Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes.

http://www.healthcare.uiowa.edu/corefacilities/esr/publications/buettnerpubs/pdf/FRBM-2001-30-1191-FQS-Redox.pdf

Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple.

Malondialdehyde is a naturally occurring product of lipid peroxidation and prostaglandin biosynthesis that is mutagenic and carcinogenic. It reacts with DNA to form adducts to deoxyguanosine and deoxyadenosine. The major adduct to DNA is a pyrimidopurinone called M1G. Site-specific mutagenesis experiments indicate that M1G is mutagenic in bacteria and is repaired by the nucleotide excision repair pathway. M1G has been detected in liver, white blood cells, pancreas, and breast from healthy human beings at levels ranging from 1–120 per 108 nucleotides. Several different assays for M1G have been described that are based on mass spectrometry, 32 P -postlabeling, or immunochemical techniques. Each technique offers advantages and disadvantages based on a combination of sensitivity and specificity. Application of each of these techniques to the analysis of M1G is reviewed and future needs for improvements are identified. M1G appears to be a major endogenous DNA adduct in human beings that may contribute significantly to cancer linked to lifestyle and dietary factors. High throughput methods for its detection and quantitation will be extremely useful for screening large populations.

Lipid peroxidation—DNA damage by malondialdehyde

Biochemistry, Biology, and Carcinogenicity of Tobacco-Specific N-Nitrosamines†

Normal human bronchial epithelial cells were infected with SV40 virus or an adenovirus 12-SV40 hybrid virus, or transfected via strontium phosphate coprecipitation with plasmids containing the SV40 early region genes. Colonies of morphologically altered cells were isolated and cultured; these cells had extended culture lifespans compared to normal human bronchial epithelial cells. All cultures eventually underwent senescence, with the exception of one which appears to have unlimited proliferative potential. Colonies arising after viral infection were screened for virus production by cocultivation with Vero cells; only viral nonproducer cultures were analyzed further. The cells retained electron microscopic features of epithelial cells, and keratin and SV40 T-antigen were detected by indirect immunofluorescence. All of the cultures were aneuploid with karyotypic abnormalities characteristic of SV40-transformed cells. No tumors formed after s.c. injection of the cells in nude mice. These cells should be useful for studies of multistage bronchial epithelial carcinogenesis.

https://cancerres.aacrjournals.org/content/canres/48/7/1904.full.pdf

Transformation of Human Bronchial Epithelial Cells by Infection with SV40 or Adenovirus-12 SV40 Hybrid Virus, or Transfection via Strontium Phosphate Coprecipitation with a Plasmid Containing SV40 Early Region Genes

Because betel quid chewing has been linked to the development of oral cancer, pathobiological effects of an aqueous areca nut extract, four areca nut alkaloids (arecoline, guvacoline, guvacine, and arecaidine), and four nitrosated derivatives [N-nitrosoguvacoline, N-nitrosoguvacine, 3-(N-nitrosomethylamino)propionaldehyde and 3-(N-nitrosomethylamino)propionitrile] have been investigated using cultured human buccal epithelial cells. Areca nut extract in a dose-dependent manner decreases cell survival, vital dye accumulation, and membrane integrity, and it causes formation of both DNA single strand breaks and DNA protein cross-links. Depletion of cellular free low-molecular-weight thiols also occurs, albeit at quite toxic concentrations. Comparisons of the areca nut-related N-nitroso compounds and their precursor alkaloids, at concentrations up to 5 mM, indicate that 3-(N-nitrosomethylamino)propionaldehyde is the most potent on a molar basis to decrease both survival and thiol content and to cause significant formation of DNA single strand breaks. Arecoline, guvacoline, or N-nitrosoguvacoline decreases survival and cellular thiols, whereas arecaidine, guvacine, N-nitrosoguvacine, and 3-(N-nitrosomethylamino)propionitrile have only minor effects on these variables. Taken together, the present studies indicate that aqueous extract and, in particular, one N-nitroso compound related to areca nut, i.e., 3-(N-nitrosomethylamino)propionaldehyde, are highly cytotoxic and genotoxic to cultured human buccal epithelial cells, of potential importance in the induction of tumors in betel quid chewers.

https://cancerres.aacrjournals.org/content/canres/49/19/5294.full.pdf

Cytotoxic and genotoxic effects of areca nut-related compounds in cultured human buccal epithelial cells.

The ability of the highly reactive aldehyde acrolein to affect growth, membrane integrity, differentiation, and thiol status and to cause DNA damage has been studied at serum- and thiol-free conditions using cultured human bronchial epithelial cells. Acrolein markedly decreases colony survival at 3 microM whereas about 10-fold higher concentrations are required to increase membrane permeability, measured as uptake of trypan blue dye. Acrolein at micromolar concentrations also causes epithelial cells to undergo squamous differentiation as indicated by decreased clonal growth rate, dose-dependent increased formation of cross-linked envelopes, and increased cell planar surface area. Acrolein causes a marked and dose-dependent cellular depletion of total and specific free low-molecular-weight thiols as well as protein thiols. Exposure to acrolein did not cause oxidation of glutathione indicating that thiol depletion occurred by direct conjugation of reduced glutathione to acrolein without concomitant generation of active oxygen species. Furthermore, acrolein is genotoxic and causes both DNA single strand breaks and DNA protein cross-links in human bronchial epithelial cells. The results indicate that acrolein causes several cytopathic effects that relate to multistage carcinogenesis in the human bronchial epithelium.

/pdf/pathobiological-effects-of-acrolein-in-cultured-human-32fblln7rh.pdf

Pathobiological Effects of Acrolein in Cultured Human Bronchial Epithelial Cells

Lipid peroxidation aldehydes of the 4-hydroxy-alpha, beta-unsaturated type, as well as the tobacco-smoke related alpha, beta-unsaturated aldehyde, acrolein, were highly cytotoxic and decreased the intracellular thiol content in cultured human bronchial fibroblasts after treatment with micromolar concentrations. In comparison, formaldehyde and acetaldehyde were less toxic and 100- to 300-fold higher doses were required to affect cell survival or thiol levels. The unsaturated aldehydes also markedly inhibited the DNA repair enzyme O6-methylguanine-DNA methyltransferase known to have a cysteine residue in its active site, but had no effect on the activity of uracil-DNA glycosylase. Our results indicate that reactive aldehydes of either exogenous or endogenous origin have direct cytotoxic effects and may also make cells more susceptible to other toxic chemicals due to an impairment in cellular defense mechanisms, e.g., DNA repair and detoxification by systems requiring glutathione.

Cytotoxicity, thiol depletion and inhibition of O6-methylguanine-DNA methyltransferase by various aldehydes in cultured human bronchial fibroblasts.

The ability of acetaldehyde, a respiratory carcinogen present in tobacco smoke and automotive emissions, to affect cell viability, thiol status and intracellular Ca2+ levels and to cause DNA damage and mutations has been studied using cultured human cells. Within a concentration range of 3-100 mM, a 1 h exposure to acetaldehyde decreases colony survival and inhibits uptake of the vital dye neutral red in bronchial epithelial cells. Acetaldehyde also causes both DNA interstrand cross-links and DNA protein cross-links whereas no DNA single strand breaks are detected. The cellular content of glutathione is also decreased by acetaldehyde, albeit, without concomitant changes in the glutathione redox status or in the content of protein thiols. Transient or sustained increases in cytosolic Ca2+ occur within minutes following exposure of cells to acetaldehyde. Moreover, acetaldehyde significantly decreases the activity of the DNA repair enzyme O6-methylguanine-DNA methyltransferase. Finally, a 5 h exposure to acetaldehyde causes significant levels of 6-thioguanine resistance mutations in an established mutagenesis model involving skin fibroblasts. The results indicate that mM concentrations of acetaldehyde cause a wide range of cytopathic effects associated with multistep carcinogenesis. The fact that acetaldehyde, in relation to its cytotoxicity, causes comparatively higher genotoxicity and inhibits DNA repair more readily than other major aldehydes in tobacco smoke and automotive emissions is discussed.

Pathobiological effects of acetaldehyde in cultured human epithelial cells and fibroblasts

The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol-reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin perturbs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein-bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low-molecular-weight thiols and protein-bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol-depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein-bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cysteine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low-molecular-weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.

K. Sundqvist

Papers

Cytotoxic and genotoxic effects of areca nut-related compounds in cultured human buccal epithelial cells.

Pathobiological Effects of Acrolein in Cultured Human Bronchial Epithelial Cells

Cytotoxicity, thiol depletion and inhibition of O6-methylguanine-DNA methyltransferase by various aldehydes in cultured human bronchial fibroblasts.

Pathobiological effects of acetaldehyde in cultured human epithelial cells and fibroblasts

Growth-associated modifications of low-molecular-weight thiols and protein sulfhydryls in human bronchial fibroblasts.