The doserepair curve for the LUC activity was first established by performing the LUC assay with three lymphoblastoid cell lines with different levels of DRC and primary lymphocytes from three donors.Assays repeated four times with one repairproficient cell line and one repairdeficient cell line indicated that the LUC assay could easily distinguish repairproficient from repairdeficient cell lines with relatively small coefficients of variation. In this assay, the frequency of in vitro bleomycininduced breaks in shortterm PBL cultures is used as a measure of cancer susceptibility hypothesized that in the general population, susceptibility to chromosome damage in response to clastogens varies along a continuum, with recognized chromosome fragility syndromes being at one extreme of the spectrum.The assay requires only ml of a fresh whole blood sample that undergoes shortterm culture.For each sample, the chromosome breaks in metaphases are counted and expressed as the mean number of bc.Chromatid gaps or attenuated regions are disregarded; only frank chromatid breaks or exchanges are recorded.We have shown, in several smaller casecontrol studies, that mutagen sensitivity is an independent risk factor for lung cancer with a doseresponse relationship to the numbers of induced chromosome breaks. Highest risk was evident in the heaviest packyear tertile.For studying tobaccorelated cancers, a more appropriate mutagen to trigger DNA damage is BPDE, the product of bioactivation in vivo by cytochrome P and epoxide hydrolase. As mentioned above, the NER pathway is responsible for removal of such bulky BPDE adducts and for the restoration of normal DNA structure. However, in vitro experiments suggest that baseexcision repair may also play an important role in repair of these lesions.The comet assay appears to have many advantages including allowing relatively highthroughput screening, requiring a few cells, and facilitating the detection of primary DNA damage in individual cells. Although it requires viable cells, it does not require growth, and is applicable to any cell line or tissue from which a single cell suspension can be obtained, and can even be applied to terminally differentiated cells. The cells are lysed by submersing the slides in freshly prepared lysis buffer for hat C to remove all of the cellular proteins.To allow for DNA denaturation, unwinding, and expression of the alkalilabile sites, the slides are next placed in buy Gallic acid alkali buffer. To separate the damaged DNA from the intact nuclei, a constant electric current is applied, and the slides are neutralized, fixed in methanol, and stored in the dark at room temperature until ready for analysis.A fluorescent dye that binds to the DNA is used for quantification of DNA damage.Under different biochemical conditions for both lysis and electrophoresis, different classes of DNA lesions can be detected.During electrophoresis, damaged DNA migrates from the nucleus toward the anode as a result of a constant electric current that forms the typical comet cell.The head of the comet relates to the nucleus of the cell, whereas the tail of the comet refers to the damaged DNA that has been liberated from the nucleus via electrophoresis.The tail mean head mean calculates the difference in the distance between the center of gravity of the DNA distribution in the comet head from the center of gravity of DNA distribution in the comet tail.