Comparative analysis of the prevalence of the glutathione S-transferase (GST) system in malignant and benign thyroid tumor cells

ABSTRACT CONTEXT AND OBJECTIVE: When null, the mu and theta genes of the glutathione S-transferase system (GSTM1 and GSTT1, respectively) are related to malignant tumors affecting the lungs, colon, prostate, bladder and head and neck. In the thyroid, the appearance of cancer has been correlated with deletion of these genes. The aim of this study was to compare the frequencies of these genes in patients with benign and malignant tumors of the thyroid gland. DESIGN AND SETTINGS: This was a cross-sectional clinical trial carried out in the Head and Neck Surgery Division, Faculdade de Medicina da Santa Casa de São Paulo. METHODS: Samples of thyroid tissue were collected from 32 patients and divided into two groups: benign tumor (A) and malignant tumor (B). After DNA extraction, the genes were amplified using PCR. RESULTS: The B group presented four cases of positive genotyping for both genes, seven positive for GSTT1 and negative for GSTM1, two negative for GSTT1 and positive for GSTM1, and only one case of double negative. The A group showed 11 cases with positive genotyping for both genes and none with the double negative genotype. CONCLUSION: In this study, there was no relationship between the presence of the GSTT1 and GSTM1 genes and the benign and malignant thyroid tumors.


INTRODUCTION
Human neoplasia occurs by means of a genetic non-lethal lesion in a single stem cell.By multiplying, this damaged cell gives rise to the tumor.These cellular abnormalities, which are advantageous to the cell itself and disadvantageous to the organism, occur because of acquired or inherited genetic alterations that alter the expression of the biochemical properties of the genes, particularly of those involved in the cycle or maintenance of cellular life. 1 The appearance of malignant lesions in the thyroid is related to the deletion of the glutathione S-transferase (GST) theta and mu genes (GSTT1 and GSTM1, respectively). 2 GST consists of a large multigenic group of detoxifi cation enzymes. 3GSTs catalyze the conjugation of toxic compounds with glutathione (GSH -γ-glutamyl cysteinyl glycine, which is a tripeptide), thereby inactivating hazardous effects in the cell.These protein dimers conjugate with electrolytic molecules and free radicals in order to reduce them to products that are nontoxic to the organism.The deletion of GSTT1 and GSTM1 increases the risk of thyroid lesions by a factor of 2.6. 4

OBJECTIVE
The aim of the present study was to compare GSTM1 and GSTT1 gene frequencies between patients with benign and malignant tumors of the thyroid gland.

Setting Setting
This was a cross-sectional study that analyzed 32 patients who underwent operations performed by the Head and Neck Surgery Group at Hospital da Irmandade de Misericórdia da Santa Casa de São Paulo between October 2005 and September 2006 after approval by the institution's Ethics Committee.

Sample Sample
Thyroid tissue samples were collected consecutively from 32 patients and evaluated with regard to the presence or absence of the GSTT1 and GSTM1 genes.There were 29 female patients (90.6%) and three male patients (9.4%).The patients were divided into two groups, benign tumors (A) and malignant tumors (B), based on the anatomopathological study.There were 18 female patients in the A group.In the B group, there were 3 males (21.4%) and 11 females (78.6%); of these 14 patients, two had follicular carcinoma and 12 had papillary carcinoma (Table 1).

Procedures Procedures
Immediately after thyroid ablation, before the surgery procedures fi nished, the nodules were divided into two halves: one fragment was stored in liquid nitrogen (N 2 ) and then in a freezer at -80° C, and the other was sent for anatomopathological study, in 10% formol.To extract the thyroid DNA, the Qiagen DNeasy ® kit was used.The GSTM1 and GSTT1 genes were amplifi ed using the polymerase chain reaction (PCR), including amplifi cation of the β-globin gene as a positive control for the DNA sample, and then the genotype was identifi ed by viewing under ultraviolet light.

Statistical analysis Statistical analysis
The statistical method used was Fisher's exact test, with a confi dence interval of 95% (p < 0.05).

RESULTS
After DNA extraction, we observed the PCR pattern that is shown in Table 2.In group A, there were no cases of double negative genotype, while 61.1% were double positive and 38.9% were positive for one of the genes.In group B, one of the cases presented double negative genotype, while the others were distributed between double positive (28.6%), positive genotyping only for GSTT1 (50%) and positive only for GSTM1 (14.3%).

DISCUSSION
The data collected for analyzing the benign tumor group showed high frequency of the double positive genotype (61.1%) and no double negative genotype.In group B there was a more homogeneous distribution.Cases with positive GSTT1 genotype and negative GSTM1 (T + M -; 50%) predominated, while the double negative genotype was observed in only one case (7.1%).The double positive genotype occurred in 28.6%.
Analysis of the GSTM1 gene alone showed that there was a relatively high fre-quency of the negative GSTM1 genotype in group B, i.e. 8 out of the 14 cases (57.1%), in comparison with group A, in which the frequency was 27.8%.However, statistical analysis showed that this was not signifi cant (p = 0.09).Analysis of the GSTT1 gene alone showed that it was present in 88.9% of the cases in group A and 78.6% of the cases in group B, and also was not statistically signifi cant (p = 0.3).
In the literature, separate analyses of the genes GSTT1 and GSTM1 are inconclusive regarding the risk of developing thyroid cancer. 5Our results from separate analyses of these genes are in accordance with the literature, although we observed the presence of GSTM1 in 72.2% (13/18) and GSTT1 in 88.9% (16/18) of the benign tumor cases.These frequencies are quite high and, although non-signifi cant, cannot be ignored.

CONCLUSION
Analysis of the data obtained, under the conditions of the present study, allows the conclusion that there is no relationship between the presence of the GSTT1 and GSTM1 genes and the benign and malignant thyroid tumors.
However, the relatively small number of cases makes it impossible to reach more substantial conclusions.In order to achieve this, a larger sample would be needed.

Table 1 .
Types of thyroid neoplasia in relation to patients' gender

Table 2 .
Results from DNA analysis of thyroid tumors patients .Namba H, Rubin SA, Fagin JA.Point mutations of ras oncogenes are an early event in thyroid tumorigenesis.Mol Endocrinol.