Cadmium induction of lipid peroxidation and effects on root tip cells and antioxidant enzyme activities in Vicia faba L.

The effects of different concentrations (1–50 μM) of Cd on root growth, cell division and nucleoli in root tip cells, protective enzyme activities and lipid peroxidation in Vicia faba were investigated in order to better understand the processes of Cd-induced senescence. The results indicated that lower concentration of Cd (1 μM) had no obviously influence on the root growth during 24–48 h treatment, but higher concentrations (5–50 μM) inhibited significantly after 48 and 72 h. The mitotic index decreased with increasing of Cd concentration and duration of treatment except for the group exposed to 1 μM Cd. Cd induced c-mitosis, chromosome bridges, chromosome stickiness and lagging chromosomes. The rate of aberrant dividing cells increased with prolonging duration of treatment and increasing of Cd concentration. On nucleolus, some particulates containing the argyrophilic proteins were extruded from the nucleus into the cytoplasm in the cells stressed by Cd and some were scattered in the nucleus. After the treatment with Cd (10 μM Cd, 48 h), the nucleolus did not disaggregate normally and still remain its characteristic structure during metaphase and the particles of similar silver-stained materials were localized on chromosomes. In leaves, Catalase (CAT) activity declined but Peroxidase (POD) activity increased with increasing of the duration of treatment. In roots, CAT activity increased with increasing of the duration of treatment, POD activity increased during early days and then declined. Superoxide dismutase (SOD) activity showed an upward trend with increasing of the duration of treatment after 3 and 6 days, then declined both in leaves and roots (9 days). SOD and POD had highest activities at 50 μM Cd in leaves. CAT activity was lowest at 50 μM Cd. Malondialdehyde (MDA) content increased with the increasing of Cd concentrations and duration of treatment in leaves. In roots, MDA content showed an upward trend with increasing of the duration of treatment at early time and then declined.     

Genotoxicity response of Vicia faba seedlings to cadmium in soils as characterized by direct soil exposure and micronucleus test

To overcome the drawbacks of the Vicia faba root tip micronucleus test in soil using the solution extract method, we conducted a potting experiment by direct soil exposure. Cadmium was spiked into 3 typical soils (brown soil, red soil, and black soil) to simulate environmental concentrations (0.625, 1.25, 2.5, 5, and 10 mg kg−1). Multiple Vicia faba tissues (primary root tips, secondary root tips, and leaf tips) were sampled, and mitotic index (MI), chromosome aberration frequency (CA), and micronucleus frequency (MN) were used as endpoints after a seedling period of 5 days. The results showed a response between Cd concentrations and multiple sampling tissues of Vicia faba, and the secondary root tips responded to Cd stress the most, followed by primary root tips and leaf tips. Soil physicochemical properties (e.g., pH, total phosphorus, total organic carbon, etc.) influenced the genotoxicity of Cd, and pH was the dominant factor, which resulted in the genetic toxicity response of Cd in soils in the order: red soil > brown soil > black soil. The lowest observable effect concentration (LOEC) of Cd was 1.25 mg kg−1 for both brown soil and red soil and 2.5 mg kg−1 for black soil. In view of this, we suggested that soil properties should be considered in evaluating genotoxicity risk of Cd in soil, especially with soil pH range, and the secondary root tips should be taken as suitable test tissues in the MN test due to its more sensible response feature to Cd stress in soil.     

光泽汀体内遗传毒性风险评价

目的 评价光泽汀小鼠体内的遗传毒性。方法 C57BL/6J小鼠分为溶剂对照（0.5% CMC-Na）组、茜草素（200 mg·kg^-1，结构对照）组、乙酰基亚硝基脲（ENU，40 mg·kg^-1，阳性对照）组、甲基磺酸乙酯（EMS，200 mg·kg^-1，阳性对照）组和光泽汀低、中、高剂量（100、200、300 mg·kg^-1）组，溶剂、光泽汀和茜草素连续7 d ig给予，给药第1天记为D1，阳性对照ENU和EMS分别连续3 d给予，均每天给药1次。于D7、D56采集约0.5 mL外周血用于血清生化检测；于D14、D28、D42、D56采集外周血开展Pig-a基因突变试验；末次给药后采集肝、肾细胞开展彗星试验，分析每只动物至少100个细胞的尾DNA百分含量；末次给药后制备骨髓细胞样本，计算嗜多染红细胞的微核发生率。解剖后取心、肝、脾、肺以及肾脏进行组织病理学检查。结果 试验期间所有动物一般症状未见明显异常，各组动物体质量未见明显差异，未见与给予受试物有关的组织病理学改变。光泽汀低、中、高剂量组及EMS组肾脏尾DNA百分率均显著高于溶剂对照组（P<0.05、0.001），光泽汀高剂量组及EMS组肝脏尾DNA百分率与溶剂对照组比较显著增加（P<0.05、0.001）。光泽汀与茜草素的小鼠骨髓微核试验、Pig-a基因突变试验均为阴性。结论 100～300 mg·kg^-1光泽汀未见对小鼠整体产生明显毒性。光泽汀可导致小鼠肝、肾细胞DNA损伤，肾细胞DNA损伤程度更为严重。     

Physico-chemical characterization of legumin-T from faba bean (Vicia faba L.)

Legumin-T, the high-molecular mass product of limited tryptic hydrolysis of faba bean legumin, was investigated using hydrodynamic methods, static light scattering, fluorescence and ultraviolet spectroscopy. The following physico-chemical parameters were determined in a high-ionic strength buffer system: molecular mass, 2.4 × 10⁵ g/mol; sedimentation coefficient, s³₁₀= 10.8 × 10^?13s_i; diffusion coefficient, D⁰₂₀= 4.1 × 10^?7 cm² s^?1; intrinsic viscosity, [n] = 3.51 mL/g; partial specific volume, v∣?= O.719 mL/g; frictional ratio, f/f_o= 1.22; shape factor, β= 2.17 × 10⁶. Conformational changes during the formation of legumin-T can be deduced from the fluorescence emission and UV spectra. © Munksgaard 1996     

Strategies to in vitro assessment of major human CYP enzyme activities by using liquid chromatography tandem mass spectrometry

At the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single candidate can be identified for development. Determining the role of CYP enzymes in the metabolism of a compound and evaluating the effect of NCEs on human CYP activities are key issues in pharmaceutical development as they may explain inter-subject variability, drug-drug interactions, non-linear pharmacokinetics and toxic effects. Reliable methods for determining enzyme activities are needed to characterize an individual CYP enzyme and to obtain a tool for the evaluation of its role in drug metabolism in humans. Different liquid chromatography tandem mass spectrometry methodologies have been developed for the fast and routine analysis of major in vivo and in vitro CYPs enzyme activities. The high sensitivity and selectivity of mass spectrometry allow traditional assays to be minimized, thus saving time, efforts and money. Therefore this technology has become the method of choice for the fast assessment of CYP enzyme activities in early drug discovery development. Our intention herein is to review the most recent approaches that have been developed to quickly assess CYPs activities using in vitro models and liquid chromatography coupled with mass spectrometry, as well as their application in early drug discovery.     

Rice busk biochar treatment to cobalt-polluted fluvo-aquic soil: speciation and enzyme activities

Rice busk biochar was mixed with cobalt (Co)-polluted soil to examine the efficacy of biochar for Co immobilization and detoxification in fluvo-aquic soil. The Co speciation (modified BCR sequential extraction), fluorescein diacetate (FDA) hydrolysis and soil enzyme activities were investigated. In soil, the Co ions (acid-soluble fraction) could be uptake by biochar due to the microporous structure on the surface, as well as the oxygen-containing functional groups and conjugated structure in the molecular structure. Therefore, when the biochar concentration was lower than the optimum concentration (~6 g·kg⁻¹), there was transformation of Co from the acid-soluble fraction to the oxidizable fraction, resulting in lower environmental risk. However, if the biochar concentration continued increasing, the distribution coefficient of Co in the acid-soluble fraction increased (P < 0.05). The biochar could also reduce the toxicity of Co, resulting in the negative correlations between soil enzyme activities (FDA hydrolysis, urease and alkaline phosphatases) and Co in the acid-soluble fraction (r = –0.816, –0.928 and –0.908, respectively, P < 0.01). When the biochar concentration ranged from 5.83 to 6.76 g·kg⁻¹, the efficacy for Co immobilization and detoxification reached the maxima. To conclude, in fluvo-aquic soil, rice busk biochar is an effective amendment for immobilizing Co ions and reducing the toxicity of Co. The biochar concentration in soil should range from 5.83 to 6.76 g·kg⁻¹ to reach the optimum efficacy.

     

Stability of 11S globulin from Vicia faba seeds

Heat denaturation of 11S globulin, a dodecameric globular protein isolated from Vicia faba seeds was studied using scanning microcalorimetry at pH 7.6 and NaCl concentrations from 0 to 1 M. The specific enthalpy of denaturation was shown to be a linear function of temperature. The ratio of the calorimetric enthalpy to the effective one (Van't-Hoff's) per protomer of 11 S globulin was 0.9 ± 0.06. It is concluded that at first approximation 11 S globulin protomers denaturated independently in conformity with the two-state model. The plotted temperature-dependent specific free energy of 11 S globulin denaturation at different NaCl concentrations demonstrated that an increase in the salt content brought about the rise in protein stability. The maximum 11 S globulin stability is reached at about 300°K. The molar free energy of denaturation at 300°K in 1 M NaCl is 918 kJ/mol.     

Risk assessment using time

The ED₀₁ study undertaken by NCTR has provided extremely valuable data which should stimulate the development of improved cancer risk assessment procedures. A general model for carcinogenic risk assessment including both dose level and time to response is given herein-induding the ED₀₁ data. The need to include time in risk assessment is clearly demonstrated. Some important new definitions of acceptable risk which more realistically reflect the time of the response are presented and illustrated. The analysis of the ED₀₁ data indicates 1. the need to use a relatively fine time partition if each animal’s observation time is not treated individually, 2. the considerable room-toroom consistency in risk assessments, and 3. estimated models which are nonlinear in both dose and time for both bladder carcinomas and liver tumors - in contrast to the linearity of the estimated liver tumor multistage quantal response model which ignores time. The capability and need to do time based risk assessment exists now.     

Biological activities of broad bean (Vicia faba L.) extracts cultivated in South Anatolia in favism sensitive subjects

Aqueous extracts of a different variety of fresh broad bean seeds obtained from a favism endemic area in Turkey, were incubated with blood from sensitive and non-sensitive (control) subjects. Red blood cells were characterized by a whole blood glutathione (GSH) and a deficiency of Glucose-6-phosphate dehydrogenase (G-6-PD) activity. As the decrease in GSH percent is taken as an index of haemolytic activity, the test results were as following: Sakiz , Milas -Region, French broad bean extracts reduced the blood GSH levels 48%, 70%, 46% and 53%, respectively, in favism sensitive subjects. Active principles which are responsible for the haemolysis ( Vicine and Convicine ) were isolated from broad beans and their effects on GSH levels of blood were 99% and 81%, respectively, in favism sensitive subjects and 33.3% and 19% in normal subjects.     

Enzyme inhibitory activities of two Leguminosae: Phaseolus vulgaris L. pods and Vicia faba L. hulls. Trypsin inhibitory activity

Trypsin inhibitory activities of Phaseolus vulgaris L. pods and Vicia faba L. bean hulls have been studied according to the extraction conditions of the powder: solvent pattern, temperature and pH. The two vegetal powders have a trypsin inhibitory activity quite similar, weak but appreciable. The trypsin inhibitor of Phaseolus vulgaris L. pods is thermosensitive but stable at low pH. Inversely the Vicia faba L. bean hulls one is thermostable but it feels low pH.     

Contribution of reactive oxygen species (ROS) to genotoxicity of Nitrobenzene on V. faba

Nitrobenzene is an important organic intermediate widely used in industry that can be hazardous to the environment. In our previous study, nitrobenzene showed genotoxic effect on soybean and tobacco plants at concentrations in the culture medium higher than 10 mg/L. The genotoxicity of nitrobenzene has been hypothesized to be multifactorial and reflective of the generation of free radicals; however, the mechanism has not been fully elucidated. The aim of this study was to investigate the relationship between the induction of genotoxicity and the production of free radicals in young seedlings of V. faba exposed to nitrobenzene, nitrobenzene + Vitamin C, and the controls (distilled water or Vitamin C). Micronucleus and chromosome aberration assays performed on root and leaf tissue of V. faba seedlings exposed to nitrobenzene (25 mg/L) demonstrated genotoxic effects which were partly reduced by Vitamin C at 25 mg/L. Increases in lipid peroxidase, O2?-, H₂O₂, superoxide dismutase and catalase activities were also observed in these tissues along with an attenuation of their induction by Vitamin C. Concomitant occurrence of genotoxicity and the generation of free radicals that are attenuated in the presence of Vitamin C, a scavenger of cellular free radicals, indicate that reactive oxygen species may contributes to genotoxicity of nitrobenzene in V. faba. These results are valuable for further understanding the genotoxicity mechanism of nitrobenzene.     

In vivo genotoxicity assessment of nerolidol

Nerolidol is a sesquiterpenoid component of essential oil used as a flavor and aroma enhancer. It has also been studied as a topical skin penetration enhancer, and has inhibitory activities against S. aureus and E. coli, among other activities. The objective of this study was to evaluate the ability of a single nerolidol treatment to induce DNA damage in peripheral blood and liver cells of mice and micronuclei in polychromatic erythrocytes of bone marrow cells of the same animals. In the dose range‐finding assays, the maximum tolerated dose was higher than 2000 mg kg^?1. The doses used in the experiments were 250, 500 and 2000 mg kg^?1, administered by gavage in a single dose. Peripheral blood cells were collected 4 and 24 h after the treatments and liver cells 24 h after. At least 100 nucleoids per cell type/animal were analyzed to determine the DNA damage scores and 2000 PCEs per animal for micronuclei in PCEs. The positive control was N‐nitroso‐N‐ethylurea 50 mg kg^?1. Cytotoxicity was assessed by scoring 200 consecutive total polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE:NCE ratio). The results showed that nerolidol induced weak levels of dose‐related DNA damage in both types of cells analyzed, and enhanced the average number of micronucleated cells in the two high doses tested. The PCE:NCE ratio showed no cytotoxicity for the three doses of the compound. The data obtained support the view that nerolidol induces clastogenicity and very weak genotoxicity in the mouse cells tested. Copyright © 2010 John Wiley & Sons, Ltd.     

Exogenous application of salicylic acid to alleviate the toxic effects of insecticides in Vicia faba L

The present study investigated the possible mediatory role of salicylic acid (SA) in protecting plants from insecticides toxicity. The seeds of Vicia faba var IIVR Selection‐1 were treated with different concentrations (1.5, 3.0, and 6.0 ppm) of the insecticides alphamethrin (AM) and endosulfan (ES) for 6 h with and without 12 h conditioning treatment of SA (0.01 mM). Insecticides treatment caused a significant decrease in mitotic index (MI) and induction of different types of chromosomal abnormalities in the meristematic cells of broad bean roots. Pretreatment of seeds with SA resulted in increased MI and significant reduction of chromosomal abnormalities. SA application also regulated proline accumulation and carotenoid content in the leaf tissues. SA resulted in the decrement of insecticides induced increase in proline content and increased the carotenoids content. These results illustrate the ameliorating effect of SA under stress conditions and reveal that SA is more effective in alleviating the toxic effects of insecticides at higher concentrations than that at lower concentrations. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 666–672, 2013.     

PCB126 enhanced the genotoxicity of BaP in HepG2 cells by modulating metabolic enzyme and DNA repair activities

Both of benzo(a)pyrene (BaP) and 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) are ubiquitous and persistent environmental pollutants. These two chemicals coexist in various environmental media and human samples and thus may have combined effects on human health. However, the toxic effects and related mechanism of co-exposure to BaP and PCB126 remain unknown. In a series of experiments using the HepG2 cells exposed to BaP (50 μM) or/and PCB126 (0.01, 0.1, 1 and 10 nM), we measured the rate of micronucleus (MN) formation, CYP1A1 activity and expression of nucleotide excision repair (NER) proteins (XPA and XPC). We found that the exposure to BaP or PCB126 alone could effectively increase the CYP1A1 activity and the XPA expression. BaP alone had a profound enhancement of MN formation. Compared with BaP alone, co-exposure to both BaP and PCB126 significantly enhanced the CYP1A1 activity and the formation of MN but reduced the expression of both XPA and XPC. The synergistic effect of PCB126 on BaP-induced MN formation was inhibited by alpha-naphthoflavone (ANF), an inhibitor of CYP1A1. Our findings suggest that PCB126 may enhance BaP-induced DNA damage and genotoxicity by increasing cytochrome P450 1A activity and decreasing the NER capacity.     

比久和赤霉素GA3对蚕豆根尖细胞的诱变作用

丁酰肼(daminozide)俗称比久,是植物生长抑制剂,其大鼠经口LD为8 400 mg/kg,属低毒[1-2].但在一定条件下它的水解产物具有致癌和致畸作用,因此各国对它的使用进行了严格限制,如在花生中我国规定不得使用,韩国要求使用限量不超过1 mg/kg[3].     

An assessment of the genotoxicity of vanadium

The activities of vanadium oxide (V₂O₃), vanadyl sulfate (VOSO₄) and ammonium metavanadate (NH₄VO₃) in inducing sister chromatid exchange (SCE) and chromosomal aberrations (CAb) were assayed in Chinese hamster ovary cells. The toxic concentrations (TC₅₀) for these compounds were found to be 25, 23 and 16 μg elemental vanadium/ml, respectively. At doses 1/50−1/4 TC₅₀, vanadium compounds were able to induce significant increases (P < 0.01) in the SCE frequency with or without the addition of rat hepatic S9 mix. These compounds also induced CAb in the cells at doses closely equivalent to the TC₅₀.     

Use of intrinsic fluorescence to follow the denaturation of vicilin,a storage protein from Vicia faba

Excitation of native vicilin protein at 274 nm resulted in a single emission peak at 304 nm attributed to tyrosine fluorescence. Changes in the quantum yield of the tyrosine fluorescence with addition of both urea and guanidine hydrochloride (GdnHCl) were only significant at intermediate denaturant concentrations, and not suitable for assessment of conformational change. Emission spectra for the tryptophan residues were obtained by excitation of the vicilin at 295 nm. The wavelength of maximum tryptophan emission shifted from 347 to 353 nm with the addition of denaturants (2.0 M GdnHCl or 3.0 M urea). At higher concentrations of denaturant, the quantum yield values for tryptophan also increased, reflecting a gradual change in the conformation of vicilin. The degree of polarization for both tyrosine and tryptophan fluorescence decreased significantly at denaturant concentrations slightly above those at which the protein showed the first signs of unfolding. This was further indication of a variety of conformational states for vicilin with increasing levels of denaturant.     

Metabolic activation of herbicide products by Vicia faba detected in human peripheral lymphocytes using alkaline single cell gel electrophoresis.

Ametryn and metribuzin S-triazines derivatives and EPTC thiocarbamate are herbicides used extensively in Mexican agriculture, for example in crops such as corn, sugar cane, tomato, wheat, and beans. The present study evaluated the DNA damage and cytotoxic effects of three herbicides after metabolism by Vicia faba roots in human peripheral lymphocytes using akaline single cell gel electrophoresis. Three parameters were scored as indicators of DNA damage: tail length, percentage of cells with DNA damage (with comet), and level DNA damage. The lymphocytes were treated for 2 h with 0.5-5.0 microg/ml ametryn or metribuzin and 1.5-10 microg/ml EPTC. Lymphocytes also were coincubated for 2 h with 20 microl V. faba roots extracts that had been treated for 4 h with 50-500 mg/l of the two triazines or with the thiocarbamate herbicide or with ethanol (3600 mg/l), as positive control. The lymphocytes treated with three pesticides without in vivo metabolic activation by V. faba root did not show significant differences in the mean values between genotoxic parameters compared with negative control. But when human cells were exposed to three herbicides after they had been metabolized the frequency of cell comet, tail length and level DNA damage all increased. At highest concentrations of the three herbicides produced severe DNA damage compared with S10 fraction and negative control. The linear regression analysis of the tail length values of three herbicides indicated that there was genotoxic effect concentration-response relationship with ametryn and ametribuzin but no EPTC. The ethanol induced major increase DNA damage compared with S10 fraction and the three pesticides. There were not effects in cell viability with treatment EPTC and metribuzin whether or not it had been metabolized. High concentrations of ametryn alone and after it had been metabolized decreased cell viability compared with the negative control. The results demonstrated that the three herbicides needed to be activated by the V. faba root metabolism to produce DNA damage in human peripheral lymphocyte. The alkaline comet technique is a rapid and sensitive assay, to quickly evaluate DNA damage the metabolic activation of herbicide products by V. faba root in human cells in vitro.     

The enzyme toxicity and genotoxicity of chlorpyrifos and its toxic metabolite TCP to zebrafish Danio rerio

Chlorpyrifos is a broad-spectrum organophosphorus insecticide (O,O-diethyl -O-3,5,6-trichloro-2-pyridyl phosphorothioate) that is used in numerous agricultural and urban pest controls. The primary metabolite of chlorpyrifos is 3,5,6-trichloro pyridine-2-phenol (TCP). Because of its strong water solubility and mobility, this harmful metabolite exists in the environment in a large amount. Although TCP has potentially harmful effects on organisms in the environment, few studies have addressed TCP pollution. Therefore, this study was undertaken to investigate the effect of chlorpyrifos and TCP on the microsomal cytochrome P450 content in the liver, on the activity of NADPH-P450 reductase and antioxidative enzymes [catalase (CAT) and superoxide dismutase (SOD)], and on reactive oxygen species (ROS) generation and DNA damage in zebrafish. Male and female zebrafish were separated and exposed to a control solution and three concentrations of chlorpyrifos (0.01, 0.1, 1 mg L^?1) and TCP (0.01, 0.1, 0.5 mg L^?1), respectively, sampled after 5, 10, 15, 20 and 25 days. The results indicated that the P450 content and the NADPH-P450 reductase and antioxidative enzyme (CAT and SOD) activities could be induced by chlorpyrifos and TCP. DNA damage of zebrafish was enhanced with increasing chlorpyrifos and TCP concentrations. Meanwhile, chlorpyrifos and TCP induced a significant increase of ROS generation in the zebrafish hepatopancreas. In conclusion, this study proved that chlorpyrifos (0.01–1 mg L^?1) and TCP (0.01–0.5 mg L^?1) are both highly toxic to zebrafish.