Mutation Research, 281 (1992) 17-23 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00
17
MUTLET 00604
Detection by fluorescence analysis of D N A unwinding and unscheduled D N A synthesis, of D N A damage and repair induced in vitro by direct-acting mutagens on human lymphocytes Lucia Celotti, Paola Ferraro and M. Raffaella Biasin Department of Biology, Padua (Italy) (Received 23 July 1991) (Revision received 2 September 1991) (Accepted 12 September 1991)
Keywords: Genotoxic agents; DNA repair; Unscheduled DNA synthesis; Fluorescence analysis of DNA unwinding; Human peripheral blood lymphocytes
Summary The sensitivity and reliability of UDS and F A D U in detecting mutagenic effects were compared by measuring DNA damage and repair in PBL treated in vitro with UV light. MMS and BPDE. The results indicate that F A D U is more sensitive than UDS, as it is able to detect DNA damage at doses 3-4-fold lower. We also determined the DNA damage and repair induced by the above agents on lymphocyte samples from different donors by F A D U and UDS, confirming that the DNA repair process in humans is characterized by interindividually variable efficiency.
A wide range of variability of DNA-repair activity exists in human populations, which may underlie the different individual susceptibility to the final effects of mutagenic and carcinogenic agents. Several methods are currently available to study D N A repair, among which the measurement of the patching step of repair (UDS) is the most commonly used on account of its simplicity and rapidity. The UDS method is based on the
Correspondence: Dr. L. Celotti, Department of Biology, University of Padua, via Trieste 75, 1-35121 Padua (Italy).
Abbreciations: BPDE, benzo[a]pyrene-diol-epoxide; EB, ethidium bromide; FADU, fluorescence analysis of DNA unwinding; 3H-TdR, tritiated thymidine; HU, hydroxyurea; MMS, methyl methanesulfonate; PBL, peripheral blood lymphocytes; UDS, unscheduled DNA synthesis.
quantitative determination of the radioactivity incorporated into the DNA of control and treated cells in the absence of D N A replication. In humans a measure of the efficiency of DNA repair can be obtained by determining the UDS induced by in vitro UV irradiation of G o PBL incubated with 3H-TdR. The different levels of UDS in PBL among exposed and control subjects are attributed to the genotoxic effect of the in vivo exposure, which is thought to interfere with the DNA repair process by inhibiting or inducing the enzymes involved (Pero et al., 1982; Benigni et al., 1984; Celotti et al., 1989, 1990). DNA repair can also be monitored on the basis of the strand breaks which appear and disappear during the different phases of the repair process. Strand breaks can be detected by F A D U as the rate of unwinding of large DNA
18 molecules, such as those of mammalian cells, increases with the presence of strand breaks. Therefore an increased rate of DNA unwinding can be considered a sensitive measure of induced strand breaks (Birnboim and Jevcak, 1981). In order to detect DNA unwinding, the fluorescent dye ethidium bromide (EB) is used as a direct probe of double-stranded (ds) DNA (Morgan and Pulleyblank, 1974). FADU has been applied to PBL in order to compare the rate of unwinding of DNA from cells treated with DNA-damaging agents (Birnboim and Jevcak, 1981; McLean et al., 1982, Parodi et al., 1989; Percy and Chipman, 1991) or of rejoining of DNA-strand breaks during UV-induced DNA repair in blood cells (Thierry et al., 1985; Munch-Petersen, 1988). In the present work we compared the sensitivity of the UDS and FADU procedures in detecting DNA damage and repair in PBL exposed in vitro to UV light, MMS and BPDE (a very active metabolite of benzo[a]pyrene). We also compared the interindividual variability among PBL samples obtained from different donors of the damage and repair values obtained by F A D U and UDS. Materials and methods
From samples of 20-30 ml of peripheral blood, obtained by venepuncture from healthy donors, PBL were isolated and cultured in Eagle's minimal essential medium supplemented with 10% fetal calf serum (MEM) as previously described (Celotti et al., 1989). PBL were suspended in MEM at 1 x 10 6 cells/ml, and 1 ml of cell suspension was irradiated in open petri dishes (5 cm diameter) by 254-nm UVS-11 mineral light giving a fluence rate of 2 J / m Z / s . The cells were exposed to different doses of UV light (2-48 j / m 2 ) . Samples of 3-5 x 106 PBL were treated in MEM with MMS (Merk, Schuchardt, Germany) or BPDE I (Chemsyn Science Laboratories, Lenexa, KS, U.S.A.) as indicated in the Results. UDS was determined in G o PBL by incubation for 3 h with 10 p~Ci/ml 3H-TdR (spec. act. 40 Ci/mmole, Amity-PC, Aylesbury, U.K.). 3H-TdR was present in the medium during the treatment with MMS or BPDE. The incorporation of ra-
dioactivity was measured in the presence of 2.5 mM hydroxyurea (HU; Sigma Chimica, Milan, Italy). UDS was calculated as the difference between the macromolecular radioactivity in treated and control cultures. The radioactivity incorporated into DNA was measured by liquid-scintillation spectroscopy in a 300 C Packard counter A, with an efficiency of 30%. Induction and rejoining of DNA-strand breaks after mutagenic treatments were measured by the FADU technique as indicated by Birnboim and Jevcak (1981) with minor modifications. 2.5 x 106 control or treated cells were incubated in the presence of the DNA-synthesis/repair inhibitor arabinosylcytosine (ara-C, Sigma Chimica; 10/zM final concentration). This made it possible to detect the total amount of breaks induced by the treatments. The removal of DNA damages was determined by FADU in parallel PBL samples incubated in the absence of ara-C. The incubation was terminated by cooling on ice. After centrifugation, the cells were suspended in icecold phosphate buffer 10 raM. Following the original procedure (Birnboim and Jevcak, 1981), each PBL suspension was divided equally into 3 sets of tubes: (1) a blank sample (B) in which DNA was completely unwound; (2) a sample (T) used for evaluating total fluorescence of native DNA; (3) a sample (P) used to estimate the unwinding rate in control (C) or treated (UV, MMS, BPDE) PBL. The percentage of ds DNA, D, was estimated by staining with ethidium bromide (0.4/.~g/ml, Sigma Chimica), which selectively binds to ds DNA. The fluorescence was read with an excitation wavelength of 520 nm and an emission wavelength of 590 nm by a RF-5000 Shimadzu spectrofluorimeter. The percentage of ds DNA (D) was determined from the fluorescence of tubes B, T and P by the equation: D = (P- B)/(T-
B) X 100.
From D values A D = D (control c e l l s ) - D (treated cells) can be calculated, which is an arbitrary measure of the amount of DNA strand breaks. AD in the presence of ara-C (AD o) can also be calculated and used as a measure of the percentage of total breaks in the absence of rejoining.
19
A
90.
b •
. . . . .
800
600
200 .
.
2
.
.
I
4
6
10
24
Jim 2
I
I
I
I
6
12
24
48
I1
il mi
I ¢J o
B
,"
90.
b
on"
"9
"
', 70
O0
a
I
I
.
.--
.300
. -
< 5o /
~
30-
a
~
17
MUTLET 00604
Detection by fluorescence analysis of D N A unwinding and unscheduled D N A synthesis, of D N A damage and repair induced in vitro by direct-acting mutagens on human lymphocytes Lucia Celotti, Paola Ferraro and M. Raffaella Biasin Department of Biology, Padua (Italy) (Received 23 July 1991) (Revision received 2 September 1991) (Accepted 12 September 1991)
Keywords: Genotoxic agents; DNA repair; Unscheduled DNA synthesis; Fluorescence analysis of DNA unwinding; Human peripheral blood lymphocytes
Summary The sensitivity and reliability of UDS and F A D U in detecting mutagenic effects were compared by measuring DNA damage and repair in PBL treated in vitro with UV light. MMS and BPDE. The results indicate that F A D U is more sensitive than UDS, as it is able to detect DNA damage at doses 3-4-fold lower. We also determined the DNA damage and repair induced by the above agents on lymphocyte samples from different donors by F A D U and UDS, confirming that the DNA repair process in humans is characterized by interindividually variable efficiency.
A wide range of variability of DNA-repair activity exists in human populations, which may underlie the different individual susceptibility to the final effects of mutagenic and carcinogenic agents. Several methods are currently available to study D N A repair, among which the measurement of the patching step of repair (UDS) is the most commonly used on account of its simplicity and rapidity. The UDS method is based on the
Correspondence: Dr. L. Celotti, Department of Biology, University of Padua, via Trieste 75, 1-35121 Padua (Italy).
Abbreciations: BPDE, benzo[a]pyrene-diol-epoxide; EB, ethidium bromide; FADU, fluorescence analysis of DNA unwinding; 3H-TdR, tritiated thymidine; HU, hydroxyurea; MMS, methyl methanesulfonate; PBL, peripheral blood lymphocytes; UDS, unscheduled DNA synthesis.
quantitative determination of the radioactivity incorporated into the DNA of control and treated cells in the absence of D N A replication. In humans a measure of the efficiency of DNA repair can be obtained by determining the UDS induced by in vitro UV irradiation of G o PBL incubated with 3H-TdR. The different levels of UDS in PBL among exposed and control subjects are attributed to the genotoxic effect of the in vivo exposure, which is thought to interfere with the DNA repair process by inhibiting or inducing the enzymes involved (Pero et al., 1982; Benigni et al., 1984; Celotti et al., 1989, 1990). DNA repair can also be monitored on the basis of the strand breaks which appear and disappear during the different phases of the repair process. Strand breaks can be detected by F A D U as the rate of unwinding of large DNA
18 molecules, such as those of mammalian cells, increases with the presence of strand breaks. Therefore an increased rate of DNA unwinding can be considered a sensitive measure of induced strand breaks (Birnboim and Jevcak, 1981). In order to detect DNA unwinding, the fluorescent dye ethidium bromide (EB) is used as a direct probe of double-stranded (ds) DNA (Morgan and Pulleyblank, 1974). FADU has been applied to PBL in order to compare the rate of unwinding of DNA from cells treated with DNA-damaging agents (Birnboim and Jevcak, 1981; McLean et al., 1982, Parodi et al., 1989; Percy and Chipman, 1991) or of rejoining of DNA-strand breaks during UV-induced DNA repair in blood cells (Thierry et al., 1985; Munch-Petersen, 1988). In the present work we compared the sensitivity of the UDS and FADU procedures in detecting DNA damage and repair in PBL exposed in vitro to UV light, MMS and BPDE (a very active metabolite of benzo[a]pyrene). We also compared the interindividual variability among PBL samples obtained from different donors of the damage and repair values obtained by F A D U and UDS. Materials and methods
From samples of 20-30 ml of peripheral blood, obtained by venepuncture from healthy donors, PBL were isolated and cultured in Eagle's minimal essential medium supplemented with 10% fetal calf serum (MEM) as previously described (Celotti et al., 1989). PBL were suspended in MEM at 1 x 10 6 cells/ml, and 1 ml of cell suspension was irradiated in open petri dishes (5 cm diameter) by 254-nm UVS-11 mineral light giving a fluence rate of 2 J / m Z / s . The cells were exposed to different doses of UV light (2-48 j / m 2 ) . Samples of 3-5 x 106 PBL were treated in MEM with MMS (Merk, Schuchardt, Germany) or BPDE I (Chemsyn Science Laboratories, Lenexa, KS, U.S.A.) as indicated in the Results. UDS was determined in G o PBL by incubation for 3 h with 10 p~Ci/ml 3H-TdR (spec. act. 40 Ci/mmole, Amity-PC, Aylesbury, U.K.). 3H-TdR was present in the medium during the treatment with MMS or BPDE. The incorporation of ra-
dioactivity was measured in the presence of 2.5 mM hydroxyurea (HU; Sigma Chimica, Milan, Italy). UDS was calculated as the difference between the macromolecular radioactivity in treated and control cultures. The radioactivity incorporated into DNA was measured by liquid-scintillation spectroscopy in a 300 C Packard counter A, with an efficiency of 30%. Induction and rejoining of DNA-strand breaks after mutagenic treatments were measured by the FADU technique as indicated by Birnboim and Jevcak (1981) with minor modifications. 2.5 x 106 control or treated cells were incubated in the presence of the DNA-synthesis/repair inhibitor arabinosylcytosine (ara-C, Sigma Chimica; 10/zM final concentration). This made it possible to detect the total amount of breaks induced by the treatments. The removal of DNA damages was determined by FADU in parallel PBL samples incubated in the absence of ara-C. The incubation was terminated by cooling on ice. After centrifugation, the cells were suspended in icecold phosphate buffer 10 raM. Following the original procedure (Birnboim and Jevcak, 1981), each PBL suspension was divided equally into 3 sets of tubes: (1) a blank sample (B) in which DNA was completely unwound; (2) a sample (T) used for evaluating total fluorescence of native DNA; (3) a sample (P) used to estimate the unwinding rate in control (C) or treated (UV, MMS, BPDE) PBL. The percentage of ds DNA, D, was estimated by staining with ethidium bromide (0.4/.~g/ml, Sigma Chimica), which selectively binds to ds DNA. The fluorescence was read with an excitation wavelength of 520 nm and an emission wavelength of 590 nm by a RF-5000 Shimadzu spectrofluorimeter. The percentage of ds DNA (D) was determined from the fluorescence of tubes B, T and P by the equation: D = (P- B)/(T-
B) X 100.
From D values A D = D (control c e l l s ) - D (treated cells) can be calculated, which is an arbitrary measure of the amount of DNA strand breaks. AD in the presence of ara-C (AD o) can also be calculated and used as a measure of the percentage of total breaks in the absence of rejoining.
19
A
90.
b •
. . . . .
800
600
200 .
.
2
.
.
I
4
6
10
24
Jim 2
I
I
I
I
6
12
24
48
I1
il mi
I ¢J o
B
,"
90.
b
on"
"9
"
', 70
O0
a
I
I
.
.--
.300
. -
< 5o /
~
30-
a
~
