Arie J. Timmerman

Physiological Doses of Ultraviolet Irradiation Induce DNA Strand Breaks in Cultured Human Melanocytes, as Detected by Means of an Immunochemical Assay

Abstract— An immunochemical assay, i.e. sandwich enzyme‐linked immunosorbent assay, has been modified to detect UV‐induced damage in cellular DNA of monolayer‐grown human melanocytes. The method is based on the binding of a monoclonal antibody to single‐stranded DNA. The melanocytes derived from human foreskin of skin type II individuals were suspended and exposed to UVA, UVB, solar‐simulated light or γ‐rays. Following physiological doses of UVA, UVB or solar‐simulated light, a dose‐related DNA unwinding comprising a considerable number of single‐strand breaks (ssb) was observed. No correlation was found between different seeded cell densities or different culturing periods and the UVA sensitivity of the cells. After UVA irradiation, 0.07 ssb/1010 Da/kJ/m2 were detected and after UVB irradiation 1.9 ssb/1010 Da/kJ/m2 were seen. One minimal erythema dose of solar‐simulated light induced 2.25 ssb/1010 Da. Our results from melanocytes expressed in ssb/Da DNA are comparable and have the same sensitivity toward UVA as well as toward UVB as nonpigmented skin cells. As low doses of UVA have already been shown to induce detectable numbers of ssb, this assay is of great interest for further investigations about the photoprotecting and/or photosensitizing effects of melanins in human melanocytes derived from different skin types.

DNA-damage processing in blood lymphocytes of head-and-neck-squamous-cell-carcinoma patients is dependent on tumor site.

It has been reported that an intrinsic susceptibility to cancer is related to the way an individual responds to DNA‐damaging agents. The aim of this study was to evaluate whether, in addition to bleomycin‐induced chromosomal instability, radiation‐induced initial DNA damage and subsequent repair is associated with the development of head‐and‐neck squamous‐cell carcinoma. In this study, 2 assays were performed to measure DNA damage in human peripheral‐blood lymphocytes. One was a chromosomal aberration assay which determines sensitivity to chromatid breaks induced by bleomycin, the other an elegant immunochemical assay which measures the level of radiation‐induced strand breaks as well as subsequent repair. Age, smoking and alcohol‐drinking behavior did not influence the number of chromatid breaks, initial DNA damage or repair capacity. As has been found in previous studies, the mean number of chromatid breaks per cell was significantly different between patients (n = 18) and control persons (n = 19), whereas the amount of initial DNA damage was not. No correlation was found between the outcome of the 2 assays in the subject groups. In contrast to laryngeal‐carcinoma patients, oral‐cavity‐carcinoma patients showed significantly slower repair capacity than controls. Our hypothesis is that the way DNA damage is processed by the patients determines at which site cancer develops in the head and neck area.

A modified immunochemical assay for the fast detection of DNA damage in human white blood cells

An immunochemical assay to detect damage in DNA has been modified to a so-called sandwich ELISA. With this assay DNA damages can be detected that give rise to a certain level of single-strandedness in DNA of white blood cells during partial unwinding of cellular DNA under alkaline conditions. The modified method includes the following steps: incubation of alkali-treated whole blood in the wells of microtiter plates precoated with antibody directed against single-stranded DNA (ssDNA), which results in selective binding of ssDNA, and the subsequent detection of bound ssDNA by incubation with anti-ssDNA antibody alkaline phosphatase conjugate. With this method the amount of damage induced by ionizing radiation in DNA in cells of human blood can be detected within 1 h, after doses as low as 0.2 Gy. The precoating of microtiter plates with anti-ssDNA antibody enables the detection of ssDNA fragments directly in alkali-treated blood samples, isolation of the nucleated cells from the blood is not necessary. Because the DNA is released somewhat faster from lymphocytes than from granulocytes upon alkali treatment, it even appeared possible to discriminate between the effect of the radiation on these cell types in the same blood sample. The method is also applicable to other cell types that can be obtained in suspension.

Detection of Single-strand Breaks and Base Damage in DNA of Blood Cells from Leukaemia Patients Receiving Chemo- and Radiotherapy

Chemotherapy combined with total-body irradiation (TBI), a conditioning regimen for bone-marrow transplantation (BMT), causes lesions in the cellular DNA of the patients treated. To understand possible consequences of the DNA damage induced during such treatment, information is required about the nature of the damage, the level of induction and its persistence, and about the importance of the various lesions for cell-lethality and/or mutation induction. Recently, we developed a sensitive immunochemical method to quantify single-strand breaks (SSB) in the DNA of mammalian cells. In addition, a modification of the so-called alkaline elution technique was introduced which allows quantification of SSB together with base damage (SSB+BD). These methods have now been applied successfully to study the in vivo induction and repair of DNA damage in WBC of leukaemia patients who prior to BMT were treated with cyclophosphamide (CY) and received TBI. SSB and SSB+BD were determined after two treatments with CY (60 mg kg-1) followed by TBI (4.5-8.6Gy). The CY treatments gave rise to rather persistent SSB. In addition to these, radiation-induced SSB and SSB+BD could be detected shortly after TBI. However, 105 min after TBI, these SSB could be observed no longer, as a result of rapid repair.

Single-Strand Breaks and Base Damage in DNA of Human White Blood Cells in Full Blood Exposed to Ionizing Radiation Detected at Biologically Relevant Doses

Both physical (UV- and X-irradiation) and chemical agents can damage cells. Of special interest are the DNA molecules, the carriers of the genetic information. Damage inflicted on DNA, unless repaired, may kill cells by interference with transcription and replication or by the induction of mutations. Non-lethal mutations may result in cells with altered properties which may contribute to ageing, heart diseases and cancer.