels are maintained throughout the study period. 30% of the total ER remains cytoplasmic in localization despite continuous hormonal exposure. Properties of ERC and ERN after 6 h of continuous hormonal exposure were investigated and found to be different from receptors found in these subcellular compartments 30 min after hormone injections. ERC from uteri 30 min after injection showed a faster sedimentation coefficient than ERC prepared 6 h after hormone treatment. ERC after 6 h of hormonal exposure showed a reduction of binding to calf thymus DNA adsorbed on cellulose in a cell-free system. ERC 30 min after [3H]E2 treatment had a biphasic dissociation pattern consistent with two different receptor populations, whereas uterine ERC obtained after 6 h of in vivo exposure to estradiol showed virtually no dissociation at 22 and 28 degrees C. In contrast to ERC, ERN 6 h after hormone injection sedimented faster than ERN obtained 30 min after treatment. KCl extractable ERN obtained either at 30 min or 6 h posthormone treatment showed biphasic dissociation kinetics at 22 and 28 degrees C, whereas KCl nonextractable ERN showed virtually no dissociation. Virtually all of the specifically bound ligand in cytosol and nuclear preparations was proven to be authentic E2. We conclude that total cellular receptor is quantitatively conserved during 6 h of continuous hormonal treatment. Nuclear receptor loss is not a requisite for receptor-mediated steroid function, although important time-dependent changes in receptor properties in both cytoplasmic and nuclear compartments do occur.     

Evaluation of the cytotoxic and mutagenic potentiality of technetium-99m in Escherichi coli.

Since technetium-99m (99mTc) was introduced in medical research it has become one of the most employed radionuclides in nuclear medicine. 99mTc is ideal for routine use on the labeling of different radiopharmaceuticals due to its favorable characteristics. However, some biological effects have been described. These effects may be related to internal conversion electron and/or Auger electron emissions from 99mTc decay that present high linear energy transfer and can generate reactive oxygen species (ROS) in the medium. We evaluated in Escherichia coli K12S and Salmonella typhimurium TA102, both proficient in DNA repair, contribution of those decay emissions on the cytotoxicity induced by 99mTc, both either by generating lesions on DNA or by inducing alterations at membrane. We also studied the genotoxic and/or mutagenic potentiality of 99mTc, in Salmonella typhimurium, using the Ames test. The results showed that: i/ 99mTc is cytotoxic to the Escherichia coli K12S strains; ii/ this effect is related to the electrons (Auger and internal conversion) emissions, and iii/ the 99mTc is not mutagenic and/or genotoxic, when measured by Ames test.     

In vitro differentiation of human calvarial suture derived cells with and without dexamethasone does not induce in vivo-like expression

Osteogenic supplements are a requirement for osteoblastic cell differentiation during in vitro culture of human calvarial suture-derived cell populations. We investigated the ability of ascorbic acid and beta-glycerophosphate with and without the addition of dexamethasone to stimulate in vivo-like osteoblastic differentiation. Cells were isolated from unfused and prematurely fused suture tissue from patients with syndromic and non-syndromic craniosynostosis and cultured in each osteogenic medium for varying lengths of time. The effect of media supplementation was investigated with respect to the ability of cells to form mineralised bone nodules and the expression of five osteodifferentiation marker genes (COL1A1, ALP, BSP, OC and RUNX2), and five genes that are differentially expressed during human premature suture fusion (GPC3, RBP4, C1QTNF3, WIF1 and FGF2). Cells from unfused sutures responded more slowly to osteogenic media but formed comparable bone nodules to fused suture-derived cells after 16 days of culture in either osteogenic media. However, gene expression differed between unfused and fused suture-derived cells, as did expression in each osteogenic medium. When compared to expression in the explant tissue of origin, neither medium induced a level or profile of gene expression similar to that seen in vivo. Overall, our results demonstrate that cells from the same suture that are isolated during different stages of morphogenesis in vivo, despite being de-differentiated to a similar level in vitro, respond uniquely and differently to each osteogenic medium. Further, we suggest that neither cell culture medium recapitulates differentiation via activation of the same genetic cascades as occurs in vivo.     

The use of technetium-99m as a radioisotopic label to assess cell-mediated immunity in vitro

We have developed a radioisotopic microassay of cell-mediated immunity employing target cells prelabeled with technetium-99m (^99mTc), a high specific activity metastable gamma emitter. Labeling kinetics, release and reutilization, subcellular localization, and effects of ^99mTc on DNA and protein synthesis have been investigated. Target cells were optimally labeled with 10 mCi of ^99mTc at 37 °C for 10 min. Cyclic freezing and thawing released less than 10% of total bound radioisotope. Spontaneous leakage of ^99mTc by monolayer cells was negligible over 48 hr and that which was released appeared to be nonreutilizable. Cell fractionation revealed that nuclear, mitochondrial, and microsomal fractions all were labeled with ^99mTc. The incorporation of ³H-thymidine and ³H-amino acids was not impaired in ^99mTc-labeled cells.The alloimmune reactivity of C57BL/6 mice which had received A/J skin allografts was studied by means of the ^99mTc microcytotoxicity assay. Cell-mediated immunity was clearly evident at 7 days postgrafting, peaked at 14 days, and had declined to background levels by 21 days. These findings correlated well with initial acceptance and ultimate rejection of the allografts. The rapid labeling time without dependence upon cell division for incorporation, high specific activity, low spontaneous release, and nonreutilizability are important advantages of ^99mTc over other radionuclides which have been employed in in vitro assays of cell-mediated immunity.     

Clastogenic effects in human lymphocytes of power frequency electric fields: In vivo and in vitro studies

Summary In vivo and in vitro studies of the clastogenic effects of power frequency electric fields and transient electric currents have been performed. For the in vivo investigation peripheral lymphocytes from twenty switchyard workers were screened for chromosome anomalies. The rates of chromatid and chromosome breaks were found to be significantly increased compared to the rates in 17 controls.Exposure of human peripheral lymphocytes, in vitro, to a 50-Hz current with 1 mA/cm² current density did not induce any chromosome damage. Exposure to ten 3 µs-long spark discharge pulses with a peak field strength in the samples of 3.5 kV/cm, however, resulted in chromosome breaks at a frequency similar to that induced in lymphocytes in vitro by ionizing radiation at 0.75 Gy.The biological significance of chromosomal damage induced in somatic cells is discussed.     

Study of the biodistribution of the amantadine labelled with technetium-99m in Wistar female rats.

Amantadine (AMA) has been described as dopamine stimulant and norepineprhine release, capable to block the N-methyl-D aspartate (NMDA) glutamatergic and nicotinic receptors, enhancing the sexual behavior of the male rats and inducing hypersexuality in humans. The use of technetium-99m (99mTc) can be justified for its physical and chemical properties. The aim of this study was to label and evaluate the bioavailability of the AMA labelled with 99mTc (99mTc-AMA) in Wistar female rats. The solution of 99mTc-AMA was administered by intraperitoneal way and the animals were sacrificed in CO2 chamber 10 min after the administration of the radiotracer. Various organs were removed, weighted, their radioactivity was determined using an auto-gamma counter and the results were expressed as the percentage of the injected activity per gram of tissue (%ATI/g). In the control group only Na99mTcO4 was administered. The analysis of results shows that the highest uptakes 99mTc-AMA treated group were: ovary (7.11 +/- 1.43), spleen (3.54 +/- 1.05), thyroid (2.67 +/- 0.15), stomach (1.56 +/- 1.10), duodenum (0.87 +/- 0.52), muscular tissue (0.57 +/- 0.06), liver (0.52 +/- 0.25), and at control group: thyroid (16.45 +/- 2.57), ovary (1.28 +/- 0.12), liver (1.10 +/- 0.04), spleen (0.57 +/- 0.07) and muscular tissue (0.26 +/- 0.03). The results obtained suggest that 99mTc-AMA may be used to study the bioavailability of amantadine and evaluate its effect in sexual behavior in female rats.     

In vivo and in vitro ethylene oxide exposure of human lymphocytes assessed by chemical stimulation of unscheduled DNA synthesis

Factory workers exposed to ethylene oxide (EO), 0.5–1.0 ppm in factory air, together with matched controls from the same factory, were examined for evidence of toxic exposure by measurement of unscheduled DNA synthesis (UDS) induced by N-acetoxy-2-acetylaminofluorene (NA-AAF) and of chromosome aberrations in peripheral lymphocytes.The total chromatid gaps plus breaks were significantly elevated and NA-AAF-induced UDS was significantly reduced in the EO-exposed group as compared with the unexposed control group. The NA-AAF-induced UDS values negatively correlated to the duration (yr) of EO exposure (r = ?0.45, p < 0.02) and the number of chromosome breaks (r = ?0.61, p < 0.05), indicating an inhibition in vivo of DNA-repair capacity by EO. These data were verified in vitro by biochemical and autoradiographic studies of EO-induced UDS in human blood cells. Above 2 mM EO, UDS was inhibited in lymphocytes whether they were cultured for 24 or 122 h after alkylation with EO. Even at the subtoxic EO dose of 0.1 mM, lymphocytes were sensitized to additional exposures of NA-AAF, so that cytotoxicity was increased to 40% compared with 5% for the controls even though UDS was unaffected.It is concluded that EO was toxic to lymphocytes, even when they were sensitized at non-toxic EO doses to the cytotoxic action of other mutagens (e.g. NA-AAF), and the cells that did survive above 2 mM EO were inhibited in their DNA-repair capacity as judged by reduced UDS.     

TP53 tumor suppressor protein in normal human fibroblasts does not respond to 837 MHz microwave exposure.

The TP53 tumor suppressor protein (formerly known as p53) responds to a wide variety of environmental insults. To evaluate the safety of cellular telephones, TP53 responses in human fibroblast cells were studied after exposure to 837 MHz microwaves. Cells were exposed in a temperature-controlled transverse electromagnetic (TEM) chamber to a specific absorption rate (SAR) of 0.9 or 9.0 W/kg at 837 MHz continuous-wave (CW) microwave irradiation for 2 h. The TP53 protein levels were measured by Western blot at 2, 8, 24 and 48 h after treatment. The TP53 protein levels in microwave-treated cells, sham-treated cells, and untreated cells remained unchanged relative to each other at all times tested (Fisher test and Student-Newman-Keuls test, P > 0.05). No morphological alterations were observed in microwave-treated cells compared to sham-treated cells. We conclude that TP53 protein expression levels in cultured human fibroblast cells do not change significantly during a 48-h period after exposure to 837 MHz continuous microwaves for 2 h at SAR levels of 0.9 or 9.0 W/kg.     

Effects of in vivo exposure to antineoplastic drugs on DNA repair and replication in human lymphocytes

In peripheral blood lymphocytes of 12 nurses and 3 patients exposed to antineoplastic drugs we determined the ability to repair DNA after UV irradiation and DNA replicative synthesis after stimulation by PHA. In nurses the levels of unscheduled DNA synthesis and DNA replication were not different than in a control group, whereas in patients significant changes were observed during and after chemotherapy in the level of both types of DNA synthesis.     

Cysteine,a chelating moiety for synthesis of technetium-99m radiopharmaceuticals: II. Attempt to synthesize renal tubular radiopharmaceuticals

N-acyl glycine residue is known to interact with renal tubular transport enzymes and thereby promotes renal tubular secretion. Recently, a similar renal property was also observed with dioxotechnetium aminocarboxy chelates. Therefore, a radiopharmaceutical was designed containing both the above groups with the expectation that an efficient ^99mTc labelled renal tubular agent could be developed by this process with which evaluation of various renal parameters will be possible. The synthesized compound, though secreted through the renal tubular pathway, did not show the expected efficiency. It was indicated that the renal property of the molecule was entirely due to chelated dioxotechnetium moiety and the expected effect of the acyl glycine residue was not observed in the molecule.     

Cytogenetic effects of cyclophosphamide on human lymphocytes in vivo and in vitro

A comparative study of cytogenetic effects in human lymphocytes caused in vivo by cyclophosphamide (CP) after intravenous injection and in vitro by exposure of plasma of the same patients was carried out. It was found that the frequency of induced chromosome aberrations (CA) and sister-chromatid exchanges (SCE) increased linearly for SCE and exponentially for CA within the 'dose' of alkylating activity of CP metabolites. Parameters of 'cytogenetic effect-dose' in vivo and in vitro coincided. The intensity of cytogenetic effects varied between individuals.