Mutation Research, 271 (1992) 49-58
49
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1161/92/$05.00
MUTENV 08808
Species differences in the genotoxicity of cyclophosphamide and styrene in three in vivo assays A.P. S i m u l a a n d B . G . P r i e s t l y Department of Clinical and Experimental Pharmacology, University of Adelaide, G.P.O. Box 498, Adelaide, 5001, South Australia (Australia) (Received 26 March 1991) (Revision received 12 August 1991) (Accepted 26 August 1991)
Keywords: Species differences; Styrene; Cyclophosphamide; Micronulei; Sister-chromatid exchanges; Sperm morphology
Summary Species differences in dispositional factors such as distribution, metabolism and excretion may often account for species differences in the toxic responses to foreign chemicals. In this study we compared the genotoxic responses of cyclophosphamide (CP) and styrene (ST) between Porton rats and LACA Swiss mice in three in vivo assays (bone marrow micronucleus (MN), sperm morphology (SM) and sister-chromatid exchange (SCE) assays). The sensitivities of the three assays were compared by the doses of the compounds required to elicit a significant genotoxic response. The baseline levels for the MN, SCE and SM assays were 1.1-1.4 and 1.2-1.3 MNPCEs/1000 PCEs, 0.23-0.24 and 0.20-0.21 SCEs/chromosome, 3.5-5.7% and 1.6-1.9% abnormal sperm in mice and rats, respectively. CP was a potent genotoxin in the MN and SCE assays but weakly genotoxic in the SM assay. At comparable doses, the rat was approximately 3-, 2.5- and 1.8-fold more sensitive to CP than mice in the MN, SM and SCE assays, respectively. ST produced weak genotoxic responses in all assays in mice and only in the SM and SCE assays in rats. The mice were more sensitive to ST in the MN and SM assays, while it was difficult to compare the species in the SCE assay. For both compounds the sensitivity of the three assays, in decreasing order, were SCE > MN >> SM. For CP the relative responses in the Porton rats and LACA Swiss mice were qualitatively similar to previous reports. Although the use of different strains may explain differences between the studies in the magnitude of the responses observed. The results for ST in the rat shows that the choice of genotoxic endpoint can determine whether a response is detectable. Moreover, the discrepancies between the results for ST in this study and others, suggest that as well as using a battery of in vivo tests, it may be prudent to select more that one strain or species to fully assess a compound's ability to produce DNA damage.
Correspondence: Dr. B.G. Priestly, Department of Clinical and Experimental Pharmacology, University of Adelaide, G.P.O. Box 498, Adelaide, 5001, South Australia (Australia).
In the last decade there has been an increased interest in the use of in vivo rather than in vitro short-term tests as a means of assessing a compound's genotoxic potential (Ashby, 1983; Ashby
50 and Purchase, 1988). In vivo tests take into account dispositional factors such as the route of exposure, distribution, metabolism and excretion, all of which may influence the response. In the development of in vivo assays it is not uncommon for one species to dominate. For example, the mouse is the species primarily used in the micronucleus (MN) and sperm morphology (SM) assays (Heddle, 1973; Wyrobek et al., 1983). Given that rats and mice are commonly used in carcinogenesis bioassays, correlations between carcinogenicity and in vivo genotoxicity may be compromised if there are marked species differences in either or both of these two endpoints. It has been shown with the mycotoxin aflatoxin B i, that the choice of an 'insensitive' test species can lead to a false negative judgement on the genotoxic potential of the test compound (Madle et al., 1986a). Many genotoxic chemicals require metabolic activation to toxic metabolites (Wright, 1980; Dipple et al., 1985). Variations in drug metabolism from one species to another may appear as qualitative differences in the actual pathways present a n d / o r quantitative differences in the level of activity of pathways common to several species (Lorenz et al., 1984; Kato, 1979). These species differences may account for the marked speciesspecific toxicities often encountered, even in species as closely related as rats, mice and hamsters (Tee et al., 1986; Williams, 1971). Moreover, species-specific tissue distribution of various xenobiotic-metabolising enzymes may be major determinants in the primary site of action of toxic chemicals. In this study we report on differences between the Porton rat and the LACA Swiss mouse in genotoxic responses to cyclophosphamide (CP) and styrene (ST) in three in vivo assays. The Porton rat is an outbred strain derived from the Wistar Albino rat, while the LACA Swiss mouse is derived from the Swiss mouse strain and is also outbred. CP is a cytostatic drug and has been implicated as the cause of a number of secondary tumours in cancer patients (Taylor and Wade, 1984). ST is a commercially important chemical widely used in the plastics and polymer industries and is a suspected human carcinogen (Valic, 1982). CP is a useful reference compound in in vivo assays, since it is a potent genotoxin in rats,
mice and hamsters (Madle et al., 1986b; Wyrobek and Bruce, 1975) and its metabolism has been investigated in rats, mice and humans. The in vivo genotoxicity of ST has only been assessed in the mouse and to a limited extent in the hamster, with equivocal findings (Norppa, 1981; Pentilla et al., 1980; Salomaa et al., 1985; Conner et al., 1979). While its metabolism has been studied extensively in rats and humans (Vainio et al., 1984; Ramsey and Young, 1978). Species variation in the in vivo genotoxicity of CP and ST are reported here with respect to the incidence of sister-chromatid exchanges (SCEs) in splenocytes, micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow and abnormal sperm head shapes. The SCE assay was conducted in vitro following in vivo exposure, while the MN and SM assays were entirely in vivo. Materials and methods
All chemicals purchased were of reagent (AR) or pharmaceutical grade quality: CP (Bristol Labs), ST (Ajax Chemicals), Wright's Stain, Giemsa, concanavalin A, 2-mercaptoethanol (Sigma), Harris's Haematoxylin, Eosin Y (BDH), RPMI 1640 (Flow Laboratories), colcemid (Calbiochem). Male LACA Swiss mice (20-30 g) and male Porton rats (200-400 g) obtained from the Adelaide University Central Animal House, were allowed food ad libitum and kept in a controlled environment of 12 h light. 4-12 animals were used per dose group. The dose volume for all i.p. administered drugs was 4 m l / k g body weight. CP solutions were made up in saline, while ST solutions were made up in peanut oil. In all experiments control animals were given comparable volumes of the vehicle. Micronucleus assay. The assay was carried out as recommended by Salamone and Heddle (1983). CP was administered to rats and mice by a single i.p. injection of 1.25, 2.5, 5, 10, 20 mg/kg. ST was administered to rats and mice by a single i.p. injection of 300, 750, 1500, 3000 and 150, 300, 450, 600 m g / k g , respectively. Bone marrow was sampled at 30, 48 and 72 h post-dosing. From freshly sacrificed animals femoral bone marrow
51 smears were air-dried and prepared for analysis by a modified method of Schmid (1975). Slides were fixed for 10 min in anhydrous methanol; stained for 5 min in undiluted Wright's solution (Wright's stain, 2.5 mg/ml in methanol); stained for 2 min in Wright's solution diluted 1:4 in 55 mM phosphate buffer, pH 6.4; stained for 10 min in Giemsa solution (7.6 mg/ml Giemsa in 1:1 methanol/glycerine) diluted 1:19 with water; destained in water; air-dried and mounted with a cover slip. Coded slides were assessed in a blind fashion at 1000 x magnification. The genotoxic response was recorded as the frequency of micronucleated polychromatic erythrocytes (MNPCEs) in 1000 polychromatic erythrocytes (PCEs). The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) in 500 total erythrocytes was also calculated to determined the cytotoxic response. Sperm morphology assay. The procedure was a modification of Wyrobek et al. (1983). Mice and rats were given single i.p. injections on five consecutive days of 50, 100, 200, 400 m g / k g / d and 250, 500, 1000, 2000 m g / k g / d of ST, respectively or 5, 10, 20, 40 m g / k g / d of CP. Mice and rats were sacrificed at 3, 5, 7 and 5, 8, 11 weeks, respectively, from commencement of dosing. The animals were, therefore, examined for the effects of the chemicals on the early spermatid, primary spermatocyte and spermatogonial stages of spermatogenesis. The contents of the vasa deferentia were expelled onto a slide smeared, air-dried and stained using conventional haematoxylin/eosin staining techniques (15 min in 0.4% Harris's haematoxylin; 1 min in 0.1% Eosin Y). The slides were assessed in a blind fashion at 400 x magnification. For each animal 1000 sperm heads were examined for the frequency of morphological abnormalities. Sister-chromatid exchange assay. Mice and rats were given a single i.p. injection of CP (0.63, 1.25, 2.5, 5 mg/kg and 0.31, 0.63, 1.25, 2.5 mg/kg, respectively) or ST (75, 150, 300, 450 mg/kg and 375, 750, 1500, 3000 mg/kg, respectively). Mice and rats treated with CP were sacrificed at 4 and 8 h post-dosing, respectively and mice and rats treated with ST were sacrificed at 24 and 48 h,
respectively. The time of sacrifice was determined from pilot experiments (data not shown). The isolation and culture of splenocytes followed a modified method of Krishna et al. (1988). Isolated cells were washed and incubated in a final volume of 1 ml supplemented RPMI 1640 at a concentration of 3 x 106 viable cells/ml for mice and in a final volume of 4 ml at a concentration of 1 x 106 viable cells/ml for rats. The mitogens used were concanavalin A (5 ~ g / m l ) and 2mercaptoethanol (5/zM). Mouse and rat splenocytes were cultured for a period of 40 and 62 h, respectively, followed by the addition of colcemid (10 /xM final concentration) for 3 h prior to harvesting and staining of slides. Harvesting of cells and preparation of slides was by the method of Krishna et al., (1988) and were stained by the method of Perry and Wolff (1974). The SCE frequency per animal was determined by analyzing 20 metaphase spreads containing a minimum of 38 sister-chromatids. The induced cytotoxicity was determined by the replicative index (R/). It is the frequency of first, second and third division metaphases in 100 metaphases and is calculated as"
R/=
1M 1 + 2M 2 + 3M 3 100
where M1, M 2 and M 3 represent percentages of first, second and third division metaphases, respectively (Schneider and Lewis, 1981). Statistical analyses. The results of the experiments were presented as the mean_+ standard error. For all analyses p < 0.05 was set as the critical level of significance. The genotoxicity data for the MN and SCE assays followed a binomial distribution and was, therefore, analyzed using Analysis of Deviance (Williams, 1978, 1982) followed by Asymptotic Tests (McCullagh and Nelder, 1983) as a means of multiple comparisons to establish threshold doses of CP and ST in each assay. The SCE data fit a normal distribution when transformed using the angular transformation, x' = arcsin ~ - as recommended by Gad and Weil (1986). It was subsequently analyzed using a 1-way Anova followed by Tukey's Multiple-comparison Test (Byrkit, 1987). Untransformed cyto-
52
toxicity data from the MN and SCE assays were analyzed by 1-way Anova and Tukey's Multiplecomparison test. Results CP was genotoxic in all assays, although the response in the SM assay was relatively weak. In the MN assay the top dose of CP increased the M N P C E incidence 10-fold and 42-fold above control levels in mice and rats, respectively (Table 1). The response was dose-, sampling time- and species-dependent ( p < 0.001). Although the threshold dose for CP was 2.5 m g / k g in both species, the magnitude of the responses were greater in the rat at higher doses ( p < 0.001). The mouse M N P C E incidence was highest at 30 h but no response was detected at 72 h. In the rat the timing of the maximal response varied in a dosedependent manner. At low doses the genotoxic effect of CP was greater at 30 h and as the dose was increased the maximal response was more delayed. Like the mouse, a significant effect was not detectable at 72 h. The P C E / N C E ratio was significantly ( p < 0.01) reduced by CP in both species (Table 2), the effect of which differed significantly between species ( p < 0.01) and time of sampling ( p < 0.01). CP was cytotoxic to rat bone marrow down to 5 m g / k g at all time points,
while in mice cytotoxicity was observed down to 10 m g / k g after 72 h. CP also produced a dose- and sampling timedependent ( p < 0.001) increase in the proportion of abnormal sperm in both species (Table 3), where the greatest effect was observed for sperm at 5 weeks. There was also a significant species difference in response to CP ( p < 0.001), where at 5 weeks, 40 m g / k g / d a y of CP induced a 12-fold and 5-fold increase in the percentage of abnormal sperm in rats and mice, respectively. The rat was also more sensitive than the mouse to CP-induced systemic toxicity, with only 3/15 rats and 14/15 mice surviving at 40 m g / k g / d a y . In the SCE assay CP significantly increased the SCE frequency ( p < 0.001) in a dose-related fashion in both species (Table 4). Like the MN and SM assays, the rat was more sensitive than the mouse at comparable doses, where the limit of detection for a genotoxic response was 1.25 m g / k g in mice and 0.63 m g / k g in rats. The ability of splenocytes to divide in culture (RI) was not compromised by in vivo exposure to CP in either species (Table 5). ST produced a weak increase in the incidence of MNPCEs in treated mice (Table 1). The effect was significant ( p < 0.05) at the top dose, at 48 h only, a dose level lethal to 50% of the mice. Genotoxicity was not demonstrated in the rat in
TABLE 1 EFFECT OF CP AND ST ON THE F R E Q U E N C Y OF MNPCEs IN M O U S E AND RAT BONE M A R R O W Compound
CP
ST
Mouse
Rat
Dose
MNPCEs/1000 PCEs
(mg/kg)
30 h
48 h
1.2±0.2 (10) 1.9+0.3 (7) 3.0_+0.4 (7) 3.9_+0.4(10) 7.7±0.6(10) 12.1_+1.0(10)
1.2-+0.2(10) 1.1_+0.2(10) 1.0+_0.4 (7) 1.0+0.3 (7) 1.6±0.4 (7) 1.3-+0.4 (7) 1.9_+0.4 (9) 0.9_+0.4 (8) 3.4+_0.4(10) a 1.6+0.3 (8) 5.3-+0.3 (6) a 1.8-+0.5 (6)
0 1.25 2.5 5 10 20 0 150 300 450 600
1.4+_0.3(10) 2.4-+0.3(10) 2.5+_0.4(10) 1.5+_0.3(10) 2.3+_0.4 (9)
a a a a
1.3+_0.3 (5) 2.6+_0.5(10) 2.5-+0.5(10) 3.0+_0.4(10) 4.2+_1.0 (9) a
72 h
Dose
MNPCEs/1000 PCEs
(mg/kg)
30 h
48 h
72 h
1.3 _+0.2 0 1.3 ±0.2 (10) 1.2_+0.2(10) 1.2_+0.4 1.25 2.2+_0.5 (6) 1.2+0.5 (6) 1.3+0.3 2.5 3.3-+0.8 (6) a 2.3+0.3 (6) 5 10.9±1.3(10)" 4.0+1.1 (5) ~' 1.8_+0.3 10 17.5±1.3 (4) ~' 18.8+2.1 (5) a 2.8±0.5 20 21.1_+2.5 (7) a 50.1+_7.2 (7) a 2.0+0.4
(10) (5) (6) (4) (4) (6)
1.2_+0.1 1.5±0.7 1.7-+0.6 O.9_+0.3 1.5 ±0.3
(5) (4) (7) (8) (4)
1.4-+0.2 (5) 0 1.4+_0.2(10) 300 1.1_+0.2(10) 750 1.0_+0.3 (9) 1500 0.6_+0.2 (5) 3000
The number of animals per treatment group are indicated in parentheses. a Indicates a significant difference from respective controls, p < 0.05.
1.3+_0.2 1.8-+0.5 1.9+_0.3 2.0+_0.5 1.8+_0.7
(8) (4) (7) (7) (5)
1.3+_0.2 2.0+_0.9 1.3+_0.6 1.6+_0.3 1.6+_0.2
(9) (4) (7) (7) (5)
0 150 300 450 600
0 1.25 2.5 5 10 20
0.58+0.04(10) 0.67-1-0.07(10) 0.66 _+0.04 (10) 0.69_+0.04(10) 0.73_+0.11 (9)
1.00+0.01 (10) 1.06 _+0.03 (7) 1.09 + 0.04 (7) 1.11_+0.03 (10) 1.07_+0.10 (10) 1.06+0.07(10)
P C E / N C E ratio
30 h
Dose
(mg/kg)
Mouse
(10) (7) (7) (9) (10) (6)
0.96_+0.06 (5) 0.78-+0.07(10) 0.89 -+ 0.06 (10) 0.63+0.09(10) 0.69_+0.08 (9)
1.00_+0.01 1.05 _+0.04 1.09 _+0.07 1.03+0.05 0.90_+0.07 0.81+0.10
48 h
a
1.07_+ 0.07 0.89_+0.05 1.04 _+0.04 0.93 + 0.04 0.89_+ 0.08
1.00_+0.01 1.00 _+0.05 1.00+0.05 0.87 + 0.05 0.86_+ 0.05 0.54_+ 0.06
72 h
The n u m b e r of animals per treatment group are indicated in parentheses. a Indicates a significant difference from respective controls, p < 0.05.
ST
CP
Compound
(5) (10) (10) (9) (5)
(10) (7) (7) (8) (8) a (6) a 0 300 750 1500 3 000
0 1.25 2.5 5 10 20
(mg/kg)
Dose
Rat
0.04 0.04 0.05 0.02 0.03 0.04
(10) (6) (6) (10) a (4) a (7)a
0.72_+ 0.61 _+ 0.48_+ 0.42 _+
0.03 0.08 0.03 0.09
(4) (7) (7) (5) a
0.70_+ 0.05 (8)
0.61_+ 0.39 -+ 0.47 _+ 0.39_+ 0.32+ 0.27_+
30 h
P C E / N C E ratio
E F F E C T O F CP A N D ST O N T H E R A T I O O F PCEs T O NCEs IN M O U S E A N D R A T B O N E M A R R O W
TABLE 2
0.70_+ 0.53_+ 0.75 _+ 0.63 + 0.51+
0.60+ 0.50_+ 0.55-+ 0.24-+ 0.19_+ 0.11_+
48 h
0.05 0.08 0.07 0.09 0.13
(9) (4) (7) (7) (5)
0.04(10) 0.12 (6) 0.12 (6) 0.15 (5) a 0.01 (5) a 0.02 (7) a
0.70_+ 0.74+ 0.37+ 0.42_+ 0.22_+
0.61 _+ 0.55 _+ 0.52+ 0.35+ 0.26 + 0.19_+
72 h
0.04 0.05 0.05 0.09 0.03
0.03 0.03 0.05 0.01 0.02 0.04
(5) (4) (7) a (8) a (4) a
(10) (5) (6) (4) a (4) a (6) a
54 TABLE 3 E F F E C T O F CP A N D ST O N T H E P E R C E N T A G E O F A B N O R M A L S P E R M IN T H E M O U S E A N D R A T Compound
Mouse
Rat
Dose
% Abnormal sperm
(mg/kg/d)
Dose
% Abnormal sperm
(mg/kg/d)
5 wk
8 wk
11 wk
1.8±0.4(5) 1.4±0.1(5) 3.3±0.1(5) 3.4±0.2(4) NS
1.6±0.3(5) 1.5±0.2(5) 2.7±0.4(5) 18.4±4.1(5) 19.8±4.4(3)
1.9±0.2(5) 2.2±0.2(5) 4.6±0.6(5) 7.6+1.1(5) NS
1.7±0.1(5) 1.9±0.1(5) 2.5±0.1(4) 2.7±0.3(5) 2.4(1)
1.6±0.1(5) 1.8±0.2(5) 1.9±0.l(5) 1.7+0.2(5) NS
3 wk
5 wk
7 wk
CP
0 5 10 20 40
3.5±0.6(5) 2.6±0.4(4) 5.7±1.9(4) 6.6±1.6(5) 6.0±1.1(4)
3.8±0.3(5) 7.0±2.2(5) 4.8±0.7(5) 7.6±2.4(5) 19.2+3.5(5)
3.7±0.4(5) 5.6±2.3(4) 4.8±1.2(5) 10.5±0.7(5) 13.1±3.4(4)
ST
0 50 100 200 400
4.1±1.0(5) 4.3±0.9(5) 5.3±0.6(4) 6.5±1.3(5) 6.9±1.4(7)
4.0±0.8(5) 5.7±1.3(5) 0 7.8±1.5(5) 8.6±1.9(5) 250 7.2±0.9(5) 17.8±6.6(5) 500 7.7±1.3(5) 20.5±4.7(5) a 10~ 11.2±3.0(8) ~ 15.3±1.1(7) a 2000
0 5 10 20 40
1.7±0.1(5) 2.1±0.2(5) 2.2±0.2(5) 2.6±0.3(5) a NS
The number of animals per treatment group are indicated in parentheses. a Indicates a significant difference from respective controls, p < 0.05. NS, No survivors.
this assay, even at a dose level (3000 m g / k g ) lethal to 40% of rats. ST was not cytotoxic to the haemopoietic tissues in the mouse, although it did decrease the P C E / N C E ratio in the rat (Table 2). The effects in mice were complicated by a possible vehicle effect. In the SM assay 400 and 1000 m g / k g / d a y of ST produced a small, but significant increase ( p < 0.001) in the proportion of abnormal sperm
in mice (2.8-fold) and rats (1.6-fold), respectively (Table 3). Like the MN assay, the mouse appeared to be more sensitive than the rat. The top dose of ST was lethal to 90% of treated rats. In the SCE assay ST produced a weak but significant ( p < 0.001) genotoxic effect at the top dose of 450 m g / k g (Table 4), which was lethal to one of five treated mice. ST significantly increased the SCE frequency ( p < 0.001) in splenocytes of
TABLE 4 E F F E C T O F CP A N D ST ON T H E SCE F R E Q U E N C Y IN T H E M O U S E A N D R A T Compound
Mouse Dose (mg/kg)
CP
ST
0 0.62 1.25 2.5 5 0 75 150 300 450
Rat SCEs/chrom 0.24 ± 0.01 (5) 0.25 +_0.01 (5) 0.33 ± 0.01 (5) a 0.53 +_0.01 (5) ~ 0.85 ± 0.03 (5) a 0.23 ± 0.23 ± 0.23 ± 0.23 ± 0.27 ±
0.02 0.01 0.01 0.02 0.02
(5) (5) (5) (5) (4) ~
The number of animals per treatment group are indicated in parentheses. a Indicates significant difference from respective controls, p < 0.05.
Dose ( m g / k g ) 0 0.31 0.62 1.25 2.5 0 375 750 1500 3 000
SCEs/chrom 0.20 _+0.01 0.21 + 0.01 0.32+0.01 0.49 ___0.04 0.79 + 0.04
(5) (5) (5) a (5) a (5) a
0.21 + 0.01 0.21 ± 0.02 0.28 + 0.03 0.45 ± 0.04 0.43 (1)
(5) (5) (5) a (5) a
55 TABLE 5 EFFECT OF CP AND ST ON THE REPLICATIVE INDEX OF CULTURED SPLENOCYTES FROM TREATED MICE AND RATS Compound CP
Mouse Dose (mg/kg) 0 0.62 1.25
2.5 5 ST
0 75 150 300 450
Replicative index 2.04 + 0.04 (5) 1.96+0.04 (5) 2.02 + 0.04 (5) 1.97 + 0.06 (5) 2.04 + 0.03 (5)
Rat Dose (mg/kg) 0 0.31 0.62 1.25 2.5
Replicative index 2.45 + 0.09 (5) 2.37+0.02 (5) 2.40 + 0.07 (5) 2.20 + 0.09 (5) 2.20 + 0.03 (5)
2.00 + 0.03 (5) 2.00+0.01 (5) 1.98+0.02 (5) 1.99 ± 0.04 (5) 2.065:0.03 (4)
0 375 750 1500 3000
2.37 + 0.07 (5) 2.37+0.08 (5) 2.305:0.11 (5) 2.32 5:0.03 (5) 2.54 (1)
The number of animals per treatment group are indicated in parentheses. treated rats down to 750 m g / k g . ST was not cytotoxic to rat- or mouse-derived splenocytes (Table 5). Discussion
CP-induced genotoxic effects in the MN assay in both species were quantitatively similar, at comparable doses, to some previously reported data (Madle et al., 1986b), where Wistar rats were shown to be more sensitive than N M R I mice. The longer plasma half-life of alkylating CP metabolites in rats compared to mice may account for the greater sensitivity of the rat (Torkelson et al., 1974; Garattini et al., 1974). Furthermore, Madle et al., (1986b) using an alternative target tissue for the SCE assay, also found it to be more sensitive than the MN assay at comparable doses. CD1 mice used by Krishna et al. (1986, 1987) to assess CP-induced SCEs in bone marrow and splenocytes appear to be less sensitive than the N M R I mice used by Madle et al. (1986b) and the L A C A Swiss mice used in this study. Likewise, a study by Trzos et al. (1978) using S p r a g u e - D a w l e y rats found that 20 m g / k g of CP induced a 7-fold increase in bone marrow micronuclei at 24 h, while 5 m g / k g of CP was able to induce a similar response in the Porton rats at 30 h. The importance of multiple sampling for the MN assay was demonstrated in the rat, where at
10 and 20 m g / k g the responses were higher at 48 h than at 30 h. This was due to the cytotoxic properties observed for CP in the rat. The marked inhibition of haemopoiesis (Table 2) can delay the progression of the D N A - d a m a g e d erythroblasts to the P C E stage and hence delay the appearance of MNPCEs (Salamone and Heddle, 1983). This effect of CP in the rat has previously been reported by other investigators (Madle et al., 1986b; Goetz et al., 1975). A similar delay in the response to CP at higher doses was not observed in mice, consistent with the relative lack of CP-induced cytotoxicity (Table 2). C o m p a r e d to the MN and SCE assays, the SM assay was insensitive. CP was only able to produce an effect at lethal or near-lethal doses in both species. Similar results with respect to mice, were also observed by Wyrobek and Bruce (1975). They found the primary spermatocyte stage of spermatogenesis in (C57 × C3H)F~ mice to be the most sensitive to CP exposure, observing approximately a 4-fold increase in the proportion of abnormal sperm at 50 m g / k g / d a y , compared to the 5-fold increase at 40 m g / k g / d a y in the L A C A Swiss mice. The weak genotoxic response to ST in the M N assay in the mouse is consistent with the observation of N o r p p a (1981), who also found weak positive responses in C 5 7 B L / 6 mice at 250 and 1000 m g / k g . In contrast, Pentilla et al. (1980) did not detect an effect of ST up to 1 g / k g in the
56 same assay in Chinese hamsters. In the SM test, the increase in abnormal sperm by ST is inconsistent with the report of no response in (C57 × C3H)F 1 mice at doses up to 700 m g / k g / d a y at 3 and 5 weeks after dosing (Salomaa et al., 1985). This may be due to strain differences in ST metabolism, as Cantoni et al., (1978) have reported marked diferrences in the ability of different species to activate and deactivate styrene in a number of tissues. Like the MN and SM assays, ST was able to induce a weak response in mice, in the SCE assay, at near lethal doses. These results agree with the observations of Conner et al., (1979), who reported a significant increase in SCE frequency in bone marrow cells of BDFI mice exposed to ST by inhalation (565 _+ 15.8 p.p.m.; 6 h / d a y ; 4 days). On the other hand, Shareif et al. (1986) failed to detect a significant increase in SCE frequency in the bone marrow of C 5 7 B 1 / 6 mice given a single i.p. dose of up to 1 g / k g of ST. A combination of the use of different mouse strains, routes of administration and target tissues may explain differences between our results and those of the other two laboratories. In contrast to the response in mice, a relatively large response was observed in the SCE assay for rats at doses where no detectable effect was observed in the MN assay. This is similar to CP, where SCEs were found to be a more sensitive endpoint than micronuclei. Although the responses to ST were weak, it was consistently positive in all assays in the mouse and two of three assays in the rat. Therefore, it has the potential to induce similar genetic damage and possibly tumours in humans. This is supported by other evidence such as; (1) the detection of S T - D N A adducts in the liver, brain, lungs, spleen and testes of exposed mice (Nordqvist et al., 1985), (2) increased frequency of lymphocyte micronuclei in workers exposed to low level ST (Hogstedt et al., 1983) and (3) the ability of styrene oxide, the putative toxic metabolite of ST, to induce tumours in rats and mice (Lijinsky, 1986; Ponomarkov et al., 1984). In the SM assay near lethal doses of both CP and ST were required to elicit a response. This may be due to the presence of the blood-testis barrier, which can restrict the access of foreign
chemicals to the germ cells (Okumura et al., 1975), or the relatively high activity of detoxifying enzymes present in germ cells (Mukhtar et al., 1978). In view of the susceptibility of the frequency of abnormal sperm to various non-mutagenic factors determined by the health of the animal (Komatsu et al., 1982; Cairnie and Leach, 1980; Topham, 1983), it is conceivable that the responses may have been an indirect result of systemic toxicity induced by the large doses of CP and ST, rather than a true genotoxic effect. The results of CP and ST in this assay should, therefore, be interpreted with care. O f the three assays the SCE assay was the most sensitive to the two genotoxins used in this study. This may be due to differences in the ability of the tissues to locally activate a n d / o r deactivate toxic metabolites or differences in the ability of the tissues to repair D N A damage. Alternatively, this may reflect differences in the potential of the two compounds to induce SCEs relative to micronuclei and abnormal sperm, as each may involve different molecular mechanisms. The low baseline levels of micronuclei, SCEs and abnormal sperm in the Porton rats and L A C A Swiss mice demonstrates the potential usefulness of these strains in in vivo genotoxicity testing. The qualitatively similar results for the reference genotoxin, CP, in this study and that of others, further illustrates this point. The results for ST in the SM and SCE assays in mice were not in complete agreement with other reports. This may be due to the use of alternative strains of mice. The response to ST in the rat indicates that the choice endpoint a n d / o r target tissue are important determinants in the ability to detect a compound's genotoxic response. Therefore, a negative result for a compound in an in vivo assay should be verified by either examining alternative endpoints or the same endpoint in a different species or strains of animal before a firm conclusion can be made on a compound's genotoxic potential.
Acknowledgements We would like to thank Dr. A. Verbyla for his advice and assistance in the statistical analysis of
57
the data, and R. Irvine, J. Edwards, P. Wright and G. Crabb for their technical assistance.
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sister chromatid exchanges in bone marrow and spleen cells in mouse and Chinese hamster, Environ. Mutagen., 8, 449-459. Krishna, G., J. Xu, J. Nath, M. Peterson and T. Ong (1987) Cyclophosphamide-induced cytogenetic effects in mouse bone marrow and spleen cells in in vivo and in v i m / i n vitro assays, Teratogen. Carcinogen. Mutagen., 7, 183-195. Krishna, G., J. Nath, M. Peterson and T. Ong (1988) In vivo and in vivo/in vitro kinetics of cyclophosphamide-induced sister-chromatid exchanges in mouse bone marrow and spleen cells, Mutation Res., 204, 297-305. Lijinsky, W. (1986) Rat and mouse forestomach tumours induced by chronic oral administration of styrene oxide, J. Natl. Cancer Inst., 77(2), 471-476. Lorenz, J., H.R. Glatt, R. Fleichmann, R. Ferlinz and F. Oesch (1984) Drug metabolism in man and its relationship to that of three rodent species: Monoxygenase, epoxide hydrolase and glutathione S-transferase activities in subcellular fractions of lung and liver, Biochem. Med., 32, 43-56. Madle, E., A. Korte and B. Beek (1986a) Species differences in mutagenicity testing, I. Micronucleus and SCE testing in rats, mice and Chinese hamsters with aflatoxin B l, Teratogen. Carcinogen. Mutagen., 6, 1-13. Madle, E., A. Korte and B. Beek (1986b) Species differences in mutagenicity testing, II. Sister chromatid exchange and micronucleus induction in rats, mice and Chinese hamsters treated with cyclophosphamide, Mutagenesis, 1(6), 419422. McCullagh, P., and J.A. Nelder (Eds.) (1983) Generalized Linear Models, Chapman and Hall, London. Mukhtar, H., I.P. Lee, G.L. Foureman and J.R. Bend (1978) Epoxide metabolizing enzyme activities in rat testes: Postnatal development and relative activity in interstitial and spermatogenic cell compartments, Chem.-Biol. Interact., 22, 153-165. Nordqvist, M.B., A. Lof, S. Osterman-Golkar and S.A.S. Walles (1985) Covalent binding of styrene and styrene oxide to plasma proteins, hemoglobin and DNA in the mouse, Chem.-Biol. Interact., 55, 63-73. Norppa, H. (1981) Styrene and vinyltoluene induce micronuclei in mouse bone marrow, Toxicol. Lett., 8, 247-251. Okumura, K., I.P. Lee and R.L. Dixon (1975) Permeability of selected drugs and chemicals across the blood-testes barrier of the rat, J. Pharmacol. Exp. Ther., 194, 89-95. Pentilla, M., M. Sorsa and H. Vainio (1980) Inability of styrene to induce nondysfunction in Drosophila or a positive micronucleus test in the Chinese hamster, Toxicol. Lett., 6, 119-123. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids, Nature (London), 251, 156-158. Ponomarkov, V., J.R.P. Cabral, J. Wahrendorf and D. Galendo (1984) A carcinogenicity study of styrene-7,8-oxide in rats, Cancer Lett., 24, 95-101. Ramsey, J.C., and J.D. Young (1978) Pharmacokinetics of inhaled styrene in rats and humans, Scand. J. Work Environ. Health, 4($2), 84-91.
58 Salamone, M.F., and J.A. Heddle (1983) The bone marrow micronucleus assay: Rationale for a revised protocol, in: F.J. de Serres (Ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 8, Plenum, New York, pp. 111-149. Salomaa, S., M. Donner and H. Norppa (1985) Inactivity of styrene in the mouse sperm morphology test, Toxicol. Lett., 24, 151-155. Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-15. Schneider, E.L., and J. Lewis (1981) Aging and the sister chromatid exchange VIII. Effect of the aging environment on sister ehromatid exchange induction and cell cycle kinetics in Ehrlich ascites tumor cells, abrief note, Mech. Age Develop., 17, 327-330. Sharief, Y., A.M. Brown, L.C. Backer, J.A. Campbell, B. Westbrook-Collins, A.G. Stead and J.W. Allen (1986) Sister chromatid exchange and chromosomal aberration analyses in mice after in vivo exposure to acrylonitrile, styrene or butadiene monoxide, Environ. Mutagen., 8, 439-448. Taylor, A.T., and A.E. Wade (1984) Chemical carcinogenicity and the antineoplastic agents, Am. J. Hosp. Pharm., 41, 1844-1848. Tee, L.B.G., D.S. Davies, C.E. Seddon and A.R. Boobis (1986) Species differences in the hepatotoxicity of paracetamol are due to differences in the rate of conversion to its cytotoxic metabolite, Biochem. Pharmacol., 36, 1041-1052. Topham, J.C. (1983) Chemically induced changes in sperm in animals and humans, in: F.J. de Serres (Ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 8, Plenum, New York, pp. 201-234. Torkelson, A.R., J.A. LaBudde and J.H. Weikel (1974) The
metabolic fate of cyclophosphamide, Drug Metab. Rev., 3(1), 131-165. Trzos, R.J., G.L. Petzold, M.N. Brunden and J.A. Swenberg (1978) The evaluation of sixteen carcinogens in the rat using the micronucleus test, Mutation Res., 58, 79-86. Vainio, H., E. Hietenen and G. Belvedere (1984) Pharmacokinctics and metabolism of styrene, in: J.W. Bridges and L.F. Chasseaud (Eds.), Progress in Drug Metabolism, Vol. 8, Taylor and Francis, London, pp. 203-239. Valic, F. (1982) Environmental health criteria document on styrene, I n t e r n a t i o n a l P r o g r a m m e on Chemical Safety/WHO. Williams, D.A. (1978) The analysis of binary responses from toxicological experiments involving reproduction and teratogenicity, Biometrics, 31,949-952. Williams, D.A. (1982) Extra-binomial variation in logistic linear models, Appl. Stat., 35, 144-148. Williams, R.T. (1971) Species variations in drug biotransformations, in: B.N LaDu, H.G. Mandel and E.L. Way (Eds.), Fundamentals of Drug Metabolism and Drug Disposition, Williams and Wilkins, Baltimore, pp. 187-205. Wright, A.S. (1980) The role of metabolism in chemical mutagenesis and chemical carcinogenesis, Mutation Res., 75, 215-241. Wyrobek, A.J., and W.R. Bruce (1975) Chemical induction of sperm abnormalities in mice, Proc. Natl. Acad. Sci. (U.S.A.)., 72, 4425-4429. Wyrobek, A.J., L.A. Gordon, J.G. Burkhart, M.W. Francis, R.W. Kapp, G. Letz, H.V. Mailing, J.C. Topham and M.D. Whorton (1983) An evaluation of the mouse sperm morphology test and other sperm tests in non-human mammals, Mutation Res., 115, 1-72.
49
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1161/92/$05.00
MUTENV 08808
Species differences in the genotoxicity of cyclophosphamide and styrene in three in vivo assays A.P. S i m u l a a n d B . G . P r i e s t l y Department of Clinical and Experimental Pharmacology, University of Adelaide, G.P.O. Box 498, Adelaide, 5001, South Australia (Australia) (Received 26 March 1991) (Revision received 12 August 1991) (Accepted 26 August 1991)
Keywords: Species differences; Styrene; Cyclophosphamide; Micronulei; Sister-chromatid exchanges; Sperm morphology
Summary Species differences in dispositional factors such as distribution, metabolism and excretion may often account for species differences in the toxic responses to foreign chemicals. In this study we compared the genotoxic responses of cyclophosphamide (CP) and styrene (ST) between Porton rats and LACA Swiss mice in three in vivo assays (bone marrow micronucleus (MN), sperm morphology (SM) and sister-chromatid exchange (SCE) assays). The sensitivities of the three assays were compared by the doses of the compounds required to elicit a significant genotoxic response. The baseline levels for the MN, SCE and SM assays were 1.1-1.4 and 1.2-1.3 MNPCEs/1000 PCEs, 0.23-0.24 and 0.20-0.21 SCEs/chromosome, 3.5-5.7% and 1.6-1.9% abnormal sperm in mice and rats, respectively. CP was a potent genotoxin in the MN and SCE assays but weakly genotoxic in the SM assay. At comparable doses, the rat was approximately 3-, 2.5- and 1.8-fold more sensitive to CP than mice in the MN, SM and SCE assays, respectively. ST produced weak genotoxic responses in all assays in mice and only in the SM and SCE assays in rats. The mice were more sensitive to ST in the MN and SM assays, while it was difficult to compare the species in the SCE assay. For both compounds the sensitivity of the three assays, in decreasing order, were SCE > MN >> SM. For CP the relative responses in the Porton rats and LACA Swiss mice were qualitatively similar to previous reports. Although the use of different strains may explain differences between the studies in the magnitude of the responses observed. The results for ST in the rat shows that the choice of genotoxic endpoint can determine whether a response is detectable. Moreover, the discrepancies between the results for ST in this study and others, suggest that as well as using a battery of in vivo tests, it may be prudent to select more that one strain or species to fully assess a compound's ability to produce DNA damage.
Correspondence: Dr. B.G. Priestly, Department of Clinical and Experimental Pharmacology, University of Adelaide, G.P.O. Box 498, Adelaide, 5001, South Australia (Australia).
In the last decade there has been an increased interest in the use of in vivo rather than in vitro short-term tests as a means of assessing a compound's genotoxic potential (Ashby, 1983; Ashby
50 and Purchase, 1988). In vivo tests take into account dispositional factors such as the route of exposure, distribution, metabolism and excretion, all of which may influence the response. In the development of in vivo assays it is not uncommon for one species to dominate. For example, the mouse is the species primarily used in the micronucleus (MN) and sperm morphology (SM) assays (Heddle, 1973; Wyrobek et al., 1983). Given that rats and mice are commonly used in carcinogenesis bioassays, correlations between carcinogenicity and in vivo genotoxicity may be compromised if there are marked species differences in either or both of these two endpoints. It has been shown with the mycotoxin aflatoxin B i, that the choice of an 'insensitive' test species can lead to a false negative judgement on the genotoxic potential of the test compound (Madle et al., 1986a). Many genotoxic chemicals require metabolic activation to toxic metabolites (Wright, 1980; Dipple et al., 1985). Variations in drug metabolism from one species to another may appear as qualitative differences in the actual pathways present a n d / o r quantitative differences in the level of activity of pathways common to several species (Lorenz et al., 1984; Kato, 1979). These species differences may account for the marked speciesspecific toxicities often encountered, even in species as closely related as rats, mice and hamsters (Tee et al., 1986; Williams, 1971). Moreover, species-specific tissue distribution of various xenobiotic-metabolising enzymes may be major determinants in the primary site of action of toxic chemicals. In this study we report on differences between the Porton rat and the LACA Swiss mouse in genotoxic responses to cyclophosphamide (CP) and styrene (ST) in three in vivo assays. The Porton rat is an outbred strain derived from the Wistar Albino rat, while the LACA Swiss mouse is derived from the Swiss mouse strain and is also outbred. CP is a cytostatic drug and has been implicated as the cause of a number of secondary tumours in cancer patients (Taylor and Wade, 1984). ST is a commercially important chemical widely used in the plastics and polymer industries and is a suspected human carcinogen (Valic, 1982). CP is a useful reference compound in in vivo assays, since it is a potent genotoxin in rats,
mice and hamsters (Madle et al., 1986b; Wyrobek and Bruce, 1975) and its metabolism has been investigated in rats, mice and humans. The in vivo genotoxicity of ST has only been assessed in the mouse and to a limited extent in the hamster, with equivocal findings (Norppa, 1981; Pentilla et al., 1980; Salomaa et al., 1985; Conner et al., 1979). While its metabolism has been studied extensively in rats and humans (Vainio et al., 1984; Ramsey and Young, 1978). Species variation in the in vivo genotoxicity of CP and ST are reported here with respect to the incidence of sister-chromatid exchanges (SCEs) in splenocytes, micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow and abnormal sperm head shapes. The SCE assay was conducted in vitro following in vivo exposure, while the MN and SM assays were entirely in vivo. Materials and methods
All chemicals purchased were of reagent (AR) or pharmaceutical grade quality: CP (Bristol Labs), ST (Ajax Chemicals), Wright's Stain, Giemsa, concanavalin A, 2-mercaptoethanol (Sigma), Harris's Haematoxylin, Eosin Y (BDH), RPMI 1640 (Flow Laboratories), colcemid (Calbiochem). Male LACA Swiss mice (20-30 g) and male Porton rats (200-400 g) obtained from the Adelaide University Central Animal House, were allowed food ad libitum and kept in a controlled environment of 12 h light. 4-12 animals were used per dose group. The dose volume for all i.p. administered drugs was 4 m l / k g body weight. CP solutions were made up in saline, while ST solutions were made up in peanut oil. In all experiments control animals were given comparable volumes of the vehicle. Micronucleus assay. The assay was carried out as recommended by Salamone and Heddle (1983). CP was administered to rats and mice by a single i.p. injection of 1.25, 2.5, 5, 10, 20 mg/kg. ST was administered to rats and mice by a single i.p. injection of 300, 750, 1500, 3000 and 150, 300, 450, 600 m g / k g , respectively. Bone marrow was sampled at 30, 48 and 72 h post-dosing. From freshly sacrificed animals femoral bone marrow
51 smears were air-dried and prepared for analysis by a modified method of Schmid (1975). Slides were fixed for 10 min in anhydrous methanol; stained for 5 min in undiluted Wright's solution (Wright's stain, 2.5 mg/ml in methanol); stained for 2 min in Wright's solution diluted 1:4 in 55 mM phosphate buffer, pH 6.4; stained for 10 min in Giemsa solution (7.6 mg/ml Giemsa in 1:1 methanol/glycerine) diluted 1:19 with water; destained in water; air-dried and mounted with a cover slip. Coded slides were assessed in a blind fashion at 1000 x magnification. The genotoxic response was recorded as the frequency of micronucleated polychromatic erythrocytes (MNPCEs) in 1000 polychromatic erythrocytes (PCEs). The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) in 500 total erythrocytes was also calculated to determined the cytotoxic response. Sperm morphology assay. The procedure was a modification of Wyrobek et al. (1983). Mice and rats were given single i.p. injections on five consecutive days of 50, 100, 200, 400 m g / k g / d and 250, 500, 1000, 2000 m g / k g / d of ST, respectively or 5, 10, 20, 40 m g / k g / d of CP. Mice and rats were sacrificed at 3, 5, 7 and 5, 8, 11 weeks, respectively, from commencement of dosing. The animals were, therefore, examined for the effects of the chemicals on the early spermatid, primary spermatocyte and spermatogonial stages of spermatogenesis. The contents of the vasa deferentia were expelled onto a slide smeared, air-dried and stained using conventional haematoxylin/eosin staining techniques (15 min in 0.4% Harris's haematoxylin; 1 min in 0.1% Eosin Y). The slides were assessed in a blind fashion at 400 x magnification. For each animal 1000 sperm heads were examined for the frequency of morphological abnormalities. Sister-chromatid exchange assay. Mice and rats were given a single i.p. injection of CP (0.63, 1.25, 2.5, 5 mg/kg and 0.31, 0.63, 1.25, 2.5 mg/kg, respectively) or ST (75, 150, 300, 450 mg/kg and 375, 750, 1500, 3000 mg/kg, respectively). Mice and rats treated with CP were sacrificed at 4 and 8 h post-dosing, respectively and mice and rats treated with ST were sacrificed at 24 and 48 h,
respectively. The time of sacrifice was determined from pilot experiments (data not shown). The isolation and culture of splenocytes followed a modified method of Krishna et al. (1988). Isolated cells were washed and incubated in a final volume of 1 ml supplemented RPMI 1640 at a concentration of 3 x 106 viable cells/ml for mice and in a final volume of 4 ml at a concentration of 1 x 106 viable cells/ml for rats. The mitogens used were concanavalin A (5 ~ g / m l ) and 2mercaptoethanol (5/zM). Mouse and rat splenocytes were cultured for a period of 40 and 62 h, respectively, followed by the addition of colcemid (10 /xM final concentration) for 3 h prior to harvesting and staining of slides. Harvesting of cells and preparation of slides was by the method of Krishna et al., (1988) and were stained by the method of Perry and Wolff (1974). The SCE frequency per animal was determined by analyzing 20 metaphase spreads containing a minimum of 38 sister-chromatids. The induced cytotoxicity was determined by the replicative index (R/). It is the frequency of first, second and third division metaphases in 100 metaphases and is calculated as"
R/=
1M 1 + 2M 2 + 3M 3 100
where M1, M 2 and M 3 represent percentages of first, second and third division metaphases, respectively (Schneider and Lewis, 1981). Statistical analyses. The results of the experiments were presented as the mean_+ standard error. For all analyses p < 0.05 was set as the critical level of significance. The genotoxicity data for the MN and SCE assays followed a binomial distribution and was, therefore, analyzed using Analysis of Deviance (Williams, 1978, 1982) followed by Asymptotic Tests (McCullagh and Nelder, 1983) as a means of multiple comparisons to establish threshold doses of CP and ST in each assay. The SCE data fit a normal distribution when transformed using the angular transformation, x' = arcsin ~ - as recommended by Gad and Weil (1986). It was subsequently analyzed using a 1-way Anova followed by Tukey's Multiple-comparison Test (Byrkit, 1987). Untransformed cyto-
52
toxicity data from the MN and SCE assays were analyzed by 1-way Anova and Tukey's Multiplecomparison test. Results CP was genotoxic in all assays, although the response in the SM assay was relatively weak. In the MN assay the top dose of CP increased the M N P C E incidence 10-fold and 42-fold above control levels in mice and rats, respectively (Table 1). The response was dose-, sampling time- and species-dependent ( p < 0.001). Although the threshold dose for CP was 2.5 m g / k g in both species, the magnitude of the responses were greater in the rat at higher doses ( p < 0.001). The mouse M N P C E incidence was highest at 30 h but no response was detected at 72 h. In the rat the timing of the maximal response varied in a dosedependent manner. At low doses the genotoxic effect of CP was greater at 30 h and as the dose was increased the maximal response was more delayed. Like the mouse, a significant effect was not detectable at 72 h. The P C E / N C E ratio was significantly ( p < 0.01) reduced by CP in both species (Table 2), the effect of which differed significantly between species ( p < 0.01) and time of sampling ( p < 0.01). CP was cytotoxic to rat bone marrow down to 5 m g / k g at all time points,
while in mice cytotoxicity was observed down to 10 m g / k g after 72 h. CP also produced a dose- and sampling timedependent ( p < 0.001) increase in the proportion of abnormal sperm in both species (Table 3), where the greatest effect was observed for sperm at 5 weeks. There was also a significant species difference in response to CP ( p < 0.001), where at 5 weeks, 40 m g / k g / d a y of CP induced a 12-fold and 5-fold increase in the percentage of abnormal sperm in rats and mice, respectively. The rat was also more sensitive than the mouse to CP-induced systemic toxicity, with only 3/15 rats and 14/15 mice surviving at 40 m g / k g / d a y . In the SCE assay CP significantly increased the SCE frequency ( p < 0.001) in a dose-related fashion in both species (Table 4). Like the MN and SM assays, the rat was more sensitive than the mouse at comparable doses, where the limit of detection for a genotoxic response was 1.25 m g / k g in mice and 0.63 m g / k g in rats. The ability of splenocytes to divide in culture (RI) was not compromised by in vivo exposure to CP in either species (Table 5). ST produced a weak increase in the incidence of MNPCEs in treated mice (Table 1). The effect was significant ( p < 0.05) at the top dose, at 48 h only, a dose level lethal to 50% of the mice. Genotoxicity was not demonstrated in the rat in
TABLE 1 EFFECT OF CP AND ST ON THE F R E Q U E N C Y OF MNPCEs IN M O U S E AND RAT BONE M A R R O W Compound
CP
ST
Mouse
Rat
Dose
MNPCEs/1000 PCEs
(mg/kg)
30 h
48 h
1.2±0.2 (10) 1.9+0.3 (7) 3.0_+0.4 (7) 3.9_+0.4(10) 7.7±0.6(10) 12.1_+1.0(10)
1.2-+0.2(10) 1.1_+0.2(10) 1.0+_0.4 (7) 1.0+0.3 (7) 1.6±0.4 (7) 1.3-+0.4 (7) 1.9_+0.4 (9) 0.9_+0.4 (8) 3.4+_0.4(10) a 1.6+0.3 (8) 5.3-+0.3 (6) a 1.8-+0.5 (6)
0 1.25 2.5 5 10 20 0 150 300 450 600
1.4+_0.3(10) 2.4-+0.3(10) 2.5+_0.4(10) 1.5+_0.3(10) 2.3+_0.4 (9)
a a a a
1.3+_0.3 (5) 2.6+_0.5(10) 2.5-+0.5(10) 3.0+_0.4(10) 4.2+_1.0 (9) a
72 h
Dose
MNPCEs/1000 PCEs
(mg/kg)
30 h
48 h
72 h
1.3 _+0.2 0 1.3 ±0.2 (10) 1.2_+0.2(10) 1.2_+0.4 1.25 2.2+_0.5 (6) 1.2+0.5 (6) 1.3+0.3 2.5 3.3-+0.8 (6) a 2.3+0.3 (6) 5 10.9±1.3(10)" 4.0+1.1 (5) ~' 1.8_+0.3 10 17.5±1.3 (4) ~' 18.8+2.1 (5) a 2.8±0.5 20 21.1_+2.5 (7) a 50.1+_7.2 (7) a 2.0+0.4
(10) (5) (6) (4) (4) (6)
1.2_+0.1 1.5±0.7 1.7-+0.6 O.9_+0.3 1.5 ±0.3
(5) (4) (7) (8) (4)
1.4-+0.2 (5) 0 1.4+_0.2(10) 300 1.1_+0.2(10) 750 1.0_+0.3 (9) 1500 0.6_+0.2 (5) 3000
The number of animals per treatment group are indicated in parentheses. a Indicates a significant difference from respective controls, p < 0.05.
1.3+_0.2 1.8-+0.5 1.9+_0.3 2.0+_0.5 1.8+_0.7
(8) (4) (7) (7) (5)
1.3+_0.2 2.0+_0.9 1.3+_0.6 1.6+_0.3 1.6+_0.2
(9) (4) (7) (7) (5)
0 150 300 450 600
0 1.25 2.5 5 10 20
0.58+0.04(10) 0.67-1-0.07(10) 0.66 _+0.04 (10) 0.69_+0.04(10) 0.73_+0.11 (9)
1.00+0.01 (10) 1.06 _+0.03 (7) 1.09 + 0.04 (7) 1.11_+0.03 (10) 1.07_+0.10 (10) 1.06+0.07(10)
P C E / N C E ratio
30 h
Dose
(mg/kg)
Mouse
(10) (7) (7) (9) (10) (6)
0.96_+0.06 (5) 0.78-+0.07(10) 0.89 -+ 0.06 (10) 0.63+0.09(10) 0.69_+0.08 (9)
1.00_+0.01 1.05 _+0.04 1.09 _+0.07 1.03+0.05 0.90_+0.07 0.81+0.10
48 h
a
1.07_+ 0.07 0.89_+0.05 1.04 _+0.04 0.93 + 0.04 0.89_+ 0.08
1.00_+0.01 1.00 _+0.05 1.00+0.05 0.87 + 0.05 0.86_+ 0.05 0.54_+ 0.06
72 h
The n u m b e r of animals per treatment group are indicated in parentheses. a Indicates a significant difference from respective controls, p < 0.05.
ST
CP
Compound
(5) (10) (10) (9) (5)
(10) (7) (7) (8) (8) a (6) a 0 300 750 1500 3 000
0 1.25 2.5 5 10 20
(mg/kg)
Dose
Rat
0.04 0.04 0.05 0.02 0.03 0.04
(10) (6) (6) (10) a (4) a (7)a
0.72_+ 0.61 _+ 0.48_+ 0.42 _+
0.03 0.08 0.03 0.09
(4) (7) (7) (5) a
0.70_+ 0.05 (8)
0.61_+ 0.39 -+ 0.47 _+ 0.39_+ 0.32+ 0.27_+
30 h
P C E / N C E ratio
E F F E C T O F CP A N D ST O N T H E R A T I O O F PCEs T O NCEs IN M O U S E A N D R A T B O N E M A R R O W
TABLE 2
0.70_+ 0.53_+ 0.75 _+ 0.63 + 0.51+
0.60+ 0.50_+ 0.55-+ 0.24-+ 0.19_+ 0.11_+
48 h
0.05 0.08 0.07 0.09 0.13
(9) (4) (7) (7) (5)
0.04(10) 0.12 (6) 0.12 (6) 0.15 (5) a 0.01 (5) a 0.02 (7) a
0.70_+ 0.74+ 0.37+ 0.42_+ 0.22_+
0.61 _+ 0.55 _+ 0.52+ 0.35+ 0.26 + 0.19_+
72 h
0.04 0.05 0.05 0.09 0.03
0.03 0.03 0.05 0.01 0.02 0.04
(5) (4) (7) a (8) a (4) a
(10) (5) (6) (4) a (4) a (6) a
54 TABLE 3 E F F E C T O F CP A N D ST O N T H E P E R C E N T A G E O F A B N O R M A L S P E R M IN T H E M O U S E A N D R A T Compound
Mouse
Rat
Dose
% Abnormal sperm
(mg/kg/d)
Dose
% Abnormal sperm
(mg/kg/d)
5 wk
8 wk
11 wk
1.8±0.4(5) 1.4±0.1(5) 3.3±0.1(5) 3.4±0.2(4) NS
1.6±0.3(5) 1.5±0.2(5) 2.7±0.4(5) 18.4±4.1(5) 19.8±4.4(3)
1.9±0.2(5) 2.2±0.2(5) 4.6±0.6(5) 7.6+1.1(5) NS
1.7±0.1(5) 1.9±0.1(5) 2.5±0.1(4) 2.7±0.3(5) 2.4(1)
1.6±0.1(5) 1.8±0.2(5) 1.9±0.l(5) 1.7+0.2(5) NS
3 wk
5 wk
7 wk
CP
0 5 10 20 40
3.5±0.6(5) 2.6±0.4(4) 5.7±1.9(4) 6.6±1.6(5) 6.0±1.1(4)
3.8±0.3(5) 7.0±2.2(5) 4.8±0.7(5) 7.6±2.4(5) 19.2+3.5(5)
3.7±0.4(5) 5.6±2.3(4) 4.8±1.2(5) 10.5±0.7(5) 13.1±3.4(4)
ST
0 50 100 200 400
4.1±1.0(5) 4.3±0.9(5) 5.3±0.6(4) 6.5±1.3(5) 6.9±1.4(7)
4.0±0.8(5) 5.7±1.3(5) 0 7.8±1.5(5) 8.6±1.9(5) 250 7.2±0.9(5) 17.8±6.6(5) 500 7.7±1.3(5) 20.5±4.7(5) a 10~ 11.2±3.0(8) ~ 15.3±1.1(7) a 2000
0 5 10 20 40
1.7±0.1(5) 2.1±0.2(5) 2.2±0.2(5) 2.6±0.3(5) a NS
The number of animals per treatment group are indicated in parentheses. a Indicates a significant difference from respective controls, p < 0.05. NS, No survivors.
this assay, even at a dose level (3000 m g / k g ) lethal to 40% of rats. ST was not cytotoxic to the haemopoietic tissues in the mouse, although it did decrease the P C E / N C E ratio in the rat (Table 2). The effects in mice were complicated by a possible vehicle effect. In the SM assay 400 and 1000 m g / k g / d a y of ST produced a small, but significant increase ( p < 0.001) in the proportion of abnormal sperm
in mice (2.8-fold) and rats (1.6-fold), respectively (Table 3). Like the MN assay, the mouse appeared to be more sensitive than the rat. The top dose of ST was lethal to 90% of treated rats. In the SCE assay ST produced a weak but significant ( p < 0.001) genotoxic effect at the top dose of 450 m g / k g (Table 4), which was lethal to one of five treated mice. ST significantly increased the SCE frequency ( p < 0.001) in splenocytes of
TABLE 4 E F F E C T O F CP A N D ST ON T H E SCE F R E Q U E N C Y IN T H E M O U S E A N D R A T Compound
Mouse Dose (mg/kg)
CP
ST
0 0.62 1.25 2.5 5 0 75 150 300 450
Rat SCEs/chrom 0.24 ± 0.01 (5) 0.25 +_0.01 (5) 0.33 ± 0.01 (5) a 0.53 +_0.01 (5) ~ 0.85 ± 0.03 (5) a 0.23 ± 0.23 ± 0.23 ± 0.23 ± 0.27 ±
0.02 0.01 0.01 0.02 0.02
(5) (5) (5) (5) (4) ~
The number of animals per treatment group are indicated in parentheses. a Indicates significant difference from respective controls, p < 0.05.
Dose ( m g / k g ) 0 0.31 0.62 1.25 2.5 0 375 750 1500 3 000
SCEs/chrom 0.20 _+0.01 0.21 + 0.01 0.32+0.01 0.49 ___0.04 0.79 + 0.04
(5) (5) (5) a (5) a (5) a
0.21 + 0.01 0.21 ± 0.02 0.28 + 0.03 0.45 ± 0.04 0.43 (1)
(5) (5) (5) a (5) a
55 TABLE 5 EFFECT OF CP AND ST ON THE REPLICATIVE INDEX OF CULTURED SPLENOCYTES FROM TREATED MICE AND RATS Compound CP
Mouse Dose (mg/kg) 0 0.62 1.25
2.5 5 ST
0 75 150 300 450
Replicative index 2.04 + 0.04 (5) 1.96+0.04 (5) 2.02 + 0.04 (5) 1.97 + 0.06 (5) 2.04 + 0.03 (5)
Rat Dose (mg/kg) 0 0.31 0.62 1.25 2.5
Replicative index 2.45 + 0.09 (5) 2.37+0.02 (5) 2.40 + 0.07 (5) 2.20 + 0.09 (5) 2.20 + 0.03 (5)
2.00 + 0.03 (5) 2.00+0.01 (5) 1.98+0.02 (5) 1.99 ± 0.04 (5) 2.065:0.03 (4)
0 375 750 1500 3000
2.37 + 0.07 (5) 2.37+0.08 (5) 2.305:0.11 (5) 2.32 5:0.03 (5) 2.54 (1)
The number of animals per treatment group are indicated in parentheses. treated rats down to 750 m g / k g . ST was not cytotoxic to rat- or mouse-derived splenocytes (Table 5). Discussion
CP-induced genotoxic effects in the MN assay in both species were quantitatively similar, at comparable doses, to some previously reported data (Madle et al., 1986b), where Wistar rats were shown to be more sensitive than N M R I mice. The longer plasma half-life of alkylating CP metabolites in rats compared to mice may account for the greater sensitivity of the rat (Torkelson et al., 1974; Garattini et al., 1974). Furthermore, Madle et al., (1986b) using an alternative target tissue for the SCE assay, also found it to be more sensitive than the MN assay at comparable doses. CD1 mice used by Krishna et al. (1986, 1987) to assess CP-induced SCEs in bone marrow and splenocytes appear to be less sensitive than the N M R I mice used by Madle et al. (1986b) and the L A C A Swiss mice used in this study. Likewise, a study by Trzos et al. (1978) using S p r a g u e - D a w l e y rats found that 20 m g / k g of CP induced a 7-fold increase in bone marrow micronuclei at 24 h, while 5 m g / k g of CP was able to induce a similar response in the Porton rats at 30 h. The importance of multiple sampling for the MN assay was demonstrated in the rat, where at
10 and 20 m g / k g the responses were higher at 48 h than at 30 h. This was due to the cytotoxic properties observed for CP in the rat. The marked inhibition of haemopoiesis (Table 2) can delay the progression of the D N A - d a m a g e d erythroblasts to the P C E stage and hence delay the appearance of MNPCEs (Salamone and Heddle, 1983). This effect of CP in the rat has previously been reported by other investigators (Madle et al., 1986b; Goetz et al., 1975). A similar delay in the response to CP at higher doses was not observed in mice, consistent with the relative lack of CP-induced cytotoxicity (Table 2). C o m p a r e d to the MN and SCE assays, the SM assay was insensitive. CP was only able to produce an effect at lethal or near-lethal doses in both species. Similar results with respect to mice, were also observed by Wyrobek and Bruce (1975). They found the primary spermatocyte stage of spermatogenesis in (C57 × C3H)F~ mice to be the most sensitive to CP exposure, observing approximately a 4-fold increase in the proportion of abnormal sperm at 50 m g / k g / d a y , compared to the 5-fold increase at 40 m g / k g / d a y in the L A C A Swiss mice. The weak genotoxic response to ST in the M N assay in the mouse is consistent with the observation of N o r p p a (1981), who also found weak positive responses in C 5 7 B L / 6 mice at 250 and 1000 m g / k g . In contrast, Pentilla et al. (1980) did not detect an effect of ST up to 1 g / k g in the
56 same assay in Chinese hamsters. In the SM test, the increase in abnormal sperm by ST is inconsistent with the report of no response in (C57 × C3H)F 1 mice at doses up to 700 m g / k g / d a y at 3 and 5 weeks after dosing (Salomaa et al., 1985). This may be due to strain differences in ST metabolism, as Cantoni et al., (1978) have reported marked diferrences in the ability of different species to activate and deactivate styrene in a number of tissues. Like the MN and SM assays, ST was able to induce a weak response in mice, in the SCE assay, at near lethal doses. These results agree with the observations of Conner et al., (1979), who reported a significant increase in SCE frequency in bone marrow cells of BDFI mice exposed to ST by inhalation (565 _+ 15.8 p.p.m.; 6 h / d a y ; 4 days). On the other hand, Shareif et al. (1986) failed to detect a significant increase in SCE frequency in the bone marrow of C 5 7 B 1 / 6 mice given a single i.p. dose of up to 1 g / k g of ST. A combination of the use of different mouse strains, routes of administration and target tissues may explain differences between our results and those of the other two laboratories. In contrast to the response in mice, a relatively large response was observed in the SCE assay for rats at doses where no detectable effect was observed in the MN assay. This is similar to CP, where SCEs were found to be a more sensitive endpoint than micronuclei. Although the responses to ST were weak, it was consistently positive in all assays in the mouse and two of three assays in the rat. Therefore, it has the potential to induce similar genetic damage and possibly tumours in humans. This is supported by other evidence such as; (1) the detection of S T - D N A adducts in the liver, brain, lungs, spleen and testes of exposed mice (Nordqvist et al., 1985), (2) increased frequency of lymphocyte micronuclei in workers exposed to low level ST (Hogstedt et al., 1983) and (3) the ability of styrene oxide, the putative toxic metabolite of ST, to induce tumours in rats and mice (Lijinsky, 1986; Ponomarkov et al., 1984). In the SM assay near lethal doses of both CP and ST were required to elicit a response. This may be due to the presence of the blood-testis barrier, which can restrict the access of foreign
chemicals to the germ cells (Okumura et al., 1975), or the relatively high activity of detoxifying enzymes present in germ cells (Mukhtar et al., 1978). In view of the susceptibility of the frequency of abnormal sperm to various non-mutagenic factors determined by the health of the animal (Komatsu et al., 1982; Cairnie and Leach, 1980; Topham, 1983), it is conceivable that the responses may have been an indirect result of systemic toxicity induced by the large doses of CP and ST, rather than a true genotoxic effect. The results of CP and ST in this assay should, therefore, be interpreted with care. O f the three assays the SCE assay was the most sensitive to the two genotoxins used in this study. This may be due to differences in the ability of the tissues to locally activate a n d / o r deactivate toxic metabolites or differences in the ability of the tissues to repair D N A damage. Alternatively, this may reflect differences in the potential of the two compounds to induce SCEs relative to micronuclei and abnormal sperm, as each may involve different molecular mechanisms. The low baseline levels of micronuclei, SCEs and abnormal sperm in the Porton rats and L A C A Swiss mice demonstrates the potential usefulness of these strains in in vivo genotoxicity testing. The qualitatively similar results for the reference genotoxin, CP, in this study and that of others, further illustrates this point. The results for ST in the SM and SCE assays in mice were not in complete agreement with other reports. This may be due to the use of alternative strains of mice. The response to ST in the rat indicates that the choice endpoint a n d / o r target tissue are important determinants in the ability to detect a compound's genotoxic response. Therefore, a negative result for a compound in an in vivo assay should be verified by either examining alternative endpoints or the same endpoint in a different species or strains of animal before a firm conclusion can be made on a compound's genotoxic potential.
Acknowledgements We would like to thank Dr. A. Verbyla for his advice and assistance in the statistical analysis of
57
the data, and R. Irvine, J. Edwards, P. Wright and G. Crabb for their technical assistance.
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