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Toxicity and chromosomal damage in fetal Syrian hamster and human pulmonary epithelial cells
41(19&3)37-48 Scientificpublishers Ireland Ltd.
CancerLettera, Fhvier
TOXICITY HAMSTER
27
AND CIIROMOSOMAL DAMAGE IN FETAL SYRIAN AND HUMAN PULMONARY EPITHELIAL CELLS
J. WEN*. M. EMURA, M. RIRBE and U. MOHR In#itutfdr Expesimentelle Patho&@, Str. 8,3t?OOHuwwver 61 f’F.R.G.j
Medieinieche Hochrchule Hannover, Konatanty-Gutschow
(Received 2 Januuy 1988) (Accepted 14 March 1988)
SUMMARY
In order to test the hypothesis that human chromosomes are more stable than those of rodents, we compared the fetal human and Syrian hamster pulmonary epithelial cell lines for their sensitivity to the induction of cytotoxicity (CT), chromosomal aberrations (CAsl and sisterchromatid exchanges WEE). One day after plating, the cells were exposed for 2 h to various doses of ENU dissolved in an exposure medium consisting of McIlvaine buffer, RPM1 1640, and bovine serum albumin. CAs and SCEs were examined‘in 24 -64 post-exposure hours by the standard methods, while CT was determined by cell counts. The frequency of CAs (particularly ehromatid exchanges) and that of SCEs showed clear dose dependency and was remarkably higher in the hamster than in the human cells. CT determined in 1 week of post-exposure incubation showed a similar dose response for both cell lines with sharply declining regressions. These results suggest that human chromosomes are indeed more resistant to ENU-inducible aberrations than hamster chromosomes and that CT may not always be directly asso ciated with chromosomal damage. Key words: Chromosome; Damage; Hamster: Human
INTRODUCTION
An important aspect of carcinogenesis research is the extrapolation of laboratory animal data to the human situation. A rapid improvement in cell culture techniques has enabled us to make more exact comparison of the Cowerpondrnce to: M. Emura. lPrerewt ad&eta: Institute of P6thoIogy. Sun Yat-Sen Univemity of Medical Sciences, Zhongshau Roul2. Gnmgxhou. The People’6 Republic of Cbim. 0 1988 Ekwier O~&SO Published 6nd Prinbd in Ireimd.
Scientific Publishers Irelmd Ltd.
response to carcinogenic agents of animal and human cells, including epithelial cells [8] from which most tumors originate. Past experience has shown that human cells are more difficult to transform using chemical carcinogens than rodent cells [3]. A tentative explanation could be the assumption that human chromosomes are more stable than those of rodents. In an effort to test this hypothesis, we compared the fetal human and Syrian hamster pulmonary epithelial cells for their sensitivity to the induction of cytotoxicity (CT), chromosoma1 aberrations (CAs) and sisterchromatid exchanges (SCEsl by short-term exposure to ethylnitrosourea (ENU). Pulmonary cells were used because we were interested in the mechanisms of lung carcinogenesis. MATERIALS AND METHODS
The human epithelial cell line (diploid, 23 h PDT) was raised from a piece of bronchus taken from an 1Sweekold fetus, as described previously [4]. The Syrian hamster epithelial cell line (pseudodiploid, 11 h PDT), was derived from the lung of a fetus on day 16 of gestation [5]. Both cell lines were grown in a growth medium (GM) consisting of RPM1 1640 (80%, Flow, Meckenheim), fetal bovine serum (20%; Flow), etc. as described previously [S]. Cultures were maintained at 37 OCin a humidified atmosphere of 10% CO, and 20% air. Autoradiographic examination with 5 pCi/ml (spec. act. 26 Ci/mmol; 30 min incubation1 of [sHjthymidine (Amersham, Braunschweigl has shown that the DNA synthesizing population of the Syrian hamster (SH) cell line comes into a steady state (at 60%) approx. 12 h after cell plating (10’ cells in &cm2 dish with GM), while in the human (Hl cell line this first occurs after 24 h (Fig. 11. Therefore, ENU treatment was performed at 24 h after cell plating, lest the different incidence of S-phase cells before 24 h should influence the extent of toxicity and chromosomal damage in the 2 cell types.
Fig. 1. Rate of DNA synthesizing (S-phase) population in fetal Syrian hamster (SH) and human (H) epithelial cells after plating. Cells (10’) were plated in &me dishes containing GM, and at 3-h intervals they were incubated with PH]thymidine for 30 min. Each point indicates the average from 3 dishes. in each of which 100 autoradiographed cella were scored. Fig. 2. Spontaneous decay of ENU in various sorta of solution with different pH at 37°C. m, McIlvaine buffer (pH 8.0); 0, McIlvaine buffer (pH 7.2); 0, exposure medium (EM) (pH 6.0).
ENU exposure Because of the remarkable reduction of chemical stability of ENU around neutral pH and in the presence of serum [12,16] a special exposure medium (EM) was developed consisting of 2 parts of 0.01 M McIlvaine buffer (pH 6.0) with 0.9% NaCl, 1 part of RPM1 1640, 0.1% bovine serum albumin (fraction V; Sigma, Munich) and 110 &ml sodium pyruvate, and the ENU was dissolved in this EM immediately prior to use. In the EM (pH 6.01the spontaneous decay of ENU was greatly reduced with about 35% retention of native-ENU in 2 h (Fig. 21,while at pH 7.2 less than 3% remained undecayed in 1 h. Exposure to ENU at the doses prescribed for each experiment was carried out for 2 h at 37 “C 24 h after plating the cells (cell no. depending on the experiment) in 25-cm2 flasks (Falcon).
Cytotoxicity To estimate the cytotoxicity by cell counts, 2 x 10” cells/flask were plated in 5 ml GM. ENU doses examined were 0, 0.1, 0.2, 0.4, 0.8 and 1.6 mg/ml, and to each dose 3 flasks were allotted. After removal of ENU the cultures were washed once with GM and further incubated in GM until the the unexposed control cultures had reached 9OW of confluency (for SH and H cells 7 and 9 days, respectively). The medium was changed twice a week. After trypsinization, viable cells were counted by the trypan blue exclusion method.
Chromosomal aberrations At 24 h after plating the cells were exposed to various doses of ENU (Fig. 41. After washing once with GM they were incubated in GM for 2,4,6 and 8 days. Chromosomes were prepared by the conventional air-dry techniques after incubation with 0.3 pg/ml of Demecolcine (Sigma, Munich) for 3 h. After Giemsa (4%) staining, chromosomal changes in well-spread metaphases were classified according to Savage [18]. Ring chromosomes, dicentrics and chromatid exchanges were considered to be caused by 2 breaks in 1 or 2 chromosomes, and deletions and minute chromosomes by 1 break, with gaps being excluded regardless of the width [l,ll]. On this basis the numbers of breaks were counted on the above-prescribed days and later added together to normalize the break counts per lo4 chromosomes.
Sister-chromatid exchange Cells (2 - 5 x lo6 per flask) were plated. After ENU exposure (for the doses see Fig. 51,further incubation with 10 ccglmlbromodeoxyuridine in GM was carried out at 24 and 48 h for SH and H cells, respectively, in the first experiment (lower dose range), and 36 and 64 h for SH and H cells, respectively, in the second experiment (higher dose range), during which period 2 rounds of replication were expected to occur. Chromosomes were prepared as for the chromosomal aberration examination and stained routinely [19] with slight modifications. The extent of SCE was expressed as average numbers per chromosome with the control values subtracted from SCE frequencies scored in at least 50 metaphases [21].
0.162
0.4
0.6 E NU
1.6
hp/ml)
Fig. 3. Dose-dependent toxicity of ENU in SH and Ii cells as measured by counting living cells whose numbers at various doses were related in percentage to tbe plated cell number (z x 10% cm% Each point indicates the mean of 9 flashs with SD.
RESULTS
For both SH and H cells the survival potential decreased dose-dependently and there were no remarkable differences between them (Fig. 3). Only at higher doses, H cells appeared to be a little more resistant to ENU than SH cells. Linear regression analysis based on the semilogarithmic plotting indicated the
9
ENU
Cmo/ml)
Exp I
;
Exp II
.. 1
ENU
(mg/ml)
Fig. 4. Dose-dependent incidence of chromatid breaks in SH and Ii cells. For the details of measurement see Materials and Methods. The double points indicate a statistically significant difference with P < 0.001 (x+tesU between SH and H. Fig. 6. Dose-dependent incidence of sister-cbromatid exchanges in SH and H cells. For the detailed measurement see Materials and Methods. Single and double points indicate a statistically significant difference with P < 0.05 and P < 0.001 (XI_test).respectively, between SH and H. Bars indicate S.D.
41
mean lethal doses of ENU to be 162 and 180 &ml for SH and H cells, respectively. By contrast, the chromosome breaks occurred far more frequently in SH cells than in H cells, showing clear dose dependencies for both (Fig. 41. The response of H cells at 0.8 mg/ml was very sharp as compared to that at lower doses. The major chromosomal alterations were of a chromatid type such as exchanges including triradials and quadriradials. The alterations of a chromosoma1 type, such as dicentrics, minutes or rings, were also significantly more frequent in SH than in H cells (data not shown). The gaps, however, showed slightly higher frequencies in H than in SH cells, although the overall rates were at any rate very low. SCE also demonstrated a significantly higher occurrence in SH cells than in H cells, particularly so in the higher dose range (experiment 2; Fig. 51. The frequencies were clearly dose-dependent for the cells of both species. DISCUSSION
This study has clearly demonstrated that a potent mutagen and eventual carcinogen, ENU, induced CAs and SCEs much more frequently in SH cells than in H cells. These results appear, at least outwardly, to support the commonly held belief that chromosomal stability [3] is the basis of the recurrently experienced greater resistance of human than rodent cells to various carcinogenic insults in culture. However, the exact mechanisms of CT, CA and SCE induction by ENU are still undetermined, although they are considered to have at least certain areas in common [17]. In this regard, mention should be made of our finding that in spite of the same extent of CT induction by ENU in SH and H cells, CA and SCE were far more intensive in the former than in the latter cells, indicating different processes for CT than for CA and SCE [17]. Very recently a study with CHO cells has also suggested that CT of ENU could be caused not only by DNA alkylation but also by reaction with other macromolecules [7] Carbomoylation of proteins [15] and formation of ethylphosphotriesters [2] may be another cause of CT, not necessarily associated directly with chromosomal damage. Numerous cytogenetic studies on individuals genetically prone to cancer (families) have pointed conclusively to the existence of a close relationship between CA and oncogenic predisposition [9]. When comparing different species there appears to be a species-dependent propensity for CA. For example, human fetal tracheal epithelial cells still retained a normal chromosomal constitution after long-term exposure to DEN, notwithstanding the later acquisition of anchorage independency [4], while Syrian hamster fetal tracheal epithelial cells showed a considerable amount of CAs after transplacental exposure to DEN 1201,later leading unequivocally to the development of papillomas of this organ. Benzo[alpyrene induced much fewer breaks and SCEs in human embryo cells than in Chinese and Syrian hamster embryo cells [lo], quite similar to our present data with ENU. It is still unknown which type of CA chromatid or chromosome type, is more closely associated with the carcinogenesis
42 processes.
Nor is it certain whether CAs and SCEs are induced by the same chains of events [lS]. However, evidence is available that indicates that chromatid-type aberrations and SCEs reflect the amount of slowly reparable DNA lesions, and chromosome-type aberrations reflect the amount of rapidly reparable DNA lesions [14]. It has also been reported that human fibroblasts are able to remove 50-750~ of 06-alkylguanine shortly after the exposure [2], while CHO and V79 cells cannot remove the same adducts over 20- 24 h after exposure [6]. In this connection, information about DNA repair will help us to interpret the results of such comparative studies between different species. Studies with different human individuals are required to draw any conclusion on species differences, since the variations in sensitivity to carcinogens are, reportedly, extremely wide among different human individuals [8]. ACKNOWLEDGEMENT
The authors manuscript.
wish to thank Ann Borchert
for her assistance
with the
REFERENCES 1 2
3 4
5
6 7 8 9 10
11 12
13
Abe, S. and Sasahi, Id. (1977) Chromosome aberrations and sister chromatid exchanges in Chinese hamster cells exposed to various chemicals. J. Natl. Cancer Inst., 58.1535- 1640. Bode& W.J., Singer, B.. Thomas, G.H. and Cleaver, J.E. (19791Evidence for removal at different rates of O-ethyl pyrimidines and ethylphosphotriesters in two human fibroblast cell lines. Nucleic Acids Res., 0,2819-2829. DiPaolo, J.A. (19831 Relative difficulties in transforming human and animal cells in vitro. J. Natl. Cancer Inst., 70.3 - 8. Emura, M., Mohr, U., Kahunaga, T. and Hilfrich, J. (19851 Growth inhibition and transformation of a human fetal tracheal epithelial cell line by long-term exposure to diethylnitrosamine. Carcinogenesis, 6,1079- 1085. Emura. M.. Richter-Reichhelm, H.-B., B5ning. W., Eichinger, R., Schoch, C., Althoff. J. and Mohr, U. (19821A fetal respiratory epithellal cell line for studying some problems of transplacental carcinogeneeis in Syrian golden hamsters. J. Cancer Res. Clin. Oncol., 104,133- 144. Goth-Goldstein, R. (1980) Inability of Chinese hamster ovary cells to excise O%lhylguanine. Cancer Res., 40.2623- 2024. Goth-Goldstein, R. and Hughes, M. (19871 Cell hilling by various monofunctional alhylating agents in Chinese hamster ovary cells. Mutat. Res., 177.267- 270. Harris, C.C. (19871Human tissues and cells in carcinogenesls research. Cancer Res., 47,1- 10. Hus, T.C. (19871Genetic predisposition to cancer with special reference to mutagen sensitivity. In Vitro Cell. Dev. Biol., 23.591-603. Iheuchi, T. and Sasahi, M. (19811Differential inducibility of chromosome aberrations and sisterchromatid exchanges by indirect mutagens in various mammalian cell lines. Mutat. Res., 90, 149- 161. ISCN (19811An international system for human cytogenetic nomenclature (1975). Cytogenet. Cell Genet.. 31,1-23. Jensen, E.M., LaPolla. R.J., Kirby, P.E. and Haworth, S.R. (1977) In vitro studies of chemical mutagens and carcinogens. I. Stability studies in cell culture medium. J. Natl. Cancer Inst., 59, 941-944. Kimball, RF. (1987) The development of ideas about the effect of DNA repair on the induction of gene mutations and chromosomal aberrations by radiation and by chemicals. Mutat. Res., 16&l- 34.
43 14
16 16
17
18 19 20
21
Kiihi, K. (1987) Effects of repair inhibition in the G, phase of ciastogen-treated human lympho cytes on the frequencies of chromosome-type and chromatid-type aberrations and sister-chromatid exchanges. Mutat. Res., 178,106- 116. Knox, P. (1976) Carcinogenic nitrosamides and ceii cultures, Nature. 289,671-673. de Kok. Al.. de Kniff, P. and Mohn, G.R. (1983) Correlation between the stabiity of ethylnitro sourea in aqueous solution at different pH and temperature and its mutagenic effectiveness for Salmon&a typhimurium strain TA 100. Mutat. Res., 12981-89. Natarajan, A.T., Simons. J.W.I.M., Vogel, E.W. and van Zeeland, A.A. (1984) Relationship between cell kiliing. chromosomai aberrations, sisterehromatid exchanges and point mutations induced by monofunctionai aIkyIating agents in Chinese hamster cells. A correlation with different ethylation products in DNA. Mutat. Res., X%,31-40. Savage, J.R.K. (1975) Classification and relationships of induced chromosomal structural changes. J. Med. Genet., 12.103-122. Speit. G. (1984) Considerations on the mechanism of differential Giemsa staining of BrdU-substituted chromosomes. Hum. Genet.. 67,264- 269. Tsuda. H., Emura, M., Richter-Reichhelm. H.-B., Biining, W. and Mohr, U. (1983). Transplacental effect of Ndiethylnitrosamine on chromosomai aberrations in fetal tracheal cells of the Syrian hamster: comparison between epithelial and mesenchymal cells. Carcinogenesis. 4,13861388. Wolff, S. (1981) The sister chromatid exchange test. In: Short-term tests for chemical carcino gens pp. 236 - 242. Editors: H.F. Stich and R.H.C. San, Springer, New York.
CancerLettera, Fhvier
TOXICITY HAMSTER
27
AND CIIROMOSOMAL DAMAGE IN FETAL SYRIAN AND HUMAN PULMONARY EPITHELIAL CELLS
J. WEN*. M. EMURA, M. RIRBE and U. MOHR In#itutfdr Expesimentelle Patho&@, Str. 8,3t?OOHuwwver 61 f’F.R.G.j
Medieinieche Hochrchule Hannover, Konatanty-Gutschow
(Received 2 Januuy 1988) (Accepted 14 March 1988)
SUMMARY
In order to test the hypothesis that human chromosomes are more stable than those of rodents, we compared the fetal human and Syrian hamster pulmonary epithelial cell lines for their sensitivity to the induction of cytotoxicity (CT), chromosomal aberrations (CAsl and sisterchromatid exchanges WEE). One day after plating, the cells were exposed for 2 h to various doses of ENU dissolved in an exposure medium consisting of McIlvaine buffer, RPM1 1640, and bovine serum albumin. CAs and SCEs were examined‘in 24 -64 post-exposure hours by the standard methods, while CT was determined by cell counts. The frequency of CAs (particularly ehromatid exchanges) and that of SCEs showed clear dose dependency and was remarkably higher in the hamster than in the human cells. CT determined in 1 week of post-exposure incubation showed a similar dose response for both cell lines with sharply declining regressions. These results suggest that human chromosomes are indeed more resistant to ENU-inducible aberrations than hamster chromosomes and that CT may not always be directly asso ciated with chromosomal damage. Key words: Chromosome; Damage; Hamster: Human
INTRODUCTION
An important aspect of carcinogenesis research is the extrapolation of laboratory animal data to the human situation. A rapid improvement in cell culture techniques has enabled us to make more exact comparison of the Cowerpondrnce to: M. Emura. lPrerewt ad&eta: Institute of P6thoIogy. Sun Yat-Sen Univemity of Medical Sciences, Zhongshau Roul2. Gnmgxhou. The People’6 Republic of Cbim. 0 1988 Ekwier O~&SO Published 6nd Prinbd in Ireimd.
Scientific Publishers Irelmd Ltd.
response to carcinogenic agents of animal and human cells, including epithelial cells [8] from which most tumors originate. Past experience has shown that human cells are more difficult to transform using chemical carcinogens than rodent cells [3]. A tentative explanation could be the assumption that human chromosomes are more stable than those of rodents. In an effort to test this hypothesis, we compared the fetal human and Syrian hamster pulmonary epithelial cells for their sensitivity to the induction of cytotoxicity (CT), chromosoma1 aberrations (CAs) and sisterchromatid exchanges (SCEsl by short-term exposure to ethylnitrosourea (ENU). Pulmonary cells were used because we were interested in the mechanisms of lung carcinogenesis. MATERIALS AND METHODS
The human epithelial cell line (diploid, 23 h PDT) was raised from a piece of bronchus taken from an 1Sweekold fetus, as described previously [4]. The Syrian hamster epithelial cell line (pseudodiploid, 11 h PDT), was derived from the lung of a fetus on day 16 of gestation [5]. Both cell lines were grown in a growth medium (GM) consisting of RPM1 1640 (80%, Flow, Meckenheim), fetal bovine serum (20%; Flow), etc. as described previously [S]. Cultures were maintained at 37 OCin a humidified atmosphere of 10% CO, and 20% air. Autoradiographic examination with 5 pCi/ml (spec. act. 26 Ci/mmol; 30 min incubation1 of [sHjthymidine (Amersham, Braunschweigl has shown that the DNA synthesizing population of the Syrian hamster (SH) cell line comes into a steady state (at 60%) approx. 12 h after cell plating (10’ cells in &cm2 dish with GM), while in the human (Hl cell line this first occurs after 24 h (Fig. 11. Therefore, ENU treatment was performed at 24 h after cell plating, lest the different incidence of S-phase cells before 24 h should influence the extent of toxicity and chromosomal damage in the 2 cell types.
Fig. 1. Rate of DNA synthesizing (S-phase) population in fetal Syrian hamster (SH) and human (H) epithelial cells after plating. Cells (10’) were plated in &me dishes containing GM, and at 3-h intervals they were incubated with PH]thymidine for 30 min. Each point indicates the average from 3 dishes. in each of which 100 autoradiographed cella were scored. Fig. 2. Spontaneous decay of ENU in various sorta of solution with different pH at 37°C. m, McIlvaine buffer (pH 8.0); 0, McIlvaine buffer (pH 7.2); 0, exposure medium (EM) (pH 6.0).
ENU exposure Because of the remarkable reduction of chemical stability of ENU around neutral pH and in the presence of serum [12,16] a special exposure medium (EM) was developed consisting of 2 parts of 0.01 M McIlvaine buffer (pH 6.0) with 0.9% NaCl, 1 part of RPM1 1640, 0.1% bovine serum albumin (fraction V; Sigma, Munich) and 110 &ml sodium pyruvate, and the ENU was dissolved in this EM immediately prior to use. In the EM (pH 6.01the spontaneous decay of ENU was greatly reduced with about 35% retention of native-ENU in 2 h (Fig. 21,while at pH 7.2 less than 3% remained undecayed in 1 h. Exposure to ENU at the doses prescribed for each experiment was carried out for 2 h at 37 “C 24 h after plating the cells (cell no. depending on the experiment) in 25-cm2 flasks (Falcon).
Cytotoxicity To estimate the cytotoxicity by cell counts, 2 x 10” cells/flask were plated in 5 ml GM. ENU doses examined were 0, 0.1, 0.2, 0.4, 0.8 and 1.6 mg/ml, and to each dose 3 flasks were allotted. After removal of ENU the cultures were washed once with GM and further incubated in GM until the the unexposed control cultures had reached 9OW of confluency (for SH and H cells 7 and 9 days, respectively). The medium was changed twice a week. After trypsinization, viable cells were counted by the trypan blue exclusion method.
Chromosomal aberrations At 24 h after plating the cells were exposed to various doses of ENU (Fig. 41. After washing once with GM they were incubated in GM for 2,4,6 and 8 days. Chromosomes were prepared by the conventional air-dry techniques after incubation with 0.3 pg/ml of Demecolcine (Sigma, Munich) for 3 h. After Giemsa (4%) staining, chromosomal changes in well-spread metaphases were classified according to Savage [18]. Ring chromosomes, dicentrics and chromatid exchanges were considered to be caused by 2 breaks in 1 or 2 chromosomes, and deletions and minute chromosomes by 1 break, with gaps being excluded regardless of the width [l,ll]. On this basis the numbers of breaks were counted on the above-prescribed days and later added together to normalize the break counts per lo4 chromosomes.
Sister-chromatid exchange Cells (2 - 5 x lo6 per flask) were plated. After ENU exposure (for the doses see Fig. 51,further incubation with 10 ccglmlbromodeoxyuridine in GM was carried out at 24 and 48 h for SH and H cells, respectively, in the first experiment (lower dose range), and 36 and 64 h for SH and H cells, respectively, in the second experiment (higher dose range), during which period 2 rounds of replication were expected to occur. Chromosomes were prepared as for the chromosomal aberration examination and stained routinely [19] with slight modifications. The extent of SCE was expressed as average numbers per chromosome with the control values subtracted from SCE frequencies scored in at least 50 metaphases [21].
0.162
0.4
0.6 E NU
1.6
hp/ml)
Fig. 3. Dose-dependent toxicity of ENU in SH and Ii cells as measured by counting living cells whose numbers at various doses were related in percentage to tbe plated cell number (z x 10% cm% Each point indicates the mean of 9 flashs with SD.
RESULTS
For both SH and H cells the survival potential decreased dose-dependently and there were no remarkable differences between them (Fig. 3). Only at higher doses, H cells appeared to be a little more resistant to ENU than SH cells. Linear regression analysis based on the semilogarithmic plotting indicated the
9
ENU
Cmo/ml)
Exp I
;
Exp II
.. 1
ENU
(mg/ml)
Fig. 4. Dose-dependent incidence of chromatid breaks in SH and Ii cells. For the details of measurement see Materials and Methods. The double points indicate a statistically significant difference with P < 0.001 (x+tesU between SH and H. Fig. 6. Dose-dependent incidence of sister-cbromatid exchanges in SH and H cells. For the detailed measurement see Materials and Methods. Single and double points indicate a statistically significant difference with P < 0.05 and P < 0.001 (XI_test).respectively, between SH and H. Bars indicate S.D.
41
mean lethal doses of ENU to be 162 and 180 &ml for SH and H cells, respectively. By contrast, the chromosome breaks occurred far more frequently in SH cells than in H cells, showing clear dose dependencies for both (Fig. 41. The response of H cells at 0.8 mg/ml was very sharp as compared to that at lower doses. The major chromosomal alterations were of a chromatid type such as exchanges including triradials and quadriradials. The alterations of a chromosoma1 type, such as dicentrics, minutes or rings, were also significantly more frequent in SH than in H cells (data not shown). The gaps, however, showed slightly higher frequencies in H than in SH cells, although the overall rates were at any rate very low. SCE also demonstrated a significantly higher occurrence in SH cells than in H cells, particularly so in the higher dose range (experiment 2; Fig. 51. The frequencies were clearly dose-dependent for the cells of both species. DISCUSSION
This study has clearly demonstrated that a potent mutagen and eventual carcinogen, ENU, induced CAs and SCEs much more frequently in SH cells than in H cells. These results appear, at least outwardly, to support the commonly held belief that chromosomal stability [3] is the basis of the recurrently experienced greater resistance of human than rodent cells to various carcinogenic insults in culture. However, the exact mechanisms of CT, CA and SCE induction by ENU are still undetermined, although they are considered to have at least certain areas in common [17]. In this regard, mention should be made of our finding that in spite of the same extent of CT induction by ENU in SH and H cells, CA and SCE were far more intensive in the former than in the latter cells, indicating different processes for CT than for CA and SCE [17]. Very recently a study with CHO cells has also suggested that CT of ENU could be caused not only by DNA alkylation but also by reaction with other macromolecules [7] Carbomoylation of proteins [15] and formation of ethylphosphotriesters [2] may be another cause of CT, not necessarily associated directly with chromosomal damage. Numerous cytogenetic studies on individuals genetically prone to cancer (families) have pointed conclusively to the existence of a close relationship between CA and oncogenic predisposition [9]. When comparing different species there appears to be a species-dependent propensity for CA. For example, human fetal tracheal epithelial cells still retained a normal chromosomal constitution after long-term exposure to DEN, notwithstanding the later acquisition of anchorage independency [4], while Syrian hamster fetal tracheal epithelial cells showed a considerable amount of CAs after transplacental exposure to DEN 1201,later leading unequivocally to the development of papillomas of this organ. Benzo[alpyrene induced much fewer breaks and SCEs in human embryo cells than in Chinese and Syrian hamster embryo cells [lo], quite similar to our present data with ENU. It is still unknown which type of CA chromatid or chromosome type, is more closely associated with the carcinogenesis
42 processes.
Nor is it certain whether CAs and SCEs are induced by the same chains of events [lS]. However, evidence is available that indicates that chromatid-type aberrations and SCEs reflect the amount of slowly reparable DNA lesions, and chromosome-type aberrations reflect the amount of rapidly reparable DNA lesions [14]. It has also been reported that human fibroblasts are able to remove 50-750~ of 06-alkylguanine shortly after the exposure [2], while CHO and V79 cells cannot remove the same adducts over 20- 24 h after exposure [6]. In this connection, information about DNA repair will help us to interpret the results of such comparative studies between different species. Studies with different human individuals are required to draw any conclusion on species differences, since the variations in sensitivity to carcinogens are, reportedly, extremely wide among different human individuals [8]. ACKNOWLEDGEMENT
The authors manuscript.
wish to thank Ann Borchert
for her assistance
with the
REFERENCES 1 2
3 4
5
6 7 8 9 10
11 12
13
Abe, S. and Sasahi, Id. (1977) Chromosome aberrations and sister chromatid exchanges in Chinese hamster cells exposed to various chemicals. J. Natl. Cancer Inst., 58.1535- 1640. Bode& W.J., Singer, B.. Thomas, G.H. and Cleaver, J.E. (19791Evidence for removal at different rates of O-ethyl pyrimidines and ethylphosphotriesters in two human fibroblast cell lines. Nucleic Acids Res., 0,2819-2829. DiPaolo, J.A. (19831 Relative difficulties in transforming human and animal cells in vitro. J. Natl. Cancer Inst., 70.3 - 8. Emura, M., Mohr, U., Kahunaga, T. and Hilfrich, J. (19851 Growth inhibition and transformation of a human fetal tracheal epithelial cell line by long-term exposure to diethylnitrosamine. Carcinogenesis, 6,1079- 1085. Emura. M.. Richter-Reichhelm, H.-B., B5ning. W., Eichinger, R., Schoch, C., Althoff. J. and Mohr, U. (19821A fetal respiratory epithellal cell line for studying some problems of transplacental carcinogeneeis in Syrian golden hamsters. J. Cancer Res. Clin. Oncol., 104,133- 144. Goth-Goldstein, R. (1980) Inability of Chinese hamster ovary cells to excise O%lhylguanine. Cancer Res., 40.2623- 2024. Goth-Goldstein, R. and Hughes, M. (19871 Cell hilling by various monofunctional alhylating agents in Chinese hamster ovary cells. Mutat. Res., 177.267- 270. Harris, C.C. (19871Human tissues and cells in carcinogenesls research. Cancer Res., 47,1- 10. Hus, T.C. (19871Genetic predisposition to cancer with special reference to mutagen sensitivity. In Vitro Cell. Dev. Biol., 23.591-603. Iheuchi, T. and Sasahi, M. (19811Differential inducibility of chromosome aberrations and sisterchromatid exchanges by indirect mutagens in various mammalian cell lines. Mutat. Res., 90, 149- 161. ISCN (19811An international system for human cytogenetic nomenclature (1975). Cytogenet. Cell Genet.. 31,1-23. Jensen, E.M., LaPolla. R.J., Kirby, P.E. and Haworth, S.R. (1977) In vitro studies of chemical mutagens and carcinogens. I. Stability studies in cell culture medium. J. Natl. Cancer Inst., 59, 941-944. Kimball, RF. (1987) The development of ideas about the effect of DNA repair on the induction of gene mutations and chromosomal aberrations by radiation and by chemicals. Mutat. Res., 16&l- 34.
43 14
16 16
17
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