A.A.W.M. van Loon

Induction and repair of DNA single-strand breaks and DNA base damage at different cellular stages of spermatogenesis of the hamster upon in vitro exposure to ionizing radiation

Alkaline elution has been used for quantitative detection of DNA damage caused by ionizing radiation in unlabeled somatic and germ cells. Both the induction and subsequent repair have been studied for two classes of DNA damage, viz. single-strand breaks (SSB), and base damage (BD) recognized by the gamma-endonuclease activity in a cell-free extract of Micrococcus luteus bacteria. The high sensitivity of the assay permitted the measurement of induction and repair of SSB and BD after in vitro exposure of hamster germ cells in different cellular stages of spermatogenesis (spermatocytes, round and elongated spermatids), and of bone-marrow cells, to biologically relevant doses (0-8 Gy) of 60Co gamma-rays. A dose-dependent increase was observed for both types of lesions, which was similar for most cell types. The elongated spermatids, however, showed a lower induction frequency of SSB (and perhaps BD). Spermatocytes, round spermatids and bone-marrow cells had normal, fast repair of the SSB when compared with the repair reported for cultured rodent cells and human lymphocytes. In contrast, the elongated spermatids showed hardly any SSB repair. The initial rate of repair of BD in spermatocytes and bone-marrow cells was in the same range as that for SSB, but only 60-70% of the initial BD was repaired within 1 h, whereas after that period no SSB were detectable. The round spermatids hardly repaired any BD within the first hour after irradiation, but after 7 h only a few BD could be detected. In elongated spermatids repair of BD could not be measured due to a high background level of this type of damage.

Detection of DNA Damage in Cells Exposed to Ionizing Radiation by Use of Anti-single-stranded DNA Monoclonal Antibody

An immunochemical method has been developed for quantitative detection of DNA damage in mammalian cells. The method is based on the binding of a monoclonal antibody to single-stranded DNA. The clone producing this antibody (D1B) was obtained as a by-product from fusion of mouse myeloma cells with spleen cells isolated from a mouse immunized with chemically modified DNA. The technique is based upon the determination of the percentage single-strandedness resulting from the time-dependent partial unwinding of cellular DNA under alkaline conditions. Single- and double-strand DNA breaks, or lesions converted into such breaks in alkaline medium, form initiation points for the unwinding. The extent of unwinding from these points under defined conditions is a measure of the number of such sites. The method is rapid, does not require radioactive labelling of DNA or physical separation of single-from double-stranded molecules, is sufficiently sensitive to detect damage induced by 1 Gy of ionizing radiation and needs only small numbers of cells. The usefulness of the technique was demonstrated in a study of the induction of DNA damage and its repair in cultured Chinese hamster cells and in human white blood cells after exposure to 60Co-gamma-rays, and in white blood cells and bone marrow cells of X-irradiated mice. A dose-related DNA unwinding was observed and repair of DNA lesions was observed up to 60 min after irradiation.

Detection of Base Damage in DNA in Human Blood Exposed to Ionizing Radiation at Biologically Relevant Doses

The alkaline elution technique for the detection of DNA damage has been adapted to allow application on unlabelled blood cells. Both the induction and subsequent repair have been studied of two classes of DNA damage, viz, single-strand breaks and base damage recognized by the gamma-endonuclease activity in a cell-free extract of Micrococcus luteus bacteria. The high sensitivity of the assay permitted the measurement of induction and repair of base damage after in vitro exposure of full blood under aerobic conditions to biologically relevant doses of gamma-rays (1.5-4.5 Gy). After a radiation dose of 3 Gy about 50% of the base damage was removed within 1.5 h of repair. Base damage could still be detected at 24 h after exposure to 15 Gy.

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6 V. The correlation of DNA strand breaks and base damage to chromosomal aberrations and sister-chromatid exchanges induced by X-irradiation

The X-ray-sensitive Chinese hamster ovary (CHO) mutant cell lines xrs 5 and xrs 6 were used to study the relation between X-ray-induced DNA lesions and biological effects. The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCE) were determined in wild-type CHO-K1 as well as mutants xrs 5 and xrs 6 cells following X-irradiation under aerobic and anaerobic conditions. Furthermore, we used a newly developed immunochemical method (based on the binding of a monoclonal antibody to single-stranded DNA) to assay DNA single-strand breaks (SSBs) induced by gamma-rays in these CHO cells, after a repair time of up to 4 h. For all cell lines tested the frequency of X-ray-induced chromosomal aberrations was strongly increased after irradiation in air compared with hypoxic conditions. When compared to the wild-type line, the xrs mutants known to have a defect in repair of DNA double-strand breaks (DSBs) exhibited a markedly enhanced sensitivity to aerobic irradiation, and a high OER (oxygen enhancement ratio) of 2.8-3.5, compared with 1.8-2 in CHO-K1 cells. The induction of SCE by X-rays was relatively little affected in CHO-K1 irradiated in air compared with hypoxic conditions (OER = 0.8), and in xrs 5 (OER = 0.7). A dose-dependent increase in the frequency of SCEs was obtained in xrs 6 cells treated with X-rays in air, and a further increase by a factor of 2 was evident under hypoxic conditions (OER = 0.4). With the immunochemical assay of SSB following gamma-irradiation, no difference was found between wild-type and mutant strains in the number of SSBs induced. The observed rate of rejoining of SSBs was also the same for all cell lines studied.

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.