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D L Mitchell

Publications and source records attributed to D L Mitchell.

At least 37 records · Page 2Linked to original sources
PubMed1999

Effect of oxidative DNA damage in promoter elements on transcription factor binding.

Reactive oxygen species produced by endogenous metabolic activity and exposure to a multitude of exogenous agents impact cells in a variety of ways. The DNA base damage 8-oxodeoxyguanosine (8-oxodG) is a prominent indicator of oxidative stress and has been well-characterized as a premutagenic lesion in mammalian cells and putative initiator of the carcinogenic process. Commensurate with the recent interest in epigenetic pathways of cancer causation we investigated how 8-oxodG alters the interaction between cis elements located on gene promoters and sequence-specific DNA binding proteins associated with these promoters. Consensus binding sequences for the transcription factors AP-1, NF-kappaB and Sp1 were modified site-specifically at guanine residues and electrophoretic mobility shift assays were performed to assess DNA-protein interactions. Our results indicate that whereas a single 8-oxodG was sufficient to inhibit transcription factor binding to AP-1 and Sp1 sequences it had no effect on binding to NF-kappaB, regardless of its position. We conclude from these data that minor alterations in base composition at a crucial position within some, but not all, promoter elements have the ability to disrupt transcription factor binding. The lack of inhibition by damaged NF-kappaB sequences suggests that DNA-protein contact sites may not be as determinative for stable p50 binding to this promoter as other, as yet undefined, structural parameters.

Base Sequence↗
PubMed1999

Effects of chronic low-dose ultraviolet B radiation on DNA damage and repair in mouse skin.

Chronic exposure to sunlight causes skin cancer in humans, yet little is known about how habitual exposure to low doses of ultraviolet B radiation (UVB) affects DNA damage in the skin. We treated Skh-1 hairless mice with daily doses of suberythemal UVB for 40 days and analyzed the amount and distribution of DNA photodamage using RIAs and immunofluorescence micrography. We found that DNA damage accumulated in mouse skin as a result of chronic irradiation and that this damage persisted in the dermis and epidermis for several weeks after the chronic treatment was terminated. Although the persistent damage was evenly distributed throughout the dermis, it remained in the epidermis as a small number of heavily damaged cells at the dermal-epidermal boundary. Rates of DNA damage induction and repair were determined at different times over the course of chronic treatment in response to a higher challenge dose of UVB light. The amount of damage induced by the challenge dose increased in response to chronic exposure, and excision repair of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone dimers was significantly reduced. The sensitization of mouse epidermal DNA to photoproduct induction, the reduction in excision repair, and the accumulation of nonrepairable DNA damage in the dermis and epidermis suggest that chronic low-dose exposure to sunlight may significantly enhance the predisposition of mammalian skin to sunlight-induced carcinogenesis.

Animals↗
PubMed1999

DNA repair and survival in human lens epithelial cells with extended lifespan.

PURPOSE: Ultraviolet-B radiation (290-320 nm) produces cataracts in animals and has been associated with human cataract formation in several epidemiological studies. UVB radiation decreases the long-term cell survival and changes the pattern of protein synthesis in cultured lens epithelial cells. However, the relationship between DNA photoproduct formation and long term cell survival in human lens epithelial cells is not known. In the present work, we used human lens epithelial cells with extended lifespan (HLE B-3 cells) to examine the kinetics of DNA repair and cell survival after UVB exposure. METHODS: Cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4) photoproducts were analyzed by radioimmunoassay. Long-term survival of the cells was determined by measuring their ability form colonies when plated at low density. RESULTS: HLE B-3 cells were repair competent after UVB (302 nm) exposure. Excision repair of the (6-4) photoproduct was more efficient than that of the cyclobutane dimer. Ninety five percent of the (6-4) photoproducts were repaired 24 h after 400 J/m2 UVB exposure, whereas 50% of the cyclobutane dimers were repaired during this time. When cells were split for the clonogenic assay immediately after irradiation, only 10% of the cells formed colonies following 7 days of culture in the serum-containing medium. When cells were split for the clonogenic assay after a 48 hour incubation in serum-containing medium, the colony-forming ability of the irradiated cells increased to 60% following culture in a serum-containing medium. CONCLUSIONS: These results indicate a close correlation between the repair of cyclobutane dimers and the increase in the long-term survival of the cells as measured by their colony-forming ability. The extended lifespan human lens epithelial cells HLE B-3 may be a useful model to investigate the mechanism and regulation of UVB-induced DNA repair in human lens cells.

Cell Survival↗
PubMed1999

Marine bacterial isolates display diverse responses to UV-B radiation.

The molecular and biological consequences of UV-B radiation were investigated by studying five species of marine bacteria and one enteric bacterium. Laboratory cultures were exposed to an artificial UV-B source and subjected to various post-UV irradiation treatments. Significant differences in survival subsequent to UV-B radiation were observed among the isolates, as measured by culturable counts. UV-B-induced DNA photodamage was investigated by using a highly specific radioimmunoassay to measure cyclobutane pyrimidine dimers (CPDs). The CPDs determined following UV-B exposure were comparable for all of the organisms except Sphingomonas sp. strain RB2256, a facultatively oligotrophic ultramicrobacterium. This organism exhibited little DNA damage and a high level of UV-B resistance. Physiological conditioning by growth phase and starvation did not change the UV-B sensitivity of marine bacteria. The rates of photoreactivation following exposure to UV-B were investigated by using different light sources (UV-A and cool white light). The rates of photoreactivation were greatest during UV-A exposure, although diverse responses were observed. The differences in sensitivity to UV-B radiation between strains were reduced after photoreactivation. The survival and CPD data obtained for Vibrio natriegens when we used two UV-B exposure periods interrupted by a repair period (photoreactivation plus dark repair) suggested that photoadaptation could occur. Our results revealed that there are wide variations in marine bacteria in their responses to UV radiation and subsequent repair strategies, suggesting that UV-B radiation may affect the microbial community structure in surface water.

Colony Count, Microbial↗
PubMed1999

Effect of N-acetylcysteine on UVB-induced apoptosis and DNA repair in human and mouse keratinocytes.

The incidence of skin cancer is increasing rapidly, particularly in the Caucasian population. Epidemiological and experimental studies demonstrated that ultraviolet radiation (UVR) is the primary cause for the increasing incidence of skin cancer. It is well known that UV irradiation induces DNA damage. If the damage is not repaired or removed in time, it can lead to mutations and skin carcinogenesis. N-acetylcysteine (NAC) has been shown to be an effective protector against UVB-induced immunosuppression and to modulate the expression of some oncogenes and tumor suppressor genes. To test further the protective effect of NAC against UVR, we used both in vitro and in vivo models to investigate the effect of NAC on UVB-induced apoptosis and repair of DNA damage in human and mouse keratinocytes. Our data indicate that the intracellular glutathione level was increased after treatment with NAC at 10-20 mM but decreased with 40 mM NAC treatment due to the toxicity. At concentrations up to 20 mM NAC did not have a significant effect on UVB-induced apoptosis of cultured human keratinocytes. In addition, in an in vivo mouse model, topical application of NAC (3 mumol cm-2) that has been shown to inhibit UVB-induced immunosuppression did not have any effect on UVB-induced apoptosis and did not reduce the formation or enhance the repair of UVB-induced cyclobutane pyrimidine dimers and (6-4) photoproducts. Our results indicate that NAC is ineffective in preserving the genomic stability of keratinocytes against UVB irradiation.

Acetylcysteine↗
PubMed1998

The effect of vitamin E acetate on ultraviolet-induced mouse skin carcinogenesis.

Despite the benefits of sunscreens, ultraviolet (UV) exposure can still lead to skin cancer. In this study we investigated the effect of topical application of the antioxidant vitamin E acetate (VEA) on the inhibition of UV-induced carcinogenesis. Hairless SKH-1 mice received 5.2 mg of VEA 30 min before (VEA/UV) or after (UV/ VEA) a single minimal erythemic dose of UV light. Vehicle-control animals received acetone 30 min before UV exposure (Ace/UV). After 24 h, cyclobutane dimer repair was twofold and 1.5-fold greater in the UVNEA and VEA/UV groups, respectively. Expression of p53 protein in the UV/VEA group was maximum at 12 h after UV exposure, whereas in the Ace/UV- and VEA/UV-treated mice, maximum p53 immunostaining was statistically higher at 15 h (P = 0.03). DNA synthesis as determined by 5-bromo-2'-deoxyuridine incorporation was twofold higher after 15 h in all groups but was not statistically different among treatment groups. Protein levels of cyclin D1 and p21 were increased in both VEA groups by 6 h. In addition, VEA treatments delayed tumor formation and yield for the first 20 wk, although this difference was lost by 30 wk. The telomerase activity of carcinomas from the UV/VEA-treated mice was statistically lower than that of the Ace/UV-treated mice (P = 0.05). This study showed that although VEA may mitigate some of the initial events associated with UV irradiation such as DNA damage and p53 expression, it has limited potential in preventing UV-induced proliferation and tumor formation.

Animals↗
PubMed1997

Photorepair mutants of Arabidopsis.

UV radiation induces two major DNA damage products, the cyclobutane pyrimidine dimer (CPD) and, at a lower frequency, the pyrimidine (6-4) pyrimidinone dimer (6-4 product). Although Escherichia coli and Saccharomyes cerevisiae produce a CPD-specific photolyase that eliminates only this class of dimer, Arabidopsis thaliana, Drosphila melanogaster, Crotalus atrox, and Xenopus laevis have recently been shown to photoreactivate both CPDs and 6-4 products. We describe the isolation and characterization of two new classes of mutants of Arabidopsis, termed uvr2 and uvr3, that are defective in the photoreactivation of CPDs and 6-4 products, respectively. We demonstrate that the CPD photolyase mutation is genetically linked to a DNA sequence encoding a type II (metazoan) CPD photolyase. In addition, we are able to generate plants in which only CPDs or 6-4 products are photoreactivated in the nuclear genome by exposing these mutants to UV light and then allowing them to repair one or the other class of dimers. This provides us with a unique opportunity to study the biological consequences of each of these two major UV-induced photoproducts in an intact living system.

Arabidopsis↗
PubMed1997

Phenotypic heterogeneity in nucleotide excision repair mutants of rodent complementation groups 1 and 4.

Rodent ultraviolet light (UV)-sensitive mutant cells in complementation groups (CGs) 1 and 4 normally are known for their extraordinary (approximately 80-100 x) sensitivity to mitomycin C (MMC), although some CG1 mutants with reduced MMC sensitivity were previously reported (Stefanini et al. (1987) Cytotechnology 1, 91). We report here new CG1 and CG4 mutants with only 1.6-10 x wild-type MMC sensitivity despite low unscheduled DNA synthesis (UDS) levels. Mutant UV140, in UV CG4, has approximately 3.8 x the UV sensitivity of parental line AA8, approximately 1.6 x wild-type MMC sensitivity, wild-type X-ray and ethyl methanesulfonate (EMS) sensitivity, and is only slightly (approximately 1.4 x) hypermutable to 8-azaadenine resistance by UV light. It has moderately decreased incision of UV-damaged DNA, has moderately decreased removal of (6-4) photoproducts, and is profoundly deficient in UDS after UV. After UV, it shows abnormally decreased DNA synthesis and persistently decreased RNA synthesis. In addition a cell-free extract of this mutant displays strongly reduced nucleotide excision repair synthesis using DNA treated with N-acetoxy-acetyl-amino-fluorene (AAF). The extract selectively fails to complement extracts of group 1 and 4 mutants consistent with the notion that the affected proteins, ERCC1 and ERCC4, are part of the same complex and that mutations in one subunit also affect the other component. Mutant UV212 is a CG1 mutant with approximately 3.3 x wild-type UV and approximately 5-10 x wild-type MMC sensitivity, with profoundly deficient UDS and hypermutability (approximately 5.8 x) by UV. Mutant UV201, probably in CG1, is only slightly (approximately 1.5 x) UV-sensitive and has near wild-type (1.02X) UV mutability. These unusual group 1 and 4 mutants demonstrate that the unique UV and MMC sensitivity phenotypes displayed by these groups can be separated and support the idea that they are the result of distinct repair functions of the corresponding ERCC1 and ERCC4 genes: nucleotide excision repair for UV lesions and a separate repair pathway for removal of interstrand crosslinks.

Acetoxyacetylaminofluorene↗
PubMed1997

The fission yeast UVDR DNA repair pathway is inducible.

In addition to nucleotide excision repair (NER), the fission yeast Schizosaccharomyces pombe possesses a UV damage endonuclease (UVDE) for the excision of cyclobutane pyrimidine dimers and 6-4 pyrimidine pyrimidones. We have previously described UVDE as part of an alternative excision repair pathway, UVDR, for UV damage repair. The existence of two excision repair processes has long been postulated to exist in S.pombe, as NER-deficient mutants are still proficient in the excision of UV photoproducts. UVDE recognizes the phosphodiester bond immediately 5'of the UV photoproducts as the initiating event in this process. We show here that UVDE activity is inducible at both the level of uve1+ mRNA and UVDE enzyme activity. Further, we show that UVDE activity is regulated by the product of the rad12 gene.

Base Sequence↗
PubMed1997

Quantitative study of bacterial colonization of dental casts.

STATEMENT OF PROBLEM: Contamination of dental casts can occur if the record bases are improperly disinfected or inadvertently not disinfected during fabrication of a prosthesis. It is essential to develop an effective means of disinfecting dental casts from professional, medical, and legal points of view. PURPOSE: This study determined whether: (1) saliva contamination on the surface of the dental cast contributed to bacterial growth over time and (2) cleaning or disinfecting of dental casts can minimize bacterial growth. MATERIAL AND METHODS: Five dental casts were contaminated with saliva. Each cast was divided into six areas and swabbed at 15, 30, 60, 120, 180, and 240 minutes. Sheep blood agar plates were inoculated and incubated at 37 degrees C for 72 hours. Standardized dental stone cylinders were contaminated with 25 microliters of saliva and treated by rinsing in tap water, scrubbing with soap and tap water, soaking in 2% glutaraldehyde, or as controls with and without saliva contamination (n = 12). The treated dental stone cylinders were placed in individual test tubes containing 2.5 ml of sterile phosphate-buffered solution and a final dilution of 10(-4) was achieved. Sheep blood agar plates were inoculated and incubated at 37 degrees C for 24 hours. RESULTS: Contamination of dental casts did not decrease, even when allowed to sit 4 hours before handling. Results also demonstrated that rinsing saliva-treated stone cylinders for 20 seconds significantly diminished bacterial contamination. Scrubbing with soap and tap water or soaking in 2% glutaraldehyde significantly reduced the bacterial contamination of saliva-treated stone cylinders when compared with rinsing with tap water. CONCLUSION: Bacterial contamination of dental casts can occur and requires an effective method of disinfecting.

Agar↗
PubMed1997

Lased and sandblasted denture base surface preparations affecting resilient liner bonding.

STATEMENT OF PROBLEM: Adhesive failure between the liner and the denture base creates an environment for potential bacterial growth and accelerated breakdown of the soft liner resulting in a deteriorating prosthesis. PURPOSE: This study evaluated the effects of a specific sandblasted or lased preparation on the interfacial bonding of polymethyl methacrylate and silicone and polyethyl methacrylate resilient liners. MATERIAL AND METHODS: Polymethyl methacrylate test specimens were fabricated and received one of three surface treatments: untreated (control), sandblasted (250 microns aluminum oxide particles), and lased (carbon dioxide). Polyethyl methacrylate and silicone resilient lining materials were applied to these surfaces and the peel strengths were determined with the American Society for Testing and Materials peelin-adhesion test. RESULTS: Altering the polymethyl methacrylate surface by sandblasting significantly reduced the peel strengths for the polymethyl methacrylate/polyethyl methacrylate and polymethyl methacrylate/silicone specimens. Altering the polymethyl methacrylate surface by delivering carbon dioxide laser energy to form a grid pattern produced lower peel strengths that were statistically significant from the controls for the polymethyl methacrylate/polyethyl methacrylate specimens, but not so for the polymethyl methacrylate/silicone specimens. Untreated polymethyl methacrylate/polyethyl methacrylate peel strengths were significantly higher than polymethyl methacrylate/silicone. CONCLUSIONS: Results of this study imply that mechanical surface preparation of denture bases before application of a resilient liner may not be warranted.

Adhesiveness↗
PubMed1997

Epidermal proliferation but not quantity of DNA photodamage is correlated with UV-induced mouse skin carcinogenesis.

The hairless SKH-1 mouse strain has a higher skin tumor incidence, shorter tumor latency, and higher tumor yield in response to ultraviolet (UV) irradiation than the SENCAR strain. In this study we assessed the differences in UV susceptibility of both strains by measuring DNA photodamage and epidermal proliferation after one UV treatment and after 1, 3, 6, and 9 wk of chronic UV irradiation. Induction rates for cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4) PDs] were significantly greater in the SKH-1 strain than the SENCAR strain, but no strain differences in repair kinetics were detected for CPDs or (6-4) PDs. With chronic UV exposure we observed the following: (i) there was an equal amount of DNA photodamage in both strains; (ii) the number of (6-4) PDs was significantly greater than the CPDs after 6 wk; (iii) there were a significantly greater number of epidermal cells (1.5-fold increase) in the SKH-1 strain; (iv) the number of cycling cells, as measured by 5-bromo-2'-deoxyuridine (BrdU), were located both basally and suprabasally and were significantly greater in the SKH-1 strain; and (v) the number of cells immunoreactive to p53 was equivalent in both strains, but immunoreactive cells were located suprabasally in the SKH-1 strain after 9 wk of UV. These results show that the etiologic role of UV in tumorigenesis is dependent on events other than the amount of DNA photodamage in mouse epidermis.

Animals↗
PubMed1997

Evaluation of stresses and forces in selected I-bars using the finite element method.

PURPOSE: Three-dimensional models of half-round, tapered and full-round, untapered I-bar clasps of varying configurations and material properties were constructed. The purpose of this study was to examine the stresses and reaction forces produced within each model upon deflection to 0.01 in (0.254 mm), 0.02 in (0.508 mm), and 0.03 in (0.762 mm) at 1 mm from the tip using the finite element method. MATERIALS AND METHODS: Three-dimensional computer models of half-round and full-round clasps were constructed using solid eight-node brick elements. The half-round, tapered I-bar clasp model was 2.4 and 1.4 mm in diameter at the base and tip, respectively. The full-round, untapered I-bar clasp model was 1 mm in diameter. Three design groups were created for each clasp form. Group A had 25% of the total length in the straight anchor end of the I-bar clasp, B had 35%, and C had 50%. All models were 31 mm in length and had a radius of curvature of 5 mm. Different material properties were incorporated into the models. Each model was deflected at a point 1 mm from the tip to 0.01 in (0.254 mm), 0.02 in (0.508 mm), and 0.03 in (0.762 mm). RESULTS: The stresses and forces produced as a result of the deflection applied to each clasp were viewed and displayed graphically. The maximum von Mises stresses in megapascals and the reaction force in newtons (N) were recorded. Stresses varied in each clasp in the range of 0 to 154.3 MPa for the half-round, tapered I-bar clasp models, and 0 to 100.9 MPa for the full-round I-bar clasp models at 0.01-in deflection. Reaction force measured near the tip of the clasp models was between 1.60 N and 6.31 N for the half-round, and between 0.22 N to 2.13 N for the full-round I-bar clasp models. For all clasps studied, as the deflection increased, the location of stress within each group remained the same regardless of the material properties; however, the stress and force values increased linearly. CONCLUSIONS: The location of maximum stress varied with the length of the anchor portion of the clasps studied. Maximum stresses were located on the flat side of the half-round, tapered I-bar clasp model.

Color↗
PubMed1997

Melanin: a two edged sword?

Melanin is both photosensitizer and photoprotector. Skin cancer rates decrease with increasing constitutive pigmentation, yet the pigment has been shown to be photoreactive and capable of producing damaging reactive oxygen species. We utilized model systems of related cells or similar cell type that vary in constitutive and in induced pigment. Induction of eumelanin in Cloudman S91 mouse melanoma cells leads to less UV-induced killing and to less mutation induction at the ouabain locus (Na+, K(+)-ATPase). Pigmented mouse melanocytes, melan-b (brown) and melan-a (black) were slightly less sensitive than melan-c (albino) melanocytes to killing after UVC and UVA but were more sensitive to killing after UVB and UVB + UVA. Pigment had a small sensitizing effect on pyrimidine dimer DNA damage in both the melanoma cells and the melanocytes. The lack of consistency in these results suggests that intracellular pigment may disregulate the milieu intérieur resulting in end effects that are unrelated to the original genomic damage.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Survival of Cloudman mouse melanoma cells after irradiation by solar wavelengths of light.

A number of variants of Cloudman S91 mouse melanoma cells that differ with respect to the amount of pigment produced are available for study. In this report, we compare the photobiological responses of S91/amel, which contains about 1 pg of melanin per cell, with S91/I3, which contains about 3 pg/cell. Earlier studies had shown that UVC induced more oxidative damage (in the form of thymine glycols) in cell line S91/I3 than in S91/amel and that cell line S91/amel was more resistant to killing by UVC than S91/I3. The present study finds that S91/amel cells are also relatively resistant to killing by near monochromatic UVB from a Philips TL01 fluorescent lamp and by near monochromatic UVA from a Philips HPW125 lamp. However, when the cells are irradiated with a Westinghouse FS20 polychromatic lamp, the S91/I3 cells are more resistant than the S91/amel cells. These findings cannot be explained on the basis of pigment induction because in S91/I3 this is about the same after UVB and FS20, although the maximum is reached earlier after UVB. Nor can our findings be explained on the basis of pyrimidine dimer formation, which is comparable in the two cell lines regardless of the type of irradiation. These results suggest that, with a pigment such as melanin, which absorbs light across the visible and ultraviolet ranges of the spectrum, cellular responses to monochromatic light do not necessarily predict responses to polychromatic mixtures.

Animals↗
PubMed1997

Comparative action spectrum for ultraviolet light killing of mouse melanocytes from different genetic coat color backgrounds.

The photobiology of mouse melanocyte lines with different pigment genotypes was studied by measuring colony-forming ability after irradiation. The cell lines were wild-type black (melan-a) and the mutants brown (melan-b) and albino (melan-c). Four lamps emitting various UV wavelengths were used. These were germicidal (UVC, 200-280 nm), 82.3% output at 254 nm, TL01 (UVB, 280-320 nm), 64.2% at 310-311 nm, FS20, broadband with peak output at 312 nm and Alisun-S (UVA, 320-400 nm), broadband with peak output at 350-354 nm. Appropriate filtration reduced the contaminating UVC to nonlethal levels for the longer waverange lamps. Wild-type melan-a was resistant to UVC and UVA compared to the other two cell lines, but the differences were small. The melan-c cell line was more resistant to UVB and markedly more resistant to FS20 than the pigmented lines. With the exception of FS20 responses, melan-b was more sensitive than melan-a to killing by the various UV lamps. There were more pyrimidine dimers (cyclobutane dimers and 6-4 photoproducts) produced in melan-a than in melan-c cells by UVC, UVB and FS20 lamps. Unlike melan-c, melan-a and melan-b showed a strong free radical signal of melanin character with a detectable contribution of pheomelanin-like centers. The contribution of pheomelanin was higher in melan-b than in melan-a, while the total melanin content in these two cell lines was comparable. The abundant melanin granules of wild-type melan-a melanocytes were well melanized and ellipsoidal, whereas those of melan-b melanocytes tended to be spherical. In the albino line (melan-c) the melanocytes contained only early-stage melanosomes, all of which were devoid of melanin. The results indicate that pigment does not protect against direct effect DNA damage in the form of pyrimidine dimers nor does it necessarily protect against cell death. High pigment content is not very protective against killing by UVC and UVA, and it may photosensitize in UVB the very wavelength range that is of greatest concern with respect to the rising incidence in skin cancer, especially melanoma. It is clear from these studies that, in pigment cells, monochromatic results cannot predict polychromatic responses and that cell death from solar irradiations is a complex phenomenon that depends on more than DNA damage.

Animals↗