Campinas, Ago sto/2014 IADMFR Chile, 2015 INVESTIGATION EXPRESSION OF PEROXIREDOXIN I IN EPITHELIAL CELLS FROM THE JUGAL MUCOSA BEFORE AND AFTER DIGITAL.

Campinas, Agosto/2014

IADMFRChile, 2015

INVESTIGATION

EXPRESSION OF PEROXIREDOXIN I IN EPITHELIAL CELLS FROM THE JUGAL MUCOSA BEFORE AND AFTER DIGITAL

PANORAMIC RADIOGRAPHY IN CHILDREN AND YOUTH FROM 3 TO 20 YEARS

Authors: Santos RRLQ, Soares MJ, Silva MBF, Araújo NS, Junqueira JLC

OBJECTIVE

The aim of this study was to investigate the cellular effect of X-rays based on the expression of peroxiredoxin 1 (PRX1) in the epithelial cells of the oral mucosa of children and adolescents (3 to 20 years old), after digital panoramic radiography and low-dose ionizing radiation, using Trizol protocol procedures and quantitative polymerase chain reaction (qPCR).

Methods

• The study was reviewed and approved by the Research Ethics Committee of the College Faculdade São Leopoldo Mandic (Protocol 2012/0242).

• All research procedures were carried out in the College Faculdade São Leopoldo Mandic - Campinas - Sao Paulo - Brazil.

INCLUSION CRITERIA:• 30 children and adolescents between 3 and 20 years old;

•Patients were referred to receive panoramic radiography in the dental radiology department;

•All patients and their parents were informed about the study objectives and signed an informed consent form.

EXCLUSION CRITERIA:•Patients who presented lesions in the oral mucosa;

•Patients who underwent radiographic examinations of the head and neck within a month prior to the investigation.

• A single operator performed the cytology of the area with a nº 24 metal spatula on the right side before and on the left side after radiographic acquisition;

• Radiographic acquisition was performed using Digital Panoramic X-ray model Orthopantomograph OP200 D, manufactured by Instrumentarium Dental (Nahkelantie 160, FI- 04300, Tuusula, Finland);

• Acquisition Protocol: 70 kVp, 10 mA, and 14 seconds.

To obtain the RNA, the collected material was stored in Trizol® at -20°C and thawed only at the time of RNA extraction.

After thawing, 0.2 ml chloroform was added to each 1.0 ml of Trizol® collected;

After vortexing for 40 seconds, the sample was incubated at room temperature for 3 minutes;

The material was centrifuged at 12,000 x g for 15 minutes at 4°C;

The aqueous phase was transferred to a new Eppendorf tube and RNA was precipitated with the addition of 0.5 ml of isopropanol;

The sample was homogenized and centrifuged;

The supernatant was discarded – the precipitate was used as the RNA sample;

Washing with 75% ethanol (1 ml);

The sample was homogenized and centrifuged again;

Ethanol was removed and the tubes were placed paper

towels for 10 minutes;

The concentration of RNA was determined by

spectrophotometry;

1. Complementary DNA synthesis was carried out by reverse transcription using SuperScript III reagent (Life Technologies).

2. The PRX1 expression levels were analyzed by qPCR.

3. The expression levels were quantified by the "SDS System" program (Life Technologies), and the relative expression levels between the samples were calculated according to the comparative cycle threshold (Ct) method based on the formula 2-ΔΔCt.

4. The results were analyzed statistically.

RESULTS

•Among the 30 samples collected, 19 did not have enough material or there was no expression detected and were thus excluded.

•Of the 11 patients, 7 had increased levels of PRX1 expression after radiation, and 4 had decreased PRX1 decreased expression (Table 1).

AGE Before digital panoramic radiography

(Control)

After digital panoramic radiography (RX)

Patient 1 03 years old1 1,649295

Patient 2 08 years old0,183083 1,343834

Patient 3 11 years old1,597787 1,112095

Patient 4 12 years old0,244349 0,152848

Patient 5 19 years old2,224096 2,65164

Patient 6 09 years old1,288668 1,990101

Patient 7 11 years old1,746689 1,498104

Patient 8 16 years old0,725991 1,29825

Patient 9 14 years old0,424816 1,209348

Patient 10 04 years old0,92778 1,994735

Patient 11 05 years old1,118687 0,571958

Table 1 - Values of PRX1 expression levels obtained in arbitrary units (AU) from the 2–ΔΔCt calculation.

• PRX1 expression was significantly increased in patients undergoing radiography (p = 0.0293).

Graph 1 – Relative PRX1 expression determined by qPCR. Gene transcription levels of PRX1 were higher after radiography (Rx) compared to patients with no radiographic exposure (C). Values are expressed as mean ± standard deviation. The symbol * indicates a statistically significant difference (paired t-test).

CONCLUSION

• Digital panoramic radiography increased the expression of PRX1 in epithelial cells of the oral mucosa, indicating an effect of radiation at the cellular level.

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Thank you - Gracias - Obrigada

College São Leopoldo Mandic - Campinas - Sao Paulo - Brazil