Wan Liu

Multiple biomarkers response in maize (Zea mays L.) during exposure to copper

Biomarkers in higher plants played an important role to estimate exposure effects of pollutants in soil ecosystem and have received increasing attention in recent years. The qualitative and quantitative modifications arising in amplified fragment length polymorphism (AFLP) profiles as a measure of DNA effects were compared with a number of parameters, namely, the root length, total soluble protein content in root tips, chlorophylls content and shoot size to select the most sensitive biomarker responding to copper stress in the range of 0-600 mg/kg. The changes occurring in AFLP profiles of root tips following Cu treatment included loss of normal bands and appearance of new bands and variation in band intensity in comparison to that of the normal seedlings. A reduction in root length was observed at the 200 mg/kg of copper, which was accompanied with a decrease in total soluble protein content. According to their sensitivity to the copper toxicity, the above indicator rank in the following order: AFLP profiles > total soluble protein content > root length > chlorophylls content > shoot. We concluded that the AFLP offered a useful alternative biomarker assay for the detection of genotoxic effects of environmental pollutants.

Degradation of pyrene by immobilized microorganisms in saline-alkaline soil.

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) is very difficult in saline-alkaline soil due to the inhibition of microbial growth under saline-alkaline stress. The microorganisms that can most effectively degrade PAHs were screened by introducing microorganisms immobilized on farm byproducts and assessing the validity of the immobilizing technique for PAHs degradation in pyrene-contaminated saline-alkaline soil. Among the microorganisms examined, it was found that Mycobacterium sp. B2 is the best, and can degrade 82.2% and 83.2% of pyrene for free and immobilized cells after 30 days of incubation. The immobilization technique could increase the degradation of pyrene significantly, especially for fungi. The degradation of pyrene by the immobilized microorganisms Mucor sp. F2, fungal consortium MF and co-cultures of MB+MF was increased by 161.7% (P < 0.05), 60.1% (P < 0.05) and 59.6% (P < 0.05) after 30 days, respectively, when compared with free F2, MF and MB+MF. Scanning electron micrographs of the immobilized microstructure proved the positive effects of the immobilized microbial technique on pyrene remediation in saline-alkaline soil, as the interspace of the carrier material structure was relatively large, providing enough space for cell growth. Co-cultures of different bacterial and fungal species showed different abilities to degrade PAHs. The present study suggests that Mycobacterium sp. B2 can be employed for in situ bioremediation of PAHs in saline-alkaline soil, and immobilization of fungi on farm byproducts and nutrients as carriers will enhance fungus PAH-degradation ability in saline-alkaline soil.

DNA mismatch repair related gene expression as potential biomarkers to assess cadmium exposure in Arabidopsis seedlings

In the current study, Arabidopsis seedlings were hydroponically grown on MS media containing cadmium (Cd) of 0-2.0 mg L(-1) for 60 h of treatment. Gene expression profiles were used to relate exposure to Cd with some altered biological responses and/or specific growth effects. RT-PCR analysis was used to quantitate mRNA expression for seven genes known to be involved in DNA mismatch repair (MMR) system and cell division. Results indicated that Cd concentrations of 0.25-2.0 mg L(-1) cause increased total soluble protein levels in shoots of Arabidopsis seedlings in an inverted U-shaped dose-response manner. Exposure to 0.25 and 0.5 mg L(-1) of Cd dramatically induced expression of four genes (i.e. proliferating cell nuclear antigen 2 (atPCNA 2), MutL1 homolog (atMLH1), MutS 2 homolog (atMSH2) and atMSH3) and five genes (i.e. atPCNA1,2, atMLH1 and atMSH2,7), respectively, in shoots of Arabidopsis seedlings; Exposure to 1.0 mg L(-1) of Cd significantly elevated expression of only two genes (atMSH6,7), but caused prominent inhibition in expression of three genes (atPCNA2, atMLH1 and atMSH3) in shoots of Arabidopsis seedlings. The expression alterations of the above genes were independent of any biological effects such as survival, fresh weight and chlorophyll level of shoots. However, shoots of Arabidopsis seedlings exposed to 2.0 mg L(-1) of Cd exhibited statistically prominent repression in expression of these seven genes, and showed incipient reduction of fresh weight and chlorophyll level. This research provides data concerning sensitivity of expression profiles of atMLH1, atMSH2,3,6,7 and atPCNA1,2 genes in Arabidopsis seedlings to Cd exposure, as well as the potential use of these gene expression patterns as representative molecular biomarkers indicative of Cd exposure and related biological effects.

Cadmium-induced DNA damage and mutations in Arabidopsis plantlet shoots identified by DNA fingerprinting

Random amplified polymorphic DNA (RAPD) test is a feasible method to evaluate the toxicity of environmental pollutants on vegetal organisms. Herein, Arabidopsis thaliana (Arabidopsis) plantlets following Cadmium (Cd) treatment for 26 d were screened for DNA genetic alterations by DNA fingerprinting. Four primers amplified 20-23 mutated RAPD fragments in 0.125-3.0 mg L(-1) Cd-treated Arabidopsis plantlets, respectively. Cloning and sequencing analysis of eight randomly selected mutated fragments revealed 99-100% homology with the genes of VARICOSE-Related, SLEEPY1 F-box, 40S ribosomal protein S3, phosphoglucomutase, and noncoding regions in Arabidopsis genome correspondingly. The results show the ability of RAPD analysis to detect significant genetic alterations in Cd-exposed seedlings. Although the exact functional importance of the other mutated bands is unknown, the presence of mutated loci in Cd-treated seedlings, prior to the onset of significant physiological effects, suggests that these altered loci are the early events in Cd-treated Arabidopsis seedlings and would greatly improve environmental risk assessment.

Cadmium stress alters gene expression of DNA mismatch repair related genes in Arabidopsis seedlings

Cadmium (Cd) is a non essential element, and is a widespread environmental pollutant. Exposure to Cd can result in a variety of adverse health effects in plant and humans. In the current study, Arabidopsis seedlings were used as a bio-indicator of Cd pollution. Seedlings were grown on MS media containing 0-6.0 mg L(-1) Cd for 18 days, and the gene expression patterns were used to link increased Cd exposure with progressive biological effects. Reduction of total soluble protein content in shoots of the Arabidopsis seedlings occurred with increase in Cd concentrations. For the gene expression patterns, seven genes known to be involved in cell division and DNA mismatch repair (MMR) system were investigated by semi-quantitative RT-PCR, and normalized using 18S rRNA gene expression. Expression of the proliferating cell nuclear antigen 2 (atPCNA 2), MutS 3 homolog (atMSH 3) and MutL1 homolog (atMLH1) genes in shoots of Arabidopsis was strongly induced by exposure to 0.75 mg L(-1) Cd, but were repressed by other Cd concentrations whereas exposure to 0.75-6 mg L(-1) of Cd resulted in a decreased expression of atPCNA1, atMSH 2, 6 and 7 genes independently of any observable biological effects, including survival, fresh weight and chlorophyll level of shoots. This work demonstrated that specific gene expression changes could serve as useful molecular biomarkers indicative of Cd exposure and related biological effects.

Cadmium-induced genomic instability in Arabidopsis: Molecular toxicological biomarkers for early diagnosis of cadmium stress.

Microsatellite instability (MSI) analysis, random-amplified polymorphic DNA (RAPD), and methylation-sensitive arbitrarily primed PCR (MSAP-PCR) are methods to evaluate the toxicity of environmental pollutants in stress-treated plants and human cancer cells. Here, we evaluate these techniques to screen for genetic and epigenetic alterations of Arabidopsis plantlets exposed to 0-5.0 mg L(-1) cadmium (Cd) for 15 d. There was a substantial increase in RAPD polymorphism of 24.5, and in genomic methylation polymorphism of 30.5-34.5 at CpG and of 14.5-20 at CHG sites under Cd stress of 5.0 mg L(-1) by RAPD and of 0.25-5.0 mg L(-1) by MSAP-PCR, respectively. However, only a tiny increase of 1.5 loci by RAPD occurred under Cd stress of 4.0 mg L(-1), and an additional high dose (8.0 mg L(-1)) resulted in one repeat by MSI analysis. MSAP-PCR detected the most significant epigenetic modifications in plantlets exposed to Cd stress, and the patterns of hypermethylation and polymorphisms were consistent with inverted U-shaped dose responses. The presence of genomic methylation polymorphism in Cd-treated seedlings, prior to the onset of RAPD polymorphism, MSI and obvious growth effects, suggests that these altered DNA methylation loci are the most sensitive biomarkers for early diagnosis and risk assessment of genotoxic effects of Cd pollution in ecotoxicology.

Short-term toxic effects of chlorobenzenes on broadbean (Vicia faba) seedlings

The root growth, changes in Superoxide dismutase (SOD, EC 1.15.1.1) activity, malonyldialdehyde (MDA) and total soluble protein level of broadbean (Vicia faba) seedlings were researched at different soil concentrations of chlorobenzene (CB), 1,2,4-trichlorobenzene (TCB) and hexachlorobenzene (HCB). The results showed that root growth of seedlings was interrupted after 5d of 50–200 μg · g−1 TCB treatment. During a 3 d of recovery period, root growth was, however, restored to some extent although there was a delay in returning to the control level. The total soluble protein content in seedlings increased with TCB concentration and duration of exposure. Effect of TCB stress on SOD activity in seedlings displayed a significant dose-effect relationship for 1–5 d of 50–200 μg · g−1 treatment. When broadbean seedlings were placed in clean tap water for 3 d following exposure to 5 d of TCB stress to clear tap water for 3 d, SOD activity at 50 μg · g−1 TCB recovered towards control level (P> 0.05) while a significant increase in SOD activity was observed at 100 and 200 μg · g−1 TCB compared to control (P< 0.05). The experiments also revealed that a significant increase of MDA level in seedlings occurred after 3 and 5 d of 100 and 200 μg · g−1 TCB treatment (P< 0.05 andP< 0.01), and there was a positive correlation between TCB concentration and MDA level. All the above results showed that SOD activity and MDA level of broadbean seedlings might be proposed as the biomarkers for short-term TCB contamination in soil. Compared to TCB, the toxicity of 50−1000 μg · g−1 CB or HCB in soil to broadbean seedlings was not observed after a 3 d exposure.

WITHDRAWN: Studies on the interaction between triphenyltin and bovine serum albumin by fluorescence and CD spectroscopy

The interaction between triphenyltin (TPT) and bovine serum albumin (BSA) in physiological buffer (pH=7.4) was investigated by the fluorescence quenching technique. The results of fluorescence titration revealed that TPT could strongly quench the intrinsic fluorescence of BSA through a static quenching procedure. The apparent binding constants K and number of binding sites n of TPT with BSA were (7.04±0.0057)×10(2) and (0.77±0.016) which were obtained by the fluorescence quenching method. The thermodynamic parameters enthalpy change (ΔH), entropy change (ΔS) were positive, which indicated that the interaction of TPT with BSA was driven mainly by hydrophobic forces. The process of binding was a spontaneous process in which Gibbs free energy change was negative. The distance r between donor (BSA) and acceptor (TPT) was calculated to be 3.05nm based on Forsters non-radiative energy transfer theory. The results of synchronous fluorescence, three-dimensional fluorescence and Circular Dichroism (CD) spectra showed that the triphenyltin induced conformational changes of BSA.

CADMIUM ACCUMULATION OF TAGETES ERECTA L. AFFECTED BY PLANT GROWTH INHIBITORS AND GLUTATHIONE

The effect of two plant growth inhibitors maleic hydrazide (MH) and cyeloheximide (CHI) on cadmium (Cd) accumulation was investigated in Tagetes erecta L. The relative growth rate (RGR) as well as Cd content in shoots of Tagetes erecta L. was obviously decreased by the two inhibitors, indicating that there was close relationship between Cd accumulation in shoots and the plant growth. When the sulfur (S)-containing compound glutathione (GSH) was applied to the leaves of Tagetes erecta L. under the MH or CHI treatment, the decreased Cd content in shoots affected by MH was alleviated, but there was no effect of the GSH on Cd content in shoots under CHI treatment, suggesting that GSH has an counter-effect on Cd accumulation in shoots of Tagetes erecta L. under the MH treatment. All the results suggested that plant growth and S-containing compounds play an important role in Cd accumulation in Tagetes erecta L.

Cell cycle arrest mediated by Cd-induced DNA damage in Arabidopsis root tips

Accumulating evidence demonstrates that the aberrant expression of cell cycle regulation and DNA repair genes can result in abnormal cell proliferation and genomic instability in eukaryotic cells under different stresses. Herein, Arabidopsis thaliana (Arabidopsis) seedlings were grown hydroponically on 0.5 × MS media containing cadmium (Cd) at 0-2.5mgL-1 for 5d of treatment. Real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed that expression of DNA damage repair and cell cycle regulation genes, including BRCA1, MRE11, WEE1, CDKA;1 and PCNA1, showed an inverted U-shaped dose-response. In contrast, notably reduced expression was observed for G1-to-S transition-related genes, Histone H4, E2Fa and PCNA2; DSB end processing, GR1; G2-to-M transition-related gene, CYCB1;1; and DNA mismatch repair, MSH2, MSH6 and MLH1 genes in root tips exposed to 0.125-2.5mg/L Cd for 5d. Flow cytometry (FCM) analysis revealed significant increases of cells with a 2C nuclear content and with a 4C and 8C nuclear content under Cd stresses of 0.125 and 1-2.5mgL-1, respectively. Our results suggest that 0.125mgL-1 Cd-induced DNA damage induced the marked G1/S arrest, leading to accelerated growth in root tips, while 1.0-2.5mgL-1 Cd-induced DNA damage caused a notable G2/M arrest in root tips, leading to reduced growth in root tips. This may be a protective mechanism that prevents cells with damaged DNA from dividing under Cd stress.