{"id":52124,"date":"2023-09-30T10:08:55","date_gmt":"2023-09-30T10:08:55","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=52124"},"modified":"2023-10-07T12:01:15","modified_gmt":"2023-10-07T12:01:15","slug":"degenerative-changes-of-buccal-cells-after-one-cycle-of-chemotherapy-on-male-breast-cancer-patients","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no3\/degenerative-changes-of-buccal-cells-after-one-cycle-of-chemotherapy-on-male-breast-cancer-patients\/","title":{"rendered":"Degenerative Changes of Buccal Cells After One Cycle of Chemotherapy on Male Breast Cancer Patients"},"content":{"rendered":"\n

Introduction\n<\/strong><\/p>\n\n\n\n

Micronuclei originate\nfrom chromosomes\u2019 fragments and\/or entire chromosomes that failed to be\nincorporated into the nucleus of the cell in the nuclear division process.\nAnalysis of the micronucleus (MN) in the exfoliative cells taken from human,\ncould potentially serve as a great tool to study the modifications of the\ngenome directly in the tumor-affected target organs 1<\/sup>.\n<\/p>\n\n\n\n

In some research, a\nsignificant correlation has been shown between the chromosomal aberration\u2019s levels\nin lymphocytes and MN in exfoliative cells of the buccal mucosa in patients\nexposed to environmental mutagens 2<\/sup>.<\/p>\n\n\n\n

There are different ways\nby which genome damage can be caused, such as by environmental genotoxins (e.g.,\nradiation and chemical substances), by micronutrient deficiencies (e.g.,\nfolates), by life habits (e.g., alcohol, tobacco, drugs, stress, etc.) and lastly\nby genetic factors which include inherited defects in metabolism and DNA repair\n3<\/sup>.\nConsidering these facts, it is assumed that the epithelial cells of the oral\nmucosa represent one preferred site for the early appearance of genotoxic modifications\ninduced by carcinogenic agents that are introduced in the body either through\nthe inhaling or by ingestion 3<\/sup>.<\/p>\n\n\n\n

Thus, in this study we\nhave analyzed the degree of change of the nuclei of exfoliative epithelial in\nthe buccal mucosa of the oral cavity in patients diagnosed with breast cancer\nand who received chemotherapy treatment.<\/p>\n\n\n\n

Material\nand Methods<\/strong><\/p>\n\n\n\n

Sample collection was done in the Clinical Center of Kosova and a total of 40 male patients were enrolled in the study. Samples were collected before and after the first cycle of chemotherapy, thus the inclusion criteria consisted on patients from the time of diagnosis and after the first chemotherapy. Male breast cancer patients who underwent more than one cycle of chemotherapy at the time of the study were excluded.  All the study procedures were approved by the Institutional Ethic Committee of the Oncology Department in the Clinical Center of Kosova.<\/p>\n\n\n\n

All the samples were taken the same way with a cytobrush. After the collection of the epithelial cells from the buccal cavity of the mouth, we added 5 ml of physiological solution NaCl 9% to the test tubes. The next step was centrifuging the samples, for 10 minutes at the 1200 rpm in a multiple purpose benchtop centrifuge by Eppendorf. After that the supernatant was carefully removed, leaving 1 ml together with the precipitate. Next, we carefully mixed the precipitate with the solution left on the tube. Then, we took a small quantity of the mixture and spread on the surface of three glasses. After that, the samples were dried for 2-3 hours at room temperature. The next step included the adding of ethanol 96% and after the complete drying of the sample we placed the glasses in Giemsa staining solution from CellaVision (Sweden) diluted 1:5 for a period of 1 hour. Next, after the coloring step, we rinsed with distilled water the glasses and dried. The samples were examined under the optical microscope. <\/p>\n\n\n\n

Results are presented in the tabular and graphic form. All statistical analyses were conducted using SPSS 22 software platform and the significant changes were shown. Changes values being P<0.05 were considered significant. <\/p>\n\n\n\n

Results\n<\/strong><\/p>\n\n\n\n

Demographic presentation of patients<\/strong><\/p>\n\n\n\n

As a starting point, demographic data from all the male\npatients diagnosed with breast carcinomas, involved in the study were analyzed (Table 1). The average\nage of our patients was 45.8\u00b115.5 years. Interestingly, looking up at the\nhistory of the patients, we have observed that a vast majority of the patients were tobacco users\nwhile a small percentage declared alcohol consumption, 54,5% and 5,5% respectively (Figure 1A, B). <\/p>\n\n\n\n

Table 1: Demographic data of patients with breast cancer treated with chemotherapy<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\n

Sex<\/strong><\/p>\n<\/td>\n

\n

Number<\/strong><\/p>\n<\/td>\n

\n

%<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Male<\/p>\n<\/td>\n

\n

40 patients<\/p>\n<\/td>\n

\n

100%<\/p>\n<\/td>\n<\/tr>\n

\n

Age<\/p>\n<\/td>\n

\n

Number<\/p>\n<\/td>\n

\n

%<\/p>\n<\/td>\n<\/tr>\n

\n

45-61 years old<\/p>\n<\/td>\n

\n

5 patients<\/p>\n<\/td>\n

\n

13.%<\/p>\n<\/td>\n<\/tr>\n

\n

61-80 years old<\/p>\n<\/td>\n

\n

35 patients<\/p>\n<\/td>\n

\n

87 %<\/p>\n<\/td>\n<\/tr>\n

\n

Age, mean (SD).<\/p>\n<\/td>\n

\n

45.8 patients<\/p>\n<\/td>\n

\n

15.5<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 1: The tobacco and alcohol use among the patients<\/strong>.<\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Figure 1A shows that a percentage of 54.5% corresponds to\nregular tobacco users while 1B only 5.5% of the patients declared of using\nalcohol.<\/p>\n\n\n\n

Changes on epithelial cells of the oral mucosa of the patients <\/strong><\/p>\n\n\n\n

The average number of\ndegenerative modifications in the exfoliative epithelial cells taken from breast\ncancer patients\u2019 buccal mucosa, was shown to be significantly increased after a\nsingle chemotherapy. We\nhave examined these types of changes; Micronuclei (MN), Pycnotic Nuclei (Pyk),\nKaryorrhectic cells (KR), Karyolysis (KL), Binuclear cells (BN) and the total\nchanges before and after the first cycle of chemotherapy treatment. With the MN\ntest we have in fact not found striking differences in Micronuclei cells\nspecifically, but we have noticed differences in other degenerative\nmodifications such as pycnotic nuclei (Pyk) cells with mean of 5.5\u00b17.7 before\nthe treatment and 11.5\u00b113.5 after the treatment and the differences were p<\/em><0.00001.\nThe differences in karyorrhectic cells, karyolysis and binuclear cells were\nalso found significant between the samples taken before and after the first\nchemotherapy cycle with p<<\/em>0.0001, p<<\/em>0.0003, p<<\/em>0.00002,\nrespectively, The mean of total changes before the treatment were 15.5\u00b122.4,\nwhile the changes after the first cycle of chemotherapy was 27.9\u00b130.1 with a\nsignificant change of p<\/em><0.0002. Table 2 summarizes the significant\nincrease for all types of the nuclear change. The numbers represent the manually counting of the\ndegenerated cells per sample.<\/p>\n\n\n\n

Moreover, we were\ncurious to see if age if a factor in having a higher number of degenerative cells,\nthus we have checked the differences in the degenerative modifications in the\nexfoliative epithelial cells in\npatients, comparing two groups of patients with differences in age. At this\npoint, we have observed that a higher, despite not significant, total\ndegenerative changes occurred in older patients (group age from 61 to 80 years\nold) rather than younger patients (group age from 45 to 61 years old), with\ndifferences being 13.24 and 8.20 respectively. Confirming that age is not a\nfactor in changes of the cells independently.  Data for all type of nuclear changes\nrepresented accordingly to the age are shown in Table 3. <\/p>\n\n\n\n

Table 2: Mean degenerative modifications in the exfoliative epithelial cells of the male breast cancer patients\u2019 buccal mucosa (n=40).<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Type of degenerative changes<\/strong><\/p>\n<\/td>\n

\n

Base line<\/strong><\/p>\n<\/td>\n

\n

After the treatment<\/strong><\/p>\n<\/td>\n

\n

Difference<\/strong><\/p>\n<\/td>\n

\n

P<\/em><\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

 <\/p>\n<\/td>\n

\n

Mean<\/strong><\/p>\n<\/td>\n

\n

Sd<\/strong><\/p>\n<\/td>\n

\n

Mean<\/strong><\/p>\n<\/td>\n

\n

Sd<\/strong><\/p>\n<\/td>\n

\n

Mean<\/strong><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

Micronuclei<\/p>\n

(MN)<\/p>\n<\/td>\n

\n

0.2<\/p>\n<\/td>\n

\n

0.0<\/p>\n<\/td>\n

\n

0.0<\/p>\n<\/td>\n

\n

0.0<\/p>\n<\/td>\n

\n

-0.1<\/p>\n<\/td>\n

\n

ns<\/p>\n<\/td>\n<\/tr>\n

\n

Pycnotic Nuclei (Pyk)<\/p>\n<\/td>\n

\n

5.5<\/p>\n<\/td>\n

\n

7.7<\/p>\n<\/td>\n

\n

11.5<\/p>\n<\/td>\n

\n

13.5<\/p>\n<\/td>\n

\n

9.0<\/p>\n<\/td>\n

\n

0.00001*<\/p>\n<\/td>\n<\/tr>\n

\n

Karyorrhectic cells (KR)<\/p>\n<\/td>\n

\n

6.9<\/p>\n<\/td>\n

\n

8.5<\/p>\n<\/td>\n

\n

17.7<\/p>\n<\/td>\n

\n

16.5<\/p>\n<\/td>\n

\n

15.6<\/p>\n<\/td>\n

\n

0.0001*<\/p>\n<\/td>\n<\/tr>\n

\n

Karyolysis (KL)<\/p>\n<\/td>\n

\n

51.9<\/p>\n<\/td>\n

\n

31.6<\/p>\n<\/td>\n

\n

65.8<\/p>\n<\/td>\n

\n

46.9<\/p>\n<\/td>\n

\n

18.9<\/p>\n<\/td>\n

\n

0.0003*<\/p>\n<\/td>\n<\/tr>\n

\n

Binuclear cells (BN)<\/p>\n<\/td>\n

\n

5.4<\/p>\n<\/td>\n

\n

4.6<\/p>\n<\/td>\n

\n

11.9<\/p>\n<\/td>\n

\n

6.6<\/p>\n<\/td>\n

\n

8.4<\/p>\n<\/td>\n

\n

0.00002*<\/p>\n<\/td>\n<\/tr>\n

\n

Changes in total<\/p>\n<\/td>\n

\n

15.5<\/p>\n<\/td>\n

\n

29.1<\/p>\n<\/td>\n

\n

27.9<\/p>\n<\/td>\n

\n

34.4<\/p>\n<\/td>\n

\n

10.2<\/p>\n<\/td>\n

\n

0.0001*<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

In the table are shown the mean and standard deviation\nnumber of micronuclei cells (MN), pycnotic nuclei cells (Pyk), karyorrhectic\ncells (KR), cells that underwent karyolysis (KL), binuclear cells (BN) and the\nchanges in total of the cells. The mean and standard deviation is shown for the\nsamples taken before the treatment, (base line) first column and after the\nfirst treatment (after treatment) second column. Third column represents the\ndifferences of the above-mentioned changes before and after treatment. The last\ncolumn shows the p<\/em> values obtained the Student\u2019s t<\/em>-test,\nwith ns presenting1<\/sup> \u2018not significant\u2019 and (*) being p<\/em><0.05.<\/p>\n\n\n\n

Table 3: Differences of degenerative modifications in exfoliative epithelial cells of buccal mucosa according to the age.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Type of degenerative changes<\/strong><\/p>\n<\/td>\n

\n

Age<\/strong><\/p>\n

45-61<\/strong><\/p>\n<\/td>\n

\n

Age<\/strong><\/p>\n

61-80<\/strong><\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n

\n

 <\/p>\n<\/td>\n

\n

Differences<\/strong><\/p>\n

Mean<\/strong><\/p>\n<\/td>\n

\n

Differences<\/strong><\/p>\n

Mean<\/strong><\/p>\n<\/td>\n

\n

T-test<\/strong><\/p>\n<\/td>\n

\n

p<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Micronuclei<\/p>\n

(MN)<\/p>\n<\/td>\n

\n

 <\/p>\n

0.10<\/p>\n<\/td>\n

\n

 <\/p>\n

0.00<\/p>\n<\/td>\n

\n

 <\/p>\n

—<\/p>\n<\/td>\n

\n

 <\/p>\n

NT<\/p>\n<\/td>\n<\/tr>\n

\n

Pycnotic Nuclei<\/p>\n

(Pyk)<\/p>\n<\/td>\n

\n

11.00<\/p>\n<\/td>\n

\n

10.40<\/p>\n<\/td>\n

\n

1.0<\/p>\n<\/td>\n

\n

0.36<\/p>\n<\/td>\n<\/tr>\n

\n

Karyorrhectic cells<\/p>\n

(KR)<\/p>\n<\/td>\n

\n

16.59<\/p>\n<\/td>\n

\n

17.00<\/p>\n<\/td>\n

\n

0.64<\/p>\n<\/td>\n

\n

0.51<\/p>\n<\/td>\n<\/tr>\n

\n

Karyolysis<\/p>\n

(KL)<\/p>\n<\/td>\n

\n

18.29<\/p>\n<\/td>\n

\n

24.00<\/p>\n<\/td>\n

\n

0.6<\/p>\n<\/td>\n

\n

0.55<\/p>\n<\/td>\n<\/tr>\n

\n

Binuclear cells<\/p>\n

(BN)<\/p>\n<\/td>\n

\n

9.36<\/p>\n<\/td>\n

\n

8.00<\/p>\n<\/td>\n

\n

2.6<\/p>\n<\/td>\n

\n

0.12<\/p>\n<\/td>\n<\/tr>\n

\n

Changes in total<\/p>\n<\/td>\n

\n

8.20<\/p>\n<\/td>\n

\n

13.24<\/p>\n<\/td>\n

\n

0.6<\/p>\n<\/td>\n

\n

0.53<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The table represents the mean number of micronuclei cells\n(MN), pycnotic nuclei cells (Pyk), karyorrhectic cells (KR), cells that\nunderwent karyolysis (KL), binuclear cells (BN) and the changes in total of the\ncells according to the age of the patients divided in two groups. The last\ncolumn shows the p<\/em> values obtained the Student\u2019s t<\/em>-test.<\/p>\n\n\n\n

Discussion <\/strong><\/p>\n\n\n\n

Micronuclei\nare fragments of chromosomes or entire chromosomes, which failed to reach the\npoles of the division axis during mitosis, and remain encapsulated as a\nseparate nucleus during the process of telophase. The chromosome losses or the\ndysfunction of the mitotic spindle caused by aneugenic mechanisms, could be\ndetected if approached with micronucleus testing 4<\/sup>.<\/p>\n\n\n\n

The oral\ncavity epithelium undergoes constant regeneration through the continuous production\nof new cells in the basal layer by mitosis and the migration of these new cells\nto the surface will replace the old ones. However, the basal layer contains\nstem cells which during nuclear division, can express genetic damages (breakage\nor loss of chromosomes) such as MN. These newly produced cells may\/or may not\ncontain MNs, eventually differentiated into the spiny cell layer. Moreover, some\nof these cells can slip into cells with fragmented nuclei (karyorrhectic\ncells), pyknotic nuclei, condensed chromatin, or even karyolysis which is the\ncomplete loss of the nuclear material 5<\/sup>. Sometimes,\nthough rarely, cells may become stuck at the binuclear stage or may show\nbud-shaped nuclei, which is known as the so-called “broken eggs” in\nbuccal mucosal cells, as a gene amplification biomarker. These biomarkers could\nbe detected both in lymphocytes and in buccal cells, which represent a broader\nassessment of genome damage than MN alone, in the context of cytotoxicity and\ncytostatic effects 6<\/sup>.<\/p>\n\n\n\n

Casartelli et\nal. analyzed the frequency of MN in buccal exfoliative cells in normal mucosa,\nin precancerous lesions and in squamous cell carcinoma 7<\/sup>. They\nconcluded that the MN levels correspond to the progression of the neoplastic\ndisease since they have shown a gradual increase of this biomarker in the steps\nstarting normal mucosa to precancerous lesions and finally to carcinoma.\nPublished biomonitoring studies with evaluated MN in the buccal mucosa have\nanalyzed the effect of many factors, including the influence of environmental\nand workplace exposure, chemoprevention, radiotherapy, the influence of life\nhabits, the influence of tumors and other diseases 8<\/sup>.<\/p>\n\n\n\n

There are some\ntypes of cells which contain two nuclei within, and these types of cells are\ncalled binuclear. These types of cells are often seen in cancer and they can be\ncaused by different factors. If during cell division the cell division groove\nbegins to regress then the cell joins again, causing the chromosomes not to\nseparate 9<\/sup>. These cells\ncan also appear during the failure of cytokinesis, in which case the cell\ndivision loop is not formed at all, thus causing the remaining of both nuclei\nin one single cell. Pyknosis or karyopyknosis represents the irreversible\ncondensation of chromatin in the nucleus of cells undergoing necrosis. This is\nfollowed by karyorrhexis or fragmentation of the nucleus 10<\/sup>. Karyorrhexis\nis the destructive fragmentation of the nucleus of the dying cell, in which the\nchromatin is distributed irregularly throughout the cytoplasm. This is usually\nfollowed by karyolysis 11<\/sup>. The last,\nrepresents the complete dissolution of chromatin by the activity of the enzyme\nDNase in the cells that are dying. In apoptosis, after karyorrhexis the nucleus\nis usually dissolved into parts called the apoptotic bodies 12<\/sup>.<\/p>\n\n\n\n

Exposure of\ncells to cytotoxic substances can result in different outcomes for those cells.\nCells can undergo necrosis, during which they lose their cell membrane\nintegrity and die rapidly as a result of cell lysis. Cells can stop their\nactive growth and division (decreased cell viability), or they can activate the\ngenetic program that controls cell death (apoptosis). Interestingly, some cells\nunergo rapid necrosis, which means that there is not enough time for them to\nactivate apoptotic mechanisms, therefore, they do not show markers of apoptosis\n13<\/sup>.<\/p>\n\n\n\n

Breast cancer\nis the most commonly diagnosed cancer in women worldwide, in 2008 alone there\nwere 1.38 million new cases diagnosed (about 23% of the total in women alone,\nand about 11% of the total, both sexes combined). The incidence rate of breast\ncarcinoma in women is highest in Western Europe and lowest in East and Central\nAfrica 14<\/sup>. Even though\nbreast cancer is a disease mainly attributed to women, it also occurs in men.\nHowever, breast cancer in man is rare as it presents 1% of the total breast\ncancer cases and 1% of cancer cases in men. This makes it difficult in approaching\nthis disease in man in terms of therapy as well as diagnosis 15<\/sup>. Thus, due to\nlimited data in male breast cancer, the treatment is primarily based on those\nused for women 16<\/sup>. <\/p>\n\n\n\n

The third-generation\ntherapeutic regimens for breast carcinoma chemotherapy follows: <\/p>\n\n\n\n

AC-paclitaxel: Doxorubicin and cyclophosphamide 60 mg\/m2  and 600 mg\/m2  respectively, both IV, on day 1 every 3 weeks for 4 cycles, followed by paclitaxel 80 mg\/m2 by 1-h IV infusion weekly for 12 weeks (more effective than AC) 17<\/sup> or<\/p>\n\n\n\n

TAC: Docetaxel 75 mg\/m2,  doxorubicin 500 mg\/m2 and cyclophosphamide 500 mg\/m2  all IV,  on day 1 every 3 weeks for 6 cycles (more effective than FAC) 18,19<\/sup> or<\/p>\n\n\n\n

FEC-docetaxel: 5-FU 500 mg\/m2,  epirubicin 100 mg\/m2 and cyclophosphamide 500 mg\/m2 all IV, on day 1 every 3 weeks for 3 cycles, followed by docetaxel 100 mg\/m2 IV every 3 weeks for 3 cycles (more effective than 6 cycles of FEC) 20<\/sup> orFEC-paclitaxel: 5-FU 600 mg\/m2, epiru<\/p>\n\n\n\n

bicin 90 mg\/m2, and cyclophosphamide 600 mg\/m2 all IV, on day 1 every 3 weeks for 4 cycles, followed by 3 weeks without treatment; continued with paclitaxel 100 mg\/m2 IV weekly for 8 cycles (more effective than 6 cycles of FEC) 21,22<\/sup>.<\/p>\n\n\n\n

On the other\nhand, as a result of the application of cytotoxic drugs, the number of\nbinuclear cells has decreased, while the karyolytic cell number has increased. In\nthe future, these parameters can be used as cytotoxic markers in the studies of\ndifferent drugs 23<\/sup>. In a\nprevious study regarding acute modifications of the nuclei of buccal cells in chemotherapy\ntreated cancer patients, our group found a significant increase in the\nfrequency of karyorrhexis, karyolysis and pyknosis compared to the control\ngroup. Moreover, the frequency of karyorrhexis was significantly higher after\nchemotherapy treatment, compared to the pre-treatment period (8.8\u00b1 3.0\nvs. 3.6\u00b14.9,\np<0.006). Accordingly with the obtained results, we have found that the acute\ntherapy does not induce damages in the chromosome, but in some cases, it may\nhave cytotoxic effect 24<\/sup>. <\/p>\n\n\n\n

In the present\nstudy, the average number of pyknotic changes (p<0.0002), karyorrhexis\n(p<0.0001), karyolysis (p<0.0002), and of the binuclear cells\n(p<0.00003) had a significant increase. A significant increase of all\nchanges in the nuclei of the exfoliating cells of the buccal mucosa was found\nin male patients with breast carcinoma treated with chemotherapy as well(p<0.0002).\nHowever, we observed that age is not a factor that significantly influenced the\nfrequency of nuclear alterations after chemotherapy in male patients with\nbreast carcinoma.<\/p>\n\n\n\n

Conclusion\n<\/strong><\/p>\n\n\n\n

The number of modifications\nin the nuclei of buccal cells of the mouth could serve as a detection test or\nimportant marker for the evaluation of the cytotoxicity of cytostatic drugs in\nmale patients with breast carcinoma. These results show differences after one single chemotherapy use,\nhowever further studies would be of benefit to examine the differences after\nother cycles of chemotherapy and better evaluate the precision of the test.\n<\/p>\n\n\n\n

The results\nshow that all protocols applied to the treatment of patients with breast\ncarcinoma have induction in cell cytotoxicity, but have not given induction in\nchromosomal changes and formation of micronuclei.<\/p>\n\n\n\n

Some patients\nmay have been affected by the time of disease detection (delay for various\nreasons).<\/p>\n\n\n\n

Acknowledgments\n                <\/strong><\/p>\n\n\n\n

We thank all study participants.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

No conflict of Interest<\/p>\n\n\n\n

Funding\nsource<\/strong><\/p>\n\n\n\n

No funding source<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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