DNA damage and cellular abnormalities in tuberculosis, lung cancer and chronic obstructive pulmonary disease

A case-control study was performed with 28 patients with COPD (age 64.21?±?8.20),
18 with LC (age 65.06?±?6.64), and 22 with TB (age 36.09?±?16.25) at the beginning
of medical treatment. The control group was composed of healthy subjects with preserved
lung functions (n?=?17, age 62.82?±?4.78). Data collection and patient sampling were performed in the
following locations: COPD – Cardio Respiratory Rehabilitation Program at Santa Cruz
Hospital (SCH, Rio Grande do Sul, Brazil); LC – Integrated Oncology Center at Ana
Nery Hospital (Rio Grande do Sul, Brazil); TB – TB Clinic at SCH.

Individuals with preserved lung functions that were exposed to the same living conditions
and were similar in terms of gender, age and body mass index (BMI) were included in
the control group. These individuals were recruited from the Family Health Units located
at Rio Pardo and Taquari Valleys (Rio Grande do Sul, Brazil). The protocol of this
study was approved by the Ethics Committee of the University of Santa Cruz do Sul
– UNISC (protocol number: 374.298). All participants answered a personal health questionnaire
and signed an informed consent form.

Buccal micronucleus cytome assay (BMCyt)

We adapted the BMCyt assay from the method described by Thomas et al. 16]. Buccal cell samples were collected and processed in accordance with the same authors.
The cells were directly fixed in methanol (an adaptation of the original protocol).
For each subject, two microtubes were prepared with methanol for left cheek and right
cheek cells. The cells were collected by rotating a cytobrush in a spiral motion 20
times against the inner surface of the cheek wall. The head of the cytobrush was placed
into its respective micro tube containing 1000 ?L of methanol 17], 18]. Then, the samples were transported to the laboratory and kept under refrigeration
(5 °C) prior to performing the micronucleus test.

The cells were centrifuged and washed with methanol after the supernatant has been
removed to concentrate a larger number of cells. Approximately 200 ? L of the cell
suspension was spread over a microscope slide and allowed to dry. Then, the slides
were placed in a staining jar containing 50 % ethanol for 1 min and immediately transferred
to a staining jar containing 20 % ethanol for 1 additional minute. After washing the
slides with deionized water for 2 min, hydrochloric acid 5 M was added for 30 min
to allow cell hydrolysis.

After cell hydrolysis, the slides were washed in tap water for 5 min and then with
distilled water for 1 min. Subsequently, the slides were maintained for 1 h and 20 min
in Schiff’s reagent for staining and then washed with distilled water. After staining,
the cells were counterstained with Fast Green for 20 seconds and then washed with
distilled water for 2 min. The slides/cells were dried at room temperature and stored
for microscopic analysis. Slide analysis was performed single-blind using a conventional
optical microscope at a 400X magnification. A total of 2,000 cells per slide were
analyzed, for a total count of 4,000 cells per sample.

Cell classification

The BMCyt can be used as an indicator of DNA damage (MN and/or nuclear buds – BUDS),
cytokinesis defects (binucleated cells – BC), proliferation potential (basal cell
frequency), apoptotic cell death (condensed chromatin – CC, karyorrhectic cells –
KR, pyknotic cells – PY) and necrosis (karyolitic cells – KL) 16]. All these cell characteristics were analyzed in the samples.

Statistical analysis

Statistical analysis was performed by descriptive methods (i.e., mean, standard deviation,
median, frequency and quartiles); multiple comparisons were accomplished using the
analysis of variance (ANOVA) with the post-hoc Tukey test. The associations between the studied parameters were evaluated using
the Spearman correlation test in the Statistical Package for the Social Sciences (SPSS)
software version 20.0. Differences were considered to be significant with p 0.05.