Risk Governance of Emerging Technologies Demonstrated in Terms of its Applicability to Nanomaterials.

Nanotechnologies have reached maturity and market penetration that require nano-specific changes in legislation and harmonization among legislation domains, such as the amendments to REACH for nanomaterials (NMs) which came into force in 2020. Thus, an assessment of the components and regulatory boundaries of NMs risk governance is timely, alongside related methods and tools, as part of the global efforts to optimise nanosafety and integrate it into product design processes, via Safe(r)-by-Design (SbD) concepts. This paper provides an overview of the state-of-the-art regarding risk governance of NMs and lays out the theoretical basis for the development and implementation of an effective, trustworthy and transparent risk governance framework for NMs. The proposed framework enables continuous integration of the evolving state of the science, leverages best practice from contiguous disciplines and facilitates responsive re-thinking of nanosafety governance to meet future needs. To achieve and operationalise such framework, a science-based Risk Governance Council (RGC) for NMs is being developed. The framework will provide a toolkit for independent NMs' risk governance and integrates needs and views of stakeholders. An extension of this framework to relevant advanced materials and emerging technologies is also envisaged, in view of future foundations of risk research in Europe and globally.

Association of endothelial nitric oxide synthase (eNOS) and norepinephrine transporter (NET) genes polymorphism with type 2 diabetes mellitus.

Genetic factors in combination with environmental factors play a critical role in the development type 2 diabetes mellitus (T2DM) which is growing as an epidemic globally. In present study we aim to assess the association of eNOS (G894T, rs1799983) and NET (G1287A, rs5569) genes polymorphism with T2DM. A case-control study including a total of 400 North Indian subjects (200 T2DM cases and 200 controls) was performed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach to analyze genetic polymorphism. Alleles/genotype frequencies between cases and controls were compared using χ2 and Student's t-tests. Odds ratios and 95% confidence intervals were calculated by logistic regression to assess the relative association between disease and genotypes. In case of NET gene, GG (P = 0.002 in T2DM males, 0.053 in overall T2DM cases) genotype and G allele (P = 0.003 in T2DM males, 0.027 in overall T2DM cases) were found to be a positive risk factors and AG genotype (P = 0.012 in T2DM males) and A allele (P = 0.003 in T2DM males, P = 0.027 in overall T2DM cases) as negative risk factor for T2DM. No association of eNOS gene polymorphism was found with T2DM (P values of all genotypes and alleles were greater than 0.05). NET gene polymorphism might be associated with the risk of T2DM whereas; eNOS gene polymorphism do not confer any risk of T2DM in North Indian Ethnic group. It is hoped that understanding genetic causes of T2DM will lead to earlier diagnosis, preventive measures and more effective and specific treatment.

Impact of graphene-based nanomaterials (GBNMs) on the structural and functional conformations of hepcidin peptide.

Graphene-based nanomaterials (GBNMs) are widely used in various industrial and biomedical applications. GBNMs of different compositions, size and shapes are being introduced without thorough toxicity evaluation due to the unavailability of regulatory guidelines. Computational toxicity prediction methods are used by regulatory bodies to quickly assess health hazards caused by newer materials. Due to increasing demand of GBNMs in various size and functional groups in industrial and consumer based applications, rapid and reliable computational toxicity assessment methods are urgently needed. In the present work, we investigate the impact of graphene and graphene oxide nanomaterials on the structural conformations of small hepcidin peptide and compare the materials for their structural and conformational changes. Our molecular dynamics simulation studies revealed conformational changes in hepcidin due to its interaction with GBMNs, which results in a loss of its functional properties. Our results indicate that hepcidin peptide undergo severe structural deformations when superimposed on the graphene sheet in comparison to graphene oxide sheet. These observations suggest that graphene is more toxic than a graphene oxide nanosheet of similar area. Overall, this study indicates that computational methods based on structural deformation, using molecular dynamics (MD) simulations, can be used for the early evaluation of toxicity potential of novel nanomaterials.

Mutation in the beta3 subunit of Guanine nucleotide-binding protein (GNB3) gene is not associated with Type II diabetes mellitus risk: a case-control study of a North Indian population.

CONTEXT: Genetics play a major role in development and pathophysiology of Type 2 diabetes mellitus (T2DM). OBJECTIVE: To asses the association of Guanine nucleotide-binding protein (GNB3) (C825T) gene's polymorphism with T2DM. MATERIALS AND METHODS: A case-control study including 400 North Indians was performed using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) approach to analyze genetic polymorphism. RESULTS: No significant difference was observed in genotype and allele frequencies of GNB3 gene on comparing cases with controls. DISCUSSION: Our study is in agreement with studies on Polish, Japanese, Hispanic-American and Danish populations who observed no significant association between GNB3 (C825T) polymorphism and T2DM. CONCLUSION: GNB3 (C825T) polymorphism is not associated with T2DM.

Synthesis, functionalization and biological activity of arylated derivatives of (+)-estrone.

Various novel arylated estrone derivatives, such as 2-aryl-, 4-aryl- and 2,4-diaryl-estrones, by Suzuki-Miyaura reactions. While the synthesis of 4-arylestrones could be carried out under standard conditions, the synthesis of 2-arylestrones and 2,4-diarylestrones required a thorough optimization of the conditions and it proved to be important to use sterically encumbered biaryl ligands. The best results were obtained by the use of RuPhos. Combination of developed Suzuki coupling reactions with subsequent cyclization reactions afforded more complex hybrid structures, containing dibenzofuran, benzocoumarin and steroid moieties. These derivatives were tested as pancreatic lipase inhibitors and it was found that most of the compounds exhibited inhibition of pancreatic lipase but the maximum inhibitory potential was shown by 4-arylestrones. All of the synthesized derivatives showed inhibitory values in the range of 0.82±0.01-59.7±3.12µM. The biological activity was also rationalized on the bases of docking studies.

Synthesis of arylated coumarins by Suzuki-Miyaura cross-coupling. Reactions and anti-HIV activity.

Arylated coumarins were prepared by site-selective Suzuki-Miyaura cross-coupling reaction of the bis(triflate) of 4-methyl-6,7-dihydroxycoumarin. Triarylated coumarins were prepared by Suzuki-Miyaura cross-coupling reactions of 3-bromo-4-methyl-2-oxo-2H-chromene-6,7-diylbis(trifluoromethanesulfonate). The in vitro anti-HIV activity of the products was investigated. Two lead structures with considerable activities were identified.

Nanoparticles in the environment: assessment using the causal diagram approach.

Nanoparticles (NPs) cause concern for health and safety as their impact on the environment and humans is not known. Relatively few studies have investigated the toxicological and environmental effects of exposure to naturally occurring NPs (NNPs) and man-made or engineered NPs (ENPs) that are known to have a wide variety of effects once taken up into an organism. A review of recent knowledge (between 2000-2010) on NP sources, and their behaviour, exposure and effects on the environment and humans was performed. An integrated approach was used to comprise available scientific information within an interdisciplinary logical framework, to identify knowledge gaps and to describe environment and health linkages for NNPs and ENPs. The causal diagram has been developed as a method to handle the complexity of issues on NP safety, from their exposure to the effects on the environment and health. It gives an overview of available scientific information starting with common sources of NPs and their interactions with various environmental processes that may pose threats to both human health and the environment. Effects of NNPs on dust cloud formation and decrease in sunlight intensity were found to be important environmental changes with direct and indirect implication in various human health problems. NNPs and ENPs exposure and their accumulation in biological matrices such as microbiota, plants and humans may result in various adverse effects. The impact of some NPs on human health by ROS generation was found to be one of the major causes to develop various diseases. A proposed cause-effects diagram for NPs is designed considering both NNPs and ENPs. It represents a valuable information package and user-friendly tool for various stakeholders including students, researchers and policy makers, to better understand and communicate on issues related to NPs.

Do Carbon Nanotubes and Asbestos Fibers Exhibit Common Toxicity Mechanisms?

During the last two decades several nanoscale materials were engineered for industrial and medical applications. Among them carbon nanotubes (CNTs) are the most exploited nanomaterials with global production of around 1000 tons/year. Besides several commercial benefits of CNTs, the fiber-like structures and their bio-persistency in lung tissues raise serious concerns about the possible adverse human health effects resembling those of asbestos fibers. In this review, we present a comparative analysis between CNTs and asbestos fibers using the following four parameters: (1) fibrous needle-like shape, (2) bio-persistent nature, (3) high surface to volume ratio and (4) capacity to adsorb toxicants/pollutants on the surface. We also compare mechanisms underlying the toxicity caused by certain diameters and lengths of CNTs and asbestos fibers using downstream pathways associated with altered gene expression data from both asbestos and CNT exposure. Our results suggest that indeed certain types of CNTs are emulating asbestos fiber as far as associated toxicity is concerned.

Cyto-genotoxicity of amphibole asbestos fibers in cultured human lung epithelial cell line: role of surface iron.

The present investigations correlate the potentials of the reactive oxygen species (ROS) generation and the cyto-genotoxicity of amphibole asbestos fibers (amosite, crocidolite and tremolite) with their surface iron, under in vitro controlled conditions, using A549 cells (human lung epithelial cell line). The mobilizable surface iron was measured by Atomic Absorption Spectroscopy; the production of ROS was investigated using 2, 7 dichloro-dihydrofluorescein-diacetate (DCFH-DA) dye; for cytotoxicity assessment, the intracellular organelles specific damages were measured, using 3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide salt (MTT) assay; and, the genotoxic potential of amphibole fibers was determined by cytokinesis block micronucleus (CBMN) assay. In the study, highest amount of ROS was generated by crocidolite followed by tremolite and minimum with amosite. In MTT assay, the time- and concentration-dependent decrease in percent cell viability was recorded with all the three amphibole fibers, tremolite being most cytotoxic, followed by crocidolite, and then amosite. In genotoxicity assay, an increase in the frequency of micronuclei (MNi) in binucleated (BN) cells was observed, where crocidolite was most genotoxic, followed by tremolite, and amosite the least.The comparison of results depicts a clear trend of cyto-genotoxic potential paralleling the ROS generation, suggesting a definite role of oxidative stress in fiber-induced toxicity. However, amosite contains maximum surface iron (28%), followed by crocidolite (27%), and tremolite carrying least (as contaminant) or no iron, the mobilizable surface iron is maximum in crocidolite followed by amosite and is minimum in tremolite. The mobilizable iron somewhat corresponds with the ROS generation capacity of these fibers. This shows that the surface iron could be mainly responsible for amphibole asbestos-induced ROS toxicity; though it may not be the only factor responsible, other factors like shape and size etc., also play role in amphibole asbestos-induced toxicity.

Monitoring and identification of airborne asbestos in unorganized sectors, India.

Rajasthan state in India is credited to cater more than 90% of total production of asbestos in this country, of which around 60% is processed there in unorganized sectors including milling and manufacturing of asbestos-based products. Unorganized asbestos units particularly mills showed unhealthy occupational conditions, therefore industrial hygiene study was carried out focusing on the prevalence of asbestos fibres in air at work zone area of asbestos milling units. Fibre levels were in the range of 2.00-5.09f/cm(3) and 4.07-15.60f/cm(3) in unorganized asbestos mills of Rajasthan located at Beawer and Deogarh districts, respectively. Like asbestos concentration, fibre type and length are also vital factors in the health risk assessment of industrial workers. Phase contrast and polarized light microscopic study of asbestos fibres showed their amphibole nature registering about 90% as tremolite and rest as anthophyllite. Fibre length measured micrometrically were sub-grouped in <10microm, 11-20microm, 21-30microm and >30microm. About 30-40% fibres belonged to sub-group <10microm. It is concluded that unorganized asbestos mills bear poor industrial unhygienic conditions reflected specifically from their manyfold higher fibre concentrations than the Indian and International standards. Poor industrial unhygienic conditions are attributable to obsolete milling technology, lack of pollution control devices and escape from regulatory control.

Association of genetic variability in enzymes metabolizing chemotherapeutic agents with treatment response in head and neck cancer cases.

AIM: The prevalence of genetic variants of thiopurine S-methyltransferse (TPMT) and dihydropyrimidine dehydrogenase (DPD) in healthy controls (500) and the treatment response in 500 cases of head and neck cancer of north Indian origin was studied. METHODS: Blood collected from all the subjects was used for isolation of DNA followed by genotyping studies. The cases received cisplatin and 5-fluorouracil (5-FU) or chemo-radiotherapy and treatment response was measured using WHO criteria. RESULTS: Low frequency of heterozygous mutant genotypes of TPMT*2 (2%), TPMT*3B (2.2%), TPMT*3C (4.6%), DPD IVS14+1G>A (3.6%) and G1601A (3%) was observed, although no homozygous mutants could be identified. Treatment response studies in cases receiving cisplatin and 5-FU or chemo-radiotherapy revealed that the number of nonresponders was higher in cases who carried variant genotypes of TPMT*3B (62.50%) or TPMT*3C (59.26%) or DPD IVS14+1G>A (61.90%). Likewise, the number of nonresponders was still higher in cases carrying combination of these genetic variants. Furthermore, the frequency of nonresponders was higher in cases who carried the variant genotypes of TPMT or DPD and were also tobacco users. CONCLUSIONS: Our data clearly show that TPMT and DPD genes are polymorphic in the north Indian population and may be important in determining the treatment response in cases. The data have also suggest tobacco may play an important role in determining the outcome of cancer therapy and there is an urgent need for assessment of drugs for their efficacy/toxicity in smokers compared to nonsmokers.

Role of GNB3, NET, KCNJ11, TCF7L2 and GRL genes single nucleotide polymorphism in the risk prediction of type 2 diabetes mellitus.

Type 2 diabetes (T2DM) is a polygenic metabolic disorder characterized by hyperglycemia occurring as a result of impaired insulin secretion or insulin resistance. Various environmental and genetic factors interact and increase the risk of T2DM and its complications. Among the various genetic factors associated with T2DM, single nucleotide polymorphism in different candidate genes have been studied intensively and the resulting genetic variants have been found to have either positive or negative association with T2DM thereby increasing or decreasing the risk of T2DM, respectively. In this review, we will focus on Guanine nucleotide-binding protein subunit beta 3 (GNB3), Norepinephrine Transporter (NET), Potassium Channel gene (KCNJ11), Transcription Factor 7-Like 2 (TCF7L2) and Glucocorticoid receptor (GRL) genes and their association with T2DM studied in different ethnic groups. The products of these genes are involved in the biochemical pathway leading to T2DM. Polymorphisms in these genes have been intensively studied in individuals of different ethnic origins. Results show that genetic variants of TCF7L2 and KCNJ11 genes have potential to emerge as a risk biomarker for T2DM whereas results of GNB3, GRL and NET genes have been controversial when studied in individuals of different ethnicities. We have tried to summarize the results generated globally in context to the selected genes which could possibly help researchers working in this field and would eventually help in understanding the mechanistic pathways of T2DM leading early diagnosis and prevention.

Clinical Management of Head and Neck Cancer Cases: Role of Pharmacogenetics of CYP2 and GSTs.

Head and neck squamous cell carcinoma (HNSCC) describes a wide range of malignant tumors which originate in the upper aerodigestive tract and have a multifactorial origin involving both genetic and lifestyle risk factors. The clinical management of head and neck cancer involves surgery, radiotherapy, and chemotherapy. With the advances in treatment strategies for HNSCC, newer targeted therapies are adding to the progress already achieved in the multimodality management of patients although the problems of differences in drug response and adverse drug reactions are still grave concerns. Cancer pharmacogenomics has fast emerged as a new and promising field for the early identification of genetic markers that can predict drug response or toxicity. This could greatly help in identifying genetic markers useful for the selection of optimal drugs, dose, and treatment duration on an individual basis resulting in improved drug efficacy and decreased toxicity. This review focuses on the various treatment modalities available for the clinical management of HNSCC followed by a description of the contribution of genetic variations to chemotherapeutic toxicity and response. Furthermore, studies addressing the association of genetic variants of drug-metabolizing enzymes with treatment response in head and neck cancer are also discussed.

Mutational analysis of 9 different tumour-associated genes in human malignant mesothelioma cell lines.

Seven tumour suppressor genes (Chk1, Chk2, Apaf1, Rb1, p53, p16(INK4a) and p14(ARF)) and two oncogenes (N-ras and BRAF) were screened in nine human malignant melanoma (HMM) cell lines for point mutations or small deletions/insertions by DGGE, TGGE and SCCP analysis. For the first time in human mesothelioma, Chk1 gene mutations were detected in two of the nine investigated HMM cell lines. P53 gene mutations were found in three cell lines and p16(INK4a) mutations in 5. Mutation of the Chk1 gene implies a novel disruption mechanism of the p53 pathway in HMM, without affecting p53 itself. According to our knowledge, this is the first mutation screening of Chk1, Chk2, Apaf1 and Rb1 in human malignant mesothelioma.

Biomarkers in risk assessment of asbestos exposure.

Developments in the field of molecular epidemiology and toxicology have given valuable tools for early detection of impending disease or toxic condition. Morbidity due to respiratory distress, which may be due to environmental and occupational exposure, has drawn attention of researchers worldwide. Among the occupational exposure to respiratory distress factors, fibers and particles have been found to be main culprits in causing diseases like asbestosis, pleural plaques, mesotheliomas and bronchogenic carcinomas. An early detection of the magnitude of exposure or its' effect using molecular end points is of growing importance. The early inflammatory responses like release of the inflammatory cells collected by non-invasive methods give an indication of the unwanted exposure and susceptibility to further complications. Since free radicals like O2-, OH, OOH, NO, NOO, etc. are involved in the progression of asbestos-related diseases and lead to cytogenetic changes, an evaluation of antioxidant states reducing equivalents like GSH and ROS generation can be a good biomarker. The cytogenetic end points like chromosomal aberration, micronucleus formation and sister chromatid exchange give indication of genetic damage, hence they are used as effective biomarkers. New techniques like fluorimetric analysis of DNA unwinding, alkaline elution test, fluorescent in situ hybridization and comet assay are powerful tools for early detection of initiation of disease process and may help in planning strategies for minimizing morbidity related to asbestos fiber exposure. The present review article covers in detail possible biomarkers for risk assessment of morbidity due to fibers/particles in exposed population.

ROS-mediated genotoxicity of asbestos-cement in mammalian lung cells in vitro.

Asbestos is a known carcinogen and co-carcinogen. It is a persisting risk in our daily life due to its use in building material as asbestos-cement powder. The present study done on V79-cells (Chinese hamster lung cells) demonstrates the cytotoxic and genotoxic potential of asbestos-cement powder (ACP) in comparison with chrysotile asbestos. A co-exposure of chrysotile and ACP was tested using the cell viability test and the micronucleus assay. The kinetochore analysis had been used to analyse the pathway causing such genotoxic effects. Thiobarbituric acid-reactive substances were determined as evidence for the production of reactive oxygen species. Both, asbestos cement as well as chrysotile formed micronuclei and induced loss of cell viability in a concentration- and time-dependent way. Results of TBARS analysis and iron chelator experiments showed induction of free radicals in ACP- and chrysotile exposed cultures. CaSO4 appeared to be a negligible entity in enhancing the toxic potential of ACP. The co-exposure of both, ACP and chrysotile, showed an additive effect in enhancing the toxicity. The overall study suggests that asbestos-cement is cytotoxic as well as genotoxic in vitro. In comparison to chrysotile the magnitude of the toxicity was less, but co-exposure increased the toxicity of both.

Multiple roles of oxidants in the pathogenesis of asbestos-induced diseases.

Exposure to asbestos causes cellular damage, leading to asbestosis, bronchogenic carcinoma, and mesothelioma in humans. The pathogenesis of asbestos-related diseases is complicated and still poorly understood. Studies on animal models and cell cultures have indicated that asbestos fibers generate reactive oxygen and nitrogen species (ROS/RNS) and cause oxidation and/or nitrosylation of proteins and DNA. The ionic state of iron and its ability to be mobilized determine the oxidant-inducing potential of pathogenic iron-containing asbestos types. In addition to their capacity to damage macromolecules, oxidants play important roles in the initiation of numerous signal transduction pathways that are linked to apoptosis, inflammation, and proliferation. There is strong evidence supporting the premise that oxidants contribute to asbestos-induced lung injury; thus, strategies for reducing oxidant stress to pulmonary cells may attenuate the deleterious effects of asbestos.

Evidence that ultrafine titanium dioxide induces micronuclei and apoptosis in Syrian hamster embryo fibroblasts.

Inhaled ultrafine titanium dioxide (UF-TiO2) particles cause pronounced pulmonary inflammation, in contrast to fine TiO2. Previous studies provide evidence for the production of reactive oxygen species by alveolar macrophages, after overloading with UF-TiO2 particles and cytotoxicity of UF-TiO2 in rat lung alveolar macrophages. UF-TiO2 also causes pulmonary fibrosis and lung tumors in rats. UF-TiO2 particles are photogenotoxic, but in general, information on the genotoxicity of UF-TiO2 is still limited. We studied the potential of UF-TiO2 (particle size less than or equal to 20 nm) and fine TiO2 (particle size > 200 nm) to induce chromosomal changes, which can be monitored by the formation of micronuclei (MN) in Syrian hamster embryo (SHE) cells. We also analyzed UF-TiO2-treated cells for apoptosis induction. The MN assay revealed a significant increase in MN induction (p less than or equal to 0.05) in SHE cells after treatment with UF-TiO2 (1.0 micro g/cm2) for 12 hr (mean, 24.5 MN/1,000 cells), 24 hr (mean, 31.13 MN/1,000 cells), 48 hr (mean, 30.8 MN/1,000 cells), 66 hr (mean, 31.2 MN/1,000 cells), and 72 hr (mean, 31.3 MN/1,000 cells). Bisbenzimide staining of the fixed cells revealed typical apoptotic structures (apoptotic bodies), and the apoptosis-specific "DNA ladder pattern" resulting from internucleosomal cleavage was identified by gel electrophoresis. Furthermore, transmission electron microscopy of the exposed cells revealed the typical chromatin compaction of apoptosis.

Mutational analysis of the nf2 tumour suppressor gene in three subtypes of primary human malignant mesotheliomas.

Fourteen primary human malignant mesothelioma (HMM) samples obtained from 14 patients were screened for point mutations and microdeletions/microinsertions in exons 1-16 of the chromosome 22q-located tumour suppressor gene neurofibromin 2 (nf2) by single strand conformation polymorphism (SSCP) analysis. In one tumour (7%) a 10 basepair microdeletion of exon 10 was detected by SSCP and subsequently characterised in detail by sequencing. Deletion of the second nf2 allele in laser-microdissected regions of the 10 bp mutation-harbouring tumour was demonstrated by denaturing gradient gel electrophoresis (DGGE) analysis. Simultaneous comparative genomic hybridisation (CGH) analysis also showed losses at chromosome 22q. Our data indicate that functional loss of the NF2 protein may be involved in the formation of a subset of HMMs.

Cytogenetic biomonitoring of Indian women cooking with biofuels: micronucleus and chromosomal aberration tests in peripheral blood lymphocytes.

India currently has the largest number of indoor air pollution-related health problems in the world, with three-quarters of its households burning wood, cowdung, or crop residues ("traditional" biomass fuels) for cooking, and the remainder using kerosene and relatively clean-burning liquefied petroleum gas (LPG). Combustion of these fuels produces various pollutants that may cause serious health effects in exposed populations. In this study, the micronucleus (MN) and chromosomal aberration (CA) assays were used to evaluate the relative amounts of DNA damage produced by the use of these cooking fuels. Cytogenetic evaluation of 179 female subjects showed a significant increase in both MN and CA frequency in blood lymphocytes from users of biomass-fuels in comparison to lymphocytes from LPG users (used as a reference population). The relative MN and CA frequencies for the users of the various fuels decreased in the order cowdung > cowdung/wood >/= wood > kerosene >/= LPG. Further, the results indicated an effect of subject age, and the duration of exposure on the MN and CA frequencies in biomass fuel users. Age had no significant effects on the genotoxicity responses in subjects with 10 years, CA and MN frequencies were higher in older individuals (>30 years of age) than younger subjects. Regardless of age, subjects burning biomass fuels had higher MN and CA frequencies than LPG users only when exposures were of at least 5 years duration. These results indicate that burning biomass-based fuels increases the frequency of cytogenetic alterations in blood lymphocytes of exposed populations, possibly because of exposure to the various noxious gases and toxic substances present in biomass fuels. These cytogenetic markers could be used in the field to assess the genotoxic consequences of burning various cooking fuels and for early detection of genetic abnormalities in people exposed to various pollutants and toxicants.