Huiying Li

Responses of antioxidant gene, protein and enzymes to salinity stress in two genotypes of perennial ryegrass (Lolium perenne) differing in salt tolerance

Salinity could damage cellular membranes through overproduction of reactive oxygen species (ROS), while antioxidant capacities play a vital role in protecting plants from salinity caused oxidative damages. The objective of this study was to investigate the toxic effect of salt on the antioxidant enzyme activities, isoforms and gene expressions in perennial ryegrass (Lolium perenne L.). Salt-tolerant Quickstart II and salt-sensitive DP1 were subjected to 0 and 250 mM NaCl for 12 d. Salt stress increased the content of lipid peroxidation (MDA), electrolyte leakage (EL) and hydrogen peroxide (H₂O₂), to a greater extent in salt-sensitive genotype. Salt-stressed plant leaves exhibited a greater activity of superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11) at 4d after treatment (DAT), but a lower level of enzyme activity at 8 and 12d, when compared to the control. Catalase (CAT, EC 1.11.1.6) activity was greater at 4 DAT and thereafter decreased in salt tolerant genotype relative to the control, whereas lower than the control during whole experiment period for salt-sensitive genotype. There were different patterns of five isoforms of SOD, POD and two isoforms of APX between two genotypes. Antioxidant gene expression was positively related to isoenzymatic and total enzymatic activities during 12-d salt-treated leaves of two genotypes, with a relatively higher level in salt-tolerant genotype. Thus, salt tolerance could be related to the constitutive/induced antioxidant gene, leading to more efficient enzyme stimulation and protection in perennial ryegrass.

In vivo study on the effects of microcystin extracts on the expression profiles of proto-oncogenes (c-fos, c-jun and c-myc) in liver, kidney and testis of male Wistar rats injected i.v. with toxins.

Microcystins (MCs) are a potent liver tumor promoter, possessing potent tumor-promoting activity and weak initiating activity. Proto-oncogenes are known to be involved in the tumor-promoting mechanisms of microcystin-LR. However, few data are available on the effects of MCs on proto-oncogenes in the whole animal. To investigate the effects of MCs on the expression profile of the proto-oncogenes in different organs, male Wistar rats were injected intravenously with microcystin extracts at a dose of 86.7 mug MC-LR eq/kg bw (MC-LR eq, MC-LR equivalents). mRNA levels of three proto-oncogenes c-fos, c-jun and c-myc in liver, kidney and testis were analyzed using quantitative real-time PCR at several time points post-injection. Significant induction of these genes at transcriptional level was observed in the three organs. In addition, the increase of mRNA expression of all three genes was much higher in liver than in kidney and testis. Meanwhile, the protein levels of c-Fos and c-Jun were investigated by western blotting. Both proteins were induced in the three organs. However, elevations of protein levels were much lower than those of mRNA levels. These findings suggest that the expression of c-fos, c-jun and c-myc might be one possible mechanism for the tumor-promoting activity and initiating activity of microcystins.

Microcystin-induced variations in transcription of GSTs in an omnivorous freshwater fish, goldfish

The glutathione S-transferases are important enzymes in the microcystin-induced detoxication processes. In this experiment, we cloned the full-length cDNA of alpha, pi and theta-class-like glutathione S-transferase genes from goldfish (Carassius auratus L). Their derived amino acid sequences were clustered with other vertebrate alpha, pi and theta-class GSTs in a phylogenetic tree and the goldfish GST sequences have the highest similarity with those from common carp and zebrafish. Goldfish were i.p. injected with microcystins extract at two doses (50 and 200microgkg(-1)BW MC-LReq) and the relative changes of the mRNA abundance in liver, kidney and intestine were analyzed by real-time PCR. The transcription of GST alpha was suppressed in both liver and intestine, but induced in the kidney. Decreased transcription of GST theta was detected in liver, kidney and intestine in the low-dose group. The transcription of GST pi was suppressed in liver and intestine post-injection in both dose groups. These results suggested that the transcription of GST isoforms varied in different ways within an organ and among organs of goldfish exposed to MCs.

The profound effects of microcystin on cardiac antioxidant enzymes, mitochondrial function and cardiac toxicity in rat

Deaths from microcystin toxication have widely been attributed to hypovolemic shock due to hepatic interstitial hemorrhage, while some recent studies suggest that cardiogenic complication is also involved. So far, information on cardiotoxic effects of MC has been rare and the underlying mechanism is still puzzling. The present study examined toxic effects of microcystins on heart muscle of rats intravenously injected with extracted MC at two doses, 0.16LD(50) (14 microg MC-LReq kg(-1) body weight) and 1LD(50) (87 microg MC-LReq kg(-1) body weight). In the dead rats, both TTC staining and maximum elevations of troponin I levels confirmed myocardial infarction after MC exposure, besides a serious interstitial hemorrhage in liver. In the 1LD(50) dose group, the coincident falls in heart rate and blood pressure were related to mitochondria dysfunction in heart, while increases in creatine kinase and troponin I levels indicated cardiac cell injury. The corresponding pathological alterations were mainly characterized as loss of adherence between cardiac myocytes and swollen or ruptured mitochondria at the ultrastructural level. MC administration at a dose of 1LD(50) not only enhanced activities and up-regulated mRNA transcription levels of antioxidant enzymes, but also increased GSH content. At both doses, level of lipid peroxides increased obviously, suggesting serious oxidative stress in mitochondria. Simultaneously, complex I and III were significantly inhibited, indicating blocks in electron flow along the mitochondrial respiratory chain in heart. In conclusion, the findings of this study implicate a role for MC-induced cardiotoxicity as a potential factor that should be considered when evaluating the mechanisms of death associated with microcystin intoxication in Brazil.

Toxic effect of NaCl on ion metabolism, antioxidative enzymes and gene expression of perennial ryegrass.

Two-month old seedlings of perennial ryegrass (Lolium perenne L.) were subjected to four different levels of salinity for 7 days. The NaCl treatments reduced turf quality and normalized transpiration rates. Both chlorophyll (Chl) a and Chl b contents decreased in the grass exposed to 255 mM relative to the control. An increase in the lipid peroxidationin was observed. The activity of leaf superoxide dismutase increased while, peroxidase and catalase activities decreased in response to NaCl treatments. The expression of Chl Cu/ZnSOD, Cyt Cu/ZnSOD, FeSOD, CAT, POD, GPX and GR was up-regulated for NaCl-treated grass. Salt stress increased accumulation of Na(+) and decreased K(+)/Na(+) ratio, Mg(2+) and P content in both shoots and roots of perennial ryegrass. The findings of this study suggest that salt stress may cause toxicity to perennial ryegrass through oxidative injury and damage to Chl and cell membrane integrity.

Acute effects of microcystins exposure on the transcription of antioxidant enzyme genes in three organs (liver, kidney, and testis) of male Wistar rats.

Microcystins (MCs) induce the production of reactive oxygen species (ROS) in various tissues in mammals, whereas the endogenous antioxidant enzymes are responsible to scavenge the ROS. ROS can modulate the antioxidant enzyme activities by regulating the mRNA levels. The present study was undertaken to find out the relationship between the transcriptional alterations of antioxidant enzymes and MCs stimulation in rats. The time‐dependent changes of relative transcription abundance of catalase (CAT), Mn‐superoxide dismutase (Mn‐SOD), Cu,Zn‐superoxide dismutase (Cu,Zn‐SOD), glutathione reductase (GR), glutathione peroxidase (GPx), and gamma‐glutamylcysteine synthetase (γ‐GCS) were investigated in three organs (liver, kidney, and testis) of male Wistar rats injected intravenously (i.v.) with 80 μg MC‐LRequivalent/kg body weight using the quantitative real‐time PCR (qPCR) method. We found that MCs could affect the transcriptional activities of these antioxidant enzymes in liver, kidney, and testis of MCs‐treated rats and we speculated the possible causation of the transcriptional change. The altered transcription of antioxidant enzymes may play an important role in counteracting the potential deleterious effects of elevated oxidative stress induced by MCs, and this will provide us new insights into the possible role of antioxidant enzymes in the toxicological mechanisms of MCs at molecular level.

Involment of p53, Bax, and Bcl-2 pathway in microcystins-induced apoptosis in rat testis

It has been reported that microcystins (MCs) could accumulate in the gonads of mammals and MCs exposure exerts obvious toxic effects on male reproductive system of mammals. We have comfirmedthat MCs could accumulate and induce apoptosis in rat testis. The p53, Bax, and Bcl‐2 protein play important roles in mitochondria‐dependent apoptotic pathway, and this study aimed to investigate whether the p53, Bax, and Bcl‐2 pathway is involved in microcystins‐induced apoptosis in rat testis and discussed the possible mechanisms. Our results show that MCs led to persistent increase of transcriptional and protein level of P53 and Bax expression but led to decrease of Bcl‐2 expression, resulting in an increased ratio of Bax to Bcl‐2, which might contribute to apoptotic cell death of rat testis following MCs treatment. The increased ratio of expression of Bax to that of Bcl‐2 induced by MCs suggests their important role in MCs‐induced apoptosis in rat testis tissue.

Antioxidant responses and gene expression in perennial ryegrass (Lolium perenne L.) under cadmium stress

Perennial ryegrass (Lolium perenne L.), widely used in temperate climates, is one of turf grasses that enrich cadmium (Cd). The objective of this study was to explore the physiological responses and candidate gene expression in perennial ryegrass to Cd stress. Grasses were subjected to three levels of 0, 0.2, and 0.5xa0mM Cd for 7xa0days in the greenhouse. The results indicated that soluble protein content was lower in the Cd-treated perennial ryegrass compared to untreated plants. The Cd-treated perennial ryegrass exhibited a greater level of malondialdehyde and activity of the peroxidase (POD), catalase, and superoxide dismutase (SOD) relative to the control. The Cd stress induced up-regulated expression of FeSOD, MnSOD, Chl Cu/ZnSOD, Cyt Cu/ZnSOD, APX, GPX, GR and POD at 4–24xa0h after treatment began for perennial ryegrass. Results suggested that the gene transcript profile was related to the enzyme activity under Cd stress.

Microcystin Extracts Induce Ultrastructural Damage and Biochemical Disturbance in Male Rabbit Testis

In the present research, the changes of ultrastructures and biochemical index in rabbit testis were examined after i.p. injection with 12.5 μg/kg microcystin (MC) extracts. Ultrastructural observation showed widened intercellular junction, distention of mitochondria, endoplasmic reticulum, and Golgi apparatus. All these changes appeared at 1, 3, and 12 h, but recovered finally. In biochemical analyses, the levels of lipid peroxidation (MDA) and H2O2 increased significantly at 1 h, indicating MC‐caused oxidative stress. Finally, H2O2 decreased to the normal levels, while MDA remained at high levels. The antioxidative enzymes (CAT, SOD, GPx, GST) and antioxidants (GSH) also increased rapidly at 1 h, demonstrating a quick response of the defense systems to the oxidative stress. Finally, the activity of CAT, SOD, and GPX recovered to the normal level, while the activity of GST and the concentration of GSH remained at a high level. This suggests that the importance of MCs detoxification by GST via GSH, and the testis of rabbit contained abundant GSH. The final recovery of ultrastructure and some biochemical indexes indicates that the defense systems finally succeeded in protecting the testis against oxidative damage. In conclusion, these results indicate that the MCs are toxic to the male rabbit reproductive system and the mechanism underlying this toxicity might to be the oxidative stress caused by MCs. Although the negative effects of MCs can be overcome by the antioxidant system of testis in this study, the potential reproductive risks of MCs should not be neglected because of their wide occurrence.

Toxic effects, uptake, and translocation of Cd and Pb in perennial ryegrass.

Cadmium (Cd) and lead (Pb) are regarded as the most toxic environmental pollutants because they are a threat not only to plants, but also to humans. Better understanding of the growth response, uptake and translocation of Cd and Pb in perennial ryegrass (Lolium perenne L.) would be helpful to evaluate the role of this grass species as a potential candidate used for phyto-remediation. Perennial ryegrass seedlings were subjected to three Pb levels (0, 0.5, and 3.2xa0mM) for 6xa0days and three Cd levels (0, 0.2, and 0.5xa0mM) for 7xa0days in a walk-in growth room. The Cd and Pb reduced the normalized transpiration and growth rate. Greater amount of Cd and Pb accumulated in roots relative to shoots. The enrichment coefficient (EC) and translocation factor (TF) were greater in plants treated with Cd than those with Pb. The dose of Cd had no effect on EC and TF. However, the plants subjected to higher dose of Pb had a greater TF and a lower EC relative to that with lower dose of Pb. Meanwhile, removal rates were decreased when the doses of Pb and Cd increased. The results indicated that perennial ryegrass preferred Cd assimilation to Pb, and can be considered as one of the plant species which accumulate high levels of Cd and Pb.