Farid E. Ahmed

Excision repair in ataxia telangiectasia, Fanconi's anemia, Cockayne syndrome, and Bloom's syndrome after treatment with ultraviolet radiation and N-acetoxy-2-acetylaminofluorene

Excision repair of damage due to ultraviolet radiation, N-acetoxy-2-acetyl-aminofluorene and a combination of both agents was studied in normal human fibroblasts and various cells from cancer prone patients (ataxia telangiectasia, Fanconis anemia, Cockayne syndrome and Blooms syndrome). Three methods giving similar results were used: unscheduled DNA synthesis by radioautography, photolysis of bromodeoxyuridine incorporated into parental DNA during repari, and loss of sites sensitive to an ultraviolet endonuclease. All cell lines were proficient in repair of ultraviolet and acetoxy acetylaminofluorene damage and at saturation doses of both agents repair was additive. We interpret these data as indicating that the rate limiting step in excision repair of ultraviolet and acetoxy acetylaminofluorene is different and that there are different enzyme(s) working on incision of both types of damages.

USE OF THE DYE HOECHST 33258 IN A MODIFICATION OF THE BROMODEOXYURIDINE PHOTOLYSIS TECHNIQUE FOR THE ANALYSIS OF DNA REPAIR

Abstract— A modification of the bromodeoxyuridine photolysis technique is described in which the cells are treated with the fluorescent dye Hoechst 33258 to enhance the bromodeoxyuridine photolysis produced by 365 nm or by black light. Use of the modified assay overcomes several difficulties encountered in the original 313 nm photolysis technique and demonstrates that excision repair of damage produced in the DNA by 7,12‐dimethylbenz(fl)anthracene 5,6‐oxide takes place by a long patch type of repair pathway.

SATURATION OF DNA REPAIR IN MAMMALIAN CELLS

Abstract—Excision repair seems to reach a plateau in normal human cells at a 254 nm dose near 20J/m2. We measured excision repair in normal human fibroblasts up to 80J/m2. The four techniques used (unscheduled DNA synthesis, photolysis of BrdUrd incorporated during repair, loss of sites sensitive to a UV endonuclease from Micrococcus luteus, and loss of pyrimidine dimers from DNA) showed little difference between the two doses. Moreover, the loss of endonuclease sites in 24 h following two 20J/m2 doses separated by 24 h was similar to the loss observed following one dose. Hence, we concluded that the observed plateau in excision repair is real and does not represent some inhibitory process at high doses but a true saturation of one of the, rate limiting steps in repair.

Toxicological effects of ethanol on human health

Moderate ethanol consumption reduces stress and increases feelings of happiness and well-being, and may reduce the risk of coronary heart disease. Heavy consumption of alcohol, however, may cause addiction and increases all types of injury and trauma. Environmental and genetic factors are involved in susceptibility to alcoholism. Ethanol can lead to malnutrition, and can exert a direct toxicological effect due to its interference with hepatic metabolism and immunological functions. A causal effect has been observed between alcohol and various cancers. Cessation of alcohol consumption and balanced nutrition are recommended primary nonspecific therapeutic measures for alcoholics. Drug therapies for alcoholics suffering from liver injury has resulted in mixed results. In end-stage liver disease, liver transplantation may be considered.

DNA excision in repair proficient and deficient human cells treated with a combination of ultraviolet radiation and acridine mustard (ICR-170) or 4-nitroquinoline 1-oxide.

Excision repair was measured in normal human and xeroderma pigmentosum group C fibroblasts treated with ultraviolet radiation and the carcinogens acridine mustard (ICR-170) or 4-nitroquinoline 1-oxide (4NQO) by the techniques of unscheduled synthesis, photolysis of bromodeoxyuridine incorporated into parental DNA during repair, and assays of sites sensitive to ultraviolet (UV)-endonuclease. Doses of ICR-170 and 4NQO, low enough not to inhibit unscheduled DNA synthesis (UDS), caused damage to DNA that was repaired by a long patch type mechanism and the rates of UDS decreased rapidly in the first 12 h after treatment. Repair after a combined action of UV plus ICR-170 or UV plus 4NQO was additive in normal cells and no inhibition of loss of endonuclease sensitive sites was detected. In xeroderma pigmentosum (XP) C cells there was less repair after UV plus ICR-170 than after each treatment separately; whereas there was an additive effect after UV plus 4NQO and no inhibition of loss of endonuclease sensitive sites. The results indicate that in normal human fibroblasts there are different rate limiting steps for removal of chemical and physical damages from DNA and that XP cells have a different repair system for ICR-170, not just a lower level, than normal cells. Possibly the same long patch repair system works on 4NQO damage in both normal and XP cells.

DNA REPAIR IN V‐79 CELLS TREATED WITH COMBINATIONS OF PHYSICAL AND CHEMICAL CARCINOGENS*

Excision repair of DNA damage was measured by the photolysis of bromodeoxyuridine incorporated into parental DNA during repair in Chinese hamster V‐79 cells treated with 254 nm of ultraviolet radiation (UV), 7,12‐dimethylbenz[a]anthracene 5,6‐oxide (DMBA‐epoxide), N‐acetoxy‐2‐acetylaminofluorene (AAAF), 4‐nitroquinoline 1‐oxide (4NQO), 2‐methoxy‐6‐chloro‐9‐[3(ethyl‐2‐chloroethyl)‐aminopropylamino]acridine dihydrochloride (ICR‐170), X‐rays, ethylmethanesulfonate (EMS), methyl methanesulfonate (MMS) and combinations of these agents. Compared to normal human cells V‐79 were defective in repair of UV lesions and the lesions induced by the UV‐mimetic chemicals. The extent of the defects varied from 10 to 50% and was similar to those in Xeroderma pigmentosum group C cells (XP C). V‐79 cells repaired X‐ray damage and damage from the alkylating agents EMS and MMS to the same extent as human cells. Repair was additive after a combination of UV plus MMS indicating, as expected, that there are different rate‐limiting steps for removal of the damages from these agents. Repair was less than additive in cells treated with UV plus ICR‐170, AAAF plus ICR‐170, AAAF plus 4NQO, and 4NQO plus ICR‐170 and approximately equal to that observed for the higher of the two agents separately, indicating that there may be similar rate‐limiting steps for removal of lesions. Although the results on repair after combinations of UV plus 4NQO, UV plus DMBA‐epoxide or X‐rays plus MMS were difficult to interpret, there was not any inhibition of repair in these combinations.

Different rates of ultraviolet-induced DNA damage in the epidermis and dermis of a platyfish model for carcinogenesis

The purpose of this study was to compare fluence-response relationships for the production of cyclobutane pyrimidine dimers in epidermal or dermal DNA of platyfishXiphophorus hybrids irradiated with UVB, and to determine photoreactivation from black light on dimers producedin situ. This was accomplished by quantitative gel electrophoresis of unlabeled DNA following extraction of the DNA and treatment with an enzyme specific for the detection of pyrimidine dimers. The dermis was the target tissue for UV-induced DNA damage inXiphophorus hybrid fish skin. Shapes of dimer-fluence response data following filtered sunlamp irradiation (λ > 290 nm) or monochromatic wavelength 302 nm in the epidermis or dermis were different. In the epidermis there was an initial steep upward slope followed by a plateau, whereas in the dermis a linear relationship was observed. The final values of dimers at the high doses were, however, nearly equal in the epidermis and dermis exposed to either radiation. These differences in fluence-response relationships are probably attributable to the intertwining of the epidermis and to the shielding effect of the epidermal layer, with scales leading to a heterogenous population of cells which are exposed to different UV doses. Photoreversal of dimers was readily observed by black light irradiation in both epidermis and dermis irradiated with eitherλ > 290 nm or 302 nm.

EXCISION REPAIR IN MAMMALIAN CELLS

ABSTRACT Excision repair after combined treatments of UV and N-acetoxy-2-acetylaminofluorene (AAAF) was studied by three different techniques in cells proficient in UV excision repair and in cells deficient in UV repair. Two patterns of repair were observed: A) in repair proficient cells total repair was additive, and B) in repair deficient cells total repair was much less than additive—usually less than observed for separate treatments—and AAAF inhibited dimer excision. We conclude that in the 1st class of cells pathways for repair of UV and AAAF lesions are not identical, and in the 2nd class the residual excision enzymes are different from those in repair proficient cells.

The combined action of chemical carcinogens on DNA repair in human cells.

SummaryExcision repair was studied in normal human and ataxia telangiectasia (AT) cells proficient in repair of UV and its mimetic chemicals, and in xeroderma pigmentosum group C (XP C) cells (deficient in repair of UV and its mimetics), after treatment with several combinations of chemical carcinogens, by the photolysis of bromodeoxyuridine incorporated into parental DNA during repair. Results indicate that repair was additive in AT, and XP C cells treated with N-acetoxy-2-acetylaminofluorene (AAAF) plus ethyl methanesulfonate (EMS) or methyl methanesulfonate (MMS) indicating that there are different rate limiting steps for removal of both types of damage. Data on the combinations of 4-nitroquinoline 1-oxide (4NQO) plus MMS or EMS are difficult to interpret, but they do not indicate inhibition of DNA repair.

DNA Repair in Human Cells Exposed to Combinations of Carcinogenic Agents

Normal human and XP2 fibroblasts were treated with UV plus UV-mimetic chemicals. The UV dose used was sufficient to saturate the UV excision repair system. Excision repair after combined treatments was estimated by unscheduled DNA synthesis, BrdUrd photolysis, and the loss of sites sensitive to a UV specific endonuclease. Since the repair of damage from UV and its mimetics is coordinately controlled we expected that there would be similar rate-limiting steps in the repair of UV and chemical damage and that after a combined treatment the total amount of repair would be the same as from UV or the chemicals separately. The expectation was not fulfilled. In normal cells repair after a combined treatment was additive whereas in XP cells repair after a combined treatment was usually less than after either agent separately. The chemicals tested were AAAF, DMBA-epoxide, 4NQO, and ICR-170.