Submitted to: Journal of Bone and Mineral Research
Publication Type: Abstract Only
Publication Acceptance Date: August 1, 2003
Publication Date: September 1, 2003
Citation: RATH, N.C. EFFECTS OF PYRROLIDINE DITHIOCARBAMATE (PDTC) ON CHICKEN CHONDROCYTES. JOURNAL OF BONE AND MINERAL RESEARCH. 2003. VOL. 18 (SUPPL. 2) Technical Abstract: Thiocarbamates are widely used as fungicides, seed fumigants. Pyrrolidine dithiocarbamate (PDTC) is used as antioxidants and metabolic inhibitors of a variety of biological reactions including transcription factor NF-kB. PDTC and other thiocarbamates for example, thiram and disulfiram induce tibial dyschondroplasia (TD), a common cartilage problem in meat-type poultry. TD is characterized by the presence of a thickened plug of cartilage in the proximal end of tibial growth plate. To understand how PDTC affects chondrocytes, we studied its effects on the expression of several chondrocyte-associated genes using avian-leukosis virus transformed chickens epiphyseal growth plate chondrocyte (CEGC) cultures. CEGC were grown at a concentration of 100,000 cells /ml/well in 24 well plate for 14 days before they were treated with 2- and 4 micromolar concentrations of PDTC for 24h. The choice of these concentrations of PDTC was based on studies which showed these to be nontoxic to cells. Total RNA extracted from control and PDTC treated cells, were reverse transcribed and cDNA was amplified using PCR and chicken gene specific primers for glyceraldehyde phosphate dehydrogenase (GAPDH), aggrecan, bone morphogenetic protein (BMP)-4 and 7, collagen types II and X, matrix metalloproteinase-2 (MMP-2), and transforming growth factor-beta (TGF-beta). The PCR products were analyzed using capillary electrophoresis and laser-induced fluorescence detection which allows both size determination and quantification of amplicons. The expression of different genes was normalized against GAPDH and the results (n=3/ treatment) were analyzed using Duncans GLM procedure. PDTC at the concentration of both 2 micromolar and 4 micromolar down regulated the expression of aggrecan and type II collagen genes in a dose dependent manner but had no effect on other genes. However, at 4 micromolar concentration PDTC also down regulated the expression of BMP-4, collagen X genes without significant effect on MMP-2, or TGF-beta. These results suggest that PDTC negatively affects chondrocyte development most likely affecting the maturation of prehypertrophic population of chondrocytes. On the other hand, the hypertrophic population of chondrocytes, appear to be less drastically affected since other studies under similar conditions showed that PDTC up to 16 micromolar was not inhibitory to alkaline phosphatase that is responsible for calcification. Extrapolating these in vitro observations to an in vivo situation of growth plate, it is likely that PDTC prevents maturation of chondrocytes eventually leading to their premature death and accumulation as an unresolved plug of dead cartilage.