Detection of carcinogenic etheno-DNA adducts in children and adolescents with non-alcoholic steatohepatitis (NASH)

Abstract

Background: Carcinogenic exocyclic-DNA adducts like 1,N(6)-etheno-2'-deoxyadenosine (εdA) are formed through reactive intermediates of 4-hydroxynonenal (4-HNE) or other lipid peroxidation (LPO) products with the DNA bases A, C, methyl-C and G. High levels of hepatic etheno-DNA adducts have been detected in cancer prone liver diseases including alcoholic liver disease (ALD). In ALD εdA levels correlated significantly with cytochrome P-450 2E1 (CYP2E1) expression which is also induced in non-alcoholic steatohepatitis (NASH). We investigated the occurrence of εdA adducts in children with NASH as a DNA damage marker.

Methods: Liver biopsies from 21 children/adolescents with histologically proven NASH were analysed for hepatic fat content, inflammation, and fibrosis. εdA levels in DNA, CYP2E1-expression and protein bound 4-hydroxynonenal (HNE) were semi-quantitatively evaluated by immunohistochemistry.

Results: Among 21 NASH children, εdA levels in the liver were high in 3, moderate in 5, weak in 9 and not elevated in 4 patients. There was a positive correlation between CYP2E1 and protein-bound 4-HNE (r=0.60; P=0.008) and a trend for a positive relationship for CYP2E1 vs. staining intensity of εdA (r=0.45; P=0.06). Inflammatory activity and fibrosis correlated significantly (r=0.49, P=0.023).

Conclusions: Our results demonstrate for the first time the presence of elevated carcinogenic etheno-DNA lesions (εdA) in the majority (17/21) of liver biopsies from young NASH patients. Our data suggest that LPO-derived etheno-adducts are implicated in NASH. Whether these adducts may serve as predictive risk markers in NASH children to develop hepatocellular cancer later in life remains to be investigated.

Keywords: Cytochrome P-450 2E; etheno-DNA adducts; hepatocellular cancer; lipidperoxidation-induced DNA damage; non-alcoholic steatohepatitis in children (NASH in children).

Figure 1

Immunohistochemistry of εdA in 21 children/adolescents with NASH (100×). Immunohistochemical staining of an exocyclic etheno-DNA adduct (etheno-deoxyadenosine, εdA) in hepatic nuclei of liver biopsies from 21 children (aged 8-17 years) using a highly specific monoclonal antibody (22). The staining intensity varied from strong (#1,5,20), moderate (#8,9,10,11,21), mild (#2, 3,4,6,7,13,14,16,17) and non-detectable background levels only in (#12,15,18,19). εdA, 1,N⁶-etheno-2'-deoxyadenosine; NASH, non-alcoholic steatohepatitis.

Figure 2

Immunohistochemistry of εdA, CYP2E1, and 4-HNE in 6 children/adolescents with NASH (100×). Immunohistochemical detection of εdA (upper panel, 1), CYP2E1 (mid panel, 2) and 4-HNE (lower panel, 3) in liver biopsies of six NASH children. The staining intensity between CYP2E1 expression and protein bound 4-HNE correlated significantly (r=0.60; P=0.008); correlation between εdA and CYP2E1 in the 21 biopsies showed a trend (r=0.45; P=0.06). εdA, 1,N⁶-etheno-2'-deoxyadenosine; CYP2E1, cytochrome P-450 2E1; 4-HNE, 4-hydroxynonenal; NASH, non-alcoholic steatohepatitis.

Figure 3

Immunohistochemical staining of εdA and alkyladenine DNA glycosylase (AAG) in liver biopsies of six NASH children (100×). While in 1 biopsy (#4) high εdA staining is associated with high AAG staining, in all other biopsies εdA stainig and AAG staining seem to be inversely related to each other. εdA, 1,N⁶-etheno-2'-deoxyadenosine.