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THE ANALYSIS OF KARIOLOGICAL INDISES OF BUCCAL EPITHELIUM SAMPLES TAKEN FROM URBAN AND RURAL MEN

E-mail
Volcova A.T., Zagidullina S.R., Sabitova E.R., Potapova I..   
25.05.2011 .
the Bashkir State Medical University (Ufa )

  

Micronucleus test is used now as biomarker of genetic damage caused by medical procedures, exposures to environmental pollutants, life-style habits. Micronuclei are small rounded extra nuclear chromatin formations that appear due to chromosome apparatus injury of dividing cells, i.e. some damages of DNA molecules. Those damages may be provoked by both external and internal causes such as some diseases. Micronuclei served as the indicator of genome instability and existing cell mutations [  ., 1988].

The non-invasive nature of micronuclei technique makes it the most useful one for the bio-monitoring of human populations or individuals. Mucosa cells structure is not equal in different areas of oral cavity. And as an adaptive response to cell damage, the normally nonkeratinizing epithelium may be keratinized. New epithelial cells appear in basal layer. Micronuclei in exfoliated epithelial cells reflect genotoxic events that occurred in the dividing basal layer 1-3 weeks earlier [Tolbert et al., 1991].

Other nuclear anomaly besides micronuclei is used now to assess degree of genome stability. Micronuclei, stimulated apoptosis and, it is possible, binucleation and broken eggs may be an indicators of genotoxic insult [ ., 2007]. Increased binucleation may occur due to inflammation, regeneration and early cancer processes [Koss, 1979, ., 1997]. Broken eggs are considered to be the special case of the nuclear buds, that are the nuclear protrusion into cytoplasm as a result of elimination from the nuclear an odd copies of the faulty amplified genes during S-phase [Miele et al., 1989; Shimizu et al., 1998].

The aim of the study was to compare frequency of occurrence of the abnormal nuclei in buccal epithelial cells of the students (male) from Ufa and countryside.

Material and method of the study

The subject of our investigation was formed of the buccal mucosa cells sampled from the BMSU first-year students (male, aged 17-19). There were analyzed 10332 cells from 9 students - the natives of Ufa-city, and 12775 cells from 12 students who lived in country before arrival in the city for study. No less than 1000 cells of each person were tested. The rural students underwent an examination in first days of their stay in a town.

The specimens of the buccal mucosa cell swabs were prepared and analyzed according to the methodical guidelines “Assessment of the cytological and cytogenetic status of human Schneiderian membrane and mouth tunica mucosa” (5) and karyological indices classification by L.P. Sychiova (6).

The following indices of encoded specimens were considered: 1) cytogenetic indices (occurrence of cells with micronuclei, protrusions, nuclear bridges and atypical nucleus form); 2) proliferation indices (cells with two, tree and paired nuclei); 3) early nuclear destruction indicators (cells with perinuclear vacuole, condensed chromatin, nuclear vacuolization); 4) late nuclear destruction indicators (karyorrhexis, karypyknosis, complete karyolysis); 5) apoptotic bodies.

Besides, the integral factors such as a total amount of cytogenetic abnormalities, index of total proliferation (sum of the cells with two or more nuclei), as well as an apoptotic index or overall rate of late nuclear destruction (sum of the cells at the stage of late nucleus destruction) were calculated.

The statistical data manipulation was done using PC Statistics Programs as Excel and BIOSTAT. The χ² test was used to compare the frequency of occurrence of the cells with different types of nuclear anomalies.

Findings and their consideration

The comparison between the males from a city and country revealed that the frequency of the cells with micronuclei (<0,001), “nuclear bud” (=0,038), total amount of cytogenetic abnormalities (=0,007), paired nuclei (=0,038), karyopyknosis (<0,001) as well as the index of nuclear destruction end (<0,001) and apoptotic bodies at p<0,001 is significantly higher in a townsmen group.

On the contrary, the index of total early nucleus destruction including nuclear vacuolization and condensed chromatin is higher in a countrymen group (p<0,001).

 

Table 1

Karyological indices of males buccal epithelial cells (per 1000 cells)

Indices

Townsmen

(M±m)

countrymen (M±m)

Total cytogenetic abnormalities including:

3,89±1,111**

1,67±0,541

Micronuclei cells

1,89±0,484***

0,08±0,083

1st type protrusion (“Nuclear buds”)

0,78±0,401*

0,09±0,91

2nd type protrusion (“broken egg”)

0,11±0,111

0,08±0,083

3d type protrusion (“tongue”)

0,22±0,147

0,17±0,167

4th type protrusion (“nuclear tail”)

0,11±0,111

0,08±0,083

Bridge

0,22±0,147

0,75±0,351

Atypical form nucleus

0,11±0,111

0,50±0,195

Index of total proliferation including:

3,44±0,648

1,83±0,490

Binucleate cell

2,44±0,689

1,75±0,509

Paired nuclei

0,78±0,278*

0,08±0,083

Three-nucleate cell

0,22±0,147

0,00

Index of total early nuclear destruction including:

130,33±43,173***

162,83±31,774

Perinuclear vacuole

10,89±7,454

9,08±2,221

Nuclear vacuolization

31,44±11,321***

41,83±10,650

Condensed chromatin

87,33±31,961***

111,92±26,888

Index of total late nuclear destruction including:

30,33±13,729***

20,25±4,040

Karyorrhexis

1,56±0,412

2,25±0,880

Pyknosis

15,33±5,645***

7,83±2,128

Karyolysis

13,44±9,400

10,17±2,458

Apoptotic bodies

7,33±3,096***

0,67±0,355

General number of examined cells

10332

12775

 

Note: * - p<0,05, ** - p<0,01, *** - p<0,001- significant differences from conditionally  control group when compared by χ2- criterion.

M – mean value, m – averaged error.

Conclusion

The increase in a total number of cytogenetic abnormalities (including micronuclei and nuclear buds) is an indicator of cytotoxic effects of the environment.

The level of these anomalies is lower in the countrymen group.

                We can presume that the level of cytogenetic abnormalities in townsmen group is higher due to adverse effect of the environment that is more mutagenic in Ufa than in country as well as to some specific factors related to study in the medicine university (for instance, close contact with formalin).

The level of the cells with paired nuclei, i.e. the cells with proliferation disorders, is also higher in a townsmen group.

In an effort to eliminate the cells with cytogenetic abnormalities the apoptosis process speeds up preceded by an increase in the early destruction index.

The results of karyological analysis allow to assess the degree of organism genetic homeostasis disorder and hence the level of environmental genotoxicity.

References

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2.                   Tolbert P.E., Shy C.M., Allen J.W. Micronuclei and other nuclear anomalies in buccal smears: a field test in snuff users // Am. J. Epidemiol. – 1991. – Vol.134, 8. – P. 840-850.

3.                   .., .., .. . // - / . , ..., . .. , ... ... – .: , 2007. – . 220-267.

4.                   Koss L.G. Diagnostic cytology and its histopathologic bases. –1979. –Ed. 3. –Vol. 1,2. –Philadelphia –Toronto: J B Lippincott Co.

5.                   .., .., .. // . – 1997. – . 31, 6. – . 3-11.

6.                   Miele M., Bonatti S., Menichini P.et al.The presence of amplified regions affects the stability of chromosomes in drug-resistant Chinese hamster cells // Mut. Res. – 1989. –Vol. 219, 3. – P. 171-178.

7.                   Shimizu N., Iton N., Utiyama H.et al. Selective entrapment of extrachromosomally amplified DNA  by nuclear budding and micronucleation during S phase // J. Cell Biol. – 1998. – Vol. 140.6. – P. 1307-1320.

8.                   Nersesyan A., Kundi M., Kambis A. et al. Effect of staining procedures on the results of micronucleus assays with exfoliated oral mucosa cells // Cancer Epidem. Biomarkers Prev. – 2006. – Vol.15, 10. – P. 1835-1840.

 

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