Shuang-Qing Peng

Enhanced toxicity and ROS generation by doxorubicin in primary cultures of cardiomyocytes from neonatal metallothionein-I/II null mice

The clinical use of doxorubicin (Dox), a potent anticancer drug, is limited by its concurrent dose-dependent cardiotoxicity. We previously found that metallothionein-I/II (MT-I/II) null mice are more vulnerable to Dox-induced cardiomyopathy, but it is unknown whether depletion of MT would sensitize cardiomyocytes to Dox toxicity in vitro since the protective effect of MT still remains controversial. In the present study, a primary culture system of cardiomyocytes from neonatal MT-I/II null (MT(-/-)) and corresponding wild type (MT(+/+)) mice was established to unequivocally determine the effect of MT deficiency on Dox-induced toxicity. MT concentrations in the MT(-/-) cardiomyocytes were about 2.5-fold lower than those in MT(+/+) cardiomyocytes. MT(-/-) cardiomyocytes were more sensitive to Dox-induced cytotoxicity than MT(+/+) cardiomyocytes as measured by morphological alterations, lactate dehydrogenase leakage, cell viability, and apoptosis. Dox time- and concentration-dependently increased reactive oxygen species (ROS) formation in MT(+/+) cardiomyocytes, and this effect was exaggerated in MT(-/-) cardiomyocytes. Antioxidant N-acetylcysteine (NAC) and glutathione (GSH) significantly rescued MT(+/+) but not MT(-/-)cardiomyocytes from Dox-induced cell death and ROS generation. These findings suggest that basal MT provide protection against Dox-induced toxicity in cardiomyocytes, particularly highlight the important role of MT as a cellular antioxidant on scavenging ROS.

Repeated exposures to chlorpyrifos lead to spatial memory retrieval impairment and motor activity alteration.

Chlorpyrifos (CPF) is one of the most commonly used insecticides throughout the world and has become one of the major pesticides detected in farm products. Chronic exposures to CPF, especially at the dosages without eliciting any systemic toxicity, require greater attention. The purpose of this study was, therefore, to evaluate the behavioral effects of repeated low doses (doses that do not produce overt signs of cholinergic toxicity) of CPF in adult rats. Male rats were given 0, 1.0, 5.0 or 10.0mg/kg of CPF through intragastric administration daily for 4 consecutive weeks. The behavioral functions were assessed in a series of behavioral tests, including water maze task, open-field test, grip strength and rotarod test. Furthermore, the present study was designed to evaluate the effects of repeated exposures to CPF on water maze recall and not acquisition. The results showed that the selected doses only had mild inhibition effects on cholinesterase activity, and have no effects on weight gain and daily food consumption. Performances in the spatial retention task (Morris water maze) were impaired after the 4-week exposure to CPF, but the performances of grip strength and rotarod test were not affected. Motor activities in the open field were changed, especially the time spent in the central zone increased. The results indicated that repeated exposures to low doses of CPF may lead to spatial recall impairments, behavioral abnormalities. However, the underlying mechanism needs further investigations.

Global gene expression profiles of MT knockout and wild-type mice in the condition of doxorubicin-induced cardiomyopathy.

Increasing evidence from in vivo and in vitro studies has indicated that MT exerts protective effects against DOX-induced cardiotoxicity; however the underlying precise mechanisms still remain an enigma. Therefore, the present study was designed using MT knockout mice in concert with genomic approaches to explore the possible molecular and cellular mechanisms in terms of the genetic network changes. MT-I/II null (MT⁻/⁻) mice and corresponding wild-type mice (MT+/+) were administrated with a single dose of DOX (15 mg/kg, i.p.) or equal volume of saline. Animals were sacrificed on the 4th day after DOX administration and samples were collected for further analyses. Global gene expression profiles of cardiac mRNA from two genotype mice revealed that 381 characteristically MT-responsive genes were identified between MT+/+ mice and MT⁻/⁻ mice in response to DOX, including fos, ucp3, car3, atf3, map3k6, etc. Functional analysis implied MAPK signaling pathway, p53 signaling pathway, Jak-STAT signaling pathway, PPAR signaling pathway, Wnt signaling pathway, etc. might be involved to mediate the protection of DOX cardiomyopathy by MT. Results from the present study not only validated the previously reported possible mechanisms of MT protection against DOX toxicity, but also provided new clues into the molecular mechanisms involved in this process.

Peroxiredoxins are involved in metallothionein protection from doxorubicin cardiotoxicity

Previous studies have shown that metallothionein (MT) can antagonize the myocardiotoxicity induced by doxorubicin (Dox), a most effective anticancer agent. However, the molecular mechanisms are not well-understood. Using a proteomics approach we have detected that major peroxiredoxins (Prxs), an important redox regulating molecule family, may be involved in this process. In the present study, we assessed a link between metallothionein and peroxiredoxins. Wild-type (MT(+/+)) and MT(-/-) mice were treated intraperitoneally with doxorubicin at a single dose of 15 mg/kg and sacrificed on the 4th day after treatment. Doxorubicin induced cardiotoxicity in both wild-type and MT(-/-) mice was manifested with increased serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities, and cardiac morphological changes. These toxic responses were stronger in the hearts of MT(-/-) mice that were more vulnerable to doxorubicin-induced oxidative injury as exhibited by increased lipid peroxidation and decreased catalase and glutathione peroxidase (GSH-Px) expression. Moreover, in the MT(-/-) mice, the deficiency of metallothionein inhibited the expression of Cu/Zn Superoxide dismutase (SOD-1) induced by doxorubicin. Doxorubicin significantly increased the mRNA levels and protein expressions of Prx-1, -2, -3, -5, and -6 in the hearts of wild-type but not MT(-/-) mice. Therefore, the present study suggests that metallothionein provides protection against doxorubicin-induced cardiotoxicity, which possibly involves regulation of peroxiredoxins.

Metallothionein-I/II null cardiomyocytes are sensitive to Fusarium mycotoxin butenolide-induced cytotoxicity and oxidative DNA damage.

Previous studies revealed butenolide (BUT), a Fusarium mycotoxin distributes extensively, induced myocardial oxidative damage, which could be abated by antioxidants such as glutathione. Metallothionein (MT) has proved to attenuate several oxidative cardiomyopathies via its potent antioxidant property. The present study is therefore undertaken to investigate the protective potential of the endogenous expression of MT against BUT-induced myocardial toxicity. Primary cultures of neonatal cardiomyocytes from MT-I/II null mice along with the corresponding wild-type mice will be utilized to determine the possible mechanistic properties of MT. BUT treatment to the cardiomyocytes evoked significant cytotoxicity as evidenced by morphological changes and concentration- and time-dependent reductions in cell viability. Additionally, BUT treatment remarkably increased reactive oxygen species (ROS) production in the cardiomyocytes of both MT-I/II null and wild-type mice. As a result, noticeable DNA damage in both cardiomyocytes was detected by alkaline comet assay. Furthermore, the comparison between the MT-I/II null and wild-type cardiomyocytes indicated that ROS production in the cardiomyocytes from the MT-I/II null mice was higher than from wild-type mice. DNA damage as evaluated by percentage of comet tail DNA, tail length and tail moment was more severe in the MT-l/II null cardiomyocytes than in wild-type myocytes. And in agreement with those results mentioned above, the MT-l/II null cardiomyocytes were more sensitive to BUT-induced cytotoxicity than wild-type cardiomyocytes. Taken together, these findings clearly show that basal MT can efficiently attenuate BUT-induced cytotoxic injuries in cardiomyocytes via the inhibition of intracellular ROS production, and associated DNA damage.

Butenolide induced cytotoxicity by disturbing the prooxidant-antioxidant balance, and antioxidants partly quench in human chondrocytes.

Butenolide (BUT), a mycotoxin produced by Fusarium species, was detected often in corns or grains from endemic Kashin-Beck disease (KBD) areas in China. In this study, we evaluated the cytotoxicity of BUT on chondrocytes and the possible toxic mechanism with the aim of understanding the pathogenesis and of directing future therapeutic interventions for KBD. Exposure of human chondrocytes and engineered cartilage to high concentration of BUT (> 1 microg/ml) resulted in significant cytotoxicity, manifested by losses in cell viability and changes in cell morphology. BUT with high concentration (> 1 microg/ml) also induced significant oxidative damage to chondrocytes in vitro evidenced by increasing both lipid peroxidation and endogenous antioxidants. Furthermore, free radical scavenging agents, such as selenium (Se), vitamin C (VC) and vitamin E (VE), partly blocked BUT-induced oxidative damage. In conclusion, this finding indicates that BUT induces cytotoxicity to human chondrocytes, and the disturbance of prooxidant-antioxidant balance may play a pivotal role in BUT-induced injuries in chondrocytes. Moreover, Se, VC or VE can quench the toxic effects of BUT to a certain extent, which will possibly direct future therapeutic interventions against KBD.

Effects of Fusarium mycotoxin butenolide on myocardial mitochondria in vitro.

Fusarium mycotoxin toxicosis has been implicated in the etiology of Keshan disease, an endemic mitochondrial cardiomyopathy prevailing in certain areas of China. Butenolide (4-acetamido-4-hydroxy-2-butenoic acid γ-lactone) is one of the Fusarium mycotoxins which are frequently detected from cereal grains in endemic areas. A recent study indicates that this mycotoxin induces rat cardiotoxicity, but its effect on the myocardial mitochondria remains unclear. The present study is therefore undertaken to explore the toxic effect potential of butenolide on the myocardial mitochondria. Exposure of cultured cardiac myocytes to 50 μg/ml of butenolide provoked dissipation of mitochondrial membrane potential. Incubation of isolated rat myocardial mitochondria with butenolide of 100 μg/ml for 60 min resulted in mitochondrial swelling, indicating the occurrence of mitochondrial permeability transition. Furthermore, marked oxidative damage in myocardial mitochondria was observed after incubation of isolated myocardial mitochondria with butenolide ranging from 0 to 50 μg/ml for 60 min, as manifested by concentration-dependent increases in the production of thiobarbituric acid reactive substances, the indicator of lipid peroxidation. Contrarily, a representative antioxidant glutathione significantly alleviated this oxidative mitochondrial damage induced by butenolide. In conclusion, these observations clearly show that butenolide can induce dysfunction of myocardial mitochondria, and oxidative damage appears to play a crucial role in these deleterious effects. The present study supports the hypothesis that mycotoxin toxicosis is a probable etiological factor of Keshan disease, the mitochondrial cardiomyopathy.

Zinc-induced metallothionein overexpression prevents doxorubicin toxicity in cardiomyocytes by regulating the peroxiredoxins.

Abstract 1. Cardiotoxicity is an important factor that limits the clinical use of doxorubicin (Dox). Metallothionein (MT) can antagonize the Dox-induced cardiotoxicity. Using a proteomics approach we have detected that major peroxiredoxins (Prxs) may be involved in this process. In the present study, we further investigate the mechanisms of the MT effects against Dox-induced cytotoxicity and the interactions between MT and Prxs. 2. We have established a primary cardiomyocyte culture system from MT-I/II null (MT−/−) and corresponding wild type (MT+/+) neonatal mice, and pretreated the MT+/+ cardiomyocytes with ZnCl2 to establish the MT overexpression cardiomyocyte model. 3. Based on the results, in MT+/+ cardiomyocytes, ZnCl2 pretreatment significantly increased the cardiomyocytes MT levels and inhibited the cardiotoxicity of Dox; it can resist LDH leakage, cardiomyocyte apoptosis, DNA damage, ROS accumulation and inhibit the decrease in activity of antioxidant enzymes induced by Dox. Moreover, ZnCl2 enhanced the expression of Prx-2, -3, -5 and -6, it can inhibit the expression of Prxs decrease in MT+/+ cardiomyocytes induced by Dox, but had no effect in MT−/− cardiomyocytes. 4. Therefore, the present study suggests that ZnCl2 can protect the cardiomyocytes from the Dox-induced oxidative injury and can inhibit the changes in Prxs expression through induced MT overexpression.

Basal expression of metallothionein suppresses butenolide-induced oxidative stress in liver homogenates in vitro

Butenolide (4-acetamido-4-hydroxy-2-butenoic acid gamma-lactone) is a Fusarium mycotoxin which is frequently detected in foodstuffs and feedstuffs for human and animal consumption. It can evoke a broad spectrum of toxicities, thus posing a potential health risk to both humans and animals. Previous study showed that this mycotoxin produced a significant oxidative stress, and several antioxidants abated this effect. Metallothionein (MT) has been proposed as a potent antioxidant, therefore, this study attempts to determine whether endogenous expression of MT protects against butenolide-induced hepatic oxidative stress by using an in vitro incubation system of liver homogenates prepared from MT-I/II null (MT-/-) mice, and the corresponding wild type (MT+/+) mice. The results showed that butenolide elicited significant oxidative stress in both MT-/- mice and MT+/+ mice; however, MT-/- mice were more sensitive than MT+/+ mice to butenolide-induced hepatic oxidative stress, as evidenced by more production of thiobarbituric acid reactive substances and nitric oxide, and by more severe reductions of glutathione, superoxide dismutase and glutathione peroxidase in the liver homogenates of MT-/- mice than those of MT+/+ mice. These findings implicated the antioxidant potency of basal expression of MT in suppression of the oxidative stress of butenolide.

Study of embryotoxicity of Fusarium mycotoxin butenolide using a whole rat embryo culture model.

Butenolide, a mycotoxin elaborated by several toxigenic Fusarium species, has been implicated as an etiological factor of Kashin-Beck disease and it is always detected in food from endemic Kashin-Beck disease areas. Although butenolide is considered as a potential health risk to humans and animals, its toxicity targets and mechanism of action have not been fully understood and the knowledge of its developmental toxicity is absent. The present study investigated butenolide embryotoxicity via an in vitro whole embryo culture system using rat embryos. Embryos exposed to butenolide at a concentration of 0.625 mg/L showed and differentiation similar to that of the control embryos (=no observed adverse effect concentration; NOAECwec). The embryonic growth and differentiation were affected, represented as reduced crown-rump length and head length, and decreased number of somites from 1.25 mg/L. Total morphological scores decreased significantly at the concentration of butenolide of 2.5 mg/L. All embryos were malformed at 3.75 mg/L and above (=ICMaxWEC), presenting growth retardation with flexion failure and irregular somite differentiation. The IC503T3 of butenolide as calculated from the balb/c 3T3 cytotoxicity test is 6.45 mg/L. Our study shows that butenolide exerts detrimental effects on embryo development in vitro by inducing growth retardation and differentiation inhibition, and the embryotoxicity effect of butenolide should be treated with caution.