Genotoxicity of five food preservatives tested on root tips of Allium cepa L.

The effects of the food preservatives sodium benzoate (SB), boric acid (BA), citric acid (CA), potassium citrate (PC) and sodium citrate (SC) have been studied on root tips of Allium cepa L. Roots of A. cepa were treated with a series of concentrations, ranging from 20 to 100 ppm for 5, 10 and 20 h. The results indicate that these food preservatives reduced mitotic division in A. cepa compared with the respective control. Mitotic index values were generally decreased with increasing concentrations and longer treatment times. Additionally, variations in the percentage of mitotic stages were observed. The total percentage of aberrations generally increased with increasing concentrations of these chemicals and the longer period of treatment. Different abnormal mitotic figures were observed in all mitotic phases. Among these abnormalities were anaphase bridges, C-mitosis, micronuclei, lagging, stickiness, breaks and unequal distiribution.     

The in vivo genotoxic effects of sodium metabisulphite in bone marrow cells of rats

This study is designed to investigate the genotoxic effect of sodium metabisulphite (SMB), which is used as an antimicrobial substance in foods on bone marrow cells of rats. Four different concentrations of SMB (250, 500, 750 and 1000 mg/kg body weight) were given rats (Rattus norvegicus var. albinos) for 6, 12 and 24 hours treatment period by intraperitoneal (IP) and gavage (GV) administrations. In this study, we found that intraperitoneal implement of SMB generally more effective increasing the percentage of abnormal cells and CA/cell in all concentrations and treatment period. In addition, mitotic index (MI) data of intraperitoneal injection are lower than gavage. It can be concluded that potential genotoxic effects of SMB by IP injection is higher than GV injection.     

Evaluating cadmium toxicity in the root meristem of <Emphasis Type="Italic">Pisum</Emphasis><Emphasis Type="Italic">sativum</Emphasis> L.

The effect of cadmium (Cd) was studied on root tips of Pisum sativum L. Seeds of P. sativum were treated with a series of concentrations ranging from 0.125, 0.250, 0.500 and 1.000 mM for 6 h. The effect of Cd was analyzed by studying the percentage seed germination, radicle length (RL), mitotic index (MI) and chromosomal aberrations (CAs) in root tip. The results revealed that Cd had significant impeding effect on the root meristem activity of P. sativum at 0.500 and 1.000 mM as noticed by reduction in seed germination percentage and RL compared to control. Furthermore, it also reduced MI in dose-related manner compared to control. Additionally, the variation in the percentage of mitotic abnormalities was observed. The overall percentage of aberrations generally increased with increasing concentrations of Cd. Among these abnormalities laggards, bridges, stickiness, precocious separation and fragments were most common. The obtained results demonstrated that the Cd treatment leads to a significant reduction in MI and increase in CAs. Overall results allow us to suggest that the Cd has clastogenic effect on the crop.     

Salicylic acid-induced aluminum tolerance by modulation of citrate efflux from roots of<Emphasis Type="Italic"> Cassia tora</Emphasis> L.

Aluminum-induced exudation of organic acids from roots has been proposed as a mechanism for Al tolerance in plants. To better understand the regulatory process leading to efflux of organic acids, the possible involvement of salicylic acid (SA) in regulating Al-induced citrate release in Cassia tora L. was identified. The response of citrate efflux to exogenous SA was concentration-dependent. Application of SA at 5 microM in solution containing 20 microM Al increased citrate efflux to levels 1.76-fold higher than in controls (20 microM Al alone). However, inhibition of citrate release was observed when SA concentrations increased to more than 20 microM. Increased citrate efflux due to the SA treatment was associated with decreased inhibition of root growth and Al content in root tips, suggesting that exogenous SA could confer Al tolerance by increasing citrate efflux. We also examined citrate synthase activities (EC 4.1.3.7) and citrate concentrations in root tips exposed to Al and/or SA. However, both citrate synthase activities and citrate accumulation remained unaffected. These results indicate that SA-promotion of Al-induced citrate efflux is not correlated with increase in citrate production. Total endogenous SA concentrations were measured in root tips and the SA concentrations were significantly enhanced by Al at levels of 10-50 microM.     

Comparison of anti-Vibrio activities of potassium sorbate, sodium benzoate, and glycerol and sucrose esters of fatty acids.

The effects of fatty acids and their glycerol and sucrose esters, potassium sorbate, and sodium benzoate on growth of Vibrio parahaemolyticus in laboratory media at pH 6.7 were evaluated. The minimum concentrations at which inhibition by esters of glycerol could be detected were lowest for monolaurin (5 microgram/ml) and monocaprin (40 microgram/ml); these concentrations were lower than those observed for inhibition by lauric and capric acids, respectively. Inhibitory action of sucrose caprylate was detected at 40 microgram/ml, whereas sucrose caprate was effective at 100 microgram/ml; sucrose esters of lauric, myristic, and palmitic acids were ineffective at 100 microgram/ml. Potassium sorbate and sodium benzoate inhibited growth at concentrations as low as 30 and 300 microgram/ml, respectively, and enhanced the rate of thermal inactivation of V. parahaemolyticus at slightly higher concentrations. Fatty acid esters of glycerol and sucrose offer potential as perservatives for slightly acid or alkaline low-fat foods which do not lend themselves to the full antimicrobial action of traditional food preservatives such as potassium sorbate and sodium benzoate.     

Effects of certain food dyes on chromosomes of Allium cepa

The effects of 4 permitted food dyes, i.e., fast green FCF, indigo carmine, orange G and tartrazine, and the non-permitted dye metanil yellow on chromosomes of Allium cepa are reported. A significant increase in polyploid cells was observed in all cases. High doses of these dyes induced chromosome breaks and micronucleus formation. Although all dyes produced mitotic aberrations, metanil yellow and fast green FCF showed comparatively stronger clastogenic activity.     

The effects of fungicide benomyl (benlate) on growth and mitosis in onion (Allium cepa L.) root apical meristem

In this study, the effects of benomyl, a systemic fungicide were investigated in the mitotic cell division in onion (Allium cepa) root tip cells during germination. For this aim, different concentrations (1, 2, 5, 10 and 20 mM) of benomyl solutions were used. All the concentrations used caused several abnormalities in mitotic cell divisions and the mitotic frequency in the onion root tip cells decreased as the concentration of benomyl solution increased. Based on our findings, it is reported that benomyl has some negative effects on mitotic divisions in onion root tip cells.     

Cytological studies on onion root-tip cells treated with water-soluble extract of tobacco smoke condensate from commercial cigarettes.

The effect of water-soluble extract of tobacco smoke condensate (TSC) from two commercially available cigarettes with dirrerent types of filters was studied on the cytology of root-tip cells of onion (Allium cepa). One of the cigarettes had a 2-cm cellulose acetate filter, and the other had a filter comprised of 1 cm of cellulose acetate and 2 cm of activated charcoal. TSC from these cigarettes induced mitotic abnormalities. To investigate whether these two commercial filters could retain cigarette smoke component (s) responsible for mitotic irregularities, the cigarettes were defiltered, and TSC was prepared and tested on the young roots of onion. Observations revealed that the cytological effect of TSC from defiltered cigarettes was not significantly different from the effect of TSC from cigarettes with filters. Thus, the filters utilized in these cigarettes do not retain compound(s) responsible for mitotic irregularities in the root-tip cells of onion. With increasing concentrations (0.01% ot 0.1%) of TSC from cigarettes with filters and defiltered, precent mitotic abnormalities increased. These abnormalities included scattering, stickiness, lagging, condensation, and breaking of chromosomes during metaphase. Bridging and lagging of chromosomes were observed during anaphase.     

Recovery of Viability and Radiation Resistance by Heat-Injured Conidia of Penicillium expansum Lk. ex Thom

Spores heated in water at 54 C for up to 1 hr were plated on nutrient agar immediately or held for 3 days in aerated water at 23 C and then plated. Under these conditions, holding was optimal for recovery, increasing survival percentage up to 20-fold over values for immediate plating. Recovery was prevented partially or completely, however, when spores were held in any of the following solutions: glucose, potassium phosphate, ammonium or sodium acetate, sodium azide, or 2,4-dinitrophenol, or in the sodium or potassium salts of pyruvate, and tricarboxylic acid cycle acids. Both anaerobiosis and incubation at 0 C prevented recovery. Survivors of a heat treatment were more sensitive to gamma radiation than were unheated spores. Conditions which affected the recovery of viability had the same effect on restoration of radiation resistance. Thus, many of the processes for restoration of radiation resistance seem involved also in recovery of viability after heating. After a 99% inactivating treatment (about 30 min at 54 C), heated spores respired as fast as unheated spores, or faster. Malate, citrate, succinate, and acetate stimulated respiration in unheated spores and inhibited it in heated spores.     

Cytotoxic and genotoxic effects of Br-containing oxaphosphole on Allium cepa L. root tip cells and mouse bone marrow cells

The continuous production and release of chemicals into the environment has led to the need to assess their genotoxicity. Numerous organophosphorus compounds with different structures have been synthesized in recent years, and several oxaphosphole derivatives are known to possess biological activity. Such chemical compounds may influence proliferating cells and cause disturbances of the genetic material. In this study, we examined the cytotoxicity and genotoxicity of 4-bromo-N,N-diethyl-5,5-dimethyl-2,5-dihydro-1,2-oxaphosphol-2-amine 2-oxide (Br-oxph). In A. cepa cells, Br-oxph (10(-9) M, 10 (-6) M and 10 (-3) M) reduced the mitotic index 48 h after treatment with the two highest concentrations, with no significant effect at earlier intervals. Mitotic cells showed abnormalities 24 h and 48 h after treatment with the two lowest concentrations but there were no consistent changes in interphase cells. Bone marrow cells from mice treated with Br-oxph (2.82 x 10 (-3) μg/kg) also showed a reduced mitotic index after 48 h and a greater percentage of cells with aberrations (principally chromatid and isochromatid breaks). These findings indicate the cytotoxicity and genotoxicity of Br-oxph in the two systems studied.     

Differential modulation of citrate synthesis and release by fatty acids in perfused working rat hearts

The objective of this study was to test the effect of increasing fatty acid concentrations on substrate fluxes through pathways leading to citrate synthesis and release in the heart. This was accomplished using semirecirculating work-performing rat hearts perfused with substrate mixtures mimicking the in situ milieu (5.5 mM glucose, 8 nM insulin, 1 mM lactate, 0.2 mM pyruvate, and 0.4 mM oleate-albumin) and 13C methods. Raising the fatty acid concentration from 0.4 to 1 mM with long-chain oleate or medium-chain octanoate resulted in a lowering ( approximately 20%) of cardiac output and efficiency with unaltered O2 consumption. At the metabolic level, beyond the expected effects of high fatty acid levels on the contribution of pyruvate decarboxylation (reduced >3-fold) and beta-oxidation (enhanced approximately 3-fold) to citrate synthesis, there was also a 2.4-fold lowering of anaplerotic pyruvate carboxylation. Despite the dual inhibitory effect of high fatty acids on pyruvate decarboxylation and carboxylation, tissue citrate levels were twofold higher, but citrate release rates remained unchanged at 11-14 nmol/min, representing <0.5% of citric acid cycle flux. A similar trend was observed for most metabolic parameters after oleate or octanoate addition. Together, these results emphasize a differential modulation of anaplerotic pyruvate carboxylation and citrate release in the heart by fatty acids. We interpret the lack of effects of high fatty acid concentrations on citrate release rates as suggesting that, under physiological conditions, this process is maximal, probably limited by the activity of its mitochondrial or plasma membrane transporter. Limited citrate release at high fatty acid concentrations may have important consequences for the heart's fuel metabolism and function.     

Fluorometric assessment of gram-negative bacterial permeabilization

Uptake of the fluorescent probe 1-N-phenylnaphthylamine (NPN), as adapted to an automated spectrofluorometer enabling multiwell reading of microtitre plates, was applied to determine permeability changes in Gram-negative bacteria. An intact outer membrane is a permeability barrier, and excludes hydrophobic substances such as NPN but, once damaged, it can allow the entry of NPN to the phospholipid layer, resulting in prominent fluorescence. With Escherichia coli O157, Pseudomonas aeruginosa, and Salmonella typhimurium as test organisms and ethylenediaminetetraacetic acid and sodium hexametaphosphate as the model permeabilizers, quantitative and highly reproducible NPN uptake levels were obtained that differed characteristically between the test bacteria. Furthermore, citric acid was shown to be a potent permeabilizer at millimolar concentrations, its effect being partly (Ps. aeruginosa, Salm. typhimurium) or almost totally (E. coli O157) abolished by MgCl2, suggesting that part of the action occurs by chelation. Sodium citrate induced weak NPN uptake, which was totally abolished by MgCl2. In conclusion, the NPN uptake assay with the automated spectrofluorometer serves as a convenient method in analysing and quantifying the effects of external agents, including potential food preservatives, on Gram-negative bacteria.     

The in vivo genotoxic effects of sodium metabisulfite in bone marrow cells of rats

This study is designed to investigate the genotoxic effect of sodium metabisulphite (SMB), which is used as an antimicrobial substance in foods on bone marrow cells of rats. Four different concentrations of SMB (250, 500, 750 and 1000 mg/kg body weight) were given rats (Rattus norvegicus var. albinos) for 6, 12 and 24 h treatment period by intraperitoneal (IP) and gavage (GV) administrations. In this study, we found that intraperitoneal implement of SMB generally more effectively increases the percentage of abnormal cells and CA/cell in all concentrations and treatment period. In addition, mitotic index (MI) data of intraperitoneal injection are lower than gavage. It can be concluded that potential genotoxic effects of SMB by IP injection are higher than GV injection. The text was submitted by the authors in English.     

Mutagenesis induced with dioxidin in the Allium-test

The influence of dioxidin in different concentrations (10-100 mg/l) on the cytogenetic parameters of Allium cepa L. has been studied. The mutagenic effect of dioxidin was shown within all the range of the studied concentrations. The curve "dose-effect" has been determined for the concentrations of 10, 20, 30, 40, 50, and 100 mg/l. The peak of the mutagenic effect and significant reduction of the mitotic index were revealed at the concentration of 100 mg/l. It was shown that the mutagenic efficiency of the dioxidin statistically correlated with reduction of the mitotic activities.     

Development of chondrocyte-laden alginate hydrogels with modulated microstructure and properties for cartilage regeneration

Alginate hydrogel is an attractive biomaterial for cell microencapsulation. The microarchitecture of hydrogels can regulate cellular functions. This study aims to investigate the applicability of sodium citrate buffer (SCB) as a culture medium supplement for modulating the microstructure of alginate microbeads to provide a favorable microenvironment for chondrogenic induction. The chondrocyte-laden microbeads, with and without TGF-β3 incorporation, were produced through an encapsulator. The obtained small-sized microbeads (~300 μm) were exposed to a treatment medium containing SCB, composed of varied concentrations of sodium citrate (1.10–1.57 mM), sodium chloride (3.00–4.29 mM), and ethylenediaminetetraacetic acid (0.60–0.86 mM) to partially degrade their crosslinked structure for 3 days, followed by culture in a normal medium until day 21. Scanning electron microscope micrographs demonstrated a loose hydrogel network with an enhanced pore size in the SCB-treated microbeads. Increasing the concentration of SCB in the treatment medium reduced the calcium content of the microbeads via a Na⁺/Ca²⁺ exchange process and improved the water absorption of the microbeads, resulting in a higher swelling ratio. All the tested SCB concentrations were non-cytotoxic. Increases in aggrecan and type II collagen gene expression and their corresponding extracellular matrix accumulation, glycosaminoglycans, and type II collagen were vividly detected in the TGF-β3-containing microbeads with increasing SCB concentrations in the treatment medium. Our findings highlighted that the combination of SCB treatment and TGF-β3 incorporation in the chondrocyte-laden microbeads is a promising strategy for enhancing cartilage regeneration, which may contribute to a versatile application in cell delivery and tissue engineering.     

Effects of cadmium on meristem activity and nucleus ploidy in roots of Pisum sativum L. cv. Frisson seedlings

The effects of cadmium (Cd) administration on primary root growth, mitotic activity of apical meristems, mitotic aberrations and percentage of nucleus ploidy classes of differentiated roots were examined in Pisum sativum L. cv. Frisson. Cadmium caused a reduction of root length related to concentration, with an almost complete block of growth in plants treated with 250 μM Cd, from 24 h of treatment. Root lengthening is generally related to apical meristem activity, however, in the examined pea plants, mitotic activity was suppressed by 2.5 and 25 μM Cd treatment, while the highest Cd concentration, 250 μM, caused the occurrence of mitotic figures consisting almost exclusively of prophases. The lack of relation between root lengthening and mitotic activity was explained by the meristematic activity in the first period of treatment and by a different cell elongation. Lower (0.25, 0.5 and 1 μM), non-blocking Cd concentrations induced a number of mitotic aberrations, mainly consisting of sticky metaphases and anaphase bridges, whose frequency increased with Cd concentration. Besides, Cd induced variations of the percentages of nucleus populations in the differentiated roots, increasing the percentage of 4C nuclei and decreasing that of 2C. The mechanisms involved in the nuclear response to Cd, and the possible relations between Cd alteration of meristem cell activity and nuclear ploidy of differentiated cells are discussed.     

Genotoxicity biomarkers in occupational exposure to formaldehyde--the case of histopathology laboratories

Oxidative stress-mediated genotoxicity of wastewaters taken from two different cities, Saharanpur (SWW) and Aligarh (AWW), were compared with a battery of short-term assays namely the Allium cepa genotoxicity test, the plasmid-nicking assay, and the Ames fluctuation test. Both test-water samples - when used undiluted - increased the frequency of chromosomal abnormalities and/or micronuclei and alterations in the mitotic index of root cells of Allium cepa. Bridges and fragmentation of the chromosome were the predominant effects of the Saharanpur water sample while the Aligarh sample induced mainly chromosome fragmentation. Single- and double-strand breaks were also observed in plasmid DNA treated with these test wastewaters. The plasmid-nicking assay performed on SWW resulted in linearization of plasmid DNA when 18μl was tested (in a total reaction volume of 20μl). However, with the same amount of AWW, all three forms of plasmid, viz. supercoiled, open circular and linear were observed. Supplementation with specific scavengers of reactive oxygen species (ROS) caused a significant decline in mutagenicity of test-water samples in all the tests, pointing at oxidative stress as the mediator of the observed genotoxicity. The role of heavy metals in the AWW-induced oxidative stress and that of phenolics in SWW cannot be ruled out.     

Effects of citrate, heparin and cation supplementation on mortality and egg production of laboratory-reared horn flies

Abstract. The effects of the blood anticoagulants sodium citrate and sodium heparin on horn fly, Haematobia irritans L., egg production were tested. Sodium citrate was added to freshly collected bovine blood to give final concentrations of 5-100mM while sodium heparin was used in concentrations of 10–70 USP units/ml blood. Small cages containing five male and ten female newly emerged laboratory-reared horn flies were maintained for 8–10 days on these blood samples, and mortality and egg production recorded daily. Results showed that as blood citrate concentration was increased, egg production decreased logarithmically. At sodium citrate concentrations of 50 mM and above, severe impacts on egg production and adult horn fly survival occurred. Although no dose-related response of egg production to increasing heparin concentrations was noted, the 25 USP units heparin/ml blood treatments gave the largest egg production, yielding approximately 28% more eggs than any other treatment. Since citrate is a known chelator of divalent metal cations, the effects of supplemental cation additions to citrated blood were tested for their ability to reverse the egg production decrease seen at 50 mM sodium citrate. Blood samples containing 50mM sodium citrate were supplemented with CaCl₂, calcium lactate, CuCl₂, cupric acetate, FeCl₃, ferric citrate, MgCl₂, magnesium acetate, MnCl₂, ZnSO₄, EGTA or EGTA plus calcium lactate, each at 1 mM except EGTA which was used at 2.5 mM. The magnesium acetate supplement and the combination of calcium lactate plus EGTA resulted in a statistically significant increase in egg production ( P < 0.05).     

Fast direct method of obtaining metaphase and prometaphase chromosomes from chorion biopsy cells and human embryos during the lst semester of pregnancy]

Samples of chorionic villi and embryonic tissues (brain, brain--sheaths) are thoroughly washed with Hank's solution, immediately subjected to hypotonic treatment (0.9% sodium citrate plus few drops of 0.01% colchicine) 37 degrees C, 30 min, prefixed 20 min with equal amount of standard fixative mixture, twice fixed in standard fixative solution (1 hour, -10 degrees C), hydrated with equal volume of distilled water (5-10 min), dried, macerated directly on the slide with 60% acetic acid. The cell suspension is then evenly spread on the slide surface, dried, postfixed and stained. The method provides sufficient amount of metaphase and prometaphase mitotic plates suitable for differentiating staining in 1.5-2 hours after sampling and might be recommended for routine chromosomal analysis in prenatal diagnosis of inherited diseases during early pregnancy.     

Effects of low-molecular-weight fractions (LMWF) from milk, egg yolk, and seminal plasma on freezability of bovine spermatozoa

The effects of the dialyzable fractions from bovine seminal plasma, egg yolk, and milk and of two buffer systems (TEST and sodium citrate) on post-thaw sperm motility were studied. Each basic salt solution was used in the experimental design. These solutions were used as extender systems in combination with egg yolk and glycerol. After collection, semen samples were extended (1:20), cooled to 5 degrees C in 1.5 hr, and frozen in 0.5-cc French straws after 3 hr of equilibration. Post-thaw samples were assayed for percentage of motile cells immediately after thawing and after 4 hr of incubation at room temperature (22 degrees C). Egg yolk (25%) provided the same protection as did the combination of colloidal material present in the skim milk-yolk extenders. The use of TEST as a buffer provided significantly higher (P less than 0.01) sperm post-thaw motility than milk salts or Na citrate. Sperm survival in extenders containing high concentrations of seminal plasma and/or egg yolk salts was significantly lower (P less than 0.01). Spermatozoa frozen in the presence of 6% glycerol resulted in sperm motility significantly (P less than 0.05) higher than that of spermatozoa frozen with 3% glycerol. However, no difference was observed between these two concentrations when TEST solution was used.