Mutation Research, 275 (1992) 41-46
41
© 1992 Elsevier Science Publishers B.V. All righls reserved 0921-8734/92/$05.00
MUTAG! 09080
Effects of mutagens on the clonal lifespan of Paramecium tetraurelia Shinichi Fukushima, Hiroshi Ogawa and Sukenari Sasagawa Department of Hygiene, School of Medicine, Kinki UnirersiO; Osakasayama 589 (Japan) (Received 25 July 1991) (Revision received 30 September 1991) (Accepted 7 October 1991)
Keywords: Clonal lifespan; Paramecium: Mutagen: N-Melhyl-N'-nitro-N-nitrosoguanidine;4-Nitroquinoline-N-oxide
Summary There has been interest in the phemonenon that a cell cannot undergo unlimited reproduction under adequate conditions and undergoes senescence, in holotrichous ciiiates, Paramecium has a limit of vegetative reproduction without sexual reproduction but Tetrahymena does not always have a limited lifespan. Comparing the two species would increase our knowledge of the mechanism of cellular clonal aging. We previously showed that mutations induced by X-rays shorten clonal lifespan, in this study, we examined whether mutagens shorten the clonal lifespan of Paramecium tetraurelia. P. tetraurelia was exposed to the alkylating agent N-methyI-N'-nitro-N.nitrosoguanidine (MNNG), 0.045 mg/ml, for 30 min. The animal was exposed to MNNG 6 times in total while young (under 80 divisions from the start of a clonal life cycle) or 4 times during the senescent stage. MNNG shortened the cional lifespan as expressed by the decrease in fission number from 186 + 55 (4 cell lines) to 136 + 21 (6 cell lines) with the first two treatments but with further exposures the lifespan increased to 182 + 15 (5 cell lines). MNNG had no effect when administered at the older age. Exposure of P. tetraurelia to 4-nitroquinoline-N-oxidc at 0.021 m g / m l twice for 12 and 15 min at the younger age reduced the mean clonal lifespan from 143 4- 28 to 125 + 21 and the maximum lifespan from 263 + 33 to 175 + 25.
Paramecium does not reproduce unlimitedly without sexual reproduction under optimal conditions, i.e., it shows a clonal lifespan despite being a unicellular organism (Sonneborn, 1954). The mechanism of aging in animals is an unresolved issue in biology. Research on cell clonal aging is an important approach to this problem (Strehler,
Correspondence: Dr. S. Fukushima, Department of Hygiene, School of Medicine, Kinki University, Osakasayama 589 (Japan).
1977; Macieira-Coelho, 1988). Paramecium has been studied as an experimental model of cellular clonal aging (Smith-Sonneborn, 1990). We have previously reported the effects of X-rays on the clonal lifespan of Paramecium aurelia (Fukushima, 1974). The cell was so resistant to X-rays that irradiation with 9000 R had almost no effect on asexual reproduction. Total repeated irradiation of over 90 kR, however, shortened clonal lifespan. We suspected that the clones stopped reproducing due to cumulative mutations at a locus in the vegetative macronucleus. The
42
mutation rate estimated from the experimental results was comparable to the mutation rates of other organisms. If the accumulated mutations in the macronucleus restricted the cell clonal lifespan, a clone with a recessive lethal mutation from the start of its cional cell cycle should have a short lifespan. However, experiments using some heterogenous clones given lethal mutations refuted this hypothesis (Fukushima, 1987). We studied the effects of mutagens on the elonal lifespan in P. tetraurelia. Materials and methods
Materials P. tetraurelia stock 51 was used. The alkylating agent N-methyI-N'-nitro-Nnitrosoguanidine (MNNG) and 4-nitroquinolineN-oxide (4NQO) were used as mutagens. Cell culture We used the conventional medium and re-iso. lation culture methods as reported in our previous study (Fukushima etal., 1990). Cells were maintained at 27 ° C. An exautogamous cell, which had been repeatedly induced, was prepared to start a new clonal life cycle. The occurrence of autogamy was ensured by the depressed frequency of exautogamous ceils among the starved population in the first surplus culture and the occurrence of autogamy during vegetative reproduction was also checked (Fukushima, 1979). Treatment with mutagens To induce mutations in the macronucleus, cells were exposed to a mutagen during the latter half of its division cycle, when DNAs in the macronucleus were duplicated (Woodard etal., 1961). We determined the timing of the mutagen treatment by observing the time of the previous cell division, since cell lines tend to maintain a constant fission rate. MNNG, 0.5 mg/ml, was diluted to 0.045 mg/ml with the culture medium. Isolated cells were submerged in this solution for 30 rain, which delayed the next division but did not kill the cell.
, Exautogamous Cell
IrT
Line No.
t [ Day
t
I [
Control
12
AJ Fig. 1. Experimental scheme of the effect of MNNG on the cell clonal lifespan. An elliptical dot represents one cell. Horizontal lines show the maintenance of a cell line with triplet daily re-isolation culture. An arrow represents the isolation of a cell, Triangles show MNNG treatment.
Isolated cells were bathed in 4NQO diluted to 0.021 mg/ml with the culture medium for 12 or 15 rain. This treatment delayed the next cell division and occasionally killed the cells.
Experimental scheme of MNNG As shown in Fig. 1, an exautogamous cell was first isolated, and was allowed to divide 4 times until the following day. Then a single cell was isolated from the population, which produced 32 cells by the next day. Among those cells, 6 were randomly isolated as a control group and 6 others wore randomly selected to be exposed to the MNNG solution. These cell lines were maintained by the triplet re-isolation culture method (TIC) (Fukushima, 1979). The 6 cells that had boon exposed to MNNG each reproduced to 8 cells on the next day. One of those 8 cells was again exposed to MNNG. The 6 cells exposed twice wore named NGI and maintained in TIC until all the cell lines had died. The sister cells from NGI wore moreover exposed twice to MNNG, that is, the 3rd exposure was made after 6-8 cell divisions from the 2rid exposure and the 4th exposure was made after 2-3 divisions from the 3rd, Those 6 cell lines which had been exposed 4 times in total were
43
Exautogamous ! Cell 4Day O I C
named NGII and maintained in TIC until all the cell lines had died. The sister cells of NGII were exposed to the MNNG solution twice more and NGIII were formed, of which ceils were exposed to the MNNG solution 6 times in total. The aged cells of the control group that had divided over 90 times from the start of the clonal life cycle were exposed to the MNNG solution twice and this experimental group was named NGXI. The sister cells of NGXI were exposed to MNNG twice more and were named NGXII.
Experimental scheme of 4NQO An exautogamous cell was first isolated and a cell line was maintained in daily re-isolation culture (DIC). Among 32 cells reproduced from the isolated cell on the 4th day, 4 cells were randomly isolated. They divided 5 times on the next day. From each population, 5 cells were randomly isolated and maintained in DIC as control cell lines. A set of 5 lines was called 'sisters'. As shown in Fig. 2, another cell was exposed to 4NQO. This cell had divided 3 times by the following day. From the 8 reproduced cells, 4 were randomly isolated and they divided 5 times in 24 h. Five cells were randomly isolated from each population of 32 and maintained in DIC as E! cell lines. Other cells were randomly selected from each population and again exposed to 4NQO. They each reproduced 8 cells on the next
Dayl~
!
S It
oar
.. e
o
e
I
I I 1""'"IIIil'" m'JJli E
I
A & i&l | | | I~, tln
!1LI! LI! I]ll]llll]
Control
Fig. 2. Experimental scheme of the effect of 4NQO on the cell clonal lifespan. An elliptical dot represents one cell. Verlical lines show the maintenance of a cell line wilh daily re-isolation culture. A bundle of 5 cell lines is a group of sister lines. An arrow represents the isolation of a cell. Triangles show 4NQO treatment.
day, Among them, 5 cells were randomly isolated and maintained in DIC as Ell cell lines. When a cell line died in DIC, it was replaced by transferring a cell from another vigorous ccU line among the same sisters. This procedure was
TABLE I EFFECTS O F MNNG ON CLONAL LIFESPAN Line
NGIll
NGll
NGI
Control
NGXI
NGXII
I 2 3 4 5 6
181 174 173 208 172
169 149 162 200 206
145 99 161 137 146 127
132 148 219 246
127 IS2 220 129 128
243 137 169 178 135 175
Mean~SD
182±15
177±25
136±21
186±55
157±42
172±39
L.._,
• ]
** lines that became autogamous en route. * Significantly different ( p < 0.05). * * Significantly different ( p < 0.01). -,
I .,,,,
|4||| 4illl
° ' IIIII ' ' IIIII
E 1[
I
,,.,., ~
e
I t l Oby I
•
I t
44
repeated until all 5 cell lines had died. The fission number of the longest lived cell line with the replacements was called the maximum lifespan.
TABLE 3
Statistical analysis
Sisters
Control
El
Ell
The computer software, MUSCOT, Statistical Analysis and Categorical Analysis Vol. 1 (Y.D.K. Co. Ltd.) was used.
1
2 3 4
241 255 267 180
179 219 185 218
180 208 158 154
Mean±SD
236±39
200±21
175±25 *
Results
EFFECTS O F 4NQO ON THE MAXIMUM CLONAL LlFESPAN
* Significantly different from the control ( p < 0.05).
Effects of MNNG The lifespans of each cell line expressed by fission number are listed in Table 1. As two cell lines of the control and one of NGII, NGIII arm NGXI were assumed to be autogamous, their lifespans were not recorded. There were no significant differences among the mean lifespans of each group except for that of NGI. Although there was no significant differ-
Effects of 4NQO
TABLE 2 EFFECTS OF 4NOO ON THE MEAN CLONAL LIFESPAN Sisters
Control
El
Ell
I
183 189 157 122
125 16S 121 120 133
100 159 122 124
2
151 86 1{)6 156 137
159 177 154 193 160
131} 72 112 131 -
3
133 140
121 175 183 108 126
132 151 141 119 145
166 -
115 4
159 157 169
141 129 121 168 79
143± 28
143±29
llll
Mean±SD
ence between NGI and the control because of the split values of the control, the lifespan of NGI was shorter. There were significant differences between NGI and NGII (t = 2.26215, p < 0.05), and between NGI and NGiII (t = 3.2498, p < 0.01). Exposing old cells to MNNG had no definite effects on the lifespan.
- . lines that became autogamous en route, * Significantly different ( p < 0,05),
138 I08
124 104
133 125±21 *
The lifespans of all the cell lines are listed in Table 2. Table 3 shows the maximum lifespans of each sister cell line. The mean lifespan of Ell was shorter than the control and E! (tm 2.0345, p < 0.05; t = 2.0281, p < 0,05). The maximum lifespan of Ell was shorter than the control (t ffi 2.6457, p < {).05). Although there was no significant difference between the maximum lifespan of Eli and El (t--1.547.5) and between El and the control (t = 1.6101), the maximum lifespan showed a tendency to decrease upon exposure to 4NQO.
Discussion There has been much controversy over the mechanism of senescence. Although we do not accept at present that only one mechanism is at work, it is presumed that mammals have common mechanisms of senescence. Mutation theory is one of the hypotheses of mammalian senescence and many experiments have been attempted to verify it. The lifespan of rodents was reduced with ionizing irradiation (Russ and Scott, 1939; Lindop
45 and Rotblat, 1961). Chemical mutagens also reduced the lifespan of mice (Alexander and Connell, 1960). The effects of ionizing irradiation on the clonal lifespan of cultured cells were variable according to the experimental conditions, the species, the cell age, the dosage and so on (Macieira-Coelho et al., 1976; Icard et al., 1979; Ban et al., 1981). These results suggest that the induced mutations more or less affect senescence. It is obscure, however, whether such life-shortening effects are caused by the accumulated mutations in the somatic cells (Walburg, 1975). P. tetraurelia has been used as an experimental model to investigate cell clonal aging because of its clear limitation of vegetative reproduction and cellular maturation. In the present experimcnts, the effects of mutagens on clonal lifespan were studied. The results with 4 N Q O are clear, The mean lifespan of El, exposed once to 4NQO, is not different from the control. The maximum lifespan of El, however, decreased a little, Both mean and maximum lifespans of Ell are clearly decreased compared with the control. These results suggest that 4 N Q O has a life-shortening effect on clonal lifespan. The results with MNNG are more complex. The values of the Control values were available from only 4 cell lines and those were split into two. So there were no significant differences between the control and NG! or NGXI, though the values were decreased compared with the control. Repeated exposure of NGI cells extended clonal lifespan. We do not know what caused these results. Smith-Sonneborn (1979)showed that UV irradiation also reduced the clonal lifespan in P. tetraurelia. Tixador et al. (1981) reported that low doses of ionizing radiation shortened the clonal lifespan in P. tetraurelia. These experimental results suggest that mutation affects cell clonai aging, but it is not as simple as Szilard's hypothesis (1959) that aging hits accumulating on somatic cells cause aging. All animals have a limited lifespan. This suggests genetic control of aging. Even if genetic control acts, it is different from the developmen-
tal process that occurs after a particular manipulation, because the difference among individuals in senescent processes is considerable. Recently Harley et al. (1990) suggested that telomere reduction in aged human fibroblasts restricts cell doublings. Though Tetrahymena is a holotrichous ciliate and its clonal life cycle is very similar to that of Paramecium, it has an indefinite cell clonal lifespan (Nanney and Preparata, 1979). In immortal Tctrahymena, the telomere is not reduced with age (Larson et al., 1987). In mortal Paramecium, the macronuclear DNA decreases with age (Schwarts and Meister, 1973; Klass and SmithSonneborn, 1976; Takagi and Kanazawa, 1982). We do not yet have information concerning telomere decrease in senescent Paramecium. If we could detect teiomere reduction in aged Paramecium cells, it would suggest that telomcre reduction with age primarily decides cell clonal lifespan in general and that mutations affect clonal senescence by modifying telomere activity. References
Alexander. P.. and D.I. Connell (1960)Shortening of the life span of mice by irradiation wilh X-rays and Ireatment with radiomimetic chemicals, Radiat. Rcs., 12, 38-48. Ban, S., I. Ikushima and T. Sup,awara (lgNI) Reduction in proliferative lifespan of human diploid cells after exposure to a ruaclor radiation beam. Radial. Res.. ~7. l-~J. Fuku.~hima, S. (Ig74) Effect of X.irradi:ilions on tile clon.'d life-span and fission raie in Peramecium merelia.Exp. Cell
Res., 84, 267-270. Fukushima, S. (1979) Effect of tempcralure on tile clonal life span in Paramecitmz tetraurelia, Acta Mud. Kinki Univ.. 4, 31-36. Fukushima. S. (1987) Chmal senescence in paramecium, Acta Mud. Kinki Univ., 12, 1-7. Fukushima, S., H. Ogawa, T. Nishikawa and S. Sasagawa (1990) CIonal lifespans cultured in chemically defined medium and conventional hacterized medium in Parame. ('ium octaurelia. Mech. Ageinlj Dev., 54, 259-27(I. Harley. C.B., A.B. Futchcr and C.W. Grcider (1990) Telomeres shorten during ageing of human fihrohlasls. Nature, 345. 458-460. Itastie, N.D., M. Dempster, M.G. Dunh)p. A.M. Thompson, D.K. Green and R.C. Allshire (1990) Teh)mere reduction in human coloreetal carcinoma and with ageing, Nature, 346,, 866-868. Icard. C., R. Beaupain0 C. Diatloff and A, Macieira-Coelho (1979) Effcct of low dose rate irradiation on the division potential of cells in vitro, IV. Changes in DNA in ra-
46 diosensitivity during aging of human fibroblasts, Mech. Ageing Dev., I I, 269-278. Klass, M.R., and J. Smith-Sonneborn (1976) Studies on DNA content, RNA synthesis, and DNA template activity in aging cells of Paramecium aurelia, Exp. Cell Res., 98, 63-72. Lirson, D.D., E.A. Spangler and E.H. Blackburn (1987) Dynamics of telomere length variation in Tetrahymena thermopbila, Cell, 50, 477-483. Lindop, P.J., and J. Rotblat (1961) Long-term effects of a single whole-body exposure of mice to ionizing radiations, Proc. R. Suc. B, 154, 332-349. Macieira-Coelho, A. (1988) Biology of Normal Proliferating Cells in Vitro. Relevance for in Vitro Aging, Karger, Basel. Macieira-Coelho, A., C. Diatloff and E. Malaise (1976) Doubling potential of fibroblasts from different species after ionizing radiation, Nature, 261,586-588. Nanney, D.L., and R.M. Preparata (1979) Genetic evidence concerning the structure of the Tetrahymena thermophila macronucleus, J. Protozool., 26, 2-9. Russ, S., and G.M. Scott (1939) Biological effects of gamma irradiation, Br. J. Radiol., 12, 441)-441. Smith-Sonneborn, J. (1979) DNA repair and longevity assurance in Parameeilon tetraurelia, Science, 203, I 115-1117. Smith-Sonneborn, J, (1990) Aging in protozoa, in: E,L.
Schneider and J.W. Rowe (Eds.), Handbook of the Biology of Aging, 3rd edn., Academic Press, San Diego, CA, pp. 24-44. Sonneborn, T.M. (1954) The relation of autogamy to senescence and rejuvenescence in Paramecium aarelia, J. Protozool., 1, 38-53. Strehler, B.L. (1977) Time, Cell, and Aging, 2nd edn., Academic Press, New York, NY. Schwartz, V., and H. Meister (1973) Eine AItersver~inderung des Makronucleus yon Paramecium, Z. Naturforsch., 28c, 232. Szilard, B.L. (1959) On the nature of the ageing process, Proc. Natl. Acad. Sci. (U.S.A.), 45, 330-345. Takagi, Y., and N. Kanazawa (1982) Age-associated change in macronuclear DNA content in Paramecium caudatum, J. Cell Sci., 54, 137-147. Tixador, R., G. Richoilley, E. Monrozies and H. Planel (1981) Effects of very low doses of ionizing radiation on the clonal life-span in Paramecium tetraurelia, Int. J. Radiat. Biol., 39, 47-54. Walburg Jr., H.E. (1975) Radiation-induced life-shortening and premature aging, Adv. Radial. Biol., 5, 145-179. Woodard, J., B. Gelber and H. Swift (1961) Nucleoprotein changes during mitotic cycle in Paramecium aurelia, Exp. Cell Res., 23, 258-264.
41
© 1992 Elsevier Science Publishers B.V. All righls reserved 0921-8734/92/$05.00
MUTAG! 09080
Effects of mutagens on the clonal lifespan of Paramecium tetraurelia Shinichi Fukushima, Hiroshi Ogawa and Sukenari Sasagawa Department of Hygiene, School of Medicine, Kinki UnirersiO; Osakasayama 589 (Japan) (Received 25 July 1991) (Revision received 30 September 1991) (Accepted 7 October 1991)
Keywords: Clonal lifespan; Paramecium: Mutagen: N-Melhyl-N'-nitro-N-nitrosoguanidine;4-Nitroquinoline-N-oxide
Summary There has been interest in the phemonenon that a cell cannot undergo unlimited reproduction under adequate conditions and undergoes senescence, in holotrichous ciiiates, Paramecium has a limit of vegetative reproduction without sexual reproduction but Tetrahymena does not always have a limited lifespan. Comparing the two species would increase our knowledge of the mechanism of cellular clonal aging. We previously showed that mutations induced by X-rays shorten clonal lifespan, in this study, we examined whether mutagens shorten the clonal lifespan of Paramecium tetraurelia. P. tetraurelia was exposed to the alkylating agent N-methyI-N'-nitro-N.nitrosoguanidine (MNNG), 0.045 mg/ml, for 30 min. The animal was exposed to MNNG 6 times in total while young (under 80 divisions from the start of a clonal life cycle) or 4 times during the senescent stage. MNNG shortened the cional lifespan as expressed by the decrease in fission number from 186 + 55 (4 cell lines) to 136 + 21 (6 cell lines) with the first two treatments but with further exposures the lifespan increased to 182 + 15 (5 cell lines). MNNG had no effect when administered at the older age. Exposure of P. tetraurelia to 4-nitroquinoline-N-oxidc at 0.021 m g / m l twice for 12 and 15 min at the younger age reduced the mean clonal lifespan from 143 4- 28 to 125 + 21 and the maximum lifespan from 263 + 33 to 175 + 25.
Paramecium does not reproduce unlimitedly without sexual reproduction under optimal conditions, i.e., it shows a clonal lifespan despite being a unicellular organism (Sonneborn, 1954). The mechanism of aging in animals is an unresolved issue in biology. Research on cell clonal aging is an important approach to this problem (Strehler,
Correspondence: Dr. S. Fukushima, Department of Hygiene, School of Medicine, Kinki University, Osakasayama 589 (Japan).
1977; Macieira-Coelho, 1988). Paramecium has been studied as an experimental model of cellular clonal aging (Smith-Sonneborn, 1990). We have previously reported the effects of X-rays on the clonal lifespan of Paramecium aurelia (Fukushima, 1974). The cell was so resistant to X-rays that irradiation with 9000 R had almost no effect on asexual reproduction. Total repeated irradiation of over 90 kR, however, shortened clonal lifespan. We suspected that the clones stopped reproducing due to cumulative mutations at a locus in the vegetative macronucleus. The
42
mutation rate estimated from the experimental results was comparable to the mutation rates of other organisms. If the accumulated mutations in the macronucleus restricted the cell clonal lifespan, a clone with a recessive lethal mutation from the start of its cional cell cycle should have a short lifespan. However, experiments using some heterogenous clones given lethal mutations refuted this hypothesis (Fukushima, 1987). We studied the effects of mutagens on the elonal lifespan in P. tetraurelia. Materials and methods
Materials P. tetraurelia stock 51 was used. The alkylating agent N-methyI-N'-nitro-Nnitrosoguanidine (MNNG) and 4-nitroquinolineN-oxide (4NQO) were used as mutagens. Cell culture We used the conventional medium and re-iso. lation culture methods as reported in our previous study (Fukushima etal., 1990). Cells were maintained at 27 ° C. An exautogamous cell, which had been repeatedly induced, was prepared to start a new clonal life cycle. The occurrence of autogamy was ensured by the depressed frequency of exautogamous ceils among the starved population in the first surplus culture and the occurrence of autogamy during vegetative reproduction was also checked (Fukushima, 1979). Treatment with mutagens To induce mutations in the macronucleus, cells were exposed to a mutagen during the latter half of its division cycle, when DNAs in the macronucleus were duplicated (Woodard etal., 1961). We determined the timing of the mutagen treatment by observing the time of the previous cell division, since cell lines tend to maintain a constant fission rate. MNNG, 0.5 mg/ml, was diluted to 0.045 mg/ml with the culture medium. Isolated cells were submerged in this solution for 30 rain, which delayed the next division but did not kill the cell.
, Exautogamous Cell
IrT
Line No.
t [ Day
t
I [
Control
12
AJ Fig. 1. Experimental scheme of the effect of MNNG on the cell clonal lifespan. An elliptical dot represents one cell. Horizontal lines show the maintenance of a cell line with triplet daily re-isolation culture. An arrow represents the isolation of a cell, Triangles show MNNG treatment.
Isolated cells were bathed in 4NQO diluted to 0.021 mg/ml with the culture medium for 12 or 15 rain. This treatment delayed the next cell division and occasionally killed the cells.
Experimental scheme of MNNG As shown in Fig. 1, an exautogamous cell was first isolated, and was allowed to divide 4 times until the following day. Then a single cell was isolated from the population, which produced 32 cells by the next day. Among those cells, 6 were randomly isolated as a control group and 6 others wore randomly selected to be exposed to the MNNG solution. These cell lines were maintained by the triplet re-isolation culture method (TIC) (Fukushima, 1979). The 6 cells that had boon exposed to MNNG each reproduced to 8 cells on the next day. One of those 8 cells was again exposed to MNNG. The 6 cells exposed twice wore named NGI and maintained in TIC until all the cell lines had died. The sister cells from NGI wore moreover exposed twice to MNNG, that is, the 3rd exposure was made after 6-8 cell divisions from the 2rid exposure and the 4th exposure was made after 2-3 divisions from the 3rd, Those 6 cell lines which had been exposed 4 times in total were
43
Exautogamous ! Cell 4Day O I C
named NGII and maintained in TIC until all the cell lines had died. The sister cells of NGII were exposed to the MNNG solution twice more and NGIII were formed, of which ceils were exposed to the MNNG solution 6 times in total. The aged cells of the control group that had divided over 90 times from the start of the clonal life cycle were exposed to the MNNG solution twice and this experimental group was named NGXI. The sister cells of NGXI were exposed to MNNG twice more and were named NGXII.
Experimental scheme of 4NQO An exautogamous cell was first isolated and a cell line was maintained in daily re-isolation culture (DIC). Among 32 cells reproduced from the isolated cell on the 4th day, 4 cells were randomly isolated. They divided 5 times on the next day. From each population, 5 cells were randomly isolated and maintained in DIC as control cell lines. A set of 5 lines was called 'sisters'. As shown in Fig. 2, another cell was exposed to 4NQO. This cell had divided 3 times by the following day. From the 8 reproduced cells, 4 were randomly isolated and they divided 5 times in 24 h. Five cells were randomly isolated from each population of 32 and maintained in DIC as E! cell lines. Other cells were randomly selected from each population and again exposed to 4NQO. They each reproduced 8 cells on the next
Dayl~
!
S It
oar
.. e
o
e
I
I I 1""'"IIIil'" m'JJli E
I
A & i&l | | | I~, tln
!1LI! LI! I]ll]llll]
Control
Fig. 2. Experimental scheme of the effect of 4NQO on the cell clonal lifespan. An elliptical dot represents one cell. Verlical lines show the maintenance of a cell line wilh daily re-isolation culture. A bundle of 5 cell lines is a group of sister lines. An arrow represents the isolation of a cell. Triangles show 4NQO treatment.
day, Among them, 5 cells were randomly isolated and maintained in DIC as Ell cell lines. When a cell line died in DIC, it was replaced by transferring a cell from another vigorous ccU line among the same sisters. This procedure was
TABLE I EFFECTS O F MNNG ON CLONAL LIFESPAN Line
NGIll
NGll
NGI
Control
NGXI
NGXII
I 2 3 4 5 6
181 174 173 208 172
169 149 162 200 206
145 99 161 137 146 127
132 148 219 246
127 IS2 220 129 128
243 137 169 178 135 175
Mean~SD
182±15
177±25
136±21
186±55
157±42
172±39
L.._,
• ]
** lines that became autogamous en route. * Significantly different ( p < 0.05). * * Significantly different ( p < 0.01). -,
I .,,,,
|4||| 4illl
° ' IIIII ' ' IIIII
E 1[
I
,,.,., ~
e
I t l Oby I
•
I t
44
repeated until all 5 cell lines had died. The fission number of the longest lived cell line with the replacements was called the maximum lifespan.
TABLE 3
Statistical analysis
Sisters
Control
El
Ell
The computer software, MUSCOT, Statistical Analysis and Categorical Analysis Vol. 1 (Y.D.K. Co. Ltd.) was used.
1
2 3 4
241 255 267 180
179 219 185 218
180 208 158 154
Mean±SD
236±39
200±21
175±25 *
Results
EFFECTS O F 4NQO ON THE MAXIMUM CLONAL LlFESPAN
* Significantly different from the control ( p < 0.05).
Effects of MNNG The lifespans of each cell line expressed by fission number are listed in Table 1. As two cell lines of the control and one of NGII, NGIII arm NGXI were assumed to be autogamous, their lifespans were not recorded. There were no significant differences among the mean lifespans of each group except for that of NGI. Although there was no significant differ-
Effects of 4NQO
TABLE 2 EFFECTS OF 4NOO ON THE MEAN CLONAL LIFESPAN Sisters
Control
El
Ell
I
183 189 157 122
125 16S 121 120 133
100 159 122 124
2
151 86 1{)6 156 137
159 177 154 193 160
131} 72 112 131 -
3
133 140
121 175 183 108 126
132 151 141 119 145
166 -
115 4
159 157 169
141 129 121 168 79
143± 28
143±29
llll
Mean±SD
ence between NGI and the control because of the split values of the control, the lifespan of NGI was shorter. There were significant differences between NGI and NGII (t = 2.26215, p < 0.05), and between NGI and NGiII (t = 3.2498, p < 0.01). Exposing old cells to MNNG had no definite effects on the lifespan.
- . lines that became autogamous en route, * Significantly different ( p < 0,05),
138 I08
124 104
133 125±21 *
The lifespans of all the cell lines are listed in Table 2. Table 3 shows the maximum lifespans of each sister cell line. The mean lifespan of Ell was shorter than the control and E! (tm 2.0345, p < 0.05; t = 2.0281, p < 0,05). The maximum lifespan of Ell was shorter than the control (t ffi 2.6457, p < {).05). Although there was no significant difference between the maximum lifespan of Eli and El (t--1.547.5) and between El and the control (t = 1.6101), the maximum lifespan showed a tendency to decrease upon exposure to 4NQO.
Discussion There has been much controversy over the mechanism of senescence. Although we do not accept at present that only one mechanism is at work, it is presumed that mammals have common mechanisms of senescence. Mutation theory is one of the hypotheses of mammalian senescence and many experiments have been attempted to verify it. The lifespan of rodents was reduced with ionizing irradiation (Russ and Scott, 1939; Lindop
45 and Rotblat, 1961). Chemical mutagens also reduced the lifespan of mice (Alexander and Connell, 1960). The effects of ionizing irradiation on the clonal lifespan of cultured cells were variable according to the experimental conditions, the species, the cell age, the dosage and so on (Macieira-Coelho et al., 1976; Icard et al., 1979; Ban et al., 1981). These results suggest that the induced mutations more or less affect senescence. It is obscure, however, whether such life-shortening effects are caused by the accumulated mutations in the somatic cells (Walburg, 1975). P. tetraurelia has been used as an experimental model to investigate cell clonal aging because of its clear limitation of vegetative reproduction and cellular maturation. In the present experimcnts, the effects of mutagens on clonal lifespan were studied. The results with 4 N Q O are clear, The mean lifespan of El, exposed once to 4NQO, is not different from the control. The maximum lifespan of El, however, decreased a little, Both mean and maximum lifespans of Ell are clearly decreased compared with the control. These results suggest that 4 N Q O has a life-shortening effect on clonal lifespan. The results with MNNG are more complex. The values of the Control values were available from only 4 cell lines and those were split into two. So there were no significant differences between the control and NG! or NGXI, though the values were decreased compared with the control. Repeated exposure of NGI cells extended clonal lifespan. We do not know what caused these results. Smith-Sonneborn (1979)showed that UV irradiation also reduced the clonal lifespan in P. tetraurelia. Tixador et al. (1981) reported that low doses of ionizing radiation shortened the clonal lifespan in P. tetraurelia. These experimental results suggest that mutation affects cell clonai aging, but it is not as simple as Szilard's hypothesis (1959) that aging hits accumulating on somatic cells cause aging. All animals have a limited lifespan. This suggests genetic control of aging. Even if genetic control acts, it is different from the developmen-
tal process that occurs after a particular manipulation, because the difference among individuals in senescent processes is considerable. Recently Harley et al. (1990) suggested that telomere reduction in aged human fibroblasts restricts cell doublings. Though Tetrahymena is a holotrichous ciliate and its clonal life cycle is very similar to that of Paramecium, it has an indefinite cell clonal lifespan (Nanney and Preparata, 1979). In immortal Tctrahymena, the telomere is not reduced with age (Larson et al., 1987). In mortal Paramecium, the macronuclear DNA decreases with age (Schwarts and Meister, 1973; Klass and SmithSonneborn, 1976; Takagi and Kanazawa, 1982). We do not yet have information concerning telomere decrease in senescent Paramecium. If we could detect teiomere reduction in aged Paramecium cells, it would suggest that telomcre reduction with age primarily decides cell clonal lifespan in general and that mutations affect clonal senescence by modifying telomere activity. References
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