Mmatio.n Researcl;, .7.68~.t992) 297- 31)5 f5 ;e~2 E l ~ i c r Science PubLishers BV. All righzs reserved 0027-5107/Y2/~5.00
2~
M'UT I)51~
Clastogenicity of low pH to various cultured mammalian cells T. Morita, T, Nagaki, i. F u k u d a and K. O k u m u r a "l~kuba Research l.aboratories, Nippon (,taro ltd.. 43 ]Vad~¢,T~ukubu.shi, Ibataki 300-42 f i l l ) (Rccci',~zd I I Oclul~r It~gl) (R~'~i~ re~;vcd 14 January 1~2) (Acccptcd L4 F',.:brua~' 10gT.)
K e y ~ r ~ : Lo~' pH: Chromtt~amal aberration; Cllin~sc harr~lcr c¢11 lin~; Human I~'~ph~Ic
Summaz7 It ha', be~n reported thai low pH itself can be clastogcnic to Chinese hamster ovary cells or mouse lynlphoma LS178Y cells. On the other hand, there was no indication that low pH is claslogenic to rat or human tymphocytes. Therefore, in order to evaluate the generality of clastogcnicity of low pH conditions, chromosomal abeffation tesls were carried out on Chinese hamster cell line cells (CHO-KI, CHL, Don and ~,"/9 379A) and human cells (HeLa and peripheral lymphocytcs used as whole-blood cultures). The cytotoxicity of lov, pH to each cell line was also evaluated by counting surviving cells. The treatment medium used was Eagle's MEM containing 15 mM MES or Bis-Tri.~ as an organic buffet to maintain the acidity of the medium for the 6-h or 24-h treatment period, and pH adjustment was done with NaOH or HCL Chromosomal aberrations were induced at pH 6.5 or below in CHO or CHL cells, and the maximum frequency was 24.7% at pH 6.0 or 34% at pH 6.3, respectively. About 5-10% of Don or HeLa ceils had aberrations over the range of pH 6.6-6.0 or pH 6.6-6.3, respectiYely. In V79 379A cells or human l~nphocytes, however, aberrant cells amounted to about 8% at near pH 6.0, where cell survival was low ( < 20%). About 90% of aberrations induced in each cell line examined were chromatid.typ¢ g a ~ and breaks. When CHO or CHL cells were treated with acidic medium for 6 h plus 18 h recovery in fresh medium, about 20% of cells had aberrations including chromatid exchanscs at pH 5.5 or pH 5.7, respectively. These results indicate that clastogcnicity of low pH is a general finding, although the cxlcnt of it varies with cell type, and that the clastogenici~ is associated with varying extents of ¢ytotox[city. The mechanisms of clastogenesis at low pH are not known, but might involve inlu~oition of DNA or protein synthesis or DNA-rcpair enzyme.
Wt; arc interested in the cffccts of non-physiological conditions, particularly low pH, in in vitro chromosomal aberration tests hecaus¢ of the need
Correspondent: Dr. T. Morita.Tsa~ba ReseaxchLabQratoriex. NipponGlaxoLid.,43 WadaLT~ekulya-~hi,Ibat-a~i3t]~ 42. J ap,ln.
to avoid fal~c-p~itivc responses. Brusick (1986) and Cifone et al. (1987) reported that h~v pH treatment conditions in the presence of $9 mL~ induced chromosomal aberrations in Chinese hamster ovary (CHO) cells and in mouse l~vmphoma LSt78Y cells, rcsgcctiv¢ly. Furthern~Te, we have shown that low pH can induce chromosomaI aberrations in CHO-K! cells both in the
208
absence and in the presence of $9 mix (Morita ct al., 1989, 1990, 1991). On the other hand, no chromo~mal aberraticms were observed during 4-h treatment at pH 2.73 in rat lymphocytes (Sinha et al., 1989) or during l-h treatment at pH 6.0 in human lyraphocytes (Kalweit ctal., 1990). However, these differences in results are presumably due not only to differences in the cell system used (e.g., species, tissue of origin, morphology, etc.), but also to differences in the test protocols (e.g., treatment time, presence or absence of metabolic activation, method of acidifyin~ the medidm, etc.). Recently, Sofuni et al. (1990) pointed out the importance of comparison using a common protocol for cytogenetic ashy in order to obtain qualitatively as well as quantitatively reliable results. Furthermore, though Chinese hamster cell line cells and human lymphocytes have been regarded as equally suitable fur analysis of the el~togenic potential of new chemicals, the clastogenic responses of these cells to chemicals are very different (Kirkland and Garner, 1987; Kirkland et al., 1989; Tweats and Gut,house, 1988). In this study, we investigated elastogcnie responses in various cultured mammalian cells expo~d to law pH medium using a common protocol in order to examine the generality of elastogenie response, and to gain some insight into the mechanisms of the claslogenicity of low pH. Chinese hamster ovary. (CHO-KI) cells, Chinese hamster lung (CHL) cells and human lymphuc3'tes were selected, as they are commonly used for in vitro chromosomal aberration tests in Japan, the USA and Europe. Other Chinese hamster lung cell lines (Don and V79 379A) widely used for in vitro cytogenctic assays (e.g., chromosomal aberration test, sister-chromatid exchange a~say or gene mutation assay) were also
select,;& Human epithclioid carcinoma (HeLa) cells were chosen for the purpose of comparison with human normal primary cells (human lymph~x3'te.~).
Materials and methods Cells and culture conditions Four kinds of Chinese hamster celt line cells, one derived from ovary (CHO-KI) attd three from lung (CHL V79 379A and Don) cells, and two kinds of human cells, epith~:lioid carcinoma (HeLa) cells and human peripheral lymphocytcs, were used. All cell lines were obtained from Dai-Nippon Pharmaceutical Co. Ltd. (Osaka, Japan). Human peripheral blood w ~ obtained by venepuncture from healthy male volunteer~ who were known to be non-smokers, not to have been exposed to cytotoxic chemicals or radiation, and not receiving medication. All cells except CHOKI cells were cultured in Eagle's MEM medium in a humidified atmosphere with 5c/c, COz at 37°C. CHO-KI cells were cultured in Ham's F12 medium. MEM and FI2 were purchased from Nissui Pharmaceutical Co. Ltd. (Tokyo, Japan). The media were supplemented with 10% fetal calf serum (FCS; Flow Labs., USA), kanamyein (60 #g/ml) and .'~dium bicarbonate (1"/-34 raM). Lyrnphoeyte~, (adjusted to about 5 × 10~ cells) were stimulated to divide by the addition of phytohemagglutinin-M (PHA-M; Difeo Labs., USA) at 0.1 ml per 10-ml culture. The approximate doubling times and the modal chromosome numbers of these cells were as follows: CHO-KI (13 h, 2n--21), CHL (14 h, 2n-~ 25), Don (13 h, 2n = 22), V79 379A (10 h, 2n = 22), HeLa (21 h, 2n = 52-85) and human lymphocytes (14 h, 2n-~ 40).
TABLE I CIIARACTER1SIlCS OF OR(JANIC BUFFERS
Organic buffer
Molecular weight
pKa
Suitable pH Tunsc fur u.,~."
("oncenlraliml u~d
p]| adjuslmen!
MES :' Bis-Tris ~
213.25 2119.24
6.[5 6.46
pH 5~S-7.0 pH 5 3 - 7 . 3
15 mM I~ mM
1 M NaOll i M HCI
* ME5, "4 N.rnorpholino,~ethancsulftmic acid. h Bis-'rzis, bi~2-hydtoxyethylJimino-lr~hydr~xymelhyl)melhane.
2'?)
Chemicals Two kinds of organic acids (Good's buffers) wcrc u~d as buffers. MEg (CAS No. 4432-31-9) and Bis-Tris (CAS No. fi97fi-374D wcle purchased from Dojin Chemi~l Co. (Kumamoto. Japan). The characteristics of these organic buffers arc summarized in Table 1. Preparation of acidic treatment medium Organic buffers instead of sodium bicarbonate were used in order to maintain the oH of the treatment medium. Eagle's MEM supplemented with 10% FCS, kanamycin (60 #g/ml) and 15 mM MES or Bis-Tfis was used, The medium containing MES or Bis-Tris was adjusted to pH 7.2-5,4 by the addition of 1 M NaOH or 1 M HCi, respectively. These media were filter-sterilized prior to use. The p[-! of the medium was measured prior to and after treatment using a pH meter (Coming, Model 245). Chromosomal aberration assay and celt survival measurements Cells ( 1 - 2 × 101/25-cm 2 flask, Falcon, USA) of Chinese hamster celt lines or HeLa (8 X 10+/Z¢-cm "~ flask) were ~ltured with 5 ml of complete medium fur 72 h before the acidic treatment, Human lymphocytes (as whole blood) were seeded at a density of ca. 5 × lff'/25-cm' flask
with 1(I ml of medium for 48 h be[ore the ~rcatment. PitA-M ~d', al~ includvd in the complete medium at the start of the 4g-h pre-ncatmea~ incubation. Culture medium was changed to the: acidic medium from the complete medium. In the case of human lymphocy.tes, 0.t mt of PHA-M was added to the acidic medium, "['11¢ceils were incubated in closed culture vessels for 24 or 4g h. A 6-h trt:atment was also Ix~rformed for CHO-K1 and CHL c~lls, and the cells g+cre r,.:cultured witI3 fresh complete medium for 18 h. Chromo.'.a>me preparations were made as fo]lov,s. Colchicine (final concentration of 0.2 v.g/ml) was added to the culture medium 2 h before cell hadeating. The ceils except for human (ymphoc3'tes were tr>lnsinized, and all ceils were incubated in 0.075 M hypotonic KCI solution for 15 rain at 37;C. The cells were then fixed 3 times with ice-cold fixative (methanol:glacial acetic acid. 3:1). [v,o drops of the fixed cell suspension were spread on each slide and stained with G[emsa solution. The number of cells with chromo~raal aberrations was counted on 100 well-spread metaphases. The t ~ e s of aberration were classified into 7 groups: chromatid gaps (ctg), ehromatid breaks (ctb), chromosome gaps(csg), chromosome breaks (cab), chromatid exchanges (ctc) chromosome exchanges (cse} and fragmentation (fig). Achro-
TABLE 2 SUMMARY RE.SUt2rS OF T H E CLASTOGENICi1"Y OF LOW pit.. ON VARIOUS CULTURED MAMMALI,LN CELLS Cells
Trcatmcm time (h) ~
Organic buffer
pli ,~ge ol('A t' ub,,erw~d
- ':; - of M,"txlmum aberrant ceib,
Breaks and gaps
T~vq CA
pH of 50~ cetI survb.,al
Survivatlevel at which
CA are |ir~% detected
ClIO-K|
C!11.
Don V~ 379A IteLa Hun~n II,mp~cytes
24- (I 24- 0 6-[g 24- 0 24.- O 6- IS 24- 0 24- 0 24- O 4~- 0
MES Bis-Ttis MF~ MES Bis-'i'ris MES IVIES MES MES MES
6.4.-t9 65-6.0 5.6 5.5 6.5-63 b.b-6 4 ~.8-5.6 6.6-6.ll 6.0 fi,6-6.2 65.6.3
24:: (pit b.0) 2~.5 tpH 6.2) 5.5 tplt 5.5} ~4+(l(pH6+3) 24.fl (pH 6.5) ~Orll (pH 5+7) 9.5 (pit b.4) 6,5 (pit b.0) |0+O(pll6.5) 13.0 (pll 63)
_24- 0
MEg
6.0-I8
8,2 tpii .5.9)
+ Treatment llmc-trcc(wer~ lin)c. b CA, chromt~>mal ah~:rratMm~..
g9+0G "/0.4'7~ 46,6¢'; 91,3¢~ 8~.0"~ ~.8c~ ~.2'T~ ~7r; gT.W; NR.h r;
I~'~
6+5 6+5 $+9 6.5 6.6 6.0 6.2 6.5 6.4 6+7
43~ 42c~ 375 5or; 45H 32% 78q ITH 71t~ 28C;
6,5
27'~
TABLE 3 CLASTOGENICITY AND CYTOTOXICITY OF LOW pit ON CtlO-KI CELLS (24-h TREAqMENT WI'IM MES BUFFER,I Org'anic
pH aflcr Ircatm¢lll
Survi-
CelI.~
'1=~9¢and number of aberralinn~
buffer
Initial
2,1,h
val
~:orcd"
clot
~'~1~
ctb
~b
ctc
~c
frg
.'cll~ (%) ~
Conlrol c
7.2 7.2 ?.0 6,~ 6.6 6.5 6.4 6.3 b.2 6,1 6,0 5.9 5,8
7.3 6.S 6.8 b,7 6.6 6..~ b.4 ft.3 {:).2 b.l b. I 6,fl 5,9
If~q 91 83 7b 61 53 43 38 3b 29
.~fJ ~RI l(10 109 .~lJ .'sO0 300 21HI 3~MI 2tKI
2b
.'tOg
21 13
100 No
I II II g 2 0 5 lI 32 25 20 2
0 I 0 0 0 0 0 l) 0 0 0 0
I 0 II O 2 2 20 211 114 41 11:3 12
0 0 0 O 0 0 1 0 I 0 1 l
0 I 0 0 2 0 4 0 17 6 18 3
0 tl I) O 0 O I (} 0 0 0 O
0 0 0 0 1~ 0 0 0 I 2 2 1
0.7 0.7 11 11 1.7 1.0 63 9,0 23.0 2_~1.0 24.7 160 No
ISmM ME5
(%)
Ab,erlant
melapha~e
met aph a.~¢
CelI~~er¢ expo~dfor 24 h to MEM supplementedwith MES at varioa~low pH valuc~. Pooleddata are gk~n:2011metaph~ from two experiment~,3110melapha~e~from 3 expcrimenls. b All struclural aberrations ir~luding gaps. ¢ CelL~cultured in MI'~M supplemented with sodium b i e a ~ n a t c were u~¢a as negative con;rols~ clg, chmmatid gap,s; csg, chn)mn.,c,me g~.l~; ctb. chromalid breaks; csb, chromosome breaks; ¢te, chmmatid exchanges; c~+ chromosome exchange~ including, di~nlric and ring chromn~om¢~; fre, [ra~menlatlons.
matte regions equal to or less than the width of the chromatid were scored as gaps. The c~¢otoxicity of each treatment was also examined by counting surviving (dye-excluding) cells at 24 h after the treatment. I leman lymphu~Ttes were counted after I~sis of red cells with a 0.83% ammonium chloride solution. Cells cultured in MEM supplemented with sodium bicarbonate in open vessels were used as negative controls. All experiment,~ except the 48-h treatment with HeLa cePs were done at least twice, and duplicate cultures were used in all treatments. The results of repeated experiments were similar.
Results The resulls of each examination are summarized in Table 2.
Clastogenicity of low pH with 24-h or 48-h treatrnent The clastogenicity of low pH was studied in various cultured mammalian cells with 24-h treat-
mcnt using MES as an orgau{c buffer. The results arc shown in Table 3 and Fig. I (a-0. The
adjusted pi-] of ~h¢ medium was quite stable during 24-h treatment at the initial pH of 6.6 or below in every case. Though no chromosomal aberration was observed at pH 6.5 or above in CHO-K1 ceils, the frequency of induced chromosomal aberrations was increased Iow-pH-dependently at pH 6.4 or below (Table 3). Aberrations were ob~rved over the pH range of 6.4--5.9 (cell survival of ca. 40-20%), and the maximum frequency of aberrations was about 25% at pH 6.0 In the case of CHL cells, chromosomal aberrations were ob~rved at pH 6.5-6.3 (cell survival of ca. 50-20%). The maximum frequency of aberrations was about 35% at pH 6.3. In Don cell~ only 7.0-9.5% o[ the cells had aberrations at pH 6.6-6.0 (cell survival of ca. 80-30%). Induction of a few chromosomal aberrations was observed at pH 6.5 or below in V79 379A cells: the ,naximum frequency of aberrations was 6.5% at pH 6.0 (cell survival of 17%). In human l~phocytes, it was found that 4.5-8.2% of cells had aberrations over the pH range of 6,0-5,8. However, a sufficient
3!J~
,c'[~
2~
M'UT I)51~
Clastogenicity of low pH to various cultured mammalian cells T. Morita, T, Nagaki, i. F u k u d a and K. O k u m u r a "l~kuba Research l.aboratories, Nippon (,taro ltd.. 43 ]Vad~¢,T~ukubu.shi, Ibataki 300-42 f i l l ) (Rccci',~zd I I Oclul~r It~gl) (R~'~i~ re~;vcd 14 January 1~2) (Acccptcd L4 F',.:brua~' 10gT.)
K e y ~ r ~ : Lo~' pH: Chromtt~amal aberration; Cllin~sc harr~lcr c¢11 lin~; Human I~'~ph~Ic
Summaz7 It ha', be~n reported thai low pH itself can be clastogcnic to Chinese hamster ovary cells or mouse lynlphoma LS178Y cells. On the other hand, there was no indication that low pH is claslogenic to rat or human tymphocytes. Therefore, in order to evaluate the generality of clastogcnicity of low pH conditions, chromosomal abeffation tesls were carried out on Chinese hamster cell line cells (CHO-KI, CHL, Don and ~,"/9 379A) and human cells (HeLa and peripheral lymphocytcs used as whole-blood cultures). The cytotoxicity of lov, pH to each cell line was also evaluated by counting surviving cells. The treatment medium used was Eagle's MEM containing 15 mM MES or Bis-Tri.~ as an organic buffet to maintain the acidity of the medium for the 6-h or 24-h treatment period, and pH adjustment was done with NaOH or HCL Chromosomal aberrations were induced at pH 6.5 or below in CHO or CHL cells, and the maximum frequency was 24.7% at pH 6.0 or 34% at pH 6.3, respectively. About 5-10% of Don or HeLa ceils had aberrations over the range of pH 6.6-6.0 or pH 6.6-6.3, respectiYely. In V79 379A cells or human l~nphocytes, however, aberrant cells amounted to about 8% at near pH 6.0, where cell survival was low ( < 20%). About 90% of aberrations induced in each cell line examined were chromatid.typ¢ g a ~ and breaks. When CHO or CHL cells were treated with acidic medium for 6 h plus 18 h recovery in fresh medium, about 20% of cells had aberrations including chromatid exchanscs at pH 5.5 or pH 5.7, respectively. These results indicate that clastogcnicity of low pH is a general finding, although the cxlcnt of it varies with cell type, and that the clastogenici~ is associated with varying extents of ¢ytotox[city. The mechanisms of clastogenesis at low pH are not known, but might involve inlu~oition of DNA or protein synthesis or DNA-rcpair enzyme.
Wt; arc interested in the cffccts of non-physiological conditions, particularly low pH, in in vitro chromosomal aberration tests hecaus¢ of the need
Correspondent: Dr. T. Morita.Tsa~ba ReseaxchLabQratoriex. NipponGlaxoLid.,43 WadaLT~ekulya-~hi,Ibat-a~i3t]~ 42. J ap,ln.
to avoid fal~c-p~itivc responses. Brusick (1986) and Cifone et al. (1987) reported that h~v pH treatment conditions in the presence of $9 mL~ induced chromosomal aberrations in Chinese hamster ovary (CHO) cells and in mouse l~vmphoma LSt78Y cells, rcsgcctiv¢ly. Furthern~Te, we have shown that low pH can induce chromosomaI aberrations in CHO-K! cells both in the
208
absence and in the presence of $9 mix (Morita ct al., 1989, 1990, 1991). On the other hand, no chromo~mal aberraticms were observed during 4-h treatment at pH 2.73 in rat lymphocytes (Sinha et al., 1989) or during l-h treatment at pH 6.0 in human lyraphocytes (Kalweit ctal., 1990). However, these differences in results are presumably due not only to differences in the cell system used (e.g., species, tissue of origin, morphology, etc.), but also to differences in the test protocols (e.g., treatment time, presence or absence of metabolic activation, method of acidifyin~ the medidm, etc.). Recently, Sofuni et al. (1990) pointed out the importance of comparison using a common protocol for cytogenetic ashy in order to obtain qualitatively as well as quantitatively reliable results. Furthermore, though Chinese hamster cell line cells and human lymphocytes have been regarded as equally suitable fur analysis of the el~togenic potential of new chemicals, the clastogenic responses of these cells to chemicals are very different (Kirkland and Garner, 1987; Kirkland et al., 1989; Tweats and Gut,house, 1988). In this study, we investigated elastogcnie responses in various cultured mammalian cells expo~d to law pH medium using a common protocol in order to examine the generality of elastogenie response, and to gain some insight into the mechanisms of the claslogenicity of low pH. Chinese hamster ovary. (CHO-KI) cells, Chinese hamster lung (CHL) cells and human lymphuc3'tes were selected, as they are commonly used for in vitro chromosomal aberration tests in Japan, the USA and Europe. Other Chinese hamster lung cell lines (Don and V79 379A) widely used for in vitro cytogenctic assays (e.g., chromosomal aberration test, sister-chromatid exchange a~say or gene mutation assay) were also
select,;& Human epithclioid carcinoma (HeLa) cells were chosen for the purpose of comparison with human normal primary cells (human lymph~x3'te.~).
Materials and methods Cells and culture conditions Four kinds of Chinese hamster celt line cells, one derived from ovary (CHO-KI) attd three from lung (CHL V79 379A and Don) cells, and two kinds of human cells, epith~:lioid carcinoma (HeLa) cells and human peripheral lymphocytcs, were used. All cell lines were obtained from Dai-Nippon Pharmaceutical Co. Ltd. (Osaka, Japan). Human peripheral blood w ~ obtained by venepuncture from healthy male volunteer~ who were known to be non-smokers, not to have been exposed to cytotoxic chemicals or radiation, and not receiving medication. All cells except CHOKI cells were cultured in Eagle's MEM medium in a humidified atmosphere with 5c/c, COz at 37°C. CHO-KI cells were cultured in Ham's F12 medium. MEM and FI2 were purchased from Nissui Pharmaceutical Co. Ltd. (Tokyo, Japan). The media were supplemented with 10% fetal calf serum (FCS; Flow Labs., USA), kanamyein (60 #g/ml) and .'~dium bicarbonate (1"/-34 raM). Lyrnphoeyte~, (adjusted to about 5 × 10~ cells) were stimulated to divide by the addition of phytohemagglutinin-M (PHA-M; Difeo Labs., USA) at 0.1 ml per 10-ml culture. The approximate doubling times and the modal chromosome numbers of these cells were as follows: CHO-KI (13 h, 2n--21), CHL (14 h, 2n-~ 25), Don (13 h, 2n = 22), V79 379A (10 h, 2n = 22), HeLa (21 h, 2n = 52-85) and human lymphocytes (14 h, 2n-~ 40).
TABLE I CIIARACTER1SIlCS OF OR(JANIC BUFFERS
Organic buffer
Molecular weight
pKa
Suitable pH Tunsc fur u.,~."
("oncenlraliml u~d
p]| adjuslmen!
MES :' Bis-Tris ~
213.25 2119.24
6.[5 6.46
pH 5~S-7.0 pH 5 3 - 7 . 3
15 mM I~ mM
1 M NaOll i M HCI
* ME5, "4 N.rnorpholino,~ethancsulftmic acid. h Bis-'rzis, bi~2-hydtoxyethylJimino-lr~hydr~xymelhyl)melhane.
2'?)
Chemicals Two kinds of organic acids (Good's buffers) wcrc u~d as buffers. MEg (CAS No. 4432-31-9) and Bis-Tris (CAS No. fi97fi-374D wcle purchased from Dojin Chemi~l Co. (Kumamoto. Japan). The characteristics of these organic buffers arc summarized in Table 1. Preparation of acidic treatment medium Organic buffers instead of sodium bicarbonate were used in order to maintain the oH of the treatment medium. Eagle's MEM supplemented with 10% FCS, kanamycin (60 #g/ml) and 15 mM MES or Bis-Tfis was used, The medium containing MES or Bis-Tris was adjusted to pH 7.2-5,4 by the addition of 1 M NaOH or 1 M HCi, respectively. These media were filter-sterilized prior to use. The p[-! of the medium was measured prior to and after treatment using a pH meter (Coming, Model 245). Chromosomal aberration assay and celt survival measurements Cells ( 1 - 2 × 101/25-cm 2 flask, Falcon, USA) of Chinese hamster celt lines or HeLa (8 X 10+/Z¢-cm "~ flask) were ~ltured with 5 ml of complete medium fur 72 h before the acidic treatment, Human lymphocytes (as whole blood) were seeded at a density of ca. 5 × lff'/25-cm' flask
with 1(I ml of medium for 48 h be[ore the ~rcatment. PitA-M ~d', al~ includvd in the complete medium at the start of the 4g-h pre-ncatmea~ incubation. Culture medium was changed to the: acidic medium from the complete medium. In the case of human lymphocy.tes, 0.t mt of PHA-M was added to the acidic medium, "['11¢ceils were incubated in closed culture vessels for 24 or 4g h. A 6-h trt:atment was also Ix~rformed for CHO-K1 and CHL c~lls, and the cells g+cre r,.:cultured witI3 fresh complete medium for 18 h. Chromo.'.a>me preparations were made as fo]lov,s. Colchicine (final concentration of 0.2 v.g/ml) was added to the culture medium 2 h before cell hadeating. The ceils except for human (ymphoc3'tes were tr>lnsinized, and all ceils were incubated in 0.075 M hypotonic KCI solution for 15 rain at 37;C. The cells were then fixed 3 times with ice-cold fixative (methanol:glacial acetic acid. 3:1). [v,o drops of the fixed cell suspension were spread on each slide and stained with G[emsa solution. The number of cells with chromo~raal aberrations was counted on 100 well-spread metaphases. The t ~ e s of aberration were classified into 7 groups: chromatid gaps (ctg), ehromatid breaks (ctb), chromosome gaps(csg), chromosome breaks (cab), chromatid exchanges (ctc) chromosome exchanges (cse} and fragmentation (fig). Achro-
TABLE 2 SUMMARY RE.SUt2rS OF T H E CLASTOGENICi1"Y OF LOW pit.. ON VARIOUS CULTURED MAMMALI,LN CELLS Cells
Trcatmcm time (h) ~
Organic buffer
pli ,~ge ol('A t' ub,,erw~d
- ':; - of M,"txlmum aberrant ceib,
Breaks and gaps
T~vq CA
pH of 50~ cetI survb.,al
Survivatlevel at which
CA are |ir~% detected
ClIO-K|
C!11.
Don V~ 379A IteLa Hun~n II,mp~cytes
24- (I 24- 0 6-[g 24- 0 24.- O 6- IS 24- 0 24- 0 24- O 4~- 0
MES Bis-Ttis MF~ MES Bis-'i'ris MES IVIES MES MES MES
6.4.-t9 65-6.0 5.6 5.5 6.5-63 b.b-6 4 ~.8-5.6 6.6-6.ll 6.0 fi,6-6.2 65.6.3
24:: (pit b.0) 2~.5 tpH 6.2) 5.5 tplt 5.5} ~4+(l(pH6+3) 24.fl (pH 6.5) ~Orll (pH 5+7) 9.5 (pit b.4) 6,5 (pit b.0) |0+O(pll6.5) 13.0 (pll 63)
_24- 0
MEg
6.0-I8
8,2 tpii .5.9)
+ Treatment llmc-trcc(wer~ lin)c. b CA, chromt~>mal ah~:rratMm~..
g9+0G "/0.4'7~ 46,6¢'; 91,3¢~ 8~.0"~ ~.8c~ ~.2'T~ ~7r; gT.W; NR.h r;
I~'~
6+5 6+5 $+9 6.5 6.6 6.0 6.2 6.5 6.4 6+7
43~ 42c~ 375 5or; 45H 32% 78q ITH 71t~ 28C;
6,5
27'~
TABLE 3 CLASTOGENICITY AND CYTOTOXICITY OF LOW pit ON CtlO-KI CELLS (24-h TREAqMENT WI'IM MES BUFFER,I Org'anic
pH aflcr Ircatm¢lll
Survi-
CelI.~
'1=~9¢and number of aberralinn~
buffer
Initial
2,1,h
val
~:orcd"
clot
~'~1~
ctb
~b
ctc
~c
frg
.'cll~ (%) ~
Conlrol c
7.2 7.2 ?.0 6,~ 6.6 6.5 6.4 6.3 b.2 6,1 6,0 5.9 5,8
7.3 6.S 6.8 b,7 6.6 6..~ b.4 ft.3 {:).2 b.l b. I 6,fl 5,9
If~q 91 83 7b 61 53 43 38 3b 29
.~fJ ~RI l(10 109 .~lJ .'sO0 300 21HI 3~MI 2tKI
2b
.'tOg
21 13
100 No
I II II g 2 0 5 lI 32 25 20 2
0 I 0 0 0 0 0 l) 0 0 0 0
I 0 II O 2 2 20 211 114 41 11:3 12
0 0 0 O 0 0 1 0 I 0 1 l
0 I 0 0 2 0 4 0 17 6 18 3
0 tl I) O 0 O I (} 0 0 0 O
0 0 0 0 1~ 0 0 0 I 2 2 1
0.7 0.7 11 11 1.7 1.0 63 9,0 23.0 2_~1.0 24.7 160 No
ISmM ME5
(%)
Ab,erlant
melapha~e
met aph a.~¢
CelI~~er¢ expo~dfor 24 h to MEM supplementedwith MES at varioa~low pH valuc~. Pooleddata are gk~n:2011metaph~ from two experiment~,3110melapha~e~from 3 expcrimenls. b All struclural aberrations ir~luding gaps. ¢ CelL~cultured in MI'~M supplemented with sodium b i e a ~ n a t c were u~¢a as negative con;rols~ clg, chmmatid gap,s; csg, chn)mn.,c,me g~.l~; ctb. chromalid breaks; csb, chromosome breaks; ¢te, chmmatid exchanges; c~+ chromosome exchange~ including, di~nlric and ring chromn~om¢~; fre, [ra~menlatlons.
matte regions equal to or less than the width of the chromatid were scored as gaps. The c~¢otoxicity of each treatment was also examined by counting surviving (dye-excluding) cells at 24 h after the treatment. I leman lymphu~Ttes were counted after I~sis of red cells with a 0.83% ammonium chloride solution. Cells cultured in MEM supplemented with sodium bicarbonate in open vessels were used as negative controls. All experiment,~ except the 48-h treatment with HeLa cePs were done at least twice, and duplicate cultures were used in all treatments. The results of repeated experiments were similar.
Results The resulls of each examination are summarized in Table 2.
Clastogenicity of low pH with 24-h or 48-h treatrnent The clastogenicity of low pH was studied in various cultured mammalian cells with 24-h treat-
mcnt using MES as an orgau{c buffer. The results arc shown in Table 3 and Fig. I (a-0. The
adjusted pi-] of ~h¢ medium was quite stable during 24-h treatment at the initial pH of 6.6 or below in every case. Though no chromosomal aberration was observed at pH 6.5 or above in CHO-K1 ceils, the frequency of induced chromosomal aberrations was increased Iow-pH-dependently at pH 6.4 or below (Table 3). Aberrations were ob~rved over the pH range of 6.4--5.9 (cell survival of ca. 40-20%), and the maximum frequency of aberrations was about 25% at pH 6.0 In the case of CHL cells, chromosomal aberrations were ob~rved at pH 6.5-6.3 (cell survival of ca. 50-20%). The maximum frequency of aberrations was about 35% at pH 6.3. In Don cell~ only 7.0-9.5% o[ the cells had aberrations at pH 6.6-6.0 (cell survival of ca. 80-30%). Induction of a few chromosomal aberrations was observed at pH 6.5 or below in V79 379A cells: the ,naximum frequency of aberrations was 6.5% at pH 6.0 (cell survival of 17%). In human l~phocytes, it was found that 4.5-8.2% of cells had aberrations over the pH range of 6,0-5,8. However, a sufficient
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