Induction of chromosomal aberrations in Chinese hamster ovary cells by triethyllead acetate

Induction of chromosomal aberrations in Chinese hamster ovary cells by triethyllead acetate

Mutation Research, 298 (1992) 1-7 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1218/92/$05.00 1 MUTGEN 01820 Induction of chrom...

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Mutation Research, 298 (1992) 1-7 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1218/92/$05.00

1

MUTGEN 01820

Induction of chromosomal aberrations in Chinese hamster ovary cells by triethyllead acetate David H. Blakey, Janet M. Bayley and George R. Douglas Mutagenesis Section, Bureau of Chemical Hazards, Environmental Health Directorate, Health Protection Branch, Department of National Health and Welfare, Ottawa, Ont. K1A OL2, Canada (Received 18 January 1992) (Accepted 15 May 1992)

Keywords: Chinese hamster ovary cells; Triethyllead acetate; Organolead compounds; Gasoline, leaded; Alkylleads

Summary Organolead compounds enter the environment primarily through the combustion of leaded gasoline and industrial discharge. Lead and lead-containing compounds have been shown to induce a broad spectrum of toxic effects, including hematopoetic, renal, neurologic, and carcinogenic effects. In this study, the mutagenic activity of triethyllead acetate (Et3PbAc) was determined by measuring the induction of chromosomal aberrations in Chinese hamster ovary cells. The results indicate that Et3PbAc is very cytotoxic and a potent clastogen. In preliminary cytotoxicity studies used to determine appropriate test concentrations for chromosomal aberration analysis, the LCs0 of Et3PbAc was approximately 10/zM in the absence of metabolic activation, and 80 /~M in the presence of metabolic activation. Et3PbAc induced a significant (p < 0.01) increase in chromosomal aberrations at 6 /xM in the absence of metabolic activation, and 40 /xM in the presence of metabolic activation. The maximal response was greater with metabolic activation than without. However, a much higher dose was required to elicit a significant response in the presence of metabolic activation than in its absence.

Lead is an ubiquitous environmental pollutant known to induce a variety of toxic effects in laboratory animals and human beings. Major sources of lead pollution include both the combustion of leaded gasoline and industrial discharge. There is also a high risk of lead exposure Correspondence: Dr. David H. Blakey, Mutagenesis Section, Bureau of Chemical Hazards, Environmental Health Directorate, Health Protection Branch, Department of National Health and Welfare, Ottawa, Ont. K1A 0L2, Canada.

associated with some occupations. The toxic effects of lead compounds on the immune system, hematopoesis, renal function, and the nervous system are well-known (for review, see Gerber et al., 1980). There is also evidence that lead is carcinogenic in rodents (Stoner et al., 1976; IARC, 1980). However, since evidence of carcinogenicity in human beings is considered to be inadequate, the International Agency for Research on Cancer classifies lead as possibly carcinogenic to human beings (IARC, 1987).

The alkylleads are an important class of lead compounds that have been used as gasoline additives since 1923. The combustion of leaded gasoline is believed to contribute to elevated levels of lead around parking garages, filling stations, at the roadside (Carson et al., 1987), and in playgrounds located near busy roadways (Day et al., 1975, 1979; Anagnostopoulos, 1983). In three large cities in the United States, where the use of leaded gasoline has declined, there is a direct relationship between the consumption of leaded gasoline and levels of lead in the blood of 2 - 3 year-old children (Billick, 1983). Although the use of leaded gasoline has been reduced in North America and some parts of Europe, it is still in use throughout much of the world. Furthermore, it is still being produced for export by North American refineries. Even if the use of leaded fuels was to end, alkyllead fallout from the combustion of leaded gasoline would continue to contaminate the environment. In addition to the combustion of leaded fuels, industrial discharge is also a major source of alkyllead contamination. For example, alkylleads have been detected in fish caught downstream from manufacturing facilities that produce these compounds along the St. Clair and the St. Lawrence Rivers (Chau and Wong, 1984). Alkylleads may be particularly hazardous because they are widely distributed in the environment, and enter mammalian cells easily. Because

TABLE 1 CYTOTOXICITY OF TRIETHYLLEAD ACETATE IN CHO CELLS a Dose (/~M) 0 10 30 60 100 300 600

- $9 % survival 100 46.7± 10.7 0 0 NT b NT NT

+ $9 % survival 100 110.5_+ 11.2 96.5± 9.1 92.3 ± 11.7 19.2± 9.7 0 0

a Mean of four plates ± standard error. % survival is expressed relative to the cells treated with the solvent control (0 dose). b NT, not tested.

of the high potential for human exposure, several studies have examined the toxicological effects of alkyllead. A 90 day feeding study demonstrated that in vivo exposure of rats to Et~PbAc induced a broad spectrum of toxic effects in several tissues, including the hematopoetic system (Yagminas et al., 1987; Franklin et al., 1987). In another study, triethyllead chloride inhibited tubule formation and caused the depolymerization of tubules in mammalian ceils ( Z i m m e r m a n n et al., 1988). Previous studies have examined the mutagenicity of alkylleads. However, none of these studies have included a direct m e a s u r e m e n t of the potential for Et3PbAc to induce chromosomal aberrations in mammalian cells. For example, triethyllead (Et3Pb) has been shown to induce sister chromatid exchanges in human lymphocytes exposed in vitro (Niebuhr and Wulf, 1984) as well as nondisjunction and chromatid breaks in Drosophila melanogaster larvae (Ahlberg et aI., 1972). However, these measurements are uncertain predictors of the potental to induce chromosomal aberrations in mammalian cells. Tetraethyllead, which is metabolized to the more toxic Et3PbAc in vivo (Cremer, 1959) did not induce dominant lethal mutations in mice (Kennedy and Arnold, 1973). Since chromosomal aberrations play a major role in dominant lethality (Green et al., 1985), it is possible that Et3Pb is unable to induce chromosomal aberrations in mammalian cells. In order to answer this question, the clastogenic activity of Et3PbAc was determined directly in this study, by measuring the induction of chromosomal aberrations in Chinese hamster ovary cells in vitro. Materials and methods

Chemicals Triethyllead acetate ( > 99.9% purity) was supplied by Phiokol Inc., Danvers, MA. Cyclophosphamide (CY) was purchased from the Sigma Chemical Co., and methyl methanesulfonate (MMS) was purchased from the Aldrich Chemical Co.

Cell culture Wild-type Chinese hamster ovary ( C H O ) cells

were maintained in Eagle MEM supplemented with 1% sodium pyruvate, 1% non-essential amino acids, and 10% fetal calf serum; (complete medium; all Gibco) at 37°C, 5% CO 2 and high humidity.

Treatment The complete medium was replaced with treatment medium consisting of the test chemical diluted in either serum-free complete medium or an exogenous metabolic activation medium prepared as follows: 82% serum-free complete medium; 5.4% 20 mM HEPES buffer pH 7.2 (Sigma); 0.2% 0.5 M MgC12 (Sigma); 0.2% 3.3 M KCI (Sigma); 2% 40 mM NADP (Sigma); 2% 50 mM glucose 6-phosphate (Sigma); and 7% Aroclor 1254-induced rat liver homogenate ($9) (Ames et al., 1975). In experiments with and without metabolic activation, 25 /zg/ml cyclophosphamide and 1 mM methyl methanesulfonate, respectively, were used for positive controis. The osmolality of the treatment solutions was determined using an Osmette osmometer. Cell survival assay 4 x 60 mm well culture dishes were inoculated with 200 CHO cells/well. The cells were allowed to attach for several hours, then treated as indicated above. Following treatment, the cells were washed three times with warm (37°C) Earle's balanced salt solution (BSS), then grown in 5 ml complete medium for 7 days. For analysis, the medium was removed, and the ceils were fixed with 3:1 ethanol-acetic acid and stained with Giemsa (Gurr R66 improved). The number of colonies was counted for each well and the % survival was based on the number of colonies relative to the number of colonies in the untreated control plates. Chromosomal aberration assay 2 x 105 CHO cells were added to 5 ml complete medium in 60-mm culture dishes and incubated as above overnight. The cells were treated in 1.5 ml of medium for 2 h, then washed 3 times with Earle's balanced salt solution (Gibco), and incubated as above in 5 ml complete medium for 10 h. The positive controls were 1.0 and 0.5 mM MMS for trials 1 and 2 without metabolic activa-

tion, respectively, and 25 /xg/ml CP for experiments with metabolic activation. Colcemid (0.1 ~ g / m l ) was added to all cultures for the final 2 h of incubation. The cells were then scraped from the dishes using a rubber spatula, centrifuged at 150 g for 5 rain, resuspended in hypotonic 0.075 M KC1 for 12 min at 37°C, centrifuged as above then resuspended in 3:1 ethanol-acetic acid. Chromosome preparations were made using standard cytological techniques then stained with 4% Giemsa (Gurr R66 improved). Normally, 100 metaphase cells were scored from each of two cultures for each treatment. For some cultures in which the incidence of chromosomal aberrations was very high or where metaphase cells were difficult to recover due to the effect of the treatment on cell division, only 50 cells were analyzed. The slides were coded and scored blind to avoid observer bias.

Statistical analysis For chromosomal aberrations, the data were analysed using the Chromosomal Aberration Assay Data management and Analysis System (version 1.4) software developed under contract to the U.S. Environmental Protection Agency (Pellom et al., 1990). Results and discussion

Cytotoxicity Preliminary cytotoxicity studies were performed to determine the test concentrations for the cytogenetic studies. The results are shown in Table 1. In the absence of metabolic activation, the LCs0 of Et3PbAc was approximately 10/xM. In the presence of metabolic activation, the cytotoxicity of Et3PbAc was reduced, and the LCso was approximately 80 ~M. Chromosomal aberrations Without metabolic activation, there was a significant (p < 0.01) increase in the percentage of cells with aberrations at doses of 6/zM in Trial 1 and 10/zM in Trial 2 (Table 2A). There was also a significant (p < 0.01) increase in the number of aberrations/cell at 6 /zM in both Trial 1 and Trial 2 (Table 2a). In the presence of metabolic activation, Et3PbAc induced a significant (p <

36.0_+ 4.5 *

150

a Mean of two cultures _+ standard error. b Osmolality in m O s m / k g H 2 0 . * Significant effect ( p < 0.01).

MMS

3.0+ 1,0 5.5_+ 1.5 7.5_+ 2.5 10.5-+ 4.5 35.3_+17.0 *

200 200 200 200 150

Trial 2 0 4 6 8 i0

321 303 303 305

76.0 *

50

MMS

2.5 1.0 0.0 0.0 1.5 * 1.0 * 1.0 *

% cells with aberrations

6.5_+ 6.0_+ 3.0_+ 2.0-+ 17.5+ 61.0_+ 55.0_+ Toxic

Cells analysed

200 200 200 200 200 100 100

Osmol. b

Trial 1 0 1 2 4 6 8 10 20

Dose (p,M)

0.480+0,075 *

0,035+0.005 0.055_+0.015 0.085_+0.015 * 0.155-+0.065 * 0.593+0.290 *

2.220 *

0.070_+ 0,030 0.060+0.010 0.035 _+0.005 0.020 + 0.000 0.250+0.000 * 1.000_+0.000 * 0.880_+0.000 * Toxic

Aberrations/ cell

16

2 5 1 5 8

15

7 8 3 2 12 16 15

5

4 3 8 2 1

8

6 3 1 2 3 4 3

2

0 1 2 1 4

6

0 0 0 0 1 4 6

0 0 0 0 7 26

24

30

18

0 3 6 25 75

0 0 0 0 2 5 2

Exchanges

1 1 2 0 33 78 66

Breaks

Gaps

Breaks

Exchanges

Chromatid

Chromosome

I N D U C T I O N OF C H R O M O S O M A L A B E R R A T I O N S BY Et3PbAc W I T H O U T METABOLIC A C T I V A T I O N a

TABLE 2A

2

0 0 1 0 12

3

0 0 0 0 2 12 9

Gaps

Heavy

100

363

CP

a Mean of two cultures +_ standard error. b Osmolality in m O s m / k g H 2 0 . * Significant effect ( p < 0.01).

44.7+ 17.5 *

0.5 1.5 0.0 2.5 9.5 * 6.9 * 1.0 *

200 200 200 200 150 150 95

297 295 295 282 302 297 301

Trial 2 0 30 40 5O 60 70 80 8.5 _+ 6.5 _+ 5.0 + 6.5 + 23.3+ 32.9_+ 49.0±

50.0+_ 0.0 *

100

* * * * *

CP

0.0 0.0 0.5 3.0 1.0 0.0 2.0 6.0

% cells with aberrations

2.0 +5.0 q-_ 4.5 +14.0 ± 13.0± 36.0± 50.0+_ 60.0+

Cells analysed

200 200 200 200 200 100 100 100 Toxic

Osmol. h

Trial 1 0 20 3O 40 5O 60 70 8O 100

Dose (/xM)

26

0.627+0.220 *

1.020_+0.080 *

10 2 8 4 8 9 6

37

* * * * *

0.090 + 0.000 0.065 + 0.015 0.050 + 0.000 0.075 + 0.025 0.313+0.200 * 0.486+0.193 * 0.740+0.040 *

3 5 6 14 20 9 36 24

0.020 ± 0.000 0.055 ± 0.005 0.060 +-0.000 0.185+0.015 0.195+0.005 0.540 +-0.020 1.190_+0.150 1.450+0.330

Breaks

cell

15

7 6 1 6 8 4 5

4

0 4 4 13 8 4 1 2

Exchanges

Chromosome

Aberrations/

4

0 0 1 1 0 5 4

4

0 0 1 1 3 4 8 24

Gaps

I N D U C T I O N OF C H R O M O S O M A L A B E R R A T I O N S BY Et3PbAc W I T H METABOLIC A C T I V A T I O N a

TABLE 2B

9

0 2 0 0 24 50 56

9

0 1 1 5 5 32 69 115

Breaks

Chromatid

45

1 2 1 1 7 8 7

21

0 1 1 5 9 8 13 5

Exchanges

0 0 0 0 0 0 0 0

0

3

7

0 1 0 0 3 7 10

0 0 0 0 0 0 0 0

Heavy

0 1 1 3 5 19 63 119

Gaps

0.01) increase in the percentage of cells with aberrations and the number of aberrations/cell at doses of 40/xM in Trial 1 and 60 p.M in Trial 2 (Table 2b). The dose-response both with and without metabolic activation was highly significant ( p < 0.001). Compared to treatment without metabolic activation, a greater number of cells with chromosomal aberrations was induced, but a nearly 10-fold higher concentration was required to elicit a significant response (Table 2b). It is interesting to note that Et3PbAc induced more chromatid breaks than chromatid exchanges. This is in contrast to the positive controls which induced more chromatid exchanges than chromatid breaks. These results may indicate that the mechanism of action of Et3PbAc differs from that of MMS or CP. Both the cytotoxicity and the clastogenicity of Et3PbAc were reduced by metabolic activation. The reason for this reduction is unclear. It is unlikely that Et3PbAc is metabolized to a less toxic compound by $9. It is possible that the test compound binds to components of the activation mix, thus reducing the dose delivered to the cells. For treatment with and without metabolic activation, there was no evidence that the osmolality of the treatment media had been altered enough to induce chromosomal aberrations by osmotic shock. The treatment medium without Et3PbAc was 297 m O s m / k g H20 (Table 2b). This is very close to the value of 300 m O s m / k g H 2 0 reported previously by Galloway et al. (1987) for culture medium. All of the Et3PbAc concentrations tested were within 8% of the osmolality of the negative control sample. Several studies have been conducted to determine the mutagenic mechanism of lead compounds. Zelikoff et al. (1988) reported that lead sulfate and lead nitrate were mutagenic at the H P R T locus of Chinese hamster V79 H P R T cells. However, neither of these compounds induced DNA strand breaks detected using the DNA alkaline elution assay or sister chromatid exchanges in the same cells. In another study, Hartwig et al. (1990) reported that exposure of Chinese hamster V79 cells to 500 txM lead acetate for 20 h did not induce mutations at the H P R T locus, sister chromatid exchanges, or D N A strand breaks. However, in the same study, lead acetate enhanced

the activity of UV irradiation in the same three endpoints. Both Zelikoff et al. (1988) and Hartwig et al. (1990) concluded that the clastogenic action of lead compounds was the result of an indirect mechanism such as the inhibition of DNA repair. In previous studies, lead ions had been shown to decrease the fidelity of DNA synthesis (Sirover and Loeb, 1976) and to inhibit DNA polymerase-beta (Popenoe and Schmaeler, 1979). In contrast to the negative in vitro results, however, lead acetate induced micronuclei in the bone marrow of rats (Tachi et al., 1985) and sister chromatid exchanges in the maternal bone marrow and the liver of the fetus following exposure of pregnant mice (Sharma et al., 1985). In this study, Et3PbAc was strongly clastogenic in C H O cells, both with and without metabolic activation. Unlike lead acetate, Et3PbAc induces chromosomal aberrations in vitro without the administration of UV irradiation. The mechanism of action of Et3PbAc may therefore differ from that of lead acetate. Et3PbAc is soluble and lipophilic. In aqueous media, it dissociates rapidly to ionic Et3Pb and, as such, can readily enter the cell. Hartwig et al. (1990) demonstrated that lead acetate was also taken up into V79 and HeLa cells, but that the amount of lead acetate taken up by the cell was not correlated with cytotoxicity. Thus, the uptake of Et3Pb into the target cells cannot alone account for the differences in activity between lead acetate and Et3PbAc. It is possible that the alkyl moiety allows Et3PbAc to react directly with D N A in a way that induces chromosomal aberrations whereas other organic and inorganic lead compounds cannot. it is clear from these experiments that Et3PbAc is a potent in vitro clastogen, capable of inducing high levels of chromosomal damage at relatively low concentrations. According to the strategy developed for the assessment of genotoxicity recommended by the Health Protection Branch of Health and Welfare Canada (Health Protection Branch, 1989), Et3PbAc is designated as an in vitro mutagen. It has been demonstrated previously that in vivo exposure of rats to Et3PbAc induces a broad spectrum of toxic effects in several tissues, including the hematopoetic system (Yagminas et al., 1987; Franklin et al., 1987). These studies provide

evidence that Et3PbAc is capable of inducing toxic responses in vivo. It is therefore possible that Et3PbAc may also be clastogenic in vivo.

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