E L S E V I E R Mutation Research 309 (1994) 263-272
Fundamental and Molecular Mechanisms of Mutagenesis
Weak genotoxicity of acrylamide on premeiotic and somatic cells of the mouse
Antonella Russo *, Gigliola Gabbani, Barbara Simoncini Department of Biology, University of Padua, Via Trieste 75, 1-35121 Padua, Italy
Received 10 December; revision received 15 March 1994; accepted 21 April 1994
Abstract
The effects of acrylamide (AA) were evaluated, under the EEC/STEP project 'Detection of Germ Cell Mutagens', by carrying out several cytogenetic assays on mouse germ and somatic cells. The spermatid micronucleus (MN) test was applied after treatment of meiotically dividing or premeiotic S phase cells. Acute treatments (50 and 100 mg/kg i.p.) as well as subchronic exposure to AA (4 x 50 mg/kg, 4 i.p. injections at 24-h intervals) were performed. A weak increase of MN was induced only by treatment with AA of cells in S phase. Sister-chromatid exchange (SCE) analysis in differentiating spermatogonia treated i.p. with 50 and 100 mg/kg confirmed the weak genotoxicity of AA in the premeiotic stages of spermatogenesis. The application of the MN test in peripheral blood reticulocytes of the same animals used for the spermatid MN assay indicated that the cytogenetic effects induced by AA in the somatic and the germ cell lines are comparable in magnitude. The results obtained in this study by applying the spermatid micronucleus assay are in very good agreement with those reported by two other laboratories with the same technique.
Acrylamide (AA) is an organic molecule widely used in the production of polymers and copoly- mers. Apar t f rom the large industrial use of AA, this compound has recently become extensively used in research laboratories as a component of electrophoresis gels.
This paper is dedicated to the memory of Prof. F.H. Sobels.
* Corresponding author.
The biological consequences of the exposure to A A have been thoroughly reviewed by Dearfield et al. (1988) a few years ago, and a number of new data were put forward more re- cently (Adler et al., 1988, 1993; Cih~ik and Von- torkov~i, 1988; Knaap et al., 1988; Backer et al., 1989; Neuh~iuser-Klaus and Schmahl, 1989; Sega et al., 1989; Adler, 1990; Russell et al., 1991; Collins et al., 1992; Ehling and Neuh~iuser-Klaus, 1992; Tsuda et al., 1993). A A may be considered a clastogenic agent: it showed positive chromoso- mal effects when tested in diverse cytogenetic assays in vivo and in vitro, although some contra- dictory results were obtained (Dearfield et al.,
264 A. Russo et al. / Mutation Research 309 (1994) 263-272
1988). No gene mutations are produced by AA in many in vitro assays on prokaryotic or mam- malian cells (Dearfield et al., 1988; Tsuda et al., 1993), but AA is clearly mutagenic in the in vivo mammalian spot test (Neuh~iuser-Klaus and Schmahl, 1989) and in the specific-locus test (Neuh~iuser-Klaus and Schmahl, 1989). Available carcinogenicity data indicate AA to be a 'prob- able human carcinogen' (Dearfield et al., 1988).
As demonstrated by pharmacokinetic investi- gations in different mammalian species, the ab- sorption and metabolism of AA is rapid, and the compound is distributed equally in different tis- sues (Dearfield et al., 1988). AA belongs to the category of alkylating agents, and its ability to bind DNA has been demonstrated in vivo (Carl- son and Weaver, 1985) and in vitro (Solomon et al., 1985). In the late stages of spermiogenesis, the alkylation detectable after AA exposure af- fects specifically the protamine fraction (Sega et al., 1989).
Cytogenetic data concerning the effects of AA on somatic mammalian cells in vivo are scanty and contradictory. Recent reports demonstrate the induction of micronuclei (MN) by AA in the bone marrow of the mouse (Adler et al., 1988; Cihak and Vontorkovfi 1988; Knaap et al., 1988), while in previous investigations negative results have been obtained with the same assay (re- viewed by Dearfield et al., 1988). The induction of chromosomal aberrations in mouse bone mar- row is also reported by Adler et al. (1988) and by Cihak and Vontorkov~i (1988). A remarkable in- duction of MN has also been observed in mouse splenocytes (Backer et al., 1989). In the same study, parallel spleen cultures showed only a weak increase of SCE, while no chromosomal aberra- tions were observed.
As far as germ ceils are concerned, AA ap- pears particularly powerful as a mutagen in late stages of spermiogenesis. Dominant lethals and heritable translocations are specifically induced in late spermatids and early spermatozoa of the mouse (Shelby et al., 1986, 1987; Adler, 1990) and the rat (Smith et al., 1986). The sensitivity of these cell stages has also been clearly demon- strated in two studies reporting data on the spe- cific-locus test (Russell et al., 1991; Ehling and
Neuh~iuser-Klaus, 1992). Other studies report the lack of induction of chromosomal aberrations in mouse spermatogonia (Smith et al., 1986; Adler et al., 1988; Backer et al., 1989), while a positive effect had been detected in the past (Shiraishi, 1978). Reciprocal translocations observed in pri- mary spermatocytes indicate that AA may have a weak effect during the meiotic prophase (Adler, 1990). In the same study no translocations were found after treatment of stem or differentiating spermatogonia, confirming the results obtained from direct chromosome aberration assays in spermatogonia (see above). However, an increase of gene mutations after exposure of stem sper- matogonia has been detected with the specific- locus test (Ehling and Neuh~iuser-Klaus, 1992). Other visible effects in spermatocytes were dam- age to the synaptonemal complex and asynapsis (Backer et al., 1989). Finally, the induction of MN has been reported after treatment of lep- totene-zygotene cells, with a prevalence of kine- tochore-carrying MN (Collins et al., 1992). Early evidence (Shiraishi, 1978) of induction of aneu- ploidy by AA was considered controversial, how- ever more recently disturbance of cell division by acrylamide was demonstrated in mammalian cells in vitro and in vivo (Adler et al., 1993).
AA was selected for evaluation under the E E C / S T E P project 'Detection of Germ Cell Mu- tagens' because of its widespread production and use. Several data gaps were perceived in the data base for this compound and the data reported here and in other manuscripts in this series were gathered to fill in the missing data. Another major aim of the experiments was to standardize a protocol for the conduct of the spermatid MN test.
In this paper, the effects of AA on mouse germ and somatic cells are presented. In particu- lar, the following endpoints are considered: (1) MN frequency in early spermatids, after
treatment of meiotically dividing cells (dia- k ines i s /MI /MII ) or after treatment of pre- meiotic S phase (preleptotene).
(2) Sister-chromatid exchanges (SCE) in differen- tiating spermatogonia.
(3) MN frequency in blood peripheral reticulo- cytes. This assay (Hayashi et al., 1990) detects
A. Russo et al. / Mutation Research 309 (1994) 263-272 265
chromosome damage induced during the last cell divisions of bone marrow differentiating erythroblasts. It offers the possibility of using the same animals employed for the spermatid MN test, thus permitting a direct comparison of treatment effects on germ and somatic cells in each animal.
2. Materials and methods
Animals and chemicals All the experiments were carried out on male
BALB/c mice, 2-4 months of age. The animals were obtained from Charles River Italy, and housed for at least 1 week under the new condi- tions (21-23°C, 40-45% humidity, 12-h dark/light cycle) before being treated.
Acrylamide (CAS No. 79-06-1), distributed by the coordinator of the project (Dr. I.-D. Adler, Neuherberg, Germany) was from Sigma, St. Louis, MO, USA. Acridine orange, collagenase (type I), and colchicine (CAS No. 64-86-8) were also from Sigma. Mitomycin C (CAS No. 50-07-7) was ob- tained from Kiowa Hakko Kogyo (Tokyo, Japan).
Spermatid micronucleus assay Evaluation of the induction of MN by AA was
carried out at 2, 14 and 16 days after i.p. treat- ment. This sampling regimen ensured the exami- nation of cells which were meiotic (2 days) or involved in the last premeiotic S phase (14 and 16 days) at the time of treatment. Acute treatments were carried out at 50 or 100 mg/kg b.w. at the three time intervals. The effects of a repeated treatment with 50 mg/kg b.w. (4 times at 24-h intervals) were assayed 16 days after the first AA injection. AA was dissolved in Hanks' balanced salt solution (HBSS) immediately before treat- ments. The working concentrations were chosen according to the AA dose to be tested, so that mice received 0.01 ml /g b.w. At least four mice were treated with each experimental condition tested. Matched negative controls were injected with HBSS. Replicate experiments were per- formed. Germ cell preparations were obtained according to a protocol developed by Tates et al. (1983), but the enzymatic digestion was carried
out only with collagenase (20 min, 33°C), omitting both trypsin and DNAase treatments. Periodic Acid Schiff (PAS) reaction followed by counter- staining with Mayer's Haemalum allowed obser- vation of the developing acrosome in early sper- matids. Spermatids were classified as belonging to the Golgi or the Cap phase according to the acrosome morphology (see also Oakberg, 1956). Golgi phase corresponds to the first developmen- tal step of spermiogenesis, and its average dura- tion is 49 h (Oakberg, 1956). The subsequent stage of spermiogenesis (Cap phase) lasts on av- erage 68.6 h (Oakberg, 1956). To evaluate the MN frequency, 1000 or 2000 Golgi phase sper- matids were scored per animal, and the number of Cap phase spermatids was recorded at the same time. MN frequencies were calculated inde- pendently for the two cell phases. MN were clas- sified as large, medium or small on the basis of their diameter (small: O < 1 /~m; medium: 1 /xm < O < 2 /zm; large: • > 2 /xm), as measured using a micrometer eyepiece. Statistical compar- isons among MN proportions in control and treated groups and among the different size dis- tributions observed were done by applying the G-test (Sokal and Rohlf, 1981). This test is based on the same general assumptions of the chi-square analysis, offering however both theoretical and computational advantages. The ratio between Golgi and Cap phase spermatids was calculated, and deviations from the control value were as- sessed using Student's t-test after square root transformation.
Sister-chromatid exchanges in spermatogonia To detect SCE in differentiating spermatogo-
nia, mice were implanted s.c. with two tablets of 5-bromodeoxyuridine (BrdU; Boehringer Mann- heim, Germany), weighing 25 mg each, and agar- coated to assure a slow release of BrdU. Treat- ments were carried out 1 h after the implantation of the tablets, and the animals were killed 55 h later. AA was diluted as reported above, and tested at 50 and 100 mg/kg b.w. Four mice per dose level and four matched controls (HBSS) were used, in the course of two replicated experi- ments. Mice were exposed to colchicine (0.5% solution, 0.3 ml per animal) 3 h before sacrifice.
266 A. Russo et a l . / Mutation Research 309 (1994) 263-272
Spermatogonia metaphases were prepared as de- scribed elsewhere (Russo and Levis, 1992a). Fluo- rescence plus Giemsa staining (FPG; Perry and Wolff, 1984) was carried out on 24 h old slides, and SCE were evaluated on 25 well-differentiated metaphases per animal. SCE per cell values were then transformed as square roots, and compar- isons between control and treated groups done using the t-test. To describe the dose-response relationship, regression analysis was done by con- sidering the transformed SCE per cell values detected in each animal, and the natural loga- rithm of the dose.
MN assay in peripheral blood reticulocytes Peripheral blood reticulocytes may be isolated
from the caudal vein of living animals, and used for a MN assay, the recommended times of up- take being 1-3 days after treatment (Hayashi et al., 1990). A subsample of mice belonging to the 14-day experimental group of the spermatid MN assay (three mice for 50 mg/kg b.w., two mice for 100 mg/kg b.w.) was used, therefore, to monitor the effects induced by AA on somatic cells (dif- ferentiating erythroblasts) of the same animals tested for germ cell effects. Slides were prepared and scored as already described (Russo et al., 1993). Briefly, reticulocytes were collected at 0 (internal control), 1, 2 and 3 days after treatment
with AA, and supravitally stained with acridine orange. 2000 reticulocytes per animal were scored, and the frequency of MN was calculated on those cells belonging to the I-I I I developmental stages only. Statistical comparisons were done using the G-test (Sokal and Rohlf, 1981).
Positive controls Matched positive controls were made for the
spermatid micronucleus assay by treating mice with mitomycin C (MMC) at a dose of 1 mg/kg i.p., and sampling spermatids at 14, 15 and 16 days after treatment. Other data on MMC ob- tained in the course of parallel experimentation in the same laboratory were used as positive controls for SCE analysis in spermatogonia (Russo et al., 1994) and MN assay in peripheral blood reticulocytes (Russo et al., 1993).
3. Results
Table 1 summarizes the results obtained by testing AA effects with the spermatid MN assay. The table gives the number of animal treated per condition tested, the total number of Golgi and Cap phase spermatids scored, and the numbers and percentages of micronucleated spermatids. The last column of the table shows the ratio
Table 1 MN induction by AA in mouse germ cells, as evaluated in early spermatids
Treatment Number Total number Golgi phase of mice of Golgi phase
spermatids
Controls (HBSS) 6 8 100
AA, 50 mg/kg 2 days 5 5 022 AA, 50 mg/kg 14 days 4 8101 AA, 50 mg/kg 16 days 4 8070
AA, 100 mg/kg 2 days 5 5 073 AA, 100 mg/kg 14 days 4 8 181 AA, 100 mg/kg 16 days 4 7966
AA, 4 × 50 mg/kg 4 8 076
MMC, 1 mg/kg 14 days 2 6053 MMC, 1 mg/kg 15 days 2 6068 MMC, 1 mg/kg 16 days 2 5982
Total number Cap phase Golgi/Cap spermatids with of Cap phase spermatids with phase ratio MN (%c ± SE) spermatids MN (%o ± SE) (mean _+ SE)
A. Russo et al. / Mutation Research 309 (1994) 263-272 267
observed between the Golgi and Cap phase sper- matids. Since Golgi and Cap phases represent two subsequent developmental cell stages, occur- ring immediately after meiotic division, induction of MN should be detected first in Golgi phase spermatids. Cytotoxic effects of the treatment, as well as meiotic delay/arres t could modify the Golg i /Cap phase ratio compared to the control value. In this experimental series, the baseline MN frequency in Golgi phase spermatids was 2.47 _+ 0.55%o. AA did not induce any increase of MN Golgi phase spermatids at any treatment condition tested, even though a borderline differ- ence between treated and control animals was observed at 100 mg/kg, at 16 days (0.10 < P < 0.05). In the Cap phase spermatids, the baseline MN proportion was 0 .95_ 0.36%o, and signifi- cant increases compared to this value were found after treatment with 50 m g / k g b.w. at 14 and 16 days ( P < 0.05), after treatment with 100 mg /kg b.w. at 16 days ( P < 0.05), and after the repeated treatment protocol (4 x 50 mg /kg at 24-h inter- vals; P < 0.001). MMC (1 mg/kg), tested as posi- tive control, produced a highly significant in- crease of MN in both spermatid developmental stages, and at all the time intervals assayed (14, 15 and 16 days).
The ratio between Golgi and Cap phase sper- matids appeared variable among different treat- ment conditions, but no statistical differences from the control value (1.08 _+ 0.04) were as- sessed, apart from the ratio observed 16 days after treatment with 100 mg A A / k g b.w. (P < 0.05).
Different size distributions of MN were found in control and treated animals. Fig. 1 summarizes the results obtained by pooling measurement data carried out in Golgi and Cap phase spermatids, and in animals killed 14-16 days after treatment. Data relating to the different dose levels were taken separately. In animals treated with AA or MMC, an excess of small sized MN was found compared to the controls. Statistical analysis demonstrated that the MN size distribution ob- served after exposure to AA at 100 mg /kg b.w. was significantly different ( P < 0.05) from the distribution observed in untreated animals. A highly significant difference (P < 0.005) was also
100 % 90 I ~ srnell MN
I m e d i u m MN 80 k~ lorge MN
7O
60 27 88
5O
4O
0
70 4,} 261
contr 50 100 4x50 MMC mg/kg mg/kg mg/kg
Fig. 1. Size distribution of MN observed 14-16 days after treatment with AA or MMC. Total numbers of MN scored are reported. The results of statistical comparisons are given in the text.
found when the distributions obtained from mice treated with 4 x 50 m g /k g and the control group were compared. Although not significantly differ- ent from the controls, the distribution found after treatment with 50 m g /k g b.w. also showed a prevalence of small MN, with borderline statisti- cal significance (0.10 < P < 0.05). MN induced by MMC showed a size distribution highly compara- ble to that produced by AA, and significantly different from the controls (P < 0.001).
Table 2 gives the number of second generation metaphases scored and the mean values of SCE per cell detected in mouse spermatogonia after treatment with two dose levels of AA. Significant increases (P < 0.001) were found at both doses
Table 2 SCE induction by AA in differentiating spermatogonia
Treatment Number of SCE per cell cells scored (mean + SE)
Four mice were used for each experimental group. *** P < 0.001 (Student's t-test).
268
Table 3 Effects of AA in the somatic assay
A. Russo et al. / Mutation Research 309 (1994) 263-272
cell line of the same animals used for the spermatid MN assay: peripheral blood reticulocyte MN
Trea tment Number Reticulocytes 1-I l l type Reticulocytes of mice scored reciculocytes with MN, N (%o)
- , day 0 3 6 154 4592 11 (2.4) 50 mg /kg , day 1 3 6 348 4 840 24 (5.0) * 50 m g / k g , day 2 3 6328 4834 28 (5.8) ** 50 m g / k g , day 3 3 6 304 5 005 23 (4.6)
- , day 0 2 4332 3 396 16 (4.7) 100 mg /kg , day 1 2 4 177 3 280 16 (4.9) 100 m g / k g , day 2 2 4 256 3 341 29 (8.7) * 100 mg /kg , day 3 2 4 060 2 986 8 (2.7)
Per dose the same animals were used from day 0 to day 3. * P < 0.05; ** P < 0.01.
tested. The effect induced by AA increased lin- early with the natural logarithm of the adminis- tered dose (b = 0.182 + 0.011; P < 0.05).
Table 3 shows the results obtained with the MN test in peripheral blood reticulocytes on a subset of animals belonging to the 14-day treat- ment group of the spermatid MN assay. At 50 mg/kg, a significant increase of MN was found at 1 (P < 0.05) and 2 days (P < 0.01), the peak fre- quency corresponding to 2.4 times the sponta- neous MN proportion. At the dose of 100 mg/kg b.w. a similar pattern of induction of MN was found, with a significant difference at 2 days after treatment (P < 0.05, 1.8 times the frequency ob- served in the same mice before treatment).
A summary of the individual response of five animals to AA in the somatic and germ cell MN test is given in Table 4. In particular, the follow- ing data are shown: the MN frequencies observed
in blood reticulocytes before and after treatment (at the peak time), and the MN frequencies ob- served in Golgi and in Cap phase spermatids. Although the MN frequencies in reticulocytes are variable among animals, the effects induced by the treatment are very similar in magnitude when compared to the individual baseline.
4. Discussion
Acrylamide was tested in the mouse by study- ing several cytogenetic endpoints in the germ and somatic cell lines. In the cell types investigated here, AA appeared as a weak mutagen, whose maximum effect, at the doses tested (50-100 mg/kg), was about 2.5 times the baseline in both germ and somatic cells.
The control MN frequency observed in this
Table 4 A summary of the cytogenetic effects of AA, as evaluated in five mice by applying the MN assay in peripheral blood reticulocytes,
and the spermatid MN test
Animal Dose tested MN baseline MN MN MN number in blood observed in observed in observed in
reticulocytes reticulocytes Golgi phase Cap phase (%c) after 2 d (%o) spermatids (%0) spermatids (%~)
1 50 m g / k g 2.6 6.1 1.5 1.8 2 50 m g / k g 0.7 4.8 3.0 2.1 3 50 m g / k g 3.8 6.5 4.0 2.4 4 100 m g / k g 3.1 6.8 2.9 0 5 100 m g / k g 6.1 10.5 1.5 2.4
Frequencies of MN in spermatids were detected 14 days after t reatment , and correspond to t reatment of premeiotic S phase.
A. Russo et al. / Mutation Research 309 (1994) 263-272 269
study in early spermatids was higher than previ- ously reported (Russo and Levis, 1992a,b). Simi- lar variations of the MN frequencies in untreated animals were also found by other authors (Collins et al., 1992). Because of the high baseline of MN observed during the experiments reported here, small increases of the MN frequency may not have been detected with the statistical analysis employed. Indeed, the only positive effects of AA in the spermatid MN assay were demonstrated by the data relating to Cap phase spermatids, where the control MN frequency was lower than 1 MN spermatid per 1000 cells.
In this study, AA was effective in inducing MN only when the time interval between treatment and sacrifice permitted sampling of cells treated in the preleptotene (S phase) stage. At the same time interval (14-16 days) a powerful increase of MN was observed after treatment with the posi- tive control MMC. In contrast, no increase of MN was detected 2 days after treatment, when germ cells were treated during diakinesis/MI/ MII. The pattern described for AA should be typical of an S-dependent clastogenic compound, with no influence on chromosome segregation events. A similar cell stage specific pattern was previously reported when the effects of MMC were evaluated, whereas the radiomimetic com- pound adriamycin induced MN at either time interval (Russo and Levis, 1992a,b).
The MN size distributions observed after treatment with AA (Fig. 1) support the hypothe- sis of a clastogenic effect of the compound, since most of the induced MN were classified as small. These distributions deviated significantly from that found in the control group. Overlapping distributions were described in addition after treatment with AA and MMC, in spite of the different efficiencies of the two compounds in inducing chromosomal damage. These data indi- cate that, although the classification of MN on the basis of their size was done by a rough estimation of the diameter, the approach may help in understanding the mechanism of action of tested chemicals.
Although the data presented in this paper are in agreement with a clastogenic mechanism of action of AA, Collins et al. (1992) suggested a
possible aneugenic effect of AA on the basis of a preferential induction of kinetochore-carrying (CREST +) MN in mouse spermatids treated dur- ing leptotene-zygotene. However, these authors did not test any cell stage engaged with synthesis of DNA, so that the possible clastogenic proper- ties of AA were not detectable. In fact, by apply- ing CREST immunostaining in mouse sper- matids, we found that the majority of MN in- duced by AA after subchronic treatment do not carry the kinetochore (unpublished data).
A major reason for the conduct of the studies reported here was to compare the results of this technique with those of other laboratories partici- pating in the STEP project 'Detection of Germ Cell Mutagens'. Two species (mouse and rat), as well as two different approaches for spermatid isolation and analysis (the 'dissection method' developed by L~ihdetie and Parvinen, 1981, and the 'suspension method', developed by Tates et al., 1983) were tested. Very good agreement was obtained among three laboratories, all of them indicating the weak and S-dependent clastogenic action of AA (L~ihdetie et al., 1994; Xiao and Tates, 1994; present paper).
As demonstrated by Tates (1992) in the rat, the frequencies of MN may vary slightly with respect to the time interval and the cell popula- tion actually scored. Thus, in this study, different time intervals (14 and 16 days) and treatment conditions (acute and subchronic exposure) were tested to verify if AA may induce chromosomal damage during the premeiotic S phase. Also, matched positive controls included treatments with MMC (1 mg/kg b.w.) at different times (14, 15 and 16 days) before sacrifice, while previous data on the effects of this compound had been obtained at the 14-day interval only (Russo and Levis, 1992b). The induction of MN by AA was demonstrated only in Cap phase spermatids but not in the Golgi phase, while after treatment with the positive control MMC, increases of MN were detected in Golgi and Cap phase spermatids at all the time intervals tested. The temporal pat- tern observed in the case of MMC showed a prevalence of MN in the Golgi phase at the 14- and 16-day intervals, but at the intermediate one (15 days) MN were mostly visible in the Cap
270 A. Russo et al. / Mutation Research 309 (1994) 263-272
phase. A certain degree of interindividual vari- ability was observed in addition when AA and MMC were tested on premeiotic cells (although the sample size was doubled compared to the 1000 Golgi phase spermatids per mouse initially scored). At the 2-day time interval the variability among animals was less pronounced.
Several factors, such as induced cell cycle de- lay, may influence the precise pattern of damage observed in in vivo systems such as described here. First, each compound may alter to a differ- ent extent the cell cycle duration in spermatogo- nia. As an example, it is known from a previous study (Russo et al., 1994) that MMC induces a pronounced delay/ar res t , as detected by calculat- ing the average generation time (31.03 h vs. 28.66 h observed in untreated animals). However, in this study AA did not appear to influence the duration of the cell cycle as strongly as MMC, with an average difference of only 1 h between treated and control animals (data not shown). This fact may be critical when chromosomal er- rors are detected at long time intervals after treatment, as necessary in the spermatid MN assay. The fact that differentiating spermatogonia are characterized by a very long cell cycle (in particular: 29.5 h for type B spermatogonia, the average duration of the S phase being 14.5 h; Monesi, 1962) may in addition have an influence when chemicals characterized by different fea- tures (metabolism, distribution, etc.) are tested. Interestingly, repeated t reatment with AA (4 x 50 m g / k g ) appeared to be a useful tool for detecting the weak effects of this compound, possibly be- cause this schedule increased the probability for a single cell to be damaged by AA during the S phase.
Finally, the appearance of AA- and MMC-in- duced MN in the Cap phase spermatids 14 days after t reatment suggests that at a time interval slightly shorter than the one tested here a peak MN frequency would possibly have been found in the earlier cell stage (Golgi phase spermatids). This would be of importance when the mutagenic potential of chemicals with a very fast diffusion in the testes and an immediate effect is under study.
On the basis of the above considerations, when the mouse spermatid MN assay is used to evalu-
ate the chromosomal effects of a test compound, at least two time intervals in the range between 13.5 and 16 days should be used. In addition, both Golgi and Cap phase spermatids should be scored.
SCE analysis in differentiating spermatogonia indicated that at the maximum AA dose tested (100 m g / k g b.w.) only a 2.5-fold increase was obtained compared to the baseline. At the same time interval tested for AA (55 h), MMC induced an increase of SCE equal to 3.4 times the base- line, after t reatment with 1 m g / k g (Russo et al., 1994). This result confirms the weak genotoxicity of AA in the premeiotic stages of spermatogene- sis. The regression analysis indicated in addition that the increase of SCE induced by AA is not a linear function of the injected dose.
The application of the peripheral blood reticu- locyte MN assay gave the opportunity to evaluate in parallel, in the same individuals, the response of the somatic and germ cell lines to the same AA treatment. This assay can sensitively monitor the temporal pattern of induction of MN after treatment, since sequential blood samples may be obtained from the same animal at different times. One of the times can be before treatment, thus allowing a determination of the spontaneous MN proportion in each mouse. In this study, we car- ried out a pilot experiment with a subset of animals belonging to the 14-day t reatment groups for the spermatid MN assay. AA was demon- strated to be a weak genotoxic agent in the so- matic cell line as well, and the two tested doses gave very similar increases of MN frequency com- pared to control values (2.4 at 50 m g / k g and 1.9 at 100 mg/kg) . Recent data obtained by testing MMC (1 and 2 mg /kg ) with the same assay indicated an increase of MN much higher than 10 times the corresponding baseline (Russo et al., 1993).
The data presented here further confirm the previous reports of MN induction by AA in mouse bone marrow (Adler et al., 1988; Cihfik and Von- torkovfi 1988; Knaap et al., 1988). The parallel application of the spermatid MN assay and the MN assay in reticulocytes will aid in giving the greatest amount of information with a minimum number of mice. Table 4 gives the individual data
A. Russo et al. / Mutation Research 309 (1994) 263-272 271
collected in the course of the two assays: al- though the small number of individuals analyzed here does not allow any fine comparison among somatic and germ cell data, this table gives an example of the potential of a combined analysis in germ and somatic cells of the same individual.
In conclusion, AA was confirmed as a geno- toxic agent in the germ cell line of the mouse, as well as in the somatic ceils, but its effects on premeiotic cells are weak compared to those in- duced in late spermiogenic stages. Although AA is able to bind DNA (Solomon et al., 1985; Carl- son and Weaver, 1985), it appears to form with high specificity adducts to protamines in spermio- genic cell stages (Sega at al., 1989). This mecha- nism may explain the different responses ob- served among diverse cell stages. The present results indicate, however, that mouse chromo- somes are weakly sensitive to the effects of AA, also during cell stages other than late spermio- genesis. The pattern of effects observed in the present study indicates that AA elicits clastogenic damage, while no evidence was obtained about the possible aneugenic potential of this chemical. However, since proteins seem to represent the primary cell target of AA, other data should be collected to clarify this point.
Acknowledgements
This work was partially supported by ENEA (Contract No. 2805 3/8/92) within the EEC/ STEP project 'Detection of Germ Cell Mutagens' (contract No. STEP-91-0144)
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