Premendra D. Dwivedi

Emerging trends of nanoparticles application in food technology: Safety paradigms

Nanotechnology has expanded its wings in various spheres of life. It has progressed from first-generation passive nanomaterial to active nanotechnology (e.g., drug delivery) and nanosystems (e.g., robotics). Although nanofood is still in its infancy; however, these particles are now finding application as a carrier of antimicrobial polypeptides required against microbial deterioration of food quality in the food industry. Another challenging area is nanoencapsulation of pesticides that releases the pesticides within the stomach of the insect, thus minimizing contamination of crops and vegetables. The current nanotechnology applications in food science provide the detection of food pathogens, through nanosensors, which are quick, sensitive and less labour-intensive procedures. With the increasing health consciousness among consumers, it is possible to use nanosensors in plastic packaging to detect gases released due to food spoilage. However, it is well known that the nanoparticles equipped with new chemical and physical properties that vary from normal macro particles of the same composition may interact with the living systems thereby causing unexpected toxicity. Limited toxicological/safety assessments have been carried out for a few nanoparticles; hence studies relevant to oral exposure risk assessment are required for particles to be used in food.

Patulin causes DNA damage leading to cell cycle arrest and apoptosis through modulation of Bax, p53 and p21/WAF1 proteins in skin of mice

Patulin (PAT), a mycotoxin found in apples, grapes, oranges, pear and peaches, is a potent genotoxic compound. WHO has highlighted the need for the study of cutaneous toxicity of PAT as manual labour is employed during pre and post harvest stages, thereby causing direct exposure to skin. In the present study cutaneous toxicity of PAT was evaluated following topical application to Swiss Albino mice. Dermal exposure of PAT, to mice for 4 h resulted in a dose (40-160 mug/animal) and time (up to 6 h) dependent enhancement of ornithine decarboxylase (ODC), a marker enzyme of cell proliferation. The ODC activity was found to be normal after 12 and 24 h treatment of patulin. Topical application of PAT (160 mug/100 mul acetone) for 24-72 h caused (a) DNA damage in skin cells showing significant increase (34-63%) in olive tail moment, a parameter of Comet assay (b) significant G 1 and S-phase arrest along with induction of apoptosis (2.8-10 folds) as shown by annexin V and PI staining assay through flow cytometer. Moreover PAT leads to over expression of p(21/WAF1) (3.6-3.9 fold), pro apoptotic protein Bax (1.3-2.6) and tumor suppressor wild type p(53) (2.8-3.9 fold) protein. It was also shown that PAT induced apoptosis was mediated through mitochondrial intrinsic pathway as revealed through the release of cytochrome C protein in cytosol leading to enhancement of caspase-3 activity in skin cells of mice. These results suggest that PAT has a potential to induce DNA damage leading to p(53) mediated cell cycle arrest along with intrinsic pathway mediated apoptosis that may also be correlated with enhanced polyamine production as evident by induction of ODC activity, which may have dermal toxicological implications.

Interactive threats of nanoparticles to the biological system.

The use of nanoscale materials is growing exponentially, but concerns rise about the human hazards cannot be ignored. Nanotechnology has penetrated deep into our lives in diversified areas as engineering, information technology and diagnostics. Nonetheless owing to their peculiar properties these new materials also present new health risks upon interacting with biological systems. This is a typical case of technology preceding toxicity and therefore, various toxicological aspects for an array of nanomaterials are just beginning to be assessed. Several deleterious effects are being noticed, particularly in vitro situations as well as in mammalian system. Nanoparticles toxicity is compellingly related to oxidative stress, alteration of calcium homeostasis, gene expression, pro-inflammatory responses and cellular signalling events. It is therefore critical to understand the nature and origin of the toxicity imposed by nanomaterials. Keeping all these points in mind, the present review provides updated information on the various aspects such as sources of production, effect of different physical properties, interaction with biological system and mechanisms of engineered nanoparticles induced toxicities.

Citrinin-Generated Reactive Oxygen Species Cause Cell Cycle Arrest Leading to Apoptosis via the Intrinsic Mitochondrial Pathway in Mouse Skin

The mycotoxin, citrinin (CTN), is a contaminant of various food and feed materials. Several in vivo and in vitro studies have demonstrated that CTN has broad toxicity spectra; however, dermal toxicity is not known. In the present investigation, dermal exposure to CTN was undertaken to study oxidative stress, DNA damage, cell cycle arrest, and apoptosis in mouse skin. A single topical application of CTN caused significant change in oxidative stress markers, such as lipid peroxidation, protein carbonyl content, glutathione (GSH) content, and antioxidant enzymes in a dose-dependent (25-100 μg/mouse) and time-dependent (12-72 h) manner. Single topical application of CTN (50 μg/mouse) for 12-72 h caused significant enhancement in (1) reactive oxygen species (ROS); (2) cell cycle arrest at the G0/G1 phase (30-71%) and G2/M phase (56-65%) along with the induction of apoptosis (3.6-27%); (3) expression of p53, p21/waf1; (4) Bax/Bcl₂ ratio and cytochome c release; and (5) activities of caspase 9 (22-46%) and 3 (42-54%) as well as increased poly(ADP-ribose) polymerase cleavage. It was also observed that pretreatment with bio-antioxidants viz butylated hydroxyanisole (55 μmol/100 μl), quercetin (10 μmol/100 μl), or α-tocopherol (40 μmol/100 μl) resulted in decreases of ROS generation, arrest in the G0/G1 phase of the cell cycle, and apoptosis. These data confirm the involvement of ROS in apoptosis and suggest that these bio-antioxidants may be useful in the prevention of CTN-induced dermal toxicity.

In vitro studies on immunotoxic potential of Orange II in splenocytes.

Orange II, an azo dye, is not permitted in food preparations, but high levels of the dye have been detected in different food commodities. Though there are reports on the toxicity of Orange II but knowledge based on the immunomodulatory properties of Orange II is scanty. The present investigation was undertaken to study the in vitro immunotoxic potential of Orange II in splenocytes. Splenocytes were isolated, cultured and subjected to immunophenotypic analysis, mixed lymphocyte reaction (MLR) assay or stimulated with lipopolysaccharide (LPS) or concanavalin A (Con A) for 72 h. The supernatant was collected for cytokine assays. Orange II showed cytotoxic effects at 100-1000μg/ml concentrations and 50μg/ml was determined as the highest non-cytotoxic dose. Orange II at the non-cytotoxic dose (50μg/ml) significantly altered the relative distribution of T and B-cells, MLR response and the mitogen induced proliferative response of T-cells and B-cells. Consistent with the hypo-responsiveness of the T and B-lymphocytes, Orange II induced a concomitant decline in the secretion of cytokines IL-2, IL-4, IL-6, IFN-γ, TNF-α and IL-17. On the contrary, there was an increase in the production of IL-10, an anti-inflammatory regulatory cytokine, which may be one of the causative factor for immunosuppressive property of Orange II. These results suggest that non-cytotoxic dose of Orange II may have immunomodulatory effects.

Topical Application of Ochratoxin A Causes DNA Damage and Tumor Initiation in Mouse Skin

Skin cancer is one of the most common forms of cancer and 2–3 million new cases are being diagnosed globally each year. Along with UV rays, environmental pollutants/chemicals including mycotoxins, contaminants of various foods and feed stuffs, could be one of the aetiological factors of skin cancer. In the present study, we evaluated the DNA damaging potential and dermal carcinogenicity of a mycotoxin, ochratoxin A (OTA), with the rationale that dermal exposure to OTA in workers may occur during their involvement in pre and post harvest stages of agriculture. A single topical application of OTA (20–80 µg/mouse) resulted in significant DNA damage along with elevated γ-H2AX level in skin. Alteration in oxidative stress markers such as lipid peroxidation, protein carbonyl, glutathione content and antioxidant enzymes was observed in a dose (20–80 µg/mouse) and time-dependent (12–72 h) manner. The oxidative stress was further emphasized by the suppression of Nrf2 translocation to nucleus following a single topical application of OTA (80 µg/mouse) after 24 h. OTA (80 µg/mouse) application for 12–72 h caused significant enhancement in- (a) reactive oxygen species generation, (b) activation of ERK1/2, p38 and JNK MAPKs, (c) cell cycle arrest at G0/G1 phase (37–67%), (d) induction of apoptosis (2.0–11.0 fold), (e) expression of p53, p21/waf1, (f) Bax/Bcl-2 ratio, (g) cytochrome c level, (h) activities of caspase 9 (1.2–1.8 fold) and 3 (1.7–2.2 fold) as well as poly ADP ribose polymerase cleavage. In a two-stage mouse skin tumorigenesis protocol, it was observed that a single topical application of OTA (80 µg/mouse) followed by twice weekly application of 12-O-tetradecanoylphorbol-13-acetate for 24 week leads to tumor formation. These results suggest that OTA has skin tumor initiating property which may be related to oxidative stress, MAPKs signaling and DNA damage.

Are nanomaterials a threat to the immune system

Nanoparticles can enter into the human body through several routes and interact with components of the immune system. This interaction leads to enhanced release of different cytokines that include both proinflammatory and inflammatory cytokines as shown by several laboratories. The use of different types of materials may have different effects on the immune system; for instance, cobalt and nickel nanoparticles have inflammogenic effects while hydroxyapatite crystals stimulate TNFa secretion from macrophages which subsequently activate other phagocytes. Nanoparticles used in cosmetics and skin care products cause low systemic toxicity on skin. Carbon nanoparticles produce allergic symptoms, while fullerene is useful in mitigating allergy. Thus, nanoparticles have immunomodulatory potential as these can stimulate or suppress the immune system. However, both the conditions are undesirable and the successful nanoparticle-based therapeutics should avoid immunostimulatory or immunosuppressive reactions to the nanomaterials once administered into the body. The interaction of nanoparticles with the immune system alters its bioavailability and prolongs exposure time. Therefore, more data should be generated through in vivo studies.

Health Risks and Benefits of Chickpea (Cicer arietinum) Consumption

Chickpeas (CPs) are one of the most commonly consumed legumes, especially in the Mediterranean area as well as in the Western world. Being one of the most nutritional elements of the human diet, CP toxicity and allergy have raised health concerns. CPs may contain various antinutritional compounds, including protease inhibitors, phytic acid, lectins, oligosaccharides, and some phenolic compounds that may impair the utilization of the nutrients by people. Also, high consumption rates of CPs have enhanced the allergic problems in sensitive individuals as they contain many allergens. On the other hand, beneficial health aspects of CP consumption have received attention from researchers recently. Phytic acid, lectins, sterols, saponins, dietary fibers, resistant starch, oligosaccharides, unsaturated fatty acids, amylase inhibitors, and certain bioactive compounds such as carotenoids and isoflavones have shown the capability of lowering the clinical complications associated with various human diseases. The aim of this paper is to unravel the health risks as well as health-promoting aspects of CP consumption and to try to fill the gaps that currently exist. The present review also focuses on various prevention strategies to avoid health risks of CP consumption using simple but promising ways.

Bt Brinjal in India: A long way to go

Brinjal occupies the major proportion amongst all vegetable crops in India and is vulnerable to many diseases caused by insect-pests, fungus, bacteria and virus. Brinjal production is extensively affected by the insect brinjal fruit and shoot borer. Use of conventional chemical pesticides not only damage environment including the biotic and abiotic components but, also affect human health. Bt Brinjal was developed to combat brinjal fruit and shoot borer that has an advantage minimizing use of chemical pesticides. Extensive biosafety investigations, nutritional studies, substantial equivalence studies, relative toxicity and allergenicity assessment using animal models like Sprague Dawley rats, Brown Norway rats, rabbit, fish, chicken, goats, etc. revealed no significant differences between genetically modified brinjal and its native counterpart. Bt brinjal could effectively control the target pest and was found to be safe for environment and human health. In spite of all the scientific studies, release of Bt Brinjal has been put under moratorium. Indian government has constituted an expert committee to address this issue. In this review we have tried to explore the facts related to Bt Brinjal including its production, use of Bt toxin, use of chemical pesticides in controlling the FSB in native brinjal, along with perspective of public opinion and government initiatives. Key words: Bt Brinjal, agriculture, insecticides, GM foods, agrobacterium, transgenic crops

Computational allergenicity prediction of transgenic proteins expressed in genetically modified crops

Development of genetically modified (GM) crops is on increase to improve food quality, increase harvest yields, and reduce the dependency on chemical pesticides. Before their release in marketplace, they should be scrutinized for their safety. Several guidelines of different regulatory agencies like ILSI, WHO Codex, OECD, and so on for allergenicity evaluation of transgenics are available and sequence homology analysis is the first test to determine the allergenic potential of inserted proteins. Therefore, to test and validate, 312 allergenic, 100 non-allergenic, and 48 inserted proteins were assessed for sequence similarity using 8-mer, 80-mer, and full FASTA search. On performing sequence homology studies, ~94% the allergenic proteins gave exact matches for 8-mer and 80-mer homology. However, 20 allergenic proteins showed non-allergenic behavior. Out of 100 non-allergenic proteins, seven qualified as allergens. None of the inserted proteins demonstrated allergenic behavior. In order to improve the predictability, proteins showing anomalous behavior were tested by Algpred and ADFS separately. Use of Algpred and ADFS softwares reduced the tendency of false prediction to a great extent (74–78%). In conclusion, routine sequence homology needs to be coupled with some other bioinformatic method like ADFS/Algpred to reduce false allergenicity prediction of novel proteins.