Protective role of CYP2E1 inhibitor diallyl disulfide (DADS) on alcohol-induced malondialdehyde-deoxyguanosine (M1dG) adduct formation

Abstract

Background: Alcohol use disorders are often associated with lung disease. Alcohol exposure leads to the production of reactive oxygen species, lipid peroxidation, and formation of malondialdehyde (MDA) as well as to induce the expression of cytochrome p450 2E1 (CYP2E1). Likewise, cigarette smoking can lead to lung lipid peroxidation and formation of MDA. MDA can bind to DNA forming MDA-deoxyguanosine (M1dG) adducts, which have been implicated in alcohol-related cancers and cardiovascular disease. Because CYP2E1 regulates MDA production, and our previous studies have shown that alcohol and cigarette smoke can lead to MDA formation, we hypothesized that CYP2E1 would modulate M1dG adduct formation and single-strand DNA damage in alcohol- and cigarette smoke-exposed lung cells and tissue.

Methods: Normal human bronchial epithelial cells (HBECs) were pretreated with 10 μM diallyl disulfide (DADS) for 1 hour and treated with 80 mM ethanol (EtOH) ± 5% cigarette smoke extract (CSE) for 3 hours for comet assay and 6 hours for CYP2E1, MDA, and M1dG adduct assays. C57BL/6 mice were administered 20% EtOH ad libitum in drinking water for 8 weeks and exposed to whole-body cigarette smoke for 5 weeks. Mice were also fed a CYP2E1 inhibitor, DADS, at 1 μM/g of feed in their daily diet for 7 weeks. Whole lung tissue homogenate was used for CYP2E1, MDA, and M1dG adduct assays.

Results: EtOH exposure significantly increased HBEC olive tail moment. DADS pretreatment of HBECs attenuated this EtOH effect. EtOH also induced MDA and M1dG adduct formation, which was also significantly reduced by DADS treatment. CSE ± EtOH did not enhance these effects. In lung tissue homogenate of 8-week alcohol-fed mice, MDA and M1dG adduct levels were significantly elevated in comparison with control mice and mice fed DADS while consuming alcohol. No increase in MDA and M1dG adduct formation was observed in 5-week cigarette smoke-exposed mice.

Conclusions: These findings suggest that CYP2E1 plays a pivotal role in alcohol-induced M1dG adducts, and the use of DADS as dietary supplement can reverse the effects of alcohol on M1dG formation.

Keywords: CYP2E1; DADS; EtOH; M1dG; Malondialdehyde.

Fig. 1. Effect of ethanol exposure on single strand DNA damage measured by comet assay in HBECs

Monolayers of human bronchial epithelial cells (HBECs) were pretreated for 1 hr with diallyl disulfide (DADS; 10 μM) and further treated for 3 hr with ethanol (EtOH; 80 mM), cigarette smoke extract (CSE; 5%) and co-exposure condition. Hydrogen peroxide (H₂0₂; 0.1 mM) was used as positive control. Exposure to ethanol caused significant DNA damage, which was reduced significantly by DADS exposure. Values represent mean ± S.E.M. of (100–150) cells scored. Experiment was performed 3 independent times.

Fig. 2. Effect of ethanol and cigarette smoke on CYP2E1 levels in HBECs and C57BL/6 mice whole lung tissue homogenate

Human bronchial epithelial cells (HBECs) lysate and crude homogenates of mouse lung tissue were assayed using a sandwich ELISA. HBECs were pretreated for 1 hr with diallyl disulfide (DADS; 10 μM) and further exposed with ethanol (EtOH; 80 mM), cigarette smoke extract (CSE; 5%) and co-exposure condition for 6h and assayed using sandwich ELISA for CYP2E1 levels (A). Mice consumed EtOH ad libitum in water and were exposed to whole body cigarette smoke. Exposure to ethanol significantly induced CYP2E1 levels, which were reduced significantly by DADS supplement (B). Values represent mean ± S.E.M. of n=4 mice per condition and n=3 independent experiments for HBECs.

Fig. 3. Effect of ethanol and cigarette smoke on MDA levels in HBECs and C57BL/6 mice whole lung tissue homogenate

Lysate of human bronchial epithelial cells (HBECs) were pretreated for 1 hr with diallyl disulfide (DADS; 10 μM) and further exposed to ethanol (EtOH; 80 mM), cigarette smoke extract (CSE; 5%) and co-exposure condition for 6h and crude homogenates of mouse lung tissue were assayed for malondialdehyde (MDA) levels using a sandwich ELISA. Ethanol alone significantly increased MDA levels. Exposure to ethanol and cigarette smoke significantly increased MDA levels in tissue homogenate, which was reduced significantly by DADS supplement. Values represent mean ± S.E.M. of n=4 mice and n=3 independent experiments for HBECs.

Fig. 4. Effect of ethanol and cigarette smoke on M1dG levels in HBECs and C57BL/6 mice whole lung tissue homogenate

Lysate of human bronchial epithelial cells (HBECs) were pretreated for 1 hr with diallyl disulfide (DADS; 10 μM) and further exposed to ethanol (EtOH; 80 mM), cigarette smoke extract (CSE; 5%) and co-exposure condition for 6h and crude homogenates of mouse lung tissue were assayed using indirect ELISA for M1dG levels. Ethanol alone and in combination with cigarette smoke significantly increased M1dG levels. Ethanol exposure significantly increased M1dG levels, which were reduced significantly by DADS supplement. Values represent mean ± S.E.M. of n=4 mice and of n=3 independent experiments for HBECs.

Fig. 5. Proposed model of M1dG adduct formation

CYP2E1 mediated metabolism of alcohol leads to generation of reactive oxygen species (ROS). ROS enhances lipid peroxidation leading to the generation of malondialdehyde (MDA), which can adduct to the guanosine base of DNA to form a M1dG adduct. Inhibition of CYP2E1 by diallyl disulfide (DADS) leads to reduction in both MDA levels and M1dG adduct formation.