383
Mutation Research, 51 (1978) 3 8 3 - - 3 9 6
© Elsevier/North-Holland Biomedical Press
INDUCTION OF LONG-LIVED CHROMOSOME DAMAGE, AS MANIFESTED BY SISTER-CHROMATID EXCHANGE, IN LYMPHOCYTES OF ANIMALS EXPOSED TO MITOMYCIN-C
D.G. STETKA, J. MINKLER and A.V. CARRANO Biomedical Division, Lawrence Livermore Laboratory, Livermore, Calif. 94550 (U.S.A.) (Received 22 December 1977} (Revision received 7 March 1978) (Accepted 8 March 1978)
Summary The cytogenetic effects of repeated vs. acute exposure to a chemical mutagen--carcinogen were determined with an in vivo system in which chemicals injected into rabbits induce sister-chromatid exchanges (SCEs). SCE induction can b e monitored when the animal's peripheral l y m p h o c y t e s are cultured in the presence of b r o m o d e o x y u r i d i n e (BrdUrd) and then scored for SCE frequency. M i t o m y c i n ~ (MMC), 0.5 mg/kg, was injected intraperitoneally once a week for 8 weeks. This t r e a t m e n t initially induced small increases in SCE frequency within one day of injection, followed by a return to control levels within 1 week. After the 4th injection, however, the frequency failed to return to normal. After the 5th injection it showed a 4-fold increase over the control which was sustained for the remaining 3 weeks of treatment and for an additional 2 weeks thereafter. The frequency then dropped to twice the control value and remained at this level for more than 4 months. All of the high SCE values after the first 4 weeks were due in part to the appearance and persistence of a population of cells with high SCE frequencies. Exposure to the same total dose given as a single injection resulted in a transient elevation in the SCE frequency and a subsequent return to lower values, with no evidence of a delayed effect such as the increase observed after 4 weeks in repeatedly exposed animals. Overall, repeated exposure is at least as effective as acute exposure in eliciting long-lived SCEs in vivo.
BrdUrd, bromodeoxyuridine; FPG, fluorescence plus Giemsa (technique); HBSS, Hanks' balanced salt solution; MMC, mitomycin-C; MMS, methyl methanesulfonate; PHA, phytohemagglutinln; SCE, sister-chromatid exchange.
Abbreviations:
384 Introduction Cytogenetic damage can be estimated in assays for chromosome aberrations, micronuclei, or heritable translocation, or by observing the frequency of sisterchromatid exchanges (SCEs), which represent exchanges of homologous chromatid segments between sister chromatids. SCEs were first demonstrated in the autoradiographic studies by Taylor [36], and have received considerable attention recently with the advent of new techniques that allow accurate determination of SCE frequencies w i t h o u t using autoradiography [5,17,20,21,39]. Autoradiographic studies showed that the frequency of SCE was increased in cells exposed to a variety of physical and chemical agents including incorporated tritium [6,13], X-rays [11], and ultraviolet light [16,29,40]. With the newer techniques it was shown that alkylating agents [18,22,26,32] and other mutagenic agents [26] could also increase the yield of SCEs markedly. Many of these studies showed that the induction of SCEs is much more sensitive to chemical-mutagen exposure than is the induction of chromosome aberrations; and some, most notably those of Perry and Evans [26], Wolff et al. [41], and Carrano et al. [7], suggested that SCEs represent mutational events, although the latter hypothesis has n o t been strictly tested. In the Perry and Evans study [26], 14 proven or suspected mutagenic chemicals were tested for their ability to induce SCEs in vitro; only those chemicals requiring metabolic activation in order to become mutagenic failed to induce SCEs. Activation in vitro, using the S-9 mix of Ames [ 4 ] , was used by Stetka and Wolff [33] and Natarajan et al. [24] to convert otherwise inactive compounds into metabolic intermediates that are capable of inducing SCEs. Activation b y metabolizing feeder layers was used by Popescu and coworkers [28]. Several in vivo systems that allow detection of SCEs resulting from exposure of animals to chemical mutagens have also been developed [ 2,3,25,30,37,38]. One such system is that devised by Stetka and Wolff [ 3 4 ] , in which chemicals injected into a rabbit induce SCEs that can be observed when the animal's peripheral l y m p h o c y t e s are subsequently cultured in the presence of 5-bromodeoxyuridine (BrdUrd) and then stained with the fluorescence plus Giemsa (FPG) technique [27,39]. In this system, it was shown that chemicals that require metabolic activation, as well as those that do not, produce significant increases in SCE frequency within one day of exposure; the frequency of SCE returned to control level within two weeks. Results obtained by Stetka and Wolff [34] with the in vivo rabbit system suggested that the culture of l y m p h o c y t e s and the detection of SCEs therein might be used as an assay for exposure to chemical mutagen--carcinogens. The transient nature of the observed SCE response, however, indicated that an individual (or his blood) would have to be available for testing within one week of exposure for the test to be conclusive. It remained to be determined if higher doses or repeated exposure might induce more persistent or even permanent increases in SCE frequency. This question is addressed in the present work. In addition, the l y m p h o c y t e response was not characterized (in the earlier work) with respect to either survival or chromosome aberrations. These questions are also considered here. The antibiotic, antineoplastic drug mitomycin C (MMC) was selected for
385
use in the present study because it was already known that it induces SCEs in vitro [7,14,19,22,26], and in vivo [2], that it induces chromosome aberrations in l y m p h o c y t e s of treated humans [31] and in spermatocytes o f treated mice [1], and that it is mutagenic [7,9,23]. Materials and m e t h o d s
Experimental design Male New Zealand rabbits (5 months old at the start of each experiment) were used as the test animals. Blood was drawn from each rabbit prior to any experimental treatment. This blood was analyzed for differential and total l e u k o c y t e counts (see below) and was cultured in the presence of BrdUrd (see below) for subsequent study of SCE frequency and aberration frequency. In this way each rabbit provided his own control. The animals then received intraperitoneal (ip) injections either of MMC (Sigma, St. Louis) dissolved at 2 mg/ml in Hanks balanced salt solution (HBSS), or of HBSS alone (the latter to be henceforth referred to as control animals). Animals were hand-restrained and no anesthetic or any other drug was used. Some received weekly injections (0.5 mg/kg) for 8 weeks while others received only one injection (2 or 4 rag/ kg). In all cases blood was drawn for analysis and culture just before and one day after each injection, and also at various times after the final injection. All animals were weighed once a week.
Ly mphocy te culture and slide preparation 2 ml of blood were obtained b y making a small incision to a marginal ear vein and collecting the drops in a sterile heparinized tube. Approximately 0.2 ml of blood was added to sterile one-ounce prescription bottles containing 5 ml of McCoy's 5A medium with 20% fetal calf serum and penicillin--streptomycin (final concs. 100 units/ml and 100 /~g/ml, respectively). Phytohemagglutinin (0.15 ml, PHA M, Gibco) was also added to each bottle. The pH was adjusted to 6.8--7.0 with 10% CO2 in air. The bottles were then tightly capped and incubated at 38.5 + 0.3°C in a water bath for 20--22 h with no agitation. BrdUrd was then added to a final concentration of 10 -s M (in one experiment, BrdUrd concentrations of 0.5 X, 2 X and 4 X 10 -s were also employed), and cultures were incubated for an additional 30 h. During the last 4 h colcemid was added to the cultures (final conc. 10 -6 M). The cells were collected by centrifugation for 5 min at 1000 rpm (approx. 150 g) and the pellet was resuspended in 4 ml of 0.075 M KC1 h y p o t o n i c solution and allowed to stand for 15 min. Centrifugation was then repeated and the pellet was resuspended in methanol/glacial acetic acid (3 : 1 v/v) fixative. The cells were spun d o w n and resuspended three times in this fixative. After the third centrifugation the pellet was resuspended in 0.4 ml of fixative and drops of cell suspension were placed on microscope slides and allowed to air dry.
Slide staining and scoring Slides were stained with Hoechst 33258 at 5/~g/ml in M/15 Sorensen's buffer, pH 6.8, for 10 min and then rinsed in distilled water. The preparations were m o u n t e d in the same buffer and were exposed to light from a 200-W
386 mercury lamp at a distance of 10 cm for 10--20 min as required for sisterchromatid differentiation. Coverslips were removed and the slides were rinsed in distilled water and then stained for 15--30 min, as required, in 10% Giemsa (Gurr's R 6 6 in M/15 Sorensen's buffer, pH 6.8). After being rinsed in water and air
Mutation Research, 51 (1978) 3 8 3 - - 3 9 6
© Elsevier/North-Holland Biomedical Press
INDUCTION OF LONG-LIVED CHROMOSOME DAMAGE, AS MANIFESTED BY SISTER-CHROMATID EXCHANGE, IN LYMPHOCYTES OF ANIMALS EXPOSED TO MITOMYCIN-C
D.G. STETKA, J. MINKLER and A.V. CARRANO Biomedical Division, Lawrence Livermore Laboratory, Livermore, Calif. 94550 (U.S.A.) (Received 22 December 1977} (Revision received 7 March 1978) (Accepted 8 March 1978)
Summary The cytogenetic effects of repeated vs. acute exposure to a chemical mutagen--carcinogen were determined with an in vivo system in which chemicals injected into rabbits induce sister-chromatid exchanges (SCEs). SCE induction can b e monitored when the animal's peripheral l y m p h o c y t e s are cultured in the presence of b r o m o d e o x y u r i d i n e (BrdUrd) and then scored for SCE frequency. M i t o m y c i n ~ (MMC), 0.5 mg/kg, was injected intraperitoneally once a week for 8 weeks. This t r e a t m e n t initially induced small increases in SCE frequency within one day of injection, followed by a return to control levels within 1 week. After the 4th injection, however, the frequency failed to return to normal. After the 5th injection it showed a 4-fold increase over the control which was sustained for the remaining 3 weeks of treatment and for an additional 2 weeks thereafter. The frequency then dropped to twice the control value and remained at this level for more than 4 months. All of the high SCE values after the first 4 weeks were due in part to the appearance and persistence of a population of cells with high SCE frequencies. Exposure to the same total dose given as a single injection resulted in a transient elevation in the SCE frequency and a subsequent return to lower values, with no evidence of a delayed effect such as the increase observed after 4 weeks in repeatedly exposed animals. Overall, repeated exposure is at least as effective as acute exposure in eliciting long-lived SCEs in vivo.
BrdUrd, bromodeoxyuridine; FPG, fluorescence plus Giemsa (technique); HBSS, Hanks' balanced salt solution; MMC, mitomycin-C; MMS, methyl methanesulfonate; PHA, phytohemagglutinln; SCE, sister-chromatid exchange.
Abbreviations:
384 Introduction Cytogenetic damage can be estimated in assays for chromosome aberrations, micronuclei, or heritable translocation, or by observing the frequency of sisterchromatid exchanges (SCEs), which represent exchanges of homologous chromatid segments between sister chromatids. SCEs were first demonstrated in the autoradiographic studies by Taylor [36], and have received considerable attention recently with the advent of new techniques that allow accurate determination of SCE frequencies w i t h o u t using autoradiography [5,17,20,21,39]. Autoradiographic studies showed that the frequency of SCE was increased in cells exposed to a variety of physical and chemical agents including incorporated tritium [6,13], X-rays [11], and ultraviolet light [16,29,40]. With the newer techniques it was shown that alkylating agents [18,22,26,32] and other mutagenic agents [26] could also increase the yield of SCEs markedly. Many of these studies showed that the induction of SCEs is much more sensitive to chemical-mutagen exposure than is the induction of chromosome aberrations; and some, most notably those of Perry and Evans [26], Wolff et al. [41], and Carrano et al. [7], suggested that SCEs represent mutational events, although the latter hypothesis has n o t been strictly tested. In the Perry and Evans study [26], 14 proven or suspected mutagenic chemicals were tested for their ability to induce SCEs in vitro; only those chemicals requiring metabolic activation in order to become mutagenic failed to induce SCEs. Activation in vitro, using the S-9 mix of Ames [ 4 ] , was used by Stetka and Wolff [33] and Natarajan et al. [24] to convert otherwise inactive compounds into metabolic intermediates that are capable of inducing SCEs. Activation b y metabolizing feeder layers was used by Popescu and coworkers [28]. Several in vivo systems that allow detection of SCEs resulting from exposure of animals to chemical mutagens have also been developed [ 2,3,25,30,37,38]. One such system is that devised by Stetka and Wolff [ 3 4 ] , in which chemicals injected into a rabbit induce SCEs that can be observed when the animal's peripheral l y m p h o c y t e s are subsequently cultured in the presence of 5-bromodeoxyuridine (BrdUrd) and then stained with the fluorescence plus Giemsa (FPG) technique [27,39]. In this system, it was shown that chemicals that require metabolic activation, as well as those that do not, produce significant increases in SCE frequency within one day of exposure; the frequency of SCE returned to control level within two weeks. Results obtained by Stetka and Wolff [34] with the in vivo rabbit system suggested that the culture of l y m p h o c y t e s and the detection of SCEs therein might be used as an assay for exposure to chemical mutagen--carcinogens. The transient nature of the observed SCE response, however, indicated that an individual (or his blood) would have to be available for testing within one week of exposure for the test to be conclusive. It remained to be determined if higher doses or repeated exposure might induce more persistent or even permanent increases in SCE frequency. This question is addressed in the present work. In addition, the l y m p h o c y t e response was not characterized (in the earlier work) with respect to either survival or chromosome aberrations. These questions are also considered here. The antibiotic, antineoplastic drug mitomycin C (MMC) was selected for
385
use in the present study because it was already known that it induces SCEs in vitro [7,14,19,22,26], and in vivo [2], that it induces chromosome aberrations in l y m p h o c y t e s of treated humans [31] and in spermatocytes o f treated mice [1], and that it is mutagenic [7,9,23]. Materials and m e t h o d s
Experimental design Male New Zealand rabbits (5 months old at the start of each experiment) were used as the test animals. Blood was drawn from each rabbit prior to any experimental treatment. This blood was analyzed for differential and total l e u k o c y t e counts (see below) and was cultured in the presence of BrdUrd (see below) for subsequent study of SCE frequency and aberration frequency. In this way each rabbit provided his own control. The animals then received intraperitoneal (ip) injections either of MMC (Sigma, St. Louis) dissolved at 2 mg/ml in Hanks balanced salt solution (HBSS), or of HBSS alone (the latter to be henceforth referred to as control animals). Animals were hand-restrained and no anesthetic or any other drug was used. Some received weekly injections (0.5 mg/kg) for 8 weeks while others received only one injection (2 or 4 rag/ kg). In all cases blood was drawn for analysis and culture just before and one day after each injection, and also at various times after the final injection. All animals were weighed once a week.
Ly mphocy te culture and slide preparation 2 ml of blood were obtained b y making a small incision to a marginal ear vein and collecting the drops in a sterile heparinized tube. Approximately 0.2 ml of blood was added to sterile one-ounce prescription bottles containing 5 ml of McCoy's 5A medium with 20% fetal calf serum and penicillin--streptomycin (final concs. 100 units/ml and 100 /~g/ml, respectively). Phytohemagglutinin (0.15 ml, PHA M, Gibco) was also added to each bottle. The pH was adjusted to 6.8--7.0 with 10% CO2 in air. The bottles were then tightly capped and incubated at 38.5 + 0.3°C in a water bath for 20--22 h with no agitation. BrdUrd was then added to a final concentration of 10 -s M (in one experiment, BrdUrd concentrations of 0.5 X, 2 X and 4 X 10 -s were also employed), and cultures were incubated for an additional 30 h. During the last 4 h colcemid was added to the cultures (final conc. 10 -6 M). The cells were collected by centrifugation for 5 min at 1000 rpm (approx. 150 g) and the pellet was resuspended in 4 ml of 0.075 M KC1 h y p o t o n i c solution and allowed to stand for 15 min. Centrifugation was then repeated and the pellet was resuspended in methanol/glacial acetic acid (3 : 1 v/v) fixative. The cells were spun d o w n and resuspended three times in this fixative. After the third centrifugation the pellet was resuspended in 0.4 ml of fixative and drops of cell suspension were placed on microscope slides and allowed to air dry.
Slide staining and scoring Slides were stained with Hoechst 33258 at 5/~g/ml in M/15 Sorensen's buffer, pH 6.8, for 10 min and then rinsed in distilled water. The preparations were m o u n t e d in the same buffer and were exposed to light from a 200-W
386 mercury lamp at a distance of 10 cm for 10--20 min as required for sisterchromatid differentiation. Coverslips were removed and the slides were rinsed in distilled water and then stained for 15--30 min, as required, in 10% Giemsa (Gurr's R 6 6 in M/15 Sorensen's buffer, pH 6.8). After being rinsed in water and air
