Mutagenicity studies with the mouse spot test

Mutatwn Research, 117 (1983) 201-212

201

Elsewer Biomedical Press

Mutagenicity studies with the mouse spot test E. Gocke *, D. Wild, K. Eckhardt ** and M.-T. King Zentrallabor fur Mutagemtdtsprafung der Deutschen Forschungsgememschaft, Brelsacher Str 33, D- 7800 Fretburg / Brelsgau (Federal R epubhc of Germany)

(Received 29 March 1982) (Revision recewed 7 September 1982) (Accepted 10 September 1982)

Summary The mammalian spot test, which detects somatic gene mutations in mouse embryos, was investigated with selected chemicals to (a) further validate this test system (ENU, EMS, 2AAF, colchicine) and (b) evaluate the mutagenic potential, in a whole-mammal system, of environmental compounds that had been previously recognized as mutagens in other mammalian or submammalian test systems (1,2-dichloroethane, hydroquinone, nitrofurantoin, o-phenylenediamine, fried sausage extract). Of these substances, ENU, EMS and 2AAF were significantly mutagenic, 1,2-dichloroethane was probably weakly mutagenic. The E N U data were used to estimate the number of pigment precursor cells present at the time of treatment (day 9.25). We also describe in this report the use of a fluorescence microscope for classification of hairs from spots on the coat of C 5 7 B L / 6 J H a n × T hybrids.

In recent years, mutation assays in vitro have been developed and validated with much success. Nevertheless, mammalian methods in vivo seem to be necessary in later stages of evaluation of the mutagenic potential of environmental compounds. The mammalian spot test, which detects somatic gene mutations in the mouse, was introduced more than 25 years ago (Russell and Major, 1957) for X-ray studies. More recently it has been used by a number of laboratories (Fahrig, 1975, 1977;

* Present address: Institute of Molecular Biology, University of Aarhus, 8000 Aarhus (Denmark). ** Present address: Hoffmann-La Roche and Co., Basel (Swttzerland). Abbrematwns: ENU, ethylnitrosourea; EMS, ethyl methanesulfonate; 2-AAF, 2-acetylarmnofluorene;

oPDA, o-phenylenedlarmne; HBSS, Hanks balanced salt solution. 0165-1218/83/0000-0000/$03.00 © 1983 Elsewer Science Pubhshers

202 Russell, 1977; Davidson and Dawson, 1977; Lang, 1978; Knudsen, 1980; Peter, 1980; Neuh~iuser-Klaus, 1981; Braun et al., 1982) for studies on chemical mutagenesis. This system has now progressed reasonably far into the stage of validation. (For review see Russell et al., 1981.) Other methods, possibly useful as screening systems for somatic gene mutations induced in vivo (Maier et al., 1978; Strauss and Albertini, 1979; Dean and Hodson-Walker, 1979), are still in the developmental stage. We used the spot test for further testing of compounds that had shown genotoxic activity in our screening programs with the Salmonella/microsome assay (Ames assay), the Drosophila sex-linked recessive lethal test, and the micronucleus test: hydroquinone, oPDA, nitrofurantoin, 1,2-dichloroethane and basic extract of fried meat. Furthermore, we tested ENU, EMS, 2-AAF and colchicine for validation purposes.

Materials and methods

Ammab. C 5 7 B L / 6 J H a n female mice were obtained from the Zentralinstitut fiar Versuchstiere, Hannover (F.R.G.). The T-stock ( a / a , b / b , cchp/cchp, d s e / d se, s / s ) was kindly supplied by U.H. Ehling, Neuherberg (F.R.G.), and is rotation bred at our institute. Chemtcals. ENU, EMS and nitrofurantom were from Serva Feinbiochemica, Heidelberg (F.R.G.); 2-AAF was from Merck-Schuchardt, Hohenbrunn (F.R.G.), 1,2-dichloroethane from Merck, Darmstadt (F.R.G.); hydroquinone from EGA Chemic, Steinheim (F.R.G.); colchicine from Roth AG, Karlsruhe (F.R.G.); oPDA from Fluka AG, Buchs (Switzerland). Sausage extract was prepared from heavily fried sausages as described (Gocke et al., 1982). Experimental procedure. C57BL vlrgm female mace 2-3 months old were caged with T-stock males m the late afternoon. Examination for vaginal plugs was done the following 3 mornings. Substances dissolved in appropriate vehicles were injected intraperitoneally on the morning of the 10th day of pregnancy. The day of the plug was designated as the first day. Our '10th day' thus corresponds to Fahrig's 'day 11' and to Russell's 'day 9.25'. Usually, all females that had shown plugs were treated. With sausage extract, owing to scarcity of material, only those mice were treated that were relatively heavy ( > 23 g on day of treatment) and showed a hint of increased girth. Examination for spots was performed 1-2 times per week from weeks 2 to 4. Animals with spots were caged separately at weaning. Hairs from the spot and adjacent (or symmetric) location were plucked, placed on a microscope slide and embedded in eukltt under a cover glass. For record keeping the pelt of the whole animal was prepared. Classification of spots is discussed below. Statistics. For statisti~cal evaluation of test results, Fisher's exact test was used. The multiple decision procedure described by Selby and Olson (1981) was also followed (see also Russell et al., 1981).

203 Results and discussion

Spot classification In the first report on the somatic coat-color mutation test (Russell and Major, 1957) It was realized that not all spots can be ascribed to mutational events in melanocyte precursor cells, because some occurred even in the absence of heterozygosity at marked loci, i.e., in offspring of C57BL × C57BL crosses. Spots not due to forward mutation-were ascribed to melanocyte killing (white midventral spots) or misdifferentiation (spots associated with areas that are normally agouti-like, e.g., nipple. Classification of spots is still a major point of discussion and divergence. (See review by Russell et al., 1981.) Microscopical examination of hairs from spots should be helpful. Fahrig (1978) introduced the use of a fluorescence microscope for evaluation but did not give a detailed description of techniques and characteristics of hairs from different types of spots. In the following, we describe our method of spot classification. A Zeiss transmitting fluorescence microscope is used with the excitation filters BG 38 and BG 3 which together allow transmission of light between 300 and 500 nm. The barrier filter (U 50) transmits wavelengths >/500 nm. The intensity of residual light is sufficient to allow examination of the pigmentation of the hairs. The medullary air spaces, which appear dark and complicate the examination under the normal light microscope, hardly show up under these conditions. Magnifications between 50 x and 800 x are used. Fig. 1 shows a drawing of hairs from representative spots. (,4) Relevant spots, i.e., those that are presumably caused by mutations at the heterozygous coat-color loci. The hairs show reduced numbers of pigment granules, different distribution of pigment granules (clumping), or morphologically different granules (smaller size). These spots can occur anywhere on the fur of the animal. Because ventral hairs usually have less pigment than dorsal hairs, the pigmentation of the left hair would be within the normal pigmentation range if taken from the belly. We do not attempt to ascribe any spot to mutation at a certain specific locus. (B) Mzsdifferentiation spots. Agouti-type hairs have a terminal or subterminal yellow band (e.g. Searle, 1968), which under the fluorescence microscope is very prominent owing to its bright yellow fluorescence. In the fluorescing area, no, or only few, dark pigment granules can be seen. This type of hair is normally present in C57BL animals and C57BL x T hybrids in the area of the female nipples, genitals of both sexes, and bases of ears. Some hairs on the throat and guard hairs at the flanks of the body also have this fluorescing band. Spots containing an increased number of these hairs occur at typical locations (~hown in Fig. 2): at the inner sides of legs, close to, but not always in connection with, nipples (also in males), on the forehead, and behind the eyes. Such spots are found in C57BL animals, as well as in offspring of the C57BL × T cross. Grossly, their color varies between grayish and beige. Since the frequency of these spots is not significantly increased even after ENU treatment (see below), we suggest that they are not the result of (dominant) mutations. They have been described by Russell (1978) and termed misdifferentiation spots, but no data on their frequency were given. These spots might be misclassified as relevant

204

F~g 1 Drawmgof hairs from representattve spots For d~scuss~onsee text

spots, if examination is done only by the naked eye; therefore, we consider it helpful to include this category in publications for reasons of inter-laboratory comparison. (C) White spots. Hairs completely devoid of pigment granules are relatively frequently encountered in white spots at or close to the midventral line. Different C57BL sub-lines, on being crossed to T, yield these spots at different frequencies (Russell, 1977). The spots are supposedly caused by insufficiency of melanocyte precursor cells. Elevated frequencies are often found among treatment groups

205

Fig. 2. Locatmns of typtcal spots containing agoutl-type hmrs. The sex-symbols indicate whether the spot was found on a male or female.

containing appreciable numbers of malformed animals, suggesting cell toxicity as the underlying mechanism (Russell, 1978). White 'Stichelhaar' (only few dispersed white hairs) are often found at the navel area and might alternatively be the result of small wounds occurring when the umbilical cord was bitten off. We also observed a few small white spots (with hairs completely empty of pigment) on the backside of animals close to the base of the tail, where biting wounds are sometimes encountered. Similar observations were reported by Knudsen (1980). We also found such a spot on a C57BL female. White hairs are also found in transplantation experiments around transplanted skin areas (Harnasch, personal communication). Thus, we suggest that some of these spots are the result of skin damage leading to loss of functioning melanocytes, although we are aware that dominant mutations or (in heterozygotes) partial or total chromatid or chromosome loss or somatic recombination might produce white or near-white spots (see also the colchicine data). During the course of this work, we observed two other types of coat-color change in F I hybrids. One was probably a black-and-tan whole-body mutation (mouse with yellowish hairs of agouti type covering the belly, black hairs on the back). This was a heritable mutation, presumably of spontaneous origin. The other type was a l~rownish spotting of the whole body of two animals observed in different experiments. The hair was .not of agouti type, and the pattern was not heritable. Neither type of aberration was counted as a spot.

Mutagenicity data Controls. In a recent survey of spot test data, Russell et al. (1981) noted that the spot incidence among corn-oil controls is higher than among HBSS or saline controls, and this is again higher than among untreated controls. The data were collected from different laboratories, and their relative contributions to each control group were not equal. Nevertheless, this shows that a vehicle control is necessary for each substance. Furthermore, the observation raises the question whether spots

206 might arise due to handling (e.g., physiological stress). Research into this aspect seems to be of utmost importance, since much of the credibility of spot-test data is at stake. Our control frequency (Table 1) of relevant spots is lower than that reported (Russell et al., 1981). Although the relative frequencies of spot incidence m our different control groups are s~milar to those reported, our data would not prohibit a cumulation of all our control results. For significance calculations, we compared test results with the cumulative and also the vehicle control. Owing to the lower numbers, the comparison with the vehicle control is of course not as powerful. Whether our lower control frequency was caused by differences in spot classification or strain differences cannot be answered. The incidence of white ventral spots was also highest in the olive-oil control, but no such tendency was found for the misdifferentiation spots. Our data obtained with test compounds are shown in Table 1. E N U is the most potent mutagen observed so far in the specific-locus test (Russell et al., 1979) and in the spot test (Russell et al., 1981). Our data agree well with those reported by Russell and Montgomery (1982). A small group of C57BL animals treated with 58 m g / k g showed only white ventral spots, indicating that probably no dominant mutations were induced. To obtain information about the number of melanocyte precursor cells at the time of ENU treatment, an analysis of spot size was performed according to the calculations of Russell and Major (1957). In accord with Russell and Major's results, we found the smallest (40) and the highest number (2500) of prospective pigment cells per embryo to be separated by about 5-6 cell divisions. A majority of embryos has about 70 precursor cells (mode of the distribution), whereas the average number of precursor cells per embryo is estimated to be about 130. As might be expected, these values are lower than those reported by Russell and Major (1957) (mode, 175-200 precursor cells per embryo) for C57BL × NB embryos of day 10.25, i.e., one day older than ours. By a different method, Fahrig (1978) estimated the majority of m u t a t e d embryos (not the average embryo!) to have about 200 precursor cells at the time of treatment. Embryos with higher numbers of precursor cells have a higher chance of receiving a spot. Thus, for calculating the cell number of the average embryo a 'correction' factor has to be used (see Russell and Major, 1957). Owing to unproven assumptions and inexact measurements these calculations can give only rough estimates. E M S has given rather variable results in the spot test in different laboratories (Russell et al., 1981). This has been attributed to different purity of the chemical purchased from different manufacturers. Our data are well within the range of observed results. 2 o A A F is mutagenic in many test systems (see Hollstein et al., 1979). Metabolic activation is necessary. The spot test result is highly significant against the cumulated control ( P < 0.001). Against the ohve-oil control the significance level is P = 0.02. We studied c o l c h w m e - - known as a spindle poison - - to test the hypothesis that spots can arise from loss of whole chromosomes. Because colchicme proved to be

207 rather toxic to the mother animals, only doses up to 1 m g / k g could be tested. But doses as low as 0.1 m g / k g of the very similar colcemide have been used (Kram et al., 1979) for metaphase arrest in SCE tests performed in embryos of days 11-15. A dose of colchicine at 0.4 m g / k g induces severe chromosome-non-disjunction in mouse oocytes (Turan, 1981) and a dose of 1.25 m g / k g severe micronucleus formation in mouse bone marrow (Tsuchimoto and Matter, 1979). These findings suggest that colchicine reaches the embryo, and that the doses we used would be suitable to cause irregular chromosome distribution in meiotic and mitotic cells. Thus it was somewhat surprising that no relevant spot was observed among 485 offspring. Furthermore, no white midventral spot was found, as would have been expected if induced hypodiploid (or hyperdiploid) melanocyte precursor cells had died. This might suggest that not enough colchicine had passed the placental barrier on the 10th day of pregnancy to cause chromosome misdistributions. So the hypothesis of spots due to chromosome loss could not be verified or disproved. 1,2oDzchloroethanehas been used as a topical antirheumatic. It is also used in the manufacture of vinyl chloride. In a previous test program (King et al., 1979), we found this substance highly mutagenic in the Drosophila sex-linked recessive lethal test, but inactive in the standard Ames assay and the micronucleus test. The mutagenicity in Drosophila had been reported earlier by Rapaport (1960). Rannug et al. (1978) observed mutagenic effects in Salmonella TA1535 under special conditions, which provided enzymic conjugation to glutathione. The spot test data show a significant ( P - - 0 . 0 3 ) effect against the cumulated control, but no significance ( P = 0.18) against the oil control. This result indicates probable mutagenic activity of dichloroethane in the mouse. Hydroquinone is used in hair dyes, for skin bleaching, and as a photographic developer. Several days after treatment of the females, we observed round gray spots on the backs of these animals. These were caused by lack of pigmentation at the base of the hairs, while granules remained at the tips. No such spots were found on the offspring, but an increase of white midventral spots suggested cellular toxicity of the compound. The frequency of relevant spots was slightly, but not significantly increased. Hydroquinone induces micronuclei in mouse bone marrow and is weakly mutagenic in Salmonella TA1535; no significant activity of hydroquinone was observed in Drosophila in the Basc test (Gocke et al., 1981). oPDA, used for hair coloring and for manufacture of dyes, is mutagenic in Salmonella TA 1538 in the presence of liver microsomes. It is also clastogenic in bone marrow of mouse, Chinese hamster and guinea pig (Wild et al., 1980). Drosophila did not show a response (unpublished). In the spot test this substance increased preand post-natal mortality at doses of 108 or 216 mg/kg. An increase of white ventral spots was observed, but there was no induction of relevant spots. Nitrofurantoin is used as an antibacterial in the treatment of urinary infections. Its mutagenicity in prokaryotes has been reported by Klemencic and Wang (1978). SCE induction in vitro has been reported by Shirai and Wang 0980). To our knowledge, no mutagenic effects in mammals in vivo have been observed (Goodman et al., 1977). Our results with the Ames test and the micronucleus test (unpublished) are in line with these reports. Nitrofurantoin did not show a significant effect in the spot test.

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210 Recent reports (Sugtmura and Nagao, 1979) about the exorbitant mutagenicity of amino acid pyrolysis products and the mutagenic principles of fried meat or fish prompted us to test the basic extract of fried sausages m some detail (Gocke et al., 1982). This extract was highly mutagenic in the Ames assay (TA98, TA1538) m the presence of $9. In Drosophila, the substance was reactive, and tins was true also m gene-mutation experiments with mammalian cells in vitro. In vivo, a comparatively weak effect in the intrasanguine host-mediated assay (with TA98) was observed. This effect was seen only when the mice had been pretreated with Aroclor. In the spot test, no significant increase of relevant spots was found after treatment with the basic extract at 500 mg/kg. Tests with one of the mutagenic principles of fried meat (Kasai et al., 1980) are in progress.

Conclusion

During the course of this work, we observed a total of 125 spots (excluding Stichelhaar). Of these, only 54 were deemed to be of mutational ongin. Whereas relevant spots constitute the majority of spots after treatment with strong mutagens, they become a minority for non-mutagens or weak mutagens. This indicates that correct classification of spots is of increasing importance with decreasing spot frequencies. If, for instance, the mutational spots were to be grouped together with an equal or higher number of unrecognized misdifferentiatlon spots, a weak mutagen which induces only a small increase in relevant spots (while the misdlfferentiation spot frequency stays constant) would yield significant results only with very large sample sizes, if at all. This could lead to false negatives. Conversely, misclassification of white ventral spots as relevant spots could lead to false positives. Because spot classification criteria may differ between laboratories, we recommend the use of the fluorescence microscope for standardization, as well as care in separating midventral white spots from relevant spots. For statistical evaluation of the test results, Russell et al. (1981) proposed to use the multiple-decision procedure developed by Selby and Olson (1981). For our data (Table 1), this procedure gave either positive results, as determined by Fisher's exact test (ENU, EMS, 2-AAF, dichloroethane), one negative result (colchicine), and several inconclusive results (all other substances). As already pointed out (Russell et al., 1981), the spot test is 'well suited for the identification of potent mutagens'. It is less suitable for detecting weak mutagens. With our control frequency it would be necessary to test more than 10000 animals to secure a doubling of the spontaneous relevant spot frequency. This illustrates that the best use of the spot test is to study selected groups of substances, for instance those that have shown mutagenic properties in previous screening assays, m order to determine whether they can produce forward mutations in a mammalian system in vivo. It has been postulated that several kinds of genetic alteration such as point mutations, deletions up to chromosome loss and somatic crossing-over can result in relevant spots (Russell, 1978). Our colchicine data did not support a contribution to

211 s p o t f o r m a t i o n o f n u m e r i c a l c h r o m o s o m e a b e r r a t i o n s , n o r c o u l d t h e y d i s p r o v e it. We found no clear increase of relevant spots to be caused by hydroquinone or oPDA, two agents that induce cytogenetic damage in mouse bone marrow. These substances did, however, increase the frequencies of white hairs (white midventral s p o t s a n d S t i c h e l h a a r ) . T h e r e f o r e , it a p p e a r s w a r r a n t e d t o s t u d y f u r t h e r w h e t h e r n u m e r i c a l a n d s t r u c t u r a l c h r o m o s o m e a n o m a l i e s c a n give rise t o c o l o r s p o t s .

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212 Rapoport, J A. (1960) Reactions of gemc proteins with 1,2-dlchloroethane, Dokl. Akad. Nauk S.S R , 134, 1214-1217. Russell, L.B. (1977) V a h d a u o n of the in vlvo somatic mutation method m the mouse as a prescreen for germinal point mutaUon, Arch. Toxlcol., 38, 75-85. Russell, L.B. (1978) Somauc cells as indicators of germinal mutations m the mouse, Environ. Health Persp, 24, 113-116 Russell, L.B. (1979) In wvo somatic mutation systems m the mouse, Genetics, 92, s153-s163 Russell, L B., and M.H Major (1957) Radlauon-mduced presumed somatic m u t a u o n s m the house mouse, Genetics, 42, 161-175 Russell, L B., and C.S Montgomery (1982) Supermutagemclty of ethylmtrosourea m the mouse spot test, Comparisons with methylmtrosourea and ethylnitrosourethane, M u t a u o n Res., 92, 193-204 Russell, L B., P B. Selby, E. von Halle, W. Sheridan and L. Valcowc (1981) Use of the mouse spot test m chenucal mutagenesls: interpretation of past data and recommendatmns for further work, M u t a u o n Res., 86, 355-379. Russell, W . L , E.M. Kelly, P.R Hunslcker, J.W Bangham, S.C Maddux and E.L. Phipps (1979) Specific-locus test shows ethylmtrosourea to be the most potent mutagen m the mouse, Proc Natl Acad. Scl. (U.S A.), 76, 5818-5819 Searle, A.G. (1978) C o m p a r a u v e Genetics of Coat Colour in Mammals, Logos, London, pp 55 ff Selby, P.B., and W.H. Olson (1981) Methods and criteria for deciding whether specd~c-locus mutation rate data m mice indicate a positive, negaUve, or inconclusive result, M u t a t m n Res., 83, 403-418 Shiraz, T., and C.Y. W a n g (1980) Enhancement of sister chromatld exchange m Chinese hamster ovary cells by nitrofurans, Mutation Res., 79, 345-350. Strauss, G . H , and R.J Albertml (1979) Enumeration of 6-thioguanme-reslstant peripheral blood lymphocytes m m a n as a potentml test for somatic cell m u t a t m n s arising m VlVO, M u t a u o n R e s , 61, 353-379. Sug~mura, T., and M. Nagao (1979) Mutagenic factors m cooked foods, C R C Crtt. Rev. Toracol, 189-209 Tsuchlmoto, T., and B.E. Matter (1979) In vlvo cytogenetic screening methods for mutagens, w~th specml reference to the micronucleus test, Arch. Toxlcol., 42, 239-248 Turan, Z., Doctoral thesis, Freiburg, 1981. Wild, D., M.-T. King and K Eckhardt (1980) Cytogenetic effect of ortho-phenylenedmrmnem the mouse, Chinese hamster and guinea pig and of denvatives, evaluated by the nucronucleus test, Arch. Toxacol, 43, 249-255