SOURCE:

Faculty: Medicine
Department: Immunology

CONTRIBUTORS:

Ezeani, M.C;
Onyenekwe, C.C;
Meludu, S.C;

ABSTRACT:

The involvement of microbial agents in human breast tumourigenesis is gaining wider acceptance but the emanating microbes and their association with global DNA methylation alteration are still under studied. Microbial antigens, as components of circulating immune complexes, could be a predisposing factor for chronic inflammation and perturbation of epigenomic mechanisms that may result in DNA methylation shift in female subjects. Thus this study was designed to evaluate global DNA methylation, Pro-inflammatory molecules and level of microbial antigenic components of circulating immune complexes, in subjects with breast tumours, attending clinics at Nnamdi Azikiwe University Teaching Hospital Nnewi, Nigeria. Ninety nine (99) female subjects, comprising 24 subjects with benign breast tumour, 25 subjects with malignant breast tumour and 50 apparently healthy female subjects without tumour, were recruited for this cohort study. The test subjects had not undergone mastectomy and were not under any tumour therapy. Ten milliliter (10ml) of fasting blood sample was collected from the subjects and the serum was used for the following analysis: determination of serum level of immune complexes (IC) by precipitation using polyethylene glycol (PEG) 6000, detection of the microbial antigens using immuno-chromatographic technique; determination of serum levels of Tumour Necrosis Factor-alpha (TNF-α), total Immunoglobulin G (IgG), 8-hydroxy-2-deoxyguanosine (8-OH2DG), oestrogen and progesterone by enzyme linked immunosorbent assay. DNA extract was used for methylated DNA quantification and estimation of Nuclear Factor kappa B (NFkB), also using Enzyme Linked Immunosorbent Assay. Distribution of microbial antigens was statistically significant between subjects with benign tumour 21(87.5%), malignant tumour 20(80%) and healthy control subjects 12(24%) p=0.000. Distribution of DNA methylation patterns was statistically significant between subjects with benign tumour 18(75%), malignant tumour 24(96%) and healthy control subjects 10(20%) p=0.000. Microbial agents implicated at different degrees of distribution in benign and malignant breast tumours include: Plasmodium falciparum, Treponema pallidum, Hepatitis B virus, Hepatitis C virus, Helicobacter pylori, Salmonella typhi. In subjects with evidence of microbial antigenic components, there were significant lower mean levels of CIC, 71.00±36.00 (p=0.001); NFkB, 0.31±0.01 (p=0.000); IgG, 19.90±5.20 (p=0.000); TNF-α, 9.20±2.80 (p=0.000); 8-OH2DG, 9.20±4.00 (p=0.000); oestrogen, 141.00± 50.00(p=0.024), in control subjects compared with the subjects with tumours. Between the subjects with malignant tumour and benign tumour, there were significant higher mean values of CIC, 139.00±51.00 (P=0.036); NFkB 0.37±0.05 (p=0.014); IgG 28.20±3.90 (p=0.002); TNF-α 20.6±2.50 (p=0.009); 8-OH2DG 27.60±8.20 (p=0.000) and significant lower mean value of progesterone, 0.6.±0.4 (P=0.000) in subjects with malignant tumour. There was a positive correlation of immune complexes and oestrogen in subjects with benign and malignant tumours r=0.487 (p=0.025) and r=0.469 (p=0.039) respectively; positive correlation of NFkB and TNF-α, NFkB and OH2DG, TNF-α and 8-OH2DG in benign and control subjects p<0.05. Higher serum expression of pro-inflammatory molecules was seen in subjects with evidence of microbial antigens. The DNA methylation patterns expressed in subjects with breast tumours are hypomethylation and unmethylation. Burden of microbial infection in breast tumours was indicated by increased presence of microbial antigenic components. Routine laboratory investigation of DNA methylation shifts using whole blood is recommended; this is suggestive of epimutation and could be an early marker for tumour development.