The alkaline elution technique was used to study repair of DNA damage caused by formaldehyde (HCHO) in human bronchial epithelial cells and fibroblasts, skin fibroblasts, and DNA excision ...repair-deficient skin fibroblasts from donors with xeroderma pigmentosum. Exposure of cells to HCHO resulted in DNA-protein cross-links (DPC) and DNA single-strand breaks (SSB) in all cell types. DPC were induced at similar levels and were also removed by all cell types, with a half removal time of 2 to 3 hr. HCHO caused more SSB in the normal cell types than in the xeroderma pigmentosum fibroblasts. However, in all cell types, including the xeroderma pigmentosum cells, HCHO-induced DNA SSB and DPC were removed at comparable rates. By excision repair of HCHO-induced DNA damage, normal cells generated SSB that were also readily repaired. HCHO was only moderately cytotoxic to normal bronchial epithelial cells and fibroblasts at concentrations that induced substantial DNA damage. HCHO enhanced the cytotoxicity of both ionizing radiation and N-methyl-N-nitrosourea in both cell types. The results indicate that most DPC caused by HCHO can be removed without the involvement of DNA excision repair. Furthermore, HCHO also directly causes DNA SSB as well as SSB generated indirectly during ultraviolet-type excision repair. These studies indicate the complexity of the HCHO-induced DNA damage and its repair and that HCHO may enhance the cytotoxicity of chemical and physical carcinogens in human cells.
Acrolein, a short-chain aldehyde encountered as a component of tobacco smoke and as a ubiquitous environmental contaminant, was tested for its toxic and mutagenic effects toward human fibroblast ...cells. We found that human cells characterized by a deficiency in DNA repair (cells from xeroderma pigmentosum (XP) patients) were much more sensitive (D37 approximately equal to 0.25 microM) to the cytotoxic effects of acrolein than were cells from normal individuals (D37 approximately equal to 0.8 microM). Acrolein was also strongly mutagenic to the XP cells (a dose response was observed between 0.2 and 0.8 microM acrolein); however acrolein did not induce an increase in the mutant frequency of normal fibroblasts. Possible reasons for this apparent lack of mutagenicity in normal human cells are discussed.
The possible role of oxygen radicals in mediating the cytopathologic effects of asbestos was studied using human mesothelial cells in culture. Electron paramagnetic resonance measurements of intact ...cells using the spin trap 5,5-dimethyl-1-pyrroline-1-oxide failed to detect any increase in oxygen radicals in mesothelial cells after exposure to amosite asbestos, although oxygen radicals were readily detected in cells exposed to menadione, an uncoupler of oxidation-reduction reactions. Cellular thiol levels were reduced after exposure to menadione, but were not affected by exposure to asbestos. Addition to the culture media of the free radical scavengers superoxide dismutase, reduced glutathione, N-acetylcysteine, or D-alpha-tocopherol had no affect on the dose-dependent cytotoxicity of amosite fibers. Furthermore, exposure of the mesothelial cells to amosite fibers resulted in no significant increase in the level of DNA single-strand breaks. These results all suggest that for cultured human mesothelial cells, oxygen free radicals are not important mediators of the cytopathic effect of asbestos.
The metabolism of benzoapyrene, aflatoxin B1, N-nitrosodimethylamine, N-nitrosoethylmethylamine, and N-nitrosopyrrolidine has been studied in cultures of normal human and rat urinary bladder ...epithelial cells. The cultures were incubated with radioactively labeled carcinogens for 24 h, and the metabolism was assayed by binding of reactive metabolites to DNa and by the release of metabolites into the medium. Only slight variation in binding level of benzoapyrene to DNa among the three human bladder cell lines was seen, the level of binding being higher than to rat DNA. The major benzoapyrene-DNA adduct (80%) in human bladder cells eluted prior to the adducts formed by reaction of 7,8-dihydroxy-9,10-epoxy - 7,8,9,10-tetrahydrobenzapyrene with guanine by high pressure liquid chromatography, but has yet to be identified. The benzoapyrene-DNA adducts were quickly removed and only about 10% of the radioactivity remained associated with human bladder DNA 72 h post-treatment with benzoapyrene. The 7,8- and 9,10-diols of benzoapyrene were the major organo-soluble metabolites formed by both rat and human bladder cells. The primary benzoapyrene metabolites were conjugated to a minor extent only. The highest level of modification of DNA was seen in the case of N-nitrosodimethylamine. N-Nitrosopyrrolidine was oxidized in both the alpha-and beta-position by all three cell lines, the oxidation at the alpha-position being predominant. No binding to DNA was detectable with N-nitrosoethylmethylamine, although this compound was metabolized as measured by the formation of CO2 and aldehydes. These results add the urinary bladder to the list of human organs which have been shown to metabolize chemical carcinogens into electropositive metabolites. However, qualitative differences exist between the data from bladder cells and those from other human organs.
The role of prostaglandin H synthase (PHS) in the metabolism of 7,8-dihydroxy-7,8-dihydrobenzoapyrene (BP-7,8-diol) has been examined in short-term explant cultures of hamster and human ...tracheobronchial tissues. Labeled BP-7,8-diol was incubated with the explants in the presence and absence of the PHS substrate arachidonic acid (20:4) and the PHS inhibitor indomethacin. The addition of 10 microM to 200 microM 20:4 to incubations of hamster trachea with 5 microM BP-7,8-diol caused significant increases in the formation of 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo apyrene (anti-BPDE). These increases were not seen when 1 microM or 20 microM BP-7,8-diol was employed. The stimulation of anti-BPDE formation was observed after incubations of from 1 to 48 h. This stimulation was inhibited to the basal level by 20 microM indomethacin, supporting the role of PHS in the response. No effect of 20:4 was seen on the uptake of BP-7,8-diol by the tracheas or on the formation of water-soluble metabolites. Significant increases in covalent binding of BP-7,8-diol metabolites to DNA of the tracheal epithelium were also elicited by the addition of 20:4, however these increases were not well correlated quantitatively with the increases in anti-BPDE formation. H.p.l.c. profiles of deoxynucleoside adducts from basal and 20:4-stimulated incubations were qualitatively identical. Far greater variability of metabolism was seen in human bronchus explants, but 20:4-dependent increases in anti-BPDE formation could be demonstrated in those tissues as well. Inhibition of this stimulation by indomethacin was either absent or incomplete. This variation in the effect of indomethacin was explained by the examination of the products of 20:4 metabolism by the two tissues. Hamster trachea produced almost exclusively PHS metabolites whereas human bronchus yielded predominantly products of lipoxygenases, enzymes insensitive to indomethacin. In conclusion, this study indicates that co-oxygenation of chemical carcinogens can occur in hamster and human tracheobronchial tissues. The concentration-dependence observed with BP-7,8-diol, however, suggests that this pathway is of minor importance in the activation of BP in these tissues.
The metabolism of chemical carcinogens has been studied in cultured human bronchus, colon, duodenum, pancreatic duct, and esophagus. Metabolite patterns and carcinogen-DNA adducts are generally ...qualitatively similar among animal species, individuals within a species, and tissues within an individual. However, wide quantitative differences are observed between individuals in outbred animal species, including humans. These interindividual differences in amounts of carcinogen-DNA adducts and in activities of enzymes that are important in the metabolism of chemical carcinogens are similar in magnitude (10-to 150-fold) to those observed in pharmacogenetic studies of drug metabolism. The role of these differences as risk factors in human cancer is being investigated.
The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA ...single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenzaanthracene; benzoapyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents.
A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for ...conducting multi-institutional clinical studies based on expression measurement. Standardized RT (StaRT)-PCR has all these characteristics. In addition, the method must be reproducible. StaRT-PCR has high intralaboratory reproducibility. The purpose of this study is to determine whether StaRT-PCR provides similar interlaboratory reproducibility.
In a blinded interlaboratory study, expression of ten genes was measured by StaRT-PCR in a complementary DNA sample provided to each of four laboratories. The average coefficient of variation for interlaboratory comparison of the nine quantifiable genes was 0.48. In all laboratories, expression of one of the genes was too low to be measured.
Because StaRT-PCR data are standardized and numerical and the method is reproducible among multiple laboratories, it will allow development of a meaningful gene expression database.
