Mutagenesis, Vol. 14, No. 1, 141-151,
January 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Review |
Transgenic mouse mutation assay systems can play an important role in regulatory mutagenicity testing in vivo for the detection of site-of-contact mutagens
1 Covance Laboratories Ltd, Otley Road, Harrogate HG3 1PY, UK, 2 Glaxo Wellcome, Park Road, Ware SG12 0DJ, UK, 3 SRI International, Toxicology Laboratory, 333 Ravenswood Avenue, Menlo Park, CA 94025-3493, USA, 4 Covance Laboratories Inc., 9200 Leesburg Pike, Vienna, VA 22182, USA, 5 CIIT, Research Triangle Park, NC 27709, USA and 6 Rhone Poulenc Rorer, Alfortville, 14 94403 Vitry-sur-Seine Cedex, France
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Abstract |
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 Top Abstract IntroductionThe transgenic assayMethodsDiscussion of the dataConclusionReferences |
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Transgenic mouse mutation assays, such as MutaTMMouse (lacZ, CD2F1) and Big Blue® (lacI, B6C3F1), afford the opportunity to evaluate the mutagenic potential of chemicals in any target organ in vivo. This paper discusses published data collected from the analysis of the skin, stomach and lung DNA after topical, oral and inhalation exposure, respectively. These data indicate that both MutaTMMouse and Big Blue® should play an important part in the evaluation of genotoxicity in vivo, particularly where the endpoint or target tissue available in the more conventional tests is inappropriate. It is concluded that there is a distinct role for this type of assay in genetic toxicology testing. For substances applied to the skin or dosed orally or by inhalation and which are unlikely to reach either the bone marrow or the liver, then data derived from these assays may be more relevant to an assessment of possible risk to man than the currently used unscheduled DNA synthesis in liver and cytogenetics assays in bone marrow or peripheral blood.
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Introduction |
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TopAbstractIntroductionThe transgenic assayMethodsDiscussion of the dataConclusionReferences |
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In vivo genotoxicity tests are an important element of the regulatory test battery used in the assessment of cancer risk to man. They supplement the in vitro tests which are designed to detect the intrinsic genotoxicity of chemical agents. The advantages of the in vivo tests are that they take into account whole-animal processes such as absorption, tissue distribution, metabolism and excretion of chemicals and their metabolites. Conventionally, we use one or both of two well-validated types of in vivo mutagenicity tests: the mouse (or rat) bone marrow or peripheral blood micronucleus or bone marrow cytogenetics test and the rat liver unscheduled DNA synthesis (UDS) assay. In all in vivo tests it is essential that the target organ be exposed, but this is not always the case, since some compounds may be so reactive that they or their metabolites reach neither the liver nor the bone marrow. Only a limited number of tests allow the detection of genotoxicity in tissues exposed at the site of first contact. Both 32P-post-labelling of DNA and single cell gel electrophoresis (COMET) assays have been described in detail and are the subject of recent comprehensive reviews (Phillips, 1997
; Schmezer, 1997). However, amongst the most promising of the new generation of test systems are the transgenic rodent mutation models.
The use of transgenic rodent mutation assay systems based on the genes of the lac operon, MutaTMMouse (Gossen et al, 1989) and Big Blue® (Kohler et al, 1991a,b), in genetic toxicology has been the subject of review in recent papers (Ashby and Tinwell, 1994; Mirsalis et al., 1994, 1995; Tennant et al., 1994; Gorelick, 1995) and a report by Morrison and Ashby (1994b) gave an evaluation of the mutagenicity data generated by both the MutaTMMouse (lacZ, CD2F1) and Big Blue® (lacI, B6C3F1 or C57BL/6) assay systems to that time. Whilst we acknowledge that other models do exist, our aim in this report is to examine the published data relevant to the use of these transgenic mouse systems where a chemical might be a direct-acting or site-of-contact mutagen. We present an analysis of the relevant transgenic mouse data from the skin, stomach and lung following topical application, oral administration or inhalation exposure, respectively. These results are compared with those previously reported in the literature: genotoxicity measured in vitro, results obtained with the conventional in vivo mutagenicity tests and carcinogenicity data, where available. The purpose is to identify a useful role for this type of assay in genetic toxicology testing, especially in cases where the more conventional mouse bone marrow micronucleus or rat liver UDS assays may not be appropriate.
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The transgenic assay |
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TopAbstractIntroductionThe transgenic assayMethodsDiscussion of the dataConclusionReferences |
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MutaTMMouse is a transgenic mouse in which a vector prepared from bacteriophage
DNA (gt10) has been stably inserted into the genome and exists within every cell. Within the DNA sequence, the bacterial gene lacZ, which encodes for ß-galactosidase, was inserted at the single EcoRI restriction site. The entire vector contains ~47,000 bp, of which the lacZ gene comprises 3126 bp. The construction and analysis of MutaTMMouse have been described, as strain 40.6, by Gossen et al. (1989).
The transgenic mouse known as Big Blue® was developed using a 47.6 kb shuttle vector containing the bacterial lacI gene (1080 bp), which encodes the repressor protein of the lacZ gene. This vector is stably inserted into the genome and exists in every cell, as with MutaTMMouse. The Big Blue® system is more fully described by Kohler et al. (1991a,b).
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Methods |
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TopAbstractIntroductionThe transgenic assayMethodsDiscussion of the dataConclusionReferences |
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The studies described herein were performed for the investigation of mutation in skin, stomach or lung and the animals were treated by skin painting, orally (in the diet or by gavage) or by the inhalation routes, respectively. The animals were sacrificed after varying times of exposure. The appropriate organs were removed, flash frozen in liquid nitrogen and stored at –80°C until processed.
A mutation assay begins with dosing the animals in order to generate the mutations. Analysis of the mutations begins with the isolation of high molecular weight DNA from the tissue under investigation. This is usually performed by the phenol extraction method, although alternative DNA extraction kits are commercially available (Rogers et al., 1996). The tissue under investigation is first thawed, minced and homogenized, or simply finely diced, prior to the extraction of DNA. In the phenol extraction method, the tissue is digested with proteinase K, gently extracted with phenol:chloroform and the DNA is precipitated in ethanol and redissolved in Tris/EDTA buffer. This preparation is stored at 1–10°C until ready for use.
The next stage of mutation analysis involves excision of the lacZ or lacI genes from the mouse DNA and packaging into preheads by the use of commercially available packaging extracts. This step produces viable phage. The infectious particles are detected as plaques that form on a bacterial (Escherichia coli strain) lawn grown on an agar surface. In the case of MutaTMMouse a positive selection system is used which involves the scoring of clear plaques on titration plates to determine the total number of plaque forming units and on selection plates to determine the number of mutants (Gossen et al., 1992; Gossen and Vijg, 1993). For Big Blue®, a non-selectable colour screening assay is used, scoring blue mutant plaques among the non-mutant clear plaques (Kohler et al., 1991a). In each case the ratio of mutants to the total population (mutants plus non-mutants) gives the mutant frequency (MF).
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Discussion of the data |
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TopAbstractIntroductionThe transgenic assayMethodsDiscussion of the dataConclusionReferences |
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Data have been assembled for a total of 14 chemicals which have been investigated in either the lacI or lacZ systems (or both). The vast majority of the data is from the published literature.
Data on mutation in skin after skin painting or in stomach (forestomach and/or glandular stomach) following oral dosing or feeding in the diet are available for agaritine (mushroom extract), 2-amino-3,4-dimethylimidazo[4,5-f] quinoline (MeIQ), benzo[a]pyrene (BP), 1-chloromethylpyrene (CMP), 7,12-dimethylbenzanthracene (DMBA) and its sulphur analogue, 6,11-dimethylbenzo[b]naphtho[2,3-d]thiophene (S-DMBA), dimethylnitrosamine (DMN), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), 4-nitroquinoline-N-oxide (4NQO), ß-propiolactone and urethane. Data on mutation in lung tissue are available from inhalation studies with benzene and 1,3-butadiene.
For each compound in turn, the published data on mutagenicity in the more conventional mutation assays, both in vitro and in vivo, as well as data on rodent carcinogenicity, are discussed. The observations in transgenic gene mutation systems are then reviewed in the context of the reported mutagenicity and carcinogenicity data for the specific route of administration. The in vitro and in vivo mutagenicity of these compounds, their carcinogenicity and transgenic mouse assay results are summarized in Table I.
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In discussing the transgenic rodent mutation data we have considered all published reports even though, with current knowledge, the designs of some experiments, in retrospect, might have been further optimized. Where a weak positive is concluded, the author's interpretation has been accepted but it is acknowledged that small effects are always open to differing interpretations depending upon experimental design and the specific criteria applied.
Agaritine
Mutagenicity data on agaritine are limited. Agaritine is an aromatic hydrazine derivative found in relatively high concentrations in the mushroom. It is structurally related to known carcinogenic hydrazines and weakly Ames positive although the mutagenicity of mushroom extracts appears to be inconsistent: mutagenicity has been detected in TA100 but not TA98 (de Flora, 1981). Unfortunately, conventional in vivo mutagenicity data do not exist for agaritine so we do not yet know if it can induce micronuclei in the bone marrow or UDS in the liver. A cancer study in outbred Swiss albino mice, treated at up to 625 mg/ml in the drinking water for life, drew a negative conclusion (Toth et al., 1981a). However, derivatives of agaratine may be carcinogenic to mice (Toth et al., 1981b) and, in a more recent study, fresh mushrooms fed for 2 years induced tumours, including forestomach tumours, in mice (Toth and Erickson, 1986). Thus, carcinogenicity studies on mushrooms and agaritine are conflicting but they may indeed be a source of weakly genotoxic compounds.
The common mushroom, Agaricus bisporus, and crude agaritine extracted from mushrooms have been tested in the transgenic (lacI) mouse by Shephard et al. (1995). Fresh mushroom (30 mg agaritine/kg/day), dried mushroom feed (80 mg agaritine/kg/day) or agaritine feed (120 mg agaritine/kg/day) were fed to transgenic mice for 105 days. Forestomach DNA was examined for mutation in all treatments and it was concluded, based upon a 1.5-fold, statistically significant increase in MF, that a weak genotoxic effect was seen in the highest agaritine treatment (agaritine feed) only. Mutations were also seen in the kidney. DNA samples from the glandular stomach, liver and lung were also examined and were negative. Thus, the evidence suggests that agaratine is weakly carcinogenic in the mouse forestomach as well as a weak mutagen in the same location in transgenic mice.
2-Amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ)
MeIQ, a heterocyclic amine found in cooked foods, is mutagenic to bacteria and mammalian cells in vitro and there is evidence that it can also induce chromosomal damage in mammalian cells (de Meester, 1989; IARC, 1993). It is also capable of inducing UDS in rat and mouse hepatocytes treated in vitro (Yoshimi et al., 1988). A peripheral blood micronucleus test using mice receiving 300 p.p.m. (mg/kg) in the diet yielded a positive response after 8 weeks feeding, though all other times (1, 4 and 12 weeks) it was negative (Suzuki et al., 1996). MeIQ is a rodent carcinogen, inducing in mice forestomach tumours following dietary exposure (up to 400 mg/kg diet), with subsequent metastasis to the liver (Ohgaki et al., 1986). In the rat, tumours of the zymbal gland, oral cavity and skin are seen (300 mg/kg diet; Kato et al., 1989).
In lacI transgenic mice, MeIQ increases MF in the forestomach and liver after feeding 300 p.p.m. (mg/kg) in the diet for 12 weeks; no increases were seen after feeding for 1 or 4 weeks (Suzuki et al., 1996). Mutations were also seen in the colon and bone marrow on all three occasions but no increases in MF were seen in the heart. These observations correlate well with the mouse carcinogenicity data as primary tumours were restricted to the forestomach. One would not perhaps expect mutation in the liver if these tumours were derived from metastasis from the primary site.
Benzene
Benzene is not mutagenic in bacteria (Dean, 1978) nor does it induce gene mutation in mouse lymphoma L5178Y TK+/– cells (Lebowitz et al., 1979) or UDS in hepatocytes treated in vitro (Williams et al., 1985). However, it is clastogenic to mammalian cells in vitro, albeit using modified protocols (Ishidate et al., 1988; Scott et al., 1991) and induces chromosomal aberrations and micronuclei in rodent bone marrow following oral or i.p. dosing (Diaz et al., 1980; Hite et al., 1980; Meyne and Legator, 1980). Inhalation exposure of mice with doses of only 100 or 200 p.p.m. over 8 weeks was shown to induce micronuclei both in the bone marrow and in peripheral blood (Farris et al., 1996). Inhalation exposure of mice to benzene shows limited evidence of increased incidence of lymphoid tumours (Snyder et al., 1980). Furthermore, exposure to 300 p.p.m. benzene for 6 h/day, 5 days/week for 16 weeks saw a marked increase in the incidence of malignant lymphoma, lung adenoma and preputial gland tumours (Farris et al., 1993). Oral exposure of p53 knockout mice also yields tumours at multiple sites, consistent with the observations following a conventional tumour bioassay (Tennant et al., 1996). The use of benzene as a vehicle and negative control in skin painting studies has provided a large amount of data which suggests that it is not a skin carcinogen in rodents (IARC, 1982, 1987a). This is in marked contrast to the results of a skin painting study in TG.AC transgenic mice (carrying a mutant v-Ha-ras gene) in which benzene was clearly positive for the induction of skin papillomas (Tennant et al., 1996). Benzene is certainly clastogenic in man and is regarded as a human carcinogen (IARC, 1982, 1987a).
In lacI transgenic mice, it has been demonstrated that there is a small but significant increase in MF in DNA from the lung after inhalation for 6 h/day, 5 days/week for 12 weeks to a target dose of 300 p.p.m. (Mullin et al., 1995), perhaps reflecting the induction of lung adenomas by a similar regime (Farris et al., 1993). There was also a small increase in MF in the spleen and, in view of the increased incidence of malignant lymphomas and the clastogenicity of benzene in the bone marrow, one might also expect to see an increase in mutation in bone marrow, although this tissue was not analysed in the study described. The organ specificity of mutation induction might be clarified if negative results were obtained for tissues, such as liver, in which tumours have not been observed.
Benzo[a]pyrene (BP)
BP is mutagenic in most bacterial and mammalian systems in vitro and is also an in vivo clastogen (Holstein and McCann, 1979; de Serres and Ashby, 1981; Ishidate et al., 1988). Although BP does induce UDS in rodent hepatocytes in vitro (Lonati-Galligani et al., 1983; Harbach et al., 1989), it does not do so in vivo in liver following oral dosing in either rats or mice (Mirsalis, 1988). However, there is evidence of the induction of UDS in the glandular stomach of rats following oral dosing (Furihata and Matsushima, 1982). In mouse carcinogenicity studies, oral dosing yields forestomach tumours although leukaemias and lung adenomas were also seen when mice were fed BP in the diet (Rigdon et al., 1969; Wattenberg and Leong, 1970). Topical treatment of mice leads to skin papillomas and carcinomas (IARC, 1973). Thus, BP is clearly a mouse carcinogen which tends to produce tumours at the site of contact. The human data are inadequate to determine carcinogenicity to man (IARC, 1973).
In a lacZ study in which mice were exposed to a single topical dose (25 or 50 µg) or multiple topical doses (5x5 µg or 5x10 µg), Dean et al. (1998) reported marked increases in MF in skin for all treatments at both 7 and 21 days after the final dose. In the same animals, there was no obvious increase in mutation in either the liver or the lung at 21 days.
The data for BP would perhaps be typical for a reactive, site-of-contact mutagen. Following oral dosing, tumours are seen in the forestomach and lung and leukaemias were also noted. In the transgenic model, oral dosing yielded UDS in the forestomach. The absence of mutation, UDS or tumours in the liver (particularly when BP does induce UDS in vitro) is further evidence that these endpoints are tissue-specific. There was no sign of increased mutation in the bone marrow, although leukaemias were seen and micronuclei induced after i.p. dosing and mutations were evident in the spleen in lacI mice after i.p. treatment (Kohler et al., 1991a).
1,3-Butadiene
The mutagenicity of 1,3-butadiene was reviewed by de Meester (1988) and Jackobson-Kram and Rosenthal (1995). It is mutagenic to bacteria and there is some evidence of weak induction of sister chromatid exchanges (SCE) in cultured CHO cells (de Meester, 1988). 1,3-Butadiene failed to induce gene mutation in mouse lymphoma cells in vitro according to McGregor et al. (1991), but a positive response was reported earlier by Sernau et al. (1986). In vivo, 1,3-butadiene induces chromosomal aberrations, micronuclei and SCEs in bone marrow or peripheral blood erythrocytes following inhalation exposure in mice but not in rats (Jacobson-Kram and Rosenthal, 1995). 1,3-Butadiene is bioactivated to at least two potentially genotoxic metabolites, 1,2-epoxybutene and 1,2,3,4-diepoxybutane; following inhalation exposure, blood levels of these metabolites are higher in mice than in rats, consistent with the increased susceptibility of the mouse to micronucleus induction by 1,3-butadiene (Himmelstein et al., 1997). However, in vivo UDS assays in both species were negative and 1,3-butadiene was also negative for UDS in rat hepatocytes exposed in vitro (Arce et al., 1990). There is evidence that it can cause germ cell mutations in exposed mice and rats (Adler and Anderson, 1994; Adler et al., 1994, 1995; Jacobson-Kram and Rosenthal, 1995). In mouse cancer studies, 1,3-butadiene induces lymphomas, haemangiosarcomas of the heart and lung adenocarcinomas, with some hepatocellular neoplasms. The rat is far less susceptible to 1,3-butadiene-induced carcinogenicity (Melnick et al., 1993), again consistent with what is known about its metabolism in rats and mice (Himmelstein et al., 1997).
Mutagenicity experiments in transgenic mice have shown that exposure to 1,3-butadiene by inhalation at levels that induce tumours in B6C3F1 mice induces in vivo mutation in multiple tissues (Recio et al., 1992, 1996, 1997; Sisk et al., 1994). In lacZ transgenic mice, exposure to 625 p.p.m. 1,3-butadiene (6 h/day for 5 days) induced a significant increase in the lung lacZ mutant frequency (Recio et al., 1992). In B6C3F1 lacI transgenic mice exposed to 62.5, 625 and 1250 p.p.m. 1,3-butadiene (6 h/day, 5 days/week for 4 weeks), significant increases in the lacI mutant frequency in the bone marrow and in the spleen were observed at all exposure levels (Sisk et al., 1994; Recio et al., 1997). A significant increase in the lacI mutant frequency also occurs in the bone marrow of lacI transgenic mice following a 5 day exposure to 625 p.p.m. 1,3-butadiene (Recio et al., 1996).
These experiments show that inhalation exposure to 1,3-butadiene induces mutation in tissues of transgenic mice (lung, bone marrow and spleen) that are known to be sites of tumour induction in chronic bioassays (lung tumours and lymphomas). 1,3-Butadiene induces mutation at exposure levels that induce tumours, however, significant mutagenicity from 1,3-butadiene exposure is observed following 5 day exposures at 625 p.p.m. (Recio et al., 1992, 1996). These data also show that 1,3-butadiene not only induces mutation at the site of contact following inhalation exposures but is also absorbed from the lung and induces mutation in other somatic tissues as well. The bone marrow clastogenicity of 1,3-butadiene is consistent with these data but neither tumours nor mutations were seen in the liver and liver UDS was not detected in either in vivo or in vitro assays.
1-Chloromethylpyrene
1-Chloromethylpyrene is a direct-acting bacterial mutagen but has so far proven to be negative in vivo in both a mouse bone marrow micronucleus test and a rat stomach UDS assay (Ashby et al., 1990; Kennelly et al., 1993b). No carcinogenicity data are available, but based on data provided by the 32P-post-labelling technique, the local lymph node assay and the sebaceous gland suppression test, it is thought likely to be a potent skin carcinogen (Ashby et al., 1990).
There was no induction of mutation in the glandular stomach DNA of lacZ transgenic mice given a single oral dose of 25 or 50 mg/kg and examined 3, 7 or 10 days after treatment (Brooks and Dean, 1996). However, after a single topical treatment to transgenic mice, 1-chloromethylpyrene did induce gene mutation in skin DNA at both 5 and 10 µg at 7, 14 and 21 days post-exposure (Brooks and Dean, 1996). In this example, the negative mutation data in the stomach are consistent with the negative stomach UDS results, but the induction of mutation in the skin tends to confirm the suspicions of Ashby et al. (1990) that 1-chloromethylpyrene may well be a skin carcinogen, although definitive tumour data do not yet exist.
7,12-Dimethylbenzanthracene (DMBA)
DMBA is clearly mutagenic to bacteria in vitro and clastogenic to mammalian cells (Ishidate et al., 1988; Scott et al., 1991). It is mutagenic to mammalian cells in the presence of metabolic activation (Mitchell et al., 1997) and also induces UDS in cultured hepatocytes in vitro (Harbach et al., 1989). In vivo, DMBA gives a variable response in the induction of micronuclei in the bone marrow in one report, following oral gavage of a dose in excess of 5,000 mg/kg, an increase in micronuclei in bone marrow and colon, though not in bladder, lung or liver, was observed (Proudlock and Allen, 1986). More recently, Tinwell et al. (1990) used a triple dose protocol and reported more effective production of micronuclei in the bone marrow by the oral route, versus i.p., at a dose of 30 mg/kg. In an in vivo UDS assay, an oral dose of 200 mg/kg and an i.p. dose of 150 mg/kg to rats were both negative (Mirsalis et al., 1982). In carcinogenicity studies, DMBA is a potent skin carcinogen in mice following topical or s.c. application (Miller and Miller, 1963; Hennings et al., 1981) and it also induces skin papillomas when topically applied to TG.AC transgenic mice (Tennant et al., 1996).
Transgenic (lacI and lacZ) mouse studies with DMBA after a single topical treatment (Myhr, 1991; Ashby et al., 1993; Hoorn et al., 1993; Gorelick et al., 1995; Brooks and Dean, 1996) have all shown clear positive increases in MF in skin DNA at 40 or 100 µg/mouse in animals examined 7 days after exposure and after 14 days at 40 µg; increases of up to 6-fold were seen 7 days after treatment with 100 µg (Gorelick et al., 1995). One report also demonstrated increased MF in the bone marrow of DMBA topically treated mice (Hoorn et al., 1993). Clearly, DMBA is a potent site-of-contact carcinogen in rodents which can induce gene mutation and (albeit inconsistently) micronuclei in the bone marrow of mice but has not been shown to induce UDS in the livers of rats. There are unpublished data showing no increase in MF in livers of lacI mice at 7 days after one topical treatment with DMBA, (N.J.Gorelick et al., unpublished observations). However, consistent positive results are seen for mutation in the skin of transgenic mice following topical application, consistent also with the carcinogenicity data for this compound.
6,11-Dimethylbenzo[b]naphtho-[2,3-d]thiophene (S-DMBA)
S-DMBA is mutagenic to bacteria (Ashby et al., 1993) but is unable to induce micronuclei in the bone marrow of mice even after three oral doses at 2.5 g/kg. There are no UDS data available. S-DMBA was considered to be a weak skin carcinogen in mice (Miller and Miller, 1963).
A lacZ mouse study has shown a single topical treatment of S-DMBA to induce mutation in skin DNA after 7 days exposure at 500 µg, but not 10 or 100 µg (Ashby et al., 1993). In the case of S-DMBA, the results of the transgenic gene mutation assay tie in well with existing data and correlate also with carcinogenicity in that this analogue of DMBA is less active for both endpoints than DMBA itself. The comparison with DMBA is discussed in some detail by Ashby et al. (1993).
Dimethylnitrosamine (DMN)
DMN has been included in this overview because it is most certainly not a site-of-contact mutagen, although it does show clear organ specificity. DMN is an in vitro mutagen which is more effective in the presence rather than the absence of metabolic activation. It is mutagenic in bacteria (Montesano and Bartsch, 1976) and induces gene mutation and chromosomal damage in mammalian cells (Ishidate et al., 1988; Scott et al., 1991). DMN also causes cytogenetic damage in vivo (Lilly et al., 1975), although it is difficult to detect micronuclei in the standard bone marrow assay (Cliet et al., 1993; Morrison and Ashby, 1994a). DMN does induce micronuclei in the liver and testis (Cliet et al., 1989, 1993) and is commonly used as a positive control for in vivo UDS in rat liver (Kennellyet al., 1993a). However, although able to induce UDS in the liver, DMN was reported unable to induce UDS in the stomach of orally dosed rats (Ohsawa et al., 1993). In carcinogenicity studies, oral dosing with the cumulative equivalent of 89 mg/kg (50 mg/l in the drinking water) gave a range of tumours in blood vessels, lung and liver but no stomach tumours were reported (IARC, 1978) and it is generally considered to be a potent liver carcinogen (Gold et al., 1984).
Transgenic mice fed DMN in the diet at 0.25 mg/kg/day for 105 days showed no increase in mutation in the forestomach or lung but did show marked increases in mutation in the liver (Morrison and Ashby, 1994b; Shephard et al., 1995). Thus, DMN demonstrates clear organotrophy, with no UDS, gene mutation or tumours in the stomach but all three endpoints, as well as micronucleus induction, manifest in the liver following oral dosing.
N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)
In mutagenicity tests in vitro, MNNG is more potent in the absence of metabolic activation and induces mutation in bacteria and mammalian cells, chromosomal damage in mammalian cells and UDS in isolated hepatocytes (IARC, 1987b; Ishidate et al., 1988). In vivo, MNNG does induce micronuclei in mouse bone marrow (IARC, 1987b). However, the effect is more marked following i.p. dosing and only a weak response, with only some animals showing increases, was seen following oral dosing (Ashby and Mirkova, 1987). It does not induce UDS in the livers of orally dosed rats (Mirsalis et al., 1982) but has been reported to induce UDS in the gastric mucosa of rats following oral administration (Bakke and Mirsalis, 1988; Burlinson, 1989; Kennelly et al., 1993b; Ohsawa et al., 1993). Following i.p. administration UDS can be detected in the liver of rats but only in a small sub-population of cells, providing evidence that DNA damaging activity in hepatocytes occurs in the liver only at the site of first contact (Mirsalis et al., 1982). Following oral exposure of mice, tumours were seen throughout the gastrointestinal tract and topical application yielded tumours in the painted area of skin and no distant tumours (IARC, 1974a, 1987b).
In the glandular stomach DNA of the lacZ transgenic mouse, both Brault et al. (1996; 7 days post-treatment) and Brooks and Dean (1996; 3, 7 and 14 days post-treatment) showed mutations induce by MNNG given orally at 100 mg/kg and 50 and 100 mg/kg, respectively. No increases in mutation were seen in either the bone marrow or the liver. More recently, Brault et al. (1997) confirmed that MNNG induces mutation in the glandular stomach at 3, 7, 14, 28 and 50 days post-treatment and that DNA damage could also be detected within a few hours of dosing using the COMET assay. A single topical treatment gave positive increases in MF in skin DNA at 300 and 600 µg, but not at 100 µg 7 days after the exposure (Myhr, 1991) and at 250 and 500 µg in another study 7, 14 and 21 days following exposure (Brooks and Dean, 1996). However, mutations were not observed in the glandular stomach after topical treatment. Thus, MNNG is a potent site-of-contact carcinogen which causes gene mutation and UDS in the stomach following oral dosing and mutation in the skin following topical application. Furthermore, it is unable to induce UDS in the liver of orally dosed rats or mutation in the liver of orally dosed mice and the weak response in the micronucleus test with orally dosed mice is consistent with the lack of mutation in the bone marrow. For both routes of administration, the transgenic gene mutation assay results were clearly positive and consistent with the carcinogenicity data.
Methylnitrosourea (MNU)
MNU is also a potent in vitro mutagen in bacterial and mammalian cell cultures and is clastogenic to mammalian cells as well as being mutagenic in both somatic and germ cells in vivo (Amacher et al., 1980; Oberly et al., 1984; Natarajan and Obe, 1986; Ishidate et al., 1988). It also induces UDS and cell proliferation in the glandular stomach of orally dosed rats (Furihata et al., 1995) and UDS in the bone marrow of mice following i.p. administration (Bond and Singh, 1987). In cancer studies, MNU is a potent skin carcinogen in mice when applied topically (Waynforth and Magee, 1975; Iversen, 1979). Via the oral route, MNU induced tumours in the forestomach but not the glandular stomach and this correlates with the induction of micronuclei only in the forestomach and not the glandular stomach (Ronen and Heddle, 1984).
MNU fed to lacI mice at 2.38 mg/kg/day for 105 days induced an increase in mutation in the liver and in glandular stomach but not forestomach, kidney or lung (Shephard et al., 1995). Thus, the forestomach exhibits cytogenetic anomalies and tumours but the glandular stomach shows UDS and mutation.
4-Nitroquinoline-N-oxide (4NQO)
4NQO is a potent bacterial and mammalian cell mutagen and is capable of inducing chromosomal damage, gene mutation and UDS in cultured cells (Ashby, 1981; Ishidate et al., 1988, Mitchell et al., 1988). It is able to induce micronuclei in the bone marrow of mice (CSGMT, 1986) but did not induce UDS in the pyloric mucosa of orally dosed rats (Ohsawa et al., 1993). There are no published liver UDS data on 4NQO, although we can now report that 4NQO is negative in a conventional in vivo rat liver UDS assay (S.Dean and M.Fellows, unpublished observation). It is carcinogenic in rodents, yielding skin tumours following skin painting (Nakahara et al., 1957; Endo, 1971). Oral or intragastric administration leads not only to tumours in the glandular stomach but also to pulmonary cancers (Endo, 1971). Thus, in addition to site-of-contact effects, 4NQO also induces tumours at other sites.
When lacZ transgenic mice are dosed once orally with 200 mg/kg 4NQO, the most marked increases in MF are seen in the glandular stomach 7, 14 and 28 days after treatment (Coates and Dean, 1998). Large increases in MF are also seen in the bone marrow and liver, with smaller increases in the lung and an equivocal result from the testes.
ß-Propiolactone
ß-Propiolactone is a very reactive, direct-acting alkylating agent and a potent in vitro mutagen (IARC, 1974b). It is mutagenic in bacteria and mutagenic and clastogenic in mammalian cells (Brusick, 1977; Ishidate et al., 1988). Following i.p. dosing, ß-propiolactone was not able to induce micronuclei in the bone marrow, presumably due to its instability, although it did induce micronuclei in the liver and testis of mice (Cliet et al., 1989, 1993). Furthermore, Steinmetz et al. (1990) reported negative results for liver UDS and peripheral blood micronucleus in mice dosed orally on four consecutive days at up to 150 mg/kg/day.
ß-Propiolactone is carcinogenic to mice after a single dose. In dietary studies with rats, ß-propiolactone mainly induced stomach carcinomas (Van Duuren, 1969). Orally dosed rats exhibited forestomach tumours whereas application to the skin of mice led to the induction of skin tumours, leading to the conclusion that it is a direct-acting rodent carcinogen/mutagen (IARC, 1974b) which is unable to reach other organs when administered by conventional routes (Brusick, 1977).
A single oral dose (150 mg/kg) to lacZ transgenic mice showed an increase in mutation in stomach DNA 7 days after exposure (Brault et al., 1996) but no obvious effects in the bone marrow or the liver. In a more comprehensive study, mutations were seen in the glandular stomach 3, 7, 14, 28 and 50 days after treatment with ß-propiolactone (Brault et al., 1997). These mutations were preceded by DNA damage as measured using the COMET assay, which were seen from a few hours post-treatment but were no longer detectable at 3 days. Thus, the data show a correlation between the major site of tumorigenicity by the oral route (stomach) and the mutation data. Although micronuclei were detected in the liver and testis, this was following i.p. dosing. However, neither UDS nor micronuclei were detected following oral dosing of mice and these data do demonstrate the sensitivity of transgenic gene mutation assays to a direct-acting mutagen which cannot be detected using the conventional in vivo assays.
Urethane
Urethane is not considered to be a bacterial mutagen under standard testing conditions but it has been shown to induce chromosomal damage in vitro, although the results from different laboratories are inconclusive (Boyland, 1968; Ishidate et al., 1988; Sotomayor and Collins, 1990). Urethane was unable to induce gene mutation in L5178Y cells but did induce UDS in cultured cells (Brookes et al., 1981; Amacher and Turner, 1982). In vivo, it induces micronuclei in peripheral blood erythrocytes following oral or i.p. administration, but not liver UDS, in the mouse (Steinmetz et al., 1990). Neither was there any indication of UDS induction in mouse spermatogenic cells and both dominant lethal and sperm abnormality tests have proved negative (Sotomayor and Collins, 1990; Sotomayor et al., 1994). Oral dosing of mice (0.4% in the drinking water) induces multiple lung tumours with some lymphomas and liver tumours. In a study in which mice were treated three times per week for 5 weeks using 0.05 ml of a 10% solution of urethane, leukaemias, lung and liver tumours were seen.
In lacI mice, feeding 130 mg/kg/day in the diet for 105 days induced a small (1.5-fold) increase in MF in the forestomach (Shephard et al., 1995). In this study, there were more marked increases in MF in lung and liver, consistent with the tumour profile.
Are the transgenic rodent mutation assays a good alternative to existing in vivo assays?
The general answer to this question is, yes. For some of the chemicals described there are too many gaps in the data to be confident that this is the case and in other cases the mutagenic response was relatively weak. However, there are several good examples in which the in vivo mutation assays may be more appropriate and relevant than either rat liver UDS or even mouse bone marrow cytogenetics (see Table II).
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Of the three compounds which were not detected using in vivo cytogenetics, the most convincing evidence is the data for ß-propiolactone (Table II
). Cancer studies have strongly indicated that this is a site-of-contact mutagen which yields stomach and skin tumours following oral and topical treatment, respectively. ß-Propiolactone is clearly able to induce mutation in the stomach (not the bone marrow nor the liver) of orally dosed lacZ mice but is unable to induce micronuclei in the bone marrow of mice or UDS in the livers of orally dosed mice, although micronuclei were detected in the liver following i.p. dosing. 1-Chloromethylpyrene is another compound that is considered to be a possible skin carcinogen which is also mutagenic in vitro but did not induce micronuclei in mouse bone marrow. A conventional liver UDS assay was not performed, but UDS could not be detected in the stomach and neither could gene mutation. If this is due to inactivation within the stomach, then perhaps a liver UDS assay would also be inappropriate. However, application of 1-chloromethylpyrene to the skin did lead to mutation in that tissue, consistent with 1-chloromethylpyrene behaving as a skin carcinogen. Similarly, DMBA is a potent in vitro mutagen which is inconsistent in the induction of micronuclei in bone marrow and clearly negative for liver UDS. However, applied to the skin of transgenic mice, it is a potent mutagen in that tissue. MeIQ induces stomach cancer when present in the diet and induces mutation in the stomach, colon and bone marrow, although it is also a weak inducer of micronuclei in the bone marrow. No UDS data are available but, in view of there being no tumours in the liver, perhaps UDS would not be expected. The mutagenicity and cancer data for BP are comprehensive and it is clearly an in vitro clastogen, though it cannot induce UDS in the liver in vivo. The skin painting study with the lacZ mouse clearly shows that BP is mutagenic at the site of contact but that mutations do not occur in the bone marrow or liver, consistent with tumour data for the topical route. One might expect a negative outcome from an oral micronucleus test, despite the i.p. dosed study being positive. 4NQO is clearly a site-of-contact mutagen and carcinogen which also affects other tissues. It is yet another example of a mutagen which is readily detected cytogenetically in vivo and is mutagenic in vivo but which fails to induce UDS in rat liver in vivo (yet it does induce UDS in vitro). Finally, MNNG is another site-of-contact mutagen which is a very effective mutagen in skin following topical application and in stomach following oral treatment but which again cannot induce UDS in the liver, although it too is able to induce micronuclei in the bone marrow. All of these examples demonstrate circumstances in which the use of an in vivo gene mutation assay would be more relevant than or more sensitive than the conventional UDS assay and, in some examples, the micronucleus test. In the case of compounds less effective than some of these, the transgenic mutation systems would probably detect in vivo mutagens that the usual tests would miss altogether. Indeed, this may well be precisely the case for ß-propiolactone and 1-chloromethylpyrene.
For many of the other chemicals, the indications are that in vivo transgenic gene mutation assays would be an improvement in our ability to detect possible carcinogens, compared with current methods. Data for urethane are interesting since tumours and mutations were detected in the lung and liver and some evidence of mutation was seen (unexpectedly) in the stomach. Tumours tend to occur in the lung, liver and as leukaemias but, unfortunately, the bone marrow was not evaluated for mutation. The induction of gene mutation by 1,3-butadiene in the lung, bone marrow and spleen is consistent with the tumour data, although the heart was not evaluated in the transgenic studies. This compound is not particularly effective in vitro, perhaps due to technical problems associated with handling gases, but a positive bone micronucleus result was reported. In this case, both the lacI/lacZ and micronucleus tests would have identified 1,3-butadiene as an in vivo mutagen and the negative results in the liver for UDS and mutation were also consistent with the site-of-contact being an important consideration. Thus, the transgenics certainly compare favourably with the conventional in vivo tests.
Benzene is tumorigenic following inhalation exposure and the appearance of leukaemias is consistent with the positive clastogenicity data. Similarly, the presence of lung tumours agrees with the observation that mutant frequencies in the lungs of transgenic mice are increased. The data for benzene would be strengthened if mutagenicity were evaluated in both the bone marrow and liver and if a skin painting study confirmed the lack of genotoxic carcinogenic activity in the skin. Agaritine is an interesting example since both the mutagenic and carcinogenic effects seen in the stomach are measurable but weak. Indeed, the effects were confined to the forestomach and were not seen in the glandular tissue. The significance of the increased mutant frequency seen in the kidney is unclear. There are few other in vivo data for comparison and no mutation data for other tissues which could help confirm organ specificity.
MNU seems to give inconsistent results in the stomach after oral dosing, being negative for mutation in forestomach DNA but positive in glandular stomach DNA, whereas the micronucleus and tumour data are precisely the reverse. Clearly, the mechanism of MNU activity warrants further investigation.
Finally, there is an example for which the results are quite the opposite of expectations for a site-of-contact mutagen and for which there is a great deal of consistent in vivo data. Oral dosing with DMN has no mutagenic or carcinogenic effect on the stomach but, once it reaches the liver then mutagenicity is manifest as UDS, micronuclei, gene mutation and tumours (principle location). Thus, metabolism specific to the liver appears to be a controlling factor. For this chemical, gene mutation in vivo may not be more useful than UDS or cytogenetics for mutagen screening, but mutation detection in the liver is arguably more relevant to the carcinogenic process in that tissue.
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TopAbstractIntroductionThe transgenic assayMethodsDiscussion of the dataConclusionReferences |
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Transgenic mutation (TGM) assays, whether lacI or lacZ, (or, indeed, mouse or rat) provide the opportunity to examine target organs for potential carcinogenicity and this ability can be exploited by the genetic toxicologist for the testing of chemical compounds in an industrial environment. It is quite clear that several potent site-of-contact mutagens were not detected in conventional in vivo tests, yet these compounds were easily detected in the TGM models. The option to use TGM models in order to evaluate the genotoxicity of compounds which yield unexpectedly positive cancer results already exists. However, we recommend that a further and more significant application be considered: the data presented in this paper support the validity of the idea that an alternative test looking at mutations at the site of contact for compounds found positive in vitro may provide a better assessment of in vivo genotoxicity than the classical in vivo tests, in particular when the chemical will not reach either the bone marrow or the liver. A positive result in a transgenic assay is confirmation of in vivo genotoxicity in the rodent. A negative result in the TGM assay is evidence for the lack of in vivo genotoxicity in the rodent at a relevant site. Occupational exposure to foreign compounds, either accidental or therapeutic, generally involves exposure to skin, stomach or lung and the transgenic mouse systems provide a method of investigating mutagenicity in these target organs after the appropriate route of exposure. Current recommended in vivo genotoxicity assays that are acceptable to regulatory/legislative authorities, namely the chromosomal aberration or micronucleus assay and the UDS assay, both use tissues (bone marrow and liver respectively) remote from the site of exposure. Transgenic mouse assays afford the opportunity to examine the tissue(s) exposed directly as well as the tissues conventionally analysed and the data discussed in this paper serve to demonstrate the power of these tests for several specific cases.
We recommend the use of TGM systems as an alternative to the rat liver UDS assay and even the mouse bone marrow micronucleus test, where this can be justified on a case-by-case basis. Compounds which are direct-acting in vitro mutagens, those that may be rapidly metabolized, are highly reactive or are poorly absorbed and those for which target tissue is determined by route of administration could all be evaluated more effectively using gene mutation in the most appropriate tissues as the genetic endpoint.
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Acknowledgments |
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The authors are grateful to Drs John Ashby, Nancy Gorelick and David Kirkland for their comments on the manuscript
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7 To whom correspondence should be addressed. Tel: +44 1 423 500011; Fax: +44 1 423 569595; Email: stephen.dean{at}covance.com
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Received on July 16, 1998; accepted on September 15, 1998.
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