Animal Maintenance
Virgin female Swiss albino mice (Mus musculus; 5-6 weeks old; weight: 23-25 gms) obtained from the Central Animal Facility of CNCI were housed in polyvinyl cages within well-ventilated rooms under ideal conditions (temperature: 22°C; relative humidity: 50-60%; 12-hour day/night cycle). Prior to treatment initiation, the animals were subjected to a two-week acclimation period during which all the female and male mice were kept in complete isolation from each other. This induced pheromone influenced estrous cycle synchrony, thereby nullifying hormonal interferences [36]. Standard guidelines laid down by the Institutional Animal Ethics Committee (IAEC) certified by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India, were strictly followed for undertaking all animal experimentation.
Experimental design
Random allocation of 100 mice into three broad groups was accomplished on the basis of body weight.Each group was further classified into subgroups separated into two batches of 10 animals [No of mice (n) =5 /cage (x2) for each subgroup]. Group I was kept as an ‘untreated’ control group where mice did not receive any treatment. Group III (groups 6-10) comprised mice that had their cervix chronically painted with 3MC (MP Biomedicals, USA) solution prepared in petroleum ether (PET, Merck, Emplura, Germany) for intervals of 6, 12, 16, 24 and 30 weeks [37]. It was specifically designated the ‘treatment’ group for studying cervical carcinogenesis over these time spans. To rule out any carcinogenic effect of the solvent (if any), Group II (groups 1-5) was assigned as a ‘vehicle control’ batch, with mice being solely treated with PET. Food and water was given ad libitum. Mice were periodically monitored for any visible health abnormalities and deaths. This purposeful procedure of mouse grouping is well represented in Table 1.
Record of body weight and carcinogenic incidences
Weekly body weight alterations in mice were recorded to assess the impact of chronic 3MC and PET treatments on their physiology with time. An orderly record of tumor incidences along with gradually acquired cervical dysplastic stages in the 3MC-treated mouse batches was also maintained.
After every mentioned treatment period, mice from all three groups were sacrificed, and their entire female reproductive tissue was excised out. To track the presence of any cervical tumor, these reproductive tissues were longitudinally opened up for examination through its upper vaginal (ectocervix) region. Cervix tissues (with or without tumor) were decapitated for comparative studies. In addition to the cervix, blood and spleen were also collected for experimental purposes.
Cytopathological study
Smears of cervical exfoliated cells suspended in phosphate-buffered saline (PBS, pH 7.4) were fixed with 100% ethyl alcohol and stained as per the protocol of Mahapatra et al. (2020) [37]. Sequential polychromatic staining with Harris hematoxylin (HH), orange G6 (OG6) and eosin azure (EA50) [Merck Millipore, Mumbai, India] followed by excess stain removal in tap water and 70% and 90% alcohol was observed. Finally, stained slides were mounted and observed under a light microscope (Zeiss). Fifty fields were randomly scanned for the presence of significant cytopathological changes in each slide.
Enumeration of differential count of cervical leukocytes
A numerical count of the microscopically identified subpopulations of cervical leukocytes was quantitated as ‘Differential Leukocyte Count’ (DLC). Over 50 fields of Pap-stained slides were randomly scanned to calculate DLC. For each field, DLC was enumerated as a percentage (%) of the specific number of leukocyte subpopulations (eosinophils, neutrophils and monocytes) observed among the total number of leukocytes [DLC= (Number of specific types of leukocytes)/(Total number of leukocytes) x 100].
Histology
Dissected cervical and spleen tissues were washed in cold normal saline (0.9%), fixed in 10% (w/v) neutral buffered formalin (NBF) for 24 h, processed through alcohol grades and xylene, and embedded in paraffin. These paraffinized tissue blocks were cut into thin (~5 μm) sections using a microtome and stretched over grease-free glass slides. These sections were further deparaffinized in xylene followed by successive staining in Delafield haematoxylin [DH; haematoxylin powder, 100% alcohol, (NH4)Al(SO4)2,glycerol, H2O] and 2% eosin. After mounting, 10-20 fields of these stained slides were examined under a light microscope (Zeiss) to document the histological changes.
Immunohistochemistry
Cervical tissue sections (~5 μm thick) were stained with primary antibodies against COX2, NFκB (p50/p65), XIAP, survivin, Ki67 and PCNA proteins to locate them within tissues by following the protocol of Basu et al. (2020) [38]. Paraffin from stretched tissue sections was removed by heating the slides at 65°C for 20 min followed by xylene treatment and rehydration through 100%, 90%, 70% and 50% alcohol downgrades. After washing these sections serially in PBS for 10 min, ‘antigen retrieval’ was carried out using preheated citrate buffer [pH-6; comprising C6H807. H2O and (CH2COONa)2.2H2O] at 85°C for 1 h, after which they were incubated with the respective primary antibodies diluted in 1% bovine serum albumin (BSA; Sigma Aldrich, USA) solution within a humid chamber overnight at 4°C. Excess primary antibodies were washed in 1X PBS. Slides were further incubated with HRP-conjugated secondary antibodies (1:500) in 1% BSA solution for 2 h at 37°C followed by immunostaining with the chromogenic substrate 3–3′ diaminobenzidine (DAB; Santa Cruz Biotechnology, USA) and counterstaining with DH. These slides were dehydrated through successive alcohol grades and xylene. Finally, they were mounted in DPX for observation under a light microscope (Zeiss). Approximately ten fields were scanned to score the positive staining intensities. Staining intensities (1 = weak, 2 = moderate, 3 = strong) were enumerated as per the percentage of positively stained cells (< 1 = 0, 1–20 = 1, 20–50 = 2, 50–80 = 3 and > 80 = 4). The final evaluation of tissue-specific protein expression was made as low (score 0-2), intermediate (2-5), and high (score 5–7) levels.
The primary antibodies employed were COX2 (GeneTex, 1:1000), NFκB (p50/p65) (GeneTex, 1:1000), Ki67 (GeneTex, 1:1000), PCNA (GeneTex, 1:1000), XIAP (GeneTex, 1:1000) and survivin (GeneTex, 1:1000).
Preparation of tissue lysates
Cervix tissues along with the adjoining tumor regions were dissected out, washed, and pooled separately from Group I, II and III mice. Tissue and tumor parts were dried, weighed and homogenized in radioimmunoprecipitation assay lysis buffer(RIPA; pH-8 comprising 5 M NaCl, 0.5 M EDTA, 1 M Tris, NP-40, 10% sodium deoxycholate, 10% SDS). The extracts were kept on ice for 30 min followed by sonication and centrifugation at 10,000 g for 20 min at 4°C. The resulting supernatants were stored in chilled vials at -20°C.
Estimation of total protein
The total protein content of the tissue extracts was spectrophotometrically (VARIAN) estimated using 1X Bradford’s reagent (HIMEDIA, PA, USA) against a standard curve of BSA. Absorbance was recorded at 595 nm, and the experiment was repeated 5 times.
Western Blot Analysis
The expression statuses of inflammatory mediators (COX2, GM-CSF1), tumor suppressor proteins [p53, acetylated-p53 (lys373), p21, Rb], prosurvival molecules (NFκB, XIAP, survivin) and proliferative antigen (Ki67) were comparatively studied by western blotting. Equitable amounts of cervical protein were loaded into each well of SDS-polyacrylamide (SRL, Mumbai, India) gels, electrophoretically separated using electrophoresis buffer (25 mM Tris, 192 mM glycine, 10% SDS) and electrotransferred to nitrocellulose membranes with the aid of transfer buffer (250 mM Tris, 192 mM glycine, 10% methanol). These membranes were blocked with 5% (w/v) BSA solution and washed with Tris-buffered saline (TBS; pH 7.5; 25 mM Tris. HCl, 150 mM NaCl) and incubated overnight with primary antibodies at 4°C under constant shaking. Blots were thereafter washed with TBST Buffer solution (TBS; Tween 20) 4 times and subsequently incubated with alkaline phosphatase conjugated secondary antibodies (GeneTex, 1:500dilutions in TBS) at 40C for 2h, followed by TBST washing (4times) and incubation with the chromogenic substrate 5-bromo, 4-chloro, 3-indoylphosphate/ Nitro-Blue tetrazolium (BCIP/NBT; SantaCruz Biotechnology, USA) for visualizing protein expressions in the form of bands. β-actin was used as a loading control protein. These experiments were performed in triplicate.
The primary antibodies used were COX2 (GeneTex, 1:1000), GM-CSF1 (Santa Cruz, 1:1000), NFκB (p50/p65) (GeneTex, 1:1000), Ki67 (GeneTex, 1:1000), PCNA (GeneTex, 1:1000), XIAP (GeneTex, 1:1000), survivin (GeneTex, 1:1000), acetyl p53 (lys 373) (Merck-Millipore, 1:1000), p53, p21, Rb (Santa Cruz, 1:1000) and β-actin (GeneTex, 1:1000).
Quantitative estimation of COX2 activity
COX2 enzyme (E.C 1.14.99.1) was spectrophotometrically quantitated at 590 nm using a COX activity assay kit (Cayman Chemical, Cat No: 760151 Ann Arbor, MI, USA) as per the provided protocols. The results are presented in nmol/min/ml (U/mg of protein).
Isolation of blood leukocytes
One volume of mouse blood collected aseptically from the heart was mixed with three volumes of Solution A (pH-7.2; 0.87% NH4Cl in 10 mM Tris HCl), incubated on ice for 20 min and centrifuged at 400 g for 20 min at 0°C. The supernatant was discarded, and the pellets were again resuspended in Solution A followed by centrifugation at 400 g for another 20 min at 0°C. The resulting pellets were suspended in Solution B (pH-7.2; 0.25 M mesoinositol, 10 mM Na2SO4, 1 mM MgCl2), cold centrifuged at 1500 rpm for 5 min at 4°C, and resuspended in HEPES-buffered saline (HBS; pH 7.4; 140 mM NaCl, 5 mM KCl, 10 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose). Cell numbers were adjusted as per experimental requirements.
ROS generation
Reactive oxygen species (ROS) generated due to chronic treatment with 3MC was quantitated according to the principle of Sinha and Roy (2010) [39]. Isolated leukocytes (106 cells/ml/point) suspended in HBS were incubated with a fluorescent probe, 2’,7’-dichlorofluorescein dihydroacetate or DCFH-DA (10 μM, Sigma-Aldrich, USA), for 45 min at room temperature in complete darkness. DCFH-DA passively diffuses within cells to transform into a diol moiety, which is further oxidized into a fluorescent compound, 2’, 7’-dichlorofluorescein (DCF), by intracellular ROS. DCF was quantitated spectrofluorimetrically (VARIAN; Excitation: 485 nm and Emission: 530 nm). For each point, readings were recorded approximately five times in triplicate attempts of the experiment.
RNS generation
Equal volumes of peritoneal macrophage suspension (106 cells/ml) in PBS and Griess reagent (1% sulphanilamide, 0.1% naphthyl ethylenediamine hydrochloride and 5% orthophosphoric acid) were incubated at 37°C for 30 min in a humidified chamber to quantify reactive nitrogen species (RNS) levels [40]. Absorbance was recorded at 550 nm with a spectrophotometer (VARIAN) against a standard blank. NO levels were enumerated against a standard curve of sodium nitrite. Readings for each point were taken approximately five times in triplicate.
Quantitative estimation of nitric oxide synthetase activity
Indirect assessment of nitric oxide synthetase (iNOS) enzyme (E.C1.14.13.39) activity was performed using a spectrophotometer by calculating the percentage of L-citrulline catalytically produced from L-arginine by means of a NOS activity assay kit (Cayman Chemical, Cat No: 781001, Ann Arbor, MI, USA) in accordance with the kit instructions.
Antioxidant Enzyme Activity
The free radical quenching capacity of the antioxidant scavengers present in the blood serum isolated using serum separating vials was assessed with the help of an antioxidant assay kit (Cayman Chemical, Cat No: 709001, Ann Arbor, MI, USA). Enzyme activity was expressed as IU/L with the help of a spectrophotometer, where readings were recorded within the absorbance range of 405-750 nm.
Single-cell gel electrophoresis (SCGE or comet assay)
The clastogenic effect of 3MC on DNA was assessed following the standard laboratory protocol [41]. Concisely, a suspension of 0.6% (w/v) low melting agarose (LMA; Sigma-Aldrich, USA) and isolated leukocytes (1x104 cells) was smeared over a frosted microscopic glass slide that was priorly coated with fixative 0.75% (w/v) normal melting agarose (NMA; Lonza, USA). Following solidification at 4°C, cell and nuclear membranes were lysed in lysis buffer (pH-10; 2.5 M NaCl, 0.1 M Na2EDTA, 10 mM Tris, 0.3 M NaOH, 1%Triton X-100, and 10%DMSO). Exposed DNA from the lysed leukocytes was unwound in highly alkaline electrophoresis buffer (pH >13.0; 300 mM NaOH, I mM Na2EDTA) prior to electrophoresis for 20 min (300 mA, 20 V). Slides were washed in neutralizing buffer (Tris 0.4 M, pH 7.5) three times, stained with ethidium bromide (final concentration 40 μg/ml) and examined under a fluorescence microscope (Leica). Image analysis, head DNA quantification, comet tail DNA length estimation and comet tail moment calculation were performed using Komet Software.
Statistical Analysis
The mean values of the control, vehicle control and treatment mouse groups were compared by factorial analysis of variance (ANOVA). The relationship between the studied parameters was analysed by calculating Pearson’s correlation coefficient using the CORREL function of Microsoft Excel. Data are expressed as the mean ± standard deviation (S.D.). p value calculations were performed using GraphPad Prism Software. *p<0.005 and **p<0.01 were considered statistically significant in comparison to control batches.