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DETECTION OF DNA DAMAGE IN GERM CELLS

Objective


Exposure of cells to ionizing radiation results in damage to the deoxyribonucleic acid (DNA) including strand breaks and base modifications. These damages may lead to mutagenesis and carcinogenesis or, when induced into germ cells, to genetic abnormalities and other hereditary effects in the offspring. It is important, therefore, to inventory and quantify the various damages to get information about their relative contribution and persistency. For this purpose sensitive immunochemical and biochemical methods to quantify single strand breaks, alkali labile sites and base damages are being developed. The immunochemical method is based on the binding of a monoclonal antibody to single stranded DNA. The technique is based upon the determination of the percentage single strandedness resulting from the partial unwinding of cellular DNA under strictly controlled alkaline conditions. Strand breaks and alkali labile sites form initiation points for the unwinding. The extent of unwinding is a measure of the number of such sites. The results are compared with those obtained with alkaline elution, which assays the extent of unwinding on the basis of the rate at which the DNA passes through the pores of a membrane filter in an alkaline elution fluid. The usefulness of these approaches to detect single strand breaks was demonstrated by detection of damage and its repair in unlabelled DNA-containing cells of the human blood after in vitro and in vivo exposure to ionizing radiation. Single strand breaks could be assayed with both techniques down to doses as low as 0.5 Gy. Subsequently, this method was applied to germ cells of the Syrian golden hamster exposed to ionizing radiation at different stages of spermatogenesis. 5 stages of spermatogenesis have been invesitaged, the mid and late spermatocytes, the early and mid spermatids and the elongated spermatids. It was found that at all stages of spermatogenesis there is a fast repair of single strand breaks except in the latest stage , the so called elongated spermatids, before the differentiation to spermatozoa. Both after in vitro and in vivo irradiation, up to 90 min after exposure, no removal of single strand breaks was observed in the elongated spermatids.

Spermatozoa, which are easily obtainable from male morning urine, probably could provide a biological indicator of radiation damage or injury that is well suited for practical application. So far it has not been possible, howeve, to use the immunochemical method with spermatozoa, because the DNA stays in the condensed nucleus when the cells are brought into the alkaline solution. Therefore modifications of the immunochemical method have to be introduced, in order to make these cells accessible for investigation of DNA damage induction and repair.

Base damages can be quantified in a similar way with alkaline unwinding, when it is preceded by treatment of the DNA with damage oriented endonucleases (eg a Micrococcus luteus extract). These enzymes recognize base damages in the DNA, upon which they will introduce a break, or remove the modified base thereby leaving an alkali labile site. This break or alkali labile site can be detected by means of alkaline elution. By this method base damage could be detected after in vitro irradiation of human blood in the dose range of 1.5 to 25 Gy. After 5 Gy, measurable base damage was still present at 1.5 h after exposure. Also leukemia patients undergoing chemotherapy and radiotherapy were investigated. These patients were exposed to Endoxan and total body irradiation. Base damage induced by doses of 4.5 to 8.6 Gy could be detected, even at 90 min after irradiation.

In the TNO Medical Biological Laboraotry a technique has been developed to detect DNA damages at the single cell level. This technique uses monoclonal antibodies directed against specific DNA damages. The antibodies have a fluorescent label which can be detected by making use of a laser scan microscope. The intensity of fluorescence is a measure of the amount of damage in the cell. It was possible to detect DNA adducts in cultured cells (V79, chinese hamster ovary (CHO) and HeLa cells) as well as in human skinbiopsies that had been exposed to genotoxic chemical agents or to ultraviolet light. The immunofluorescence method can be used to study DNA damage induction and repair in oocytes. The advantage of the immunofluorescence method is that only a few cells are required which is a prerequisite when a study of mammalian oocytes is intended (mice and monkey).
Exposure of cells to ionizing radiation results in damage to the DNA. This damage comprises strand breaks and base modifications. These damages might lead to mutagenesis and carcinogenesis or, when induced in germ cells, to genetic abnormalities and other hereditary effects in the offspring. It is important, therefore, to inventorize and quantify the various damages to get information about their relative contribution and persistence. To this purpose we are developing sensitive immunochemical and biochemical methods to quantify single-strand breaks, alkali-labile sites and base damage. The immunochemical method is based on the binding of a monoclonal antibody to single-stranded DNA.

The technique is based upon the determination of the percentage single-strandedness resulting from the partial unwinding of cellular DNA under strictly controlled alkaline conditions. Strand breaks and alkali-labile sites form initiation points for the unwinding.

The extent of unwinding is a measure of the number of such sites. The results are compared with those obtained with alkaline elution Base damage can be quantified in a similar way when alkaline unwinding is preceded by treatment of the DNA with damage oriented endonuclease (eg a Micrococcus luteus extract).

The usefulness of these approaches to detect single-strand breaks and base damage was demonstrated by detection of damage and its repair in unlabelled DNA-containing cells of human blood after in vitro and in vivo exposure to ionizing radiation. Single-strand breaks could be assayed down to doses as low as 0.5 Gy. Base damage could be detected after in vitro irradiation in the dose range of 1.5 to 25 Gy. After 5 Gy, measurable base damage was still present at 1.5 hours after exposure.

Also leukemia patients undergoing chemotherapy and radiotherapy were investigated. These patients were exposed to Endoxan and total body irradiation. Base damage induced by doses of 4.5 to 8.6 Gy could be detected, even at 90 min after irradiation.

We are now trying to apply these techniques to germ cells of the Syrian golden hamster. Preliminary results showed that, after a dose of 4 GY, base damage could be detected in the round spermatids 30 min after exposure.

This is in contrast to the single-strand breaks that were repaired almost completely within this period. Furthermore, it was found that in all stages of the spermatogenesis there is a fast repair of single-strand breaks except in the latest stage, the so called elongated spermatids, before the differentiation to spermatozoa. After both in vitro and in vivo irradiation, up to 90 min after exposure, no removal of single-strand breaks was observed in the elongated spermatids.

So far it has not been possible to study the induction and repair of single-strand breaks in the spermatozoa. To this aim modifications of the immunochemical method have to be made to make these cells accessible for investigation of DNA-damage induction and repair.

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