Study On The Mechanism Of Toxicity Of T-2 Toxin On Primary Cultured Chondrocytes From Chicken Tibial Growth Plate

Endochondral ossification is a process by which growth plate chondrocytes of the embryonic cartilaginous model of most bones contributing to longitudinal growth progressively proliferate and differentiate and ultimately are replaced by bone. T-2 toxin, the most toxic non-macrocyclic type A trichothecene mycotoxin, is a secondary metabolite of the Fusarium fungi that is now considered to be related to bone malformation such as the tibial dyschondroplasia and Kaschin-Beck disease. A lot of relevant studies have shown that T-2 toxin interferes with the normal development of chondrocytes involving cell apoptosis. Previous studies have shown that T-2 toxin will trigger abnormal bone development, manifesting, for instance, as tibial dyschondroplasia, a disease reminiscent of human osteoarthropathy and characterized by an abnormal plug of nonvascularized, unmineralized cartilage persisting in the proximal metaphysis of the tibiotarsus, through interrupting endochondral ossification. However, previous studies examining the effects of T-2 toxin mainly were limited to the immune and hemogenesis systems or the in vitro toxicity of T-2 on skeletal metabolism of articular cartilage chondrocytes. In our study, therefore, a very pure preparation of isolated proximal growth plate chondrocytes from chicken tibia was brought into culture to study the mechanism of toxicity of T-2 toxin on chicken growth plate chondrocytes.Experiment I Investigation of isolation, culture, and biological characteristics of chicken growth plate chondrocytesIsolating and culturing primary chondrocytes such that they retain their cell phenotype and differentiate to a hypertrophic state is important to many investigations of skeletal growth and its regulation. The ability of chondrocyte proliferation and differentiation in vitro has additional scientific and tissue engineering interest. Investigations of growth plate dysplasias in chicken skeletal disease led us to adapt and modify a culture method of chicken growth plate chondrocytes. In this experiment, chondrocytes were isolated from tibial growth plate of 6-week-old chicken by collagenase and hyaluronidase digestions. Cells were plated in flat-bottom wells and allowed to adhere and grow under DMEM medium containing 10%FBS. Cell growth was monitored, as was expression of several relevant genes and protein:collagen types II and X, aggrecan, and others. Mineralization was measured by alizarin red binding, and cultures were also examined by light and electron microscopy. Results showed that cells retained their chondrocyte phenotype and ability to differentiate and mineralize the collagen-rich extracellular matrix. RT-PCR showed retention of chondrocyte-specific gene expression of X collagen. These cells had a flattened appearance initially, and by 2 weeks post-confluence, exhibited swelling and other salient features of hypertrophic cells seen in vivo. Collagen fibrils were abundant in the extracellularmatrix, along with matrix vesicles. If the ascorbate concentration was maintained below 25μg·mL-1, this apparent dedifferentiation to fibroblastic morphology was not seen. The transition from proliferating to hypertrophic chondrocytes is associated with both an induction of alkaline phosphatase. Conclusions:This method should prove valuable as a means of studying chondrocyte regulation and has the advantages of simpler initial dissection, yields of a purer chondrocyte population, and the ability to proliferate and differentiate for subsequent studies.ExperimentⅡEffect of inorganic phosphate on calcification of chicken growth plate chondrocytesInorganic phosphate (Pi) plays an important role in bone metabolism and terminal differentiation of growth plate chondrocytes. To explore the effects of Pi on the gowth plate chondnrocytes (GPCs) in vitro, Pi-supplemented media (0-7 mmol·L-1) were used in the present study. Our results based on biochemical analysis of these cultures showed that (1) Pi decreased the number of viable cells based on MTT assay (P<0.05) and promote the cell differentiation. (2) Calcification of GPC cultures can be induced by addition of Pi. It may be that Pi is rate-limiting for the calcification of chondrocyte cultures. Pi-induced calcification of GPCs was time-and dose-dependent and was correlated to the maturation of GPCs. (3) Alkaline phosphatase (ALP) activity reaches a maximal value at the time when mineral deposition is initiated. (4)The highest Pi concentration markedly decreased the cell viability. Therefore, Pi-induced terminal differentiation and calcification responded to an appropriated Pi levels. In summary, these findings are consistent with the following mechanism of calcification in chondrocyte cultures:Mineral deposition requires a critical extracellular Pi concentration. This critical concentration can be achieved by supplementation of the culture medium with Pi. This method provides us a possibility to explore the mechanism of Pi-induced calcification in vivo. Meanwhile, this kind of calcified culture system can be used to investigate some factors intervening endochondral ossification.ExperimentⅢEffect of T-2 toxin on proliferation, differentiation, and calcification of primary cultures of chicken growth plate chondrocytesT-2 toxin, the most toxic trichothecene, exerts a profound effect on chicken tibial growth and development. In this study, primary cultures of chicken growth plate chondrocytes (GPCs) were exposed to different concentrations of T-2 toxin to investigate the hypothesis that T-2 toxin interferes with the fate of GPCs by altering the genetic and biochemical modulators of terminal differentiation and calcification. GPCs from the proximal growth plate of avian tibia were isolated, cultured to confluence, and incubated in mineral medium. Added continually to cultures from confluence to harvest, T-2 toxin exerted significant effects on cell viability, mineralization, cell layer calcium and phosphate content, ALP activity, proteoglycan and collagen synthesis, as measured spectrophotometrically (P<0.05). The gene expression of type X collagen, vascular endothelial growth factor, and runt-related transcription factor 2, which are closely related to hypertrophy and sequential initial calcification in chondrocytes, were studied by quantitative real-time PCR. Results revealed that mRNA levels of these genes were significantly inhibited by T-2 toxin in a dose-dependent manner (P<0.05). These results suggest that T-2 toxin inhibits the proliferation, differentiation, and sequential calcification of GPCs in vitro by altering cellular metabolism and, in part, by depressing development-related gene expression.Experiment IV Effect of T-2 toxin on apoptosis and caspase-3 gene expression of chicken growth plate chondrocytes exposed to T-2 toxinPrevious studies have shown that T-2 toxin interferes with the normal development of chondrocytes. Apoptosis induced by T-2 toxin has been postulated to be one of the important mechanisms of its toxic effect. In this study, primary cultures of chicken growth plate chondrocytes (GPCs) were exposed to different concentrations of T-2 toxin to investigate its cytotoxic mechanism. T-2 toxin exerted a significant inhibitory effect on cell viability as assessed by MTT assay. Apoptotic morphological changes induced by T-2 toxin exposure in chondrocytes were demonstrated by Hoechst 33324 staining. Simultaneously, elevation of reactive oxygen species levels and intracellular [Ca2+] i, depletion of mitochondrial membrane potential and intracellular glutathione was observed during the T-2 toxin exposure. In addition, glutathion peroxidase activitiy and capase-3 mRNA level in the living cells increased significantly (P<0.05). These results suggest that T-2 toxin exerts its cytotoxic effects on of GPCs in vitro by altering cellular homeostasis and, in part, by up-regulation of caspase-3 gene expression.Experiment V N-acetyl-cysteine protects chicken growth plate chondrocytes from T-2 toxin-induced oxidative stressT-2 toxin is now considered to be related to bone malformation such as incomplete ossification, absence of bones, and fused bones. In this study, primary cultures of chicken tibial growth plate chondrocytes were treated with various concentrations of T-2 toxin (5, 50, and 500 nmol·L-1) in the absence and presence of N-acetyl-cysteine (NAC) to investigate the effect of the antioxidant NAC on T-2 toxin-induced toxicity. Our results showed that T-2 toxin markedly decreased cell viability, ALP activity, glutathione (GSH) content (P<0.05). Simultaneously, T-2 toxin significantly increased reactive oxygen species (ROS) levels and malondialdehyde (MDA) in a dose-dependent manner. In addition, the living cells increased the activities of catalase (CAT) and superoxide dismutase (SOD) to protect them from T-2 toxin-induced oxidative stress. However, the T-2 toxin-induced cytotoxicity was reversed, in part, by antioxidant NAC (P<0.05). These results suggest that T-2 toxin inhibits the proliferation and differentiation of GPCs in vitro by altering cellular homeostasis and NAC can protect GPCs against T-2 toxin cytotoxicity by reducing the T-2 toxin-induced oxidative stress.