Silviana Farrah Diba

The Number of Karyorrhexis in Patients

Undergoing Repeated Periapical or Panoramic
Radiography in RSGM Prof. Soedomo FKG-UGM
Silviana Farrah Diba; Munakhir Mudjosemedi; Rurie Ratna Shantiningsih
Department of Dentomaxillofacial Radiology, Faculty of Dentistry
Gadjah Mada University
Yogyakarta, Indonesia
s.farrah.d@gmail.com

Abstract
Radiography plays important roles in diagnostic, treatment planning, and evaluation in dentistry.
Periapical and panoramic radiography are the most frequent used techniques. Radiation exposure to the
body tissue may induce cellular damage which results in cellular death. Karyorrhexis is one of the cell
death patterns, and can be used as a biomarker for radiation that induces cellular damage. The objective
of this study was to identify the differences in the increasing number of karyorrhexis in exfoliated buccal
gingival epithelial cells after repeated periapical or panoramic radiograph exposures. Thirty patients who
underwent radiographs as requested by their dentist were divided into two groups. First group required
a panoramic radiograph; second group required a repeated periapical radiograph due to failure in the
first exposure, or during the root canal treatment procedure. Exfoliated epithelial gingival cells of the
upper dental arch were collected prior to or immediately after the exposure and 10 days after the
exposure. Smears were stained with Feulgen-Rossenbeck reaction and then karyorrhexis was scored.
Paired t-test and Independent t-test were use to analyze the frequency of karyorrhexis among the
groups. The results revealed a significant difference (p<0.05) in the frequency of karyorrhexis in gingival
cells between two groups. The increase of karyorrhexis was higher in the patients underwent panoramic
radiography than that of repeated periapical radiography. In conclusion, the present study shows that
dental radiography may increase the number of karyorrhexis in exfoliated cells of gingival. Since
karyorrhexis is one of the indicators of cellular death, dental radiography should be only used if
necessary.
Keywords: karyorrhexis, periapical, panoramic

Introduction

Nowadays, X-ray becomes applicable in many fields mainly in dentistry. Dental radiography has
several functions, not only observe the undisclosed oral cavities part, but also it is helpful in
determining diagnosis and treatment evaluation [1, 2]. Dental radiography divided into two
categories, named intra and extra oral. Above all, periapical and panoramic radiography are
the most frequent radiography technique that requested by the dentist [3].
Although dental exposure has many utilities, radiation to the tissues could be hazardous [4].
Radiation to human body may induce damaged, begin at molecular level to cellular damages
[5]. Cellular response to radiation exposure has variation in each individual [6].
X-ray radiation is a cytotoxic agent and this exposure may cause cell death [7]. Increase in the
number of cell death may increase the cell population that susceptible to malignant
transformation and it may affect the risk of cancer [8]. Cell death occurs when damages of the
cells couldnt be healed. Pyknosis, karyorrhexis, and karyolysis are the main characteristic of

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cell death. Karyorrhexis was initiated by a nuclease called DNA Fragmentation Factor (DFF)
which results fragmentation in nucleus [9, 10]. This fragmentation occurs inside an intact
membrane nucleus [11].
Regeneration always occurs in oral epithelial cells. This process called turnover, which makes
the mitotic cells in basal layer migrate to the surface, replacing the superficial cells. Nuclear
change become pyknosis, karyorrhexis, or karyolysis was expressed at basal layer [12].
Calculation to the number of karyorrhexis could enhance the sensitivity to detect cell damage
[13]. Exfoliated cell is a reliable technique to detect cell damages in oral epithelial. The
advantages of this technique are simple, non-invasive to human tissues, easy to perform, and
not time consuming [12].
In experiment conducted by Cerqueira et al, the number of karyorrhexis was significantly
higher in patients exposed to panoramic radiography [14]. The difference increasing number of
karyorrhexis between periapical and panoramic techniques has not been investigated. The aim
of the present study is to investigate the difference increasing number of karyorrhexis
between panoramic or repeated periapical radiography. Karyorrhexis cells were submitted
from gingival of upper dental arch.

Materials and Methods

Subjects
This study has been approved by the Ethics Commission of Faculty of Dentistry Universitas
Gadjah Mada (FKG-UGM). Thirty healthy subjects (8 male and 22 female) who underwent
radiography procedure were patients of RSGM Prof. Soedomo FKG UGM. Radiography
exposure to the patients was requested by their dentist. First group required panoramic
radiography. Usually, panoramic radiography was needed for orthodontic occasion. Second
group required repeated periapical radiography due to failure in the first exposure, or during
the root canal treatment procedure. Entire participants in this study signed an informed
consent form and answered a questionnaire about alcohol consumption, smoking, and use of
oral antiseptic solution, systemic disease, and previous radiographic exposure. Periapical
radiography were performed with EndosACP (70 kV, 8 mA, 0.5 s), whereas panoramic
radiography were performed with Yoshida Panoura (80-90 kV, 8-10 mA, 13 s). Repeated
periapical radiography was performed directly in the same day. Effective dose for repeated
periapical is 2 Sv, and for panoramic is 16-26 Sv [3].
Collection of Cells
Exfoliated cells were obtained from gingival mucosa of the upper dental arch by gentle
scraping with a sterile cytobrush, prior to or immediately after the exposure and 10 days
afterward. Cells were smeared into object glass containing two drops of physiological solutions
(NaCl 0.09%) and then stained with modified Feulgen-Rossenbeck reaction. A methanol-acetic
acid (3:1) was used to fix the cells. Samples were put in 5 M HCl for 15 minutes, soaked in
distilled water for 15 minutes, stained with Schiffs reagent for 90 minutes, and then
counterstained with Fast Green 1% for 1 minute [14]. Karyorrhexis were observed under a light
microscope at 400X magnification, accompanied with Optilab viewer then karyorrhexis cells
were counted. For each sample, 1000 cells were scored according to the criteria used by [15].
Karyorrhexis were defined as a fragmented nucleus because of the disintegration nucleus in
cell with an intact cell membrane.
Data Analysis
The Shapiro Wilk test was used previous to parametric paired t-test and independent t-test.
Paired t-test was adopted to compare the frequency of karyorrhexis among the samples

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before and after X-ray procedure, separately in panoramic or repeated periapical radigraphy.
To compare differences in the increasing number of karyorrhexis after repeated periapical or
panoramic radiograph exposures, the independent t-test was adopt. This entire parametric
test was performed using SPSS 16 for Windows.

Results and Discussion

Results
Table 1 shows the frequency of karyorrhexis cell, before and after exposure in patients
underwent panoramic radiography or repeated periapical radiography. The number of
karyorrhexis cell were increase after exposure in panoramic, also in repeated periapical. There
were statistical significant differences between before and after exposure in both groups
(p<0.05). Figure 1 presents the karyorrhexis cells. The increasing level of karyorrhexis after
exposure to dental X-ray was 72.1% for repeated periapical, and 96% for panoramic. Table 2
shows the number of karyorrhexis was significantly (p<0.05) higher in patients underwent
panoramic radiography rather than repeated periapical radiograph.
TABLE 1. FREQUENCY OF KARYORRHEXIS BEFORE AND AFTER RADIOGRAPHY PROCEDURES
(PARAMETRIC PAIRED T-TEST)
Groups

Exposure

Mean SD

Repeated
Periapical

Before

15

13.51 2.04

After

15

23.26 6.28

Panoramic
a.

Before

15

14.51 3.03

After

15

28.44 5.92

Raising Level
(%)

0.000a

72.1

0.000a

96

Significant difference between values before and after exposure.

TABLE 2. DIFFERENCE IN INCREASING NUMBER OF KARYORRHEXIS BETWEEN THE GROUPS

(PARAMETRIC INDEPENDENT T-TEST)
Groups

Difference in means
SD

Repeated
periapical

15

9.75 5.83

P
0.029a

Panoramic
15
13.93 3.81
a.
Significant difference between the groups.

Discussion
In this study, exfoliated gingival cells from individuals were obtained immediately prior to and
10 days after dental exposures in both groups. Epithelial turnover takes time 7-16 days. Ten
days were the appropriate time to detect nuclear change results from radiation exposure since
the turnover process brings the damage cell in basal layer to the surface where they exfoliate
[14].

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Figure 1. karyorrhexis cells (arrow); 400x magnification; modified Feulgen-Rossenbeck technique.

Karyorrhexis is an early sign of apoptosis and this nuclear change can be used as biomarker
due to radiation exposures [6,15]. Based on the morphology, apoptotic cells become
condensed starts peripherally along nucleus membrane. The next stage, nucleus starts to
break up in an intact nucleus membrane [11]. Quantity of karyorrhexis was evaluated to
monitor cytotoxic effect of radiation [16].
There was a significant difference number between before and after exposure in both groups.
The number of karyorrhexis was significantly higher after exposure, this result was in line with
Cerqueira et al [14]. In dental radiography procedure, radiation was absorbed to gingival
epithelial cells since gingival was exposed directly by the X-ray beam [14]. Orthodontic patients
who underwent panoramic only or panoramic and cephalometric radiography altogether had a
significant increase number of karyorrhexis after exposure. The cells were obtained from
buccal mucosa [16,17]. Those studies proved that X-rays are cytotoxicant agent which induces
cell death [17].
In the present study, the increase number of karyorrhexis after exposure was significantly
higher in panoramic rather than repeated periapical when the two groups were compared.
Radiation dose determined the number of cells undergoing damage and cell death as well [18].
Effective dose to repeat periapical is 2 Sv whereas for panoramic is 16-26 Sv [3]. Dental
radiography doses are lower, compared with other complex imaging [19]. Since X-ray beam is a
cytotoxic agent, the higher exposure dose may increase the number of cell death in tissues
[16].
Da silva et al investigated lateral tongue cells from two groups of dental radiography patients
consist of panoramic and repeated panoramic. They found significant difference between two
groups. The higher number of karyorrhexis in repeat panoramic groups proved that increase in
radiation dose may increase the frequency of karyorrhexis [13]. Our findings were in
accordance with those authors, that increase in radiation dose would increase the number of
cell death. In low dose radiation, energy was deposited in tissues for a few days. Radiation
effect to the tissues may decrease as time goes by [20].

Conclusion

The present study suggests that both of dental radiographys techniques which have been
used in this experiment are able to increase the number of karyorhhexis in exfoliated cells of
gingival. The number of karyorrhexis increases after panoramic exposure or repeated
periapical exposure as well. The increasing number of karyorrhexis is higher in panoramic
radiography, rather than repeated periapical radiography.

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