PubMed Health⌕ Search

Biomedical subjects

J J McCormick

Publications and source records attributed to J J McCormick.

At least 109 records · Page 6Linked to original sources
PubMed1984

Cytotoxicity and mutagenicity of aflatoxin dichloride in normal and repair deficient diploid human fibroblasts.

The cytotoxic and mutagenic effect of aflatoxin B1-dichloride (AFB1-Cl2), a direct-acting carcinogen which is a model for the proposed ultimate reactive metabolite of AFB1 (the 2,3-epoxide), was compared in normal, repair-proficient, diploid human fibroblasts and in complementation Group A xeroderma pigmentosum cells (XP12BE) which are virtually incapable of excision repair of DNA damage induced by ultraviolet radiation, the 7,8-diol-9,10-epoxide of benzo[alpha]pyrene, and several reactive aromatic amide derivatives. The XP cells were significantly more sensitive than normal to the cytotoxic and mutagenic effects of AFB1-Cl2, not only as a function of concentration administered but also of the number of AFB1-Cl2 residues initially bound to DNA. Cytotoxicity was determined from survival of colony-forming ability; resistance to 6-thioguanine was the genetic marker used for mutagenicity. We compared the rate of loss of AFB1-Cl2-DNA adducts from cells treated and held in the non-dividing state (confluent) over several days, as well as their ability to recover from the potentially mutagenic and/or cytotoxic effects of the agent. AFB1-Cl2 residues were lost from both strains of cells and both exhibited a gradual increase in survival. However, the rate of loss of adducts from the DNA in the normal cells was more rapid than in XP cells and they exhibited recovery from higher doses of AFB1-Cl2 than XP cells. The major primary DNA adduct formed in the human cells and in isolated DNA was a chemically unstable guanine derivative which could undergo a change in structure with time posttreatment to form a more stable secondary adduct. The cytotoxic effect of AFB1-Cl2 was highly correlated with the presence of either of these guanine adducts. Evidence suggests that the primary adduct is an N7-guanine adduct. The kinetics of the loss of this guanine and its transformation into the more stable secondary adduct resembled that reported recently for the major primary DNA adduct formed by the reaction of AFB1 at the N-7 position of guanine in the DNA of normal and XP cells and its transformation into the putative AFB1-ring opened triamino pyrimidyl structure.

Aflatoxin B1↗
PubMed1984

Comparison of the frequency of diphtheria toxin and thioguanine resistance induced by a series of carcinogens to analyze their mutational specificities in diploid human fibroblasts.

The mutagenic specificities of ethylnitrosourea (ENU), X-rays (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7, 8,9,10-tetrahydrobenzo[a]pyrene (BPDE), ICR-191, and N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) were analyzed and compared in diploid human fibroblasts and Salmonella typhimurium. In the human fibroblasts, we compared the frequency of diphtheria toxin (DT)-resistant mutants, presumably induced in the gene coding for elongation factor-2, with the frequency of 6-thioguanine (TG) resistance induced by mutations in the gene coding for hypoxanthine(guanine)phosphoribosyltransferase (HPRT). Recovery of DT-resistant (DTr) cells requires that the mutant EF-2 retain the ability to carry on protein synthesis since the normal EF-2 will be inactivated by DT selection. Therefore, the DTr mutation cannot involve major changes in the gene. In contrast, cells can acquire TG resistance by any mechanism which eliminates HPRT activity, e.g., base substitution, frameshift, deletion, loss of chromosomes. Each agent was assessed by calculating the ratio of the slopes of the dose-response plots (induced variant frequency as a function of dose of the agent used) for the two markers (DTr/TGr variants.). In S. typhimurium we examined the reversion frequency in four histidine-requiring strains bearing forward mutations of the frameshift (TA1538, TA98) or missense (TA1535, TA100) type. ENU, which was predominantly a base substitution mutagen in the bacteria, gave a ratio of DTr to TGr variants of 1.5. As expected of an agent inducing gross chromosomal changes, X-rays induced no revertants in bacteria and in human cells gave a ratio of 0.1. ICR-191 which was predominantly a frameshift mutagen in bacteria gave a ratio of 0.15. In the set of bacterial strains containing the plasmid pKM101, BPDE reverted both frameshift and base substitution mutations. It did not cause reversions in the other set of strains. In human cells BPDE gave a response similar to ENU, i.e., a ratio of DTr/TGr variants of 1.5. As reported by others, N-AcO-AAF was predominantly a frameshift mutagen in bacteria. However, in the human cells it gave a ratio of DTr/TGr variants of 1.5, similar to ENU and BPDE. These results suggest that in human cells, BPDE and N-AcO-AAF, like ENU, yield predominantly base substitutions, while ICR-191 and X-rays largely produce mutations by mechanisms which result in more extensive alterations in the gene.

Carcinogens↗
PubMed1984

Fibroblasts from patients with hereditary cutaneous malignant melanoma are abnormally sensitive to the mutagenic effect of simulated sunlight and 4-nitroquinoline 1-oxide.

Because of a possible etiologic link between mutations and carcinogenesis, we compared fibroblasts derived from skin biopsies of several patients with hereditary cutaneous malignant melanoma and the dysplastic nevus syndrome for sensitivity to the mutagenic and/or cytotoxic effect of broad-spectrum simulated sunlight and of a UV mimetic carcinogen, 4-nitroquinoline 1-oxide (4NQO). The genetic marker was resistance to 6-thioguanine; loss of colony-forming ability was the assay for cytotoxicity. All five strains tested were more sensitive than normal to the killing effect of 4NQO (slopes of survival curves were 2- to 3-fold steeper), but only one strain was hypersensitive to killing by Sun Lamp radiation. Two strains were tested for mutagenicity. The response of each to the mutagenic action of these agents corresponded to its response to cell killing. Both strains were hypermutable after exposure to 4NQO, but only one showed a higher than normal frequency of mutants induced by simulated sunlight. The finding that nonmalignant fibroblasts from patients with a hereditary variant of malignant melanoma are abnormally susceptible to carcinogen-induced mutations suggests that hypersensitivity to mutagens contributes to risk of melanoma in patients. It also supports the somatic cell mutation hypothesis for the origin of cancer.

4-Nitroquinoline-1-oxide↗
PubMed1984

Correlation between O6-methylguanine-DNA-methyltransferase activity and resistance of human cells to the cytotoxic and mutagenic effect of N-methyl-N'-nitro-N-nitrosoguanidine.

Cells from Gardner's syndrome (GS) and familial polyposis coli (FP) patients, persons with a hereditary predisposition to colon cancer, were compared to those of normal persons for sensitivity to the cytotoxic and mutagenic action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a model compound chosen because methylating agents have been implicated in colon carcinogenesis. FP cell line GM2355 and GS cell lines 2938 and GM3948 exhibited normal sensitivity to the cytotoxic and mutagenic effects of MNNG. In contrast, GS cell line GM3314 and cells from an apparently normal fetus GM0011 showed extreme sensitivity to the killing and mutagenic effect of this alkylating agent. To determine if the resistance of the various cell lines to MNNG correlated with their ability to remove methyl groups from the O6-position of guanine, we measured their O6-methylguanine-DNA methyltransferase (MT) activity. The resistant cell lines exhibited normal levels of MT; the sensitive strains showed virtually non-detectable levels of this activity. We also compared fibroblasts from a xeroderma pigmentosum (XP) patient (XP12BE, complementation group A), an SV40 virus-transformed XP cell line (XP12ROSV) and a normal cell line transformed by this virus (GM637) for their response to the cytotoxic and mutagenic effect of MNNG and for MT activity. XP12BE cells showed normal sensitivity and a normal level of MT; GM637 cells showed an intermediate level of sensitivity and a reduced level of MT activity; XP12ROSV cells were extremely sensitive to the cytotoxic and mutagenic effect of MNNG and showed virtually non-detectable levels of MT activity. The MT did not remove methyl group from O4-methyl-thymine. These results suggest that O6-methylguanine and/or any other adduct repaired by the methyltransferase, is a potentially cytotoxic and mutagenic lesion. They also indicate that the predisposition to colon cancer of FP and GS patients is not necessarily correlated with an increased sensitivity of their fibroblasts to mutations induced by methylating carcinogens.

Cell Line↗
PubMed1984

Endothelial cell cycle kinetics. Changes in culture and correlation with endothelial properties.

Endothelial cells in culture have emerged as a model system for evaluation of endothelial properties. Culture conditions may affect expression of these endothelial properties or growth characteristics. Therefore, we evaluated cell cycle kinetics, prostacyclin release, and angiotensin-converting enzyme activity in primary and subcultured cells from bovine pulmonary and systemic vessels. Prostacyclin release was highly variable during passage in culture. However, release following stimulation with ionophore A23187 declined with serial passage. Angiotensin-converting enzyme activity also declined with repeated subculturing. Greater than 10% of primary confluent bovine endothelial cells were cycling compared to 2 to 8% of human umbilical vein endothelial cells. With passage in culture, 25% of bovine endothelial cells were cycling at confluence. Occasional strains of subcultured bovine endothelium arrested in G2 but would reenter G1 upon subculturing. Exposure to trypsin did not appear to contribute to this phenomenon. These studies have demonstrated variability in endothelial cell properties and growth characteristics during in vitro propagation. The existence of this variability must be considered when studying properties or responses of subcultured endothelium.

Animals↗
PubMed1984

Immunologic injury to vascular endothelial cells: effects on release of prostacyclin.

Prostacyclin is released from cultured and ex vivo bovine vascular endothelium following sublethal immunologic injury by a heterologous antibody to endothelial cells developed in rabbits. This release was dependent on calcium and complement and was not enhanced by the presence of platelets. Prostacyclin release was diminished 1-2 hr after the injury, but recovered fully following reculture of the endothelial cells for 72 hr.

Animals↗
PubMed1983

Biological effects of incorporation of O6-methyldeoxyguanosine into Chinese hamster V79 cells.

Analysis of the biological effects of specific DNA alkylations by simple alkylating agents is complicated by the variety of sites involved. It is, therefore, of value to be able to incorporate into cellular DNA nucleosides alkylated in a single position, e.g., O6-methyldeoxyguanosine. Such cellular incorporation is particularly difficult to achieve because this nucleoside is rapidly demethylated by adenosine deaminase. We have attempted to achieve such incorporation into the DNA of V79 cells by using coformycin, an inhibitor of adenosine deaminase, and by forcing the cells to depend on exogenous purines by the use of medium containing aminopterin. The DNA of V79 cells exposed to O6-methyl-[8-3H]deoxyguanosine (2.4 microM, sp. act. 14500 Ci/mole) showed an incorporation level of 4 X 10(-8) nucleotides. When 1000-fold higher concentrations were employed (3-15 mM, sp. act. 1.6 Ci/mole), significant cytotoxicity and inhibition of DNA synthesis was observed. However, because it was not economically feasible to administer high specific activity O6-methyldeoxyguanosine to the cells at these concentrations, we could not determine the amount of labeled nucleoside incorporated into DNA. Examination of the frequency of 6-thioguanine-resistant cells in these treated populations showed no significant increase above the background level. Comparison of the cytotoxic effect of O6-methyldeoxyguanosine with deoxyadenosine showed that the toxicity induced by O6-methyldeoxyguanosine could have resulted from mimicry of deoxyadenosine, rather than by incorporation of the alkylated nucleoside itself.

Adenosine Deaminase Inhibitors↗
PubMed1982

An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts.

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on cell growth status. Exponentially growing cells treated with 10(-3)-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 x 10(-4). However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10(-3)-0.6 LF/ml), the survival decreased to 2 x 10(-6) and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10(-3) mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.

Cell Division↗
PubMed1982

Extent of excision repair before DNA synthesis determines the mutagenic but not the lethal effect of UV radiation.

Excision repair-proficient diploid fibroblasts from normal persons (NF) and repair-deficient cells from a xeroderma pigmentosum patient (XP12BE, group A) were grown to confluence and allowed to enter the G0 state. Autoradiography studies of cells released from G0 after 72 h and replated at lower densities (3-9 x 10(3) cells/cm2) in fresh medium containing 15% fetal bovine serum showed that semi-conservative DNA synthesis (S phage) began approximately 24 h after the replating. To determine whether the time available for DNA excision repair between ultraviolet irradiation (254 nm) and the onset of DNA synthesis was critical in determining the cytotoxic and/or mutagenic effect of UV in human fibroblasts, we released cultures of NF or XP12BE cells from G0, allowed them to reattach at lower densities, irradiated them in early G1 (approximately 18 h prior to the onset of S) or just prior to S phase, and assayed the frequency of mutations to 6-thioguanine resistance and the survival of colony-forming ability. The XP12BE cells, which are virtually incapable of excising UV-induced DNA lesions, showed approximately the same frequency of mutations and survival regardless of the time of UV irradiation. In NF cells, the slope of the dose response for mutations induced in cells irradiated just prior to S was about 7-fold steeper than that of cells irradiated 18 h earlier. However, the two sets of NF cells showed no significant difference in survival. Neither were there significant differences in the survival of NF cells released from G0, plated at cloning densities and irradiated as soon as they had attached and flattened out. (approximately 20 h prior to S) or 4, 8, 12, 16, 20, or 24 h later. We conclude that the frequency of mutations induced by UV is dependent upon the number of unexcised lesions remaining at the time of semi-conservative DNA replication. However, the amount of time available for excision of potentially cytotoxic lesions is not determined primarily by the period between irradiation and the onset of S phase.

Cell Line↗
PubMed1982

Relationship between excision repair and the cytotoxic and mutagenic effect of the 'anti' 7,8-diol-9,10-epoxide of benzo[a]pyrene in human cells.

The cytotoxic and mutagenic effect of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE) in normally excising diploid human cells treated just prior to onset of S was compared with that of cells allowed approximately 16 h for excision repair before onset of S and with that observed in excision-deficient xeroderma pigmentosum (XP12BE) cells. The cells were synchronized by release from density inhibition of cell replication. DNA synthesis began approximately 22 h after the cells were plated at lower density (i.e., 1.4 x 10(4) cells/cm2). The frequency of thioguanine-resistant mutants induced in normal cells treated just prior to onset of S was approximately 12- to 16-fold higher than that observed in cells treated in early G1 or treated in G0 (confluence) and then plated at lower density. The frequency approximated that expected for XP12BE cells from extrapolation of data obtained at lower doses. The frequency of mutants measured in normal cells treated in exponential growth was also much higher than that in the cells treated in early G1 or in G0. No such difference could be seen in XP12BE cells treated in exponential growth or in G0. In contrast to the mutagenicity data in the normal cells, there was no significant difference in the slope of the survival curve of normal cells treated at various times prior to S phase at low densities. However, normal cells treated even at the onset of S exhibited survival equal to XP12BE cells given a 4- to 5-fold lower dose. The data support the hypothesis that DNA synthesis is the cellular event which converts unexcised DNA lesions into mutations. However, they indicate that S is not the event primarily responsible for translating DNA damage into cell death. Accompanying studies on the rate of excision of anti BPDE adducts from the normal cells during the period prior to S support the conclusions.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1982

Frequency of UV-induced neoplastic transformation of diploid human fibroblasts is higher in xeroderma pigmentosum cells than in normal cells.

If neoplastic transformation of diploid human cells results from carcinogen-induced mutations, cells deficient in excision repair of UV-induced DNA damage should be significantly more sensitive to transformation by UV light than normal cells. We tested this hypothesis by irradiating fibroblasts from a xeroderma pigmentosum patient (XP7BE, complementation group D) with low doses of Uv light (254 nm) and cells from a normal person with much higher doses and comparing the frequency of transformation to anchorage independence. Both sets of cells exhibited a dose-dependent increase in transformation which corresponded to a dose-dependent decrease in survival. At doses that caused equal cell killing, the frequency of anchorage-independent cells was approximately equal. Colonies of XP7BE and normal cells isolated from agar, propagated, and injected into X-irradiated athymic mice produced fibrosarcomas in 100% of the animals. Normal cells irradiated shortly before the onset of DNA synthesis exhibited a high frequency of anchorage-independent cells; cells irradiated in early G1 showed no increase over background. These results agree with those we observed for UV induction of 6-thioguanine-resistant mutants in these cells and support the hypothesis that anchorage independence results from mutations induced by DNA replication on a damaged template.

Cell Cycle↗
PubMed1981

Identifying human cells capable of metabolizing various classes of carcinogens.

Human cells that appear capable of metabolizing various classes of carcinogens have been identified using one of two methods: metabolism of tritiated benzo(a)pyrene to aqueous-acetone soluble forms or inhibition of cellular DNA synthesis. Each of the assay systems was optimized and the results on 15 human epithelial cell lines were compared. One or more cell lines were found to activate each of four classes of carcinogens examined: polycyclic hydrocarbons, aromatic amines, heterocyclic hydrocarbons, and nitrosamines. Cells that appeared capable of metabolizing polycyclic hydrocarbons or aromatic amines by these methods were also found to produce metabolites which were cytotoxic to cocultivated human xeroderma pigmentosum fibroblasts after a 48-hr exposure to the carcinogen.

Benzo(a)pyrene↗
PubMed1981

Enhanced cell killing and mutagenesis by ethylnitrosourea in xeroderma pigmentosum cells.

It was shown previously that following treatment with ethylnitrosourea (ENU) SV40-transformed cells from xeroderma pigmentosum patient XP12RO, complementation group A remove O6-ethylguanine from their DNA 2 to 3 times more slowly than do such cells from a normal individual (GM637), but that each of the several other DNA adducts studied are removed at the same rate by both cell lines. If O6-ethylguanine contributes significantly to the cytotoxic effect of ENU, a population which can excise this lesion more rapidly than another during the posttreatment period should exhibit a higher survival. We compared these two cell lines for survival of colony-forming ability following exposure to ENU. XP12RO-SV40 cells proved approximately 3.5-fold more sensitive than GM637. To extend this study to non-transformed diploid human fibroblasts, we compared the survival of cells derived from normal individuals (NF) with that of fibroblasts from several XP patients: XP12BE (group A), XP7BE (group D), and XP4BE (an XP variant). The survival curve of NF, XP7BE, and XP4BE cells, but not XP12BE cells, exhibited a distinct shoulder. The slope of the survival curve for XP12BE cells was 3-fold steeper than that of the exponential part of the curve for NF; the slope of the curve for XP7BE cells, 2-fold steeper; and that of XP4BE cells, 1.7-fold steeper. This enhanced cytotoxicity was not observed in XP12BE cells treated with methylating agents such as methylnitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine. We compared NF and XP12BE cells for sensitivity to the mutagenic action of ENU and found approximately 3-fold higher frequencies of 6-thioguanine resistant mutants in the XP cells. This result is expected if the XP cells are slower than normal in excising O6-ethylguanine and if this adduct is mutagenic. When we compared the frequency of mutations induced by ENU and u.v. radiation at doses which caused equal cell killing, ENU was 4-fold more efficient a mutagen than u.v.

Cell Survival↗