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Induction of mutations and chromosome aberrations in lung cells following in vivo exposure of rats to nitrogen oxides
Mutation Research, 136 (1984) 119-125 Elsevier
119
MTR 00861
Induction of mutations and chromosome aberrations in lung cells following in vivo exposure of rats to nitrogen oxides Kimio Isomura, Masatsugu Chikahira, Kiyoshi Teranishi and Kokichi Hamada Public Health Institute of Hyogo Prefecture, 2-1 Arata cho, Hyogo ku, Kobe 652 (Japan) (Received 12 January 1983) (Revision received 24 October 1983) (Accepted 8 December 1983)
Summary In order to investigate the mutagenic effects of nitrogen oxides (NOx), induced mutations and chromosome aberrations were examined using primary lung cells obtained from rats exposed in vivo to NO 2 and NO. Rats were exposed to nitrogen oxide gases at concentrations of 8-27 ppm for 3 h in a stainless steel chamber. Over the range 15-27 ppm, NO 2 significantly increased mutation to ouabain resistance. Over a similar dose range, NO significantly increased mutation only at the highest concentration (27 ppm). F o l l o w i n g N O 2 exposure, chromosome aberrations (mainly chromatid type) were induced in chromatid breaks, 2.5-11.6-fold over the control at 8 and 27 ppm, respectively.
The genetic effects caused by ozone, one of the principal oxidants in urban photochemical smog, have been reported by several workers. These include chromosome aberrations in plant root meristems (Fetner, 1958), in mammalian cells in culture (Fetner, 1962), and in lymphocytes from animals (Zelac et al., 1971a, b) or human subjects (Mertz et al., 1975) as well as sister-chromatid exchanges in mammalian cells in culture (Guerrero et al., 1979). In contrast, little experimental evidence is available for the genetic effects caused by nitrogen oxides (NOx), another class of oxidants in smog, although nitrogen dioxide (NOz) has been reported to be highly cytotoxic in vivo (Hine et al., 1970). Tsuda et al. (1981) reported that chromosome aberrations and sister-chromatid exchanges were induced in cultured Chinese hamster cells by NO 2. Previously we reported the mutagenicity of NO z and nitric oxide (NO) in bacterial cells (Teranishi et al., 1977) and that of NO in Chinese hamster cells in culture (Isomura et al., 1976). 0165-1218/84/$03.00 © 1984 Elsevier Science Publishers B.V.
In the present study, we describe the induction of mutation to ouabain resistance and the increase in number of chromosome aberrations in lung cells from rats exposed in vivo to NO and N O 2. Materials and methods
(1) NOx exposure of rats The standard gases, NO z and NO (about 2000 ppm in N2; Seitetsu Kagaku Co., Tokyo), were diluted with filtered air and introduced into a stainless steel chamber for exposure (0.35 m3). Before introduction, NO was passed through granular soda lime. The concentration of NO 2 in the chamber was measured by the method of Saltzman (1954). The concentration of N O in the chamber was similarly measured after NO had been oxidized to NO 2 using KMnO 4. During the exposure of NO, the N O z / N O ratio in the chamber routinely obtained was less than 0.05. When the concentration of gas had reached a constant value, animals to be exposed were placed in the chamber.
120
3-week-old male rats (Sprague-Dawley JCL, 2n = 42 in chromosome number), weighing 45-55 g, were employed. Groups of 5 rats were exposed to NOx for 3 h at 23-25°C. The concentrations used were 8-27 p p m for N O 2 and 9-27 p p m for NO. As a control, the rats were exposed to filtered air.
sion, obtained by vigorous pipetting, were distributed into plastic dishes (100 mm in diameter; Corning Glass Works, NY) and incubated at 37°C in a CO 2 incubator. A sufficient number of cells for the mutagenicity tests and analysis of chromosome aberrations was usually obtained after a 5-day period of incubation; medium changes were made twice during the period.
(2) Preparation of lung cells and their primary culture After exposure, animals were removed from the chamber and maintained overnight (18 h) before sacrifice. The lung cells were prepared by procedures outlined by Grover (1962) with minor modifications. For every rat the lung was excised, washed with Hanks' balanced salt solution, and finally minced with iris scissors. The tissues were then incubated with stirring in a solution of 0.8% NaC1, 0.04% KC1, and 1.0% glucose containing collagenase (type 1; Sigma Co., MO) for 40 min at room temperature. After 10% fetal bovine serum (Gibco R773321; Gibco, NY) was added to the suspension, undissociated particles were removed by several sheets of gauze. The cell suspension was centrifuged, washed once with balanced salt solution (Grover, 1962), and the resultant cell pellet was employed for primary culture. 50 ml of a-medium (Flow Laboratories Inc., VA) supplemented with 10% fetal bovine serum (growth medium) was added to the pelleted lung cells from each rat. 10-ml aliquots of the suspen-
(3) Cell surviual and mutagenicity test Cell survival was determined by examining the colony-forming ability of primary cultured lung cells from each rat. 4 inocula of 200 cells from each rat were transferred to 5 ml of growth medium in 60-mm plastic dishes (Corning Glass Works, NY). After incubation for 7 days at 37°C in a CO 2 incubator, the colonies were fixed, stained with Giemsa, and counted. 8 inocula of 2 × 105 primary cultured cells from each rat were transferred to 10 ml of growth medium in 100-mm plastic dishes. After incubation for 5 h at 37°C, medium containing the selective agent, ouabain (Merck and Co. Inc., N J) was added and the cells were refed with the selection medium 2, 5 and 9 days thereafter. After 14 days the colonies were fixed, stained with Giemsa, and counted. The mutation frequency was calculated for 105 surviving cells. The experimental design of the mutagenicity and cell survival tests is summarized in Fig. 1.
Primary Culture of
Medium
Lung Cel Is
Change
[ 2 or 3 Days ~terva[s.
Medium, 1 mM Ouabain ]
IB:-:.:-:.:.-I////////////////A Days
0
1
Exposure of Rats to Gas
2
3
/,
5
6
7
8
9
10
Inoculation " 200 or 2 x 10 5 Cells per Dish [ Medium. 1 mM Ouabain]
14
20
Fixation and counting for
Fixation and counting for
P.E.
M.F.
Expression Time in vivo [18 hours] F77"~ Expression Time in vitro
[5
days ]
Fig. 1. Experimental schema for mutagenicity tests. P.E. and M.F. show plating efficiency and mutation frequency, respectively.
121
from each rat were all combined. The resultant suspensions were then seeded in 10 ml of growth medium in 3 plastic dishes (100 mm) and incubated for 3 days at 37°C in a CO 2 incubator. Colcemid (Gibco, NY) was added at a level of 0.05 F g / m l during the last 5 h prior to harvesting. The harvested cells were treated with hypotonic Dulbecco's phosphate buffered saline : water (1 : 3.5) and fixed in acetic acid : methanol (1 : 3). They were then placed on a wet glass microslide, flame-dried, and stained with Giemsa. A large number of metaphase plates (450-550) were examined for each point.
O [0"1
Q
I0 -z
iO-~
g 10-4
o\:
.
Results
IO-5 •
0.3
0.5
0.75
I
2
Ouoboin concentrotion
(mM)
Fig. 2. Dose-response curve for PE of rat lung cells as a function of ouabain concentration. Lung cells from each of 5 pre-exposed rats were seeded at densities of 200 (0, 0.1 and 0.3 mM) or 2 x 1 0 s cells (0.5, 0.75, 1.0 and 2.0 mM) in growth medium in 100-mm dishes in quadruplicate, and 7 days later the number of colonies was counted. The medium was estimated to contain about 6 mM potassium ion together with those in 9 parts of a-medium (5.4 mM) and in 1 part of serum (10-12 mM, Baker et al., 1974). Different symbols show the data obtained in each rat.
(4) Chromos'ome preparation and staining procedure For every exposure dose of N O 2 , small aliquots of the suspension containing primary lung cells
To determine the optimal selective concentration for ouabain, the plating efficiency (PE) of rat lung cells was examined as a function of ouabain concentration (Fig. 2). The PE declined rapidly up to 0.75 mM; a plateau level in PE reflected a spontaneous mutation frequency for drug-resistant mutants of about 2 x 10 -5. Baker et al. (1974) also indicated a similar relationship between the PE and increasing concentrations of ouabain in C H O and L cells. In rodent cells in culture, the frequency of spontaneous forward mutations to ouabain resistance of the order of 10 -5 agreed with that reported by Baker et al. (1974) or with that to 8-azaguanine (8-AG) resistance reported earlier ( H a m a d a et al., 1978). For these reasons, 1 mM
TABLE 1 OUABAIN-RESISTANT MUTATIONS I N D U C E D IN L U N G CELLS BY EXPOSURE OF RATS TO NO 2 A N D NO Exposed to
Concentration (ppm)
Number of rats exposed
Plating efficiency (%)
Mutation frequency/10 s survivors Mean _+S.D. a
± S.E. b
Mean +_S.D. a
±S.E. b
40
13.3±4.8
±0.8
2.6± 1.9
±0.3
Air
Control
NO 2 NO 2 NO 2 NO 2
8 15 21 27
5 5 5 5
15.7±4.3 11.1±5.7 10.6±3.4 9.7±1.4
±1.9 ±2.5 ±1.5 ±0.6
4.4± 1.8 31.6±21.6 29.1±12.0 55.4±13.3
±0.8 ±9.4 ±5.4 ±5.9
NO NO NO
9 19 27
5 5 5
12.7±3.2 9.2±2.2 14.8±3.3
±1.4 ±1.0 ±1.5
6.2± 5.6 5.2± 1.8 9.5± 3.0
±2.5 ±0.8 ±1.4
a S.D., standard deviation. b S.E., standard error.
122
700-
A
"700
•
500.
(D
500-
B
-500
t
500
0 0.
o. n x
300-
= 300
o
_=J., __ =o O
O o
J
0
~
• -----'--'-
E 0
I00
II:lO0
o
IO0-
300
• I00
0__._~0.0~ 0 /
o
O- ,'
"0
0
2 • •
4
6
NO=
(pprn)
TAIO0
8
0
I0
0 0
5
I0
15
20
25
30
NO (pprn)
OZX
TAI535
Fig. 3. Mutagenic activity of NO 2 and NO in Salmonella typhimurium. The mutagenicity test was performed essentially as reported by Ames et al. (1973) and McCann et al. (1975). About 107 (for mutation) or 2-3 x 102 (for survival) histidine-requiring bacterial cells were seeded on minimal agar plates with or without histidine, respectively and were exposed to gases for 40 min at a flow rate of 1 l / m i n in a desiccator. After a 2-day incubation at 37°C, prototrophic revertants or survival colonies were counted. (A) Mutagenic activity of NO 2 in strains TA100 and TA1535. No mutagenic activity was found in TA98 and TA1538 over a similar dose range of NO 2. e, mutagenic activity in TA100; A, survival in TA100; O, mutagenic activity in TA1535; zx, survival in TA1535. (B) Mutagenic activity of NO in strain TA100. NO 2 concentration being contained in NO exposure was as follows: 0.1 ppm (at 5.1 ppm of NO), 0.2 ppm (at 8.4 ppm), 0.5 ppm (at 13.4 ppm), 1.1 ppm (at 18.1 ppm), 2.1 ppm (at 23.6 ppm) and 2.5 ppm (at 29.6 ppm). II, mutagenic activity; O, survival.
151.00
° ' ~ o ~'- o oOO o_~_o_.a8 oO_..___o_
o
o
% -
lO-
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0.10
c o m
o
'.9
ab
tS ,
o
,
" ,
,
,
,
.
"0.01 ,
,
,
IO Concentration of NO (pprn)
,
20
,
Fig. 4. Cytotoxicity and mutagenic activity of NO in cultured Chinese hamster Don cells. Cells were grown in Eagle's MEM containing 10% fetal calf serum (Microbiological Associates Inc., MD), 4 mM L-glutamine, 1 mM sodium pyruvate, and 0.2 mM L-serine at 37°C in a CO 2 incubator. Exposure of cells to gas was essentially the same as reported by Fetner (1962). After an 18-h incubation of 106 cells in TD40 culture bottle (Ikemoto Rika Co. Ltd., Tokyo), cells were washed 3 times with 10 ml of Hanks' balanced salt solution, and the bottle, upside-down, was exposed to gases for 10 min at a flow rate of 100 ml/min. For cytotoxicity, 3 inocula of 150 cells were seeded in 60-mm glass plates (Tyston, Tokyo) after exposure, incubated for 7 days at 37°C, and the number of colonies counted. For mutation frequency, immediately after the exposure the cells in the culture bottle were incubated in fresh medium for 48 h (expression time). 4 inocula of 3 x l0 s cells were seeded in growth medium in 90-mm glass plates, and 5-6 h later the medium was replaced with selection medium containing 10 ~ g / m l of 8,AG (Shigma). Cells were refed with the selective agent at 3, 7 and 11 days thereafter, and 12-14 days after seeding 8-AG-resistant colonies were counted. The colony-forming ability after expression time was determined as in cytotoxicity. O, cytotoxicity of NO; e, mutagenic activity of NO.
123 TABLE2 C H R O M O S O M E A B E R R A T I O N S I N D U C E D IN L U N G CELLS BY EXPOSURE OF RATS TO NO 2 Concentra-
Number
Abnormal
Aberrations/100metaphases
tion (ppm)
of cells scored
metaphases
Chromatid
Chromoso-
Chromatid
Chromoso-
Chromatid
gaps
me gaps
breaks
me breaks
exchanges
Multiple aberrations a
440 550 550
26 67 195
3.2 4.4 16.5
0 0.2 0.4
2.3 5.8 26.7
0.2 0.2 1.6
0 0.4 0.9
0 0 0.5
0 (air) 8 27
Metaphases with multiple aberrations; types of aberration could not be distinguished.
ouabain was used as selective agent in subsequent mutagenicity tests. Enhanced mutations to ouabain resistance in lung cells of rats exposed to N O 2 and N O are shown in Table 1. In these experiments, the average PE for each point of exposure was similar to the air-exposed control (about 13%). A dose-related increase in the mutation frequency was clearly observed following N O 2 exposure. That is, N O 2 significantly increased the mutation frequencies compared to the control with increasing doses, up to 21-fold at 27 ppm. The mutagenic activity of N O was much less than that of NO2; the increase in mutation over the control was significant only at the 27-ppm dose level. Statistical analysis by t-test revealed that the frequency shown at 27 p p m was significantly different from that shown ha the control', whereas the values at 9 and 19 p p m were indistinguishable from that in the control ( P < 0.05). Chromosome aberrations in lung cells from rats exposed to N O 2 were also investigated in order to see whether the aberrations would parallel mutation (Table 2). Lung cells from rats exposed to N O 2 showed a significant dose-related increase in aberrations (chromatid type). For example, chromatid breaks were increased 2.5- and 12-fold over the control at 8 and 27 ppm, respectively. Thus it was clearly demonstrated that N O 2 and N O can induce mutations and that N O 2 also has the ability to induce chromosome aberrations in lung cells of rats in vivo. Discussion
We have previously investigated the mutagenicity of N O 2 and N O in Salmonella typhimurium
strains (TA1535 and TA100) (Teranishi et al., 1977) and in Chinese hamster Don cells (Isomura et al., 1976). In the bacterial cells, increasing doses of N O 2 increased the frequency of base-pair change mutations (at concentrations of about 2-10 p p m for 40 min) (Fig. 3A); in contrast, N O exposure over 20 p p m for the same period increased the mutation frequency only slightly (Fig. 3B). These results agree with those observed in lung cells from rats exposed in vivo. However, N O induced mutations to 8-AG resistance in cultured Chinese hamster cells (Fig. 4). Little or no mutations were induced by N O 2, but a very short expression time (48 h) was used (Isomura et al., 1976). It was shown later that 8-AG- or 6-thioguanine (6-TG)resistant mutations require a longer expression time than ouabain-resistant mutations (Trosko et al., 1977; Chang et al., 1978; Bonatti et al., 1980). Thus, the 48 h employed for NOx-induced mutations to 8-AG resistance in the earlier report, was probably too short for the optimal expression of NO2-induced mutations. The response to N O may also have been underestimated. Induced chromosome aberrations in lung cells following N O 2 exposure of rats were mainly of the chromatid type. Tsuda et al. (1981) also indicated that a significant number of chromosome aberrations (mainly chromatid gaps and breaks) were induced in Chinese hamster V79 cells by N O 2 exposure at concentrations of 10-50 ppm. For detection of presumptive clastogens, it is generally accepted and recommended that the chromosome preparations be made before the first cell division. These recommendations are derived from results following treatment with ionizing radiation, chemical agents, and viruses (Evans and
124
O'Riordan, 1975). In the present study, chromosome aberrations were detected in cells after several (at least 4-5) divisions following NO 2 exposure in vivo. It has clearly been established that the chromosome is not multistranded, but is a single DNA double helix (Wolff and Perry, 1975). From this, a likely explanation of the persistence of aberrations is that the lesions induced by NO 2 or something produced by it in the cell, are longlived, i.e., not easily repaired by these cells. According to the mechanism of NO 2 toxicity described by Menzel (1980), NO 2 (a partially free inorganic radical) reacts with fatty acids to give rise to an alkyl-free radical (R), and a peroxyl-free radical (RO~) is subsequently produced via oxygen scavenging of the alkyl free radical. The resulting peroxyl-free radical can produce lipid peroxides (Thomas et al., 1968), abstracting hydrogen from fatty acids to produce the alkyl-free radical again. This reaction is reduced by antioxidants such as vitamin E (Thomas et al., 1968) through the inactivation of the peroxyl-free radical. The peroxyl-free radical can also lead to epoxides and alkoxy free radicals ( R O ) considered to be responsible for the alkylation of DNA (Menzel, 1980). The mechanism whereby N O 2 induces mutations and chromosome aberrations is not clear, but may involve the alkylation of DNA by NO 2. The reactivity of NO with cellular constituents is more poorly understood than that of N O 2. Several authors have described some substances that decrease the biological effects of gases on mammalian cells in culture. Fetner (1962) and Tsuda et al. (1979) indicated that chromosome aberrations in culture cells were not induced by ozone and N O 2 gases, respectively, if cells were not washed with balanced salt solution prior to exposure. The latter authors also suggested that certain materials, protective against gas effects, might be present in the culture medium or cell surface. Hurst et al. (1971) reported the loss of the oxidizing property of ozone in the presence of glutathione or serum protein. Guerrero et al. (1979) reported the lack of sister-chromatid exchanges in the lymphocytes exposed to ozone in vivo, in contrast to the presence of exchanges in cells exposed in vitro to ozone. They suggested that the lack of sister-chromatid exchanges in the lymphocytes could be attributed to the protective effect of
serum a n d / o r other agents. The lymphocyte system offers many advantages for chromosome analysis, including the availability of large numbers of synchronized cells (Evans and O'Riordan, 1975). Lymphocytes obtained from animals exposed to gases in vivo, may, however, reflect biological effects which have been reduced by substances in the blood. The lung cells are affected to a much lesser extent by blood constituents, and may assess more accurately direct effects of gases on cells. In this respect, the use of lung cells reported here may provide reliable information on the genetic effects of gases. In the present study, we clearly demonstrated the presence of cells with induced mutations, or with increased chromosome aberrations in animals exposed in vivo to NO 2 or NO. We believe that this is the first report on the mutagenicity of gas, studied from the practical viewpoint of determining biological effects of gases on individuals in the environment. Other gases such as SO 2 (Ma et al., 1973) or mixtures of gases should be studied to determine their potential.
Acknowledgement A part of this work was supported by a Grantsin-Aid for Scientific Research from the Ministry of Education, Science and Culture (1978), Japan.
References Ames, B.N., F.D. Lee and W.E. Durston (1973) An improved bacterial test system for the detection and classification of mutagens and carcinogens, Proc. Natl. Acad. Sci. (U.S.A.), 70, 782-786. Baker, R.M., D.M. Brunette, R. Mankovitz, L.H. Thompson, G.F. Whitmore, L. Siminovitch and J.E. Till (1974) Ouabain-resistant mutants of mouse and hamster cells in culture, Cell, 1, 9 21. Bonatti, S., A. Abbondandolo, A. Mazzaccaro and R. Fiorio (1980) Experiments on the effects of the medium in mutation tests with the H G P R T system in cultured mammalian cells, Mutation Res., 72, 475-482. Chang, C.C., J.E. Trosko and T. Akera (1978) Characterization of ultraviolet light-induced ouab~/in-resistant mutations in Chinese hamster cells, Mutation Res., 51, 85-98. Evans, H.J., and M.L. O'Riordan (1975) H u m a n peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests, Mutation Res., 31, 135-148. Fetner, R.H. (1958) Chromosome breakage in Vicia faba by ozone, Nature (London), 18, 504-505.
125 Fetner, R.H. (1962) Ozone-induced chromosome breakage in human cell culture, Nature (London), 194, 793-794. Grover, J.W. (1962) The influence of age and environmental factors on the behavior of reaggregated embryonic lung cells in culture, Exp. Cell Res., 26, 344-359. Guerrero, R.R., D.E. Rounds, R.S. Olson and J.D. Hackeny (1979) Mutagenic effects of ozone on human cells exposed in vivo and in vitro based on sister chromatid exchange analysis, Environ. Res., 18, 336-346. Hamada, K., K. Isomura, K. Teranishi and H. Watanabe (1978) Induction and isolation of frameshift mutations in cultured Chinese hamster Don cells, Mutation Res., 54, 61-72. Hine, C.H., F.H. Meyers and R.W. Wright (1970) Pulmonary changes in animals exposed to nitrogen dioxide, Effects of acute exposures, Toxicol. Appl. Pharmacol., 16, 201-213. Hurst, D.J., and D.E. Coffin (1971) Ozone effect on lysosomal hydrolases of alveolar macrophages in vitro, Arch. Intern. Med., 27, 1059-1063. lsomura, K., O. Fukase and H. Watanabe (1976) Cytotoxicities and mutagenicities of gaseous air pollutants on cultured cells, Taikiosen Kenkyu, 11, 59-64 (in Japanese). Ma, T., D. Isbandi, S. Khan and Y. Tseng (1973) Low level of SO 2 enhanced chromatid aberrations in Tradescantia pollen tubes and seasonal variation of the aberration rates, Mutation Res., 21, 93-100. McCann, J., N.E. Spingarn, J. Kobori and B.N. Ames (1975) Detection of carcinogens as mutagens: Bacterial tester strains with R factor plasmids, Proc. Natl. Acad. Sci. (U.S.A.), 72, 979-983. Menzel, D.B. (1980) Pharmacological mechanisms in the toxicity of nitrogen dioxide and its relation to obstructive respiratory disease, in: S.D. Lee (Ed.), Nitrogen Oxides and their Effects on Health, Ann Arbor Science, Ann Arbor, MI. Mertz, T., M.A. Bender, H.D. Kerr and T.J. Kulle (1975) Observations of aberrations in chromosomes of lympho-
cytes from human subjects exposed to ozone at a concentration of 0.5 ppm for 6 and 10 hours, Mutation Res., 31, 299-302. Saltzman, B.E. (1954) Colorimetric microdetermination of nitrogen dioxide in the atmosphere, Anal. Chem., 26, 1949-1955. Teranishi, K., O. Fukase, K. Hamada, N. Takeda and H. Watanabe (1977) Mutagenicity of the gaseous air pollutants, in: Abstracts of Papers Presented at the Annual Meeting of Japan Society of Air Pollution, 18, 228 (in Japanese). Thomas, H.V., P.K. Mueller and R.L. Lyman (1968) Lipoperoxidation of lung lipids of rats exposed to nitrogen dioxide, Science, 159, 532-534. Trosko, J.E., C.C. Chang, L.P. Yotti and H.Y. Chu (1977) Effect of phorbol myristate acetate on the recovery of spontaneous and ultraviolet light-induced 6-thioguanine and ouabain-resistant Chinese hamster cells, Cancer Res., 37, 188-193. Tsuda, H., A. Kushi, D. Yoshida and F. Goto (1981) Chromosomal aberrations and sister-chromatid exchanges induced by gaseous nitrogen dioxide in cultured Chinese hamster cells, Mutation Res., 89, 303-309. Wolff, S.H., and P. Perry (1975) Insights on chromosome structure from sister chromatid exchange ratios and the lack of both isolabelling and heterolabelling as determined by the FPG technique, Exp. Cell Res., 93, 23-30. Zelac, R.E., H.L. Cromroy, W.E. Bloch Jr., B.G. Dunavant and H.A. Bevis (1971a) Inhaled ozone as a mutagen, I. Chromosome aberrations induced in Chinese hamster lymphocytes, Environ. Res., 4, 262-282. Zelac, R.E., H.L. Cromroy, W.E. Bolch Jr., B.G. Dunavant and H.A. Bevis (1971b) Inhaled ozone as a mutagen, II. Effect on the frequency of chromosome aberrations observed in irradiated Chinese hamsters, Environ. Res., 4, 325-342.
119
MTR 00861
Induction of mutations and chromosome aberrations in lung cells following in vivo exposure of rats to nitrogen oxides Kimio Isomura, Masatsugu Chikahira, Kiyoshi Teranishi and Kokichi Hamada Public Health Institute of Hyogo Prefecture, 2-1 Arata cho, Hyogo ku, Kobe 652 (Japan) (Received 12 January 1983) (Revision received 24 October 1983) (Accepted 8 December 1983)
Summary In order to investigate the mutagenic effects of nitrogen oxides (NOx), induced mutations and chromosome aberrations were examined using primary lung cells obtained from rats exposed in vivo to NO 2 and NO. Rats were exposed to nitrogen oxide gases at concentrations of 8-27 ppm for 3 h in a stainless steel chamber. Over the range 15-27 ppm, NO 2 significantly increased mutation to ouabain resistance. Over a similar dose range, NO significantly increased mutation only at the highest concentration (27 ppm). F o l l o w i n g N O 2 exposure, chromosome aberrations (mainly chromatid type) were induced in chromatid breaks, 2.5-11.6-fold over the control at 8 and 27 ppm, respectively.
The genetic effects caused by ozone, one of the principal oxidants in urban photochemical smog, have been reported by several workers. These include chromosome aberrations in plant root meristems (Fetner, 1958), in mammalian cells in culture (Fetner, 1962), and in lymphocytes from animals (Zelac et al., 1971a, b) or human subjects (Mertz et al., 1975) as well as sister-chromatid exchanges in mammalian cells in culture (Guerrero et al., 1979). In contrast, little experimental evidence is available for the genetic effects caused by nitrogen oxides (NOx), another class of oxidants in smog, although nitrogen dioxide (NOz) has been reported to be highly cytotoxic in vivo (Hine et al., 1970). Tsuda et al. (1981) reported that chromosome aberrations and sister-chromatid exchanges were induced in cultured Chinese hamster cells by NO 2. Previously we reported the mutagenicity of NO z and nitric oxide (NO) in bacterial cells (Teranishi et al., 1977) and that of NO in Chinese hamster cells in culture (Isomura et al., 1976). 0165-1218/84/$03.00 © 1984 Elsevier Science Publishers B.V.
In the present study, we describe the induction of mutation to ouabain resistance and the increase in number of chromosome aberrations in lung cells from rats exposed in vivo to NO and N O 2. Materials and methods
(1) NOx exposure of rats The standard gases, NO z and NO (about 2000 ppm in N2; Seitetsu Kagaku Co., Tokyo), were diluted with filtered air and introduced into a stainless steel chamber for exposure (0.35 m3). Before introduction, NO was passed through granular soda lime. The concentration of NO 2 in the chamber was measured by the method of Saltzman (1954). The concentration of N O in the chamber was similarly measured after NO had been oxidized to NO 2 using KMnO 4. During the exposure of NO, the N O z / N O ratio in the chamber routinely obtained was less than 0.05. When the concentration of gas had reached a constant value, animals to be exposed were placed in the chamber.
120
3-week-old male rats (Sprague-Dawley JCL, 2n = 42 in chromosome number), weighing 45-55 g, were employed. Groups of 5 rats were exposed to NOx for 3 h at 23-25°C. The concentrations used were 8-27 p p m for N O 2 and 9-27 p p m for NO. As a control, the rats were exposed to filtered air.
sion, obtained by vigorous pipetting, were distributed into plastic dishes (100 mm in diameter; Corning Glass Works, NY) and incubated at 37°C in a CO 2 incubator. A sufficient number of cells for the mutagenicity tests and analysis of chromosome aberrations was usually obtained after a 5-day period of incubation; medium changes were made twice during the period.
(2) Preparation of lung cells and their primary culture After exposure, animals were removed from the chamber and maintained overnight (18 h) before sacrifice. The lung cells were prepared by procedures outlined by Grover (1962) with minor modifications. For every rat the lung was excised, washed with Hanks' balanced salt solution, and finally minced with iris scissors. The tissues were then incubated with stirring in a solution of 0.8% NaC1, 0.04% KC1, and 1.0% glucose containing collagenase (type 1; Sigma Co., MO) for 40 min at room temperature. After 10% fetal bovine serum (Gibco R773321; Gibco, NY) was added to the suspension, undissociated particles were removed by several sheets of gauze. The cell suspension was centrifuged, washed once with balanced salt solution (Grover, 1962), and the resultant cell pellet was employed for primary culture. 50 ml of a-medium (Flow Laboratories Inc., VA) supplemented with 10% fetal bovine serum (growth medium) was added to the pelleted lung cells from each rat. 10-ml aliquots of the suspen-
(3) Cell surviual and mutagenicity test Cell survival was determined by examining the colony-forming ability of primary cultured lung cells from each rat. 4 inocula of 200 cells from each rat were transferred to 5 ml of growth medium in 60-mm plastic dishes (Corning Glass Works, NY). After incubation for 7 days at 37°C in a CO 2 incubator, the colonies were fixed, stained with Giemsa, and counted. 8 inocula of 2 × 105 primary cultured cells from each rat were transferred to 10 ml of growth medium in 100-mm plastic dishes. After incubation for 5 h at 37°C, medium containing the selective agent, ouabain (Merck and Co. Inc., N J) was added and the cells were refed with the selection medium 2, 5 and 9 days thereafter. After 14 days the colonies were fixed, stained with Giemsa, and counted. The mutation frequency was calculated for 105 surviving cells. The experimental design of the mutagenicity and cell survival tests is summarized in Fig. 1.
Primary Culture of
Medium
Lung Cel Is
Change
[ 2 or 3 Days ~terva[s.
Medium, 1 mM Ouabain ]
IB:-:.:-:.:.-I////////////////A Days
0
1
Exposure of Rats to Gas
2
3
/,
5
6
7
8
9
10
Inoculation " 200 or 2 x 10 5 Cells per Dish [ Medium. 1 mM Ouabain]
14
20
Fixation and counting for
Fixation and counting for
P.E.
M.F.
Expression Time in vivo [18 hours] F77"~ Expression Time in vitro
[5
days ]
Fig. 1. Experimental schema for mutagenicity tests. P.E. and M.F. show plating efficiency and mutation frequency, respectively.
121
from each rat were all combined. The resultant suspensions were then seeded in 10 ml of growth medium in 3 plastic dishes (100 mm) and incubated for 3 days at 37°C in a CO 2 incubator. Colcemid (Gibco, NY) was added at a level of 0.05 F g / m l during the last 5 h prior to harvesting. The harvested cells were treated with hypotonic Dulbecco's phosphate buffered saline : water (1 : 3.5) and fixed in acetic acid : methanol (1 : 3). They were then placed on a wet glass microslide, flame-dried, and stained with Giemsa. A large number of metaphase plates (450-550) were examined for each point.
O [0"1
Q
I0 -z
iO-~
g 10-4
o\:
.
Results
IO-5 •
0.3
0.5
0.75
I
2
Ouoboin concentrotion
(mM)
Fig. 2. Dose-response curve for PE of rat lung cells as a function of ouabain concentration. Lung cells from each of 5 pre-exposed rats were seeded at densities of 200 (0, 0.1 and 0.3 mM) or 2 x 1 0 s cells (0.5, 0.75, 1.0 and 2.0 mM) in growth medium in 100-mm dishes in quadruplicate, and 7 days later the number of colonies was counted. The medium was estimated to contain about 6 mM potassium ion together with those in 9 parts of a-medium (5.4 mM) and in 1 part of serum (10-12 mM, Baker et al., 1974). Different symbols show the data obtained in each rat.
(4) Chromos'ome preparation and staining procedure For every exposure dose of N O 2 , small aliquots of the suspension containing primary lung cells
To determine the optimal selective concentration for ouabain, the plating efficiency (PE) of rat lung cells was examined as a function of ouabain concentration (Fig. 2). The PE declined rapidly up to 0.75 mM; a plateau level in PE reflected a spontaneous mutation frequency for drug-resistant mutants of about 2 x 10 -5. Baker et al. (1974) also indicated a similar relationship between the PE and increasing concentrations of ouabain in C H O and L cells. In rodent cells in culture, the frequency of spontaneous forward mutations to ouabain resistance of the order of 10 -5 agreed with that reported by Baker et al. (1974) or with that to 8-azaguanine (8-AG) resistance reported earlier ( H a m a d a et al., 1978). For these reasons, 1 mM
TABLE 1 OUABAIN-RESISTANT MUTATIONS I N D U C E D IN L U N G CELLS BY EXPOSURE OF RATS TO NO 2 A N D NO Exposed to
Concentration (ppm)
Number of rats exposed
Plating efficiency (%)
Mutation frequency/10 s survivors Mean _+S.D. a
± S.E. b
Mean +_S.D. a
±S.E. b
40
13.3±4.8
±0.8
2.6± 1.9
±0.3
Air
Control
NO 2 NO 2 NO 2 NO 2
8 15 21 27
5 5 5 5
15.7±4.3 11.1±5.7 10.6±3.4 9.7±1.4
±1.9 ±2.5 ±1.5 ±0.6
4.4± 1.8 31.6±21.6 29.1±12.0 55.4±13.3
±0.8 ±9.4 ±5.4 ±5.9
NO NO NO
9 19 27
5 5 5
12.7±3.2 9.2±2.2 14.8±3.3
±1.4 ±1.0 ±1.5
6.2± 5.6 5.2± 1.8 9.5± 3.0
±2.5 ±0.8 ±1.4
a S.D., standard deviation. b S.E., standard error.
122
700-
A
"700
•
500.
(D
500-
B
-500
t
500
0 0.
o. n x
300-
= 300
o
_=J., __ =o O
O o
J
0
~
• -----'--'-
E 0
I00
II:lO0
o
IO0-
300
• I00
0__._~0.0~ 0 /
o
O- ,'
"0
0
2 • •
4
6
NO=
(pprn)
TAIO0
8
0
I0
0 0
5
I0
15
20
25
30
NO (pprn)
OZX
TAI535
Fig. 3. Mutagenic activity of NO 2 and NO in Salmonella typhimurium. The mutagenicity test was performed essentially as reported by Ames et al. (1973) and McCann et al. (1975). About 107 (for mutation) or 2-3 x 102 (for survival) histidine-requiring bacterial cells were seeded on minimal agar plates with or without histidine, respectively and were exposed to gases for 40 min at a flow rate of 1 l / m i n in a desiccator. After a 2-day incubation at 37°C, prototrophic revertants or survival colonies were counted. (A) Mutagenic activity of NO 2 in strains TA100 and TA1535. No mutagenic activity was found in TA98 and TA1538 over a similar dose range of NO 2. e, mutagenic activity in TA100; A, survival in TA100; O, mutagenic activity in TA1535; zx, survival in TA1535. (B) Mutagenic activity of NO in strain TA100. NO 2 concentration being contained in NO exposure was as follows: 0.1 ppm (at 5.1 ppm of NO), 0.2 ppm (at 8.4 ppm), 0.5 ppm (at 13.4 ppm), 1.1 ppm (at 18.1 ppm), 2.1 ppm (at 23.6 ppm) and 2.5 ppm (at 29.6 ppm). II, mutagenic activity; O, survival.
151.00
° ' ~ o ~'- o oOO o_~_o_.a8 oO_..___o_
o
o
% -
lO-
of~l--
0.10
c o m
o
'.9
ab
tS ,
o
,
" ,
,
,
,
.
"0.01 ,
,
,
IO Concentration of NO (pprn)
,
20
,
Fig. 4. Cytotoxicity and mutagenic activity of NO in cultured Chinese hamster Don cells. Cells were grown in Eagle's MEM containing 10% fetal calf serum (Microbiological Associates Inc., MD), 4 mM L-glutamine, 1 mM sodium pyruvate, and 0.2 mM L-serine at 37°C in a CO 2 incubator. Exposure of cells to gas was essentially the same as reported by Fetner (1962). After an 18-h incubation of 106 cells in TD40 culture bottle (Ikemoto Rika Co. Ltd., Tokyo), cells were washed 3 times with 10 ml of Hanks' balanced salt solution, and the bottle, upside-down, was exposed to gases for 10 min at a flow rate of 100 ml/min. For cytotoxicity, 3 inocula of 150 cells were seeded in 60-mm glass plates (Tyston, Tokyo) after exposure, incubated for 7 days at 37°C, and the number of colonies counted. For mutation frequency, immediately after the exposure the cells in the culture bottle were incubated in fresh medium for 48 h (expression time). 4 inocula of 3 x l0 s cells were seeded in growth medium in 90-mm glass plates, and 5-6 h later the medium was replaced with selection medium containing 10 ~ g / m l of 8,AG (Shigma). Cells were refed with the selective agent at 3, 7 and 11 days thereafter, and 12-14 days after seeding 8-AG-resistant colonies were counted. The colony-forming ability after expression time was determined as in cytotoxicity. O, cytotoxicity of NO; e, mutagenic activity of NO.
123 TABLE2 C H R O M O S O M E A B E R R A T I O N S I N D U C E D IN L U N G CELLS BY EXPOSURE OF RATS TO NO 2 Concentra-
Number
Abnormal
Aberrations/100metaphases
tion (ppm)
of cells scored
metaphases
Chromatid
Chromoso-
Chromatid
Chromoso-
Chromatid
gaps
me gaps
breaks
me breaks
exchanges
Multiple aberrations a
440 550 550
26 67 195
3.2 4.4 16.5
0 0.2 0.4
2.3 5.8 26.7
0.2 0.2 1.6
0 0.4 0.9
0 0 0.5
0 (air) 8 27
Metaphases with multiple aberrations; types of aberration could not be distinguished.
ouabain was used as selective agent in subsequent mutagenicity tests. Enhanced mutations to ouabain resistance in lung cells of rats exposed to N O 2 and N O are shown in Table 1. In these experiments, the average PE for each point of exposure was similar to the air-exposed control (about 13%). A dose-related increase in the mutation frequency was clearly observed following N O 2 exposure. That is, N O 2 significantly increased the mutation frequencies compared to the control with increasing doses, up to 21-fold at 27 ppm. The mutagenic activity of N O was much less than that of NO2; the increase in mutation over the control was significant only at the 27-ppm dose level. Statistical analysis by t-test revealed that the frequency shown at 27 p p m was significantly different from that shown ha the control', whereas the values at 9 and 19 p p m were indistinguishable from that in the control ( P < 0.05). Chromosome aberrations in lung cells from rats exposed to N O 2 were also investigated in order to see whether the aberrations would parallel mutation (Table 2). Lung cells from rats exposed to N O 2 showed a significant dose-related increase in aberrations (chromatid type). For example, chromatid breaks were increased 2.5- and 12-fold over the control at 8 and 27 ppm, respectively. Thus it was clearly demonstrated that N O 2 and N O can induce mutations and that N O 2 also has the ability to induce chromosome aberrations in lung cells of rats in vivo. Discussion
We have previously investigated the mutagenicity of N O 2 and N O in Salmonella typhimurium
strains (TA1535 and TA100) (Teranishi et al., 1977) and in Chinese hamster Don cells (Isomura et al., 1976). In the bacterial cells, increasing doses of N O 2 increased the frequency of base-pair change mutations (at concentrations of about 2-10 p p m for 40 min) (Fig. 3A); in contrast, N O exposure over 20 p p m for the same period increased the mutation frequency only slightly (Fig. 3B). These results agree with those observed in lung cells from rats exposed in vivo. However, N O induced mutations to 8-AG resistance in cultured Chinese hamster cells (Fig. 4). Little or no mutations were induced by N O 2, but a very short expression time (48 h) was used (Isomura et al., 1976). It was shown later that 8-AG- or 6-thioguanine (6-TG)resistant mutations require a longer expression time than ouabain-resistant mutations (Trosko et al., 1977; Chang et al., 1978; Bonatti et al., 1980). Thus, the 48 h employed for NOx-induced mutations to 8-AG resistance in the earlier report, was probably too short for the optimal expression of NO2-induced mutations. The response to N O may also have been underestimated. Induced chromosome aberrations in lung cells following N O 2 exposure of rats were mainly of the chromatid type. Tsuda et al. (1981) also indicated that a significant number of chromosome aberrations (mainly chromatid gaps and breaks) were induced in Chinese hamster V79 cells by N O 2 exposure at concentrations of 10-50 ppm. For detection of presumptive clastogens, it is generally accepted and recommended that the chromosome preparations be made before the first cell division. These recommendations are derived from results following treatment with ionizing radiation, chemical agents, and viruses (Evans and
124
O'Riordan, 1975). In the present study, chromosome aberrations were detected in cells after several (at least 4-5) divisions following NO 2 exposure in vivo. It has clearly been established that the chromosome is not multistranded, but is a single DNA double helix (Wolff and Perry, 1975). From this, a likely explanation of the persistence of aberrations is that the lesions induced by NO 2 or something produced by it in the cell, are longlived, i.e., not easily repaired by these cells. According to the mechanism of NO 2 toxicity described by Menzel (1980), NO 2 (a partially free inorganic radical) reacts with fatty acids to give rise to an alkyl-free radical (R), and a peroxyl-free radical (RO~) is subsequently produced via oxygen scavenging of the alkyl free radical. The resulting peroxyl-free radical can produce lipid peroxides (Thomas et al., 1968), abstracting hydrogen from fatty acids to produce the alkyl-free radical again. This reaction is reduced by antioxidants such as vitamin E (Thomas et al., 1968) through the inactivation of the peroxyl-free radical. The peroxyl-free radical can also lead to epoxides and alkoxy free radicals ( R O ) considered to be responsible for the alkylation of DNA (Menzel, 1980). The mechanism whereby N O 2 induces mutations and chromosome aberrations is not clear, but may involve the alkylation of DNA by NO 2. The reactivity of NO with cellular constituents is more poorly understood than that of N O 2. Several authors have described some substances that decrease the biological effects of gases on mammalian cells in culture. Fetner (1962) and Tsuda et al. (1979) indicated that chromosome aberrations in culture cells were not induced by ozone and N O 2 gases, respectively, if cells were not washed with balanced salt solution prior to exposure. The latter authors also suggested that certain materials, protective against gas effects, might be present in the culture medium or cell surface. Hurst et al. (1971) reported the loss of the oxidizing property of ozone in the presence of glutathione or serum protein. Guerrero et al. (1979) reported the lack of sister-chromatid exchanges in the lymphocytes exposed to ozone in vivo, in contrast to the presence of exchanges in cells exposed in vitro to ozone. They suggested that the lack of sister-chromatid exchanges in the lymphocytes could be attributed to the protective effect of
serum a n d / o r other agents. The lymphocyte system offers many advantages for chromosome analysis, including the availability of large numbers of synchronized cells (Evans and O'Riordan, 1975). Lymphocytes obtained from animals exposed to gases in vivo, may, however, reflect biological effects which have been reduced by substances in the blood. The lung cells are affected to a much lesser extent by blood constituents, and may assess more accurately direct effects of gases on cells. In this respect, the use of lung cells reported here may provide reliable information on the genetic effects of gases. In the present study, we clearly demonstrated the presence of cells with induced mutations, or with increased chromosome aberrations in animals exposed in vivo to NO 2 or NO. We believe that this is the first report on the mutagenicity of gas, studied from the practical viewpoint of determining biological effects of gases on individuals in the environment. Other gases such as SO 2 (Ma et al., 1973) or mixtures of gases should be studied to determine their potential.
Acknowledgement A part of this work was supported by a Grantsin-Aid for Scientific Research from the Ministry of Education, Science and Culture (1978), Japan.
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