Study of the genotoxic activity of five chlorinated propanones using the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test

Genetic Toxicology

ELSEVIER

Mutation Research 341 (1994) l-15

Study of the genotoxic activity of five chlorinated propanones using the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test Frank Le Curieux apb,Daniel Marzin alb~*,Frangoise Erb b aLaboratory of Genetic Toxicology, Pasteur Institute of Lille, 1 rue du Professeur Calmette, BP 245, 59019 Lille Ceder, France b Department Toxicology-Hydrology-Hygiene, Faculty of Pharmacy, 3 rue du Professeur Laguesse, BP 83, 59006 Lille Cedex, France Received 12 April 1994; revision received 7 June 1994; accepted 7 June 1994

Abstract Three short-term assays (the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test) were carried out to evaluate the genotoxicity of five chlorinated propanones identified in several chlorinated waters (monochloropropanone, l,l-dichloropropanone, 1,3-dichloropropanone, l,l,l-trichloropropanone and 1,1,3-trichloropropanone). In the SOS chromotest, all the compounds except monochloropropanone were found to induce primary DNA damage in Escherichiu c&i. With the fluctuation test, all five chloropropanones showed mutagenic activity on Salmonella typhimurium strain TAlOO. The newt micronucleus assay detected a clastogenic effect on the peripheral blood erythrocytes of Pleurodeles Walt1 larvae only for 1,3-dichloropropanone and 1,1,3-trichloropropanone. Moreover, two structure-activity relationships are noticeable: (1) chloropropanones with chlorine substituents on both carbon positions (1,3-DCP and 1,1,3-TCP) are by far more genotoxic than chloropropanones substituted only on one carbon position (l,l-DCP and l,l,l-TCP); (2) the increase of the number of chlorine substituents decreases the mutagenic activity (fluctuation test) of the chlorinated propanones studied. Keywords: Chloropropanone;

Chlorination

by-product;

SOS chromotest; Ames-fluctuation

test; Newt micronucleus

test; Sensitivity; Isomer

1. Introduction

MCP, monochloropropanone; l,l-DCP, l,l-dichloropropanone; 1,3-DCP, 1,3-dichloropropanone; l,l,l-TCP, l,l,l-trichloropropanone; 1,1,3-TCP, 1,1,3-trichloropropanone; DMSO, dirnethylsulfoxide; 4-NQO, 4-nitroquinoline l-oxide; CPA, cyclophosphamide; S9-mix, metabolic system. * Corresponding author. Tel. (33) 20 87 79 14; Fax: (33) 20 87 73 10. 0165-1218/94/$07.00

Since the identification of halogenated organits in drinking water by Rook (1974) and Bellar et al. (1974), numerous studies have shown the genotoxic

0 1994 Elsevier Science B.V. All rights reserved

SSDZ 0165-1218(94)00043-3

activity

of chlorinated

drinking

water

extracts (Kool et al., 1982; Horth, 1989) and of by-products of chlorination (Bull et al., 1985; Cheh, 1986; Pommery et al., 1989; Tikkanen and Kronberg, 1990; Brunborg et al., 1991; Jansson et

2

F. Le Curieux et al. /Mutation

al., 1993). It is now widely accepted that the genotoxicity detected in drinking water mainly originates from the reaction of chlorine with natural organic substances (i.e. humic substances) which lead to the formation of organohalogenated derivatives. This issue was reviewed by Meier (1988). In the present work, we performed three short-term genotoxicity tests in order to investigate the genotoxicity of five chlorinated propanones, a group of chemicals likely to be found in drinking water samples (Meier et al., 1985; Krasner et al., 1989; Jacangelo et al., 1989). The SOS chromotest, the Ames-fluctuation test and the newt micronucleus test were the three assays implemented in this study. In previous papers (Le Curieux et al., 1992; 19931, these three tests were carried out to determine the genotoxic activity of reference genotoxic chemicals. They appeared to be very well suited for the study and the testing of genotoxicity in water samples.

2. Materials and methods Chemicals

The main characteristics of the five chloropropanones studied are shown in Table 1. Genotoxicity tests SOS chromotest. The tester strain, Escherichia coli PQ37, was kindly given by M. Hofnung (In-

stitut Pasteur, Paris, France). Its genetic features were described by Quillardet and Hofnung (1985).

Table 1 Characteristics of the five chloropropanones

Research 341 (1994) I-15

The SOS chromotest was performed as recommended by these researchers and following the adaptation made by Marzin et al. (1986). After incubation, the mixtures were divided into two series, one for /3-galactosidase activity measurement (an induction assay) and the other for alkaline phosphatase (a control of protein synthesis). In our study, a chemical is considered as toxic when it induces a decrease of more than 50% in the alkaline phosphatase activity compared to the solvent control. The measurement of enzymatic activities was performed using microplates and an automatic microplate reader as described by Xu et al. 0989). The genotoxic activity for concentration c may be expressed in the ratio R, =/3/p where /I represents p-galactosidase activity (in mIU) and p, phosphatase alkaline activity (in mIU). The induction factor for a compound at concentration c is defined as I, = RJR,, in which R, is the spontaneous ratio measured in the blank test (solvent control). Experiments were performed with and without metabolic activation for every chorinated propanone studied, To ensure the validity of the assay, a positive control was included in each experiment. The positive controls used were 4-NQO (1 pg/ml) without S9-mix and B[a]P (30 pg/ml) with S9-mix. All the chloropropanones studied were tested with and without the metabolic system. Compounds were tested at least twice (two independent assays) using 6 experimental points for each dose. A compound is considered as an SOS repair system inducer in E. coli if the four following conditions are fulfilled (Olivier and Marzin, 1987):

studied

Name

Abbreviation

Molecular formula

Molecular Density Solubility weight (p = powder) (mg/H,O at 20°C) (g/mol.)

Monochloropropanone l,l-Dichloropropanone 1,3-Dichloropropanone l,l,l-Trichloropropanone 1,1,3-Trichloropropanone

MCP l,l-DCP 1,3-DCP l,l,l-TCP 1,1,3-TCP

CH,COCH,Cl CHsCOCHCl, ClCH,COCH,Cl CHsCOCCl, ClCHsCOCHCl,

92.53 126.97 126.97 161.42 161.42

Chemicals

1.15 1.327 p 1.435 1.53

100000 32000 27 900 -

Solvent Supplier Purity CAS used (%I No.

DMSO DMSO DMSO DMSO DMSO

Merck Aldrich Aldrich Aldrich Merck

> 95 98 > 95 97 95

78-95-5 513-88-2 534-07-6 918-00-3 921-03-9

F. le Curieux et al. /Mutation Research 341 (1994) l-15

3

(1) the induction factor is higher than 1.5, (2) the P-galactosidase activity is significantly increased compared to the solvent control, (3) the induction factor versus concentration graph shows a doseeffect relationship and (4) the result is reproducible.

significant increase in the number of positive wells compared to the solvent control, (2) if a dose-effect relationship is noticeable and (3) if the result is reproducible.

test. The tester strain, Salmonella typhimurium TAlOO, was kindly provided by

de Biologie du Developpement, University P. Sabatier (Toulouse, France) and acclimatized for one week before the experiment. The assay was performed as described by Jaylet et al. (1986) and according to the recommendations of the French standard AFNOR T-90-325 (1987) revised in 1992 (AFNOR, 1992). Briefly, larvae at stage 53 of the developmental table established by Gallien and Durocher (1957) were used for the experiment, since at this stage the mitotic index is at a maximum (Deparis, 1973). The highest concentration to be used in the 1Zday micronucleus assay is defined as half the minimum concentration that led to detectable physiological disturbances (weight loss, swelling, reduction in food intake, swimming in circles, etc.) in a 6-day preliminary toxicity test. In every glass container, the water (containing the dissolved chemical at the required concentration) was renewed daily. After 12 days of treatment, blood samples were taken from every animal (15 to 20 larvae per concentration) by cardiac puncture into an heparinized micropipette. The slides (one for each animal) were stained with Masson acid Hemalun and the number of micronucleated erythocytes was counted in a sample of 1000 erythrocytes. As there is not a normal distribution of micronucleus frequencies, median values and quartiles were calculated instead of mean values. The statistical method of Mac Gill et al. (1978), a quick and reliable test adapted to small sample sizes and values that do not have normal distribution, was used to analyse the results. A compound is considered as genotoxic: (1) if it induces a statstically significant increase in the number of micronucleated erythrocytes compared to the control (p < 0.05) and (2) if the median for the treated group is at least twice as high as the control median. In case the second condition is not fulfilled, the chemical is considered as weakly genotoxic.

Ames-fluctuation

B.N. Ames (University of California, Berkeley, USA). The genetic features of the strain were described by Maron and Ames (1983). We used strain TAlOO during all the experiment because it is considered as the most sensitive strain to water mutagens and particularly to chlorinated by-products (Loper, 1980; Forster et al., 1983; Harrington et al,, 1983; Vartianen and Liimatainen, 1986; Fielding and Horth, 1987; Meier, 1988). The Ames-fluctuation test was performed as described by Hubbard et al. (1984). This assay is a modification of the Ames test: briefly, the compound under study is exposed to bacteria in a liquid medium in many replicate cultures (96-well microplate) instead of the agar plate used in the Ames assay. After the 3-day incubation, bromothymol blue (600 pug/ml) was added. Positive wells (containing prototrophic mutants) turned yellow whereas negative wells remained green. For each experiment, spontaneous reversion in response to the solvent used (DMSO) was included. The positive response to a standard mutagen was also performed to ensure the validity of the test, i.e. sensitivity of the bacteria and of the metabolizing system when used. The positive control used was 1 ng/ml4-NQO without metabolic activation and 60 pg/ml CPA with metabolic activation. All the chloropropanones studied were tested with and without the metabolic system. Compounds were tested at least twice (two independent assays) using 3 experimental points for each dose. The statisical significance of the results was assessed with the x2 test: p < 0.05 for x2 > 3.84 and p < 0.01 for x2 > 6.63 (Green et al., 1976). The compounds were tested at least twice (two independent assays), using triplicate microplates for every concentration. A compound is considered as mutagenic: (1) if it induces a statistically

Newt micronucleus test. The test-organisms, Pleurodeles waltl larvae, were provided by the Centre

4

F. le Curieux et al. /Mutation

Metabolic system (SPmk). In the in vitro assays (SOS chromotest and fluctuation test), the genotoxic activity of promutagens was studied by adding a metabolic system (S9-mix) to the incubation medium. The S9-mix is prepared from S9, an Aroclor induced Sprague-Dawley rat liver extract. The S9-mix contains 10% of S9 (1 ml of S9-mix is 0.1 ml of S9 + 0.01 ml of MgCl, 0.4 M + 0.01 ml of KC1 1.65 M + 0.5 ml of phosphate buffer 0.2 M pH 7.4 + 0.04 ml of NADP 0.1 M + 0.005 ml of Glucose-6-phosphate 1 M + 0.335 ml of H,O). The same batch of S9 was used during the whole of this experimentation.

Research 341 (1994) l-15

a

l2

I

3. Results The results of the three genotoxicity tests are summarized in Tables 2 to 4 and results of chemicals inducing significant genotoxic activity are shown in Figs. 1 to 3. The detailed results for every test are reported on Tables 6 to 8. In the SOS chromotest, all the chlorinated propanones studied, except monochloropropanone, show a genotoxic effect (Table 2). l,l-dichloropropanone (l,l-DCP) and l,l,l-trichloropropanone (l,l,l-TCP) are genotoxic on E. coli, only in the absence of SPmix. 1,3-dichloropropanone (1,3-DCP) and 1,1,3-trichloropropanone (1,1,3-TCP) induce a genotoxic effect both with and without S9-mix. The results indicate that the metabolic system increases the genotoxicity of 1,3-DCP, decreases the activity of 1,1,3-TCP and cancels the action of l,l-DCP and l,l,l-TCP. If we consider the maximum induction factor obtained with or without metabolic activation, the following genotoxic order can be established: 1,1,3-TCP > 1,3-DCP > l,l,l-TCP > l,l-DCP. Among the substances studied, 1,1,3-trichloropropanone appears to be the strongest SOS repair system inducer with a maximum induction factor of 10.52 in the absence of SPmix. For l,l-dichloropropanone and l,l,l-trichloropropanone, the induction factor, although significant, never exceeds 2.25. We can note that the toxic effect of every chloropropanone studied on E. coli (i.e. a decrease in phosphatase alkaline activity) is reduced in the presence of the metabolic system.

0.1

0.01

10

1

CONCENTRATIONS

100

1000

10000

(&ml)

6- b 5.5 .

5.

4.5 .

4.

0

20

‘lo

NJ

80

100

CONCENTRATIONS

120

140

160

180

200

@g/ml)

Fig. 1. Results for the chemicals inducing a significant genotoxic activity in the SOS chromotest (a) without SPmix; (b) with S9-mix).

F. le Curieux et al. /Mutation Research 341 (1994) 1-15

0.1

0.01

1

CONCENTRATIONS

30

10

100

1000

(&ml)

b

20 26 24

5

In the fluctuation test, all the five chloropropanones studied show a mutagenic activity in this test with or without metabolic activation (Table 3). All the compounds except monochloropropanone induced mutagenic activity in the absence of S9-mix. On the contrary, the mutagenic properties of monochloropropanone only appear in the presence of the metabolic system, but metabolic activation decreased bacterial toxicity in all the compounds. The results also demonstrate that the toxicity of chlorinated propanones decreases when the number of chlorine substituents increases. The newt micronucleus test is only positive for 1,3-DCP and 1,1,3-TCP (Table 4). Monochloropropanone, l,l-dichloropropanone and l,l,l-trichloropropanone, tested up to sublethal concentrations, demonstrate no clastogenic activity on newt peripheral blood erythrocytes. Table 5 indicates, for each compound and each test, the lowest concentration inducing a significant genotoxic effet. As already described in a previous study (Le Curieux et al., 1993), the sensitivity of a test is defined as follows: for a given compound, test A is more sensitive than test B if the minimum concentration inducing a significant genotoxic effect in test A is lower than the minimum concentration producing a significant genotoxic effect in test B.

4. Discussion

6 4 2 0 0

5

10 CONCENTRATIONS

15

20

(Irglml)

Fig. 2. Results for the chemicals inducing a significant genotoxic activity in the Ames-fluctuation test (a) without S9-mix, (b) with S9-mix).

Monochloropropanone (MCP) was identified in drinking water samples (Meier et al., 1985). Our results do not demonstrate any genotoxic activity of this chemical on E. coli (SOS chromotest) or on newt peripheral blood erythrocytes (newt micronucleus test). On the contrary, the fluctuation test indicates that MCP is mutagenic, only with metabolic activation, on S. typhimurium TAlOO at the lowest genotoxic concentration of 6 pg/ml. Using the Ames test in solid medium (Ames Salmonella/microsome assay) without S9-mix, Cheh and Carlson (1981) showed the lack of mutagenicity of this compound on strain TAlOO. Our results show that the fluctuation test is the only test, among the three performed, able to

F. le Curieux et al. /Mutation Research 341 (1994) l-15

6

Table 2 Results of the SOS chromotest on the five choropropanones Chemicals

s9

Range of concentrations studied (pg/mI in the assay)

Monochloropropanone (MCP) l,l-Dichloropropanone (l,l-DCP) 1,3-Dichloropropanone (1,3-DCP) l,l,l-Trichloropropanone (l,l,l-TCP) 1,1,3-Trichloropropanone (1,1,3-TCP)

_

0.01-3 000 0.1-100 3-l 000 3-l 000 0.001-10 0.1-100 3-2000 3-3 000 0.03-3 000 0.1-300

+ _ + + + +

studied Threshold toxic concentrations &g/ml in the assay) 10 30 100

1000 1 100 1500 3000 3 100

Range of genotoxic concentrations (pg/ml in the assay) _a _=

30-80 _a 0.3-0.5 20-100 300-l 000 _= 0.3-10 20-150

Maximum Induction Factor 1.02 1.08 2.01 1.00 2.01 4.62 2.29 1.00 10.52 4.97

‘-: no genotoxic effect detected on Escherichia coli PQ37.

TAlOO using the Ames test in solid medium (i.e. agar) with and without S9-mix. The result obtained without metabolic activation corresponds to ours. On the contrary, when the assay is performed in the presence of SPmix, our sudy displays an absence of mutagenic activity while Meier et al. (1985) do not demonstrate any significant modification in genotoxicity compared to the test without S9-mix. This divergence can be explained by the relative inefficiency (detoxification) of the metabolic system used by Meier et al. (1985). In both studies, S9-mix was prepared from the same

demonstrate a genotoxic activity of MCP, but in the presence of metabolic activation only. 1,1-Dichloropropanone (l,l-DCP) was detected in drinking water (Meier et al., 1985) and in solutions of chlorinated humic substances (Kopfler et al., 1985; Meier et al., 1985). In the United States, this compound was found in drinking water at concentrations around the median values of 0.5 pg/l (Krasner et al., 1989) and 0.2 pug/l (Jacangelo et al., 1989). The study of Meier et al. (1985) showed the mutagenicity of l,l-DCP on S. typhimurium strain Table 3 Results of the Ames-fluctuation

test on the five choropropanones

studied

Chemicals

s9

Range of concentrations studied @g/ml in the assay)

Threshold toxic concentrations a &g/ml in the assay)

Range of mutagenic concentrations @g/ml in the assay)

Maximum X2 value ’

Monochloropropanone (MCP) l,l-Dichloropropanone (l,l-DCP) 1,3-Dichloropropanone (1,3-DCP) l,l,l-Trichloropropanone (l,l,l-TCP) 1,1,3-Trichloropropanone (1,1,3-TCP)

+ + + + +

0.03-10 l-30 0.03-300 l-l 000 0.001-l 0.1-30 3-10000 10-10000 0.01-30 l-300

10 30 100 1000 1 30 1000 10000 30 300

_b 6-10 10-100 _b 0.03-0.3 _b 100-300 _b 0.3-10 _b

0.03 6.91 ** 34.51 ** 0.60 83.84 ** 0.10 88.19 ** 1.32 138.38 ** 0.12

a Lowest concentration leading to a bacteriostatic effect b-: no mutagenic effect detected on Salmonella typhimurium strain TAlOO ‘* p < 0.05 (X2 > 3.841; ** p < 0.01 (X2 > 6.63).

F. le Curieux et al. /Mutation

Table 4 Results of the newt micronucleus test on the five chloropropanones Threshold toxic concentration @g/ml in the assay)

Chemicals

Research 341 (1994) 1-15

studied Concentrations studied &g/ml in the assay)

Monochloropropanone (MCP) 0.8 0.1-0.2-0.4 1,1-Dichloropropanone (l,l-DCP) 5 0.5-l-2 1,3-Dichloropropanone (1,3-DCP) 0.2 0.025-0.05-0.1 l,l,l-Trichlorpropanone (l,l,l-TCP) 20 2.5-5-10 1,1,3-Trichloropropanone (1,1,3-TCP) 0.5 0.5-l-2 a - = no genotoxic effect detected on newt Pkurodeles waltl. b C = (maximum frequency of polynucleated erythrocytes for the treated groups)/(frequency control).

type of liver extracts: S9 from Aroclor 1254 induced Sprague-Dawley rat livers. But, the ratio of S9 per ml of S9-mix is 50 pg/ml for Meier et al. (1985) and 100 pg/ml in our study. Several other parameters have their place in the explanation: compared to the liquid medium used in the fluctuation test (our study), the agar medium used in the Ames test (Meier et al., 1985) can induce a decrease in the efficiency of the metabolic system. Our results show that, for l,l-dichloropropanone, the fluctuation test is more sensitive than the SOS chromotest and that the newt micronucleus test is negative (Table 5). 1,3-Dichloropropanone (1,3-DCP) was identified in chlorinated solutions of humic substances

MCP

Sas9+

l,l-DCP

S, s9+

1,3-DCP

S, s9+

l,l,l-TCP

S, s9+

1,1,3-TCP S, s9

+

_ +30 + 0.3 +20 + 300 + 0.3 +20

_a _a 0.05-0.1 _a 2

1.16 1.38 2.46 1.15 2

of polynucleated erythrocytes for the

studied Conclusions b

Tests a SOS chromotest

C Factor b

(Kopfler et al., 1985; Meier et al., 1985) and in chlorinated wood pulp effluents (Rapson et al., 1985). This compound has not yet been detected in drinking water. The results available in other publications describe the genotoxicity of 1,3-DCP on E. coli and the mutagenic activity of this compound on S. typhimurium. Unlike the results in our study, Von Der Hude et al. (1988) did not detect the genotoxic activity of 1,3-DCP using the SOS chromotest (with or without S9-mix). At least two main differences can be noted between the protocole followed by Von der Hude et al. (1988) and our procedure: the final volume of the incubation medium and the procedure followed to determine enzymatic activities. Nevertheless, these dif-

Table 5 Summary of the results obtained with the three tests on the five chloropropanones Chemicals

Clastogenic concentrations &g/ml in the assay)

Fluctuation

Newt micronucleus

-

fluctu positive SOS and MN negative fluctu > SOS MN negative MN = fluctu > SOS

+6 +10 _ + 0.03 + 100 + 0.3 -

a + , Significant genotoxic activity; the lowest concentration -, No significant genotoxic activity. b MN, net micronucleus test.

+ 0.025

fluctu > SOS _

MN negative fluctu = SOS > MN

+1 inducing a genotoxic effect is reported @g/ml

in the assay).

8

F. le Curieux et al. /Mutation Research 341 (1994) l-15

ferences can not be considered responsible for the divergence in the results obtained in the two studies. Meier et al. (1985) showed a mutagenic activity of 1,3-dichloropropanone on strain TAlOO without S9-mix (the mutagenicity was not studied in

the presence of S9-mix). On the contrary, Majeska and Matheson (1983) concluded that this compound is mutagenic on TAlOO in the Ames test with S9-mix. These researchers used S9-mix prepared from Aroclor induced rat liver S9 (as in our study) as well as phenobarbital induced mouse

Table 6 Detailed results of the SOS chromotest on the five chloropropanones Chemicals

Concentrations (wz/mI)

Monochloropropanone (MCP)

Monochloropropanone (MCP)

1,1-Dichloropropanone (ll-DCP)

l,l-Dichloropropanone (ll-DCP)

1,3-Dichloropropanone (13-DCP)

studied

Pal b

S9 P-Gal a

IF ’

Result d

Assay 1 * Assay 2 * Mean Assay 1 * Assay 2 * Mean Assay 1 * Assay 2 * Mean

0 0.1 0.3 1 3 6 8 10

-

3.2 3.2 3.3 3.3 3.2 1.6 1.1 0.8

3.5 3.5 3.4 3.4 3.2 1.4 1.1 0.8

3.4 3.4 3.4 3.4 3.2 1.5 1.1 0.8

58.0 56.3 60.3 59.0 57.8 27.1 23.5 18.8

53.4 52.3 54.6 57.2 56.2 26.5 23.5 21.7

55.7 54.3 57.5 58.1 57.0 26.8 23.5 20.3

1.00 1.02 1.00 1.02 0.99 0.88 0.73 0.74

1.00 1.02 0.95 0.92 0.89 0.80 0.67 0.59

1.00 1.02 0.98 0.97 0.94 0.84 0.70 0.67

T T T

0 0.1 0.3 1 3 10 30 100

+ + + + + + + +

3.4 2.8 2.6 3.2 3.6 3.4 0.4 0.4

3.1 2.9 3.1 2.7 3.3 3.8 0.4 0.3

3.2 2.8 2.9 2.9 3.5 3.6 0.4 0.4

25.6 20.8 19.6 23.6 24.3 24.1 8.1 6.7

25.2 24.5 26.4 20.9 26.3 31.0 9.0 6.4

25.4 22.7 23.0 22.3 25.3 27.5 8.6 6.5

1.00 1.00 1.02 1.02 1.13 1.08 0.41 0.50

1.00 0.96 0.96 1.05 1.03 0.98 0.40 0.41

1.00 0.98 0.99 1.04 1.08 1.03 0.41 0.45

T T

0 10 20 30 50 60 80 100

-

2.9 4.3 5.3 6.0 5.5 5.0 3.6 2.7

4.3 7.0 7.8 8.7 6.5 5.1 4.5 3.7

3.6 5.7 6.5 7.3 6.0 5.0 4.1 3.2

35.4 43.7 49.3 48.3 34.8 30.2 22.5 17.5

67.4 98.1 91.7 90.3 52.9 42.3 34.3 26.9

51.4 70.9 70.5 69.3 43.9 36.3 28.4 22.2

1.00 1.19 1.29 1.49 1.92 1.99 1.93 1.88

1.00 1.13 1.34 1.52 1.93 1.89 2.09 2.20

1.00 1.16 1.32 1.50 1.92 1.94 2.01 2.04

+ + + + T

0

+ + + + + + +

3.3 3.5 5.2 3.6 3.9 2.7 0.3

4.7 5.0 3.6 3.8 3.7 2.1 0.5

4.0 4.3 4.4 3.7 3.8 2.4 0.4

27.4 30.4 42.5 32.9 34.2 29.6 5.9

38.0 42.4 30.8 32.7 29.7 22.4 6.3

32.7 36.4 36.7 32.8 32.0 26.0 6.1

1.00 0.96 1.00 0.91 0.94 0.76 0.42

1.00 0.95 0.94 0.93 1.00 0.76 0.68

1.00 0.95 0.97 0.92 0.97 0.76 0.55

T

-

4.3 8.1 10.5 9.7 4.3 3.6 2.3 1.9 1.7

3.0 3.6 4.2 3.9 2.7 2.4 1.9 1.6 1.4

3.6 5.9 7.4 6.8 3.5 3.0 2.1 1.7 1.6

67.4 96.9 91.8 63.9 31.1 24.1 20.8 18.2 17.8

37.7 42.4 40.3 30.5 17.4 13.7 13.4 12.6 11.6

52.5 69.6 66.0 47.2 24.3 18.9 17.1 15.4 14.7

1.00 1.33 1.81 2.39 2.20 2.36 1.77 1.62 1.54

1.00 1.06 1.31 1.62 1.91 2.17 1.80 1.58 1.53

1.00 1.19 1.56 2.01 2.06 2.26 1.78 1.60 1.53

+ + T T T T T

1: 30 100 300 1000 0 0.1 0.3 0.5 0.75 1 1.5 2 2.5

9

F. k Curieux et al. /Mutation Research 341 (1994) 1-15 Table 6 (continued) Detailed results of the SOS chromotest on the five chloropropanones Chemicals

Concentrations

S9 P-Gal a

&g/ml) 1,3-Dichloropropanone (13-DCP)

l,l,l-Trichloropropanone (ill-TCP)

l,l,l-Trichloropropanone (ill-TCP)

1,1,3-Trichloropropanone (113-TCP)

1,1,3-Trichloropropanone (113-TCP)

0 10 20 30 40 50 60 80 100 0 100 300 600 800 1000 1500 2000 0 3 10 30 100 300 1000 3000 0 0.03 0.1 0.3 1 3 10 30 0 10 20 30 50 75 100 150 200 300

studied

Pal b

IF ’

Result d

Assay 1 * Assay 2 * Mean Assay 1* Assay 2 * Mean Assay 1* Assay 2 * Mean + + + + + + + + + + + + + + + + + + + + + + + + + + +

3.1 3.3 5.3 1.1 8.0 6.6 7.8 7.3 3.2 3.5 5.3 7.3 8.2 7.0 4.1 1.1 0.7 3.3 4.0 4.9 3.8 3.7 3.5 2.6 0.4 3.4 3.6 3.9 5.0 9.6 10.8 6.9 0.5 3.1 3.6 4.9 4.4 6.2 8.6 1.6 4.2 1.6 0.5

4.0 7.0 8.1 11.5 13.2 12.6 14.7 9.1 6.4 3.4 4.4 5.3 5.4 4.6 4.2 1.0 0.6 4.1 4.0 3.9 4.2 4.1 3.5 2.3 0.4 3.7 3.6 3.8 5.5 10.3 11.8 6.5 0.5 4.0 3.8 4.6 6.8 12.0 12.5 9.9 7.1 2.6 0.5

3.6 5.2 6.7 9.3 10.6 9.6 11.3 8.2 4.8 3.4 4.8 6.3 6.8 5.8 4.1 1.1 0.7 4.0 4.0 4.4 4.0 3.9 3.5 2.5 0.4 3.6 3.6 3.9 5.3 10.0 11.3 6.7 0.5 3.6 3.7 4.8 5.6 9.1 10.6 8.8 5.7 2.1 0.5

25.2 24.4 25.1 23.2 20.0 16.5 15.7 13.6 8.4 57.8 65.0 61.8 51.5 44.2 32.2 17.9 14.4 27.4 34.4 41.8 33.6 33.2 35.4 22.9 4.1 57.1 56.1 54.2 55.6 52.4 17.3 9.9 8.7 25.3 23.8 19.7 20.0 19.7 17.6 12.2 8.5 6.4 4.1

31.3 36.0 32.1 31.2 28.2 23.4 24.0 14.5 11.5 48.1 52.1 49.2 39.9 33.5 33.3 17.7 11.8 38.0 33.5 34.2 35.1 35.8 33.5 22.0 3.9 55.2 52.1 49.2 52.6 46.7 17.6 10.4 9.5 31.3 27.1 25.9 21.9 24.6 20.2 15.8 11.5 6.4 3.6

28.3 30.2 28.6 27.2 24.1 20.0 19.8 14.1 10.0 52.9 58.5 55.5 45.7 38.9 32.8 17.8 13.1 32.1 33.9 38.0 34.3 34.5 34.5 22.4 4.0 56.2 54.1 51.7 54.1 49.6 17.5 10.2 9.1 28.3 25.5 22.8 23.9 22.1 18.9 14.0 10.0 6.4 4.1

1.00 1.09 1.73 2.48 3.25 3.25 4.06 4.36 3.10 1.00 1.35 1.97 2.64 2.63 2.11 0.88 0.77 1.00 0.95 0.96 0.92 0.91 0.82 0.92 0.92 1.00 1.08 1.22 1.52 3.06 10.53 11.66 1.03 1 .oo 1.22 2.01 1.79 2.53 3.94 5.04 4.00 2.02 0.82

1.00 1.53 1.97 2.87 3.66 4.19 4.79 4.88 4.30 1.00 1.19 1.53 1.90 1.95 1.77 0.83 0.78 1 .oo 0.95 0.92 0.95 0.93 0.84 0.85 0.80 1.00 1.04 1.16 1.55 3.31 9.96 9.38 0.79 1.00 1.08 1.40 1.90 3.80 4.84 4.90 4.83 3.13 1.10

1.00 1.31 1.85 2.67 3.45 3.72 4.42 4.62 3.70 1.00 1.27 1.75 2.27 2.29 1.94 0.85 0.77 1.00 0.95 0.94 0.94 0.92 0.83 0.89 0.86 1.00 1.06 1.19 1.54 3.18 10.25 10.52 0.91 1.00 1.15 1.70 1.84 3.17 4.39 4.97 4.41 2.57 0.96

+ + + + + +, T +,T + + + + T T T + + +, T +, T T + + + + +, T +, T T T

a P-galactosidase activity; b Alkaline phosphatase activity; ’ Induction factor; * Every value is the mean of 6 individual values. d + , significant genotoxic activity; -, no genotoxic activity detected; T, toxicity.

liver S9. As the protocole followed by Majeska and Matheson (1983) is not very precise, we cannot give any explanation for the divergence between these results and ours. The results obtained in our study show that for

1,3-dichloropropanone, the fluctuation test and the newt micronucleus test are of comparable sensitivity and more sensitive than the SOS chromotest (Table 5). I,I,l-Trichloropropanone (l,l,l-TCP) was de-

10

F. le Curieux et al. /Mutation

Table 7 Detailed results of the Ames-fluctuation Chemicals

Research 341 (1994) l-15

test on the five chloropropanones

Concentrations kVm0

S9

Number of positive wells (out of 96) Assay I*

Monochloropropanone (MCP)

Monochloropropanone (MCP)

l,l-Dichloropropanone (ll-DCP)

1,3-Dichloropropanone (13-DCP)

1,3-Dichloropropanone (13-DCP)

l,l,l-Trichloropropanone (ill-TCP)

Assay 2 *

X2

Result a

Mean

_ _ -

_ -

7 8 8 8 5 6 0

9 4 6 7 10 6 0

8 6 7 8 7 6 0

0.00

+ + + + + + +

9 6 8 24 20 14 0

11 10 13 25 23 17 0

10 8 10 24 21 15 0

0.00 0.28 0.00 6.91 4.58 1.13 10.91

3 10 30 100 300

_ _ _

9 9 14 20 47 28 0

10 10 12 24 46 25 0

10 10 13 22 47 27 0

0.00 0.00 0.62 5.93 34.51 9.88 9.99

0 3 10 30 100 300 1000

+ + + + + + +

13 21 16 17 16 10 0

11 10 12 12 16 4 0

12 16 14 15 16 7 0

0.00 0.46 0.14 0.23 0.60 1.45 13.03

0 0.001 0.003 0.01 0.03 0.1 0.3

_ -

8 9 9 12 18 48 67 0

9 11 9 12 19 38 75 8

8 10 9 12 19 43 71 4

0.00 0.13 0.02 0.60 4.68 31.86 83.84 1.80

0

3 10 30

+ + + + + + +

9 12 9 11 10 4 1

9 7 8 10 7 4 0

9 10 9 10 9 4 0

0.00 0.02 0.01 0.10 0.01 1.98 8.32

_ T

0 3 10 30 100 300 1000

_ _ _ _

8 11 11 17 30 69 1

10 11 11 20 41 77 1

9 11 11 19 35 73 1

0.00 0.31 0.31 3.79 20.97 88.19 5.67

_

0

0.03 0.1 0.3 1 3 10 0 3 6 10 20 30

l,l-Dichloropropanone (ll-DCP)

studied

0

0.1 0.3

0.35 0.09 0.03 0.04 0.47 8.48

T _ ++ + T T _ + ++ ++,T T _ _ T T _ _ _ + ++ ++ T

(+) ++ ++ T

F. le Curieux et al. /Mutation Research 341 (1994) l-15

Table 7 Detailed results of the Ames-fluctuation

test on the five chloropropanones

11

studied

Number of positive wells (out of 96)

Chemicals

Concentrations (pg/ml)

S9

Assay 1 *

Assay 2 *

Mean

l,l,l-Trichloropropanone (ill-TCP)

0 10 30 100 300 1000 3000 10000

+ + + + + + + +

13 11 20 14 13 17 5 0

11 15 15 11 10 19 2 0

12 13 18 13 12 18 4 0

0.02 1.12 0.00 0.02 1.32 5.00 13.03

_ _ -

5 6 7 15 25 67 88 85 0

9 6 7 10 29 58 89 88 0

6 7 13 27 63 89 87 0

0.00 0.08 0.00 1.73 14.73 69.47 138.38 131.76 7.26

0

11 12 11 13 15 4 0

10 10 9 11 11 2 0

0.00 0.02 0.00 0.07 0.12 6.05 10.23

1,1,3-Trichloropropanone (113-TCP)

1,1,3-Trichloropropanone (113-TCP)

0 0.01 0.03 0.1 0.3 1 3 10 30 0 1 3 10 30 100 300

+ + + + + + +

X2

0.00

Result a

_ _ _ T _ _ _ ++ ++ ++ ++ T _ T T

* Every value is the mean of 3 individal values. a Not mutagenicon strain TAlOO; + , mutagenic (p < 0.05); + + , mutagenic (p < 0.01); T, toxic or bacteriostatic effect. -7

tected in drinking water samples (Meier et al., 1985) and in solutions of chlorinated humic substances (Kopfler et al., 1985; Meier et al., 1985). This chemical, is, after chloroform, dichloro- and trichloroacetic acids, the fourth most abundant substance identified in solutions of chlorinated humic acids (Meier et al., 1985). In the USA, l,l,l-TCP was found in drinking water samples at concentrations around the median values of 0.40.8 pg/l (Krasner et al., 1989) and 1.1-1.8 pg/l (Jacangelo et al., 1989). It must be noted that Nicholson et al. (1984) detected a concentration of 20 pg of l,l,l-TCP per liter in some Australian drinking water samples. The mutagenic activity of this compound was previously studied by Meier et al. (1985) who demonstrated the mutagenicity of l,l,l-trichloropropanone on TAlOO with and without S9-mix.

As for l,l-dichloropropanone, Meier et al. (1985) showed the persistance of the mutagenic activity of l,l,l-TCP with S9-mix, whereas our results indicte that the addition of S9-mix induces the disappearance of its mutagenic effect on strain TAlOO. The comments concerning the efficiency of the metabolic system used by Meier et al. (1985), exposed in the paragraph related to l,lDCP, are also applicable here. The results obtained point out that for l,l,ltrichloropropanone, the fluctuation test is more sensitive than the SOS chromotest while the newt micronucleus test is negative (Table 5). 1,1,3-Trichloropropanone (1,1,3-TCP) was identified in solutions of chlorinated humic substances (Kopfler et al., 1985; Meier et al., 1985). This compound has not been detected in drinking water yet.

12

F. le Curieux et al. /Mutation

Research 341 (1994) 1-15

Articles available regarding the genotoxic effects of 1,1,3-TCP are limited to two, dealing with its mutagenicity: using the Ames test, Douglas et al. (1983) and Nestmann et al. (1985) showed a strong mutagenic activity of this compound on S. typhimurium strain TAlOO. These results concord with ours. The results obtained with the three tests implemented indicate that the fluctuation test and SOS chromotest are of comparable sensitivity and slightly more sensitive than the newt micronucleus test for the detection of 1,1,3-TCP (Table 5). Considering the number of compounds detected, the fluctuation test is the most interesting, as it is able to detect the genotoxic activity of all the five chlorinated propanones studied. The SOS chromotest does not detect the genotoxic effect of monochloropropanone. The newt micronucleus test does not demonstrate the clastogenic activity of monochloropropanone, l,l-dichloropropanone and l,l,l-trichloropropanone. The fact

that the corresponding isomers (1,3-dichloropropanone and 1,1,3-trichloropropanone) induce clear genotoxic effects in the newt micronucleus test must be underlined. It should also be noted that the fluctuation test is the most sensitive assay for every chloropropanone tested. Moreover, an interesting structure-activity relationship is apparent concerning the genotoxic effect of the chloropropanones studied. The position of the chlorine substituents seems to be determining: in the SOS chromotest, 1,3-DCP is 100-fold more genotoxic than l,l-DCP and 1,1,3TCP is lOOO-fold more active than l,l,l-TCP. The same pattern is observed for the fluctuation test: in this point mutation assay, 1,3-DCP is about 300 times more mutagenic than l,l-DCP and 1,1,3-TCP is about 300 times more genotoxic than l,l,l-CP. The most striking result is obtained with the newt micronucleus test: 1,3-DCP and 1,1,3-TCP induce the formation of micronuclei in peripheral blood erythrocytes while this is not the case for l,l-DCP and l,l,l-TCP. As de-

Table 8 Detailed results of the newt micronucleus test on the five chloropropanones Chemicals

Monochloropropanone (MCP)

Toxicity @g/ml) 0.8

l,l-Dichloropropanone (l,l-DCPI

5

1,3-Dichloropropanone (1,3-DCP)

0.2

l,l,l-Trichloropropanone (l,l,l-DCP)

1,1,3-Trichloropropanone (1,1,3-DCPI

20

4

Concentrations studied (pg/mlI 0 0.1 0.2 0.4 0 0.5 1 2* 0 0.025 0.05 0.1 0 2.5 5 10 0 0.5 1 2*

studied

Micronuclei frequency (%) Median

Result a

Observations

+ Icf

6 7

k1.05 + 1.82

_

7 7 6.5 8 9

k1.62 + 2.43 *0.70 +2.03 f 2.23

_ _ m

6.5 12 17 16 6.5 7 9 7.5

+ 0.70 f 2.03 f 3.45 + 1.42 kO.70 f 1.62 k1.82 f 1.47

6.5 8 13

kO.70 f 1.01 + 2.43

All the larvae died after a 8-day exposure at 2 pg/ml

(+I + + _ _ _ + m

a + , genotoxic; ( + ), weakly genotoxic; -, no genotoxic activity detected; m, mortality of the larvae. * Refer to the corresponding observations.

8 of the 15 larvae died after the 12-day exposure at 2 &g/ml

F. le Curieux et al. /Mutation Research 341 (1994) 1-15

20 . 8 0 B

18.

4 0 m ‘6.

) 1,3-DCP

F 1,1,3-TCP

65 4

B 1

2

1

OI 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

CONCENTRATIONS (whl)

Fig. 3. Results for the chemicals inducing a significant genotoxic activity in the newt micronucleus test.

scribed by Meier et al. (1985), this structure-activity relationship can be explained by the fact that chloropropanones with chlorine substituents at both carbon positions (Id-DCP and 1,1,3-TCP) behave like poly- or bifunctional genotoxic agents (Leonard, 1990): these compunds can produce, via substitution reactions, the alkylation of DNA in two nucleophilic sites. Thus, the action of 1,3-DCP and 1,1,3-TCP would lead to the formation of intra- and interstrand DNA cross-links which cause more serious damage than one-site alkylations induced by l,l-DCP and l,l,l-TCP. This genotoxicity mechanism is particularly foreseeable in the case of clastogenic activity as demonstrated in the newt micronucleus test. The fact that monochloropropanone (a monofunctional chlorinate propanone) is negative in the newt micronucleus test is a new argument in favour of this theory. Besides, a correlation is noticeable between the number of chlorine substituents and genotoxic activity. As far as bifunctional chloro-

13

propanones (1,3-dichloropropanone and 1,1,3-trichloropropanone) are concerned, the results obtained with the fluctuation test and the newt micronucleus test indicate that genotoxicity decreases as the number of chlorine substituents increases (1,3-DCP > 1,1,3-TCP). The SOS chromotest does not give any useful information on that particular point. Similarly, for monofunctional chloropropanones (monochloropropanone, l,l-dichloropropanone and l,l,l-trichloropropanone), the SOS chromotest and the fluctuation test demonstrate that the increase in the number of chlorine substituents decreases the genotoxic activity observed (except that MCP is negative in the SOS chromotest). As MCP, l,l-DCP and l,l,l-TCP are not clastogenic on the peripheral blood erythroctes of newt larvae, the results of the newt micronucleus test do not offer any further information. It is interesting to comment on the effect of the metabolic system on the genotoxic activity of the compounds tested. In the fluctuation test, it appears that the addition of S9-mix leads to the disappearance of mutagenicity of chemicals containing two or three chlorine substitutions (l,lDCP, 1,3-DCP, l,l,l-TCP and 1,1,3-TCP). In the SOS chromotest, the addition of S9-mix also induces the disappearance of genotoxicity for monofunctional chloropropanones (l,l-DCP and l,l,l-TCP) but only leads to a decrease in genotoxic effect for bifunctional chloropropanones (1,3-DCP and 1,1,3-TCP). Thus, except for MCP, the S9-mix appears to have efficient detoxification properties on the four other chlorinated propanones. This point is rather comforting, as far as potential human hazard is concerned: indeed, these chemicals are likely to be metabolised and undergo detoxification that will lessen or cancel their genotoxic activity. The chloropropanone group is cited by the World Health Organisation (WHO) as being among the chemicals found in drinking water that could represent some hazard for public health (WHO, 1994). But, as the data concerning health hazards associated with this group of chemicals is inadequate, WHO has not yet given any guideline values as to the occurence of chloropropanones in drinking water. None of the five chlorinated

14

F. le Curieux et al. /Mutation Research 341 (1994) 1-15

propannes tested has been studied by the International Agency for Research on Cancer (IARC) with regard to its carcinogenic risks for humans. In order to corroborate the results of our study and to investigate more thoroughly the genotoxic activity of these substances, other in vitro and in vivo short-term tests should probably be implemented. In case such experimentations would confirm the genotoxic potencies of chloropropanones, the study of the carcinogenic activities of these chemicals should eventually be considered. Indeed, animal carcinogenicity assays would help to assess the health risks associated with the presence of chloropropanones in chlorinated waters.

Acknowledgements This research was carried out in the programme of the Groupe d’Etude Scientifique sur le Test Micronoyau-Triton and was partially supported by grants from the Compagnie GCnCrale des Eaux, the Lyonnaise des Eaux and the S.A.U.R. (France). The authors wish to thank the personnel of the Laboratoire de Toxicologe GCnCtique (Institut Pasteur de Lille, France) and of the Departement Toxicologie-Hydrologie-Hygitne (Fact&C de Pharmacie de Lille, France) for their skilful assistance.

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