PubMed · 1938253
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W T Ulmer. 1991. [Cough].. https://pubmed.ncbi.nlm.nih.gov/1938253/
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When human respiratory tract epithelial cells were exposed to 100 microM H2O2, there was rapid induction of DNA strand breakage and chemical modifications to all 4 DNA bases suggestive of attack by OH.. The major products were FAPy-adenine, FAPy-guanine, and 8-OH-guanine. Some of the base modifications were removed very quickly from the DNA (e.g., 8-OH-guanine), whereas others persisted for longer (e.g., thymine glycol), probably due to differential activity of different repair enzymes. By contrast, strand breaks continued to increase over the time course of the experiment, perhaps because strand breakage is also implicated in the repair process. One should therefore be cautious in using strand breakage as a sole measure of oxidative DNA damage, and when drawing conclusions about the pattern and biological significance of oxidative DNA damage in cells the relative persistence of different lesions must be considered.
To investigate the role of human bronchial epithelial cells (HBECs) in the maintenance and remodeling of the extracellular matrix, we evaluated the expression by HBECs of 72- and 92-kDa gelatinases under basal conditions and after exposure to bacterial lipopolysaccharides (LPS). Confluent HBECs from explants were cultured in plastic dishes coated with type I and III collagens. Gelatin zymography of HBEC-conditioned media showed constitutive major 92-kDa and minor 72-kDa gelatinases recognized by specific human antibodies and totally inhibited by the metalloproteinase inhibitor EDTA. The identification of the two matrix metalloproteinases was confirmed by quantitative reverse transcription-polymerase chain reaction. Identical patterns of gelatinase expression were observed with repetitive primary cultures issued from the same explants. Zymography showed that exposure of HBECs to LPS induced 2- and 20-fold increases in 92-kDa gelatinase production and activation, respectively, as well as a smaller increase in activated 68-kDa gelatinase. With [3H]gelatin substrate, elevated metallogelatinolytic activity (138 microgram of hydrolyzed gelatin/48 h/10(6) cells) was also observed, whereas no activity was detected in the absence of LPS. A human epithelial cell line (16HBE14o-) exhibited the same basal profile of gelatinase activity, but this profile remained unchanged after exposure to LPS. Quantitative reverse transcription-polymerase chain reaction demonstrated only minimal changes in 92-kDa mRNA levels in response to LPS, but the half-life of 92-kDa gelatinase mRNA was increased with exposure to LPS. In contrast, concomitant slight increases in 72-kDa gelatinase protein and mRNA were found, suggesting that the control mechanisms regulating the expression of 92- and 72-kDa gelatinases by HBECs in response to LPS are divergent. All these data allowed us to propose that HBECs may be actively involved in the physiological and physiopathological remodeling of the airway basement membrane.
We have examined various radiobiological parameters using commercially-available primary normal human bronchial epithelial (NHBE) cells, which can be subcultured more than 20 population doublings, and have established the mutation system in order to characterize the molecular changes in gamma-irradiated primary cells. The survival curve, obtained after irradiation of cells with 137Cs gamma-rays, indicates that the D0, Dq, and n values are 1.34 Gy, 1.12 Gy, and 2.3, respectively. The induction of HPRT- mutation was dose-dependent and the mutant fraction increased in a non-linear fashion. Since the doubling number of NHBE cells is limited, DNA was extracted directly from the single mutant colonies and alteration in the HPRT gene locus was analyzed using multiplex PCR technique. Among spontaneous mutants, the proportion with total and partial deletions of the gene was 10.0% (2/20) and 60.0% (12/20), respectively, while 30.0% (6/20) did not have any detectable changes in the nine exons examined. On the other hand, the fraction of total deletion increased by more than 2-fold among mutants induced by gamma-rays in that 26.3% (10/38) of them showed the total gene deletions. Twenty-five out of 38 gamma-induced mutants (65.8%) had partial deletions and 3 mutants (7.9%) had no detectable alteration. The present results showed that gamma-irradiation efficiently induced HPRT gene mutation in primary human epithelial cells and that most of the induced mutants suffered larger deletions compared to that observed in spontaneous mutants. This system provides an useful tool for determination of mutagenicity and understanding the molecular mechanisms of environmental carcinogens in primary human bronchial cells.