Mutation Research , 245 (1990) 145-150
145
Elsevier MUTLET 0410
Induction of DNA-repair synthesis in primary rat hepatocytes by epoxides Wilhelm vonder
Hude, Ralf Mateblowski
and Armin Basler 1
lhstitut fiir Allgemeine Genetik, Freie Universitiit Berlin and ~Max yon Pettenkofer-lnstitut des Bundesgesundheitsamtes, D-IO00 Berlin 33 (Germany)
(Received 28 March 1990) (Accepted 11 June 1990)
Keyword:~: Unscheduled DNA synthesis; Epoxides; Primary rat hepatocytes
Summary The genotoxicity of 10 epoxides was investigated in the UDS test with primary rat hepatocytes. The sensitivity of the assay was demonstrated using 2-acetylaminofluorene. The epoxides 1,2-epoxyoctane, 1,2-epoxydecane, epoxycyclooctane, epoxycyclododecane, (+)-limoneoxide, a-pinaneoxide, t r a n s stilbeneoxide, and c i s - 2 , 3 - e p o x y s u c c i n i c acid, which are known to be non-mutagenic in the Ames test, as well as the bacterial mutagen, 1,2-epoxyphenoxypropane did not induce UDS in primary hepatocytes of the rat. However, a positive UDS response obtained with glycidyltrimethylammonium chloride showed that metabolic inactivation of the oxirane ring in hepatocytes is influenced by further structural substituents.
Several epoxides are known to induce mutations in bacterial test systems without metabolic activation. In most cases, their mutagenic activity is reduced by $9 mix (Canter et al., 1986; E1-Tantawy and H a m m o c k , 1980) due to enzymes in the liver homogenate which are known to inactivate the electrophilic oxirane ring (Oesch, 1988; Meijer and Depierre, 1988). Because these biotransformation capabilities are also present in intact liver ceils the mutagenic epoxides 1,2:3,4-diepoxybutane, 1,2epoxybutane and epichlorohydrin gave negative results in the UDS test with primary rat hepatocytes (Williams et al., 1982; Probst et al., 1981). Some epoxides, however, developed mutagenic
activity in the Ames test only in the presence of $9 mix ( v o n d e r H u d e et al., 1985; Canter et al., 1987), or mutagenic activity was increased in the presence o f $9 mix (Thompson et al., 1981). Recently Williams et al. (1989) reported a positive result in the P R H / U D S test with the direct alkylating butadiene monoepoxide which obviously is not inactivated in the liver cells. Because so far only 4 direct alkylating epoxides have been tested in the P R H / U D S test, 10 epoxides, some mutagenic and some non-mutagenic in the Ames test, were tested in this study with the P R H / U D S assay in order to obtain further information on the response of the P R H / U D S test to chemicals of this class.
Correspondence: Dr. W. v. d. Hude, Max von Pettenkoferlnstitut des Bundesgesundheitsamtes, Postfach 33 00 13, D-1000 Berlin 33 (Germany). 0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
146 TABLE I RESULTS OF THE PRH/UDS Substance
TEST PERFORMED
WITH EPOXIDES
Dose
Experiment I
E x p e r i m e n t II
(mole/l)
NG a
pG b
Net g r a i n s
% pos.
NG a
PG b
Net g r a i n s
% pos.
DMSO
1070
18.1 ± 4.5
26.7 + 7.1
-
8.6 ± 4.5
0
7.7 ± 2.3
8.1 + _ 2 . 2
-0.4
1,2-Epoxy-
3 x 1 0 -4
37.1 ± 6.4
38.6 ± 8.1
-
1.5 ± 5.7
0
5.6 ± 1.4
6.8 +_ 1.2
- 1.2 +
1.5
2
octane
3 × 1 0 -3
32.1 _+ 4.7
32.7 ± 3.8
-
0.6 ± 3.7
42
5.8 ± 1.9
4.0 ± 0.8
+ 1.8 ± 2.1
10
3 x 10-2
toxic
± 2.6
8
toxic
DMSO
1070
18.1 + 4.5
26.7 + 7.1
-
8.6 + 4.5
0
6.2 ± 2.1
10.7 ± 1.9
-4.5
± 2.0
2
1,2-Epoxy-
3×10 -6
33.0 + 6.3
31.1 ± 5.9
+
1.9 +_ 5.1
5
5.4 + 1.3
8.3 + 1.4
-2.9
___ 1.3
0
decane
3 x 1 0 -5
33.8 ± 4.7
34.4 ± 4.9
-
0.6 ___ 4.6
8
4.1 ± 1.4
7.5 _+ 1.2
- 3.4
+
1.4
0
3 × 10- 4
toxic
toxic
H20
1°70
35.5 + 6.3
46.4 + 6.1
-10.9
cis-2,3-Epoxy-
10 -3
36.9 + 5.4
44.0 + 5.5
-
± 5.8
0
5.7 + 1.2
8.1 _+ 1.5
- 2.4
±
1.5
3
7.1 + 3.6
0
4.2 + 1.1
7.3 ± 1.1
-3.1
± 1.1
0
succinic
10 - 2
35.3 ± 5.0
46.8 + 5.0
-11.5
acid
10- ~
toxic
3.6 +_ 1.1
6.5 ± 1.3
- 2.9
±
1.2
0
0
_+ 4.5
0
toxic
DMSO
1070
37.5 + 6.2
49.4 ± 5.9
-11.9
Epoxycyclo-
3 x 1 0 -5
40.8 _+ 7.0
49.9 + 7.5
-
± 5.7
0
6.2 ± 2.1
10.7 _+ 1.9
-4.5
± 2.0
9.1 + 3.8
0
4.4 _+ 1.3
6.6 ± 1.2
-2.2
+
1.2
octane
3x10 -4
37.7 ± 6.4
48.9 ± 7.8
-11.2
2
0
5.4 ± 1.3
8.6 ± 1.4
- 3.2
_+ 1.3
0
3 × 10- 3
toxic 18.1 ± 4.5
± 4.1
toxic
DMSO
1070
Epoxycyclo-
3 × 10- 7
26.7 ± 7.1
-
8.6 ± 4.5
0
dodecane
3x10 -6
29.3 +_ 4.1
36.8 ± 4.5
-
7.5 ± 3.7
2
3 x 1 0 -5
38.4 ± 4.7
38.1 ± 5.2
-
0.3 ± 4.2
8
3x10-4
toxic
6.2 ± 2.1
10.7 ± 1.9
-4.5
_+ 2.0
0
3.2 + 0.9
5.2 ± 0.9
-2.0
± 0.9
2
4.4 + 2.6
9.9 ± 2.2
-5.5
± 2.2
0
toxic
DMSO
1°70
18.1 ± 4.5
26.7 +_ 7.1
-
8.6 + 4.5
0
7.7 + 2.3
8.1 + 2.2
-0.4
_+ 2.6
8
c~-Pinane-
6 x 1 0 -5
28.0 ___ 4.8
33.5 +_ 5.3
-
5.5 ± 4.2
0
3.7 ± 1.3
6.6 ___ 1.3
-2.9
± 1.5
2
oxide
6 × 10 - 4
38.2 ± 7.1
30.9 +_ 6.7
+
8.7 ± 7.2
42
9.6 +_ 1.9
8.8 ± 1.6
+ 0 . 8 ± 1.9
5
6 × 10- 3
toxic
DMSO
1070
37.5 ± 6.2
49.4 _+ 5.9
-11.9
+ 5.7
0
6.2 ± 2.1
10.7 + 1.9
-4.5
± 2.0
0
(+)-Limone-
10 -5
37.6 ± 6.1
40.6 ± 5.5
-
3.0 ± 4.6
5
5.8 ± 1.6
9.2 _+ 1.1
- 3.4
±
1.4
0
oxide
10 - 4
36.1 ± 6.9
43.8 ___ 8.1
-
7.7 +_ 5.2
0
4.8 ± 1.1
7.5 ± 1.1
-2.7
± 1.1
2
10 - 3
toxic 35.5 + 6.3
46.4 + 6.1
-2.4
Glycidyltri-
2x10 -6
47.7 ± 5.5
44.3 ± 4.3
methylammo-
2 x 1 0 -5
58.8 ± 6.7
45.8 + 6.1
13.0 ± 6.4
niumchloride
2 × 1 0 -4
77.3 ± 9.1
33.4 ± 4.9
43.9 ± 5.3
DMSO 1,2-Epoxy-
107o 3x10 -6
phenoxy-
3x10-5
propane
3 × 10- 4
H20
toxic
toxic -10.9
+ 5.8
0
5.7 ± 1.2
8.1 ± 1.5
3.4 ± 4.6
19
9.5 ± 2.4
8.4 _+ 2.2
65
15.5 ± 3.1
7.7 ± 7.8
100
43.1 ± 6.6
4.0 ± 1.2
± 1.5
3
1.1 ± 2.5
15
7.8 _+ 2.8
70
39.1 +_ 6.5 100
7.7 ± 2.3 5.4 ± 1.5
8.1 + 2 . 2
-0.4
± 2.6
8
7.0±
- 1 . 6 --. 1.5
0
5.5 ± 1.4
8.5 ± 1.5
- 3.0
±
1.6
0
1.1
toxic
DMSO
1 070
6.2 + 2.1
10.7 ± 1.9
-4.5
___ 2.0
2
1,2-Epoxy-
3×10 -6
6.4 ± 1.3
8.6 _+ 1.3
-4.2
±
1.7
0
phenoxy-
3 × 10- 5
6.2 ± 1.9
10.4 ± 1.5
-4.2
±
1.7
2
propane
3 x 10 - 4
toxic
147 TABLE I (continued) Substance
Acetone
Dose
Experiment I
(mole/l)
NG a
trans-
1°70 2 × 10 - 7
Stilbeneoxide
2 × 10 - 6 2 × 10- 5
E x p e r i m e n t II pG b
Net grains
°70 p o s .
NG a 6 . 0 ± 1.7 6 . 0 ± 1.5 6.1 ± 1.6 toxic
pG b
Net grains
% pos.
7.7 ± 1.5
-
1.7
±
1.5
5
9.3 + 1.3
- 3.3
+
1.7
2
7.7 _+ 1.6
-
+
1.5
3
+ 1.1
2
1.6
Acetone
lO70
3.6 + 1.1
7.0+
trans-
2 × 10 - 7
5 . 0 _+ 1.3
8.6 + 1.4
Stilbene-
2 × 10 - 6
7.4 + 1.8
10.6 + 2.8
oxide
2 x 10- 5
DMSO AAF
1°70 10 - 9
18.1 _+ 4.5 nd
2 6 . 7 _+ 7.1
7.7 + 2.2 9 . 0 _+ 2.3
8.1 ± 2.2 7.5 + 1.5
1.5 ± 2 . 2
3
AAF
10 - 8
64.4 + 7.4
4 5 . 8 + 7.3
18.6 ± 7.9
95
2 1 . 6 _+ 4 . 0
5.8 _+ 1.7
15.8 _+ 3.9
73
AAF
10 - 7
100#
55.3 ± 9.8
nc
100
56.8 + 6 . 6
3.9 _+ 1.0
5 2 . 9 + 6.7 100
AAF
10 - 6
150#
4 5 . 7 _+ 7.7
nc
100
7 8 . 4 ± 13.2
6.0 + 1.6
7 2 . 4 _+ 1 2 . 8 1 0 0
DMSO AAF
lo70 10 - 9
37.5 + 6 . 2 nd
49.3 + 5.9
-11.8
6 . 2 + 2.1 10.8 + 3.0
10.7 + 1.9
AAF
10 - 8
5 0 . 0 _+ 6.7
4 5 . 8 ± 5.9
4 . 2 + 5.9
33
10.7 + 2 . 9
8.3 + 1.6
AAF
10 - 7
100#
39.7 _ 5.5
nc
100
4 3 . 7 + 6.2
7.3 + 1.5
3 6 . 4 _+ 5.8 100
AAF
10 - 6
150#
4 1 . 4 _ 5.0
nc
100
110.0 + 14.3
6 . 0 + 1.4
104.0 ___ 1 4 . 3 1 0 0
1.1
-3.4 -
3.6 +_
1.5
3
- 3.2
_+ 1 . 6
2
-0.4
_+ 2 . 6
2
toxic 8.6 + 7.1
0
± 5.7 2
10.5 + 2 . 0
-4.5 + 2.0 0.3 +_ 2.8
2 25
2 . 4 + 2.5
37
T h e e x p e r i m e n t s o f series I w e r e p e r f o r m e d w i t h 5 / z C i 3 H - d T / m l a n d t h o s e o f series II w i t h 1 /~Ci 3 H - d T / m l . a N u c l e a r g r a i n s , m e a n o f 60 cells ± s t a n d a r d d e v i a t i o n . b p l a s m a t i c g r a i n s , m e a n o f 6 0 cells _ s t a n d a r d d e v i a t i o n . # U n d e r e s t i m a t e d g r a i n c o u n t s d u e to o v e r l a p p i n g g r a i n s . nc, not calculated, because nuclear grain counts were not exact. nd, not done.
Materials and methods
Chemicals The test substances glycidyltrimethylammonium chloride (CAS No. 3033-77-0, 97o70) and cis-2,3-epoxysuccinic acid (16533-72-5, 90o70) were obtained from Fluka (Switzerland), 1,2-epoxydecane (2404-44-6, 98%), epoxycyclooctane (286-62-4, 98%), epoxycyclododecane (286-99-7, 95%), (+)-limoneoxide (1195-92-2, 97%), transstilbeneoxide (1439-07-2, 99°70) and a-pinaneoxide (1686-14-2, 98o7o) from Aldrich (U.S.A.), and 1,2-epoxyoctane (2984-50-1, 98°70), 1,2-epoxyphenoxypropane (122-60-1, 98%) from Merck (F.R.G.). 2-acetylaminofluorene (AAF) served as the positive control substance.
Methods Primary rat hepatocytes (PRH) were prepared from male Wistar rats (10 weeks, 180-200 g) and cultured by the method of Williams et al. (1982) with slight modifications as described by Butterworth et al. (1987). PRH (5 × 105 viable cells, determined by trypan blue exclusion) were seeded onto coverslips (25 mm, Miles Scientific, U.S.A.). The unattached cells were removed by washing after 2 h. The attached PRH were incubated at 37°C in the presence of the test compounds and of [methyl)H]thymidine (3H-dT, 90 Ci/mmole, Amersham, U.K.) with 5 #Ci/ml or 1/~Ci/ml for 20 h. Thereafter the coverslips were processed by hypotonic treatment as described by Butterworth et al. (1987) and mounted with a 1:1 diluted Ilford K5
148
emulsion. The slides were developed after 2 days in Kodak D19 developer and the PRH were stained with hematoxylin/eosin (HE). The grains were counted with a Biotran II colony counter (New Brunswick, U.S.A.) interfaced with a Zeiss microscope (100× objective). 20 cells of normal morphology were counted per slide and 3 slides per experimental point. A cell was judged to be UDSpositive if the net grains were more than twice the standard deviation (SD) of the solvent control. A substance was judged as UDS-positive if there was a dose-response increase in net grains and more than 50% of the investigated cells were found to be UDS-positive. Results The results of the PRH/UDS test are shown in Table 1. The clear-cut positive results obtained with the known UDS inducer AAF demonstrate the sensitivity of the P R H / U D S test and the reliability of the Ilford K5 film emulsion. Although in the first experimental series with 5/~Ci/ml 3H-dT the nuclear grains obtained after AAF treatment increased dose-dependently, exact grain counts could not be established at high AAF concentrations because of overlapping grains. Accordingly, the grain numbers were underestimated by the counter. The values given in Table 1 without standard deviation (SD) represent the smallest counts within 60 cells. In further experimental series the concentration of ~H-dT was reduced to 1 #Ci/ml. Again, a clearly positive result was obtained with AAF and exact grain counting was possible at all concentrations. In the experimental series with 1 t~Ci/ml 3H-dT as well as with 5 /~Ci/ml, 1,2-epoxyoctane, 1,2epoxydecane, epoxycyclooctane, epoxycyclododecane, (+)-limoneoxide, a-pinaneoxide, and cis-2,3-epoxysuccinic acid did not produce increased net grain counts in the PRH/UDS. Glycidyltrimethylammonium chloride, on the other hand, yielded genotoxic responses in both experiments of the P R H / U D S test. Trans-stilbeneoxide and 1,2-epoxyphenoxypropane were tested twice in independent ex-
periments in the series with 1/zCi/ml 3H-dT. Both substances yielded reproducible negative results in the P R H / U D S test. Discussion Ten epoxide-bearing chemicals were tested for their genotoxic potential with the PRH/UDS test. Nine epoxides yielded negative results and 1 was positive in this test system. The aliphatic oxiranes epoxyoctane and epoxydecane, the cycloaliphatic oxiranes epoxycyclooctane and epoxycyclododecane, the disubstituted oxiranes cis-2,3-epoxysuccinic acid and transstilbeneoxide and the 1,1,2-trisubstituted oxiranes a-pinaneoxide and (+)-limoneoxide did not show mutagenic properties in the Ames test (Wade et al., 1978; Watabe et al., 1980; Frantz and Sinsheimer, 1981; Canter et al., 1986; v o n d e r Hude et al., 1990). They also did not induce UDS in the mammalian PRH/UDS test. In contrast to the above-mentioned epoxides epoxyphenoxypropane is a direct-acting bacterial mutagen. This compound has been shown to be detoxified by mammalian epoxide hydrolases and glutathione-S-transferases (Sinsheimer et al., 1987). This agrees with the negative results reported for the in vivo micronucleus test in mice (Seiler, 1984) and the negative results obtained in the present PRH/UDS test. These data, in addition to the negative results in the PRH/UDS test obtained with the directly acting mutagens 1,2:3,4-diepoxybutane, 1,2-epoxybutane and epichlorohydrin (Williams et al., 1982; Probst et al., 1981), indicate that hepatocytes are capable of inactivating mutagenic epoxides. Nevertheless, glycidyltrimethylammonium chloride, in contrast to those compounds, gave positive results in the PRH/UDS test. This agrees with the results obtained from other in vitro mutagenicity tests. Glycidyltrimethylammonium chloride has been shown to induce point mutations in Klebsiella pneumoniae, Salmonella typhimurium and Escherichia coli strains and chromosomal aberrations in mammalian cells without external metabolic activation. The mutagenic properties of
149
this epoxide were not affected by the addition of $9 mix (Voogd et al., 1981; Vleminckx et al., 1987; Dean et al., 1985). The results obtained with glycidyltrimethylammonium chloride are of special interest because it inserts a cationic group into the DNA and the influence of such compounds on DNA damage is not known. The presence of DNA-damaging properties of electrophilic organic cations may be assumed, but with this special compound steric hindrances preventing DNA attack would seem to be more probable. This hypothesis is supported by the negative results obtained in the Ames test without $9 mix testing the cationic phenyltrimethylammonium chloride (Heimann and Huber, 1989). On the other hand, because this compound is apparently not inactivated by liver homogenate and whole liver cells, it is concluded that charged compounds are not efficient substrates for epoxidedetoxifying enzymes. Furthermore, the positive results obtained with glycidyltrimethylammonium chloride in this study and with butadiene monoepoxide observed by Williams et al. (1989) in the P R H / U D S test demonstrate that enzymes present in freshly isolated liver cells do not necessarily inactivate chemicals carrying an oxirane ring. The inactivation seems to be affected by substituents adjacent to the epoxide group. Because glycidyltrimethylammonium chloride is a directly mutagenic epoxide which is not inactivated by mammalian detoxifying systems, $9 mix and whole liver cells, carcinogenic properties have to be assumed. Considering its widespread use in the textile, electronic and cosmetic industries (for an overview see Vleminckx et al., 1987), evaluation of the possible carinogenic potency of this compound is recommended.
Acknowledgement Part of this work was supported by the Umweltbundesamt (Federal Environmental Agency), Project Number 106 03 067.
References Butterworth, B.E., J. Ashby, E. Bermudez, D. Casciano, J. Mirsalis, G. Probst and G. Williams (1987) A protocol and guide for the in vitro hepatocyte DNA-repair assay, Mutation Res., 189, 113-121. Canter, D.A., E. Zeiger, S. Haworth, T. Lawlor, K. Mortelmans and W. Speck (1986) Comparative mutagenicity of aliphatic epoxides in Salmonella, Mutation Res., 172, 105-138. Dean, B.J., T.M. Brooks, G. Hodson-Walker and D.H. Hutson (1985) Genetic toxicology testing of 41 industrial chemicals, Mutation Res., 153, 57-77. E1-Tantawy, M.A., and B.D. Hammock (1980) The effect of hepatic microsomal and cytosolic subcellular fractions on the mutagenic activity of epoxide-containing compounds in the Salmonella assay, Mutation Res., 79, 59-71. Frantz, S.W., and J.E. Sinsheimer (1981) Bacterial mutagenicity and toxicity of cycloaliphatic epoxides, Mutation Res., 90, 67-78. Heimann, K.-G., and W. Huber (1989) Mutagenit~itspriifungen von Arbeitsstoffen mit wesentlicher Bedeutung und Verbreitung, Z. Ges. Umwelt-Mutationsforsch., 4, 2-6. Meijer, J., and J.W. Depierre (1988) Cytosolic epoxide hydrolase, Chem.-Biol. Interact., 64, 207-249. Oesch, F. (1988) Antimutagenesis by shift in monooxygenase isoenzymes and induction of epoxide hydrolase, Mutation Res., 202, 335-342. Probst, G.S., R.E. McMahon, L.E. Hill, C.T. Thompson, J.K. Epp and S.B. Neal (1981) Chemically-induced unscheduled DNA synthesis in primary rat hepatocyte cultures: a comparison with bacterial mutagenicity using 218 compounds, Environ. Mutagen., 3, 11-32. Seiler, J.P. (1984) The mutagenicity of mono- and di-functional aromatic glycidyl compounds, Mutation Res., 135, 159-167. Sinsheimer, J.E., E. van den Eeckbout, B.H. Hooberman and V.G. Beylin (1987) Detoxification of aliphatic epoxides by diol formation and glutathione conjugation, Chem.-Biol. Interact., 63, 75-90. Thompson, E.D., W.J. Coppinger, C.E. Piper, N. McCarroll, T.J. Oberly and D. Robinson (1981) Mutagenicity of alkyl glycidyl ethers in three short-term assays, Mutation Res., 90, 213-231. Vleminckx, C., J. Arany, B. Hendrickx and W. Moens (1987) Genotoxic effects of glycidyltrimethylammonium chloride, Mutation Res., 189, 387-394. v o n d e r Hude, W., M. Scheutwinkel-Reich, R. Braun and W. Dittmar (1985) In vitro mutagenicity of valepotriates, Arch. Toxicol., 56, 267-271. Von der Hude, W., A. Seelbach and A. Basler (1990) Epoxides: comparison of the induction of SOS-repair in Escherichia coli PQ37 and the bacterial mutagenicity in the Ames test, Mutation Res, in press. Voogd, C.E., J.J. van der Stel and J.J.J.A.A. Jacobs (1981)
150 The mutagenic action of aliphatic epoxides, Mutation Res., 89, 269-282. Wade, D.R., S.C. Airy and J.E. Sinsheimer (1978) Mutagenicity of aliphatic epoxides, Mutation Res., 58,217-223. Watabe, T., A. Hiratsuka, M. Isobe and N. Ozawa (1980) Metabolism of d-limonene by hepatic microsomes to nonmutagenic epoxides toward Salmonella typhimurium, Biochem. Pharmacol., 29, 1068-1071. Williams, G.M. (1976) Carcinogen-induced DNA repair in primary rat liver cell cultures; a possible screen for chemical carcinogens, Cancer Lett., 1,231-236.
Williams, G.M. (1978) Further improvements in the hepatocyte primary culture DNA repair test for carcinogens: detection of carcinogenic biphenyl derivatives, Cancer Lett., 4, 69-75. Williams, G.M., M.F. Laspia and V.C. Dunkel (1982) Reliability of the hepatocyte primary culture/DNA repair test in testing coded carcinogens and noncarcinogens, Mutation Res., 97, 359-370. Williams, G.M., H. Mori and C.A. McQueen (1989) Structureactivity relationships in the rat hepatocyte DNA-repair test for 300 chemicals, Mutation Res., 221,263-286. Communicated by H. Greim
145
Elsevier MUTLET 0410
Induction of DNA-repair synthesis in primary rat hepatocytes by epoxides Wilhelm vonder
Hude, Ralf Mateblowski
and Armin Basler 1
lhstitut fiir Allgemeine Genetik, Freie Universitiit Berlin and ~Max yon Pettenkofer-lnstitut des Bundesgesundheitsamtes, D-IO00 Berlin 33 (Germany)
(Received 28 March 1990) (Accepted 11 June 1990)
Keyword:~: Unscheduled DNA synthesis; Epoxides; Primary rat hepatocytes
Summary The genotoxicity of 10 epoxides was investigated in the UDS test with primary rat hepatocytes. The sensitivity of the assay was demonstrated using 2-acetylaminofluorene. The epoxides 1,2-epoxyoctane, 1,2-epoxydecane, epoxycyclooctane, epoxycyclododecane, (+)-limoneoxide, a-pinaneoxide, t r a n s stilbeneoxide, and c i s - 2 , 3 - e p o x y s u c c i n i c acid, which are known to be non-mutagenic in the Ames test, as well as the bacterial mutagen, 1,2-epoxyphenoxypropane did not induce UDS in primary hepatocytes of the rat. However, a positive UDS response obtained with glycidyltrimethylammonium chloride showed that metabolic inactivation of the oxirane ring in hepatocytes is influenced by further structural substituents.
Several epoxides are known to induce mutations in bacterial test systems without metabolic activation. In most cases, their mutagenic activity is reduced by $9 mix (Canter et al., 1986; E1-Tantawy and H a m m o c k , 1980) due to enzymes in the liver homogenate which are known to inactivate the electrophilic oxirane ring (Oesch, 1988; Meijer and Depierre, 1988). Because these biotransformation capabilities are also present in intact liver ceils the mutagenic epoxides 1,2:3,4-diepoxybutane, 1,2epoxybutane and epichlorohydrin gave negative results in the UDS test with primary rat hepatocytes (Williams et al., 1982; Probst et al., 1981). Some epoxides, however, developed mutagenic
activity in the Ames test only in the presence of $9 mix ( v o n d e r H u d e et al., 1985; Canter et al., 1987), or mutagenic activity was increased in the presence o f $9 mix (Thompson et al., 1981). Recently Williams et al. (1989) reported a positive result in the P R H / U D S test with the direct alkylating butadiene monoepoxide which obviously is not inactivated in the liver cells. Because so far only 4 direct alkylating epoxides have been tested in the P R H / U D S test, 10 epoxides, some mutagenic and some non-mutagenic in the Ames test, were tested in this study with the P R H / U D S assay in order to obtain further information on the response of the P R H / U D S test to chemicals of this class.
Correspondence: Dr. W. v. d. Hude, Max von Pettenkoferlnstitut des Bundesgesundheitsamtes, Postfach 33 00 13, D-1000 Berlin 33 (Germany). 0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
146 TABLE I RESULTS OF THE PRH/UDS Substance
TEST PERFORMED
WITH EPOXIDES
Dose
Experiment I
E x p e r i m e n t II
(mole/l)
NG a
pG b
Net g r a i n s
% pos.
NG a
PG b
Net g r a i n s
% pos.
DMSO
1070
18.1 ± 4.5
26.7 + 7.1
-
8.6 ± 4.5
0
7.7 ± 2.3
8.1 + _ 2 . 2
-0.4
1,2-Epoxy-
3 x 1 0 -4
37.1 ± 6.4
38.6 ± 8.1
-
1.5 ± 5.7
0
5.6 ± 1.4
6.8 +_ 1.2
- 1.2 +
1.5
2
octane
3 × 1 0 -3
32.1 _+ 4.7
32.7 ± 3.8
-
0.6 ± 3.7
42
5.8 ± 1.9
4.0 ± 0.8
+ 1.8 ± 2.1
10
3 x 10-2
toxic
± 2.6
8
toxic
DMSO
1070
18.1 + 4.5
26.7 + 7.1
-
8.6 + 4.5
0
6.2 ± 2.1
10.7 ± 1.9
-4.5
± 2.0
2
1,2-Epoxy-
3×10 -6
33.0 + 6.3
31.1 ± 5.9
+
1.9 +_ 5.1
5
5.4 + 1.3
8.3 + 1.4
-2.9
___ 1.3
0
decane
3 x 1 0 -5
33.8 ± 4.7
34.4 ± 4.9
-
0.6 ___ 4.6
8
4.1 ± 1.4
7.5 _+ 1.2
- 3.4
+
1.4
0
3 × 10- 4
toxic
toxic
H20
1°70
35.5 + 6.3
46.4 + 6.1
-10.9
cis-2,3-Epoxy-
10 -3
36.9 + 5.4
44.0 + 5.5
-
± 5.8
0
5.7 + 1.2
8.1 _+ 1.5
- 2.4
±
1.5
3
7.1 + 3.6
0
4.2 + 1.1
7.3 ± 1.1
-3.1
± 1.1
0
succinic
10 - 2
35.3 ± 5.0
46.8 + 5.0
-11.5
acid
10- ~
toxic
3.6 +_ 1.1
6.5 ± 1.3
- 2.9
±
1.2
0
0
_+ 4.5
0
toxic
DMSO
1070
37.5 + 6.2
49.4 ± 5.9
-11.9
Epoxycyclo-
3 x 1 0 -5
40.8 _+ 7.0
49.9 + 7.5
-
± 5.7
0
6.2 ± 2.1
10.7 _+ 1.9
-4.5
± 2.0
9.1 + 3.8
0
4.4 _+ 1.3
6.6 ± 1.2
-2.2
+
1.2
octane
3x10 -4
37.7 ± 6.4
48.9 ± 7.8
-11.2
2
0
5.4 ± 1.3
8.6 ± 1.4
- 3.2
_+ 1.3
0
3 × 10- 3
toxic 18.1 ± 4.5
± 4.1
toxic
DMSO
1070
Epoxycyclo-
3 × 10- 7
26.7 ± 7.1
-
8.6 ± 4.5
0
dodecane
3x10 -6
29.3 +_ 4.1
36.8 ± 4.5
-
7.5 ± 3.7
2
3 x 1 0 -5
38.4 ± 4.7
38.1 ± 5.2
-
0.3 ± 4.2
8
3x10-4
toxic
6.2 ± 2.1
10.7 ± 1.9
-4.5
_+ 2.0
0
3.2 + 0.9
5.2 ± 0.9
-2.0
± 0.9
2
4.4 + 2.6
9.9 ± 2.2
-5.5
± 2.2
0
toxic
DMSO
1°70
18.1 ± 4.5
26.7 +_ 7.1
-
8.6 + 4.5
0
7.7 + 2.3
8.1 + 2.2
-0.4
_+ 2.6
8
c~-Pinane-
6 x 1 0 -5
28.0 ___ 4.8
33.5 +_ 5.3
-
5.5 ± 4.2
0
3.7 ± 1.3
6.6 ___ 1.3
-2.9
± 1.5
2
oxide
6 × 10 - 4
38.2 ± 7.1
30.9 +_ 6.7
+
8.7 ± 7.2
42
9.6 +_ 1.9
8.8 ± 1.6
+ 0 . 8 ± 1.9
5
6 × 10- 3
toxic
DMSO
1070
37.5 ± 6.2
49.4 _+ 5.9
-11.9
+ 5.7
0
6.2 ± 2.1
10.7 + 1.9
-4.5
± 2.0
0
(+)-Limone-
10 -5
37.6 ± 6.1
40.6 ± 5.5
-
3.0 ± 4.6
5
5.8 ± 1.6
9.2 _+ 1.1
- 3.4
±
1.4
0
oxide
10 - 4
36.1 ± 6.9
43.8 ___ 8.1
-
7.7 +_ 5.2
0
4.8 ± 1.1
7.5 ± 1.1
-2.7
± 1.1
2
10 - 3
toxic 35.5 + 6.3
46.4 + 6.1
-2.4
Glycidyltri-
2x10 -6
47.7 ± 5.5
44.3 ± 4.3
methylammo-
2 x 1 0 -5
58.8 ± 6.7
45.8 + 6.1
13.0 ± 6.4
niumchloride
2 × 1 0 -4
77.3 ± 9.1
33.4 ± 4.9
43.9 ± 5.3
DMSO 1,2-Epoxy-
107o 3x10 -6
phenoxy-
3x10-5
propane
3 × 10- 4
H20
toxic
toxic -10.9
+ 5.8
0
5.7 ± 1.2
8.1 ± 1.5
3.4 ± 4.6
19
9.5 ± 2.4
8.4 _+ 2.2
65
15.5 ± 3.1
7.7 ± 7.8
100
43.1 ± 6.6
4.0 ± 1.2
± 1.5
3
1.1 ± 2.5
15
7.8 _+ 2.8
70
39.1 +_ 6.5 100
7.7 ± 2.3 5.4 ± 1.5
8.1 + 2 . 2
-0.4
± 2.6
8
7.0±
- 1 . 6 --. 1.5
0
5.5 ± 1.4
8.5 ± 1.5
- 3.0
±
1.6
0
1.1
toxic
DMSO
1 070
6.2 + 2.1
10.7 ± 1.9
-4.5
___ 2.0
2
1,2-Epoxy-
3×10 -6
6.4 ± 1.3
8.6 _+ 1.3
-4.2
±
1.7
0
phenoxy-
3 × 10- 5
6.2 ± 1.9
10.4 ± 1.5
-4.2
±
1.7
2
propane
3 x 10 - 4
toxic
147 TABLE I (continued) Substance
Acetone
Dose
Experiment I
(mole/l)
NG a
trans-
1°70 2 × 10 - 7
Stilbeneoxide
2 × 10 - 6 2 × 10- 5
E x p e r i m e n t II pG b
Net grains
°70 p o s .
NG a 6 . 0 ± 1.7 6 . 0 ± 1.5 6.1 ± 1.6 toxic
pG b
Net grains
% pos.
7.7 ± 1.5
-
1.7
±
1.5
5
9.3 + 1.3
- 3.3
+
1.7
2
7.7 _+ 1.6
-
+
1.5
3
+ 1.1
2
1.6
Acetone
lO70
3.6 + 1.1
7.0+
trans-
2 × 10 - 7
5 . 0 _+ 1.3
8.6 + 1.4
Stilbene-
2 × 10 - 6
7.4 + 1.8
10.6 + 2.8
oxide
2 x 10- 5
DMSO AAF
1°70 10 - 9
18.1 _+ 4.5 nd
2 6 . 7 _+ 7.1
7.7 + 2.2 9 . 0 _+ 2.3
8.1 ± 2.2 7.5 + 1.5
1.5 ± 2 . 2
3
AAF
10 - 8
64.4 + 7.4
4 5 . 8 + 7.3
18.6 ± 7.9
95
2 1 . 6 _+ 4 . 0
5.8 _+ 1.7
15.8 _+ 3.9
73
AAF
10 - 7
100#
55.3 ± 9.8
nc
100
56.8 + 6 . 6
3.9 _+ 1.0
5 2 . 9 + 6.7 100
AAF
10 - 6
150#
4 5 . 7 _+ 7.7
nc
100
7 8 . 4 ± 13.2
6.0 + 1.6
7 2 . 4 _+ 1 2 . 8 1 0 0
DMSO AAF
lo70 10 - 9
37.5 + 6 . 2 nd
49.3 + 5.9
-11.8
6 . 2 + 2.1 10.8 + 3.0
10.7 + 1.9
AAF
10 - 8
5 0 . 0 _+ 6.7
4 5 . 8 ± 5.9
4 . 2 + 5.9
33
10.7 + 2 . 9
8.3 + 1.6
AAF
10 - 7
100#
39.7 _ 5.5
nc
100
4 3 . 7 + 6.2
7.3 + 1.5
3 6 . 4 _+ 5.8 100
AAF
10 - 6
150#
4 1 . 4 _ 5.0
nc
100
110.0 + 14.3
6 . 0 + 1.4
104.0 ___ 1 4 . 3 1 0 0
1.1
-3.4 -
3.6 +_
1.5
3
- 3.2
_+ 1 . 6
2
-0.4
_+ 2 . 6
2
toxic 8.6 + 7.1
0
± 5.7 2
10.5 + 2 . 0
-4.5 + 2.0 0.3 +_ 2.8
2 25
2 . 4 + 2.5
37
T h e e x p e r i m e n t s o f series I w e r e p e r f o r m e d w i t h 5 / z C i 3 H - d T / m l a n d t h o s e o f series II w i t h 1 /~Ci 3 H - d T / m l . a N u c l e a r g r a i n s , m e a n o f 60 cells ± s t a n d a r d d e v i a t i o n . b p l a s m a t i c g r a i n s , m e a n o f 6 0 cells _ s t a n d a r d d e v i a t i o n . # U n d e r e s t i m a t e d g r a i n c o u n t s d u e to o v e r l a p p i n g g r a i n s . nc, not calculated, because nuclear grain counts were not exact. nd, not done.
Materials and methods
Chemicals The test substances glycidyltrimethylammonium chloride (CAS No. 3033-77-0, 97o70) and cis-2,3-epoxysuccinic acid (16533-72-5, 90o70) were obtained from Fluka (Switzerland), 1,2-epoxydecane (2404-44-6, 98%), epoxycyclooctane (286-62-4, 98%), epoxycyclododecane (286-99-7, 95%), (+)-limoneoxide (1195-92-2, 97%), transstilbeneoxide (1439-07-2, 99°70) and a-pinaneoxide (1686-14-2, 98o7o) from Aldrich (U.S.A.), and 1,2-epoxyoctane (2984-50-1, 98°70), 1,2-epoxyphenoxypropane (122-60-1, 98%) from Merck (F.R.G.). 2-acetylaminofluorene (AAF) served as the positive control substance.
Methods Primary rat hepatocytes (PRH) were prepared from male Wistar rats (10 weeks, 180-200 g) and cultured by the method of Williams et al. (1982) with slight modifications as described by Butterworth et al. (1987). PRH (5 × 105 viable cells, determined by trypan blue exclusion) were seeded onto coverslips (25 mm, Miles Scientific, U.S.A.). The unattached cells were removed by washing after 2 h. The attached PRH were incubated at 37°C in the presence of the test compounds and of [methyl)H]thymidine (3H-dT, 90 Ci/mmole, Amersham, U.K.) with 5 #Ci/ml or 1/~Ci/ml for 20 h. Thereafter the coverslips were processed by hypotonic treatment as described by Butterworth et al. (1987) and mounted with a 1:1 diluted Ilford K5
148
emulsion. The slides were developed after 2 days in Kodak D19 developer and the PRH were stained with hematoxylin/eosin (HE). The grains were counted with a Biotran II colony counter (New Brunswick, U.S.A.) interfaced with a Zeiss microscope (100× objective). 20 cells of normal morphology were counted per slide and 3 slides per experimental point. A cell was judged to be UDSpositive if the net grains were more than twice the standard deviation (SD) of the solvent control. A substance was judged as UDS-positive if there was a dose-response increase in net grains and more than 50% of the investigated cells were found to be UDS-positive. Results The results of the PRH/UDS test are shown in Table 1. The clear-cut positive results obtained with the known UDS inducer AAF demonstrate the sensitivity of the P R H / U D S test and the reliability of the Ilford K5 film emulsion. Although in the first experimental series with 5/~Ci/ml 3H-dT the nuclear grains obtained after AAF treatment increased dose-dependently, exact grain counts could not be established at high AAF concentrations because of overlapping grains. Accordingly, the grain numbers were underestimated by the counter. The values given in Table 1 without standard deviation (SD) represent the smallest counts within 60 cells. In further experimental series the concentration of ~H-dT was reduced to 1 #Ci/ml. Again, a clearly positive result was obtained with AAF and exact grain counting was possible at all concentrations. In the experimental series with 1 t~Ci/ml 3H-dT as well as with 5 /~Ci/ml, 1,2-epoxyoctane, 1,2epoxydecane, epoxycyclooctane, epoxycyclododecane, (+)-limoneoxide, a-pinaneoxide, and cis-2,3-epoxysuccinic acid did not produce increased net grain counts in the PRH/UDS. Glycidyltrimethylammonium chloride, on the other hand, yielded genotoxic responses in both experiments of the P R H / U D S test. Trans-stilbeneoxide and 1,2-epoxyphenoxypropane were tested twice in independent ex-
periments in the series with 1/zCi/ml 3H-dT. Both substances yielded reproducible negative results in the P R H / U D S test. Discussion Ten epoxide-bearing chemicals were tested for their genotoxic potential with the PRH/UDS test. Nine epoxides yielded negative results and 1 was positive in this test system. The aliphatic oxiranes epoxyoctane and epoxydecane, the cycloaliphatic oxiranes epoxycyclooctane and epoxycyclododecane, the disubstituted oxiranes cis-2,3-epoxysuccinic acid and transstilbeneoxide and the 1,1,2-trisubstituted oxiranes a-pinaneoxide and (+)-limoneoxide did not show mutagenic properties in the Ames test (Wade et al., 1978; Watabe et al., 1980; Frantz and Sinsheimer, 1981; Canter et al., 1986; v o n d e r Hude et al., 1990). They also did not induce UDS in the mammalian PRH/UDS test. In contrast to the above-mentioned epoxides epoxyphenoxypropane is a direct-acting bacterial mutagen. This compound has been shown to be detoxified by mammalian epoxide hydrolases and glutathione-S-transferases (Sinsheimer et al., 1987). This agrees with the negative results reported for the in vivo micronucleus test in mice (Seiler, 1984) and the negative results obtained in the present PRH/UDS test. These data, in addition to the negative results in the PRH/UDS test obtained with the directly acting mutagens 1,2:3,4-diepoxybutane, 1,2-epoxybutane and epichlorohydrin (Williams et al., 1982; Probst et al., 1981), indicate that hepatocytes are capable of inactivating mutagenic epoxides. Nevertheless, glycidyltrimethylammonium chloride, in contrast to those compounds, gave positive results in the PRH/UDS test. This agrees with the results obtained from other in vitro mutagenicity tests. Glycidyltrimethylammonium chloride has been shown to induce point mutations in Klebsiella pneumoniae, Salmonella typhimurium and Escherichia coli strains and chromosomal aberrations in mammalian cells without external metabolic activation. The mutagenic properties of
149
this epoxide were not affected by the addition of $9 mix (Voogd et al., 1981; Vleminckx et al., 1987; Dean et al., 1985). The results obtained with glycidyltrimethylammonium chloride are of special interest because it inserts a cationic group into the DNA and the influence of such compounds on DNA damage is not known. The presence of DNA-damaging properties of electrophilic organic cations may be assumed, but with this special compound steric hindrances preventing DNA attack would seem to be more probable. This hypothesis is supported by the negative results obtained in the Ames test without $9 mix testing the cationic phenyltrimethylammonium chloride (Heimann and Huber, 1989). On the other hand, because this compound is apparently not inactivated by liver homogenate and whole liver cells, it is concluded that charged compounds are not efficient substrates for epoxidedetoxifying enzymes. Furthermore, the positive results obtained with glycidyltrimethylammonium chloride in this study and with butadiene monoepoxide observed by Williams et al. (1989) in the P R H / U D S test demonstrate that enzymes present in freshly isolated liver cells do not necessarily inactivate chemicals carrying an oxirane ring. The inactivation seems to be affected by substituents adjacent to the epoxide group. Because glycidyltrimethylammonium chloride is a directly mutagenic epoxide which is not inactivated by mammalian detoxifying systems, $9 mix and whole liver cells, carcinogenic properties have to be assumed. Considering its widespread use in the textile, electronic and cosmetic industries (for an overview see Vleminckx et al., 1987), evaluation of the possible carinogenic potency of this compound is recommended.
Acknowledgement Part of this work was supported by the Umweltbundesamt (Federal Environmental Agency), Project Number 106 03 067.
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150 The mutagenic action of aliphatic epoxides, Mutation Res., 89, 269-282. Wade, D.R., S.C. Airy and J.E. Sinsheimer (1978) Mutagenicity of aliphatic epoxides, Mutation Res., 58,217-223. Watabe, T., A. Hiratsuka, M. Isobe and N. Ozawa (1980) Metabolism of d-limonene by hepatic microsomes to nonmutagenic epoxides toward Salmonella typhimurium, Biochem. Pharmacol., 29, 1068-1071. Williams, G.M. (1976) Carcinogen-induced DNA repair in primary rat liver cell cultures; a possible screen for chemical carcinogens, Cancer Lett., 1,231-236.
Williams, G.M. (1978) Further improvements in the hepatocyte primary culture DNA repair test for carcinogens: detection of carcinogenic biphenyl derivatives, Cancer Lett., 4, 69-75. Williams, G.M., M.F. Laspia and V.C. Dunkel (1982) Reliability of the hepatocyte primary culture/DNA repair test in testing coded carcinogens and noncarcinogens, Mutation Res., 97, 359-370. Williams, G.M., H. Mori and C.A. McQueen (1989) Structureactivity relationships in the rat hepatocyte DNA-repair test for 300 chemicals, Mutation Res., 221,263-286. Communicated by H. Greim
