AFLP analysis as biomarker of exposure to organic and inorganic genotoxic substances in plants

In recent years several plant species have been in use as bioindicators and several tests have been developed to evaluate the toxicity of environmental pollutants in vegetal organisms. In the present paper Arabidopsis thaliana (L.) Heynh. (ecotype Wassilewskija) was used as bioindicators of two genotoxic substances: potassium dichromate and dihydrophenanthrene. Inhibition of seed germination was observed with both pollutants. AFLP analysis (i) indicated that both substances are genotoxic, (ii) showed that dihydrophenanthrene induces DNA changes in different target sequences than potassium dichromate, (iii) quantified the genotoxic effect using cluster analysis by comparing DNA from treated plants with that of control plants. On the basis of these considerations we suggest that AFLP method is a powerful tool for measuring qualitative and quantitative genotoxic activity due to environmental pollutants. AFLP method can be applied to a wide range of bioindicator organisms and may become a universal methodology to identify target genes for specific genotoxic agents. This could open up possibilities for designing specifically targeted assays and new approaches to risk assessment.     

Zea mays L. protein changes in response to potassium dichromate treatments

The plant metabolic response to heavy metal stress is largely unknown. The present investigation was undertaken to examine the influence of different concentrations of potassium dichromate on the Zea mays L. plantlets. A clear effect of chromium on maize plantlets growth and seed germination was observed strating from 100-300 ppm up to 1500 ppm. In this concentration range, chromium uptake was dependent on the concentration in the medium. Metallothioneins, involved in heavy metal binding, were measured by capillary electrophoresis (CE), and showed a dose-response induction. Protein profile analyzed by two-dimensional gel electrophoresis showed differential expression of several proteins. Identification of spots of upregulated proteins was performed by MALDI mass spectrometry. Results showed that proteins induced by heavy metal exposure are principally involved in oxidative stress tolerance or in other stress pathways. Induction of proteins implicated in sugar metabolism was also observed. Identification of factors involved in plant response may lead to a better understanding of the mechanisms involved in cell protection and tolerance. This information could be used to improve agricultural production and environmental quality.     

Potassium dichromate increases the micronucleus frequency in the crayfish Procambarus clarkii

The assessment of biological effects on aquatic vertebrate and invertebrate species is frequently employed to monitor water pollution because it provides meaningful information on bioavailability and effective concentration levels. Of special concern are genotoxic agents that induce DNA alterations at subtoxic exposure levels. With the objective of developing a field-assay for the detection of genotoxic pollutants in water, we investigated the effects of hexavalent chromium in the haemolymph cells of Procambarus clarkii using the micronuclei (MN) test. The frequency of micronucleated cells significantly correlated with the amount of potassium dichromate in water and with the amount of chromium found in gills.     

Molecular evidence for benzo[a]pyrene and naphthalene genotoxicity in Trifolium repens L

Polycyclic aromatic hydrocarbons (PAHs) are among the most dangerous environmental contaminants due to their toxic, carcinogenic and mutagenic effects. Although there are many data in literature that detail the effects of PAHs on animals, little is known about their action on higher plants which are often used as bioindicators. The aim of the present study was to evaluate the genotoxicity of two different PAHs, benzo[a]pyrene (BaP) and naphthalene (Naph), on Trifolium repens L. Clover plants were exposed to soil which had been artificially contaminated with three concentrations of BaP (5, 10 and 20 microg g-1) or Naph (25, 50 and 100 microg g-1). After 15 days, changes in the DNA content and sequence of roots and shoots were evaluated by flow cytometry (FCM) and amplified fragment length polymorphism (AFLP). Root and shoot dry weight were also determined to assess plant growth. Results showed that BaP and Naph were both genotoxic for white clover, inducing significant changes in root and shoot DNA sequence. Damage was more severe in the root than in the shoot suggesting that the translocation of these compounds and their genotoxic metabolites was limited. Ploidy alterations were not detected and the extent of damage caused by all the tested PAH concentrations was not sufficient to affect plant development.     

Excess chromium alters uptake and translocation of certain nutrients in citrullus

Citrullus plants were grown in refined sand with varying levels of chromium to determine their tolerance limit to excess chromium. The plants were maintained in control nutrient solution for 24 days and on the 25th day chromium as dichromate was added at 0.05, 0.1, 0.2, 0.3 and 0.4 mM. A control set of plants was grown in the same nutrient solution without chromium. At chromium levels >0.2 mM plants showed growth depression, with chlorosis and loss of turgor of middle leaves. Affected leaves had narrow lamina; tendrils were thin, short and did not have coiling property. Later chlorosis became severe and changed to necrosis in patches. Petiole along with lamina became wilted, rugged and hung down due to complete loss of water. At lower chromium concentration, (0.05, 0.1 and 0.2 mM) only depression in growth was observed. With increase in chromium concentration of nutrient solution accumulation of chromium in different parts of Citrullus was increased. Increase in concentrations of phosphorus, manganese and decrease in iron, copper, zinc and sulphur were observed in leaves. Toxicity of chromium was greater at 0.2-0.4 mM, compared to lower concentrations. Threshold of toxicity and toxicity of Cr in old leaves were, respectively, 0.9 and 3.9 microg g(-1) dry matter of citrullus.     

Tolerance of transgenic canola plants (Brassica napus) amended with plant growth-promoting bacteria to flooding stress at a metal-contaminated field site

The growth of transgenic canola (Brassica napus) expressing a gene for the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase was compared to non-transformed canola exposed to flooding and elevated soil Ni concentration, in situ. In addition, the ability of the plant growth-promoting bacterium Pseudomonas putida UW4, which also expresses ACC deaminase, to facilitate the growth of non-transformed and transgenic canola under the above mentioned conditions was examined. Transgenic canola and/or canola treated with P. putida UW4 had greater shoot biomass compared to non-transformed canola under low flood-stress conditions. Under high flood-stress conditions, shoot biomass was reduced and Ni accumulation was increased in all instances relative to low flood-stress conditions. This is the first field study to document the increase in plant tolerance utilizing transgenic plants and plant growth-promoting bacteria exposed to multiple stressors.     

Influence of plant growth promoting bacteria and Cr6+ on the growth of Indian mustard

The Cr(6+) resistant plant growth promoting bacteria (PGPB), Pseudomonas sp. PsA4 and Bacillus sp. Ba32 were isolated from heavy metal contaminated soils and their plant growth promoting activity on the Indian mustard (Brassica juncea) were assessed with different concentrations of Cr(6+) in soil. Production of siderophores and the solubilization of phosphate were observed in both strains, PsA4 and Ba32. Production of IAA was only observed in strain PsA4. Inoculation of PsA4 or Ba32 promoted the growth of plants at 95.3 and 198.3 microg of Cr(6+)g(-1) soil. The maximum growth was observed in the plants inoculated with strain PsA4. Both strains, PsA4 and Ba32 did not influence the quantity of accumulation of chromium in root and shoot system. The present observations showed that the strains PsA4 and Ba32 protect the plants against the inhibitory effects of chromium, probably due to the production of IAA, siderophores and solubilization of phosphate.     

Photochemical reduction of hexavalent chromium in glycerol-containing solutions

Hexavalent chromium [Cr(VI)] in the form of potassium dichromate was photochemically reduced to trivalent chromium [Cr(III)] in aqueous solutions containing glycerol. This reaction occurred rapidly during irradiation with either unfiltered sunlight or a UVA-emitting light source. Photochemical reduction of Cr(VI) was pH-dependent and did not occur in dilute solutions of sodium hydroxide. In acidified solutions, the reduction occurred at elevated rates and at lower concentrations of glycerol. This reaction was found to be dependent on the unsubstituted alcohol groups of glycerol since alpha-phosphoglycerol and beta-phosphoglycerol did not support the photochemical reduction of Cr(VI). These findings suggest that glycerol or related polyols can be used for the remediation of hexavalent (toxic) chromium at contaminated environmental sites.     

Phytoextraction with Brassica napus L.: a tool for sustainable management of heavy metal contaminated soils

Phytoextraction is a promising tool to extract metals from contaminated soils and Brassica napus L. seems to be a possible candidate species for this purpose. To select accessions with the ability to accumulate cadmium, hydroponically grown 21 day old seedlings of 77 B. napus L. accessions were exposed to 0.2 microM CdSO(4) for an additional 10 days. The effects of Cd on several parameters were quantified i.e.; shoot Cd concentration ([Cd](shoot)), total amount of Cd in shoots (Total Cd) and the shoot to root Cd concentration ratio (S/R ratio). Though generally natural variation was low for [Cd](shoot), Total Cd and S/R ratio, a number of accessions could be selected. Our results indicated that Total Cd and S/R ratio are independent parameters for Cd accumulation and translocation. The selected varieties were then tested in field experiments on two locations nearby metal smelters. The two locations differed in extractable soil Cd, Zn, Ca concentration and pH levels. On both locations B. napus L. accessions showed significant differences in [Cd](shoot) and Total Cd. Furthermore we found significant correlations between Cd and Zn accumulation in shoots. There were site-specific effects with respect to Cd accumulation in the B. napus L. accessions, however, two accessions seem to perform equally well on both sites. The results of the field experiment suggest that certain B. napus L. accessions are suitable for phytoextraction of moderately heavy metal contaminated soils.     

Growth responses of crop and weed species to heavy metals in pot and field experiments

Greenhouse and field studies were performed to examine the growth responses and possible phytoremediation capacity towards heavy metals of several Brassicaceae (Brassica alba, Brassica carinata, Brassica napus and Brassica nigra) and Poaceae (durum wheat and barley). Soils used featured total concentrations of Cr, Cu, Pb and Zn largely exceeding the maximum levels permitted by the Italian laws. Different organic amendments were tested such as a compost and the plant growth-promoting rhizobacterium Bacillus licheniformis. In the greenhouse experiment, plant length, leaf area index and shoots dry matter were evaluated periodically for the Brassicaceae examined. Whereas plant length, grains production, weight of 1,000 seeds, ear fertility and tiller density were determined under field conditions at the end of the crop cycle for wheat and barley. In general, the species tested appeared to be tolerant to high heavy metal concentrations in soil, and slightly significant differences were found for all parameters considered. A marked growth increase was shown to occur for Brassicaceae cultivated on compost- and bacillus-amended contaminated soils, with respect to non-amended contaminated soils. With some exception, higher growth parameters were measured for wheat and barley plants cropped from contaminated soils in comparison to non-contaminated soils. Further, bacillus amendment enhanced the length of wheat and barley plants in both non-contaminated and contaminated soils, while different effects were observed for the other parameters evaluated.     

Spectrophotometric determination of sulphur dioxide

Sulphur dioxide, an important industrial gas and air pollutant, is usually estimated using mercury salts. The authors have developed a method in which hazardous mercury salts are avoided. Sulphur dioxide is trapped in aqueous morpholine and mixed with the excess of dichromate solution in acidic medium. The hexavalent chromium in dichromate is reduced to trivalent chromium by sulphur dioxide and the excess of hexavalent chromium is determined with diphenylcarbazide which yields a soluble red-violet complex with an absorption maximum at 540 nm. The decrease in the absorbance values of the red-violet complex formed after reduction, when compared to that of a reagent control, is proportional to the concentration of sulphur dioxide used for reduction. Beer's law operates between 0.4 and 4 microg ml(-1) concentration of sulphur dioxide in solution.     

Limitations in the use of potassium dichromate as a blood preservative for the analysis of organohalogenated compounds: two month results

For analysis of organochlorine contaminants in human tissue, the "gold standard" for preservation, storage, and shipping is usually freezing. However, this method can be difficult, if samples are taken in remote areas, and costly, when the samples must be shipped on dry ice. Therefore, a more simple and cost effective method of preservation is essential for remote field work. Potassium dichromate (K(2)Cr(2)O(7)) has been successfully employed in the preservation of human and cows' milk as well as chicken eggs. Our previous studies described the use of potassium dichromate for preservation of whole blood for analysis of dioxins, dibenzofurans, and PCBs. Potassium dichromate was found to successfully preserve blood at room temperature for 34 d with no significant differences in the measured concentrations of chemical contaminants or blood lipid level when compared to frozen samples. However, in a follow-up study, 3 months and 6 months of potassium dichromate preservation proved inadequate to preserve the samples for organic pollutant analysis. We noted that the lipid portion of the blood in the chemically preserved samples was declining in level or degrading, while the persistent organic pollutants remained intact at the same levels on a whole weight basis. To narrow down the window of efficacy for the use of potassium dichromate to preserve blood samples for analysis, the present study compared chemical preservation to freezing for an intermediate time period, 2 months. Similar to our previous findings at 3 and 6 months, at 2 months significant lipid degradation was observed in the chemically preserved samples. Chemically preserved samples had significantly higher levels of organochlorine contaminants (dioxins, dibenzofurans, and PCBs) when measured on a blood lipid basis but not on a wet weight basis compared to frozen samples. While 2 months of potassium dichromate preservation was not useful for obtaining accurate measure of dioxins, furans, and PCBs on a lipid basis, previous studies found this method of preservation to be useful for at least one month (Schecter, A., Pavuk, M., P?pke, O., Malisch, R., 2004. The use of potassium dichromate and ethyl alcohol as blood preservatives for analysis of organochlorine contaminants. Chemosphere 57, 1-7). However blood stored at -70 degrees C and at 22 degrees C with potassium dichromate gave similar results when expressed on a wet weight basis.     

Antiradical activity of natural honeys and antifungal effect against Penicillium genera

The purpose of the study was to examine the antiradical activity of 11 natural honeys and to evaluate the antifungal properties of honey. Honey samples (10) were collected from different locations of Slovak Republic. Honeys were native to different plant species of Robinia pseudoacaccia, Brassica napus subs. napus, Castanea sativa Mill. Thymus serpyllum vulgaris and the other samples had multifloral origin. The low antiradical activitity in honey samples was determined. The best results were found in thyme honey from Rhodos (11.84 %) and Castanea honey from Nitra (10.61 %). The lowest antiradical activity was found in Acacia honey and determined to be 7.62 %. Statistically significant differences (P< 0.001) were found among thyme/Rhodos and Castanea/Nitra. The antifungal activities of honey samples were tested by 10 %, 25 % and 50 % (by mass per volume) concentration against fungi Penicillium crustosum, P. expansum, P. griseofulvum, P. raistrickii and P. verrucosum and by the agar well diffusion method. The solutions containing 10 % (by mass per volume) of honey did not have any effect on the growth of fungi. The strongest antifungal effect was shown by 50 % honey concentration against P. raistrickii.     

Protective role of humic acids against picloram-induced genomic instability and DNA methylation in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>

Picloram (4-amino-3,5,6-trichloropicolinic acid) is a liquid auxinic herbicide used to control broad-leaved weeds. Picloram is representing a possible hazard to ecosystems and human health. Therefore, in this study, DNA methylation changes and DNA damage levels in Phaseolus vulgaris exposed to picloram, as well as whether humic acid (HA) has preventive effects on these changes were investigated. Random amplified polymorphic DNA (RAPD) techniques were used for identification of DNA damage and coupled restriction enzyme digestion-random amplification (CRED-RA) techniques were used to detect the changed pattern of DNA methylation. According to the obtained results, picloram (5, 10, 20, and 40 mg/l) caused DNA damage profile changes (RAPDs) increasing, DNA hypomethylation and genomic template stability (GTS) decreasing. On the other hand, different concentrations of applied HA (2, 4, 6, 8, and 10%) reduced hazardous effects of picloram. The results of the experiment have explicitly indicated that HAs could be an alternative for reducing genetic damage in plants. In addition to the alleviate effects of humic acid on genetic damage, its epigenetic effect is hypomethylation.     

Proteomic analysis of chromate-induced modifications in Pseudokirchneriella subcapitata

In this work, we have analyzed the changes in the protein expression profile elicited by chromium (Cr) exposure in the freshwater green alga Pseudokirchneriella subcapitata, a well known bio-indicator of water pollution. We tested two experimental conditions, namely 0.2 and 1 ppm of potassium dichromate; this concentration range includes the environmentally-relevant concentrations. Results show that neither concentration of potassium dichromate tested inhibited algal growth. However, the proteomic approach allowed the identification of relevant modifications in protein expression. In fact, among 800 protein spots detected by two-dimensional electrophoresis, 16 Cr-regulated proteins, including predicted and novel ones, were identified using tandem mass spectromic protein analysis.The results demonstrate a Cr-specific action in altering several photosynthetic proteins, such as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), RuBisCO activase, Light Harvesting Chla/b protein complex, and stress related Chla/b binding protein1. Although Cr toxicity with respect to photosynthesis has been already documented, here we have identified, for the first time, the target proteins of this toxicity. Cr also induced a modulation of some proteins involved in the metabolism of the amino acids glutamine, arginine and methionine. These data are supported by changes in cellular polyamine (PA) accumulation. Present findings provide new insight into the molecular mechanisms underlying Cr toxicity in P. subcapitata.     

The use of potassium dichromate and ethyl alcohol as blood preservatives for analysis of organochlorine contaminants

The "gold standard" for preserving and shipping of human tissue samples for analysis of organochlorine contaminants is freezing. This method can be difficult, costly if using heavy dry ice for shipping, and often unfeasible, especially in less developed countries where electricity and dry ice are frequently rare or absent. Therefore, it is essential that more convenient and practical methods for preservation of blood samples are found. As an alternative to freezing, there have been studies employing potassium dichromate as a preservative for human or cow's milk or ethyl alcohol preservation of blood for dioxin analysis. In this study, four methods were compared to investigate the effectiveness of ethyl alcohol and potassium dichromate as blood preservatives for analysis of dioxins, dibenzofuran, and polychlorinated biphenyl (PCB) congeners. Samples of whole blood from a Dallas, Texas hospital were collected and pooled. Freezing, ethyl alcohol in two concentrations (20% and 40% per volume of sample), and potassium dichromate were used for blood preservation. The blood samples containing potassium dichromate or ethyl alcohol were stored and sent to ERGO laboratory for dioxin analysis and comparison with results from the frozen sample, which was kept frozen at all times until analyzed. This study suggests that potassium dichromate is a suitable alternative to freezing for preservation of whole blood for dioxin, dibenzofuran, and PCB measurements when either lipid or wet weight based results are reported. Potassium dichromate tablets were very easy and convenient to use--two 100 mg tablets (with a dichromate content of about 33 mg each) were added to each bottle containing 65 ml of blood. However, ethyl alcohol at 20% and 40% concentration under the conditions of this pilot study and the analytical method employed did not appear to provide satisfactory preservation when lipid based results are given or when the fat content has to be determined (wet or whole weight). Further research with a larger number of samples, inclusion also of other groups of persistent organic pollutants such as organochlorine pesticides or brominated flame retardants, a longer duration of storage time, and at temperatures greater than US or German room temperature is indicated in order to recommend the routine use of potassium dichromate as preservative for whole blood intended for dioxin, dibenzofuran, and PCB analysis.     

Reduction of hexavalent chromium by ascorbic acid in aqueous solutions

Hexavalent chromium is a priority pollutant in the USA and many other countries. Reduction of Cr(VI) to Cr(III) is environmentally favorable as the latter species is not toxic to most living organisms and also has a low mobility and bioavailability. Reduction of Cr(VI) by ascorbic acid (vitamin C) as a reductant was studied using potassium dichromate solution as the model pollutant. Effects of concentration of vitamin C, pH, temperature, irradiation and reaction time on the reduction of Cr(VI) were examined. Cr(VI) might be reduced by vitamin C not only in acidic conditions but also in weakly alkaline solutions. The reduction of Cr(VI) by vitamin C might occur not only under irradiation but also in the dark. Vitamin C is an important biological reductant in humans and animals, and not toxic. It is water-soluble and can easily permeate through various types of soils. The results indicate that vitamin C could be used in effective remediation of Cr(VI)-contaminated soils and groundwater in a wide range of pH, with or without sunlight.     

Genetic polymorphisms in XRCC1, OGG1, and XRCC3 DNA repair genes and DNA damage in radiotherapy workers

DNA damage may develop at any dose of ionizing radiation. DNA damage activates pathways that regulate cell growth and division or coordinate its replication and repair. The repair pathways, base excision repair (BER) and single-strand break repair (SSBR), can repair such damages efficiently and maintain genome integrity. Loss of this repair process or alteration of its control will be associated with serious outcomes for cells and individuals. This study aimed to determine the relationship between XRCC1 (Arg194Trp, Arg280His, and Arg399Gln), OGG1 (Ser326Cys), and XRCC3 (Thr241Met) SNPs and DNA damage and to identify high-risk individuals with reduced DNA repair capacity. This case-control study was conducted on 80 subjects; 50 subjects working in Clinical Oncology and Nuclear Medicine Department in Assiut University Hospital along with 30 controls. A total of 1 mL blood samples were collected for Single-Cell Gel Electrophoresis Technique (Comet Assay) for detection of DNA damage in those subjects. A total of 3 mL fresh blood samples were collected and analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)–based technique. DNA damage detected by comet test was significantly high in IR-exposed workers than control. Statistically high significant difference was found in exposed subjects versus control subjects regarding the frequencies of the variant alleles of hOGG1³²⁶, XRCC1^{280 & 399}, and XRCC3²⁴¹. The level of DNA damage was not affected by OGG1³²⁶ SNPs when comparing subjects of wild genotype with those of (pooled) variants either in the exposed staff or in the control group while XRCC1^{280, 399} and XRCC3²⁴¹ variant alleles had an influence on the studied DNA damage biomarker. Moreover, genotyping distribution pattern was highly variable in relation to gender. The present study indicated a relationship between DNA damage detected by comet test and single nucleotide polymorphisms in genes coding for DNA certain repair enzymes. Individuals occupationally exposed to low doses of ionizing radiation could be at great risk and more susceptible to the increased DNA damage if they have inherited genetic polymorphism.

     

Improvement of rape (Brassica napus) plant growth and cadmium uptake by cadmium-resistant bacteria

This study focuses on the screening of cadmium-resistance bacterial strains from heavy metal-polluted soils to examine their plant growth promotion and cadmium uptake in rape (Brassica napus). A large number of bacteria were isolated from heavy metal-polluted soil in Nanjing, China. Thirty isolates showing cadmium-resistance on Cd-amended medium were selected and evaluated for their potential to solubilize cadmium carbonate in solution culture. Atomic absorption spectrometer analysis showed variable amounts of water-soluble Cd (ranging from 24 to 117 mg l(-1)) released by the cadmium-resistant bacterial strains from cadmium carbonate. Qualitative analysis confirmed the presence of indole acetic acid as the auxin in the culture of these cadmium-resistant bacterial strains. Root elongation assay conducted on rape under gnotobiotic conditions demonstrated increases (up to 31%) in root elongation of inoculated rape seedlings compared to the control plants. Based upon cadmium-resistance, bio-activation of CdCO3 and growth-promoting activity, three isolates were selected for promoting plant growth and uptake of cadmium from cadmium-amended soil in pot experiments. Inoculation with the isolates was found to increase root dry weight (ranging from 8% to 20%) and shoot dry weight (ranging from 6% to 25%) of rape. An increase in cadmium content varying from 16 to 74%, compared to the non-inoculated control, was observed in rape plants cultivated in soil treated with 100 mgCd kg(-1) (as CdCl2) and inoculated with the isolates. The bacterial isolates were also able to colonize and develop in the rhizosphere soil of rape after root inoculation.