An improved terminal deoxynucleotidyl transferase- mediated dUTP-biotin nick end labeling method for the quantification of DNA damage in tissues and cultured cells was developed. Many reports have revealed that histochemistry of DNA damage can be visualized using immunohistochemistry for the terminal deoxynucleotidyl transferase reaction in tissue sections. However, few reports have described quantification of DNA damage in tissues or cells. In this study, to estimate the degree of DNA damage, the confirmed method for histochemistry using biotinylated dUTP and deoxynucleotidyl transferase was applied to label the cleaved DNA ends caused by DNA damage in tissues or cells. After end-labeling, avidin-conjugated peroxidase was reacted. A significant correlation was observed between numbers of cleaved DNA ends and peroxidase activity after the reaction. The obtained signals for presented method showed higher than those for ordinary method, and correlate with degree of DNA damage caused by serum deprivation and chemical dose. In addition, DNA damage caused by apoptosis in cells treated with 6-hydroxydopamine or Cu and in the tissues of rats administered a diet containing no Zn could be evaluated quantitatively using the present method.
Many different methods have been used to evaluate DNA damage caused by apoptosis and/or necrosis. As a marker of fragmented DNA, the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) method [
Apoptosis is a representative morphology of programmed cell death that occurs within a developmental context in response to a definable physiologic stimulus [
We estimated that color development by the enzymatic reaction using ordinary TUNEL method in the sections can be applied reactions in vitro to measure the DNA damage. In this study, to develop an improved method for the quantification of DNA damage caused by apoptosis and necrosis, an ordinary method using the TdT reaction for visualization of DNA damages in tissue sections (TUNEL method) was applied to measure the numbers of cleaved DNA ends caused by DNA damage in tissues and cultured cells. This novel method is expected to be used widely for studying the mechanisms of apoptosis and DNA damage caused by chemicals.
PC12 cells, a cell line of rat pheochromocytoma cell, were purchased from the American Type Culture Collection (USA). Dulbecco’s modified Eagle’s medium (DMEM), streptavidin-conjugated peroxidase, and o-phenylenediamine dihydrochloride (OPD) were obtained from SigmaAldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was from HyClone (Rockville, MD, USA). TdT, HindIII and HinfI were purchased from Toyobo (Osaka, Japan). Biotin-16-2’-dUTP, the blocking reagent and a high pure PCR template preparation kit were obtained from Roche Diagnostics (Mannheim, Germany). Other chemicals were of analytical regent grade.
PC12 cells in 35 cm2 flasks or on sterilized cover glass in 6-well plates were maintained in a humidified incubator with 5% CO2 at 37˚C. The cells were incubated in DMEM with and without 10% FBS for 2 hr to 3 days. When the medium was changed to serum-free medium, cells in the flask were washed twice with serum-free DMEM.
Cultured cells were divided into 5 groups. The cells in the control group were cultured in medium containing serum without chemicals. After extraction of DNA from control cells, part of the DNA was supplied for digestion of restriction enzymes. The second group of cells was cultured in serum-free medium without chemicals for 2 - 72 hr. It is well known that apoptosis is induced by removing the serum from whole culture medium [
On the other hand, genomic DNA was extracted from about 0.3 g of testis tissue from rats administered with 0 or 20 mg zinc/100 g diet (equivalent to an ordinary diet) [
DNA (each 1 mg) was cleaved with 1 ml of HindIII or HinfI under the condition of 10 mM Tris-HCl, pH7.5, 10 mM MgCl2, 1 mM dithiothreitol and 50 mM NaCl in total 20 ml solution for 1 hr at 37˚C. After reaction, DNA was precipitated by 70% ethanol and 0.2 M sodium acetate buffer, pH 4.5.
To quantify the DNA damage, an equal amount of genomic DNA (0.02 to 2 mg) prepared from the cells or tissues was placed on a 96-well plate, and the plate was incubated at 4˚C overnight. The wells were washed twice with 40 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl. Nonspecific binding sites were blocked with blocking reagent. The TdT reaction was performed in a reaction mixture consisting of 30 mM Tris-HCl buffer, pH 7.4, containing 140 mM sodium cacodylate, 1 mM cobalt chloride, 0.1 units TdT/mL, and 4 mM biotinylated dUTP at 37˚C for 1 hr. The wells were washed twice with the same buffer. After adding of streptavidin-conjugated peroxidase (diluted 1:200 with same buffer), the plates were incubated for 1 hr at 37˚C, and then washed twice with the same buffer. Then, 0.1% OPD in 50 mM phosphate-citrate buffer, pH 5.0, containing 0.03% sodium perborate was added to the wells. After 30 min, HCl was added to stop the enzyme reaction. Absorbance at 495 nm was measured with a Microplate Reader model 450 (Bio-Rad, USA).
To examine whether DNA damage may be evaluated quantitatively using our improved TUNEL method, the level of TUNEL positivity, which is dependent on DNA cleavage by restriction enzymes, was measured (
increased. In addition, level of TUNEL positivity in DNA cleaved with HinfI was about 10-times greater than that in DNA cleaved with HindIII. These values were as expected from the viewpoint of the appearance frequency of each restriction enzyme, because the number of DNA ends from DNA cleaved with HinfI was about 16-times greater compared with that from DNA cleaved with HindIII. A strong significant correlation was observed between TUNEL positivity levels and DNA cleavage sites (
To examine whether ladder fragmentation due to apoptosis is reflected by TUNEL-positive levels measured by the presented method, DNA damage in cells cultured in medium without serum was evaluated (
without serum for 48 - 72 hr was indicated by electrophoresis. Thus, the serum deprivation in PC12 cells also induces apoptosis (
Finally, we examined whether the present method could be applied to cell and animal models. Cu and 6OHDA are well-known inducers of apoptosis in PC12 cells [9,10]. As shown in
On the other hand, it was reported that the functional and morphologic changes observed in the testes of rats administered a 0 mg Zn/100 g diet were caused by increased apoptosis [
to an ordinary diet). From these results, it was suggested that the present method is effective for evaluating the degree of apoptosis in tissues.
We have developed an improved method for the quantification of DNA damage caused by apoptosis and/or necrosis. Linde et al. also reported a new TUNEL assay using a scintillating microplate [
This research was supported by Grants-in-Aid from the Japan Society for the Promotion of Science (No. 20310017 and No. 23655139 for Kurasaki). The authors are indebted to Prof. H. Yanagisawa from the Department of Public Health and Environmental Medicine, Jikei University School of Medicine, Tokyo, Japan for kindly providing the tissue samples from the rats administered the 0 and 20 mg Zn/100 g diet. The authors also thank Ms. Ikumi Yanagiuchi for her technical assistance.