Uranium induces genomic instability and slows cell cycle progression in human lymphocytes in acute toxicity study.

In the situation of radiation triage, accidental exposure to uranium, or uranium contamination in food or water; haematopoietic decline or bone marrow sickness is observed in the aftermath followed by other systemic effects. Most studies done previously have been on cytogenetic analysis in blood lymphocytes of uranium miners wherein causal relationship was difficult to be established. This study provides new insights into the minimum risk level of uranium to human lymphocytes, DNA damage induced and alterations in the cell cycle progression through 96-h acute toxicity study. Cytotoxicity studies by MTT assay and flow cytometry showed that uranyl nitrate concentration of 1280 µM lead to 50% cell death, 640 µM caused 25% death, 250 µM caused 10% cell death and 5 µM was the NOAEL. Uranium caused DNA damages in a dose dependent manner as evident from comet and CBMN assays. A marked increase in G2/M phase cells was observed in the test culture groups. Halting of cell cycle at G2/M checkpoint also signified the extent of double strand breaks and genetic instability with increasing uranium dose in this study. Better cell cycle responses and lower genetic damage index observed in lower dosage of exposure, suggests adaptability and repair responses in human lymphocytes. Together these results advance our understanding of uranium effects on mammalian cells.

HOXD10 expression in human breast cancer.

Breast cancer is the most frequent malignancy among females. In this study, we analyzed the expression pattern of a homeobox gene (HOXD10) in human invasive ductal breast cancer tissues and normal tissues. With the ACTB (ß-actin) gene as a reference, HOXD10 was detected in 60 breast cancer tissues by using the quantitative real-time PCR (qPCR) method with the Relative Expression Software Tool (REST). We found that the HOXD10 expression level was significantly different between cancerous and normal tissues. Downregulation of the HOXD10 gene expression was examined in high-grade samples. Low-grade tissue showed no difference from the control group. HOXD10 expression was reduced in grade II breast carcinoma tissues. This data reveal that misexpression of the HOXD10 gene supports the development and involvement in breast cancer and may serve as a potential biomarker for the diagnosis of human ductal invasive breast carcinoma.

Prevalence of RET/PTC1 and RET/PTC3 gene rearrangements in Chennai population and its correlation with clinical parameters.

Thyroid cancer is one of the most common endocrine disorders in the world. In India, about 42 million people suffer from various thyroid disorders. However, based on population-based cancer registry (PBCR) and Chennai cancer registry, thyroid cancer is emerging as a common cancer particularly in Chennai. Papillary thyroid carcinoma is considered as the most prevalent cancer constituting about 80-85 % of thyroid malignancies. Rearranged during transfection (RET)/papillary thyroid carcinoma (PTC) gene rearrangements are one of the major genetic alterations found in papillary thyroid carcinoma. This present study aims at estimating the frequency of RET/PTC1 and RET/PTC3 gene rearrangements in Chennai population and investigating the correlation between RET/PTC gene expressions with clinical parameters. Formalin-fixed paraffin-embedded (FFPE) tumor tissues obtained from 30 patients with papillary thyroid carcinoma were analyzed. Initially, to differentiate classic and follicular variants of papillary thyroid carcinoma samples, immunohistochemistry was performed. Thereafter, total RNA was isolated, and quantitative evaluation of RET/PTC1 and RET/PTC3 gene rearrangements by real-time PCR was performed. Chi-square test was performed to understand the correlation between positive and negative mutations of RET/PTC messenger RNA (mRNA) expression with clinical parameters. RET/PTC3 gene rearrangements were identified in 26/30 (86.67 %) cases, and none of the patient in our series had RET/PTC1 gene rearrangements. There was no statistically significant difference observed between positive and negative mutations of RET/PTC3 mRNA expression with clinic pathological parameters. Our results indicate that RET/PTC3 gene rearrangements are the most prevalent form of rearrangements in PTCs of Chennai population.

Regulation of BMI1 Polycomb gene expression in histological grades of invasive ductal breast carcinomas and its correlation with hormone receptor status.

BMI1 is the first functional mammalian Polycomb group (PcG) proto-oncogene involved in multiple biological processes. Regulation of B cell-specific Moloney murine leukaemia virus integration site 1 (BMI1) expression with increase in histological grades of breast carcinoma in correlation with hormone receptor status was studied in 60 Indian breast cancer patient's formalin-fixed paraffin-embedded tissue blocks. Relative expression of BMI1 was studied using real-time PCR. Immunohistochemistry explained the distribution of hormone receptor markers. Correlation of BMI1 gene expression with oestrogen receptor, progesterone receptor (PR) and human epidermal growth factor receptor 2/neu status was analysed using Hex-protein docking tool. The hormone receptor expression was reduced with increasing grades of breast tumour. BMI1 gene expression was downregulated (real-time polymerase chain reaction analysis). Docking analysis explained the correlation between BMI1 and PR expression. BMI1 gene was co-regulated (down) with PR in the invasive ductal breast carcinoma with relative progression explicating it a diagnostic biomarker for ductal carcinoma of the breast.

Assessment of in vitro skin irritation potential of nanoparticles: RHE model.

The skin irritation test is designed for the prediction of acute skin irritation of nanoparticles by measurement of its cytotoxic effect, as reflected in the MTT assay, on the Reconstructed Human Epidermis (RHE) model. RHE tissues are commercially available.

Oxidative stress and inflammatory responses of rat following acute inhalation exposure to iron oxide nanoparticles.

In this research, we investigated the toxicity responses of rat following a continuous 4 h inhalation exposure of only the head and nose to iron oxide nanoparticles (Fe(3)O(4) NPs, size = 15-20 nm). The rats for the investigation were exposed to a concentration of 640 mg/m(3) Fe(3)O(4) NPs. Markers of lung injury and proinflammatory cytokines (interleukin-1ß, tumor necrosis factor-α, and interleukin-6) in bronchoalveolar lavage fluid (BALF) and blood, oxidative stress in lungs, and histopathology were assessed on 24 h, 48 h, and 14 days of postexposure periods. Our results showed a significant decrease in the cell viability, with the increase in the levels of lactate dehydrogenase, total protein, and alkaline phosphatase in the BALF. Total leukocyte count and the percentage of neutrophils in BALF increased within 24 h of postexposure. Immediately following acute exposure, rats showed increased inflammation with significantly higher levels of lavage and blood proinflammatory cytokines and were consistent throughout the observation period. Fe(3)O(4) NPs exposure markedly increased malondialdehyde concentration, while intracellular reduced glutathione and antioxidant enzyme activities were significantly decreased in lung tissue within 24-h postexposure period. On histological observation, the lung showed an early activation of pulmonary clearance and a size-dependant biphasic nature of the Fe(3)O(4) NPs in causing the structural alteration. Collectively, our data illustrate that Fe(3)O(4) NPs inhalation exposure may induce cytotoxicity via oxidative stress and lead to biphasic inflammatory responses in Wistar rat.

Repeated dose dermal toxicity study of nano zinc oxide with Sprague-Dawley rats.

CONTEXT: In light of the increased use of zinc oxide nanoparticles in cosumer products such as sunscreens, there is a need for screening the potential dermal toxicity of these nanoparticles. OBJECTIVE: The aim of this study is to identify the risk associated with the nano zinc oxide at realistic exposure levels through dermal route. This study is to understand the toxic potential of nano zinc oxide through repeated dermal exposure for a period of 28 days. MATERIALS AND METHODS: Six- to 8-week-old Sprague-Dawley rats were applied with three different doses (75, 180, and 360 mg/kg body weight) of nano zinc oxide (20 nm) at 5 days/week basis for a period of 28 days. The dose levels were calculated taking into consideration the percentage of nanomaterial in the sunscreen, number of application times, and average weight of the consumer in order to assess the realistic risk related to it. Control group animals were applied with distilled water alone. The collagen content was estimated in skin and tail of all the treated and control animals. RESULTS: The content was significantly decreased in all the nano zinc oxide-treated groups with an inverse dose relationship. DISCUSSION AND CONCLUSION: The percentage collagen loss was high in skin when compared with tail. This may be due to the site of application where in the nano zinc oxide may be passed through skin due to their small size and may induce oxidative stress. Hence, we suggest that regulators and industry need to address the toxicity of nanomaterials with a realistic exposure assessment rather following conventional dose measurements following existing protocols.

Nanoemulsified ethanolic extract of Pyllanthus amarus Schum & Thonn ameliorates CCl4 induced hepatotoxicity in Wistar rats.

Phyllanthus amarus (PA) is commonly used in traditional medicine for hepatoprotectivity. The major limitation is that, treatment requires a large quantity of herbal extract for a longer duration. Aim of the present study was to encapsulate ethanolic plant extract for sustained release of constituents in intestine and facilitate maximum absorption. The efficacy was compared for the hepatoprotective activity of nanoencapsulated ethanolic extract of P. amarus (NPA) and PA in carbon tetrachloride (CCl4) induced hepatotoxic male rats. Based on total phenol content (TPC), the loading efficiency of nanocapsules was 89% (pH 7.0) and optimum concentration was 2:18 (mg/mL) for plant extract: olive oil. Scanning electron microscopy (SEM) showed a spherical morphology, photon correlation spectroscopy (PCS) identified mean particle diameter as 213 nm and Fourier transform infrared spectroscopy (FT-IR) revealed that the phytoconstituents were stable. An oral dose of NPA (20 mg/kg body wt.) showed a better hepatoprotective activity than PA (100 mg/kg body wt.) and also repeated dose oral toxicity proved to be safe. These biochemical assessments were supported by rat biopsy examinations. In conclusion, the nanoemulsification method may be applied for poor water-soluble ethanolic herbal extracts to reduce the dosage and time.

Transplacental genotoxicity evaluation of cypermethrin using alkaline comet assay.

Transplacental genotoxic effect of cypermethrin technical was investigated. Three doses (25, 50 and 75 mg/kg body weight) were administered to groups of pregnant Wistar rats during 6-15 days of gestation. Animals were killed on gestation day 20. Fetal blood and liver samples were evaluated for DNA damage using alkaline comet assay. A marginal increase in the mean percentage of DNA damage was recorded in both blood and liver samples of fetuses from cypermethrin-treated dams, but the values were not statistically significant. No skeletal or visceral fetal abnormalities were recorded in treated groups. Nevertheless, the results lead to an understanding that transplacental exposure to cypermethrin can induce low levels of DNA damage in fetuses. This observation could be an explanation for the teratogenic effect exhibited by this chemical in many other studies. The results indicate that cypermethrin may be transplacentally genotoxic. The authors propose more detailed investigations for validating the current findings.

Toxicity Assessment of Expired Pesticides to Green Algae Pseudokirchneriella subcapitata.

In order to investigate the effect of expired pesticides on the yield and growth rate of green algae Pseudokirchneriella subcapitata, a study was conducted as per the Organisation for Economic Cooperation and Development (OECD) guideline number 201. Fifteen expired pesticide formulations, most commonly used in Indian agriculture, were tested in comparison with their unexpired counterparts. The expired pesticide formulations studied belonged to various class and functional groups: organophosphate, pyrethroid-based insecticides; azole-based fungicides; acetamide, propionate, acetic acid-based herbicides; fungicides mixtures containing two actives-azole and dithiocarbamate. The toxicity endpoints of yield (EyC50: 0-72 h) and growth rate (ErC50: 0-72 h) of Pseudokirchneriella subcapitata for each pesticide formulation (both expired and unexpired pesticides) were determined statistically using TOXSTAT 3.5 version software. The results pointed out that some expired pesticide formulations exhibited higher toxicity to tested algal species, as compared to the corresponding unexpired pesticides. These data thus stress the need for greater care to dispose expired pesticides to water bodies, to avoid the effects on aquatic ecospecies tested.

Acute inhalation toxicity of cerium oxide nanoparticles in rats.

The aim of the present study was to assess the acute toxic potential of cerium oxide nanoparticles (CeO(2) NPs) in rats when exposed through the head and nose inhalation route. The rats were exposed to CeO(2) NPs and the resultant effects if any, to cause cytotoxicity, oxidative stress and inflammation in the lungs were evaluated on a 24h, 48h and 14 day post exposure period. Our results showed a significant decrease in the cell viability, with the increase of lactate dehydogenase, total protein and alkaline phosphatase levels in the bronchoalveolar lavage fluid (BALF) of the exposed rats. Total leukocyte count and the percentage of neutrophils in BALF were elevated within 24h of post exposure. The concentrations of pro-inflammatory cytokines (IL-1ß, TNF-α, and IL-6) were significantly increased in the BALF and in the blood throughout the observation period. The level of malondialdehyde was elevated with the decreased levels of intracellular reduced glutathione (GSH) in the lung after exposure. The alveolar macrophages (AMs) and neutrophils overloaded with phagocytosed CeO(2) NPs were observed along with non-phagocytosed free CeO(2) NPs that were deposited over the epithelial surfaces of the bronchi, bronchiole and alveolar regions of lungs within 24h of post exposure and were consistent throughout the observation period. A well distributed, multifocal pulmonary microgranulomas due to impairment of clearance mechanism leading to biopersistence of CeO(2) NPs for an extended period of time were observed at the end of the 14 day post exposure period. These results suggest that acute exposure of CeO(2) NPs through inhalation route may induce cytotoxicity via oxidative stress and may lead to a chronic inflammatory response.

Developmental toxicity of homobrassinolide in Wistar rats.

Brassinosteroids (BRs) are close analogues of animal cholesterol. Brassinosteroids have shown their great value as yield promoters of a variety of plants. In view of its steroidal moiety and recent use in agriculture in many countries, the teratogenic potential of homobrassinolide (HBR) was evaluated in Wistar rats. Homobrassinolide was administered by oral gavage at doses 0, 100, and 1000 mg/kg body weight in water during gestation days (GD) 6 to 15 in groups of 20 mated females. Maternal and embryo-fetal toxicity was analyzed by studying the effects such as clinical signs, mortality/morbidity, abortions, body weight, feed consumption, and pregnancy data, gravid uterine weights, implantation losses, litter size, external, visceral, and skeletal malformations. No treatment-related effect was observed on any of the maternal/fetal end points in any dose group. From the results, it can be concluded that HBR is nonteratogenic at doses as high as up to 1000 mg/kg body weight in Wistar rats.

Assessment of the dermal and ocular irritation potential of multi-walled carbon nanotubes by using in vitro and in vivo methods.

In view of increase in the manufacture of various nanomaterials runs the risk of increased human exposure, in vitro screening will serve as a preliminary method to assess possible risk in animal studies. We attempted to know whether the validated in vitro alternative models established for chemicals, drugs, pesticides are suitable for nanomaterials, since these materials differ largely and may interfere with commonly used test systems. In vitro and in vivo studies on ocular and dermal irritation were carried out with two different sizes of multi-walled carbon nanotubes (MWCNT). The results of acute eye irritation toxicity studies with two different sizes of MWCNT in rabbits demonstrated reversible conjunctival redness and discharge and exhibited minimal concern while acute dermal irritation studies indicated that MWCNT of two sizes were non-irritant to the skin of rabbits. Both the sizes of MWCNT revealed non-irritant result in HE-CAM test. In vitro skin (EPISKIN) irritation studies revealed that two sizes of MWCNT are non-irritant to skin. In conclusion this work purports, the future of alternative research lies in the validation of the methodology for nanomaterials.

Lambda cyhalothrin induced alterations in Clarias batrachus.

The present study was undertaken to find out the toxic effect of synthetic pyrethroid lambda cyhalothrin on the functioning of endocrine glands in freshwater catfish, Clarias batrachus. The fish were exposed to the pesticide for a period of 45 days at a sublethal concentration of 5.768 ppm. Analysis of hormone profile was carried out on the 15, 30 and 45 days of exposure to find out the alteration in hormone secretion and the response of the fish to the compound. The results obtained showed a significant decline (p<0.05) in levels of thyroid hormones and testosterone while a significant increase (p<0.05) was observed in cortisol levels during the different days of exposure of the fish to lambda cyhalothrin.

Larvicidal activity of neem and karanja oil cakes against mosquito vectors, Culex quinquefasciatus (say), Aedes aegypti (L.) and Anopheles stephensi (L.).

Larvicidal effect of neem (Azadirachta indica) and karanja (Pongamia glabra) oil cakes (individuals and combination) was studied against mosquito species. Both the oil cakes showed larvicidal activity against the mosquito species tested. The combination of neem and karanja oil cakes in equal proportion proved to have better effect than the individual treatments. The combination of the two oil cakes recorded an LC95 of 0.93, 0.54 and 0.77% against the mosquitoes, Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi respectively The increase in efficacy of the combination treatment over individuals in all the mosquito larvae tested was found to range about 4 to 10 fold in terms of LC50 and 2 to 6 fold in terms of LC95.

Broccoli flower head extract reduces mitomycin-C induced sister chromatid exchange in cultured human lymphocytes.

This study is a continuation of our previous work [Murugan, S.S., Balakrishnamurthy, P., Mathew, Y.J., 2007. Antimutagenic effect of broccoli flower head by the Ames Salmonella reverse mutation assay. Phytother. Res. 21, 545-547], in search of possible antimutagenic properties in broccoli flower head extracts. In the present investigation, the effect of addition of ethanol extract of broccoli flower head on mitomycin-C (MMC) induced sister chromatid exchange (SCE) in cultured human peripheral blood lymphocytes was investigated. Broccoli flower head was extracted in ethanol using either acetone or ethanol as solvents. The extract was tested at final concentrations of 200 and 400 microg/ml culture and set for SCE assay. MMC at a concentration of 1 microg/ml and the test concentrations of broccoli flower head were added to the culture following 48 h from the initiation of culture. Enumeration of SCE in second division mitotic cells indicated that broccoli flower head extract significantly reduced MMC induced SCEs at both the concentrations tested. This observation is in line with our earlier finding and confirms to the presence of antimutagenic principles in broccoli flower head extract.

Hen egg chorioallantoic membrane bioassay: an in vitro alternative to draize eye irritation test for pesticide screening.

As an alternative to the standard Draize eye irritation test, the potential irritancy of compounds was evaluated by observing adverse changes that occur in chorioallantoic membrane CAM) of the hen egg (HECAM) after exposure to a test chemical placed directly on the CAM. The occurrence of hemorrhage, coagulation, and lysis in response to a test compound is the basis for employing this technique to evaluate its potential for in vivo damage to mucous membrane, in particular the eye. Irritancy is scored according to the severity and speed at which damage occurs. In the present study, five different classes of pesticides were screened for irritation potential. There was good correlation between the HECAM assay and the in vivo Draize eye irritation test. The proposed HECAM assay, which reduces the requirement for laboratory animals, could be a painless alternative to the Draize test.

Screening of hepatoprotective effect of a herbal mixture against CCl4 induced hepatotoxicity in Swiss albino mice.

The hepatoprotective potential of a herbal mixture was evaluated against CCl4 induced liver injury in Swiss albino mice. Liv 52, a commercially available polyherbal hepatoprotective drug was evaluated for comparison. The potential toxicity of the above herbal hepatoprotective agents was also compared. It was observed that there was a reduction in the enzyme biomarkers (Aspartate and Alanine Transaminase) of liver injury in the herbal mixture treated groups, which was similar to the reduction initiated by Liv 52. An increase in glutathione was observed in the herbal mixture treated groups and it was assumed that the herbal mixture protects the liver by virtue of its antioxidant nature along with high regeneration initiation potential. From the study it is also concluded that the herbal mixture is safer than Liv 52.

Comparative efficacy and persistency of permethrin in Olyset net and conventionally treated net against Aedes aegypti and Anopheles stephensi.

The efficacy of the Olyset net was compared to a net treated conventionally with permethrin 10% emulsifiable concentrate at the World Health Organization recommended dose. The nets were assessed under laboratory conditions against the mosquito vectors Aedes aegypti and Anopheles stephensi. Knockdown efficacy for the Olyset net was assessed after the first 10 washings and then after the 15th and 20th washings. Data were not collected for the conventionally treated netting after 5 washings because the conventional treatment was no longer effective. The results of the study indicate permethrin effectively persisted on Olyset net for at least 20 washings, confirming the regeneration of pesticide after each wash.