The effect of malondialdehyde is modified by simian virus 40 transformation in human lung fibroblast cells.

The effects of malondialdehyde (MDA), a product of oxidative stress, on normal lung fibroblast cells (MRC5) and transformed cells (MRC5 SV2) showed differing responses between the two cell lines. MRC5 cells showed lower viability at low MDA concentrations (<250 µM) but had better viability at higher concentrations than the transformed cells. Both cell lines showed an increase in the number of micronuclei, nuclear size and a relocation of p53 to the nucleus with increasing MDA. The expression of p53 was higher in the MRC5 cells at 24 h; 2-8 fold induction vs 1-2.5 fold in the MRC5 SV2 cells, but reduced to almost zero at 48 h in the MRC5 cells. Mutation sequencing of the PCR products of a 689 bp region (residues 4640-5328) of the TP53 gene revealed MRC5 had more mutations than MRC5 SV2 cells (n = 21 and 11 respectively) and that they were predominantly insertions (MRC5 81%, MRC5 SV2 100%). A common mutation was observed in both cell lines; a G insertion at residue 4724 (n = 7) which could prove to be a mutational hotspot. These results indicate that the transformed cells are slower to respond to oxidative stress and/or mutagenic compounds. The mutation spectrum of predominantly frameshift mutations (insertions) suggests that oxidative stress plays a minimal role in smoking related lung cancer, but could be of greater importance to other lung diseases and cancer caused by exposures such as passive smokers, passive vapers and atmospheric pollutants.

3-(2-deoxy-ß-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine adducts of workers exposed to asbestos fibers.

Asbestos is the commercial name for a group of silicate minerals naturally occurring in the environment and widely used in the industry. Asbestos exposure has been associated with pulmonary fibrosis, mesothelioma, and malignancies, which may appear after a period of latency of 20-40 years. Mechanisms involved in the carcinogenic effects of asbestos are still not fully elucidated, although the oxidative stress theory suggests that phagocytic cells produce large amounts of reactive oxygen species, due to their inability to digest asbestos fiber. We have conducted a mechanistic study to evaluate the association between 3-(2-deoxy-ß-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) adducts, a biomarker of oxidative stress and lipid peroxidation, and asbestos exposure in the peripheral blood of 327 subjects living in Tuscany and Liguria, Italy, stratified by occupational exposure to asbestos. Adduct frequency was significantly greater into exposed subjects with respect to the controls. M1dG per 108 normal nucleotides were 4.0±0.5 (SE) in 156 asbestos workers, employed in mechanic, naval, petrochemical, building industries, and in pottery and ceramic plants, versus a value of 2.3±0.1 (SE) in 171 controls (p<0.001). After stratification for occupational history, the effects persisted in 54 current asbestos workers, mainly employed in building renovation industry (2.9±0.3 (SE)), and in 102 former asbestos workers (4.5±0.7 (SE)), with p-values of 0.033, and <0.001, respectively. A significant effect of smoking on heavy smokers was found (p=0.005). Our study gives additional support to the oxidative stress theory, where M1dG may reflect an additional potential mechanism of asbestos-induced toxicity.

Exocycilic DNA Adducts in a Murine Model of Non-alcoholic Steatohepatitis.

INTRODUCTION: Non-alcoholic fatty liver disease is the most common hepatic disorder in Western countries. The transition from abnormal accumulation of lipids toward non-alcoholic steatohepatitis (NASH) represents a key step in the development of chronic liver pathologies. Oxidative stress and lipid peroxidation have often been proposed as mechanisms in the progression to steatohepatitis. METHODS: We have examined the hepatic levels of exocyclic DNA adducts, indicated from 3-(2-deoxy-ß-D-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) adduct, a biomarker of oxidative stress and lipid peroxidation, in a murine model of NASH using the 32P-DNA postlabeling assay. RESULTS: Our findings show that C57BL/6 mice fed with high-fat and cholesterol diet developed signs associated with NASH after eight weeks, whereas there was no evidence of steatosis in control mice. The score for steatohepatitis ranged from grade 2 to 3 for steatosis, inflammation, and fibrosis, showing that the experimental diet was able to induce pathologic alterations of the parenchyma in eight weeks. Higher levels of M1dG adducts were detected in the livers of C57BL/6 mice which developed experimental NASH after eight weeks of high-fat and cholesterol feed, 5.6 M 1dG ± 0.4 (SE) per 106 total nucleotides, as compared to control mice, 1.6 M1dG ± 0.4 (SE). The statistical analysis showed that the increment of oxidatively damaged DNA in mice with NASH raised on high-fat and cholesterol diet was statistically significant as compared to control mice, P=0.006. CONCLUSIONS: Our report suggests a link between NASH and M1dG in experimental animals fed with a diet rich in saturated fats and cholesterol. High-fat and cholesterol may act together in inducing a broader spectrum of oxidatively damaged DNA, including exocyclic DNA adducts, that may contribute to the decline of hepatocyte functions, from disturbance of critical pathways, such as transcription and replication, triggering transient or permanent cell-cycle arrest and cell-death, up to chromosomal instability.