Maumita Bandyopadhyay

Genotoxicity of titanium dioxide (TiO2) nanoparticles at two trophic levels: Plant and human lymphocytes

The environmental fate and behaviour of titanium dioxide (TiO(2)) nanoparticles is a rapidly expanding area of research. There is a paucity of information regarding toxic effect of TiO(2) nanoparticles in plants and to certain extent in humans. The present study focuses on the effect of exposure of TiO(2) nanoparticles in two trophic levels, plant and human lymphocytes. The genotoxicity of TiO(2) nanoparticles was evaluated using classical genotoxic endpoints, comet assay and DNA laddering technique. DNA damaging potential of TiO(2) nanoparticles in Allium cepa and Nicotiana tabacum as representative of plant system could be confirmed in the comet assay and DNA laddering experiments. In Allium micronuclei and chromosomal aberrations correlated with the reduction in root growth. We detected increased level of malondialdehyde (MDA) concentration at 4mM (0.9 μM) treatment dose of TiO(2) nanoparticles in Allium cepa. This indicated that lipid peroxidation could be involved as one of the mechanism leading to DNA damage. A comparative study of the cytotoxic and genotoxic potential of TiO(2) nanoparticles and bulk TiO(2) particles in human lymphocytes also reveal interesting results. While TiO(2) nanoparticles were found to be genotoxic at a low dose of 0.25 mM followed by a decrease in extent of DNA damage at higher concentrations; bulk TiO(2) particles reveal a more or less dose dependent effect, genotoxic only at dose 1.25 mM and above. The study thus confirms the genotoxic potential of TiO(2) nanoparticles in both plant and human lymphocytes.

In vitro and in vivo genotoxicity of silver nanoparticles

The biocidal effect of silver nanoparticles (Ag-np) has resulted in their incorporation into consumer products. While the population exposed to Ag-np continues to increase with ever new applications, Ag-np remains a controversial research area with regard to their toxicity in biological systems. Here a genotoxic and cytotoxic approach was employed to elucidate the activity of Ag-np in vitro and in vivo. Characterization of Ag-np using scanning electron microscopy revealed a size range of 90-180nm. Cytotoxic potential of Ag-np was evaluated in human lymphocytes via cell viability assay (Trypan blue dye exclusion method, MTT and WST assay). The uptake and incorporation of Ag-np into the lymphocytes was confirmed by flow cytometry. Additionally apoptosis (AnnexinV-FITC-PI staining) and DNA strand breaks (comet assay) in human lymphocytes revealed that Ag-np at concentration 25μg/ml can cause genotoxicity. In vivo experiments on plants (Allium cepa and Nicotiana tabacum) and animal (Swiss albino male mice) showed impairment of nuclear DNA. Induction of oxidative stress was also studied. The DNA damage and chromosomal aberrations raise the concern about the safety associated with applications of the Ag-np. A single ip administration of Ag-np gave a significant (P≤0.05) increase in the frequency of aberrant cells and Tail DNA percent at concentrations 10mg/kg body weight and above. Results of comet assay in A. cepa and N. tabacum demonstrated that the genotoxic effect of Ag-np was more pronounced in root than shoot/leaf of the plants. The present study indicated a good correlation between the in vitro and in vivo experiments. Therefore the biological applications employing Ag-np should be given special attention besides adapting the antimicrobial potential.

Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of Withania somnifera

Developmental variability was introduced into Withania somnifera using genetic transformation by Agrobacterium rhizogenes, with the aim of changing withasteroid production. Inoculation of W. somnifera with A. rhizogenes strains LBA 9402 and A4 produced typical transformed root lines, transformed callus lines, and rooty callus lines with simultaneous root dedifferentiation and redifferentiation. These morphologically distinct transformed lines varied in T-DNA content, growth rates, and withasteroid accumulation. All of the lines with the typical transformed root morphology contained the TL T-DNA, and 90% of them carried the TR T-DNA, irrespective of the strain used for infection. Accumulation of withaferin A was maximum (0.44% dry weight) in the transformed root line WSKHRL-1. This is the first detection of withaferin A in the roots of W. somnifera. All of the rooty callus lines induced by strain A4 contained both the TL and the TR-DNAs. In contrast, 50% of the rooty-callus lines obtained with strain LBA 9402 contained only the TR T-DNA. All the rooty callus lines accumulated both withaferin A and withanolide D. The callusing lines induced by LBA 9402 lacked the TL T-DNA genes, while all the callusing lines induced by strain A4 contained the TL DNA. Four of these callus lines produced both withaferin A (0.15–0.21% dry weight) and withanolide D (0.08–0.11% dry weight), and they grew faster than the transformed root lines. This is the first report of the presence of withasteroids in undifferentiated callus cultures of W. somnifera.

Multi-walled carbon nanotubes (MWCNT): induction of DNA damage in plant and mammalian cells.

Increasing use of multiwalled carbon nanotubes (MWCNT) necessitates an improved understanding of their potential impact on environment health. In the present study we evaluated the genotoxicity of MWCNT on plant and mammalian test systems. Genotoxic responses such as chromosomal aberrations and DNA strand breakages were studied in Allium cepa, human lymphocytes, mouse bone marrow cells and pBR322 plasmid DNA. Results showed that MWCNT could cause chromosomal aberrations, DNA fragmentation and apoptosis in Allium root cells that could be correlated with the internalization of MWCNT in the plant cells. In human lymphocytes significant genotoxic response was observed at the concentration 2 μg/ml. Higher concentrations led to a decrease in values of the tail DNA percent that may be due to the formation of crosslinks. Annexin V-FITC-PI staining indicated only a small percentage of cells were undergoing apoptosis. Genotoxic effects were shown by micronuclei (MN) frequencies in experiments on mouse bone marrow cells. In the cell free DNA system (plasmid pBR322), a strong correlation between DNA strand break and concentration was observed. Based on the findings of the present study MWCNT may have significant impact on genomic activities.

MWCNT uptake in Allium cepa root cells induces cytotoxic and genotoxic responses and results in DNA hyper-methylation.

Advances in nanotechnology have led to the large-scale production of nanoparticles, which, in turn, increases the chances of environmental exposure. While humans (consumers/workers) are primarily at risk of being exposed to the adverse effect of nanoparticles, the effect on plants and other components of the environment cannot be ignored. The present work investigates the cytotoxic, genotoxic, and epigenetic (DNA methylation) effect of MWCNT on the plant system- Allium cepa. MWCNT uptake in root cells significantly altered cellular morphology. Membrane integrity and mitochondrial function were also compromised. The nanotubes induced significant DNA damage, micronucleus formation and chromosome aberration. DNA laddering assay revealed the formation of internucleosomal fragments, which is indicative of apoptotic cell death. This finding was confirmed by an accumulation of cells in the sub-G0 phase of the cell cycle. An increase in CpG methylation was observed using the isoschizomers MspI/HpaII. HPLC analysis of DNA samples revealed a significant increase in the levels of 5-methyl-deoxy-cytidine (5mdC). These results confirm the cyto-genotoxic effect of MWCNT in the plant system and simultaneously highlight the importance of this epigenetic study in nanoparticle toxicity.

Biotechnological approaches for the production of forskolin, withanolides, colchicine and tylophorine

Plantsarecapableofsynthesizingavarietyoflow-molecu-lar-weight organic compounds, called secondary metab-olites, usually with unique and complex structures. Manyof these compounds are differentially distributed amonglimited taxonomic groups within the plant kingdom and,conversely, each plant species has a distinct profile ofsecondary metabolites. Plant secondary metabolites areof tremendous importance, both for the plant itself (forplant–environment interactions) and to humans, for theirbiological activities that can have therapeutic value. Com-paredtothemainmoleculesfoundintheplants,thesesec-ondary metabolites were soon defined by their lowabundance, often less than 1% of the total carbon, or sto-rage, usually occurring in dedicated cells or organs.Plants are probably the best cell factories on this earthfrom which more than 100000 secondary metaboliteshave been discovered, with the estimated total number inplants exceeding 500000 (Hadacek, 2002). Due to theirwide biological activities, plant secondary metaboliteshave been used for centuries in traditional medicine.Nowadays, they correspond to valuable compounds suchas pharmaceuticals, cosmetics, fine chemicals or morerecently, nutraceutics (Bourgaud et al., 2001).Medicinal plants are the world’s oldest health-care pro-ducts. Plant species are used as medicinal products in twoways; traditional medicines, singly or in formulations,such as those prepared and dispensed bytraditional medical practitioners, which may or may notattract a market price; and commercial products, dis-pensed by prescription or over-the-counter sales, suchas patented or licensed medical products of allopathy ortraditional systems of medicine. The world market formedicinal and aromatic plants (MAPs) is huge. The largestglobal markets for MAPs are China, France, Germany,Italy, Japan, Spain, the UK and the USA. Japan has thehighest per capita consumption of botanical medicinesin the world (Laird, 1999). The International Council forMedicinal and Aromatic Plants expects world growthduring 2001 and 2002 to be approximately 8–10% a year(Srivastava, 2000). India is a major exporter of raw MAPsand processed plant-based drugs (Lambert et al., 1997);75% of the total exports from India are sent to sixcountries, namely France, Germany, Japan, Switzerland,theUKandtheUSA.India’s boomingexporttradein med-icinal plants has risen almost three-fold during the lastdecade. This boom in local use and export trade is deplet-ingmanyspeciesfromthewild,bringingsometotheedgeofextinction.Themajorprobleminconventionalprocure-ment ofMAPsis thatonlyafewarecultivated; over95%ofthe medicinal plants used in India are collected from thewild. Data on threatened species are rare but all overthe world, some species are becoming difficult to obtainin sufficient amounts to meet increasing demands.Destruction of natural habitats and technical difficultiesin cultivation are also driving the drastic reductions inplant availability.In the search for alternatives to production of desirablemedicinal compounds from plants, biotechnological

Comparative karyomorphological studies of three edible locally important species of Allium from India

Karyomorphological studies of three locally important species of Allium, namely, A. tuberosum, A. chinense and A. schoenoprasum, were performed to highlight the chromosomal variations between them, despite the fact that all of them are known as “chives” in commerce, due to their overall morphological similarities. Only critical morphological analyses reveal several important phenotypic dissimilarities between the three species. Karyomorphological investigations show that A. tuberosum and A. chinense are both 2n = 32, and A. schoenoprasum is 2n = 16. A. chinense has maximum karyotype symmetry among the three, and most of its chromosomes are of either median or submedian types. The karyotype of A. tuberosum is characterized by the presence of different types of secondary constrictions, while A. schoenoprasum, apart from showing the maximum karyotype asymmetry among the three, also reveals the presence of at least one pair of acrocentric chromosomes.

Engineered Nickel Oxide Nanoparticle Causes Substantial Physicochemical Perturbation in Plants

Concentration of engineered nickel oxide nanoparticle (NiO-NP) in nature is on the rise, owing to large scale industrial uses, which have accreted the scope of its exposure to plants, the primary producers of the ecosystem. Though an essential micronutrient for the animal system, supported by numerous studies confirming its toxicity at higher dosages, nickel oxide is graded as a human carcinogen by WHO. A few studies do depict toxicity and bioaccumulation of nickel in plants; however, interaction of NiO-NP with plants is not well-elucidated. It is known that exposure to NiO-NP can incite stress response, leading to cytotoxicity and growth retardation in some plants, but a defined work on the intricate physicochemical cellular responses and genotoxic challenges is wanting. The present study was planned to explore cytotoxicity of NiO-NP in the model plant, Allium cepa L., its internalization in the tissue and concomitant furore created in the antioxidant enzyme system of the plant. The prospect of the NiO-NP causing genotoxicity was also investigated. Detailed assessments biochemical profiles and genotoxicity potential of NiO-NP on A. cepa L. was performed and extended to four of its closest economically important relatives, Allium sativum L., Allium schoenoprasum L., Allium porrum L., and Allium fistulosum L. Growing root tips were treated with seven different concentrations of NiO-NP suspension (10–500 mg L−1), with deionised distilled water as negative control and 0.4 mM EMS solution as positive control. Study of genotoxic endpoints, like, mitotic indices (MI), chromosomal aberrations (CAs), and chromosome breaks confirmed NiO-NP induced genotoxicity in plants, even at a very low dose (10 mg L−1). That NiO-NP also perturbs biochemical homeostasis, disrupting normal physiology of the cell, was confirmed through changes in state of lipid peroxidation malonaldehyde (MDA), as well as, in oxidation marker enzymes, like catalase (CAT), super oxide dismutase (SOD), and guiacol peroxidase (POD) activities. It was evident that increase in NiO-NP concentration led to decrease in MIs in all the study materials, concomitant with a spike of stress-alleviating, antioxidant enzymes-CAT, POD, SOD, and significant increase in MDA formation. Hence, it can be confirmed that NiO-NP should be treated as an environmental hazard.

New chromosome number counts and karyotype analyses in three important genera of Zingiberaceae

Zingiberaceae has reportedly undergone frequent polyploidization and hybridization events. Here we describe the new chromosome counts and karyomorphological details for the three important members of the family. Root tip cells of plants of Alpinia zerumbet (earlier reported to be 2n = 48, 40) and Globba marantina (reportedly 2n = 44 to 2n = 96) showed 2n = 52 chromosomes, both being aneuploids, Hedychium spicatum plants (2n = 34 according to previous reports) were found to be tetraploid with somatic chromosome number of 2n = 68. Detailed karyomorphological analyses performed using various parameters revealed that all the three novel cytotypes had moderately symmetrical karyotypes with low levels of intra– and interchromosomal variation in terms of both chromosome lengths and centromeric indices.

Karyomorphological investigations on some economically important members of Zingiberaceae from Eastern India

In this paper, karyological investigations of four economically and medicinally important Indian species of Zingiberaceae belonging to the genera Curcuma and Zingiber were performed. The somatic chromosome number of Curcuma amada was 2n = 42 and that of Curcuma longa was 2n = 63, while the chromosome numbers of both Zingiber officinale and Zingiber zerumbet were 2n = 22. Chromosome morphology of the two species of Curcuma studied showed predominance of constrictions in the median region and absence of secondary constriction. In contrast, the two species of Zingiber studied showed a wide variation of constrictions from median to subterminal and six chromosomes with secondary constrictions. The results indicated presence of more symmetric karyotype in Curcuma spp. with respect to that of the Zingiber spp. studied. Detailed karyomorphological studies were undertaken and the results obtained were analyzed with respect to the published karyotype data for the four species.