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Poult Sci 2007. 86:2390-2395. doi:10.3382/ps.2007-00219
© 2007 Poultry Science Association
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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION

Thiram-Induced Changes in the Expression of Genes Relating to Vascularization and Tibial Dyschondroplasia1

N. C. Rath2, W. E. Huff and G. R. Huff

Poultry Production and Product Safety Research Unit, Agricultural Research Service, USDA, Fayetteville, AR 72701

2 Corresponding author: nrath{at}uark.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tibial dyschondroplasia (TD), a major metabolic cartilage disease in poultry, is characterized by the distension of proximal growth plates of tibia that fail to form bone, lack blood vessels, and contain nonviable cells. Thiram, a carbamate pesticide, when fed to young broiler chicks induces TD with high regularity and precision. We used this experimental model to understand the cause of the defects associated with TD by determining the expression of selective candidate genes associated with vascularization and cell survival. Week-old broiler chickens were fed 100 ppm thiram for 48 h between d 8 and 10 posthatch and the expression of the genes for vascular endothelial growth factor (VEGF), its receptors (VEGFR1 and VEGFR2), and an antiapoptotic protein (Bcl-2) were determined in the growth plate cartilage at 48 and 166 h after feeding thiram. Reverse transcription PCR and capillary electrophoresis were used to determine the expression of these genes relative to the 18S gene as an internal standard. There was an increase in the expression of the VEGF gene by thiram at 48 h, which remained elevated above the control level at 166 h. A suppression of genes encoding both VEGF receptors and Bcl-2 was evident at 48 h in thiram-fed chickens when there was no visible distension of growth plate indicative of TD. At 166 h, however, there was a significant distension of growth plates in thiram-treated birds, with a high percentage of cells derived from these tissues exhibiting characteristics of dead cells. Although the expressions of VEGF receptors were low at 166 h in thiram-treated birds, they were not statistically different from controls; the Bcl-2 gene expression, however, remained significantly downregulated in those birds. It appears that some of the early effects of thiram on the growth plate may be the failure of genes encoding VEGF receptors and Bcl-2 resulting from endothelial cell death, which compromise vascularization, cartilage remodeling, and the removal of dead chondrocytes leading to TD lesions.

Key Words: tibial dyschondroplasia • thiram • angiogenesis • gene expression


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tibial dyschondroplasia (TD) is a metabolic cartilage disease in meat-type poultry that causes lameness, bone breakage, and infection (Leach and Nesheim, 1965; Edwards, 1990; Thorp et al., 1991; Orth and Cook, 1994; Farquharson, 2002; Pines et al., 2005). An atypical accumulation of dead chondrocytes in the proximal growth plates of tibia and tibiotarsal bones is accompanied by the failure of cartilage remodeling and bone formation and eventually leads to the pathogenesis of TD (Rath et al., 1998, 2005). Long bone growth is a complex process that entails replacement of cartilage template by bone facilitated by vascular invasion and angiogenesis (Gerber et al., 1999; Provot and Schipani, 2005). Vascular endothelial growth factor and its receptors are some of the major factors behind the angiogenesis process (Neufeld et al., 1999; Ferrara, 2004). Targeted disruption of VEGF expression has been shown to impair proper growth plate development in mammals (Gerber et al., 1999; Maes et al., 2004). Dithiocarbamate pesticides are environmental chemicals widely used in many agricultural and household applications as fungicides, herbicides, and insect and rodent repellants (USEPA, 2001). Chronic exposures to some dithiocarbamate pesticides such as the tetramethyl or tetraethyl thiuram disulfide (thiram and disulfiram, respectively) increase the incidence of TD in chickens (Vargas et al., 1983; Edwards, 1990). We have shown that feeding week-old broiler chickens diets containing 50 to 100 ppm thiram for a day or two leads to the pathogenesis of severe TD with good precision and regularity (Rath et al., 2004, 2007). Therefore, this experimental model is amenable to study the mechanism of TD and to assay for the factors that may prevent the disease. Because some of the major hallmark features of TD lesions are the avascularity of the cartilage plug and the presence of dead chondrocytes (Rath et al., 1998, 2005; Praul et al., 2000), the objective of this study was to determine whether thiram alters the expression of genes associated with angiogenesis and cell survival. Using selective candidate genes associated with angiogenesis, vascular endothelial growth factor (VEGF), its receptors (VEGFR1 and VEGFR2), and an antiapoptotic protein, Bcl-2, that is crucial to cell survival (Eguchi et al., 1992), the current report examines their expression in the growth plates of chickens at an early and a late time interval following treatment with thiram.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All animal experiments were conducted according to applicable institutional animal care and use guidelines. Unless specified, all reagents and chemicals were purchased from Sigma Chemical Company (St. Louis, MO).

Induction of TD

Nonvaccinated male broiler chicks were raised from d 1 of hatch in Petersime batteries at a density of 10 to 12 birds/cage under a constant schedule of 23L:1D. The birds were given chick starter diet prepared according to NRC specifications (NRC, 1994) and free access to water. On d 7, the feed was removed overnight for a period of approximately 14 h before the birds were given either a regular diet (control) or a diet containing 100 ppm tetramethylthiuram disulfide (experimental) for 48 h. Thereafter, all chicks in both control and experimental groups received the same regular feed for the rest of the experimental period. Using this protocol, >90% of chickens fed thiram have been shown to be affected by TD, with lameness discernible by d 15, whereas controls show no growth plate defects (Rath et al., 2004, 2007). Five chickens from each of the control and thiramfed groups were euthanized by cervical dislocation after 48 h (d 10) and 166 h (d 15) of thiram treatment, and the proximal growth plates of each tibia under articular cartilage were exposed. Cylindrical wedges of growth plates from the left tibia of each bird were harvested longitudinally until the metaphyseal junction using a 5-mm curette. The tissues were immediately placed in TRI Reagent (Sigma) and homogenized using plastic pestles. Because of different sizes of the tissues derived from the growth plates of birds, particularly on d 15, the volume of TRI Reagent was adjusted to contain approximately 100 mg of tissue/mL. The tissues from growth plates from the right tibia were similarly harvested and digested in Dulbecco’s modified Eagle’s minimum essential medium containing antibiotics and antimycotics, 2 mM glutamine, 10 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid, 10% fetal bovine serum, and 1 mg each of bacterial collagenase, type IV (Worthington Biochemicals, Lakewood, NJ), and testicular hyaluronidase per milliliter of medium for 20 h to release chondrocytes according to an earlier procedure (Rath et al., 1994).

RNA Extraction, Reverse Transcription, and PCR

Ribonucleic acid extracted using TRI Reagent according to the manufacturer suggested protocol was subjected to gel electrophoresis using 2% agarose to determine the integrity of RNA, after which it was treated with DNase using a Qiagen on-column DNase digestion kit (Qiagen Inc, Chatsworth, CA). The DNA-free RNA was quantified with Ribogreen reagent (Molecular Probe, Eugene, OR), after which, RNA from each sample was reverse-transcribed using a RETROscript kit (Ambion, Austin, TX) to generate cDNA.

The PCR was performed using a multiplex PCR assay master mix (Ambion) to amplify the genes for this study. The dynamic range of the PCR amplification was determined for all the genes using pooled cDNA and was found to be linear in the exponential region of the curve. A set of 2 genes was amplified using a QuantumRNA amplification kit (Ambion), 1 consisting of universal 18S RNA and a competimer at the ratio of 3:7 as internal standard along with one of the other gene primers for VEGF, VEGFR1, VEGFR2, and Bcl-2. The cDNA equivalent of 2 µg of RNA was used in each reaction volume of 20 µL. Preliminary experiments showed suitability of 35 cycles of PCR reaction for each of the 4 genes under study. The primers were designed using Primer 3 software (Rozen and Skaletsky, 2000) from the coding sequences of respective chicken genes in the National Center for Biotechnology Information database and were synthesized by Invitrogen (Invitrogen, Carlsbad, CA; Table 1Go). A PTC 200 gradient cycler (MJ Research, Watertown, MA) was used, and the reaction proceeded for 35 cycles, which involved denaturation at 94° C for 1 min, annealing at 60° C for 30 s, and the extension at 72° C for 30 s, respectively (Rath et al., 2005). The PCR products were diluted 100-fold with water and analyzed using a P/ACE 5500 capillary electrophoresis system equipped with a laser-induced fluorescence detector (Beckman-Coulter, Fuller-ton, CA) using procedures described earlier (Richards and Poch, 2002; Rath et al., 2003, 2005). The sizes of the amplicons were determined from a standard curve obtained using a low-mass DNA ladder (Fermentas, Hanover, MD). A capillary filled with a 70% concentration of DNA gel buffer containing a DNA intercalating dye, SYBR green (Molecular Probe), was used to develop the electropherogram (Richards and Poch, 2002). The samples were injected electrokinetically at 3 kV for 10 s at the cathode end of a 75-µm internal diameter uSIL DNA capillary (Agilent Technology, San Jose, CA) and separated at a constant current of 15 kV for 5 min. In between separations, the capillary was rinsed for 1 min with methanol followed by a 1-min rinse with a fresh gel buffer. The total separating distance from the inlet to the detector was 18 cm. The PCR products were detected as peaks. The relative changes in the expression of different genes were calculated by dividing the peak areas with coamplified 18S peak area. The average expression was based on the estimation of the results from 4 to 5 individual samples per group. The percentage of CV for molecular size estimates of different amplicons was within 10% of the range using capillary electrophoresis.


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  		Table 1. The primer sequences of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptors (VEGFR), and Bcl-2
 
Growth Plate Morphology and Cell Treatments

Growth plate cells were harvested by collagenase-hyaluronidase digestion, as described above, were removed of debris by gravity sedimentation for 15 min, and an aliquot of supernatant from each sample was mixed with an equal volume of trypan blue to determine the percentage of dead cells. It was calculated by counting the number of trypan blue-positive cells relative to the total number of cells in 2 aliquots of each preparation. Between 1 to 1.5 x 103 cells from 10 different fields were counted for statistical determinations. Except for the erythrocytes that were not counted, no other distinction was made with respect to cell types and time factor that may have affected the survival of certain cells. Aliquots of cells were also attached to the slides using a Wescor Cytopro centrifuge (Wescor, Logan, UT) and fixed with 4% p-formaldehyde for 30 min for subsequent staining. The morphological assessment of cells was done first staining the nuclei with 5 µg of propidium iodide/mL of PBS followed by a Sypro orange stain (Molecular Probe) in PBS. The cells were photographed using a fluorescent Olympus microscope.

TD Index

The TD indices of the birds were determined by examining the proximal tibial growth plates according to an earlier procedure (Rath et al., 2004) at 166 h after thiram feeding, because it was not possible to discern differences at 48 h.

Statistics

Quantitative results were expressed as mean ± SEM, analyzed by the GLM procedure and separated using Duncan’s multiple range tests with SAS statistical software (SAS Institute, 1994). Differences were considered significant at P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TD Induction and Gene Expression

There was no visible distinction in the growth plate width at 48 h between the birds that received either control or thiram diets, but at 166 h, there were clear differences with all birds in the thiram-fed group showing a significant increase in growth plate width and severe TD with fragility of the cartilage plug. The gene expression results showed no negative effect of thiram on VEGF either at 48 or 166 h after feeding. On the contrary, an increase in the expression of VEGF was observed at 48 h in thiram-fed birds (Figure 1Go), whereas the expressions of both VEGF receptors were significantly downregulated in thiram-treated chickens only at 48 h. At 166 h, the expression of the genes for the VEGF receptors were low but not significantly different from their age-matched controls. The expression of antiapoptotic protein Bcl-2 remained downregulated in thiram-treated birds at both time intervals (Figure 1Go).


Figure 1
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  		Figure 1. Changes in the expression of candidate genes associated with growth plate vascularization and cell survival at 48 and 166 h after feeding 100 ppm thiram. a–cDissimilar letters over each bar type, symbolic of a specific gene, denote significant differences (P ≤ 0.05). The gene expression changes are represented by the peak area ratio of individual gene product with an 18S internal standard. VEGF = vascular endothelial growth factor; VEGFR = vascular endothelial growth factor receptor.

 
Growth Plate Cell Viability

Comparative evaluation of viability of cells isolated from the growth plates of control and thiram-treated chickens showed a dramatic increase in trypan blue-positive nonviable cells at 166 h in thiram-treated birds (Figure 2Go). The percentage of trypan blue-excluding cells released from the growth plates of birds treated with thiram were not different at 48 h nor showed many morphological differences. Cytocentrifuged cells from d 15 thiram-treated growth plates, however, showed many small cells with pyknotic nuclei that were different from normal healthy cells (Figure 3Go).


Figure 2
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  		Figure 2. Percentage of nonviable chondrocytes released from normal and thiram-treated growth plates of chickens at 48 and 166 h, respectively. Asterisk indicates significant difference with other groups (P ≤ 0.05).

 

Figure 3
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  		Figure 3. Chondrocytes derived from d 15 normal (left) and thiram-induced tibial dyschondroplasia growth plates of birds stained with fluorescent dyes Sypro orange and a nuclear stain propidium iodide. Arrows show typical dead population of cells with invisible to shrunken nuclei. Bar = 20 µ m.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Angiogenesis, a process of new vessel formation from preexisting blood vessel (Folkman, 1990), is essential for endochondral bone formation (Gerber et al., 1999). The new vessels act as portals for the recruitment of osteo-blasts and osteoclasts for bone formation and mediate breakdown of hypertrophying cartilage template and their removal (Maes et al., 2004; Provot and Schipani, 2005). Vascular endothelial growth factor is 1 of the key factors in the process of angiogenesis, which, acting through its receptors, VEGFR1, or Flt-1, and VEGFR2, or Flk-1, stimulates endothelial cell mitogenesis and blood vessel growth (Neufeld et al., 1999; Ferrara, 2004). The prehypertrophic and hypertrophic chondrocytes are principal sources of VEGF in the growth plate (Carlevaro et al., 2000; Karsenty and Wagner, 2002). The results of our study show that the expression of the VEGF gene was not impaired by thiram treatment either at 48 or at 166 h; rather, it showed an increase at 48 h. This is consistent with our earlier observation using the same primer but with β-actin as the reference gene (Rath et al., 2005). However, thiram treatment decreased the expression of both VEGF receptor genes at the 48-h time point, and they recovered to control levels at 166 h. The expression of Bcl-2, a prosurvival and antiapoptotic gene (Eguchi et al., 1992), however, remained suppressed in thiram-treated birds at both time points, whereas a significant increase in the nonviable chondrocyte population was observed at 166 h. These results suggest that the survival and differentiation of prehypertrophic chondrocytes to hypertrophic chondrocytes may not be affected by thiram, but the survival of the hypertrophic chondrocytes is affected in the absence of angiogenesis. It is also supported by the fact that in both naturally occurring and experimentally induced disease, regardless of the severity of the TD, the long bone growth is not impaired, and the proliferative region of the growth plates is not affected (Rath et al., 2007). Besides, an increase in the population of dying chondrocyte is temporally followed by capillary cell death (Rath et al., 2005).

A major difference between an avian and a mammalian growth plate is the vascularity of the cartilaginous growth plate, which in birds contains sporadically distributed blood vessels compared with a completely avascular mammalian growth plate (Pines and Hurwitz, 1991). Both thiram and disulfiram have been shown to be antiangiogenic (Marilkovsky, 2002). Angiogenesis is a required process for chondrocyte hypertrophy and growth plate remodeling (Poole, 1991). In an earlier study, we observed thiram having toxicity to aortic endothelial cells at a much lower dose than chondrocytes (Rath et al., 1995). The death of endothelial cells is likely to affect the expression of VEGF receptor genes, because these receptors are localized in these cells (Ferrara, 2004). Therefore, regardless of the expression of VEGF gene or its corresponding protein by the chondrocytes, the angiogenesis process is likely to be compromised. A recent histochemical study using thiram-induced TD in chickens (C. V. Gay, V. R. Gilman, and R. M. Leach Jr., Department of Poultry Science, Pennsylvania State University, University Park) showed the presence of blunted capillary vessels in the metaphyseal regions of growth plate bordering the TD lesion, although they did not observe any change in the expression of VEGF protein. Impairment of the vascularization process by surgical interference has been shown to prevent bone formation (Trueta and Trias, 1961), and in chickens, lead to a TD-like condition (Riddell, 1977). Also, the arrest of neovascularization of growth plate can preclude removal of apoptotic chondrocytes resulting from the terminal differentiation of hypertrophic chondrocytes, which occurs in the developing growth plate (Zenmyo et al., 1996; Roach, 2002). The absence of removal of apoptotic chondrocytes over a period can result in the accumulation of cartilage containing nonviable chondrocytes leading to the distension of growth plate.

In conclusion, it appears that one of the early effects of thiram may be on the vascular endothelial cells triggering their death, which prevents the expression of genes associated with VEGF receptors and neovascularization of growth plates. These changes can subsequently affect the survival of hypertrophic zone chondrocytes and lead to their accumulation and the distension of growth plate and the TD lesion.


   ACKNOWLEDGMENTS
 
We thank David Horlick, Scott Zornes, Dana Bassi, Sonia Tsai, and Wally McDonner for technical assistance.


   FOOTNOTES
 
1 Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. Back

Received for publication May 31, 2007. Accepted for publication July 16, 2007.


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