- Email: [email protected]
Measurement of DNA single-strand breaks by alkaline elution and fluorometric DNA quantification
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 326 (2004) 146–152 www.elsevier.com/locate/yabio
Measurement of DNA single-strand breaks by alkaline elution and fluorometric DNA quantification Marina Goumenou and Kyriaki Machera* Laboratory of Pesticides Toxicology, Benaki Phytopathological Institute, Kifissia, GR-145 61 Athens, Greece Received 23 June 2003
Abstract The method presented is based on the alkaline elution procedure for the determination of DNA single-stand (ss) breaks developed by Kohn and on the principles of DNA quantification after binding with the dye Hoechst 33258. In the present study, modification of the alkaline elution procedure with regard to the elution solution volume was performed. The influences of the DNA strandedness, the ethylenediaminetetraacetate/tetraethylammonium hydroxide denaturation and elution solution presence, the DNA solution pH, the dye amount, and the incubation time for the formation of the dye–ssDNA complex on the DNA fluorometric quantification were also studied. The modified DNA alkaline elution procedure followed by the optimized fluorometric determination of the ssDNA was applied on liver tissue from both untreated and treated (N -nitroso-N -methylurea- administered) Wistar rats. The criteria for the selection of the appropriate estimator and statistical analysis of the obtained results are also presented. The method of the DNA alkaline elution followed by fluorometric determination of ssDNA as modified and evaluated is an accurate and reliable approach for the determination of in vivo induced ssDNA strand breaks. Ó 2003 Elsevier Inc. All rights reserved. Keywords: DNA; ssBs; Alkaline elution; Fluorometric ssDNA quantification
A great number of chemicals such as pesticides, drugs, and environmental contaminants exhibit mutagenic activity while most of the carcinogens can be detected as mutagens. Consequently, the detection of the mutagenic activity of a compound is essential for hazard identification. The underlying basis for mutagenesis is the damage in the structure of affected DNA [1]. A major class of DNA damages concerns DNA strand breaks, divided into single strand breaks (ssBs)1 and double-strand breaks (dsBs). ssBs occur during different processes including direct scission of the sugar–phosphate backbone by chemical or radical attack, hydrolysis of alkali–labile sites, enzymatic incision during base
*
Corresponding author. Fax: +0030-210-8077506. E-mail address: [email protected] (K. Machera). 1 Abbreviations used: ssBs, single-strand breaks; dsBs, double-strand breaks; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; TEA, tetraethylammonium hydroxide; SSC, saline-citrate buffer solution; MNU, N -nitroso-N -methylurea; RFI, relative fluorescence intensity; LOD, limit of detection; LOQ, limit of quantification. 0003-2697/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2003.11.019
and nucleotide excision repair, post replicational or recombinational repair and enzymatic scissions due to the action of topoisomerase or lysosomic hydrolases [2]. dsBs are also produced by direct scissions, during repair processes, or during the replication of DNA containing ssBs. For the determination of ssBs on DNA, several methods have been presented in the literature. One of them is the alkaline elution technique that was originally developed by Kohn et al. [3,4] targeting the replacement of the DNA sedimentation with alkaline sucrose gradients. According to this method, cells are lysed on membrane filter and large nonelutable dsDNA is released. The filter is then eluted with an alkaline solution, ssDNA is released and slowly pumped through while fractions are collected to determine the elution kinetics. The elution rate of the ssDNA was proven to be size dependent and used as a measure of the DNA ssBs. During the following years and until nowadays several modifications of the original first procedure have been published [5–8].
M. Goumenou, K. Machera / Analytical Biochemistry 326 (2004) 146–152
Initially, for quantification of the DNA amount eluted during the alkaline elution procedure, prelabeling of cells with radioactive probes was used [3,4]. Soon after, DNA quantification using fluorometric methods [9–11] was considered preferable to avoid the use of radioactive materials [5,12–14]. In the present study, DNA quantification is based on the principles of the method described by Cesarone et al. [10]. According to this method, enhancement of the Hoechst 33258 dye fluorescence yield is observed when it is complexed with DNA. No information with regard to possible differences in relative fluorescence intensity (RFI) in dsDNA and ssDNA as a function of dye/DNA ratio was reported [10]. In the first part of the present study, the influence of the DNA strandedness upon fluorescence intensity as a function of the dye/dsDNA and dye/ssDNA ratios was studied. A reduction of the fluorescence intensity of the dye–ssDNA complex with regard to that of the dye– dsDNA complex was observed at the same dye/DNA ratios. Further reduction of the dye–ssDNA complex fluorescence was observed in the presence of the denaturation solution used (EDTA/TEA). Furthermore, the influence of other ssDNA quantification parameters, such as pH, dye concentration, and incubation time for the dye–DNA binding, was studied with appropriate fluorescence calibration plots. In the second part, adaptation and modifications of the procedure presented by Kohn for the DNA alkaline elution were performed. The main modifications were related to the elution time and the extraction of the DNA retained on the filter after elution. The performances of both modified approaches were evaluated on liver tissue from both untreated and treated Wistar rats. Additionally, examination of different estimators for the DNA ssBs evaluation and statistics for data analysis were performed. Finally, the historical control data of the ssBs in rat liver DNA from our laboratory are presented.
Materials and methods Chemicals and equipment Na3 EDTA, EDTA (acid form), N -lauroylsarcosine (sarcosyl), tetraethylamonium hydroxide (20% in water), Hoechst 33258, saline-citrate buffer solution (SSC, pH 7,0), N -nitroso-N -methylurea (MNU), and calf thymus DNA were purchased from Sigma. The fluorometric DNA quantification was performed with a Jasco FP-6200 spectrofluorometer. Animals The alkaline elution technique was performed in liver tissue obtained from young adult Wistar rats from the
147
breeding colony of our laboratory. All animals were housed in groups of five in stainless steel wire cages under controlled environmental conditions, with ad libitum consumption of feed and water. Evaluation of influence and optimization of the experimental parameters for the fluorometric DNA quantification For the study of the parameters affecting the ssDNA quantification (DNA strandedness, EDTA/TEA solution, pH, dye concentration, and incubation time) appropriate fluorescence calibration plots (RFI vs. ssDNA conc.) were established and evaluated with regard to linearity, sensitivity, etc. Stock solutions of standard calf thymus dsDNA at a concentration of 1 mg/ml in SSC buffer solution and of Hoechst 33258 at a concentration of 1.5 104 M in water were prepared. Both solutions were stored at 4 °C. Working solutions of the dye and the dsDNA were prepared by appropriate dilution with SSC buffer solution (pH 7.0) and the final concentrations were confirmed spectrophotometrically at 260 and 338 nm, respectively. Two series of ssDNA working solutions were prepared. For the first series, 50 lg dsDNA was denaturated by boiling for 15 min followed by immediate cooling and dilution with SSC buffer solution (pH 7.0). In the second series, the DNA denaturation was performed by the addition of EDTA/TEA solution (0.04 M acid EDTA and TEA solution up to pH 12.3) to 50 lg dsDNA and dilutions were made by addition of the EDTA/TEA solution to simulate the composition of the fractions produced from the alkaline elution procedure. For the preparation of all calibration plots, 1 ml of the appropriate DNA working solution was taken and its pH was adjusted with KH2 PO4 (0.2 M, pH 4.4). The solutions were diluted with water up to 2 ml and 1 ml of dye working solution was added. The final solutions remained undisrupted in the dark and the fluorescence was measured at excitation wavelength 355 nm and emission wavelength 475 nm. The above procedures were performed under reduced-light conditions. For the study of the influence of the DNA strandedness and the TEA/EDTA denaturation solution on the dye–DNA complex fluorescence intensity, six calibration plots, using neutralized dsDNA and ssDNA (with and without EDTA/TEA) working solutions, the same concentration range (0.17 to 7.68 lg/ml) and two different dye concentrations, 1.5 106 and 4.5 107 M, were established. For the determination of the optimum pH, five calibration plots at the above-mentioned ssDNA concentration range were established in the pH range 6.5 to 9. The dye concentration was 1.5 106 M. For the study of the dye concentration influence on the RFI of the ssDNA, three different calibration plots at dye concentrations 0.5 107 , 4.5 107 , and 1.5 106 M were established. Dye concentrations
148
M. Goumenou, K. Machera / Analytical Biochemistry 326 (2004) 146–152
outside of this range have been reported to be insufficient for DNA quantification [10,11]. The pH of the working solutions was 7.0. The incubation time of all the above-described studies was ca. 30 min. Finally, for the study of the incubation time for the formation on the dye–ssDNA complex, calibration plots at incubation times of 2, 5, 10, 20, 30, and 60 min were established. Fluorometric quantification of the DNA in the alkaline elution fractions The modified method based on the above-determined optimum experimental parameters was applied on fractions obtained from alkaline elution of liver tissue. The pH of 1 ml of each fraction was adjusted in the optimum range 6.8–7.2 with KH2 PO4 (0.2 M, pH 4.4). The neutralized fractions were diluted with water up to 2 ml, 1 ml of Hoechst 33258 solution of concentration 1.5 106 M was added, and the mixture was incubated in darkness for ca. 30 min. The fluorescence intensity was measured at excitation wavelength 355 nm and emission wavelength 475 nm. Alkaline elution of DNA The influence of elution volume on the dsDNA unwinding was studied using 50 lg of calf thymus dsDNA (approximately the expected DNA amount in nuclei used) denaturated with 5, 10, and 20 ml of EDTA/TEA. For the determination of the appropriate dsDNA unwinding time, 10 ml of the EDTA/TEA solution (pH 12.3) was added to 50 lg of dsDNA and the unwinding was interrupted by addition of KH2 PO4 solution (0.2 M, pH 4.4) after 1, 10, and 20 min at a final pH of 7.0. Solutions of dsDNA and ssDNA after boiling of the dsDNA were also prepared at the same final concentration for comparison with the chemically denaturated ssDNA. For the quantitative extraction of DNA retained on the filter after completion of the alkaline elution procedure, the filter was extracted twice with 4 ml of the denaturation solution and the DNA concentration in the two extracts was measured separately. Following the study of the above parameters, the modified alkaline elution method was applied and evaluated on liver tissue derived from untreated controls and MNU-treated (positive control) Wistar rats. For the evaluation of the method performance and of the variability of the results as affected by the different experimental parameter variations such as filter, temperature, pH, and buffer composition, the use of negative and positive controls was considered necessary. For this purpose, the modified DNA alkaline elution method was applied on liver tissue from untreated and MNU-treated Wistar rats of both sexes. MNU was administered at 200 mg/kg body weight by single oral intubation. Water was used as vehicle in all treated groups. The animals were anes-
thetized with CO2 and sacrificed by exsanguination at 4 and 24 h after administration. The livers were immediately removed and rinsed with saline and approximately 1/3 of the tissue was minced in 10 ml precooled saline EDTA solution (pH 7.4). Larger and adhering tissue pieces were removed through a stainless steal wire net (mesh 60 mm). The homogenate rested for ca. 15 min; the supernatant was decanted and centrifuged at 50 g for 2 min at 2 °C. The formed pellet was resuspended in 2 ml saline EDTA solution. The above procedure was performed at temperature below 4 °C while the following steps were performed under reduced-light conditions. A polycarbonate membrane filter (25-mm diameter, 5-lm pore size) was placed on a filter holder connected through a peristaltic pump to the fraction collector. The nuclei suspension (2 ml, ca. 2 106 nuclei) was placed in the funnel and rinsed with saline EDTA solution. The nuclei were lysed with 5 ml of lysis solution (2.0 M NaCl, 0.02 M Na3 EDTA, and 0.2% sarkosyl, pH 10.2) and the filter was rinsed with 2.5 ml of 0.02 M Na3 EDTA solution (pH 10.2). Afterward, 5 ml of EDTA/TEA solution (0.04 M acid EDTA, pH 12.3, adjusted with TEA solution) was added to the funnel and the ssDNA was eluted at a flow rate of 0.17 ml/min. The ssDNA retained on the filter was extracted twice with 4 ml of the EDTA/ TEA solution. The two extracts were analyzed separately to verify the accomplishment of the DNA quantitative recovery. Finally, the funnel and the line were washed with 4 ml of the EDTA/TEA solution. The ssDNA contained in all fractions was determined fluorometrically as described above. Data analysis For the study of the elution kinetics, plots (elution profiles) of the % ssDNA fraction retained on the filter in relation to the total ssDNA amount vs elution volume were established for each DNA sample. After verification of the goodness of fit to the first order kinetics, the elution constant (k) was estimated for each sample as the slope of the plot of the natural logarithm of the retained DNA on the filter vs elution volume. The distribution of the k values of the historical control data of our laboratory was the criterion for the selection of appropriate statistical tests for data analysis.
Results and discussion Influence of the DNA strandedness and the EDTA/TEA solution on the fluorescence intensity From the study of the influence of DNA strandedness upon RFI, at dye concentrations of 1.5 106 M (Fig. 1A) and 4.5 107 M (Fig. 1 B), it is observed that the RFI obtained from the ssDNA was 59 and
M. Goumenou, K. Machera / Analytical Biochemistry 326 (2004) 146–152
149
Fig. 1. Influence of the DNA strandedness and the EDTA/TEA solution on the fluorescence of the DNA/dye complex. (A) Dye concentration 1.5 106 M; (B) dye concentration 4.5 107 M (m) dsDNA; (j) ssDNA denaturated by boiling; (r) ssDNA denaturated by EDTA/TEA solution.
61% of the RFI for the dsDNA at the respective dye concentrations, as estimated from the slopes of the respective plots. The RFI of the ssDNA was further reduced by 15 and 17% in the presence of the EDTA/ TEA solution at the high and low dye concentrations, respectively. The significance of this reduction was confirmed by regression analysis. Plot linearity in the studied concentration range was satisfactory except for the dsDNA plot at dye concentration 4.5 107 M where it became insufficient (R2 ¼ 0:969). Based on the above observations, the study of the influence of the dye concentration, the pH, and the incubation time for the formation of the dye–ssDNA complex on the ssDNA quantification in the presence of the EDTA/ TEA solution and the optimization of these parameters were considered necessary.
optimum. In addition, satisfactory linearity was observed (Fig. 2) at the two highest dye concentration levels (R2 > 0:98). Influence of the pH From the study of the influence of the ssDNA solution pH on the RFI it is shown that the linearity of the RFI vs ssDNA concentration plots was not affected (R2 > 0:99) in any of the studied pH values. However, considerable influence of pH was observed on sensitivity (slope) and background signal (intercept) according to second order polynomial models (R2 > 0:99). As shown in Fig. 3, the sensitivity was higher in the pH range of 6.5 )7.5 while minimum background signal was observed at pH 7.0.
Influence of the dye concentration
Kinetics of the ssDNA–dye binding
From the study of the dye concentration influence on the ssDNA quantification in the presence of the EDTA/ TEA solution it is concluded that the highest sensitivity is observed at 1.5 106 M Hoechst 33258 (Fig. 2) and consequently this concentration is considered the
From the study of the optimum incubation time the dye–ssDNA formation and the slopes of the spective calibration plots (Fig. 4) it is indicated that maximum sensitivity was achieved after 30 min incubation.
Fig. 2. Influence of the Hoechst 33258 dye concentration on linearity and sensitivity of the ssDNA determination. Dye concentration: (m) 1.5 106 M; (j) 4.5 107 M; (r) 0.5 107 M.
Fig. 3. Influence of pH on sensitivity and background signal of ssDNA quantification. (d) Slope; (j) intercept.
for rethe of
150
M. Goumenou, K. Machera / Analytical Biochemistry 326 (2004) 146–152
Fig. 4. Influence of the incubation time on the ssDNA quantification sensitivity.
Determination of LOD and LOQ The limit of detection and the limit of quantification of the ssDNA were calculated from the following equations: LOD ¼ (3 Sb )/slope, and LOQ ¼ (10 Sb )/ slope. The slope and the standard error of the intercept (Sb ) were calculated from a calibration plot established for concentrations close to the expected LOD (data not shown) [17]. The values determined were LOD ¼ 0.10 lg/ml and LOQ ¼ 0.33 lg/ml. These limits were shown to be adequate for the quantification of the DNA obtained in the alkaline elution fractions. Evaluation of the alkaline elution modifications No significant influence on the RFI of the ssDNA from the reduction of elution volume from 20 to 5 ml was observed. Consequently 5 ml of the solution was considered sufficient for the unwinding of 50 lg dsDNA and was fractioned to five fractions of 1 ml each, which is a sufficient number of fractions for the establishment of an elution plot. The pump flow rate was set to 0.17 ml/min and consequently the elution time was ca. 30 min. This change reduces the duration of the elution by at least 50% in relation to those durations in previously reported procedures [2,3,5,13,15,16]. No significant change in the ssDNA RFI after the reduction of the dsDNA denaturation time from 20 to 1 min was observed. In addition, the relationship between the RFI of the ssDNA obtained after 1 min of denaturation and the dsDNA or the ssDNA obtained by thermal denaturation was the same as that previously observed (Fig. 1). Further prolongation of the denaturation time (ca. 24 h) resulted in significant reduction of the dye–ssDNA complex RFI. These observations indicate that the dsDNA denaturation under the present experimental conditions is completed in ca. 1 min. Consequently, the 30 min of denaturation and elution
Fig. 5. Elution profile of liver ssDNA from untreated (r) and MNUtreated (j) Wistar rats.
under the aforementioned experimental conditions are considered satisfactory. For the recovery of the DNA retained on the filter, double extraction of the filter and separate measurements of the two extracts were performed. The detected DNA amount in the second extract was always in the range from LOD to LOQ. Consequently, the DNA recovery obtained from the above-mentioned procedure is considered satisfactory. As shown by the elution profile of negative controls, linearity was observed for the undamaged DNA (Fig. 5), indicating relative length uniformity of the undisrupted DNA strands. Relative linearity was also observed in the profile of the MNU-treated samples, indicating no significant inference from alkali-induced ssBs due to alkali-labile sites or DNA cross-linking with proteins. In addition, the relatively high percentage of the eluted DNA in the case of MNUtreated rats (ca. 70–80%) indicates that the presented experimental conditions are appropriate for the sufficient elution of damaged DNA. ssBs estimators and data analysis The number of DNA strand breaks in the studied DNA samples is reflected by the relative amount of ssDNA eluted from the filter. In the present study, DNA elution followed first order kinetics and consequently the elution constant, k, was considered the most appropriate estimator for the expression of the DNA ssBs. The elution constant, k, has been used by Kohn et al. [4,18] for the evaluation of the DNA ssBs from untreated samples and samples treated with X-rays or chemicals. Another estimator used by Kohn [19] is the percentage of the DNA retained on the elution filter after a given elution time. Obviously, this estimator can be used without consideration of the elution profile kinetics and can be more appropriate in case of declination from the first order kinetics (e.g., induction of
M. Goumenou, K. Machera / Analytical Biochemistry 326 (2004) 146–152
Fig. 6. Box plots of the k distributions of control and MNU-treated liver samples from Wistar rats. (m) Mean of MNU k; (j) mean of control k (N ¼ 71), boxes, mean SD; whiskers, mean 1.96 SD.
alkali-labile sites or cross-linkage). Both negative and positive (MNU) control data of the present study were evaluated by both estimators. From the comparison of the two estimators it is indicated that increased repeatability (RSD) of the results was achieved when the % DNA retained on the elution filter was used. However, a reduced ability of this estimator to detect differences between positive and negative results using the t test was observed. Other estimators proposed in the literature are the normalized area above the elution curve [7] and the ‘‘scission factor’’ [20] defined as the ratio of the logarithm of the DNA from treated samples retained on the filter after a given elution time to the respective logarithm for the control samples. However, the use of the ‘‘scission factor’’ as estimator in in vivo experiments can be inappropriate, due to the high variability of the control values. The results from the MNU-treated rats were clearly positive in all tested cases. No significant difference between sexes was observed. However, the elution constant, k, was higher at the 4-h sampling time, reflecting a slight repair of the ssBs at 24-h. Nevertheless, for practical reasons we selected the 24 h sampling time for ‘‘positive control’’ measurements. Although relatively high variability from the positive results was observed (mean ¼ 0.294, SD ¼ 0.083, %RSD ¼ 28, N ¼ 21), no overlapping between the negative and the positive control data distributions was observed at the 95% confidence limit (Fig. 6). The distribution of the k values of the historical control data (mean ¼ 0.037, SD ¼ 0.010, N ¼ 71) (Fig. 7) and of those of the positive controls was well fitted to the normal distribution. Consequently, parametric statistics such as the t test, ANOVA, and regression analysis were more powerful than non parametric statistics and were considered the most appropriate for the statistical analysis of k values.
151
Fig. 7. Distribution of the k values of the concurrent historical control data (—, expected normal distribution).
References [1] R.J. Preston, G.R. Hoffmann, Genetic toxicology, in: C.D. Klaassen (Ed.), Casarett and DoullÕs Toxicology. The Basic Science of Poissons, McGraw-Hill, USA, 2001, pp. 321–350. [2] M.O. Bradley, G. Dysart, K. Fitzsimmons, P. Harbach, J. Lewin, G. Wolf, Measurements by filter elution of DNA Single- and double-strand breaks in rat hepatocytes: effects of nitrosamines and g-irradiation, Cancer Res. 42 (1982) 2592–2597. [3] K.W. Kohn, R.A. Grimek-Ewig, Alkaline elution analysis, a new approach to the study of DNA single-strand interruptions in cells, Cancer Res. 33 (1973) 1849–1853. [4] K.W. Kohn, L.C. Erickson, R.A. Grimek-Ewig, C.A. Friedman, Fractionation of DNA from mammalian cells by alkaline elution, Biochemistry 15 (1976) 4629–4637. [5] S. Parodi, M. Taningher, L. Santi, M. Cavanna, L. Sciaba, A. Maura, G. Brambilla, A practical procedure for testing DNA damage in vivo proposed for a pre-screening of chemical carcinogens, Mutat. Res. 54 (1978) 39–46. [6] C. Bolognesi, C.F. Cesarone, L. Santi, Standardization of the alkaline elution procedure using X-ray-damaged nuclear DNA, Tumori 31 (1979) 511–516. [7] A.S.S. Walles, K. Victorin, M. Lundborg, DNA damage in lung cells in vitro by 1,3-butadiene and nitrogen dioxide and their photochemical reaction products, Mutat. Res. 328 (1995) 11–19. [8] J.G. Hengstler, A. Hengst, J. Fuchs, B. Tanner, J. Pohl, F. Oesch, Induction of DNA crosslinks and DNA strand lesions by cyclophosphamide after activation by cytochrome P450 2B1, Mutat. Res. 373 (1997) 215–223. [9] J.M. Kissane, E. Robins, The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system, J. Biol. Chem. 233 (1958) 184–188. [10] C.F. Cesarone, C. Bolognesi, L. Santi, Improved microfluorometric DNA determination in biological material using 33258 Hoechst, Anal. Biochem. 100 (1979) 188–197. [11] D.L. Stout, F.F. Becker, Fluorometric quantification of singlestranded DNA: a method applicable to the technique of alkaline elution, Anal. Biochem. 127 (1982) 302–307. [12] M.O. Bradley, L.C. Ericson, K.W. Kohn, Non-enzymatic DNA strand breaks induced in mammalian cells by fluorescent light, Biochim. Biophys. Acta 520 (1978) 11–20. [13] L.C. Erikson, R. Osieka, N.A. Sharkey, K.W. Kohn, Measurement of DNA Damage in unlabeled mammalian cells analyzed by alkaline elution and a fluorometric DNA assay, Anal. Biochem. 106 (1980) 169–174.
152
M. Goumenou, K. Machera / Analytical Biochemistry 326 (2004) 146–152
[14] C. Bolognesi, C.F. Cesarone, L. Santi, Evaluation of DNA damage by alkaline elution technique after in vivo treatment with aromatic amines, Carcinogenesis 2 (1981) 265–268. [15] C. Bolognesi, C. Rossi, O. Barbieri, L. Santi, Benzo[a]pyrene— induced DNA damage in mouse fetal tissues, Carcinogenesis 6 (1985) 1091–1096. [16] C. Bolognesi, S. Bonatti, P. Degan, E. Gallerani, M. Peluso, R. Rabboni, P. Roggieri, A. Abbondandolo, Genotoxic activity of glyphosate and its technical formulation roundup, J. Agric. Food Chem. 45 (1997) 1957–1962. [17] J.N. Miller, J.C. Miller, Statistics and chemometrics for analytical chemistry, Prentice Hall, Essex, 2000.
[18] K.W. Kohn, R.A. Grimek-Ewig, L.C. Erickson, L.A. Zwelling, Measurement of strand breaks and cross—links by alkaline elution, in: E.C. Friedberg, P.C. Hanawalt (Eds.), DNA Repair. A Laboratory Manual of Research Procedures vol 1, Part B, Dekker, New York, 1981, pp. 379–402. [19] K.W. Kohn, DNA as a target in cancer chemotherapy: measurement of macromolecular DNA damage produced in mammalian cells by anticancer agents and carcinogens, in: H. Bush (Ed.), Methods in Cancer Research, Academic Press, New York, 1979, pp. 291–345. [20] R.E. Meyn, W.T. Jenkins, Variation in normal and tumor tissue sensitivity of mice to ionizing radiation-induced DNA strand breaks in vivo, Cancer Res. 43 (1983) 5668–5673.
Measurement of DNA single-strand breaks by alkaline elution and fluorometric DNA quantification Marina Goumenou and Kyriaki Machera* Laboratory of Pesticides Toxicology, Benaki Phytopathological Institute, Kifissia, GR-145 61 Athens, Greece Received 23 June 2003
Abstract The method presented is based on the alkaline elution procedure for the determination of DNA single-stand (ss) breaks developed by Kohn and on the principles of DNA quantification after binding with the dye Hoechst 33258. In the present study, modification of the alkaline elution procedure with regard to the elution solution volume was performed. The influences of the DNA strandedness, the ethylenediaminetetraacetate/tetraethylammonium hydroxide denaturation and elution solution presence, the DNA solution pH, the dye amount, and the incubation time for the formation of the dye–ssDNA complex on the DNA fluorometric quantification were also studied. The modified DNA alkaline elution procedure followed by the optimized fluorometric determination of the ssDNA was applied on liver tissue from both untreated and treated (N -nitroso-N -methylurea- administered) Wistar rats. The criteria for the selection of the appropriate estimator and statistical analysis of the obtained results are also presented. The method of the DNA alkaline elution followed by fluorometric determination of ssDNA as modified and evaluated is an accurate and reliable approach for the determination of in vivo induced ssDNA strand breaks. Ó 2003 Elsevier Inc. All rights reserved. Keywords: DNA; ssBs; Alkaline elution; Fluorometric ssDNA quantification
A great number of chemicals such as pesticides, drugs, and environmental contaminants exhibit mutagenic activity while most of the carcinogens can be detected as mutagens. Consequently, the detection of the mutagenic activity of a compound is essential for hazard identification. The underlying basis for mutagenesis is the damage in the structure of affected DNA [1]. A major class of DNA damages concerns DNA strand breaks, divided into single strand breaks (ssBs)1 and double-strand breaks (dsBs). ssBs occur during different processes including direct scission of the sugar–phosphate backbone by chemical or radical attack, hydrolysis of alkali–labile sites, enzymatic incision during base
*
Corresponding author. Fax: +0030-210-8077506. E-mail address: [email protected] (K. Machera). 1 Abbreviations used: ssBs, single-strand breaks; dsBs, double-strand breaks; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; TEA, tetraethylammonium hydroxide; SSC, saline-citrate buffer solution; MNU, N -nitroso-N -methylurea; RFI, relative fluorescence intensity; LOD, limit of detection; LOQ, limit of quantification. 0003-2697/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2003.11.019
and nucleotide excision repair, post replicational or recombinational repair and enzymatic scissions due to the action of topoisomerase or lysosomic hydrolases [2]. dsBs are also produced by direct scissions, during repair processes, or during the replication of DNA containing ssBs. For the determination of ssBs on DNA, several methods have been presented in the literature. One of them is the alkaline elution technique that was originally developed by Kohn et al. [3,4] targeting the replacement of the DNA sedimentation with alkaline sucrose gradients. According to this method, cells are lysed on membrane filter and large nonelutable dsDNA is released. The filter is then eluted with an alkaline solution, ssDNA is released and slowly pumped through while fractions are collected to determine the elution kinetics. The elution rate of the ssDNA was proven to be size dependent and used as a measure of the DNA ssBs. During the following years and until nowadays several modifications of the original first procedure have been published [5–8].
M. Goumenou, K. Machera / Analytical Biochemistry 326 (2004) 146–152
Initially, for quantification of the DNA amount eluted during the alkaline elution procedure, prelabeling of cells with radioactive probes was used [3,4]. Soon after, DNA quantification using fluorometric methods [9–11] was considered preferable to avoid the use of radioactive materials [5,12–14]. In the present study, DNA quantification is based on the principles of the method described by Cesarone et al. [10]. According to this method, enhancement of the Hoechst 33258 dye fluorescence yield is observed when it is complexed with DNA. No information with regard to possible differences in relative fluorescence intensity (RFI) in dsDNA and ssDNA as a function of dye/DNA ratio was reported [10]. In the first part of the present study, the influence of the DNA strandedness upon fluorescence intensity as a function of the dye/dsDNA and dye/ssDNA ratios was studied. A reduction of the fluorescence intensity of the dye–ssDNA complex with regard to that of the dye– dsDNA complex was observed at the same dye/DNA ratios. Further reduction of the dye–ssDNA complex fluorescence was observed in the presence of the denaturation solution used (EDTA/TEA). Furthermore, the influence of other ssDNA quantification parameters, such as pH, dye concentration, and incubation time for the dye–DNA binding, was studied with appropriate fluorescence calibration plots. In the second part, adaptation and modifications of the procedure presented by Kohn for the DNA alkaline elution were performed. The main modifications were related to the elution time and the extraction of the DNA retained on the filter after elution. The performances of both modified approaches were evaluated on liver tissue from both untreated and treated Wistar rats. Additionally, examination of different estimators for the DNA ssBs evaluation and statistics for data analysis were performed. Finally, the historical control data of the ssBs in rat liver DNA from our laboratory are presented.
Materials and methods Chemicals and equipment Na3 EDTA, EDTA (acid form), N -lauroylsarcosine (sarcosyl), tetraethylamonium hydroxide (20% in water), Hoechst 33258, saline-citrate buffer solution (SSC, pH 7,0), N -nitroso-N -methylurea (MNU), and calf thymus DNA were purchased from Sigma. The fluorometric DNA quantification was performed with a Jasco FP-6200 spectrofluorometer. Animals The alkaline elution technique was performed in liver tissue obtained from young adult Wistar rats from the
147
breeding colony of our laboratory. All animals were housed in groups of five in stainless steel wire cages under controlled environmental conditions, with ad libitum consumption of feed and water. Evaluation of influence and optimization of the experimental parameters for the fluorometric DNA quantification For the study of the parameters affecting the ssDNA quantification (DNA strandedness, EDTA/TEA solution, pH, dye concentration, and incubation time) appropriate fluorescence calibration plots (RFI vs. ssDNA conc.) were established and evaluated with regard to linearity, sensitivity, etc. Stock solutions of standard calf thymus dsDNA at a concentration of 1 mg/ml in SSC buffer solution and of Hoechst 33258 at a concentration of 1.5 104 M in water were prepared. Both solutions were stored at 4 °C. Working solutions of the dye and the dsDNA were prepared by appropriate dilution with SSC buffer solution (pH 7.0) and the final concentrations were confirmed spectrophotometrically at 260 and 338 nm, respectively. Two series of ssDNA working solutions were prepared. For the first series, 50 lg dsDNA was denaturated by boiling for 15 min followed by immediate cooling and dilution with SSC buffer solution (pH 7.0). In the second series, the DNA denaturation was performed by the addition of EDTA/TEA solution (0.04 M acid EDTA and TEA solution up to pH 12.3) to 50 lg dsDNA and dilutions were made by addition of the EDTA/TEA solution to simulate the composition of the fractions produced from the alkaline elution procedure. For the preparation of all calibration plots, 1 ml of the appropriate DNA working solution was taken and its pH was adjusted with KH2 PO4 (0.2 M, pH 4.4). The solutions were diluted with water up to 2 ml and 1 ml of dye working solution was added. The final solutions remained undisrupted in the dark and the fluorescence was measured at excitation wavelength 355 nm and emission wavelength 475 nm. The above procedures were performed under reduced-light conditions. For the study of the influence of the DNA strandedness and the TEA/EDTA denaturation solution on the dye–DNA complex fluorescence intensity, six calibration plots, using neutralized dsDNA and ssDNA (with and without EDTA/TEA) working solutions, the same concentration range (0.17 to 7.68 lg/ml) and two different dye concentrations, 1.5 106 and 4.5 107 M, were established. For the determination of the optimum pH, five calibration plots at the above-mentioned ssDNA concentration range were established in the pH range 6.5 to 9. The dye concentration was 1.5 106 M. For the study of the dye concentration influence on the RFI of the ssDNA, three different calibration plots at dye concentrations 0.5 107 , 4.5 107 , and 1.5 106 M were established. Dye concentrations
148
M. Goumenou, K. Machera / Analytical Biochemistry 326 (2004) 146–152
outside of this range have been reported to be insufficient for DNA quantification [10,11]. The pH of the working solutions was 7.0. The incubation time of all the above-described studies was ca. 30 min. Finally, for the study of the incubation time for the formation on the dye–ssDNA complex, calibration plots at incubation times of 2, 5, 10, 20, 30, and 60 min were established. Fluorometric quantification of the DNA in the alkaline elution fractions The modified method based on the above-determined optimum experimental parameters was applied on fractions obtained from alkaline elution of liver tissue. The pH of 1 ml of each fraction was adjusted in the optimum range 6.8–7.2 with KH2 PO4 (0.2 M, pH 4.4). The neutralized fractions were diluted with water up to 2 ml, 1 ml of Hoechst 33258 solution of concentration 1.5 106 M was added, and the mixture was incubated in darkness for ca. 30 min. The fluorescence intensity was measured at excitation wavelength 355 nm and emission wavelength 475 nm. Alkaline elution of DNA The influence of elution volume on the dsDNA unwinding was studied using 50 lg of calf thymus dsDNA (approximately the expected DNA amount in nuclei used) denaturated with 5, 10, and 20 ml of EDTA/TEA. For the determination of the appropriate dsDNA unwinding time, 10 ml of the EDTA/TEA solution (pH 12.3) was added to 50 lg of dsDNA and the unwinding was interrupted by addition of KH2 PO4 solution (0.2 M, pH 4.4) after 1, 10, and 20 min at a final pH of 7.0. Solutions of dsDNA and ssDNA after boiling of the dsDNA were also prepared at the same final concentration for comparison with the chemically denaturated ssDNA. For the quantitative extraction of DNA retained on the filter after completion of the alkaline elution procedure, the filter was extracted twice with 4 ml of the denaturation solution and the DNA concentration in the two extracts was measured separately. Following the study of the above parameters, the modified alkaline elution method was applied and evaluated on liver tissue derived from untreated controls and MNU-treated (positive control) Wistar rats. For the evaluation of the method performance and of the variability of the results as affected by the different experimental parameter variations such as filter, temperature, pH, and buffer composition, the use of negative and positive controls was considered necessary. For this purpose, the modified DNA alkaline elution method was applied on liver tissue from untreated and MNU-treated Wistar rats of both sexes. MNU was administered at 200 mg/kg body weight by single oral intubation. Water was used as vehicle in all treated groups. The animals were anes-
thetized with CO2 and sacrificed by exsanguination at 4 and 24 h after administration. The livers were immediately removed and rinsed with saline and approximately 1/3 of the tissue was minced in 10 ml precooled saline EDTA solution (pH 7.4). Larger and adhering tissue pieces were removed through a stainless steal wire net (mesh 60 mm). The homogenate rested for ca. 15 min; the supernatant was decanted and centrifuged at 50 g for 2 min at 2 °C. The formed pellet was resuspended in 2 ml saline EDTA solution. The above procedure was performed at temperature below 4 °C while the following steps were performed under reduced-light conditions. A polycarbonate membrane filter (25-mm diameter, 5-lm pore size) was placed on a filter holder connected through a peristaltic pump to the fraction collector. The nuclei suspension (2 ml, ca. 2 106 nuclei) was placed in the funnel and rinsed with saline EDTA solution. The nuclei were lysed with 5 ml of lysis solution (2.0 M NaCl, 0.02 M Na3 EDTA, and 0.2% sarkosyl, pH 10.2) and the filter was rinsed with 2.5 ml of 0.02 M Na3 EDTA solution (pH 10.2). Afterward, 5 ml of EDTA/TEA solution (0.04 M acid EDTA, pH 12.3, adjusted with TEA solution) was added to the funnel and the ssDNA was eluted at a flow rate of 0.17 ml/min. The ssDNA retained on the filter was extracted twice with 4 ml of the EDTA/ TEA solution. The two extracts were analyzed separately to verify the accomplishment of the DNA quantitative recovery. Finally, the funnel and the line were washed with 4 ml of the EDTA/TEA solution. The ssDNA contained in all fractions was determined fluorometrically as described above. Data analysis For the study of the elution kinetics, plots (elution profiles) of the % ssDNA fraction retained on the filter in relation to the total ssDNA amount vs elution volume were established for each DNA sample. After verification of the goodness of fit to the first order kinetics, the elution constant (k) was estimated for each sample as the slope of the plot of the natural logarithm of the retained DNA on the filter vs elution volume. The distribution of the k values of the historical control data of our laboratory was the criterion for the selection of appropriate statistical tests for data analysis.
Results and discussion Influence of the DNA strandedness and the EDTA/TEA solution on the fluorescence intensity From the study of the influence of DNA strandedness upon RFI, at dye concentrations of 1.5 106 M (Fig. 1A) and 4.5 107 M (Fig. 1 B), it is observed that the RFI obtained from the ssDNA was 59 and
M. Goumenou, K. Machera / Analytical Biochemistry 326 (2004) 146–152
149
Fig. 1. Influence of the DNA strandedness and the EDTA/TEA solution on the fluorescence of the DNA/dye complex. (A) Dye concentration 1.5 106 M; (B) dye concentration 4.5 107 M (m) dsDNA; (j) ssDNA denaturated by boiling; (r) ssDNA denaturated by EDTA/TEA solution.
61% of the RFI for the dsDNA at the respective dye concentrations, as estimated from the slopes of the respective plots. The RFI of the ssDNA was further reduced by 15 and 17% in the presence of the EDTA/ TEA solution at the high and low dye concentrations, respectively. The significance of this reduction was confirmed by regression analysis. Plot linearity in the studied concentration range was satisfactory except for the dsDNA plot at dye concentration 4.5 107 M where it became insufficient (R2 ¼ 0:969). Based on the above observations, the study of the influence of the dye concentration, the pH, and the incubation time for the formation of the dye–ssDNA complex on the ssDNA quantification in the presence of the EDTA/ TEA solution and the optimization of these parameters were considered necessary.
optimum. In addition, satisfactory linearity was observed (Fig. 2) at the two highest dye concentration levels (R2 > 0:98). Influence of the pH From the study of the influence of the ssDNA solution pH on the RFI it is shown that the linearity of the RFI vs ssDNA concentration plots was not affected (R2 > 0:99) in any of the studied pH values. However, considerable influence of pH was observed on sensitivity (slope) and background signal (intercept) according to second order polynomial models (R2 > 0:99). As shown in Fig. 3, the sensitivity was higher in the pH range of 6.5 )7.5 while minimum background signal was observed at pH 7.0.
Influence of the dye concentration
Kinetics of the ssDNA–dye binding
From the study of the dye concentration influence on the ssDNA quantification in the presence of the EDTA/ TEA solution it is concluded that the highest sensitivity is observed at 1.5 106 M Hoechst 33258 (Fig. 2) and consequently this concentration is considered the
From the study of the optimum incubation time the dye–ssDNA formation and the slopes of the spective calibration plots (Fig. 4) it is indicated that maximum sensitivity was achieved after 30 min incubation.
Fig. 2. Influence of the Hoechst 33258 dye concentration on linearity and sensitivity of the ssDNA determination. Dye concentration: (m) 1.5 106 M; (j) 4.5 107 M; (r) 0.5 107 M.
Fig. 3. Influence of pH on sensitivity and background signal of ssDNA quantification. (d) Slope; (j) intercept.
for rethe of
150
M. Goumenou, K. Machera / Analytical Biochemistry 326 (2004) 146–152
Fig. 4. Influence of the incubation time on the ssDNA quantification sensitivity.
Determination of LOD and LOQ The limit of detection and the limit of quantification of the ssDNA were calculated from the following equations: LOD ¼ (3 Sb )/slope, and LOQ ¼ (10 Sb )/ slope. The slope and the standard error of the intercept (Sb ) were calculated from a calibration plot established for concentrations close to the expected LOD (data not shown) [17]. The values determined were LOD ¼ 0.10 lg/ml and LOQ ¼ 0.33 lg/ml. These limits were shown to be adequate for the quantification of the DNA obtained in the alkaline elution fractions. Evaluation of the alkaline elution modifications No significant influence on the RFI of the ssDNA from the reduction of elution volume from 20 to 5 ml was observed. Consequently 5 ml of the solution was considered sufficient for the unwinding of 50 lg dsDNA and was fractioned to five fractions of 1 ml each, which is a sufficient number of fractions for the establishment of an elution plot. The pump flow rate was set to 0.17 ml/min and consequently the elution time was ca. 30 min. This change reduces the duration of the elution by at least 50% in relation to those durations in previously reported procedures [2,3,5,13,15,16]. No significant change in the ssDNA RFI after the reduction of the dsDNA denaturation time from 20 to 1 min was observed. In addition, the relationship between the RFI of the ssDNA obtained after 1 min of denaturation and the dsDNA or the ssDNA obtained by thermal denaturation was the same as that previously observed (Fig. 1). Further prolongation of the denaturation time (ca. 24 h) resulted in significant reduction of the dye–ssDNA complex RFI. These observations indicate that the dsDNA denaturation under the present experimental conditions is completed in ca. 1 min. Consequently, the 30 min of denaturation and elution
Fig. 5. Elution profile of liver ssDNA from untreated (r) and MNUtreated (j) Wistar rats.
under the aforementioned experimental conditions are considered satisfactory. For the recovery of the DNA retained on the filter, double extraction of the filter and separate measurements of the two extracts were performed. The detected DNA amount in the second extract was always in the range from LOD to LOQ. Consequently, the DNA recovery obtained from the above-mentioned procedure is considered satisfactory. As shown by the elution profile of negative controls, linearity was observed for the undamaged DNA (Fig. 5), indicating relative length uniformity of the undisrupted DNA strands. Relative linearity was also observed in the profile of the MNU-treated samples, indicating no significant inference from alkali-induced ssBs due to alkali-labile sites or DNA cross-linking with proteins. In addition, the relatively high percentage of the eluted DNA in the case of MNUtreated rats (ca. 70–80%) indicates that the presented experimental conditions are appropriate for the sufficient elution of damaged DNA. ssBs estimators and data analysis The number of DNA strand breaks in the studied DNA samples is reflected by the relative amount of ssDNA eluted from the filter. In the present study, DNA elution followed first order kinetics and consequently the elution constant, k, was considered the most appropriate estimator for the expression of the DNA ssBs. The elution constant, k, has been used by Kohn et al. [4,18] for the evaluation of the DNA ssBs from untreated samples and samples treated with X-rays or chemicals. Another estimator used by Kohn [19] is the percentage of the DNA retained on the elution filter after a given elution time. Obviously, this estimator can be used without consideration of the elution profile kinetics and can be more appropriate in case of declination from the first order kinetics (e.g., induction of
M. Goumenou, K. Machera / Analytical Biochemistry 326 (2004) 146–152
Fig. 6. Box plots of the k distributions of control and MNU-treated liver samples from Wistar rats. (m) Mean of MNU k; (j) mean of control k (N ¼ 71), boxes, mean SD; whiskers, mean 1.96 SD.
alkali-labile sites or cross-linkage). Both negative and positive (MNU) control data of the present study were evaluated by both estimators. From the comparison of the two estimators it is indicated that increased repeatability (RSD) of the results was achieved when the % DNA retained on the elution filter was used. However, a reduced ability of this estimator to detect differences between positive and negative results using the t test was observed. Other estimators proposed in the literature are the normalized area above the elution curve [7] and the ‘‘scission factor’’ [20] defined as the ratio of the logarithm of the DNA from treated samples retained on the filter after a given elution time to the respective logarithm for the control samples. However, the use of the ‘‘scission factor’’ as estimator in in vivo experiments can be inappropriate, due to the high variability of the control values. The results from the MNU-treated rats were clearly positive in all tested cases. No significant difference between sexes was observed. However, the elution constant, k, was higher at the 4-h sampling time, reflecting a slight repair of the ssBs at 24-h. Nevertheless, for practical reasons we selected the 24 h sampling time for ‘‘positive control’’ measurements. Although relatively high variability from the positive results was observed (mean ¼ 0.294, SD ¼ 0.083, %RSD ¼ 28, N ¼ 21), no overlapping between the negative and the positive control data distributions was observed at the 95% confidence limit (Fig. 6). The distribution of the k values of the historical control data (mean ¼ 0.037, SD ¼ 0.010, N ¼ 71) (Fig. 7) and of those of the positive controls was well fitted to the normal distribution. Consequently, parametric statistics such as the t test, ANOVA, and regression analysis were more powerful than non parametric statistics and were considered the most appropriate for the statistical analysis of k values.
151
Fig. 7. Distribution of the k values of the concurrent historical control data (—, expected normal distribution).
References [1] R.J. Preston, G.R. Hoffmann, Genetic toxicology, in: C.D. Klaassen (Ed.), Casarett and DoullÕs Toxicology. The Basic Science of Poissons, McGraw-Hill, USA, 2001, pp. 321–350. [2] M.O. Bradley, G. Dysart, K. Fitzsimmons, P. Harbach, J. Lewin, G. Wolf, Measurements by filter elution of DNA Single- and double-strand breaks in rat hepatocytes: effects of nitrosamines and g-irradiation, Cancer Res. 42 (1982) 2592–2597. [3] K.W. Kohn, R.A. Grimek-Ewig, Alkaline elution analysis, a new approach to the study of DNA single-strand interruptions in cells, Cancer Res. 33 (1973) 1849–1853. [4] K.W. Kohn, L.C. Erickson, R.A. Grimek-Ewig, C.A. Friedman, Fractionation of DNA from mammalian cells by alkaline elution, Biochemistry 15 (1976) 4629–4637. [5] S. Parodi, M. Taningher, L. Santi, M. Cavanna, L. Sciaba, A. Maura, G. Brambilla, A practical procedure for testing DNA damage in vivo proposed for a pre-screening of chemical carcinogens, Mutat. Res. 54 (1978) 39–46. [6] C. Bolognesi, C.F. Cesarone, L. Santi, Standardization of the alkaline elution procedure using X-ray-damaged nuclear DNA, Tumori 31 (1979) 511–516. [7] A.S.S. Walles, K. Victorin, M. Lundborg, DNA damage in lung cells in vitro by 1,3-butadiene and nitrogen dioxide and their photochemical reaction products, Mutat. Res. 328 (1995) 11–19. [8] J.G. Hengstler, A. Hengst, J. Fuchs, B. Tanner, J. Pohl, F. Oesch, Induction of DNA crosslinks and DNA strand lesions by cyclophosphamide after activation by cytochrome P450 2B1, Mutat. Res. 373 (1997) 215–223. [9] J.M. Kissane, E. Robins, The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system, J. Biol. Chem. 233 (1958) 184–188. [10] C.F. Cesarone, C. Bolognesi, L. Santi, Improved microfluorometric DNA determination in biological material using 33258 Hoechst, Anal. Biochem. 100 (1979) 188–197. [11] D.L. Stout, F.F. Becker, Fluorometric quantification of singlestranded DNA: a method applicable to the technique of alkaline elution, Anal. Biochem. 127 (1982) 302–307. [12] M.O. Bradley, L.C. Ericson, K.W. Kohn, Non-enzymatic DNA strand breaks induced in mammalian cells by fluorescent light, Biochim. Biophys. Acta 520 (1978) 11–20. [13] L.C. Erikson, R. Osieka, N.A. Sharkey, K.W. Kohn, Measurement of DNA Damage in unlabeled mammalian cells analyzed by alkaline elution and a fluorometric DNA assay, Anal. Biochem. 106 (1980) 169–174.
152
M. Goumenou, K. Machera / Analytical Biochemistry 326 (2004) 146–152
[14] C. Bolognesi, C.F. Cesarone, L. Santi, Evaluation of DNA damage by alkaline elution technique after in vivo treatment with aromatic amines, Carcinogenesis 2 (1981) 265–268. [15] C. Bolognesi, C. Rossi, O. Barbieri, L. Santi, Benzo[a]pyrene— induced DNA damage in mouse fetal tissues, Carcinogenesis 6 (1985) 1091–1096. [16] C. Bolognesi, S. Bonatti, P. Degan, E. Gallerani, M. Peluso, R. Rabboni, P. Roggieri, A. Abbondandolo, Genotoxic activity of glyphosate and its technical formulation roundup, J. Agric. Food Chem. 45 (1997) 1957–1962. [17] J.N. Miller, J.C. Miller, Statistics and chemometrics for analytical chemistry, Prentice Hall, Essex, 2000.
[18] K.W. Kohn, R.A. Grimek-Ewig, L.C. Erickson, L.A. Zwelling, Measurement of strand breaks and cross—links by alkaline elution, in: E.C. Friedberg, P.C. Hanawalt (Eds.), DNA Repair. A Laboratory Manual of Research Procedures vol 1, Part B, Dekker, New York, 1981, pp. 379–402. [19] K.W. Kohn, DNA as a target in cancer chemotherapy: measurement of macromolecular DNA damage produced in mammalian cells by anticancer agents and carcinogens, in: H. Bush (Ed.), Methods in Cancer Research, Academic Press, New York, 1979, pp. 291–345. [20] R.E. Meyn, W.T. Jenkins, Variation in normal and tumor tissue sensitivity of mice to ionizing radiation-induced DNA strand breaks in vivo, Cancer Res. 43 (1983) 5668–5673.