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DNA damage in lung cells in vivo and in vitro by 1,3-butadiene and nitrogen dioxide and their photochemical reaction products
Fundamental and Molecular Mechanisms of Mutagenesis
ELSEVIER
Mutation Research 328 (1995) 11-19
DNA damage in lung cells in vivo and in vitro by 1,3-butadiene and nitrogen dioxide and their photochemical reaction products Solveig A.S. Walles a,* , Katarina
Victorin
b, Margot
Lundborg
b
aDepartmentof Toxicology, National Institute of Occupational Health, S-l 7184 Solna, Sweden b Institute of Environmental Medicine, Karolinska Institute, Box 210, S-l 71 77 Stockholm. Sweden Received 19 August 1994; revision received 27 September 1994; accepted 4 October 1994
Abstract A UV-irradiated mixture of 1,3-butadiene and nitrogen dioxide (NO,) was tested for its potency to induce DNA damage measured as single-strand breaks (SSB) in lungs of mice. Both gases were also tested separately. After 16 h exposure a UV-irradiated mixture of 40 ppm butadiene + 20 ppm NO,, but not 20 ppm butadiene + 10 ppm NO, + UV, induced a significant increase in SSB as measured by the alkaline unwinding technique. There was no increase in the level of SSB using the alkaline elution technique during the same testing conditions. However, after 5 h exposure to 60 ppm butadiene + 30 ppm NO, + UV both methods demonstrated a significant increase in SSB. Mice were also exposed to butadiene at 80 and 200 ppm for 16 h and at 500 ppm for 5 h. DNA damage was demonstrated in both liver and lung after 5 and 16 h (only at 200 ppm) of exposure using the unwinding technique. Using the alkaline elution assay, a significant increase in the level of SSB in lung and liver was found only after 5 h of exposure. When mice were exposed to 30 ppm NO, for 16 h or 50 ppm for 5 h, a significant increase in SSB was found with the unwinding technique. Alveolar macrophages from mice were also exposed in vitro to the gas mixture and to butadiene and NO, separately. In these experiments, the DNA damage was studied with the unwinding technique. A significant effect was demonstrated with 40 ppm butadiene + 20 ppm NOz + UV. NO, itself contributed to some extent to the increase. Reasons for the discrepancies between the unwinding and the alkaline elution techniques are discussed. Keywords: Butadiene; macrophages; (Mice)
Nitrogen
dioxide;
Photochemical
1. Introduction
tant
Nitrogen dioxide (NO,) is a major in urban areas, mainly due to
* Corresponding 7303312. 0027-5107/95/$09.50 SSDI
author.
air pollumotor ex-
Tel. 46-S-7309100; Fax 46-8
products;
breaks;
Lung; Liver; Alveolar
hausts. Low molecular weight alkenes from combustion sources as well as chemical industries. It has been propene, together with NO, after UV
are emitted from petroshown that irradiation,
may fort-n reaction products that are mutagenic in Salmonella and cause sister-chromatid exchanges in Chinese hamster V79 cells (Kleindienst et al., 1985b; Shiraishi and Bandow, 1985; Victorin and
0 1995 Elsevier Science B.V. All rights reserved
0027-5107(94)00086-7
DNA single-strand
S.A.S. Walles et al. /Mutation
12
Stlhlberg, 1988b, 1989). In experiments by Victorin and Stshlberg (19891, 1,3-butadiene + NO, + UV gave a much stronger response in Salmonella, strain TAlOO, than did propene + NOz + UV after a 40-min reaction time. The strong, direct-acting, mutagenic activity of the photochemical products in Salmonella was, however, not confirmed in the somatic mutation and recombination test in Drosophila (the wing spot test) or in the mouse bone marrow micronucleus assay (Victorin et al., 1990). As the lung is the primary target for inhaled air pollutants, it was decided to study genotoxic effects in lung tissue of mice after in vivo exposure, along with alveolar macrophages exposed in vitro. Induction of DNA single-strand breaks (SSB) was chosen as endpoint, because of the relative sensitivity and straightforwardness of the method, which does not include cultivation of the cells. Induction of SSB is an indication of a genotoxic effect on the cells. Most of these damages will be repaired, but if not they may cause mutations. Two different methods for determination of SSB were used, the alkaline unwinding technique and the alkaline elution assay. Butadiene was chosen as a test compound because it is more reactive than propene. Moreover it is recognized as an important toxic air pollutant (EPA, 1990).
2. Materials
and methods
Chemicals
Proteinase K was obtained from Merck, Darmstadt, Germany. Hoechst 33258 was purchased from Fluka Chemie AG, Buchs, Switzerland and hydroxylapatite (Bio-Gel HTP and DNA-grade Bio-Gel HTP) from Bio-Rad Laboratories, CA, USA. Exposure
The flow-through exposure system used to expose the animals and alveolar macrophages has been described earlier, when used for experiments with Salmonella bacteria (Victorin and Stihlberg, 1988a,b). Butadiene (Aga Specialgas, Stockholm, 1% in nitrogen gas) was mixed with
Resew& 328 (1995) II-19
synthetic air (Aga Specialgas, < 1 ppm hydrocarbons) to the desired concentration and then let into a 20-dm3 Pyrex glass reaction vessel, surrounded by six UVA 40-W solarium lamps. The flow rate was 500 ml/min, giving a mean reaction time of 40 min. After the reaction chamber, the gas mixture was let into a 3-dm” glass exposure chamber and then out to the ventilation system. In the experiments with mice, male NMRI mice weighing 35-40 g in groups of 3-5 animals were exposed at a time during 5 or 16 h. Exposure to butadiene-NO, mixtures was performed simuItaneously with and without UV irradiation. The same number of mice was kept caged and used as controls for each experiment. In experiments with macrophages, cultures of alveolar macrophages from male mice were prepared in the following way. NMRI mice (35-40 g) were killed with an overdose of sodium pentobarbital and the lungs were excised. Macrophages were obtained by lung lavage with Hanks’ balanced salt solution (HBSS) without Ca2+ and Mg’+, pH 7.4, 37°C. The cell suspension was centrifuged at 300 x g for 10 min at room temperature and the resulting cell pellet was resuspended in HBSS with Ca2+ and Mg’+, pH 7.4, 4°C. 0.3-0.9 x lo6 macrophages in 2 ml HBSS was added to each culture dish (Falcon 35 x 10 mm). The culture dishes were placed on crushed ice and the cells were allowed to attach for about 1 h. Just before exposure, the culture dishes were rinsed with cold HBSS and then 0.5 ml was added to each plate (0.5 ml thickness of the fluid layer). Four culture dishes at a time were exposed without lids for 2 h with an extra culture dish serving as check for toxic effects by counting the number of viable cells. The exposure chambers were put on a rocking table during exposure. The viability of the macrophages was evaluated before and after the exposure of the gas by Trypan blue. As in the in vivo experiments, exposure was performed simultaneously with and without UV irradiation with the same number of unexposed plates as controls. Preparation
of cell nuclei
The mice were killed by cervical dislocation within 1 h after termination of the exposure. Cell
S.A.S. Walles et al/Mutation
Research 328 (1995) II-19
nuclei were prepared by the method of Cox et al. (1973). Briefly, the lungs were removed and homogenized by pressing through a stainless steel screen (0.4 mm) in ice-cold Merchant’s solution (0.14 M NaCl, 1.47 mM KI-IzPO,, 2.7 mM KCl, 1 10 mM EDTA, pH 7.4). The mM Na,HPO,, suspension was centrifuged at 10 X g at 4°C for 2 min. The supernatant was then centrifuged at 250 x g at 4°C for 5 min. The pellet, which consisted of cell nuclei, was suspended in 2 ml of phosphate buffered saline (pH 7.3).
solution was added. Then the same procedure was followed as for the cell nuclei. Single- and double-stranded DNA were separated on a column of hydroxylapatite. Briefly, single-stranded and double-stranded DNA were eluted with 110 mM and 250 mM phosphate buffer (pH 6.8), respectively. The amount of DNA was determined by fluorometry after addition of the fluorochrome Hoechst 33258 (Cesarone et al., 1979). The fraction of double-stranded DNA (Fds) was calculated as the ratio between the amount of double-stranded DNA and the total amount of DNA. The negative logarithm of Fds ( - log Fds) is a linear function of the number of SSB (Erixon and Ahnstrom, 1979). Erixon and Ahnstrom (1979) found that 1 krad of y-irradiation will induce 3.1 SSB/109 Da and cause an increase of about 0.10 units of -log Fds for isotope labeled human fibroblasts. The alkaline elution technique. As an alternative method for determination of DNA damage the alkaline elution assay according to Kohn and Grimek-Ewig (1973) was used. The concentration of DNA in the samples was determined by lysing and subsequent neutralization (Erixon and AhnStrom, 1979) of a small part of the cell suspension followed by analysis by fluorometry using Hoechst 33258. Cell nuclei corresponding to about 10 pg of DNA (corresponding to about 0.5 X lo6 cells) were loaded on membrane filters (polycarbonate 2 pm, Nucleopore Corp., Pleasanton, CA). After
Determination of single-strand breaks The alkaline unwinding technique. Induction of SSB in DNA was determined according to the method of Ahnstrom and Erixon (1973, 19811, which was applied to cell nuclei from organs of mice by Walles and Erixon (1984). Briefly, to 0.3 ml of the suspension of the cell nuclei, 1 ml of lysing solution (30 mM NaOH in 0.15 M NaCl, pH > 12) was added; the resulting solution was left in the dark at 20°C for 30 min. The solution was neutralized by addition of 2 ml 0.02 M NaH,PO, and then immediately sonicated for 10 s at 40 W. Finally, 0.3 ml of 1.25% sodium dodecyl sulfate was added to the solution. The samples were deep frozen and stored before analysis. In the experiments with the alveolar macrophages, the exposed cell cultures were rinsed once with 2 ml of HBSS and 1 ml of lysing
Table 1 DNA damage (-log unwinding technique)
Fds) in lungs of mice exposed
13
to different
concentration
mixtures
of 1,3-butadiene
(BUT) + NO, f UV” (the
Concentration
Time of exposure
-tog
(ppm) BUT + NO,
(h)
+uv
-uv
Control
20+
10
16
0.21 f 0.02 (4)
0.20 f 0.01 (4)
0.19 f 0.01 (4)
40 + 20 40 + 20 40 + 20
16 16 16
0.38 * 0.03 * (4) 0.37 + 0.07 (3) 0.28 f 0.02 * (4)
0.27 f 0.01 (4) 0.24 f 0.00 (3) 0.18 f 0.02 (4)
0.24 I 0.03 (4) 0.27 + 0.04 (3) 0.22 + 0.01 (4)
0.34 f 0.03 b.’ (11)
0.23 k 0.01 h (11)
0.24 + 0.02 ’ (11)
0.14 i 0.02 (3)
0.14 i 0.01 (3)
60 + 30
5
Fds
0.19 2 0.00
a Data are expressed as mean + SE. Number of animals b Mean + SE of all mice from the different groups. * p < 0.05; * * p < 0.01.
- * (4)
in each group
in parentheses.
14
S.A.S. Walles et al. /Mutation Research 328 (1995) II-19
lysing for 1 h with sarcosyl EDTA solution (2% sarcosyl in 0.02 M EDTA, pH 9.6) with or without 0.5 mg proteinase K per ml and washing with 0.02 M EDTA (pH 9.6), single-stranded DNA was eluted from the fibers for 16 h with 0.02 M EDTA, adjusted to pH 12.15 with NaOH, at a flow rate of 0.03 ml/min. Both the elution and the measurement of the samples were performed by means of an automated system (Brunborg et al., 1988). The amount of DNA was measured by fluorometry by means of Hoechst 33258. The damage of DNA was calculated as the normalized area above the curve (NAAC) according to Brunborg et al. (1990). Statististical analysis The significance of differences values was tested with Student’s pendent observations.
between mean t-test for inde-
3. Results In ho exposure of mice In Table 1 the DNA damage from mixtures of butadiene and NO, is presented as determined by the alkaline unwinding technique. When mice were exposed to a mixture of 20 ppm butadiene and 10 ppm NO, there was no increase in the level of SSB in lung either with or without UV irradiation. At higher concentrations, 40 ppm butadiene + 20 ppm NOz f UV, the level of SSB increased significantly in two out of three repeated experiments with UV irradiation. When the results from all animals in the three experiments were pooled, the UV-irradiated mixture increased the level of SSB 1.4 times compared to the control (p < 0.05). When a group of mice was exposed to 60 ppm butadiene + 30 ppm NO, for 5 h, there was an increase of 1.4 times the control value (p < 0.01) by the UV-irradiated gas mixture. The alkaline elution technique did not show any DNA damage after exposure to 40 ppm butadiene + 20 ppm NO? + UV for 16 h, either with or without proteinase K added to the Iysing solution (Table 2). However, the DNA damage increased 1.6 times (p < 0.05) compared to the
Table 2 DNA damage (NAAC, IOM3/h) in lung DNA of mice exposed to different concentration mixtures of 1,3-butadiene (BUT)+NO; (the alkaline elution technique) Concentration BUT+NO,
Time of exposure (h)
NAAC (10m3/h) + UV -uv
40+20h 40+20 c
16 16
20+ 20*2
hd
60+30
b
5
1(4) (4)
19*2*
a Data are expressed as mean+ each group in parentheses. h Lysing with proteinase K. ’ Lysing without proteinase K. * p < 0.05.
Control
2Oi2(3) 18rt l(4)
24&3(4) 20+ l(4)
14+0(3)
12&l(3)
(4)
SE. Number
of animals
in
control after exposure to a UV-irradiated gas mixture of 60 ppm butadiene + 30 ppm NO, for 5 h. Fig. 1 shows an example of aIkaline elution of DNA from cell nuclei from lungs of mice
100
0
*
Control
+
-uv
*
+uv
4
8
Time
of elution
12
16
(h)
Fig. 1. Alkaline elution profiles of DNA from lung of mice exposed to 60 ppm butadiene + 30 ppm nitrogen dioxide + UV for 5 h and control.
S.A.S. Walles et ai. /Mutation Table 3 DNA damage
(-log
F,,) in lung and liver of mice exposed
Research 328 (19951 II-19
to 1,3-butadienea
15
(the unwinding
technique)
Concentration
Time of exposure
- log F,s
(ppm)
(h)
Lung Exposed
Control
Exposed
Control
80 200 500
16 16 5
0.22 f 0.05 (3) 0.20 f 0.02 * (5) 0.28 f 0.02 * (4)
0.17 + 0.01 (4) 0.12 f 0.02 (3) 0.19 f 0.02 (4)
0.20 f 0.02 * (5) 0.34 + 0.04 * * (4)
0.12 * 0.01 (3) 0.15 * 0.02 (4)
a Data are expressed as mean f SE. Number * p < 0.05; * * p < 0.01.
Table 4 DNA damage
NAAC
(10U3/h)
Liver
of animals
in each group
in lung and liver of mice exposed
Concentration
Time of exposure
NAAC
(ppm)
(h)
Lung
in parentheses.
to 1,3-butadienea
Control
16
25 + 1 (4)
19 * 4 (3)
200 500
16 5
34 + 4 (4) 47 * 2 * * (4)
34 f 2 (3) 35 f 2 (4)
in each group
exposed to 60 ppm butadiene + 30 ppm NO, f UV for 5 h and control. The NAAC value for the control mouse was 13 x 10e3/h, for a mouse treated with non-irradiated gas mixture the NAAC value was 14 x 10-3/h and for a mouse treated with UV-irradiated gas mixture the NAAC value was 26 X 10P3/h. The mice exposed to non-irradiated mixtures of butadiene and NO, did not show any increase in SSB compared to the control mice in any experiment (Tables 1 and 2).
Table 5 DNA damage
(-log
Fds) in lung of mice exposed
elution
technique)
Liver
Exposed
of animals
(the alkaline
(1O-3/h)
80
a Data are expressed as mean f SE. Number * p < 0.05; * * p < 0.01.
_
to nitrogen
Control
Exposed _ 28 + 4 (4) 36 f 4 * (4)
25 f l(3) 19 f 2 (3)
in parentheses.
Mice were also exposed to butadiene as a single compound at a concentration of 500 ppm in air for 5 h or 80 or 200 ppm for 16 h. There was no increase of the DNA damage when mice were exposed to 80 ppm for 16 h (Tables 3 and 41, neither at 200 ppm for 16 h with the alkaline elution technique, but an increase of 1.7 times the control in both liver and lung was found with the unwinding technique (p < 0.05). After 5 h of exposure to 500 ppm both methods demonstrated a significant increase in SSB in liver and lung.
dioxide”
(the unwinding
technique)
Concentration
Time of exposure
- tog Fds
(ppm)
(h)
Exposed
Control
30 30 30
16 16 16
0.32 + 0.03 * (5) 0.31 + 0.02 (5) 0.24 rfi 0.02 (5)
0.20 _Ir0.04 (3) 0.27 + 0.02 (3) 0.19 + 0.02 (3)
0.30 2 0.02 b, * * (15)
0.23 + 0.02 b (9)
0.19 + 0.01 * * * (5)
0.12 f 0.01 (7)
50
5
a Data are expressed as mean + SE. Number of animals ’ Mean f SE of all mice from the different groups. * p < 0.05; * * p < 0.01; * * * p < 0.001.
in each group
in parentheses.
I6
S.A.S. W&es et al. / Mntation
Mice were exposed to 30 ppm of NO, for 16 h in three different experiments. Using the unwinding technique, there was an increase in SSB in all experiments, although only one experiment was statistically significant at the p < 0.05 level (Table 5). However, when the results from all individual animals were pooled, a higher significance was obtained (p < 0.01). No increase of the SSB level was found using the alkaline elution technique (data not shown). When mice were exposed to 50 ppm for 5 h, there was a highly significant increase (1.6 times the control) in the level of SSB (p < 0.001) tested with the unwinding technique. In vitro exposure of alrleolar macrophages Alveolar macrophages from the same strain of mice were exposed to 40 ppm butadiene + 20 ppm NO, for 2 h with and without UV irradiation (Table 6). DNA damage was analyzed with the unwinding technique. The UV-irradiated gas mixture increased the level of SSB 4.3 times compared to the control (p < 0.001). Without UV irradiation the level of SSB increased 2.5 times compared to the control (p < 0.01). The viability of the macrophages was 95% before exposure. After exposure to the UVirradiated gas mixture, the viability decreased to 75% and for the non-irradiated mixture the viability was 85%. The macrophages were also exposed to pure butadiene and pure NO, in air for 2 h (Table 7). The level of SSB increased about twice compared to the control (p < 0.01) at an exposure to 20 ppm NO,. After exposure to 40 ppm butadiene
Table 6 DNA damage t-log Fds) in alveolar macrophages of mice exposed in vitro to 1,3-butadiene (BUT) + NO, f UV for 2 h” (the unwinding technique) Concentration
-log
bpm)
+uv
BUT+NO, 40+20
Fds -uv
0.34rtO.02 (3)
a Data are expressed group in parentheses. * * p < 0.01; * * * p
***
0.20+0.01 (3)
as mean f SE. Number
< 0.001.
Control jr *
0.08~0.02 (3)
of dishes in each
Reseurc[z 328 (1995) I I - 19 Table I DNA damage (-log Fds) in alveolar macrophages exposed in vitro to 1,3-butadiene (BUT) and NO2 (the unwinding technique) Concentration
Gas
(ppm)
- tog F,,a Exposed
Control 0.05 f 0.00 (3)
40
BUT
0.07 + 0.01 (4)
70
NOz
0.09~0.01
* * (3)
a Data are expressed as means+SE. each group in parentheses. * * p < 0.01.
there SSB.
of mice for 2 h”
was no statistically
0.04i_0.00
Number
significant
(3)
of dishes
increase
in
in
4. Discussion The present investigation has shown that photochemically formed reaction products from butadiene and NO, can cause SSB in DNA in lung cells both in vitro and in vivo. Using the alkaline unwinding technique it was shown that 40 ppm butadiene + 20 ppm NO, + UV induced SSB both after 16 h inhalation in vivo and after 2 h exposure of alveolar macrophages in vitro. The in vivo effects were most probably caused by photochemical reaction products, as the nonirradiated mixtures did not cause any increase in SSB (Table 1). Butadiene by itself did not cause a significant effect at 80 ppm (Table 3). NO, at 30 ppm caused a significant effect in one out of three repeated experiments only (Table 5). As the gas mixture will contain lower concentrations of butadiene and NO, after the 40-min UV irradiation period than before, due to photochemical reactions, we concluded that these compounds probably did not contribute to the DNA damage observed in the experiments with UV irradiation. However, in the in vitro experiment, NO, may have contributed to the increase in SSB observed in macrophages exposed to the UV-irradiated gas mixture, as NOz at 20 ppm induced SSB when tested as a single compound (Tables 6 and 7). The UV-irradiated gas mixture induced more SSB than the non-irradiated mixture, which indicates that both NO, and the photochemical reaction
S.A.S. Walles et al./Mutation
products cause the DNA damage observed. Butadiene at 40 ppm did not induce SSB when tested as a single compound (Table 7). When mice were exposed to the UV-irradiated gas mixture, DNA damage measured by the unwinding technique was induced to the same extent after 5 and 16 h of exposure (Table 1). There might be different explanations for this phenomenon. A steady state might be formed already after 5 h of exposure. Another explanation could be that cross-links will be formed over time, which will decrease the possibility to detect SSB. In the present study we had no possibility to test if cross-links really were induced. That would have required irradiation of the cell nuclei with X-rays and we did not have access to such equipment. However, as DNA damage could be detected when the alkaline elution assay was used for analysis after 5 h of exposure but not after 16 h, this fact might also be an indication that crosslinks were formed. There are two different kinds of cross-links, namely DNA-protein cross-links and DNA inter-strand cross-links. In the present study DNA-protein cross-links can be excluded as the results were the same whether proteinase K was present or not during the lysing time (Table 2). In the procedure of the unwinding technique the strands are shortened by ultrasonic treatment. If cross-links are rare, only few pieces will contain cross-links, allowing for most of the SSB to be able to be detected. In contrast, DNA inter-strand cross-links will obscure the SSB in the alkaline elution technique, because the strands cannot separate. When mice were treated with butadiene the unwinding technique showed a significant effect both at 200 ppm for 16 h and at 500 ppm for 5 h (Table 3). With the alkaline elution technique there was no increase of DNA damage after 16 h of exposure, but there was an effect after 5 h of exposure with the higher dose (Table 4). Most probably this result is due to induction of DNA inter-strand cross-links. 1,3-Butadiene is at first metabolized to the monofunctional component 1,2-epoxybutene (EB) (de Meester, 1988) and further to the difunctional agent 1,2 : 3,4-diepoxybutane (DEB) (Malvoisin et al., 1979). EB will react with guanine in DNA (Jelitto et al., 1989) and
Research 328 (1995) II-19
17
subsequently induce SSB. DEB will induce DNA inter-strand and DNA-protein cross-links. In an investigation by Vangala et al. (1993), mice and rats were exposed to butadiene at 100-2000 ppm for 7 h. The alkaline elution technique was used for detection of DNA damage. Both types of cross-links were observed in mouse lung and liver. The formation of cross-links was higher in lung than in liver. It has earlier been shown that DEB induced complete reversibility of heat denaturation of DNA, which was interpreted as formation of cross-links in DNA (Lawley and Brookes, 1967). The present study has shown that DNA damage can be induced by NO, at sufficiently high concentrations, both in lung cells in vivo and in alveolar macrophages in vitro (Tables 5 and 71. Very few data on genotoxic effects of NOz in vivo (for review, see Victorin, 1994) have been presented before. Only one in vivo study on effects in lung cells was found in the literature (Isomura et al., 1984). In that study, exposure to 15 ppm NO, for 3 h induced mutations and chromatidtype chromosomal aberrations in cultured lung cells from exposed rats. NO, has been shown to induce SSB in V79 hamster cells in vitro (Giirsdorf et al., 19901, but no other data on SSB were found in the literature for comparison with our results in lung cells in vitro and in vivo. The reaction products formed in the present experiments have not been chemically identified. Generally, the photochemical smog reactions of alkenes proceed via radical reactions and organic peroxides (Butler, 1979). Acrolein is the major product in photochemical NO,-butadiene reactions and formaldehyde is also formed (Maldotti et al., 1980). Both formaldehyde and acrolein give rise to SSB, DNA inter-strand cross-links and DNA-protein cross-links at near-toxic concentrations in cultured bronchial cells, as measured with the alkaline elution technique (Grafstrom, 1990). Formaldehyde gives more DNA-protein cross-links than acrolein does. Formaldehyde has also been shown to form DNA-protein cross-links in vivo (Casanova-Schmitz and Heck, 1983; Ross and Shipley, 1980). In experiments by Lam et al. (1985), acrolein induced DNA-protein cross-links in rat nasal mucosa in vitro, but not in vivo. Acrolein depletes glutathione in vitro and forms
18
S.A.S.
Walles et al./Mutation
adducts with guanine of calf thymus DNA (Chung et al., 1984). According to the cited literature data on formaldehyde and acrolein, at least acrolein probably contributed to the induction of SSB in the present study. However, acrolein and formaldehyde are only weakly mutagenic in Salmonella strain TAlOO (IARC, 1985; Sasaki and Endo, 1978). Thus, it seems less likely that acrolein or formaldehyde was responsible for the strong direct-acting mutagenic activity in Salmonella TAlOO that was demonstrated in earlier experiments (Victorin and Stihlberg, 1989). In earlier in vivo studies using the same exposure system as in the present study, we obtained equivocal results in the somatic mutation and recombination test in Drosophila and no genotoxicity in the mouse bone marrow micronucleus assay (Victorin et al., 1990). The results probably reflect the fact that the photochemical reaction products are reactive, probably short-lived compounds that may be inactivated in vivo before they reach target cells in the bone marrow. In the present study we wanted to study the effect on lung cells simultaneously in vivo and in vitro, using the same endpoint. According to our previous results, one might have expected a much higher response in the in vitro exposure situation. However, according to the present results, the reaction products can cause detectable DNA damage in lung tissue upon inhalation of high concentrations to a similar extent as in isolated alveolar macrophages. Our studies show similarities with the studies by Heddle et al. (1993) on peroxyacetyl nitrate (PAN). PAN is formed in atmospheric photochemical reactions between hydrocarbons and NO?, and was suggested as one contributor to the direct-acting mutagenicity in SalmoneIla TAlOO found in photochemical experiments with propene and NO, (Kleindienst et al., 1985a,b). When tested in inhalation experiments with Chinese “0 clearly significant effects were hamsters, demonstrated concerning mutations or micronuclei in lung fibroblasts. PAN is a toxic compound, which prevents the testing of high concentrations. This is a property common to several air pollutants that are direct-acting mutagens in vitro,
Research 328 (1995) II-19
although it has been difficult to demonstrate genotoxic effects in vivo. For example, the in vivo genotoxic hazard of ozone and NO, is difficult to evaluate partly due to this reason (Victorin, 1992, 1994). A genotoxic hazard to lung tissue of inhalation of photochemical reaction products from alkenes and NO, has been demonstrated at high concentrations in the present study, but our results and those of Lam et al. (1985) on acrolein and of Heddle et al. (1993) on PAN indicate that the risk is far less than was previously indicated by mutagenicity in Salmonella bacteria.
Acknowledgements The authors wish to thank Rolf Stridde for skilful technical assistance. This work was supported by a grant from the Swedish OK environmental research foundation.
References AhnstrGm, G. and K. Erixon (1973) Radiation induced strand breakage in DNA from mammalian cells. Strand separation in alkaline solution, Int. J. Radiat. Biol., 23, 285-289. Ahnstram, G. and K. Erixon (1981) Measurement of strand breaks by alkaline denaturation and hydroxyapatite chromatography, in: E.C. Friedberg and P.C. Hanawalt (Eds.), DNA Repair, Marcel Dekker. New York, pp. 403-418. Brunborg, G., J.A. Holme, E. SGderlund, J.G. Omichinski and E. Dybing (1988) An automated alkaline elution system: DNA damage induced by 1,2-dibromo-3-chloropropane in vivo and in vitro, Anal. Biochem., 174, 522-536. Brunborg. G., J. Holme. E. Siiderlund and E. Dybing (1990) Organ-specific genotoxic effects of chemicals: The use of alkaline elution to detect DNA damage in various organs of in vivo-exposed animals, in: M.L. Mendelsohn and R.J. Albertini (Eds.), Mutation and the Environment. Part D: Carcinogenesis. Wiley-Liss, New York, pp. 43-52. Butler. J.D. (1979) Atmospheric reactions, in: J.D. Butler (Ed.), Air Pollution Chemistry, Academic Press, London, pp. 267-319. Casanova-Schmitz. M. and H.D. Heck (1983) Effects of formaldehyde exposure on the extractability of DNA from proteins in the rat nasal mucosa, Appl. Pharmacol., 70, 121-132. Cesarone. C.F.. C. Bolognesi and L. Santi (1979) Improved microfluorometric DNA determination in biological material using 33258 Hoechst, Anal. Biochem.. 100, 188-197.
S.A.S. Walles et al. /Mutation Chung, F., R. Young and S.S. Hecht (1984) Formation of cyclic l,N’-propanodeoxyguanosine adducts in DNA upon reaction with acrolein or crotonaldehyde, Cancer Res., 44, 990-99s. Cox, R., 1. Damjanow, S.E. Abanobi and D.S.R. Sarma (1973) A method for measuring DNA damage and repair in the liver in vivo, Cancer Res., 33, 2114-2121. de Meester, C. (1988) Genotoxic properties of 1,3-butadiene, Mutation Res., 195, 273-281. EPA (1990) Cancer Risk from Outdoor Exposure to Air Toxics, Vol. 1, United States Environmental Protection Agency, EPA-450/l-9000. Erixon. K. and G. Ahnstrom (1979) Single-strand breaks in DNA during repair of UV-induced damage in normal human and xeroderma pigmentosum ceils as determined by alkaline DNA unwinding and hydroxylapatite chromatography, Mutation Res., 59, 257-271. Grafstrom, R.C. (1990) In vitro studies of aldehyde effects related to human respiratory carcinogenesis, Mutation Res.. 238, 175-184. Giirsdorf, S.. E. Appel, C. Engeholm and G. Obe (19901 Nitrogen dioxide induces DNA single-strand breaks in cultured Chinese hamster cells, Carcinogenesis, 11, 37-41. Heddle, J.A., P.B. Shepson, J.D. Gingerich and K.W. So (1993) Mutagenicity of peroxyacetyl nitrate (PAN) in vivo, Environ. Mol. Mutagen., 21, 58-66. IARC (1985) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 36, Ally1 Compounds, Aldehydes, Epoxides and Peroxides, International Agency for Research on Cancer, Lyon. Isomura, K., K.T. Chikahira, K. Teranishi and K. Hamada (1984) Induction of mutations and chromosome aberrations in lung cells following in vivo exposure of rats to nitrogen oxides, Mutation Res.. 136, 119-125. Jelitto, B., R.R. Vangala and R.J. Laib (1989) Species differences in DNA damage by butadiene: Role of diepoxybutane, Arch. Toxicol.. Suppl. 13, 246-249. Kleindienst, T.E., P.B. Shepson, E.O. Edney and L.D. Claxton (1985a) Peroxyacetyl nitrate: Measurement of the mutagenic activity using the SalmoneIIa/mammalian microsome reversion assay, Mutation Res., 157, 123-128. Kleindienst, T.E., P.B. Shepson, E.O. Edney, L.T. Cupitt and L.D. Claxton (1985b) The mutagenic activity products of propylene photooxidation, Environ. Sci. Technol., 19, 620627. Kohn, K.W. and R.A. Grimek-Ewig (1973) Alkaline elution analysis, a new approach to the study of DNA single-strand interruptions in cells, Cancer Res., 33, 1849-1853. Lam, C.W., M. Casanova and H.D. Heck (1985) Depletion of nasal mucosal glutathione by acrolein and enhancement of formaldehyde-induced DNA-protein cross-linking by simultaneous exposure to acrolein, Arch. Toxicol., 58.67-71.
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19
Lawley, P.D. and P. Brookes (1967) Interstrand cross-linking of DNA by difunctional alkylating agents, J. Mol. Biol., 25. 143-160. Maldotti, A., C. Chiorboli, C.A. Bignozzi, C. Bartocci and V. Carassiti (1980) Photooxidation of 1,3-butadiene containing systems: Rate constant determination for the reaction of acrolein with OH radicals, Int. J. Chem. Kinet., 12, 905-913. Malvoisin, E., G. Lhoest, F. Poncelet, M. Roberfroid and M. Mercier (1979) Identification and quantification of 1,2epoxy-3-butene as the primary metabolite of 1,3-butadiene, J. Chromatogr., 178, 419-425. Ross, W.E. and N. Shipley (1980) Relationship between DNA damage and survival in formaldehyde-treated mouse cells, Mutation Res., 79, 277-283. Sasaki, Y. and R. Endo (1978) Mutagenicity of aldehydes in Salmonella, Mutation Res., 54. 251-252. Shiraishi, F. and H. Bandow (1985) The genetic effects of the photochemical reaction products of propylene plus NO, on cultured Chinese hamster cells exposed in vitro, J. Toxicol. Environ. Health, 15, 531-538. Vangala, R.R., R.J. Laib and H.M. Bolt (1993) Evaluation of DNA damage by alkaline elution technique after inhalation exposure of rats and mice to 1,3-butadiene, Arch. Toxicol., 67, 34-38. Victorin, K. (1992) Review of the genotoxicity of ozone, Mutation Res., 277, 221-238. Victorin, K. (1994) Review of the genotoxicity of nitrogen oxides, Mutation Res., 317, 43-55. Victorin, K. and M. Stihlberg (1988aJ A method for studying the mutagenicity of some gaseous compounds in Salmonella typhimurium, Environ. Mol. Mutagen., 11, 6577. Victorin, K. and M. Stihlberg (1988b) Photochemical formation of mutagenic compounds from alkenes and ozone or nitrogen dioxide, Environ. Mol. Mutagen., 11, 79-90. Victorin, K. and M. Stihlberg (1989) Mutagenic activity of ultraviolet-irradiated mixtures of nitrogen dioxide and propene or butadiene, Environ. Res., 49, 271-282. Victorin, K., L. Busk, H. Cederberg and J. Magnusson (1990) Genotoxic activity of 1,3-butadiene and nitrogen dioxide and their photochemical reaction products in Drosophila and in the mouse bone marrow micronucleus assay, Mutation Res.. 228, 203-209. Walles, S.A.S. and K. Erixon (1984) Single-strand breaks in DNA of various organs of mice induced by methyl methanesulfonate and dimethylsulfoxide determined by the alkaline unwinding technique, Carcinogenesis, 5, 319323.
ELSEVIER
Mutation Research 328 (1995) 11-19
DNA damage in lung cells in vivo and in vitro by 1,3-butadiene and nitrogen dioxide and their photochemical reaction products Solveig A.S. Walles a,* , Katarina
Victorin
b, Margot
Lundborg
b
aDepartmentof Toxicology, National Institute of Occupational Health, S-l 7184 Solna, Sweden b Institute of Environmental Medicine, Karolinska Institute, Box 210, S-l 71 77 Stockholm. Sweden Received 19 August 1994; revision received 27 September 1994; accepted 4 October 1994
Abstract A UV-irradiated mixture of 1,3-butadiene and nitrogen dioxide (NO,) was tested for its potency to induce DNA damage measured as single-strand breaks (SSB) in lungs of mice. Both gases were also tested separately. After 16 h exposure a UV-irradiated mixture of 40 ppm butadiene + 20 ppm NO,, but not 20 ppm butadiene + 10 ppm NO, + UV, induced a significant increase in SSB as measured by the alkaline unwinding technique. There was no increase in the level of SSB using the alkaline elution technique during the same testing conditions. However, after 5 h exposure to 60 ppm butadiene + 30 ppm NO, + UV both methods demonstrated a significant increase in SSB. Mice were also exposed to butadiene at 80 and 200 ppm for 16 h and at 500 ppm for 5 h. DNA damage was demonstrated in both liver and lung after 5 and 16 h (only at 200 ppm) of exposure using the unwinding technique. Using the alkaline elution assay, a significant increase in the level of SSB in lung and liver was found only after 5 h of exposure. When mice were exposed to 30 ppm NO, for 16 h or 50 ppm for 5 h, a significant increase in SSB was found with the unwinding technique. Alveolar macrophages from mice were also exposed in vitro to the gas mixture and to butadiene and NO, separately. In these experiments, the DNA damage was studied with the unwinding technique. A significant effect was demonstrated with 40 ppm butadiene + 20 ppm NOz + UV. NO, itself contributed to some extent to the increase. Reasons for the discrepancies between the unwinding and the alkaline elution techniques are discussed. Keywords: Butadiene; macrophages; (Mice)
Nitrogen
dioxide;
Photochemical
1. Introduction
tant
Nitrogen dioxide (NO,) is a major in urban areas, mainly due to
* Corresponding 7303312. 0027-5107/95/$09.50 SSDI
author.
air pollumotor ex-
Tel. 46-S-7309100; Fax 46-8
products;
breaks;
Lung; Liver; Alveolar
hausts. Low molecular weight alkenes from combustion sources as well as chemical industries. It has been propene, together with NO, after UV
are emitted from petroshown that irradiation,
may fort-n reaction products that are mutagenic in Salmonella and cause sister-chromatid exchanges in Chinese hamster V79 cells (Kleindienst et al., 1985b; Shiraishi and Bandow, 1985; Victorin and
0 1995 Elsevier Science B.V. All rights reserved
0027-5107(94)00086-7
DNA single-strand
S.A.S. Walles et al. /Mutation
12
Stlhlberg, 1988b, 1989). In experiments by Victorin and Stshlberg (19891, 1,3-butadiene + NO, + UV gave a much stronger response in Salmonella, strain TAlOO, than did propene + NOz + UV after a 40-min reaction time. The strong, direct-acting, mutagenic activity of the photochemical products in Salmonella was, however, not confirmed in the somatic mutation and recombination test in Drosophila (the wing spot test) or in the mouse bone marrow micronucleus assay (Victorin et al., 1990). As the lung is the primary target for inhaled air pollutants, it was decided to study genotoxic effects in lung tissue of mice after in vivo exposure, along with alveolar macrophages exposed in vitro. Induction of DNA single-strand breaks (SSB) was chosen as endpoint, because of the relative sensitivity and straightforwardness of the method, which does not include cultivation of the cells. Induction of SSB is an indication of a genotoxic effect on the cells. Most of these damages will be repaired, but if not they may cause mutations. Two different methods for determination of SSB were used, the alkaline unwinding technique and the alkaline elution assay. Butadiene was chosen as a test compound because it is more reactive than propene. Moreover it is recognized as an important toxic air pollutant (EPA, 1990).
2. Materials
and methods
Chemicals
Proteinase K was obtained from Merck, Darmstadt, Germany. Hoechst 33258 was purchased from Fluka Chemie AG, Buchs, Switzerland and hydroxylapatite (Bio-Gel HTP and DNA-grade Bio-Gel HTP) from Bio-Rad Laboratories, CA, USA. Exposure
The flow-through exposure system used to expose the animals and alveolar macrophages has been described earlier, when used for experiments with Salmonella bacteria (Victorin and Stihlberg, 1988a,b). Butadiene (Aga Specialgas, Stockholm, 1% in nitrogen gas) was mixed with
Resew& 328 (1995) II-19
synthetic air (Aga Specialgas, < 1 ppm hydrocarbons) to the desired concentration and then let into a 20-dm3 Pyrex glass reaction vessel, surrounded by six UVA 40-W solarium lamps. The flow rate was 500 ml/min, giving a mean reaction time of 40 min. After the reaction chamber, the gas mixture was let into a 3-dm” glass exposure chamber and then out to the ventilation system. In the experiments with mice, male NMRI mice weighing 35-40 g in groups of 3-5 animals were exposed at a time during 5 or 16 h. Exposure to butadiene-NO, mixtures was performed simuItaneously with and without UV irradiation. The same number of mice was kept caged and used as controls for each experiment. In experiments with macrophages, cultures of alveolar macrophages from male mice were prepared in the following way. NMRI mice (35-40 g) were killed with an overdose of sodium pentobarbital and the lungs were excised. Macrophages were obtained by lung lavage with Hanks’ balanced salt solution (HBSS) without Ca2+ and Mg’+, pH 7.4, 37°C. The cell suspension was centrifuged at 300 x g for 10 min at room temperature and the resulting cell pellet was resuspended in HBSS with Ca2+ and Mg’+, pH 7.4, 4°C. 0.3-0.9 x lo6 macrophages in 2 ml HBSS was added to each culture dish (Falcon 35 x 10 mm). The culture dishes were placed on crushed ice and the cells were allowed to attach for about 1 h. Just before exposure, the culture dishes were rinsed with cold HBSS and then 0.5 ml was added to each plate (0.5 ml thickness of the fluid layer). Four culture dishes at a time were exposed without lids for 2 h with an extra culture dish serving as check for toxic effects by counting the number of viable cells. The exposure chambers were put on a rocking table during exposure. The viability of the macrophages was evaluated before and after the exposure of the gas by Trypan blue. As in the in vivo experiments, exposure was performed simultaneously with and without UV irradiation with the same number of unexposed plates as controls. Preparation
of cell nuclei
The mice were killed by cervical dislocation within 1 h after termination of the exposure. Cell
S.A.S. Walles et al/Mutation
Research 328 (1995) II-19
nuclei were prepared by the method of Cox et al. (1973). Briefly, the lungs were removed and homogenized by pressing through a stainless steel screen (0.4 mm) in ice-cold Merchant’s solution (0.14 M NaCl, 1.47 mM KI-IzPO,, 2.7 mM KCl, 1 10 mM EDTA, pH 7.4). The mM Na,HPO,, suspension was centrifuged at 10 X g at 4°C for 2 min. The supernatant was then centrifuged at 250 x g at 4°C for 5 min. The pellet, which consisted of cell nuclei, was suspended in 2 ml of phosphate buffered saline (pH 7.3).
solution was added. Then the same procedure was followed as for the cell nuclei. Single- and double-stranded DNA were separated on a column of hydroxylapatite. Briefly, single-stranded and double-stranded DNA were eluted with 110 mM and 250 mM phosphate buffer (pH 6.8), respectively. The amount of DNA was determined by fluorometry after addition of the fluorochrome Hoechst 33258 (Cesarone et al., 1979). The fraction of double-stranded DNA (Fds) was calculated as the ratio between the amount of double-stranded DNA and the total amount of DNA. The negative logarithm of Fds ( - log Fds) is a linear function of the number of SSB (Erixon and Ahnstrom, 1979). Erixon and Ahnstrom (1979) found that 1 krad of y-irradiation will induce 3.1 SSB/109 Da and cause an increase of about 0.10 units of -log Fds for isotope labeled human fibroblasts. The alkaline elution technique. As an alternative method for determination of DNA damage the alkaline elution assay according to Kohn and Grimek-Ewig (1973) was used. The concentration of DNA in the samples was determined by lysing and subsequent neutralization (Erixon and AhnStrom, 1979) of a small part of the cell suspension followed by analysis by fluorometry using Hoechst 33258. Cell nuclei corresponding to about 10 pg of DNA (corresponding to about 0.5 X lo6 cells) were loaded on membrane filters (polycarbonate 2 pm, Nucleopore Corp., Pleasanton, CA). After
Determination of single-strand breaks The alkaline unwinding technique. Induction of SSB in DNA was determined according to the method of Ahnstrom and Erixon (1973, 19811, which was applied to cell nuclei from organs of mice by Walles and Erixon (1984). Briefly, to 0.3 ml of the suspension of the cell nuclei, 1 ml of lysing solution (30 mM NaOH in 0.15 M NaCl, pH > 12) was added; the resulting solution was left in the dark at 20°C for 30 min. The solution was neutralized by addition of 2 ml 0.02 M NaH,PO, and then immediately sonicated for 10 s at 40 W. Finally, 0.3 ml of 1.25% sodium dodecyl sulfate was added to the solution. The samples were deep frozen and stored before analysis. In the experiments with the alveolar macrophages, the exposed cell cultures were rinsed once with 2 ml of HBSS and 1 ml of lysing
Table 1 DNA damage (-log unwinding technique)
Fds) in lungs of mice exposed
13
to different
concentration
mixtures
of 1,3-butadiene
(BUT) + NO, f UV” (the
Concentration
Time of exposure
-tog
(ppm) BUT + NO,
(h)
+uv
-uv
Control
20+
10
16
0.21 f 0.02 (4)
0.20 f 0.01 (4)
0.19 f 0.01 (4)
40 + 20 40 + 20 40 + 20
16 16 16
0.38 * 0.03 * (4) 0.37 + 0.07 (3) 0.28 f 0.02 * (4)
0.27 f 0.01 (4) 0.24 f 0.00 (3) 0.18 f 0.02 (4)
0.24 I 0.03 (4) 0.27 + 0.04 (3) 0.22 + 0.01 (4)
0.34 f 0.03 b.’ (11)
0.23 k 0.01 h (11)
0.24 + 0.02 ’ (11)
0.14 i 0.02 (3)
0.14 i 0.01 (3)
60 + 30
5
Fds
0.19 2 0.00
a Data are expressed as mean + SE. Number of animals b Mean + SE of all mice from the different groups. * p < 0.05; * * p < 0.01.
- * (4)
in each group
in parentheses.
14
S.A.S. Walles et al. /Mutation Research 328 (1995) II-19
lysing for 1 h with sarcosyl EDTA solution (2% sarcosyl in 0.02 M EDTA, pH 9.6) with or without 0.5 mg proteinase K per ml and washing with 0.02 M EDTA (pH 9.6), single-stranded DNA was eluted from the fibers for 16 h with 0.02 M EDTA, adjusted to pH 12.15 with NaOH, at a flow rate of 0.03 ml/min. Both the elution and the measurement of the samples were performed by means of an automated system (Brunborg et al., 1988). The amount of DNA was measured by fluorometry by means of Hoechst 33258. The damage of DNA was calculated as the normalized area above the curve (NAAC) according to Brunborg et al. (1990). Statististical analysis The significance of differences values was tested with Student’s pendent observations.
between mean t-test for inde-
3. Results In ho exposure of mice In Table 1 the DNA damage from mixtures of butadiene and NO, is presented as determined by the alkaline unwinding technique. When mice were exposed to a mixture of 20 ppm butadiene and 10 ppm NO, there was no increase in the level of SSB in lung either with or without UV irradiation. At higher concentrations, 40 ppm butadiene + 20 ppm NOz f UV, the level of SSB increased significantly in two out of three repeated experiments with UV irradiation. When the results from all animals in the three experiments were pooled, the UV-irradiated mixture increased the level of SSB 1.4 times compared to the control (p < 0.05). When a group of mice was exposed to 60 ppm butadiene + 30 ppm NO, for 5 h, there was an increase of 1.4 times the control value (p < 0.01) by the UV-irradiated gas mixture. The alkaline elution technique did not show any DNA damage after exposure to 40 ppm butadiene + 20 ppm NO? + UV for 16 h, either with or without proteinase K added to the Iysing solution (Table 2). However, the DNA damage increased 1.6 times (p < 0.05) compared to the
Table 2 DNA damage (NAAC, IOM3/h) in lung DNA of mice exposed to different concentration mixtures of 1,3-butadiene (BUT)+NO; (the alkaline elution technique) Concentration BUT+NO,
Time of exposure (h)
NAAC (10m3/h) + UV -uv
40+20h 40+20 c
16 16
20+ 20*2
hd
60+30
b
5
1(4) (4)
19*2*
a Data are expressed as mean+ each group in parentheses. h Lysing with proteinase K. ’ Lysing without proteinase K. * p < 0.05.
Control
2Oi2(3) 18rt l(4)
24&3(4) 20+ l(4)
14+0(3)
12&l(3)
(4)
SE. Number
of animals
in
control after exposure to a UV-irradiated gas mixture of 60 ppm butadiene + 30 ppm NO, for 5 h. Fig. 1 shows an example of aIkaline elution of DNA from cell nuclei from lungs of mice
100
0
*
Control
+
-uv
*
+uv
4
8
Time
of elution
12
16
(h)
Fig. 1. Alkaline elution profiles of DNA from lung of mice exposed to 60 ppm butadiene + 30 ppm nitrogen dioxide + UV for 5 h and control.
S.A.S. Walles et ai. /Mutation Table 3 DNA damage
(-log
F,,) in lung and liver of mice exposed
Research 328 (19951 II-19
to 1,3-butadienea
15
(the unwinding
technique)
Concentration
Time of exposure
- log F,s
(ppm)
(h)
Lung Exposed
Control
Exposed
Control
80 200 500
16 16 5
0.22 f 0.05 (3) 0.20 f 0.02 * (5) 0.28 f 0.02 * (4)
0.17 + 0.01 (4) 0.12 f 0.02 (3) 0.19 f 0.02 (4)
0.20 f 0.02 * (5) 0.34 + 0.04 * * (4)
0.12 * 0.01 (3) 0.15 * 0.02 (4)
a Data are expressed as mean f SE. Number * p < 0.05; * * p < 0.01.
Table 4 DNA damage
NAAC
(10U3/h)
Liver
of animals
in each group
in lung and liver of mice exposed
Concentration
Time of exposure
NAAC
(ppm)
(h)
Lung
in parentheses.
to 1,3-butadienea
Control
16
25 + 1 (4)
19 * 4 (3)
200 500
16 5
34 + 4 (4) 47 * 2 * * (4)
34 f 2 (3) 35 f 2 (4)
in each group
exposed to 60 ppm butadiene + 30 ppm NO, f UV for 5 h and control. The NAAC value for the control mouse was 13 x 10e3/h, for a mouse treated with non-irradiated gas mixture the NAAC value was 14 x 10-3/h and for a mouse treated with UV-irradiated gas mixture the NAAC value was 26 X 10P3/h. The mice exposed to non-irradiated mixtures of butadiene and NO, did not show any increase in SSB compared to the control mice in any experiment (Tables 1 and 2).
Table 5 DNA damage
(-log
Fds) in lung of mice exposed
elution
technique)
Liver
Exposed
of animals
(the alkaline
(1O-3/h)
80
a Data are expressed as mean f SE. Number * p < 0.05; * * p < 0.01.
_
to nitrogen
Control
Exposed _ 28 + 4 (4) 36 f 4 * (4)
25 f l(3) 19 f 2 (3)
in parentheses.
Mice were also exposed to butadiene as a single compound at a concentration of 500 ppm in air for 5 h or 80 or 200 ppm for 16 h. There was no increase of the DNA damage when mice were exposed to 80 ppm for 16 h (Tables 3 and 41, neither at 200 ppm for 16 h with the alkaline elution technique, but an increase of 1.7 times the control in both liver and lung was found with the unwinding technique (p < 0.05). After 5 h of exposure to 500 ppm both methods demonstrated a significant increase in SSB in liver and lung.
dioxide”
(the unwinding
technique)
Concentration
Time of exposure
- tog Fds
(ppm)
(h)
Exposed
Control
30 30 30
16 16 16
0.32 + 0.03 * (5) 0.31 + 0.02 (5) 0.24 rfi 0.02 (5)
0.20 _Ir0.04 (3) 0.27 + 0.02 (3) 0.19 + 0.02 (3)
0.30 2 0.02 b, * * (15)
0.23 + 0.02 b (9)
0.19 + 0.01 * * * (5)
0.12 f 0.01 (7)
50
5
a Data are expressed as mean + SE. Number of animals ’ Mean f SE of all mice from the different groups. * p < 0.05; * * p < 0.01; * * * p < 0.001.
in each group
in parentheses.
I6
S.A.S. W&es et al. / Mntation
Mice were exposed to 30 ppm of NO, for 16 h in three different experiments. Using the unwinding technique, there was an increase in SSB in all experiments, although only one experiment was statistically significant at the p < 0.05 level (Table 5). However, when the results from all individual animals were pooled, a higher significance was obtained (p < 0.01). No increase of the SSB level was found using the alkaline elution technique (data not shown). When mice were exposed to 50 ppm for 5 h, there was a highly significant increase (1.6 times the control) in the level of SSB (p < 0.001) tested with the unwinding technique. In vitro exposure of alrleolar macrophages Alveolar macrophages from the same strain of mice were exposed to 40 ppm butadiene + 20 ppm NO, for 2 h with and without UV irradiation (Table 6). DNA damage was analyzed with the unwinding technique. The UV-irradiated gas mixture increased the level of SSB 4.3 times compared to the control (p < 0.001). Without UV irradiation the level of SSB increased 2.5 times compared to the control (p < 0.01). The viability of the macrophages was 95% before exposure. After exposure to the UVirradiated gas mixture, the viability decreased to 75% and for the non-irradiated mixture the viability was 85%. The macrophages were also exposed to pure butadiene and pure NO, in air for 2 h (Table 7). The level of SSB increased about twice compared to the control (p < 0.01) at an exposure to 20 ppm NO,. After exposure to 40 ppm butadiene
Table 6 DNA damage t-log Fds) in alveolar macrophages of mice exposed in vitro to 1,3-butadiene (BUT) + NO, f UV for 2 h” (the unwinding technique) Concentration
-log
bpm)
+uv
BUT+NO, 40+20
Fds -uv
0.34rtO.02 (3)
a Data are expressed group in parentheses. * * p < 0.01; * * * p
***
0.20+0.01 (3)
as mean f SE. Number
< 0.001.
Control jr *
0.08~0.02 (3)
of dishes in each
Reseurc[z 328 (1995) I I - 19 Table I DNA damage (-log Fds) in alveolar macrophages exposed in vitro to 1,3-butadiene (BUT) and NO2 (the unwinding technique) Concentration
Gas
(ppm)
- tog F,,a Exposed
Control 0.05 f 0.00 (3)
40
BUT
0.07 + 0.01 (4)
70
NOz
0.09~0.01
* * (3)
a Data are expressed as means+SE. each group in parentheses. * * p < 0.01.
there SSB.
of mice for 2 h”
was no statistically
0.04i_0.00
Number
significant
(3)
of dishes
increase
in
in
4. Discussion The present investigation has shown that photochemically formed reaction products from butadiene and NO, can cause SSB in DNA in lung cells both in vitro and in vivo. Using the alkaline unwinding technique it was shown that 40 ppm butadiene + 20 ppm NO, + UV induced SSB both after 16 h inhalation in vivo and after 2 h exposure of alveolar macrophages in vitro. The in vivo effects were most probably caused by photochemical reaction products, as the nonirradiated mixtures did not cause any increase in SSB (Table 1). Butadiene by itself did not cause a significant effect at 80 ppm (Table 3). NO, at 30 ppm caused a significant effect in one out of three repeated experiments only (Table 5). As the gas mixture will contain lower concentrations of butadiene and NO, after the 40-min UV irradiation period than before, due to photochemical reactions, we concluded that these compounds probably did not contribute to the DNA damage observed in the experiments with UV irradiation. However, in the in vitro experiment, NO, may have contributed to the increase in SSB observed in macrophages exposed to the UV-irradiated gas mixture, as NOz at 20 ppm induced SSB when tested as a single compound (Tables 6 and 7). The UV-irradiated gas mixture induced more SSB than the non-irradiated mixture, which indicates that both NO, and the photochemical reaction
S.A.S. Walles et al./Mutation
products cause the DNA damage observed. Butadiene at 40 ppm did not induce SSB when tested as a single compound (Table 7). When mice were exposed to the UV-irradiated gas mixture, DNA damage measured by the unwinding technique was induced to the same extent after 5 and 16 h of exposure (Table 1). There might be different explanations for this phenomenon. A steady state might be formed already after 5 h of exposure. Another explanation could be that cross-links will be formed over time, which will decrease the possibility to detect SSB. In the present study we had no possibility to test if cross-links really were induced. That would have required irradiation of the cell nuclei with X-rays and we did not have access to such equipment. However, as DNA damage could be detected when the alkaline elution assay was used for analysis after 5 h of exposure but not after 16 h, this fact might also be an indication that crosslinks were formed. There are two different kinds of cross-links, namely DNA-protein cross-links and DNA inter-strand cross-links. In the present study DNA-protein cross-links can be excluded as the results were the same whether proteinase K was present or not during the lysing time (Table 2). In the procedure of the unwinding technique the strands are shortened by ultrasonic treatment. If cross-links are rare, only few pieces will contain cross-links, allowing for most of the SSB to be able to be detected. In contrast, DNA inter-strand cross-links will obscure the SSB in the alkaline elution technique, because the strands cannot separate. When mice were treated with butadiene the unwinding technique showed a significant effect both at 200 ppm for 16 h and at 500 ppm for 5 h (Table 3). With the alkaline elution technique there was no increase of DNA damage after 16 h of exposure, but there was an effect after 5 h of exposure with the higher dose (Table 4). Most probably this result is due to induction of DNA inter-strand cross-links. 1,3-Butadiene is at first metabolized to the monofunctional component 1,2-epoxybutene (EB) (de Meester, 1988) and further to the difunctional agent 1,2 : 3,4-diepoxybutane (DEB) (Malvoisin et al., 1979). EB will react with guanine in DNA (Jelitto et al., 1989) and
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subsequently induce SSB. DEB will induce DNA inter-strand and DNA-protein cross-links. In an investigation by Vangala et al. (1993), mice and rats were exposed to butadiene at 100-2000 ppm for 7 h. The alkaline elution technique was used for detection of DNA damage. Both types of cross-links were observed in mouse lung and liver. The formation of cross-links was higher in lung than in liver. It has earlier been shown that DEB induced complete reversibility of heat denaturation of DNA, which was interpreted as formation of cross-links in DNA (Lawley and Brookes, 1967). The present study has shown that DNA damage can be induced by NO, at sufficiently high concentrations, both in lung cells in vivo and in alveolar macrophages in vitro (Tables 5 and 71. Very few data on genotoxic effects of NOz in vivo (for review, see Victorin, 1994) have been presented before. Only one in vivo study on effects in lung cells was found in the literature (Isomura et al., 1984). In that study, exposure to 15 ppm NO, for 3 h induced mutations and chromatidtype chromosomal aberrations in cultured lung cells from exposed rats. NO, has been shown to induce SSB in V79 hamster cells in vitro (Giirsdorf et al., 19901, but no other data on SSB were found in the literature for comparison with our results in lung cells in vitro and in vivo. The reaction products formed in the present experiments have not been chemically identified. Generally, the photochemical smog reactions of alkenes proceed via radical reactions and organic peroxides (Butler, 1979). Acrolein is the major product in photochemical NO,-butadiene reactions and formaldehyde is also formed (Maldotti et al., 1980). Both formaldehyde and acrolein give rise to SSB, DNA inter-strand cross-links and DNA-protein cross-links at near-toxic concentrations in cultured bronchial cells, as measured with the alkaline elution technique (Grafstrom, 1990). Formaldehyde gives more DNA-protein cross-links than acrolein does. Formaldehyde has also been shown to form DNA-protein cross-links in vivo (Casanova-Schmitz and Heck, 1983; Ross and Shipley, 1980). In experiments by Lam et al. (1985), acrolein induced DNA-protein cross-links in rat nasal mucosa in vitro, but not in vivo. Acrolein depletes glutathione in vitro and forms
18
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adducts with guanine of calf thymus DNA (Chung et al., 1984). According to the cited literature data on formaldehyde and acrolein, at least acrolein probably contributed to the induction of SSB in the present study. However, acrolein and formaldehyde are only weakly mutagenic in Salmonella strain TAlOO (IARC, 1985; Sasaki and Endo, 1978). Thus, it seems less likely that acrolein or formaldehyde was responsible for the strong direct-acting mutagenic activity in Salmonella TAlOO that was demonstrated in earlier experiments (Victorin and Stihlberg, 1989). In earlier in vivo studies using the same exposure system as in the present study, we obtained equivocal results in the somatic mutation and recombination test in Drosophila and no genotoxicity in the mouse bone marrow micronucleus assay (Victorin et al., 1990). The results probably reflect the fact that the photochemical reaction products are reactive, probably short-lived compounds that may be inactivated in vivo before they reach target cells in the bone marrow. In the present study we wanted to study the effect on lung cells simultaneously in vivo and in vitro, using the same endpoint. According to our previous results, one might have expected a much higher response in the in vitro exposure situation. However, according to the present results, the reaction products can cause detectable DNA damage in lung tissue upon inhalation of high concentrations to a similar extent as in isolated alveolar macrophages. Our studies show similarities with the studies by Heddle et al. (1993) on peroxyacetyl nitrate (PAN). PAN is formed in atmospheric photochemical reactions between hydrocarbons and NO?, and was suggested as one contributor to the direct-acting mutagenicity in SalmoneIla TAlOO found in photochemical experiments with propene and NO, (Kleindienst et al., 1985a,b). When tested in inhalation experiments with Chinese “0 clearly significant effects were hamsters, demonstrated concerning mutations or micronuclei in lung fibroblasts. PAN is a toxic compound, which prevents the testing of high concentrations. This is a property common to several air pollutants that are direct-acting mutagens in vitro,
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although it has been difficult to demonstrate genotoxic effects in vivo. For example, the in vivo genotoxic hazard of ozone and NO, is difficult to evaluate partly due to this reason (Victorin, 1992, 1994). A genotoxic hazard to lung tissue of inhalation of photochemical reaction products from alkenes and NO, has been demonstrated at high concentrations in the present study, but our results and those of Lam et al. (1985) on acrolein and of Heddle et al. (1993) on PAN indicate that the risk is far less than was previously indicated by mutagenicity in Salmonella bacteria.
Acknowledgements The authors wish to thank Rolf Stridde for skilful technical assistance. This work was supported by a grant from the Swedish OK environmental research foundation.
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