Qian Xiong

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.

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.

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.

The proteomic study on cellular responses of the testes of zebrafish (Danio rerio) exposed to microcystin‐RR

Microcystin‐RR (MC‐RR) is a commonly encountered cyanotoxin and receives increasing attention due to the risk of its bioaccumulation in aquatic animals like fish. This study investigated the protein profiles of zebrafish (Danio rerio) testes after intraperitoneal injection (i.p.) with 0.5 LD50 (2000u2009μg/kg). MC‐RR caused a noticeable damage to testicular ultrastructure, showing widened intercellular junction, distention of mitochondria. The testes showed a rapid response of its defense systems to the oxidative stress caused by MC‐RR. This is the first to use a proteomic approach to obtain an overview of the effects of MC‐RR on the testes of zebrafish. The proteomic results revealed that toxin exposure remarkably altered the abundance of 24 proteins that were involved in cytoskeleton assembly, oxidative stress, glycolysis metabolism, calcium ion binding and other biological functions. In conclusion, MC‐RR damaged the testes and was toxic to the reproductive system of male zebrafish mainly through causing oxidative stress.

The effect of cyanobacterial crude extract on the transcription of GST mu, GST kappa and GST rho in different organs of goldfish (Carassius auratus).

The glutathione S-transferases play important roles in the detoxification of microcystin. Core-sequences of three classes of GST (mu, kappa and rho) were cloned from goldfish (Carassius auratus L.) i.p. injected with cyanobacterial crude extract at two doses (50 and 200 microg MC-LReq kg(-1)BW). The relative changes of the mRNA abundance in liver, kidney and intestine were analyzed by real-time PCR. The transcription of GST mu was inhibited in intestine at both doses and the transcription of GST kappa was inhibited from 12 to 48h in kidney at both doses. The decreased transcription of GST rho was detected in all three organs at the high dose. It is suggested that transcription inhibition of GST rho might be significant in MCs toxicity at higher toxin concentration in omnivorous freshwater fish. Alteration in transcription of GSTs stimulated by MCs implicates an increased health risk to fish.

Transcriptional alteration of cytoskeletal genes induced by microcystins in three organs of rats

This study explored the mechanisms of toxicity of microcystins by measuring the transcription levels of nine cytoskeletal genes (actin, tubulin, vimentin, ezrin, radixin, moesin, MAP1b, tau, stathmin) in the liver, kidney and spleen of male Wistar rats treated with microcystins at a dose of 80 microg MC-LReq kg(-1) bw. Microcystins disrupted the transcriptional homeostasis of cytoskeletal genes in these organs. Changes in the transcription of four genes (beta-actin, ezrin, radixin and tau) in liver, one gene (stathmin) in kidney, and one gene (radixin) in spleen were significantly correlated with the tissue concentration of microcystins. However, the influences on the transcription of most genes we studied were greater in the liver than in the kidney or spleen. The effects of microcystins on the transcription of cytoskeletal genes may explain some of the morphological and pathological changes observed in these organs and provide new information on the hepatotoxicity of these compounds. Additionally, transcriptional changes in tumor-associated cytoskeletal genes (ezrin, moesin and stathmin) that were observed in the present study provide a possible clue to the tumor-promoting potential of microcystins and their influences on the transcription of MAP1b and tau imply possible neurological toxicity of microcystins in vertebrates.

Quantitative profiling of mRNA expression of glutathione S-transferase superfamily genes in various tissues of bighead carp (Aristichthys nobilis).

The expression of glutathione S‐transferase (GST) is a crucial factor in determining the sensitivity of cells and organs in response to a variety of toxicants. In this study, we cloned the core nucleotide of alpha, kappa, mu, mGST, pi, rho, and theta‐like GST genes from bighead carp (Aristichthys nobilis). Their derived amino acid sequences were clustered with other vertebrate GSTs in a phylogenetic tree, and the bighead carp GST sequences have the highest similarity with those from common carp and zebrafish. We quantified the constitutive mRNA transcription of GST isoforms in eight different tissues (liver, kidney, spleen, intestine, muscle, heart, brain, and gill). The information obtained from the present study could be distilled into a few generalized principles: multiple GST isoenzymes were ubiquitously expressed in all tissues; majority of GSTs had high constitutive expression in intestine, liver, and kidney. These findings are in agreement with the roles of these tissues in xenobiotic metabolism. At the same time, some unique findings were detected in the current experiment: (1) higher expression of most GSTs was observed in spleen; (2) the expression of GST pi was highest in almost all the studied tissues except muscle; the other two isoforms, GST alpha and rho, were also highly expressed in liver, kidney, intestine, spleen, heart, and brain of bighead carp. All these results strongly imply an important role of these GST isoforms in detoxification of ingested xenobiotics.