Differential efficiency of mutagenesis at three genetic loci in CHO cells by a benzo[a]pyrene diol epoxide

The formation of DNA adducts by the ultimate carcinogen 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene (BPDE-I) has been implicated in the process of carcinogenesis. In a line of Chinese hamster ovary (CHO) cells designated AT3-2 and in two derivative mutant lines, UVL-1 and UVL-10, originally selected for hypersensitivity to UV-irradiation, we have measured the formation of BPDE-I: DNA adducts and the production of biological damage. The quantity and quality of BPDE-I: DNA adducts formed initially in the 3 cell lines are identical over a wide range of BPDE-I doses. However, the UVL lines are unable to remove adducts from their DNA, while the AT3-2 cells remove about 50% of the BPDE-I: DNA adducts in a 24-h incubation. Correlated with this, the UVL lines are more sensitive to the lethal effects of BPDE-I than are the AT3-2 cells. Mutant frequencies were measured at the aprt, hprt and oua loci and were found to increase linearly with BPDE-I: DNA adduct formation at doses which gave greater than 50% survival. At the hprt and oua loci, the efficiency of mutation induction was similar for AT3-2 and UVL-10 cells. UVL-1 cells showed slightly higher (within a factor of 2-3) mutant frequencies in response to BPDE-I compared to AT3-2 at these two loci. However, at the aprt locus the repair-deficient cells were much more highly mutable (9-15-fold) than the repair-proficient AT3-2 cells. Based on the measured average level of adduct formation, it is calculated that 15% of the BPDE-I: DNA adducts in the aprt gene are converted into mutations. However, the possibility exists that the aprt locus is subject to higher levels of modification by BPDE-I than is the bulk DNA, which would lead to an artifactually high apparent conversion frequency.     

Development of captive bongos (Tragelaphus eurycerus): Activity budgets and developmental milestones

The development of changes in visual orientation, posture, activity budgets, and behavioral milestones was examined in five bongo calves which were observed from birth to 4 or 6 months of age. The only significant change in the calves' visual orientation was an increase in orientation to food. Reclining declined significantly over weeks, while standing increased and moving did not change. Overall, stationary behaviors dominated the activity budget, but decreased significantly over the study. The category that included locomotion, object exploration, and auto-play did not change, while forage and rumination increased significantly. Contrary to expectation, the decline in suckling was not significant. Affiliative interactions with the dam decreased significantly during the study, but increases in affiliation with other bongos were not significant. Levels of agonistic behavior between the calves and their dams and other herd members were low throughout the study, and did not change significantly. One female calf achieved several developmental milestones later than the others. It is suggested that these differences may have been influenced by the dominance status of her dam. The results conform to the general developmental pattern of “hider” bovids. Bongos, however, show somewhat different patterns of social development than eland, which are reported to be more precocial in expression of aggressive and sexual behaviors. © 1992 Wiley-Liss, Inc.     

Low absolute mutagenic efficiency but high cytotoxicity of a non-bay region diol epoxide derived from benzo[a]pyrene.

Insights into the mechanisms of chemical carcinogenesis can sometimes be gained by comparing the effects of closely related chemicals which differ in carcinogenic potency. We have treated Chinese hamster ovary (CHO) cells with a non-carcinogenic metabolite of benzo[a]pyrene, 9r,10t-dihydroxy-7c,8c-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-III), and measured the formation and persistence of DNA adducts. We have correlated this binding data with cytotoxicity and mutagenicity in a DNA-repair-proficient CHO cell line (AT3-2) and in two derived lines, UVL-1 and UVL-10, which are unable to repair bulky DNA adducts. These data are compared with similar studies of the effects of the carcinogenic metabolite, 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Synchronous fluorescence spectroscopy was used to measure the levels of BPDE-III-DNA adducts in treated cells. Adduct levels increased linearly with dose, but the absolute binding levels were about 30-fold lower than in comparable incubations with BPDE-I. Measurements of the removal of adducts derived from these two diol epoxides indicated no significant difference in the rate of repair measured 24 h post-treatment. When cells were treated with increasing doses of BPDE-III, survival curves were obtained which exhibited a shoulder region at low doses and an exponential decrease in plating efficiency at higher doses. By comparison of the D0's, the DNA-repair-deficient cell lines were found to be 4-5-fold more sensitive to the killing effects of BPDE-III than were the repair-proficient AT3-2 cells.     

Differences in the rate of DNA adduct removal and the efficiency of mutagenesis for two benzo[a]pyrene diol epoxides in CHO cells.

The initiation of carcinogenesis by carcinogens such as 7r,8t-dihydroxy-9,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) is thought to involve the formation of DNA adducts. However, the diastereomeric diol epoxide, 7r,8t-dihydroxy-9,10c-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-II), also forms DNA adducts but is inactive in standard carcinogenesis models. We have measured the formation and loss of DNA adducts derived from BPDE-II in a DNA-repair-proficient line of Chinese hamster ovary (CHO) cells, AT3-2, and in two derived mutant cell lines, UVL-1 and UVL-10, which are unable to repair bulky DNA adducts. BPDE-II adducts were lost from cellular DNA in AT3-2 cells with a half-life of 13.8 h; this was about twice the rate found for BPDE-I adducts. BPDE-II adducts were also lost from DNA in UVL-1 and UVL-10 cells, but at a much slower rate. When purified DNA was modified in vitro with BPDE-II and then held at 37 degrees C, DNA adducts were removed at a rate identical to that seen in UVL-1 and UVL-10 cells, suggesting that the loss in these cells was not due to enzymatic DNA-repair processes but to chemical lability of the adducts. Mutant frequencies at the APRT and HPRT loci were measured at BPDE-II doses that resulted in greater than 20% survival, and were found to increase linearly with dose. In the DNA-repair-deficient cells, the HPRT locus was moderately hypermutable compared with AT3-2 cells (about 5-fold); the APRT locus was extremely hypermutable, giving about 25-fold higher mutant fractions in UVL-1 and UVL-10 than in AT3-2 cells at equal initial levels of binding. When we compared the mutational efficiency of BPDE-II at both loci in AT3-2 cells (the mutant frequency in mutants/10(6) survivors at a dose that resulted in one adduct per 10(6) base pairs) with our previous studies of BPDE-1, we found that BPDE-II was 4-5 times less efficient as a mutagen than BPDE-I. This difference in mutational efficiency could be explained in part by the increased rate of loss of BPDE-II adducts from the cellular DNA, part of which was due to an increased rate of enzymatic removal of these lesions compared with the removal of BPDE-I adducts.     

Transformation of UV-hypersensitive Chinese hamster ovary cell mutants with UV-irradiated plasmids

Transfection of UV-hypersensitive, DNA repair-deficient Chinese hamster ovary (CHO) cell lines and parental, repair-proficient CHO cells with UV-irradiated pHaprt-1 or pSV2gpt plasmids resulted in different responses by recipient cell lines to UV damage in transfected DNA. Unlike results that have been reported for human cells, UV irradiation of transfecting DNA did not stimulate the genetic transformation of CHO recipient cells. In repair-deficient CHO cells, proportionally fewer transformants were produced with increasing UV damage than in repair-proficient cells in transfections with the UV-irradiated hamster adenine phosphoribosyltransferase (APRT) gene contained in plasmid pHaprt-1. However, transfection of CHO cells with UV-irradiated pSV2gpt resulted in neither decline in transformation frequencies in repair-deficient cell lines relative to repair-proficient cells nor stimulation of genetic transformation by UV damage in the plasmid. Blot hybridization analysis of DNA samples isolated from transformed cells showed no dramatic changes in copy number or arrangement of transfected plasmid DNA with increasing UV dose. We conclude that the responses of recipient cells to UV-damaged transfecting plasmids depend both on the type of recipient cell and the characteristics of the genetic sequence used for transfection.     

Behavioral treatment of irritable bowel syndrome: A 1-year follow-up study

Sixteen clients afflicted with irritable bowel syndrome (IBS) were reassessed 1 year following completion of a multicomponent treatment package incorporating progressive muscle relaxation, thermal biofeedback, cognitive therapy, and IBS education. For the 14 patients who kept a 2-week symptom diary, significant reductions in ratings of abdominal pain and tenderness, diarrhea, and flatulence were obtained comparing pretreatment and follow-up symptom-diary ratings. Eleven of 14 clients were improved over pretreatment levels, 57% met the criteria for clinical improvement of at least a 50% reduction in major symptom scores, and all but 1 of 16 rated themselves as subjectively improved.     

New Method for Visualization of Vascular Networks in Nonperfused Fixed Tissues

A new method of visualizing the angioarchitecture of tissues has been developed that uses blood components in nonperfused materials. Tissue blocks are fixed in 4% paraformaldehyde and cut with a vibratome into 50-60 μm sections. Endogenous peroxidase in red blood cells is then reduced in the presence of hydrogen peroxide with the resultant oxidation of the chromogen 3,3'-diaminobenzidine (DAB). This generates a dark, highly insoluble reaction product throughout the vascular system. The visualization of vascular components can be further enhanced by exposing the sections to peroxidase-conjugated IgG to increase the background staining of the blood plasma. The technique minimizes preparation artifact and permits the application of morphometric analytical methods, thus allowing parameters such as the volume density of the vascular bed to be quantified.